il 17a  (R&D Systems)

 
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    Name:
    Recombinant Canine IL 17A Protein CF
    Description:
    The Recombinant Canine IL 17A Protein from R D Systems is derived from E coli The Recombinant Canine IL 17A Protein has been validated for the following applications Bioactivity
    Catalog Number:
    5848-cl-025/cf
    Price:
    329
    Applications:
    Bioactivity
    Purity:
    >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
    Conjugate:
    Unconjugated
    Size:
    25 ug
    Category:
    Proteins and Enzymes
    Source:
    E. coli-derived Recombinant Canine IL-17A Protein
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    Structured Review

    R&D Systems il 17a
    SFB flagellins promote a swift, transient, Th17-related response in vivo . Mice ( n = 6) were i.p. injected with SFB flagellins or other antigens. The distal ileum and serum cytokine were assayed 2 h later. (A) Cytokine <t>IL-17A</t> concentrations detected in serum and supernatants of the distal ileum isolated from either mouse, which were i.p. with SFB flagellins or other antigens for 2 h ( n = 6). (B) Serum and intestinal IL-6 concentrations in conventional and experimental mice that received i.p. immunizations for 2 h ( n = 6). See statistical significance in Figure 1A . (C) The mRNA expression of IL-17A, IL-6 relative to Gapdh in intestinal of the indicated mice. Data are expressed as the mean ± SEM of three independent experiments. Error bars indicate median values. See statistical significance in Figure 1C .
    The Recombinant Canine IL 17A Protein from R D Systems is derived from E coli The Recombinant Canine IL 17A Protein has been validated for the following applications Bioactivity
    https://www.bioz.com/result/il 17a/product/R&D Systems
    Average 99 stars, based on 388 article reviews
    Price from $9.99 to $1999.99
    il 17a - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Induction of Intestinal Th17 Cells by Flagellins From Segmented Filamentous Bacteria"

    Article Title: Induction of Intestinal Th17 Cells by Flagellins From Segmented Filamentous Bacteria

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02750

    SFB flagellins promote a swift, transient, Th17-related response in vivo . Mice ( n = 6) were i.p. injected with SFB flagellins or other antigens. The distal ileum and serum cytokine were assayed 2 h later. (A) Cytokine IL-17A concentrations detected in serum and supernatants of the distal ileum isolated from either mouse, which were i.p. with SFB flagellins or other antigens for 2 h ( n = 6). (B) Serum and intestinal IL-6 concentrations in conventional and experimental mice that received i.p. immunizations for 2 h ( n = 6). See statistical significance in Figure 1A . (C) The mRNA expression of IL-17A, IL-6 relative to Gapdh in intestinal of the indicated mice. Data are expressed as the mean ± SEM of three independent experiments. Error bars indicate median values. See statistical significance in Figure 1C .
    Figure Legend Snippet: SFB flagellins promote a swift, transient, Th17-related response in vivo . Mice ( n = 6) were i.p. injected with SFB flagellins or other antigens. The distal ileum and serum cytokine were assayed 2 h later. (A) Cytokine IL-17A concentrations detected in serum and supernatants of the distal ileum isolated from either mouse, which were i.p. with SFB flagellins or other antigens for 2 h ( n = 6). (B) Serum and intestinal IL-6 concentrations in conventional and experimental mice that received i.p. immunizations for 2 h ( n = 6). See statistical significance in Figure 1A . (C) The mRNA expression of IL-17A, IL-6 relative to Gapdh in intestinal of the indicated mice. Data are expressed as the mean ± SEM of three independent experiments. Error bars indicate median values. See statistical significance in Figure 1C .

    Techniques Used: In Vivo, Mouse Assay, Injection, Isolation, Expressing

    Cytokine IL-17A Production by Small Intestinal Lamina Propria Cells after stimulated ex vivo with SFB flagellins and other antigens. (A) Activation of SILP CD4+ T cells by SFB-mFliC3, SFB-rFliC3, SFB-m5i-FliC3, sal-FliC3, anti-CD3, or PBS. IL-17A ELISA assay was evaluated after 24, 48, 72, and 96 h. (a–f) Represent statistical significance relative to the control group; anti-CD3 group; mFliC3 group; m5i-FliC3 group; rFliC3 group; sal-FliC3 group, respectively. (B) IL-17A ELISPOT assay of SILP CD4+ T cells from WT mice treated with SFB flagellins and other antigens. (Left) Representative ELISPOT images. (Right) Compilation of results from multiple animals. Each symbol represents cells from a separate animal. (C) The mRNA expression of IL-17A, IL-17F, IL-22, RORγt, AhR, and IL-1β relative to Gapdh in a co-culture system stimulated with SFB flagellins. Data are expressed as the mean ± SEM of three independent experiments. Error bars indicate median values. (a–e) Represent statistical significance relative to the control group; mFliC3 group; m5i-FliC3 group; rFliC3 group; sal-FliC3 group, respectively.
    Figure Legend Snippet: Cytokine IL-17A Production by Small Intestinal Lamina Propria Cells after stimulated ex vivo with SFB flagellins and other antigens. (A) Activation of SILP CD4+ T cells by SFB-mFliC3, SFB-rFliC3, SFB-m5i-FliC3, sal-FliC3, anti-CD3, or PBS. IL-17A ELISA assay was evaluated after 24, 48, 72, and 96 h. (a–f) Represent statistical significance relative to the control group; anti-CD3 group; mFliC3 group; m5i-FliC3 group; rFliC3 group; sal-FliC3 group, respectively. (B) IL-17A ELISPOT assay of SILP CD4+ T cells from WT mice treated with SFB flagellins and other antigens. (Left) Representative ELISPOT images. (Right) Compilation of results from multiple animals. Each symbol represents cells from a separate animal. (C) The mRNA expression of IL-17A, IL-17F, IL-22, RORγt, AhR, and IL-1β relative to Gapdh in a co-culture system stimulated with SFB flagellins. Data are expressed as the mean ± SEM of three independent experiments. Error bars indicate median values. (a–e) Represent statistical significance relative to the control group; mFliC3 group; m5i-FliC3 group; rFliC3 group; sal-FliC3 group, respectively.

    Techniques Used: Ex Vivo, Activation Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Mouse Assay, Expressing, Co-Culture Assay

    2) Product Images from "Increased interleukin-17A levels promote rituximab resistance by suppressing p53 expression and predict an unfavorable prognosis in patients with diffuse large B cell lymphoma"

    Article Title: Increased interleukin-17A levels promote rituximab resistance by suppressing p53 expression and predict an unfavorable prognosis in patients with diffuse large B cell lymphoma

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2018.4299

    IL-17A suppresses p53 expression in DLBCL cells. The SU-DHL-4 cells were co-cultured under 6 different conditions as described in the Materials and methods and defined as groups a-f. (A and B) Graphs of the mRNA and protein levels of p53 RT-qPCR and western blot analysis, respectively). (C) Representative western blots of p53 protein. The plots and bars in (B and C) are labeled in the same manner (groups a-g) as in (A); groups a-g in (B and C) represent the same groups as those in (A). Error bars represent standard deviation (SD). Significance was determined using single-factor analysis of variance (one-way ANOVA) with Student-Newman-Keuls/Dunnett's T3 test (3 groups). * P
    Figure Legend Snippet: IL-17A suppresses p53 expression in DLBCL cells. The SU-DHL-4 cells were co-cultured under 6 different conditions as described in the Materials and methods and defined as groups a-f. (A and B) Graphs of the mRNA and protein levels of p53 RT-qPCR and western blot analysis, respectively). (C) Representative western blots of p53 protein. The plots and bars in (B and C) are labeled in the same manner (groups a-g) as in (A); groups a-g in (B and C) represent the same groups as those in (A). Error bars represent standard deviation (SD). Significance was determined using single-factor analysis of variance (one-way ANOVA) with Student-Newman-Keuls/Dunnett's T3 test (3 groups). * P

    Techniques Used: Expressing, Cell Culture, Quantitative RT-PCR, Western Blot, Labeling, Standard Deviation

    Rituximab upregulates the level of IL-17A and the percentages of Th17 cells and IL-17 + Foxp3 + Treg cells in vitro . Co-cultures under 7 different conditions as described in the Materials and methods were defined as groups a-g. (A and B) Representative FACS plots of Th17 cells and IL-17 + Foxp3 + Treg cells in the co-cultures from each group. The numbers displayed are the percentages of Th17 cells and IL-17 + Foxp3 + Treg cells. (C-E) Graphs of the Th17 cells andIL-17 + Foxp3 + Treg cell percentages, and IL-17A in each group. The plots and bars in (B-E) are labeled in the same manner (groups a-g) as in (A); groups a-g in (B-E) represent the same groups as those in (A). Error bars represent standard deviation (SD). Significance was determined using single-factor analysis of variance (one-way ANOVA) with Student-Newman-Keuls/Dunnett's T3 test (3 groups). * P
    Figure Legend Snippet: Rituximab upregulates the level of IL-17A and the percentages of Th17 cells and IL-17 + Foxp3 + Treg cells in vitro . Co-cultures under 7 different conditions as described in the Materials and methods were defined as groups a-g. (A and B) Representative FACS plots of Th17 cells and IL-17 + Foxp3 + Treg cells in the co-cultures from each group. The numbers displayed are the percentages of Th17 cells and IL-17 + Foxp3 + Treg cells. (C-E) Graphs of the Th17 cells andIL-17 + Foxp3 + Treg cell percentages, and IL-17A in each group. The plots and bars in (B-E) are labeled in the same manner (groups a-g) as in (A); groups a-g in (B-E) represent the same groups as those in (A). Error bars represent standard deviation (SD). Significance was determined using single-factor analysis of variance (one-way ANOVA) with Student-Newman-Keuls/Dunnett's T3 test (3 groups). * P

    Techniques Used: In Vitro, FACS, Labeling, Standard Deviation

    Mechanistic outline of rituximab resistance in diffuse large B cell lymphoma (DLBCL). Rituximab induces DLBCL cells to secrete IL-6, which promotes the differentiation of Th17 and IL-17 + Foxp3 + Treg cells from CD4 + T cells, and these two types of cells then secrete IL-17A. IL-17A prevents rituximab-induced apoptosis and promotes the proliferation of diffuse large B cell lymphoma (DLBCL) cells by suppressing p53 expression.
    Figure Legend Snippet: Mechanistic outline of rituximab resistance in diffuse large B cell lymphoma (DLBCL). Rituximab induces DLBCL cells to secrete IL-6, which promotes the differentiation of Th17 and IL-17 + Foxp3 + Treg cells from CD4 + T cells, and these two types of cells then secrete IL-17A. IL-17A prevents rituximab-induced apoptosis and promotes the proliferation of diffuse large B cell lymphoma (DLBCL) cells by suppressing p53 expression.

    Techniques Used: Expressing

    Rituximab elevates the level of IL-17A and the percentages of Th17 cells and IL-17 + Foxp3 + Treg cells in the peripheral blood mononuclear cells (PBMCs) from patients with diffuse large B cell lymphoma (DLBCL). (A and B) Representative FACS plots of Th17 cells and IL-17 + Foxp3 + Treg cells in the PBMCs from each group. The numbers displayed are the percentages of Th17 cells and IL-17 + Foxp3 + Treg cells. (C and D) Graphs of the Th17 cells and IL-17 + Foxp3 + Treg cell percentages in each group. (E-H) Graphs of the levels of IL-17A, IL-6, IL-21 and TGF-β in each group. Error bars represent standard deviation (SD). Significance was determined using single-factor analysis of variance (one-way ANOVA) with Student-Newman-Keuls/Dunnett's T3 test (3 groups). * P
    Figure Legend Snippet: Rituximab elevates the level of IL-17A and the percentages of Th17 cells and IL-17 + Foxp3 + Treg cells in the peripheral blood mononuclear cells (PBMCs) from patients with diffuse large B cell lymphoma (DLBCL). (A and B) Representative FACS plots of Th17 cells and IL-17 + Foxp3 + Treg cells in the PBMCs from each group. The numbers displayed are the percentages of Th17 cells and IL-17 + Foxp3 + Treg cells. (C and D) Graphs of the Th17 cells and IL-17 + Foxp3 + Treg cell percentages in each group. (E-H) Graphs of the levels of IL-17A, IL-6, IL-21 and TGF-β in each group. Error bars represent standard deviation (SD). Significance was determined using single-factor analysis of variance (one-way ANOVA) with Student-Newman-Keuls/Dunnett's T3 test (3 groups). * P

    Techniques Used: FACS, Standard Deviation

    IL-17A prevents rituximab-induced SU-DHL-4 cell apoptosis and promotes SU-DHL-4 cell proliferation. Wild-type and IL-17 receptor-null SU-DHL-4 cells were co-cultured with rituximab (100 μ g/ml) and various concentrations of recombinant IL-17A as denoted in the graphs. (A) Representative FACS plots of Annexin V + /PI − and Annexin V + /PI + SU-DHL-4 cells in the co-cultures of each group. (B) Graphs of the frequency of apoptotic cells. (C) Western blot analysis was used to determine the knockdown efficacy of IL-17R in the SU-DHL-4 cells. (D) Graph of the frequency of SU-DHL-4 cell proliferation rates in each group. In (B and D) the bar labeled 10# indicates cells in which IL-17R was knocked down (IL-17R-KD). Error bars represent standard deviation (SD). Significance was determined using single-factor analysis of variance (one-way ANOVA) with Student-Newman-Keuls/Dunnett's T3 test (3 groups). * P
    Figure Legend Snippet: IL-17A prevents rituximab-induced SU-DHL-4 cell apoptosis and promotes SU-DHL-4 cell proliferation. Wild-type and IL-17 receptor-null SU-DHL-4 cells were co-cultured with rituximab (100 μ g/ml) and various concentrations of recombinant IL-17A as denoted in the graphs. (A) Representative FACS plots of Annexin V + /PI − and Annexin V + /PI + SU-DHL-4 cells in the co-cultures of each group. (B) Graphs of the frequency of apoptotic cells. (C) Western blot analysis was used to determine the knockdown efficacy of IL-17R in the SU-DHL-4 cells. (D) Graph of the frequency of SU-DHL-4 cell proliferation rates in each group. In (B and D) the bar labeled 10# indicates cells in which IL-17R was knocked down (IL-17R-KD). Error bars represent standard deviation (SD). Significance was determined using single-factor analysis of variance (one-way ANOVA) with Student-Newman-Keuls/Dunnett's T3 test (3 groups). * P

    Techniques Used: Cell Culture, Recombinant, FACS, Western Blot, Labeling, Standard Deviation

    3) Product Images from "The immunomodulatory effects of TNF-α inhibitors on human Th17 cells via RORγt histone acetylation"

    Article Title: The immunomodulatory effects of TNF-α inhibitors on human Th17 cells via RORγt histone acetylation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.13791

    The in vitro effects of etanercept and adalimumab on Th17-related cytokine production by Th17-polarized cells from an RA patient undergoing Enbrel™ treatment Th17-polarized cells induced from CD4 + T cells that were collected from an RA patient 2 h before (at the trough level) and 48 h after (at the peak level) of Enbrel™ injection. The in vitro effects of etanercept (1 μg/mL) and adalimumab (1 and 10 μg/mL) on IL-17A, IL-17F and IL-22 production in Th17-polarized cells were determined by ELISA. P -values indicate comparison of the cytokine levels before and after Enbrel™ injection. ( A ) IL-17A production by Th17-polarized cells isolated from the RA patient 2 h before and 48 h after Enbrel™ injection was significantly suppressed by in vitro treatment with adalimumab (1 and 10 μg/mL) but not etanercept (1 μg/mL). ( B ) IL-17F and ( C ) IL-22 production was significantly suppressed by in vitro treatment with etanercept (1 μg/mL) and adalimumab (1 μg/mL) when cells were isolated from the RA patient 2 h before but not 48 h after Enbrel™ injection. In vitro pretreatment with adalimumab (10 μg/mL) significantly suppressed IL-17F and IL-22 production both before and after Enbrel™ injection. The bars represent the means ± standard deviations from three independent experiments. * P
    Figure Legend Snippet: The in vitro effects of etanercept and adalimumab on Th17-related cytokine production by Th17-polarized cells from an RA patient undergoing Enbrel™ treatment Th17-polarized cells induced from CD4 + T cells that were collected from an RA patient 2 h before (at the trough level) and 48 h after (at the peak level) of Enbrel™ injection. The in vitro effects of etanercept (1 μg/mL) and adalimumab (1 and 10 μg/mL) on IL-17A, IL-17F and IL-22 production in Th17-polarized cells were determined by ELISA. P -values indicate comparison of the cytokine levels before and after Enbrel™ injection. ( A ) IL-17A production by Th17-polarized cells isolated from the RA patient 2 h before and 48 h after Enbrel™ injection was significantly suppressed by in vitro treatment with adalimumab (1 and 10 μg/mL) but not etanercept (1 μg/mL). ( B ) IL-17F and ( C ) IL-22 production was significantly suppressed by in vitro treatment with etanercept (1 μg/mL) and adalimumab (1 μg/mL) when cells were isolated from the RA patient 2 h before but not 48 h after Enbrel™ injection. In vitro pretreatment with adalimumab (10 μg/mL) significantly suppressed IL-17F and IL-22 production both before and after Enbrel™ injection. The bars represent the means ± standard deviations from three independent experiments. * P

    Techniques Used: In Vitro, Injection, Enzyme-linked Immunosorbent Assay, Isolation

    The effects of etanercept and adalimumab on IL-17A, IL-17F and IL-22 production in Th17-polarized cells from patients with RA The levels of Th17-related cytokines, including ( A ) IL-17A, ( B ) IL-17F and ( C ) IL-22, in the supernatants of Th17-polarized cells from four healthy donors (HD) and six patients with RA that were pretreated in vitro with or without etanercept (1 μg/ml) or adalimumab (1 or 10 μg/ml) were determined by ELISA. Horizontal bars indicate the median. * P
    Figure Legend Snippet: The effects of etanercept and adalimumab on IL-17A, IL-17F and IL-22 production in Th17-polarized cells from patients with RA The levels of Th17-related cytokines, including ( A ) IL-17A, ( B ) IL-17F and ( C ) IL-22, in the supernatants of Th17-polarized cells from four healthy donors (HD) and six patients with RA that were pretreated in vitro with or without etanercept (1 μg/ml) or adalimumab (1 or 10 μg/ml) were determined by ELISA. Horizontal bars indicate the median. * P

    Techniques Used: In Vitro, Enzyme-linked Immunosorbent Assay

    The suppressive effects of etanercept and adalimumab on IL-17A, IL-17F and IL-22 expression in human Th17-polarized cells through MAPK pathways ( A ) Human Th17-polarized cells were induced from purified CD4 + T cells from healthy subjects. Pretreatment with SB203580 (a p38 inhibitor, 10 −6 –10 −5 M), SP600125 (a JNK inhibitor, 10 -5 M) or PD98059 (an ERK inhibitor, 10 −5 M) significantly suppressed IL-17A expression in Th17-polarized cells. ( B ) SB203580 (10 -6 M) and SP600125 (10 −5 M) significantly suppressed IL-17F expression in human Th17-polarized cells. ( C ) Pretreatment with SB203580 (10 −5 M), SP600125 (10 −6 –10 −5 M) and PD98059 (10 −6 –10 −5 M) could significantly suppress IL-22 expression in Th17-polarized cells. # P
    Figure Legend Snippet: The suppressive effects of etanercept and adalimumab on IL-17A, IL-17F and IL-22 expression in human Th17-polarized cells through MAPK pathways ( A ) Human Th17-polarized cells were induced from purified CD4 + T cells from healthy subjects. Pretreatment with SB203580 (a p38 inhibitor, 10 −6 –10 −5 M), SP600125 (a JNK inhibitor, 10 -5 M) or PD98059 (an ERK inhibitor, 10 −5 M) significantly suppressed IL-17A expression in Th17-polarized cells. ( B ) SB203580 (10 -6 M) and SP600125 (10 −5 M) significantly suppressed IL-17F expression in human Th17-polarized cells. ( C ) Pretreatment with SB203580 (10 −5 M), SP600125 (10 −6 –10 −5 M) and PD98059 (10 −6 –10 −5 M) could significantly suppress IL-22 expression in Th17-polarized cells. # P

    Techniques Used: Expressing, Purification

    Etanercept and adalimumab suppress IL-17A and IL-17F production in human Th17-polarized T cells Human naïve CD4 + T cells polarized towards the Th17 phenotype in the presence of rhIL-2, rhIL-1β, rhIL-23, rhTGF- β and rhIL-6 with anti-hCD3, anti-hCD28, anti-hIL-4, and anti-hINF-γ, and then were cultured for 3 days or 5 days. Control naïve T cells were cultured only in the presence of anti-hCD3, anti-hCD28 and rhIL-2. The supernatants were collected for ( A ) IL-17A and ( B ) IL-17F detection by ELISA. The results represent the means ± standard deviations of three independent experiments. Pretreatment with etanercept (0.1 and 1 μg/ml) or adalimumab (1 and 10 μg/ml) suppressed ( C ) IL-17A and ( D ) IL-17F production in human CD4 + T cells from a healthy individual after 5 days of Th17-polarized conditions. The results represent the means ± standard deviations of three experiments. * P
    Figure Legend Snippet: Etanercept and adalimumab suppress IL-17A and IL-17F production in human Th17-polarized T cells Human naïve CD4 + T cells polarized towards the Th17 phenotype in the presence of rhIL-2, rhIL-1β, rhIL-23, rhTGF- β and rhIL-6 with anti-hCD3, anti-hCD28, anti-hIL-4, and anti-hINF-γ, and then were cultured for 3 days or 5 days. Control naïve T cells were cultured only in the presence of anti-hCD3, anti-hCD28 and rhIL-2. The supernatants were collected for ( A ) IL-17A and ( B ) IL-17F detection by ELISA. The results represent the means ± standard deviations of three independent experiments. Pretreatment with etanercept (0.1 and 1 μg/ml) or adalimumab (1 and 10 μg/ml) suppressed ( C ) IL-17A and ( D ) IL-17F production in human CD4 + T cells from a healthy individual after 5 days of Th17-polarized conditions. The results represent the means ± standard deviations of three experiments. * P

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    4) Product Images from "IL-17A and TNF-α Increase the Expression of the Antiapoptotic Adhesion Molecule Amigo-2 in Arthritis Synoviocytes"

    Article Title: IL-17A and TNF-α Increase the Expression of the Antiapoptotic Adhesion Molecule Amigo-2 in Arthritis Synoviocytes

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2016.00254

    Amigo2 expression levels correlate with cell death . Healthy, OA and RA synoviocytes were preexposed to a combination of IL-17A and TNF-α overnight before addition of a low dose of the proapoptotic agent Cd (A,B,D) . Amigo2 expression was then evaluated 6 h after Cd addition by quantitative real-time PCR and is expressed as fold changes compared to healthy synoviocytes exposed to vehicle (A) . Cell death was determined by cell cycle analysis after a week (B) . To evaluate the effect of HMGB1 on cell death, RA synoviocytes were preexposed to a combination of IL-17A and TNF-α in the presence or not of HMGB1 followed by Cd addition. Cell death was quantified by cell cycle analysis after a week and is represented as fold changes compared to cells treated with Cd and cytokines (C) . Similarly to Amigo2, Bcl2 expression was evaluated by quantitative real-time PCR and is expressed as fold changes compared to healthy synoviocytes exposed to vehicle (D) . Data are the mean of at least three independent experiments ± SEM. Cyt, cytokines; H, HMGB1. # Comparison with control situation, *comparison with cytokine treated cells, § comparison between cell types. *,# P ≤ 0.05, **,§§ P ≤ 0.01, ***,###,§§§ P ≤ 0.001.
    Figure Legend Snippet: Amigo2 expression levels correlate with cell death . Healthy, OA and RA synoviocytes were preexposed to a combination of IL-17A and TNF-α overnight before addition of a low dose of the proapoptotic agent Cd (A,B,D) . Amigo2 expression was then evaluated 6 h after Cd addition by quantitative real-time PCR and is expressed as fold changes compared to healthy synoviocytes exposed to vehicle (A) . Cell death was determined by cell cycle analysis after a week (B) . To evaluate the effect of HMGB1 on cell death, RA synoviocytes were preexposed to a combination of IL-17A and TNF-α in the presence or not of HMGB1 followed by Cd addition. Cell death was quantified by cell cycle analysis after a week and is represented as fold changes compared to cells treated with Cd and cytokines (C) . Similarly to Amigo2, Bcl2 expression was evaluated by quantitative real-time PCR and is expressed as fold changes compared to healthy synoviocytes exposed to vehicle (D) . Data are the mean of at least three independent experiments ± SEM. Cyt, cytokines; H, HMGB1. # Comparison with control situation, *comparison with cytokine treated cells, § comparison between cell types. *,# P ≤ 0.05, **,§§ P ≤ 0.01, ***,###,§§§ P ≤ 0.001.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Cell Cycle Assay

    Coculture of RA synoviocytes with immune cells increases Amigo2 expression in both cell types . RA synoviocytes were cocultured with PBMC from healthy donors in the presence or not of PHA for 24 h. In the cocultures, PBMC were separated from synoviocytes by EDTA addition prior cell lysis. Amigo2 expression was assessed by quantitative real-time PCR and was expressed as fold changes compared to synoviocytes cultured alone and exposed to vehicle (A,B) . Amigo2 expression was evaluated in both synoviocytes (A) and PBMC (B) cultured alone or together. The production of IL-17A (C) and TNF-α (D) by the cocultures was quantified by ELISA. The production of these cytokines was not detectable in synoviocytes cultured alone. Data are the mean of at least three independent experiments ± SEM. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.
    Figure Legend Snippet: Coculture of RA synoviocytes with immune cells increases Amigo2 expression in both cell types . RA synoviocytes were cocultured with PBMC from healthy donors in the presence or not of PHA for 24 h. In the cocultures, PBMC were separated from synoviocytes by EDTA addition prior cell lysis. Amigo2 expression was assessed by quantitative real-time PCR and was expressed as fold changes compared to synoviocytes cultured alone and exposed to vehicle (A,B) . Amigo2 expression was evaluated in both synoviocytes (A) and PBMC (B) cultured alone or together. The production of IL-17A (C) and TNF-α (D) by the cocultures was quantified by ELISA. The production of these cytokines was not detectable in synoviocytes cultured alone. Data are the mean of at least three independent experiments ± SEM. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

    Techniques Used: Expressing, Lysis, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

    Amigo2 induction in RA synoviocytes cocultured with immune cells remains stable even after immune cell removal . RA synoviocytes were cocultured with PBMC from healthy donors in the presence or not of PHA for 24 h. In the cocultures, PBMC were separated from synoviocytes by EDTA addition. Synoviocytes were cultured for 24 or 48 h more after PBMC removal. Amigo2 expression was assessed at different time-points by quantitative real-time PCR and was expressed as fold changes compared to RA synoviocytes cultured alone and exposed to vehicle (A) . The production of IL-17A (B) and TNF-α (C) by the cocultures was quantified by ELISA. The production of these cytokines was measured in cocultures with no immune cell removal (cocultures) or with immune cell removal by EDTA (cocultures EDTA). Their production was not detectable in synoviocytes cultured alone. Data are the mean of at least three independent experiments ± SEM. # comparison with control situation, *comparison between synoviocytes (A) or PBMC (B) alone and cocultures, § comparison between cocultures with or without EDTA addition. *,#,§ P ≤ 0.05, **,##,§§ P ≤ 0.01, ***, ### P ≤ 0.001.
    Figure Legend Snippet: Amigo2 induction in RA synoviocytes cocultured with immune cells remains stable even after immune cell removal . RA synoviocytes were cocultured with PBMC from healthy donors in the presence or not of PHA for 24 h. In the cocultures, PBMC were separated from synoviocytes by EDTA addition. Synoviocytes were cultured for 24 or 48 h more after PBMC removal. Amigo2 expression was assessed at different time-points by quantitative real-time PCR and was expressed as fold changes compared to RA synoviocytes cultured alone and exposed to vehicle (A) . The production of IL-17A (B) and TNF-α (C) by the cocultures was quantified by ELISA. The production of these cytokines was measured in cocultures with no immune cell removal (cocultures) or with immune cell removal by EDTA (cocultures EDTA). Their production was not detectable in synoviocytes cultured alone. Data are the mean of at least three independent experiments ± SEM. # comparison with control situation, *comparison between synoviocytes (A) or PBMC (B) alone and cocultures, § comparison between cocultures with or without EDTA addition. *,#,§ P ≤ 0.05, **,##,§§ P ≤ 0.01, ***, ### P ≤ 0.001.

    Techniques Used: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Amigo2 expression is regulated by MAPKs and HMGB1 . RA synoviocytes were preexposed to MAPKs inhibitors for 1 h followed by the addition of a combination of IL-17A and TNF-α (A) or co-exposed to HMGB1 and the TNF/IL-17A combination (B) . After 12 h, Amigo2 expression was assessed by quantitative real-time PCR and is expressed as fold changes compared to control situation. SP6000125, JNK inhibitor; SB203580, p38 inhibitor; U0125, ERK inhibitor; Ctl, control; Cyt, cytokines; H, HMGB1. Data are the mean of at least three independent experiments ± SEM. # Comparison with control situation, *comparison with cytokine-treated cells. *, # P ≤ 0.05, ** P ≤ 0.01.
    Figure Legend Snippet: Amigo2 expression is regulated by MAPKs and HMGB1 . RA synoviocytes were preexposed to MAPKs inhibitors for 1 h followed by the addition of a combination of IL-17A and TNF-α (A) or co-exposed to HMGB1 and the TNF/IL-17A combination (B) . After 12 h, Amigo2 expression was assessed by quantitative real-time PCR and is expressed as fold changes compared to control situation. SP6000125, JNK inhibitor; SB203580, p38 inhibitor; U0125, ERK inhibitor; Ctl, control; Cyt, cytokines; H, HMGB1. Data are the mean of at least three independent experiments ± SEM. # Comparison with control situation, *comparison with cytokine-treated cells. *, # P ≤ 0.05, ** P ≤ 0.01.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, CTL Assay

    Model of the regulation of Amigo2 expression which promotes cell survival and proliferation . A model was proposed whereby cellular interaction with immune cells and exposure of the cells to the pro-inflammatory cytokines IL-17A and TNF-α as well as to HMGB1 increases Amigo2 expression in an ERK-dependent manner leading to enhanced cellular adhesion and promoting cell survival and proliferation.
    Figure Legend Snippet: Model of the regulation of Amigo2 expression which promotes cell survival and proliferation . A model was proposed whereby cellular interaction with immune cells and exposure of the cells to the pro-inflammatory cytokines IL-17A and TNF-α as well as to HMGB1 increases Amigo2 expression in an ERK-dependent manner leading to enhanced cellular adhesion and promoting cell survival and proliferation.

    Techniques Used: Expressing

    Amigo2 is upregulated more specifically in RA synoviocytes in inflammatory conditions . Synoviocytes were exposed to IL-17A or TNF-α alone or to a combination of both for 12 h. Amigo2 and Amigo3 expressions were assessed by transcriptomic analysis (A,B) and Amigo2 expression was also assessed by quantitative real-time PCR (C,D) . Gene expression was compared between the different treatments and is represented as fold changes compared to control in RA synoviocytes (A–C) . Amigo2 expression was also evaluated in synoviocytes from different clinical settings (healthy, OA and RA) and is expressed as fold changes compared to healthy synoviocytes exposed to vehicle (D) . Data are the mean of at least three independent experiments ± SEM. # Comparison with control situation, *comparison between different cytokine combinations (C) or between cell types (D) . * P ≤ 0.05, ** P ≤ 0.01, ***, ### P ≤ 0.001.
    Figure Legend Snippet: Amigo2 is upregulated more specifically in RA synoviocytes in inflammatory conditions . Synoviocytes were exposed to IL-17A or TNF-α alone or to a combination of both for 12 h. Amigo2 and Amigo3 expressions were assessed by transcriptomic analysis (A,B) and Amigo2 expression was also assessed by quantitative real-time PCR (C,D) . Gene expression was compared between the different treatments and is represented as fold changes compared to control in RA synoviocytes (A–C) . Amigo2 expression was also evaluated in synoviocytes from different clinical settings (healthy, OA and RA) and is expressed as fold changes compared to healthy synoviocytes exposed to vehicle (D) . Data are the mean of at least three independent experiments ± SEM. # Comparison with control situation, *comparison between different cytokine combinations (C) or between cell types (D) . * P ≤ 0.05, ** P ≤ 0.01, ***, ### P ≤ 0.001.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    5) Product Images from "ROCK insufficiency attenuates ozone-induced airway hyperresponsiveness in mice"

    Article Title: ROCK insufficiency attenuates ozone-induced airway hyperresponsiveness in mice

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00372.2014

    Effect of ROCK1- and ROCK2-haploinsufficient mice on factors contributing to O 3 -induced AHR in WT mice. Bronchoalveolar lavage (BAL) concentrations of IL-17A ( A ), TNFα ( B ), osteopontin ( C ), hyaluronic acid (HA; D ), and pulmonary mRNA abundance
    Figure Legend Snippet: Effect of ROCK1- and ROCK2-haploinsufficient mice on factors contributing to O 3 -induced AHR in WT mice. Bronchoalveolar lavage (BAL) concentrations of IL-17A ( A ), TNFα ( B ), osteopontin ( C ), hyaluronic acid (HA; D ), and pulmonary mRNA abundance

    Techniques Used: Mouse Assay

    6) Product Images from "IL-17A Inhibits Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Wnt Signaling Pathway"

    Article Title: IL-17A Inhibits Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Wnt Signaling Pathway

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.903027

    Knockdown of the expression of sFRP1 abolished the inhibition effect of IL-17A in osteogenic differentiation of BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by the qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; ( E ) The ALP staining and Alizarin Red staining were used to analyze osteogenic differentiation level of BMSCs; O – osteogenic differentiation medium; O+I – osteogenic differentiation medium with IL-17A; O+I+shRNA – osteogenic differentiation medium with IL-17A+ sFRP1-shRNA; * P value
    Figure Legend Snippet: Knockdown of the expression of sFRP1 abolished the inhibition effect of IL-17A in osteogenic differentiation of BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by the qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; ( E ) The ALP staining and Alizarin Red staining were used to analyze osteogenic differentiation level of BMSCs; O – osteogenic differentiation medium; O+I – osteogenic differentiation medium with IL-17A; O+I+shRNA – osteogenic differentiation medium with IL-17A+ sFRP1-shRNA; * P value

    Techniques Used: Expressing, Inhibition, Quantitative RT-PCR, Western Blot, ALP Assay, Staining, shRNA

    IL-17A inhibited osteogenic differentiation of BMSCs; ( A–C ) The osteoblastic markers were detected by qRT-PCR method; ( D ) The osteoblastic markers were detected by Western blotting; ( E ) ALP staining and Alizarin Red staining were used to assess osteogenic differentiation level of BMSCs; OM – osteogenic differentiation medium; OM+IL-17A – osteogenic differentiation medium with IL-17A; D1 – 1 day; D15 – 15 days; * compared with before induction of differentiation, P value
    Figure Legend Snippet: IL-17A inhibited osteogenic differentiation of BMSCs; ( A–C ) The osteoblastic markers were detected by qRT-PCR method; ( D ) The osteoblastic markers were detected by Western blotting; ( E ) ALP staining and Alizarin Red staining were used to assess osteogenic differentiation level of BMSCs; OM – osteogenic differentiation medium; OM+IL-17A – osteogenic differentiation medium with IL-17A; D1 – 1 day; D15 – 15 days; * compared with before induction of differentiation, P value

    Techniques Used: Quantitative RT-PCR, Western Blot, ALP Assay, Staining

    IL-17A blocked the Wnt signaling pathway in BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; D1 – 1 day; D3 – 3 days; D7 – 7 days; D15 – 15 days; *: P value
    Figure Legend Snippet: IL-17A blocked the Wnt signaling pathway in BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; D1 – 1 day; D3 – 3 days; D7 – 7 days; D15 – 15 days; *: P value

    Techniques Used: Quantitative RT-PCR, Western Blot

    7) Product Images from "IL-17A Inhibits Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Wnt Signaling Pathway"

    Article Title: IL-17A Inhibits Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Wnt Signaling Pathway

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.903027

    Knockdown of the expression of sFRP1 abolished the inhibition effect of IL-17A in osteogenic differentiation of BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by the qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; ( E ) The ALP staining and Alizarin Red staining were used to analyze osteogenic differentiation level of BMSCs; O – osteogenic differentiation medium; O+I – osteogenic differentiation medium with IL-17A; O+I+shRNA – osteogenic differentiation medium with IL-17A+ sFRP1-shRNA; * P value
    Figure Legend Snippet: Knockdown of the expression of sFRP1 abolished the inhibition effect of IL-17A in osteogenic differentiation of BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by the qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; ( E ) The ALP staining and Alizarin Red staining were used to analyze osteogenic differentiation level of BMSCs; O – osteogenic differentiation medium; O+I – osteogenic differentiation medium with IL-17A; O+I+shRNA – osteogenic differentiation medium with IL-17A+ sFRP1-shRNA; * P value

    Techniques Used: Expressing, Inhibition, Quantitative RT-PCR, Western Blot, ALP Assay, Staining, shRNA

    IL-17A inhibited osteogenic differentiation of BMSCs; ( A–C ) The osteoblastic markers were detected by qRT-PCR method; ( D ) The osteoblastic markers were detected by Western blotting; ( E ) ALP staining and Alizarin Red staining were used to assess osteogenic differentiation level of BMSCs; OM – osteogenic differentiation medium; OM+IL-17A – osteogenic differentiation medium with IL-17A; D1 – 1 day; D15 – 15 days; * compared with before induction of differentiation, P value
    Figure Legend Snippet: IL-17A inhibited osteogenic differentiation of BMSCs; ( A–C ) The osteoblastic markers were detected by qRT-PCR method; ( D ) The osteoblastic markers were detected by Western blotting; ( E ) ALP staining and Alizarin Red staining were used to assess osteogenic differentiation level of BMSCs; OM – osteogenic differentiation medium; OM+IL-17A – osteogenic differentiation medium with IL-17A; D1 – 1 day; D15 – 15 days; * compared with before induction of differentiation, P value

    Techniques Used: Quantitative RT-PCR, Western Blot, ALP Assay, Staining

    IL-17A blocked the Wnt signaling pathway in BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; D1 – 1 day; D3 – 3 days; D7 – 7 days; D15 – 15 days; *: P value
    Figure Legend Snippet: IL-17A blocked the Wnt signaling pathway in BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; D1 – 1 day; D3 – 3 days; D7 – 7 days; D15 – 15 days; *: P value

    Techniques Used: Quantitative RT-PCR, Western Blot

    8) Product Images from "IL-17A Inhibits Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Wnt Signaling Pathway"

    Article Title: IL-17A Inhibits Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Wnt Signaling Pathway

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.903027

    Knockdown of the expression of sFRP1 abolished the inhibition effect of IL-17A in osteogenic differentiation of BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by the qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; ( E ) The ALP staining and Alizarin Red staining were used to analyze osteogenic differentiation level of BMSCs; O – osteogenic differentiation medium; O+I – osteogenic differentiation medium with IL-17A; O+I+shRNA – osteogenic differentiation medium with IL-17A+ sFRP1-shRNA; * P value
    Figure Legend Snippet: Knockdown of the expression of sFRP1 abolished the inhibition effect of IL-17A in osteogenic differentiation of BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by the qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; ( E ) The ALP staining and Alizarin Red staining were used to analyze osteogenic differentiation level of BMSCs; O – osteogenic differentiation medium; O+I – osteogenic differentiation medium with IL-17A; O+I+shRNA – osteogenic differentiation medium with IL-17A+ sFRP1-shRNA; * P value

    Techniques Used: Expressing, Inhibition, Quantitative RT-PCR, Western Blot, ALP Assay, Staining, shRNA

    IL-17A inhibited osteogenic differentiation of BMSCs; ( A–C ) The osteoblastic markers were detected by qRT-PCR method; ( D ) The osteoblastic markers were detected by Western blotting; ( E ) ALP staining and Alizarin Red staining were used to assess osteogenic differentiation level of BMSCs; OM – osteogenic differentiation medium; OM+IL-17A – osteogenic differentiation medium with IL-17A; D1 – 1 day; D15 – 15 days; * compared with before induction of differentiation, P value
    Figure Legend Snippet: IL-17A inhibited osteogenic differentiation of BMSCs; ( A–C ) The osteoblastic markers were detected by qRT-PCR method; ( D ) The osteoblastic markers were detected by Western blotting; ( E ) ALP staining and Alizarin Red staining were used to assess osteogenic differentiation level of BMSCs; OM – osteogenic differentiation medium; OM+IL-17A – osteogenic differentiation medium with IL-17A; D1 – 1 day; D15 – 15 days; * compared with before induction of differentiation, P value

    Techniques Used: Quantitative RT-PCR, Western Blot, ALP Assay, Staining

    IL-17A blocked the Wnt signaling pathway in BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; D1 – 1 day; D3 – 3 days; D7 – 7 days; D15 – 15 days; *: P value
    Figure Legend Snippet: IL-17A blocked the Wnt signaling pathway in BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; D1 – 1 day; D3 – 3 days; D7 – 7 days; D15 – 15 days; *: P value

    Techniques Used: Quantitative RT-PCR, Western Blot

    9) Product Images from "IL-17A Inhibits Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Wnt Signaling Pathway"

    Article Title: IL-17A Inhibits Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Wnt Signaling Pathway

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.903027

    Knockdown of the expression of sFRP1 abolished the inhibition effect of IL-17A in osteogenic differentiation of BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by the qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; ( E ) The ALP staining and Alizarin Red staining were used to analyze osteogenic differentiation level of BMSCs; O – osteogenic differentiation medium; O+I – osteogenic differentiation medium with IL-17A; O+I+shRNA – osteogenic differentiation medium with IL-17A+ sFRP1-shRNA; * P value
    Figure Legend Snippet: Knockdown of the expression of sFRP1 abolished the inhibition effect of IL-17A in osteogenic differentiation of BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by the qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; ( E ) The ALP staining and Alizarin Red staining were used to analyze osteogenic differentiation level of BMSCs; O – osteogenic differentiation medium; O+I – osteogenic differentiation medium with IL-17A; O+I+shRNA – osteogenic differentiation medium with IL-17A+ sFRP1-shRNA; * P value

    Techniques Used: Expressing, Inhibition, Quantitative RT-PCR, Western Blot, ALP Assay, Staining, shRNA

    IL-17A inhibited osteogenic differentiation of BMSCs; ( A–C ) The osteoblastic markers were detected by qRT-PCR method; ( D ) The osteoblastic markers were detected by Western blotting; ( E ) ALP staining and Alizarin Red staining were used to assess osteogenic differentiation level of BMSCs; OM – osteogenic differentiation medium; OM+IL-17A – osteogenic differentiation medium with IL-17A; D1 – 1 day; D15 – 15 days; * compared with before induction of differentiation, P value
    Figure Legend Snippet: IL-17A inhibited osteogenic differentiation of BMSCs; ( A–C ) The osteoblastic markers were detected by qRT-PCR method; ( D ) The osteoblastic markers were detected by Western blotting; ( E ) ALP staining and Alizarin Red staining were used to assess osteogenic differentiation level of BMSCs; OM – osteogenic differentiation medium; OM+IL-17A – osteogenic differentiation medium with IL-17A; D1 – 1 day; D15 – 15 days; * compared with before induction of differentiation, P value

    Techniques Used: Quantitative RT-PCR, Western Blot, ALP Assay, Staining

    IL-17A blocked the Wnt signaling pathway in BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; D1 – 1 day; D3 – 3 days; D7 – 7 days; D15 – 15 days; *: P value
    Figure Legend Snippet: IL-17A blocked the Wnt signaling pathway in BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; D1 – 1 day; D3 – 3 days; D7 – 7 days; D15 – 15 days; *: P value

    Techniques Used: Quantitative RT-PCR, Western Blot

    10) Product Images from "IL-17A Inhibits Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Wnt Signaling Pathway"

    Article Title: IL-17A Inhibits Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Wnt Signaling Pathway

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.903027

    Knockdown of the expression of sFRP1 abolished the inhibition effect of IL-17A in osteogenic differentiation of BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by the qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; ( E ) The ALP staining and Alizarin Red staining were used to analyze osteogenic differentiation level of BMSCs; O – osteogenic differentiation medium; O+I – osteogenic differentiation medium with IL-17A; O+I+shRNA – osteogenic differentiation medium with IL-17A+ sFRP1-shRNA; * P value
    Figure Legend Snippet: Knockdown of the expression of sFRP1 abolished the inhibition effect of IL-17A in osteogenic differentiation of BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by the qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; ( E ) The ALP staining and Alizarin Red staining were used to analyze osteogenic differentiation level of BMSCs; O – osteogenic differentiation medium; O+I – osteogenic differentiation medium with IL-17A; O+I+shRNA – osteogenic differentiation medium with IL-17A+ sFRP1-shRNA; * P value

    Techniques Used: Expressing, Inhibition, Quantitative RT-PCR, Western Blot, ALP Assay, Staining, shRNA

    IL-17A inhibited osteogenic differentiation of BMSCs; ( A–C ) The osteoblastic markers were detected by qRT-PCR method; ( D ) The osteoblastic markers were detected by Western blotting; ( E ) ALP staining and Alizarin Red staining were used to assess osteogenic differentiation level of BMSCs; OM – osteogenic differentiation medium; OM+IL-17A – osteogenic differentiation medium with IL-17A; D1 – 1 day; D15 – 15 days; * compared with before induction of differentiation, P value
    Figure Legend Snippet: IL-17A inhibited osteogenic differentiation of BMSCs; ( A–C ) The osteoblastic markers were detected by qRT-PCR method; ( D ) The osteoblastic markers were detected by Western blotting; ( E ) ALP staining and Alizarin Red staining were used to assess osteogenic differentiation level of BMSCs; OM – osteogenic differentiation medium; OM+IL-17A – osteogenic differentiation medium with IL-17A; D1 – 1 day; D15 – 15 days; * compared with before induction of differentiation, P value

    Techniques Used: Quantitative RT-PCR, Western Blot, ALP Assay, Staining

    IL-17A blocked the Wnt signaling pathway in BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; D1 – 1 day; D3 – 3 days; D7 – 7 days; D15 – 15 days; *: P value
    Figure Legend Snippet: IL-17A blocked the Wnt signaling pathway in BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; D1 – 1 day; D3 – 3 days; D7 – 7 days; D15 – 15 days; *: P value

    Techniques Used: Quantitative RT-PCR, Western Blot

    11) Product Images from "IL-17A Inhibits Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Wnt Signaling Pathway"

    Article Title: IL-17A Inhibits Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Wnt Signaling Pathway

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.903027

    Knockdown of the expression of sFRP1 abolished the inhibition effect of IL-17A in osteogenic differentiation of BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by the qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; ( E ) The ALP staining and Alizarin Red staining were used to analyze osteogenic differentiation level of BMSCs; O – osteogenic differentiation medium; O+I – osteogenic differentiation medium with IL-17A; O+I+shRNA – osteogenic differentiation medium with IL-17A+ sFRP1-shRNA; * P value
    Figure Legend Snippet: Knockdown of the expression of sFRP1 abolished the inhibition effect of IL-17A in osteogenic differentiation of BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by the qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; ( E ) The ALP staining and Alizarin Red staining were used to analyze osteogenic differentiation level of BMSCs; O – osteogenic differentiation medium; O+I – osteogenic differentiation medium with IL-17A; O+I+shRNA – osteogenic differentiation medium with IL-17A+ sFRP1-shRNA; * P value

    Techniques Used: Expressing, Inhibition, Quantitative RT-PCR, Western Blot, ALP Assay, Staining, shRNA

    IL-17A inhibited osteogenic differentiation of BMSCs; ( A–C ) The osteoblastic markers were detected by qRT-PCR method; ( D ) The osteoblastic markers were detected by Western blotting; ( E ) ALP staining and Alizarin Red staining were used to assess osteogenic differentiation level of BMSCs; OM – osteogenic differentiation medium; OM+IL-17A – osteogenic differentiation medium with IL-17A; D1 – 1 day; D15 – 15 days; * compared with before induction of differentiation, P value
    Figure Legend Snippet: IL-17A inhibited osteogenic differentiation of BMSCs; ( A–C ) The osteoblastic markers were detected by qRT-PCR method; ( D ) The osteoblastic markers were detected by Western blotting; ( E ) ALP staining and Alizarin Red staining were used to assess osteogenic differentiation level of BMSCs; OM – osteogenic differentiation medium; OM+IL-17A – osteogenic differentiation medium with IL-17A; D1 – 1 day; D15 – 15 days; * compared with before induction of differentiation, P value

    Techniques Used: Quantitative RT-PCR, Western Blot, ALP Assay, Staining

    IL-17A blocked the Wnt signaling pathway in BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; D1 – 1 day; D3 – 3 days; D7 – 7 days; D15 – 15 days; *: P value
    Figure Legend Snippet: IL-17A blocked the Wnt signaling pathway in BMSCs; ( A–C ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by qRT-PCR method; ( D ) The Wnt signaling pathway inhibitor (sFRP1) and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by Western blotting; D1 – 1 day; D3 – 3 days; D7 – 7 days; D15 – 15 days; *: P value

    Techniques Used: Quantitative RT-PCR, Western Blot

    12) Product Images from "Brain micro-inflammation at specific vessels dysregulates organ-homeostasis via the activation of a new neural circuit"

    Article Title: Brain micro-inflammation at specific vessels dysregulates organ-homeostasis via the activation of a new neural circuit

    Journal: eLife

    doi: 10.7554/eLife.25517

    The mortality was not affected by anti-CCL5 antibody treatment in cytokines-microinjected mice under stress condition. Percentages of mortality in mice under stress condition 2 days after mic roinjection of IL-6 and IL-17A at specific vessels of the boundary area of the third ventricle region, thalamus, and dentate gyrus with or without anti-CCL5 antibody treatment. Mean scores ± SEM are shown. Statistical significance was determined by ANOVA tests. (n.s.: not significant). Experiments were performed at least three times; representative data are shown. DOI: http://dx.doi.org/10.7554/eLife.25517.017
    Figure Legend Snippet: The mortality was not affected by anti-CCL5 antibody treatment in cytokines-microinjected mice under stress condition. Percentages of mortality in mice under stress condition 2 days after mic roinjection of IL-6 and IL-17A at specific vessels of the boundary area of the third ventricle region, thalamus, and dentate gyrus with or without anti-CCL5 antibody treatment. Mean scores ± SEM are shown. Statistical significance was determined by ANOVA tests. (n.s.: not significant). Experiments were performed at least three times; representative data are shown. DOI: http://dx.doi.org/10.7554/eLife.25517.017

    Techniques Used: Mouse Assay

    The mortality was not affected by corticosteroid receptor antagonist treatment in cytokine-microinjected mice under stress condition. Bloody stool scores and percentages of mortality in mice under stress condition 2 days after direct microinjection of IL-6 and IL-17A at the specific vessels of boundary area of third-ventricle, thalamus, and dentate-gyrus (see Figure 4G) with or without corticosteroid receptor antagonist (mifepristone and guggulsterone) treatment. Mean scores ± SEM are shown. Statistical significance was determined by ANOVA tests. (n.s.: not significant). Experiments were performed at least three times; representative data are shown. DOI: http://dx.doi.org/10.7554/eLife.25517.005
    Figure Legend Snippet: The mortality was not affected by corticosteroid receptor antagonist treatment in cytokine-microinjected mice under stress condition. Bloody stool scores and percentages of mortality in mice under stress condition 2 days after direct microinjection of IL-6 and IL-17A at the specific vessels of boundary area of third-ventricle, thalamus, and dentate-gyrus (see Figure 4G) with or without corticosteroid receptor antagonist (mifepristone and guggulsterone) treatment. Mean scores ± SEM are shown. Statistical significance was determined by ANOVA tests. (n.s.: not significant). Experiments were performed at least three times; representative data are shown. DOI: http://dx.doi.org/10.7554/eLife.25517.005

    Techniques Used: Mouse Assay

    Pathogenic CD4+ T cells derived from IL-17A deficient or IFN-γ deficient mice inhibited the severe phenotypes. ( A ) Immunohistochemical staining for CD4 and MHC class II at specific vessels of the boundary area of the third ventricle region, thalamus, and dentate gyrus of mice with IL-17A deficient or IFN-γ deficient pathogenic CD4+ T cell transfer under stress condition (n = 3–5 per group). (right) Quantification of the histological analysis. Number of cells per picture (10x). ( B ) Percentages of mortality of mice with IL-17A deficient or IFN-γ deficient pathogenic CD4+ T cell transfer under stress condition (n = 3–5 per group). Mean scores ± SEM are shown. Statistical significance was determined by ANOVA tests. Statistical significance is denoted by asterisks (***p
    Figure Legend Snippet: Pathogenic CD4+ T cells derived from IL-17A deficient or IFN-γ deficient mice inhibited the severe phenotypes. ( A ) Immunohistochemical staining for CD4 and MHC class II at specific vessels of the boundary area of the third ventricle region, thalamus, and dentate gyrus of mice with IL-17A deficient or IFN-γ deficient pathogenic CD4+ T cell transfer under stress condition (n = 3–5 per group). (right) Quantification of the histological analysis. Number of cells per picture (10x). ( B ) Percentages of mortality of mice with IL-17A deficient or IFN-γ deficient pathogenic CD4+ T cell transfer under stress condition (n = 3–5 per group). Mean scores ± SEM are shown. Statistical significance was determined by ANOVA tests. Statistical significance is denoted by asterisks (***p

    Techniques Used: Derivative Assay, Mouse Assay, Immunohistochemistry, Staining

    13) Product Images from "Frequency of circulating topoisomerase-I-specific CD4 T cells predicts presence and progression of interstitial lung disease in scleroderma"

    Article Title: Frequency of circulating topoisomerase-I-specific CD4 T cells predicts presence and progression of interstitial lung disease in scleroderma

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-016-0993-2

    Topoisomerase-I-specific CD4+ T cells show a Th17 polarized functional phenotype. a CD4+ T cell subsets were identified by flow cytometry using the combined surface expression of chemokine receptors CCR6, CXCR3, and CCR4. b After sorting, the different T helper subsets were stimulated with anti-CD3/CD28 beads for 48 h and secretion of IFNγ, IL-4, and IL-17A was measured in their supernatants by enzyme-linked immunosorbent assay ( bars represent mean ± SE, n = 3). c The distribution of polarized T helper subsets within topo-I-specific CD4+ T cells ( closed circles ) is shown in comparison to the general CD4+ population ( open circles ). T test with Bonferroni correction was used for multiple comparisons. Lines indicate mean ± SD. d After sorting topo-I-specific CD4+ T cells according to their polarized T helper phenotype (range 16–2064 cells), mRNA expression levels of IFNγ, IL-4, and IL-17A were measured by qPCR using the Arcturus® PicoPure® RNA Isolation system, which is designed to recover high-quality RNA from a very low number of cells (10–500 cells) and to accomplish cDNA synthesis from minimal amounts of cDNA. IFNγ and IL-17A expression was calculated as fold change in reference to a mix of topo-I-reactive CD4+ T cells from three patients; IL-4 expression is reported as delta Ct from actin expression since it was not detectable in the reference population. Data are representative of four separate patients (mean ± SE)
    Figure Legend Snippet: Topoisomerase-I-specific CD4+ T cells show a Th17 polarized functional phenotype. a CD4+ T cell subsets were identified by flow cytometry using the combined surface expression of chemokine receptors CCR6, CXCR3, and CCR4. b After sorting, the different T helper subsets were stimulated with anti-CD3/CD28 beads for 48 h and secretion of IFNγ, IL-4, and IL-17A was measured in their supernatants by enzyme-linked immunosorbent assay ( bars represent mean ± SE, n = 3). c The distribution of polarized T helper subsets within topo-I-specific CD4+ T cells ( closed circles ) is shown in comparison to the general CD4+ population ( open circles ). T test with Bonferroni correction was used for multiple comparisons. Lines indicate mean ± SD. d After sorting topo-I-specific CD4+ T cells according to their polarized T helper phenotype (range 16–2064 cells), mRNA expression levels of IFNγ, IL-4, and IL-17A were measured by qPCR using the Arcturus® PicoPure® RNA Isolation system, which is designed to recover high-quality RNA from a very low number of cells (10–500 cells) and to accomplish cDNA synthesis from minimal amounts of cDNA. IFNγ and IL-17A expression was calculated as fold change in reference to a mix of topo-I-reactive CD4+ T cells from three patients; IL-4 expression is reported as delta Ct from actin expression since it was not detectable in the reference population. Data are representative of four separate patients (mean ± SE)

    Techniques Used: Functional Assay, Flow Cytometry, Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Isolation

    14) Product Images from "MicroRNA-22 Inhibits Histone Deacetylase 4 to Promote T Helper-17 Cell-Dependent Emphysema"

    Article Title: MicroRNA-22 Inhibits Histone Deacetylase 4 to Promote T Helper-17 Cell-Dependent Emphysema

    Journal: Nature immunology

    doi: 10.1038/ni.3292

    HDAC4 inhibits APC activation and emphysema progression. ( a,b ) HDAC4 expression in human monocyte derived dendritic cells ( a ) and mouse BMDCs ( b ) before and after stimulation with 1000ng nCB (n=4). RQ: relative quantity to 18S. ( c-e ) BMDCs generated from CD11c–specific conditional HDAC4 deletion mice ( Hdac4 CD11c ) and control mice carrying HDAC4-loxp alleles ( Hdac4 flox ) were stimulated with LPS ( c ) or αCD40 ( d ) overnight. Secreted IL-6 ( c,d ) was quantitated by ELISA (n=4–5). ( e,f ) HDAC4 deficient BMDCs ( e ) or splenic DCs ( f ) were primed with or without 1000ng nCB and co-cultured with WT naïve CD4 + T cells for 3 days and secreted IL-17A was measured by ELISA (n=4–5). ( g ) Expression of c-Jun in HDAC4 deficient BMDCs treated with αCD40 at different time points was determined by immunoblot. Representative blot shown at bottom and the top bar graph shows cumulative density measurements of each band from three comparable experiments. ( h–k ) Hdac4 CD11c and Hdac4 flox mice were challenged with nCB for one month. ( h ) Micro-CT quantification of lung volume. ( i ) Representative H E stained lung sections (of 4 total; 50× magnification; insets: 400×; Scale bars: 200 µm; insets: 25 µm). Macrophages ( j ) and neutrophils ( k ) in BALF. *: p
    Figure Legend Snippet: HDAC4 inhibits APC activation and emphysema progression. ( a,b ) HDAC4 expression in human monocyte derived dendritic cells ( a ) and mouse BMDCs ( b ) before and after stimulation with 1000ng nCB (n=4). RQ: relative quantity to 18S. ( c-e ) BMDCs generated from CD11c–specific conditional HDAC4 deletion mice ( Hdac4 CD11c ) and control mice carrying HDAC4-loxp alleles ( Hdac4 flox ) were stimulated with LPS ( c ) or αCD40 ( d ) overnight. Secreted IL-6 ( c,d ) was quantitated by ELISA (n=4–5). ( e,f ) HDAC4 deficient BMDCs ( e ) or splenic DCs ( f ) were primed with or without 1000ng nCB and co-cultured with WT naïve CD4 + T cells for 3 days and secreted IL-17A was measured by ELISA (n=4–5). ( g ) Expression of c-Jun in HDAC4 deficient BMDCs treated with αCD40 at different time points was determined by immunoblot. Representative blot shown at bottom and the top bar graph shows cumulative density measurements of each band from three comparable experiments. ( h–k ) Hdac4 CD11c and Hdac4 flox mice were challenged with nCB for one month. ( h ) Micro-CT quantification of lung volume. ( i ) Representative H E stained lung sections (of 4 total; 50× magnification; insets: 400×; Scale bars: 200 µm; insets: 25 µm). Macrophages ( j ) and neutrophils ( k ) in BALF. *: p

    Techniques Used: Activation Assay, Expressing, Derivative Assay, Generated, Mouse Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Micro-CT, Staining

    MiR-22 deficiency protects against nCB-induced emphysema in mice. WT and Mir22 −/− mice were exposed to vehicle (PBS) or nCB for one month. ( a ) Representative H E stained lung sections (of 4 total, 50× magnification; insets: 400×; scale bars: 200 µm; insets: 25 µm). ( b ) Micro-CT quantification of lung volume. Macrophages ( c ) and neutrophils ( d ) were quantified from BALF and total lung IL-17A + T H 17 cell ( e ) were assessed by flow cytometry. N.D.: not detected. Lung CD11c + cells were isolated and cultured ex vivo overnight and supernatants assayed for secreted IL-1β ( f ) and IL-6 ( g ) by ELISA. ( h ) Lung CD11c + APCs isolated from nCB or PBS exposed mice were co-cultured with naïve syngeneic CD4 + T cells ex vivo for 3 days and secreted IL-17A was measured by ELISA. ( i–k ) Mice lacking miR-22 in CD11c + APC ( Mir22 CD11c ) and control mice ( Mir22 flox/flox ) were exposed to PBS or nCB for one month after which macrophages ( i ) and neutrophils ( j ) were quantified from BALF and total IL-17A + T H 17 cells ( k ) were enumerated from whole lung. *: p
    Figure Legend Snippet: MiR-22 deficiency protects against nCB-induced emphysema in mice. WT and Mir22 −/− mice were exposed to vehicle (PBS) or nCB for one month. ( a ) Representative H E stained lung sections (of 4 total, 50× magnification; insets: 400×; scale bars: 200 µm; insets: 25 µm). ( b ) Micro-CT quantification of lung volume. Macrophages ( c ) and neutrophils ( d ) were quantified from BALF and total lung IL-17A + T H 17 cell ( e ) were assessed by flow cytometry. N.D.: not detected. Lung CD11c + cells were isolated and cultured ex vivo overnight and supernatants assayed for secreted IL-1β ( f ) and IL-6 ( g ) by ELISA. ( h ) Lung CD11c + APCs isolated from nCB or PBS exposed mice were co-cultured with naïve syngeneic CD4 + T cells ex vivo for 3 days and secreted IL-17A was measured by ELISA. ( i–k ) Mice lacking miR-22 in CD11c + APC ( Mir22 CD11c ) and control mice ( Mir22 flox/flox ) were exposed to PBS or nCB for one month after which macrophages ( i ) and neutrophils ( j ) were quantified from BALF and total IL-17A + T H 17 cells ( k ) were enumerated from whole lung. *: p

    Techniques Used: Mouse Assay, Staining, Micro-CT, Flow Cytometry, Cytometry, Isolation, Cell Culture, Ex Vivo, Enzyme-linked Immunosorbent Assay

    MiR-22 targets HDAC4 in APCs to promote IL-6 production. ( a ) Heat map depicting changes in the expression of miR-22 predicted target genes in wild type (WT) and Mir22 −/− CD11c + APCs stimulated with either vehicle (PBS) or nCB (Carbon). ( b ) WT or Mir22 −/− BMDCs were transfected with either scrambled control siRNA or indicated gene specific siRNAs and were stimulated with αCD40 antibody overnight. IL-6 production was measured by ELISA (n=5). ( c,d ) WT BMDCs were transfected with scrambled LNA (Scram) or miR-22 antisense LNA (miR-22 LNA). Expression of HDAC4 mRNA ( c , n=3, RQ: relative quantity to 18S) and protein ( d ) were determined by RT-qPCR and immunoblot. Numbers indicate the HDAC4/β-actin density ratio. ( e ) WT or Mir22 −/− BMDCs were stimulated with αCD40 antibody for the indicated time (hrs) and HDAC4 expression was determined by immunoblot. ( f ) WT and Mir22 −/− BMDCs were transfected with scrambled or HDAC4 siRNA (siHDAC4). ( g ) WT BMDCs were transfected with scrambled, miR-22 antisense LNA (MiR-22 LNA) and/or HDAC4 siRNA (siHDAC4). All BMDCs were primed with or without 1000ng nCB and co-cultured with WT naïve CD4 + T cells for 3 days and secreted IL-17A was measured by ELISA (n=4). *: p
    Figure Legend Snippet: MiR-22 targets HDAC4 in APCs to promote IL-6 production. ( a ) Heat map depicting changes in the expression of miR-22 predicted target genes in wild type (WT) and Mir22 −/− CD11c + APCs stimulated with either vehicle (PBS) or nCB (Carbon). ( b ) WT or Mir22 −/− BMDCs were transfected with either scrambled control siRNA or indicated gene specific siRNAs and were stimulated with αCD40 antibody overnight. IL-6 production was measured by ELISA (n=5). ( c,d ) WT BMDCs were transfected with scrambled LNA (Scram) or miR-22 antisense LNA (miR-22 LNA). Expression of HDAC4 mRNA ( c , n=3, RQ: relative quantity to 18S) and protein ( d ) were determined by RT-qPCR and immunoblot. Numbers indicate the HDAC4/β-actin density ratio. ( e ) WT or Mir22 −/− BMDCs were stimulated with αCD40 antibody for the indicated time (hrs) and HDAC4 expression was determined by immunoblot. ( f ) WT and Mir22 −/− BMDCs were transfected with scrambled or HDAC4 siRNA (siHDAC4). ( g ) WT BMDCs were transfected with scrambled, miR-22 antisense LNA (MiR-22 LNA) and/or HDAC4 siRNA (siHDAC4). All BMDCs were primed with or without 1000ng nCB and co-cultured with WT naïve CD4 + T cells for 3 days and secreted IL-17A was measured by ELISA (n=4). *: p

    Techniques Used: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Cell Culture

    Enhanced miR-22 expression in human emphysema and cigarette smoke-activated lung APCs. ( a ) Expression of miR-22 in lung CD1a + mDCs from control ( n =8) and emphysema patients ( n =20) as determined by RT-qPCR (RQ: relative quantity relative to U6 snRNA). ( b ) Correlation of human miR-22 expression with percent predicted forced expiratory volume in one second (FEV1). ( n =25) ( c ) MiR-22 expression in lung CD11c + cells from mice exposed to cigarette smoke for 4 months. ( n =3, represent two independent experiments) ( d–h ) Wild-type (WT) and Mir22 −/− mice were exposed to air or cigarette smoke (SMK) for 4 months. ( d ) Representative H E stained lung sections (of 4 total, 50× magnification; insets: 400×; scale bars: 200 µm; insets: 25 µm). ( e ) Micro-CT quantification of lung volume. Total macrophages ( f ) and neutrophils ( g ) from BALF and total lung IL-17A + T H 17 cells ( h ) as assessed by flow cytometry. (d–h) n =4–5 mice/group as indicated in (e). Data represent one of four comparable experiments. *: p
    Figure Legend Snippet: Enhanced miR-22 expression in human emphysema and cigarette smoke-activated lung APCs. ( a ) Expression of miR-22 in lung CD1a + mDCs from control ( n =8) and emphysema patients ( n =20) as determined by RT-qPCR (RQ: relative quantity relative to U6 snRNA). ( b ) Correlation of human miR-22 expression with percent predicted forced expiratory volume in one second (FEV1). ( n =25) ( c ) MiR-22 expression in lung CD11c + cells from mice exposed to cigarette smoke for 4 months. ( n =3, represent two independent experiments) ( d–h ) Wild-type (WT) and Mir22 −/− mice were exposed to air or cigarette smoke (SMK) for 4 months. ( d ) Representative H E stained lung sections (of 4 total, 50× magnification; insets: 400×; scale bars: 200 µm; insets: 25 µm). ( e ) Micro-CT quantification of lung volume. Total macrophages ( f ) and neutrophils ( g ) from BALF and total lung IL-17A + T H 17 cells ( h ) as assessed by flow cytometry. (d–h) n =4–5 mice/group as indicated in (e). Data represent one of four comparable experiments. *: p

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay, Staining, Micro-CT, Flow Cytometry, Cytometry

    15) Product Images from "IL-17 drives psoriatic inflammation via distinct, target cell-specific mechanisms"

    Article Title: IL-17 drives psoriatic inflammation via distinct, target cell-specific mechanisms

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1400513111

    IL-17–stimulated dermal fibroblasts promote production of IL-17 by γδT cells. ( A ) Relative mRNA expression for indicated genes in primary dermal fibroblast (FB) cultures from WT and CIKS-KO mice, stimulated with recombinant IL-17A,
    Figure Legend Snippet: IL-17–stimulated dermal fibroblasts promote production of IL-17 by γδT cells. ( A ) Relative mRNA expression for indicated genes in primary dermal fibroblast (FB) cultures from WT and CIKS-KO mice, stimulated with recombinant IL-17A,

    Techniques Used: Expressing, Mouse Assay, Recombinant

    IL-17A regulates the proliferation and differentiation of primary keratinocytes. ( A and B ) Relative mRNA expression of indicated differentiation markers in primary WT keratinocytes cultured in low Ca 2+ or 0.12 mM Ca 2+ and left without stimulation or stimulated
    Figure Legend Snippet: IL-17A regulates the proliferation and differentiation of primary keratinocytes. ( A and B ) Relative mRNA expression of indicated differentiation markers in primary WT keratinocytes cultured in low Ca 2+ or 0.12 mM Ca 2+ and left without stimulation or stimulated

    Techniques Used: Expressing, Cell Culture

    16) Product Images from "IL-17A Activates ERK1/2 and Enhances Differentiation of Oligodendrocyte Progenitor Cells"

    Article Title: IL-17A Activates ERK1/2 and Enhances Differentiation of Oligodendrocyte Progenitor Cells

    Journal: Glia

    doi: 10.1002/glia.22783

    IL-17A-stimulated OPCs do not undergo apoptosis. OPCs from C57BL/6 and IL-17RA −/− pups were stimulated with 0, 1, or 100 ng/mL recombinant IL-17A for two days. ( A ) Total cells collected from each plate and supernatant were compared by
    Figure Legend Snippet: IL-17A-stimulated OPCs do not undergo apoptosis. OPCs from C57BL/6 and IL-17RA −/− pups were stimulated with 0, 1, or 100 ng/mL recombinant IL-17A for two days. ( A ) Total cells collected from each plate and supernatant were compared by

    Techniques Used: Recombinant

    IL-17A-stimulated OPCs exit the cell cycle. OPCs from C57BL/6 pups were cultured with 0, 0.01, 0.1, 1, 10, 100 ng/mL recombinant IL-17A for two days and mitotic cell division was measured by Ki67 staining ( A : Ki67, green; nuclei, blue) and blindly quantified
    Figure Legend Snippet: IL-17A-stimulated OPCs exit the cell cycle. OPCs from C57BL/6 pups were cultured with 0, 0.01, 0.1, 1, 10, 100 ng/mL recombinant IL-17A for two days and mitotic cell division was measured by Ki67 staining ( A : Ki67, green; nuclei, blue) and blindly quantified

    Techniques Used: Cell Culture, Recombinant, Staining

    ERK1/2 MAPK is activated in IL-17A-stimulated OPCs. OPCs from C57BL/6 and IL-17RA −/− pups were stimulated with 0, 1, or 100 ng/mL recombinant IL-17A for two days and protein for total ERK ( A ), p38 ( B ), and NFkB (p65) ( C ) were quantified
    Figure Legend Snippet: ERK1/2 MAPK is activated in IL-17A-stimulated OPCs. OPCs from C57BL/6 and IL-17RA −/− pups were stimulated with 0, 1, or 100 ng/mL recombinant IL-17A for two days and protein for total ERK ( A ), p38 ( B ), and NFkB (p65) ( C ) were quantified

    Techniques Used: Recombinant

    IL-17A enhanced OPC differentiation. ( A–C ) OPCs from postnatal rats were cultured with varying concentrations of recombinant IL-17A in the absence ( A ) or presence ( B ) of PDGF, stained with MBP by immunocytochemistry, and expression for each group
    Figure Legend Snippet: IL-17A enhanced OPC differentiation. ( A–C ) OPCs from postnatal rats were cultured with varying concentrations of recombinant IL-17A in the absence ( A ) or presence ( B ) of PDGF, stained with MBP by immunocytochemistry, and expression for each group

    Techniques Used: Cell Culture, Recombinant, Staining, Immunocytochemistry, Expressing

    IL-17A stimulated OPCs express a specific set of chemokines and cytokines. OPCs from C57BL/6 and IL-17RA −/− pups were stimulated with 0, 1, or 100 ng/mL recombinant IL-17A or 100 ng/mL IFN-γ for 2 days. Transcript levels for CNTF
    Figure Legend Snippet: IL-17A stimulated OPCs express a specific set of chemokines and cytokines. OPCs from C57BL/6 and IL-17RA −/− pups were stimulated with 0, 1, or 100 ng/mL recombinant IL-17A or 100 ng/mL IFN-γ for 2 days. Transcript levels for CNTF

    Techniques Used: Recombinant

    17) Product Images from "IL-17A Activates ERK1/2 and Enhances Differentiation of Oligodendrocyte Progenitor Cells"

    Article Title: IL-17A Activates ERK1/2 and Enhances Differentiation of Oligodendrocyte Progenitor Cells

    Journal: Glia

    doi: 10.1002/glia.22783

    IL-17A-stimulated OPCs do not undergo apoptosis. OPCs from C57BL/6 and IL-17RA −/− pups were stimulated with 0, 1, or 100 ng/mL recombinant IL-17A for two days. ( A ) Total cells collected from each plate and supernatant were compared by
    Figure Legend Snippet: IL-17A-stimulated OPCs do not undergo apoptosis. OPCs from C57BL/6 and IL-17RA −/− pups were stimulated with 0, 1, or 100 ng/mL recombinant IL-17A for two days. ( A ) Total cells collected from each plate and supernatant were compared by

    Techniques Used: Recombinant

    IL-17A-stimulated OPCs exit the cell cycle. OPCs from C57BL/6 pups were cultured with 0, 0.01, 0.1, 1, 10, 100 ng/mL recombinant IL-17A for two days and mitotic cell division was measured by Ki67 staining ( A : Ki67, green; nuclei, blue) and blindly quantified
    Figure Legend Snippet: IL-17A-stimulated OPCs exit the cell cycle. OPCs from C57BL/6 pups were cultured with 0, 0.01, 0.1, 1, 10, 100 ng/mL recombinant IL-17A for two days and mitotic cell division was measured by Ki67 staining ( A : Ki67, green; nuclei, blue) and blindly quantified

    Techniques Used: Cell Culture, Recombinant, Staining

    ERK1/2 MAPK is activated in IL-17A-stimulated OPCs. OPCs from C57BL/6 and IL-17RA −/− pups were stimulated with 0, 1, or 100 ng/mL recombinant IL-17A for two days and protein for total ERK ( A ), p38 ( B ), and NFkB (p65) ( C ) were quantified
    Figure Legend Snippet: ERK1/2 MAPK is activated in IL-17A-stimulated OPCs. OPCs from C57BL/6 and IL-17RA −/− pups were stimulated with 0, 1, or 100 ng/mL recombinant IL-17A for two days and protein for total ERK ( A ), p38 ( B ), and NFkB (p65) ( C ) were quantified

    Techniques Used: Recombinant

    IL-17A enhanced OPC differentiation. ( A–C ) OPCs from postnatal rats were cultured with varying concentrations of recombinant IL-17A in the absence ( A ) or presence ( B ) of PDGF, stained with MBP by immunocytochemistry, and expression for each group
    Figure Legend Snippet: IL-17A enhanced OPC differentiation. ( A–C ) OPCs from postnatal rats were cultured with varying concentrations of recombinant IL-17A in the absence ( A ) or presence ( B ) of PDGF, stained with MBP by immunocytochemistry, and expression for each group

    Techniques Used: Cell Culture, Recombinant, Staining, Immunocytochemistry, Expressing

    IL-17A stimulated OPCs express a specific set of chemokines and cytokines. OPCs from C57BL/6 and IL-17RA −/− pups were stimulated with 0, 1, or 100 ng/mL recombinant IL-17A or 100 ng/mL IFN-γ for 2 days. Transcript levels for CNTF
    Figure Legend Snippet: IL-17A stimulated OPCs express a specific set of chemokines and cytokines. OPCs from C57BL/6 and IL-17RA −/− pups were stimulated with 0, 1, or 100 ng/mL recombinant IL-17A or 100 ng/mL IFN-γ for 2 days. Transcript levels for CNTF

    Techniques Used: Recombinant

    18) Product Images from "Interleukin-27 ameliorates coxsackievirus-B3-induced viral myocarditis by inhibiting Th17 cells"

    Article Title: Interleukin-27 ameliorates coxsackievirus-B3-induced viral myocarditis by inhibiting Th17 cells

    Journal: Virology Journal

    doi: 10.1186/s12985-015-0418-x

    IL-27 ameliorates CVB3-induced myocarditis by inhibiting Th17 cells. a Representative FACS profile of Th17 cells in the spleen of CVB3 infection mice treated with rmIL-27 or Anti-IL-27 on day 7 post infection. Cells were gated on CD3 + CD4 + T cells. b Statistical analysis of the percentage of Th17 cells. c Expression of Th17 cells characteristic cytokines, IL-17A, IL-22, and key transcription factor ROR-γt in cardiac tissues on day 7 after CVB3 infection were detected by qRT-PCR. d The serum levels of proinflammatory cytokines IL-17A, IFN-γ, IL-6 and TNF-α in CVB3 infection mice were determined by ELISA. Normal, mice not-infected with the virus. Data are showed as the means ± SEM. * P
    Figure Legend Snippet: IL-27 ameliorates CVB3-induced myocarditis by inhibiting Th17 cells. a Representative FACS profile of Th17 cells in the spleen of CVB3 infection mice treated with rmIL-27 or Anti-IL-27 on day 7 post infection. Cells were gated on CD3 + CD4 + T cells. b Statistical analysis of the percentage of Th17 cells. c Expression of Th17 cells characteristic cytokines, IL-17A, IL-22, and key transcription factor ROR-γt in cardiac tissues on day 7 after CVB3 infection were detected by qRT-PCR. d The serum levels of proinflammatory cytokines IL-17A, IFN-γ, IL-6 and TNF-α in CVB3 infection mice were determined by ELISA. Normal, mice not-infected with the virus. Data are showed as the means ± SEM. * P

    Techniques Used: FACS, Infection, Mouse Assay, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    19) Product Images from "Th17 cells mediate pulmonary collateral priming"

    Article Title: Th17 cells mediate pulmonary collateral priming

    Journal: The Journal of allergy and clinical immunology

    doi: 10.1016/j.jaci.2011.01.067

    Role of IL-17A in Th1 collateral priming
    Figure Legend Snippet: Role of IL-17A in Th1 collateral priming

    Techniques Used:

    20) Product Images from "Hypoxia regulates IL-17A secretion from nasal polyp epithelial cells"

    Article Title: Hypoxia regulates IL-17A secretion from nasal polyp epithelial cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22189

    (A) In normoxia condition IL-17A and HIF1α mRNA levels were not related as time extended both in control and nasal polyps epithelial cells. In hypoxia condition the two mRNA levels changing trend over time were consistent in the two kinds of epithelial cells. (B) Correlation analysis between IL-17A secretion after hypoxia exposure for 24h or 48h in nasal polyps epithelial cells and the same patients’ endoscopic or CT score. (C) IL-17A and HIF1α dual labelled immunofluorescence on IT(CONTROL), IT(CRS) and NP(CRS) epithelial cells. The two protein were co-localized. (scale bars are 50 um) (D) IL-17A and HIF1α dual labelled immunofluorescence on NP(CRS) epithelial cells after hypoxia exposure for 0h,24h or 48h. (scale bars are 50 um)
    Figure Legend Snippet: (A) In normoxia condition IL-17A and HIF1α mRNA levels were not related as time extended both in control and nasal polyps epithelial cells. In hypoxia condition the two mRNA levels changing trend over time were consistent in the two kinds of epithelial cells. (B) Correlation analysis between IL-17A secretion after hypoxia exposure for 24h or 48h in nasal polyps epithelial cells and the same patients’ endoscopic or CT score. (C) IL-17A and HIF1α dual labelled immunofluorescence on IT(CONTROL), IT(CRS) and NP(CRS) epithelial cells. The two protein were co-localized. (scale bars are 50 um) (D) IL-17A and HIF1α dual labelled immunofluorescence on NP(CRS) epithelial cells after hypoxia exposure for 0h,24h or 48h. (scale bars are 50 um)

    Techniques Used: Immunofluorescence

    IL-5, TSLP, IL-17A and IL-17A receptor (IL-17AR)expression in epithelial cells derived from all four groups: inferior turbinate (IT) of control, inferior turbinate (IT) of chronic rhinosinusitis with nasal polyps (CRS)eosinophilic (Eos) and noneosinophilic (Non-Eos) chronic rhinosinusitis with nasal polyps NP(CRS) groups A-C show protein expression in epithelial cells quantified by immunohistochemistry (original magnification: 200×) and representative photomicrographs depicting protein immunostaining. (A) IL-5 protein expression in epithelial cells and the lamina propria. (B) TSLP protein expression in epithelial cells and the lamina propria. (C) IL-17A protein expression in epithelial cells and the lamina propria. (D) IL-17AR protein expression in epithelial cells and the lamina propria.
    Figure Legend Snippet: IL-5, TSLP, IL-17A and IL-17A receptor (IL-17AR)expression in epithelial cells derived from all four groups: inferior turbinate (IT) of control, inferior turbinate (IT) of chronic rhinosinusitis with nasal polyps (CRS)eosinophilic (Eos) and noneosinophilic (Non-Eos) chronic rhinosinusitis with nasal polyps NP(CRS) groups A-C show protein expression in epithelial cells quantified by immunohistochemistry (original magnification: 200×) and representative photomicrographs depicting protein immunostaining. (A) IL-5 protein expression in epithelial cells and the lamina propria. (B) TSLP protein expression in epithelial cells and the lamina propria. (C) IL-17A protein expression in epithelial cells and the lamina propria. (D) IL-17AR protein expression in epithelial cells and the lamina propria.

    Techniques Used: Derivative Assay, Expressing, Immunohistochemistry, Immunostaining

    After stimulated with normoxia(21% O 2 ) and hypoxia (1% O 2 ), protein levels for the various cytokines/chemokines: IFN-γ, IL-5, TSLP and IL-17A were determined by ELISA A-D Left column Epithelial cells were under normoxia exposure. Right column Epithelial cells were under hypoxia exposure. (A) IFN-γ secreted from epithelial cells after stimulation for 0 hours, 24 hours, and 48 hours. (B) IL-5 secreted from epithelial cells after stimulation for 0 hours, 24 hours, and 48 hours. (C) TSLP secreted from epithelial cells after stimulation for 0 hours, 24 hours, and 48 hours. (D) IL-17A secreted from epithelial cells after stimulation for 0 hours, 24 hours, and 48 hours. (E) After stimulated with hypoxia (1% O 2 ), different IL-17A secretion levels from four types of epithelial cells derived from inferior turbinate (IT) of control, inferior turbinate (IT) of chronic rhinosinusitis with nasal polyps (CRS), eosinophilic (Eos) and noneosinophilic (Non-Eos) chronic rhinosinusitis with nasal polyps NP(CRS) groups were examined by ELISA. (F) IL-17A protein expression in IT(CONTROL), IT(CRS) and NP(CRS) epithelial cells was quantified by immunofluorescent staining and high-content analysis. (scale bars are 50 μm) Statistic result of immunofluorescent staining and high-content analysis.
    Figure Legend Snippet: After stimulated with normoxia(21% O 2 ) and hypoxia (1% O 2 ), protein levels for the various cytokines/chemokines: IFN-γ, IL-5, TSLP and IL-17A were determined by ELISA A-D Left column Epithelial cells were under normoxia exposure. Right column Epithelial cells were under hypoxia exposure. (A) IFN-γ secreted from epithelial cells after stimulation for 0 hours, 24 hours, and 48 hours. (B) IL-5 secreted from epithelial cells after stimulation for 0 hours, 24 hours, and 48 hours. (C) TSLP secreted from epithelial cells after stimulation for 0 hours, 24 hours, and 48 hours. (D) IL-17A secreted from epithelial cells after stimulation for 0 hours, 24 hours, and 48 hours. (E) After stimulated with hypoxia (1% O 2 ), different IL-17A secretion levels from four types of epithelial cells derived from inferior turbinate (IT) of control, inferior turbinate (IT) of chronic rhinosinusitis with nasal polyps (CRS), eosinophilic (Eos) and noneosinophilic (Non-Eos) chronic rhinosinusitis with nasal polyps NP(CRS) groups were examined by ELISA. (F) IL-17A protein expression in IT(CONTROL), IT(CRS) and NP(CRS) epithelial cells was quantified by immunofluorescent staining and high-content analysis. (scale bars are 50 μm) Statistic result of immunofluorescent staining and high-content analysis.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Derivative Assay, Expressing, Staining, High Content Screening

    (A) Hypoxia stimulated IT(N) epithelial cells, no differences in IL-5, TSLP, IFN-γ secretion when compared with normoxia. IL-17A secretion appeared changes under hypoxia. HIF1α promoted IL-17A secretion in epithelial cells under hypoxia. (B) Hypoxia stimulated NP epithelial cells, no differences in IL-5, TSLP, IFN-γsecretion stimulated by hypoxia when compared with normoxia. IL-17A secretion appeared changes under hypoxia. Decreased HIF1α inhibited IL-17A secretion in epithelial cells under hypoxia.
    Figure Legend Snippet: (A) Hypoxia stimulated IT(N) epithelial cells, no differences in IL-5, TSLP, IFN-γ secretion when compared with normoxia. IL-17A secretion appeared changes under hypoxia. HIF1α promoted IL-17A secretion in epithelial cells under hypoxia. (B) Hypoxia stimulated NP epithelial cells, no differences in IL-5, TSLP, IFN-γsecretion stimulated by hypoxia when compared with normoxia. IL-17A secretion appeared changes under hypoxia. Decreased HIF1α inhibited IL-17A secretion in epithelial cells under hypoxia.

    Techniques Used:

    21) Product Images from "NLRC4 inflammasome-mediated production of IL-1? modulates mucosal immunity in the lung against Gram-negative bacterial infection"

    Article Title: NLRC4 inflammasome-mediated production of IL-1? modulates mucosal immunity in the lung against Gram-negative bacterial infection

    Journal: Journal of Immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1200195

    Importance of NLRC4 in IL-17A producing cells in the lung in response to Kp infection A. Shown are representative FACS plots of lung homogenates after Kp infection showing IFN-γ- and IL-17A-producing γδ, CD4, CD8, and NK T cells. Lung T cells were isolated 48 h after Kp infection and immunostained for surface CD8, and intracellular IFN-γ and IL-17A. Shown is a representative of three separate experiments. B. NLRC4 deficiency decreases the number of of IL-17A-expressing T cells. Data shown are representative of three separate experiments (* P
    Figure Legend Snippet: Importance of NLRC4 in IL-17A producing cells in the lung in response to Kp infection A. Shown are representative FACS plots of lung homogenates after Kp infection showing IFN-γ- and IL-17A-producing γδ, CD4, CD8, and NK T cells. Lung T cells were isolated 48 h after Kp infection and immunostained for surface CD8, and intracellular IFN-γ and IL-17A. Shown is a representative of three separate experiments. B. NLRC4 deficiency decreases the number of of IL-17A-expressing T cells. Data shown are representative of three separate experiments (* P

    Techniques Used: Infection, FACS, Isolation, Expressing

    22) Product Images from "Interleukin‐33 contributes to disease severity in Dengue virus infection in mice"

    Article Title: Interleukin‐33 contributes to disease severity in Dengue virus infection in mice

    Journal: Immunology

    doi: 10.1111/imm.12988

    Pharmacological inhibition of neutrophil recruitments prevents the pathogenic effects of interleukin‐33 (IL‐33). Untreated wild‐type (WT) C57BL/6 (dengue virus serotype 2; DENV2), recombinant murine (rm) IL‐33‐treated (DENV2 + rmIL‐33, 200 ng/mouse) mice and mice treated with both rmIL‐33 and DF2156A (DENV2 + rmIL‐33 + DF2156A, 10 mg/kg of body weight) were inoculated with 1 median lethal dose (LD 50 ) [200 plaque‐forming units (PFU)] of DENV2 and monitored for survival (a) and weight loss (b) for 14 days post‐infection (p.i.). In parallel experiments, groups of mice were killed on day 6 p.i. for evaluation of blood parameters: platelet count (c), haematocrit index (d) and percentages of circulating granulocytes (e). Serum samples were used for assessment of alanine aminotransferase (ALT) levels (f). Liver samples were processed and assessed for myeloperoxidase (MPO) activity (g), and the concentrations of CXCL1 (h), CXCL2 (i), CXCL6 (j), IL‐6 (k) and IL‐17A (l) were assessed by ELISA. Data are mean ± SEM, exception (a), where percentage of survival is presented. Data are representative of at least two independent experiments. NI, non‐infected. * P
    Figure Legend Snippet: Pharmacological inhibition of neutrophil recruitments prevents the pathogenic effects of interleukin‐33 (IL‐33). Untreated wild‐type (WT) C57BL/6 (dengue virus serotype 2; DENV2), recombinant murine (rm) IL‐33‐treated (DENV2 + rmIL‐33, 200 ng/mouse) mice and mice treated with both rmIL‐33 and DF2156A (DENV2 + rmIL‐33 + DF2156A, 10 mg/kg of body weight) were inoculated with 1 median lethal dose (LD 50 ) [200 plaque‐forming units (PFU)] of DENV2 and monitored for survival (a) and weight loss (b) for 14 days post‐infection (p.i.). In parallel experiments, groups of mice were killed on day 6 p.i. for evaluation of blood parameters: platelet count (c), haematocrit index (d) and percentages of circulating granulocytes (e). Serum samples were used for assessment of alanine aminotransferase (ALT) levels (f). Liver samples were processed and assessed for myeloperoxidase (MPO) activity (g), and the concentrations of CXCL1 (h), CXCL2 (i), CXCL6 (j), IL‐6 (k) and IL‐17A (l) were assessed by ELISA. Data are mean ± SEM, exception (a), where percentage of survival is presented. Data are representative of at least two independent experiments. NI, non‐infected. * P

    Techniques Used: Inhibition, Recombinant, Mouse Assay, Infection, Activity Assay, Enzyme-linked Immunosorbent Assay

    23) Product Images from "MUC1-induced immunosuppression in colon cancer can be reversed by blocking the PD1/PDL1 signaling pathway"

    Article Title: MUC1-induced immunosuppression in colon cancer can be reversed by blocking the PD1/PDL1 signaling pathway

    Journal: Oncology Letters

    doi: 10.3892/ol.2020.12180

    PDL1 expression on MDSCs, TAMs and tumor cells is greater in MUC1 high tumor tissues from mice and patients. (A) Tumor cells from tumor-bearing mice or patients with colon cancer were isolated and PDL1 expression on the surface of tumor cells was assessed using flow cytometry. (B) CT26 and (C) SW480 tumor cells were transfected with pcDNA3.1(−)/Myc-His-MUC1 . Semi-quantitative analysis of western blots of PDL1 expression in tumor cells after the transfection of pcDNA3.1(−)/Myc-His-MUC1 using an anti-PDL1 mAb was performed. β-actin was used as an internal control. (D) CT26/vector and CT26/MUC1 cells were stimulated with mouse IFN-γ (20 ng/ml) and IL-17A (10 ng/ml) for 48 h and surface PDL1 expression was assessed. PDL1 expression on the surface of (E) mouse and (F) human MDSCs and (G) mouse and (H) human TAMs in tumor tissues. PD1 expression on the surface of (I) mouse and (J) human CD8 + T cells in tumor tissues from tumor-bearing mice (n=8) or patients with colon cancer (n=12). Data are representative of four experiments. Error bars represent the standard error of the mean. *P
    Figure Legend Snippet: PDL1 expression on MDSCs, TAMs and tumor cells is greater in MUC1 high tumor tissues from mice and patients. (A) Tumor cells from tumor-bearing mice or patients with colon cancer were isolated and PDL1 expression on the surface of tumor cells was assessed using flow cytometry. (B) CT26 and (C) SW480 tumor cells were transfected with pcDNA3.1(−)/Myc-His-MUC1 . Semi-quantitative analysis of western blots of PDL1 expression in tumor cells after the transfection of pcDNA3.1(−)/Myc-His-MUC1 using an anti-PDL1 mAb was performed. β-actin was used as an internal control. (D) CT26/vector and CT26/MUC1 cells were stimulated with mouse IFN-γ (20 ng/ml) and IL-17A (10 ng/ml) for 48 h and surface PDL1 expression was assessed. PDL1 expression on the surface of (E) mouse and (F) human MDSCs and (G) mouse and (H) human TAMs in tumor tissues. PD1 expression on the surface of (I) mouse and (J) human CD8 + T cells in tumor tissues from tumor-bearing mice (n=8) or patients with colon cancer (n=12). Data are representative of four experiments. Error bars represent the standard error of the mean. *P

    Techniques Used: Expressing, Mouse Assay, Isolation, Flow Cytometry, Transfection, Western Blot, Plasmid Preparation

    24) Product Images from "MUC1-induced immunosuppression in colon cancer can be reversed by blocking the PD1/PDL1 signaling pathway"

    Article Title: MUC1-induced immunosuppression in colon cancer can be reversed by blocking the PD1/PDL1 signaling pathway

    Journal: Oncology Letters

    doi: 10.3892/ol.2020.12180

    PDL1 expression on MDSCs, TAMs and tumor cells is greater in MUC1 high tumor tissues from mice and patients. (A) Tumor cells from tumor-bearing mice or patients with colon cancer were isolated and PDL1 expression on the surface of tumor cells was assessed using flow cytometry. (B) CT26 and (C) SW480 tumor cells were transfected with pcDNA3.1(−)/Myc-His-MUC1 . Semi-quantitative analysis of western blots of PDL1 expression in tumor cells after the transfection of pcDNA3.1(−)/Myc-His-MUC1 using an anti-PDL1 mAb was performed. β-actin was used as an internal control. (D) CT26/vector and CT26/MUC1 cells were stimulated with mouse IFN-γ (20 ng/ml) and IL-17A (10 ng/ml) for 48 h and surface PDL1 expression was assessed. PDL1 expression on the surface of (E) mouse and (F) human MDSCs and (G) mouse and (H) human TAMs in tumor tissues. PD1 expression on the surface of (I) mouse and (J) human CD8 + T cells in tumor tissues from tumor-bearing mice (n=8) or patients with colon cancer (n=12). Data are representative of four experiments. Error bars represent the standard error of the mean. *P
    Figure Legend Snippet: PDL1 expression on MDSCs, TAMs and tumor cells is greater in MUC1 high tumor tissues from mice and patients. (A) Tumor cells from tumor-bearing mice or patients with colon cancer were isolated and PDL1 expression on the surface of tumor cells was assessed using flow cytometry. (B) CT26 and (C) SW480 tumor cells were transfected with pcDNA3.1(−)/Myc-His-MUC1 . Semi-quantitative analysis of western blots of PDL1 expression in tumor cells after the transfection of pcDNA3.1(−)/Myc-His-MUC1 using an anti-PDL1 mAb was performed. β-actin was used as an internal control. (D) CT26/vector and CT26/MUC1 cells were stimulated with mouse IFN-γ (20 ng/ml) and IL-17A (10 ng/ml) for 48 h and surface PDL1 expression was assessed. PDL1 expression on the surface of (E) mouse and (F) human MDSCs and (G) mouse and (H) human TAMs in tumor tissues. PD1 expression on the surface of (I) mouse and (J) human CD8 + T cells in tumor tissues from tumor-bearing mice (n=8) or patients with colon cancer (n=12). Data are representative of four experiments. Error bars represent the standard error of the mean. *P

    Techniques Used: Expressing, Mouse Assay, Isolation, Flow Cytometry, Transfection, Western Blot, Plasmid Preparation

    25) Product Images from "A Reappraisal on the Potential Ability of Human Neutrophils to Express and Produce IL-17 Family Members In Vitro: Failure to Reproducibly Detect It"

    Article Title: A Reappraisal on the Potential Ability of Human Neutrophils to Express and Produce IL-17 Family Members In Vitro: Failure to Reproducibly Detect It

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00795

    Staining human neutrophils by anti-IL-17A (AF-317-NA) polyclonal antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) stainings of two FFPE cases of human pustular psoriasis using anti-IL-17A (AF-317-NA) and anti-CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils, either untreated (top panels) or treated with 5 µM R848 (bottom panels) for 3 h, were stained with anti-IL-17A (AF-317-NA, left panels) and anti-CXCL8 (right panels) Abs. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF-317-NA immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17A (rhIL-17A) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.
    Figure Legend Snippet: Staining human neutrophils by anti-IL-17A (AF-317-NA) polyclonal antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) stainings of two FFPE cases of human pustular psoriasis using anti-IL-17A (AF-317-NA) and anti-CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils, either untreated (top panels) or treated with 5 µM R848 (bottom panels) for 3 h, were stained with anti-IL-17A (AF-317-NA, left panels) and anti-CXCL8 (right panels) Abs. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF-317-NA immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17A (rhIL-17A) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.

    Techniques Used: Staining, Immunofluorescence, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Labeling, Marker, Isolation, Incubation, Recombinant, Positive Control

    IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression, as well as IL-17R surface expression, in neutrophils from patients with psoriasis. (A) Neutrophils isolated from healthy donors (HDs) ( n = 3) or psoriatic patients ( n = 3) were cultured for 20 h with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848, or 500 ng/ml IL-17A to evaluate IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. (B) Surface IL-17RA and IL-17RC expression evaluated by flow cytometry in human neutrophils from HDs or psoriatic patients. Values represent the mean ± SEM ( n = 3). For the data of panels (A,B) no significant differences between HDs or psoriatic patients were observed by two-way ANOVA followed by Bonferroni’s post-test.
    Figure Legend Snippet: IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression, as well as IL-17R surface expression, in neutrophils from patients with psoriasis. (A) Neutrophils isolated from healthy donors (HDs) ( n = 3) or psoriatic patients ( n = 3) were cultured for 20 h with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848, or 500 ng/ml IL-17A to evaluate IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. (B) Surface IL-17RA and IL-17RC expression evaluated by flow cytometry in human neutrophils from HDs or psoriatic patients. Values represent the mean ± SEM ( n = 3). For the data of panels (A,B) no significant differences between HDs or psoriatic patients were observed by two-way ANOVA followed by Bonferroni’s post-test.

    Techniques Used: Expressing, Isolation, Cell Culture, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry

    Levels of IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, IL-17RA, azurocidin, neutrophil elastase, and myeloperoxidase (MPO) mRNA expression in neutrophils at different stages of maturation. mRNA expression data derive from Gene Expression Omnibus database (accession number GSE42519) ( 65 ). (A) IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, and IL-17RA or (B) azurocidin (AZU1), neutrophil elastase (ELANE), and MPO mRNA expression levels were measured in the following cell types: hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), early and late promyelocytes (PMs), myelocytes (MYs), metamyelocytes (MMs), band cells (BCs), and bone marrow polymorphonuclear neutrophil granulocytes. Values represent the mean ± SEM as calculated from data of the biological replicates present in the database.
    Figure Legend Snippet: Levels of IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, IL-17RA, azurocidin, neutrophil elastase, and myeloperoxidase (MPO) mRNA expression in neutrophils at different stages of maturation. mRNA expression data derive from Gene Expression Omnibus database (accession number GSE42519) ( 65 ). (A) IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, and IL-17RA or (B) azurocidin (AZU1), neutrophil elastase (ELANE), and MPO mRNA expression levels were measured in the following cell types: hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), early and late promyelocytes (PMs), myelocytes (MYs), metamyelocytes (MMs), band cells (BCs), and bone marrow polymorphonuclear neutrophil granulocytes. Values represent the mean ± SEM as calculated from data of the biological replicates present in the database.

    Techniques Used: Expressing

    No induction of IL-17A, IL-17F, and IL-17RC mRNA expression in neutrophils incubated with IL-6 plus IL-23, in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated either with 100 ng/ml rIL-17A for 2 h or with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 for 1 h, prior to adding, or not, inactivated A. fumigatus conidia (1:5 neutrophils/conidia ratio) and hyphae (1:1 neutrophils/hyphae ratio) for additional 1 h. Neutrophils were then harvested for RNA extraction to evaluate IL-17A (A) , IL-17F (B) , IL-17RC (C) , IL-17RA (D) , and SOCS3 (F) mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization (mean ± SEM, n = 4). Asterisks stand for significant differences as compared to untreated cells: * P
    Figure Legend Snippet: No induction of IL-17A, IL-17F, and IL-17RC mRNA expression in neutrophils incubated with IL-6 plus IL-23, in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated either with 100 ng/ml rIL-17A for 2 h or with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 for 1 h, prior to adding, or not, inactivated A. fumigatus conidia (1:5 neutrophils/conidia ratio) and hyphae (1:1 neutrophils/hyphae ratio) for additional 1 h. Neutrophils were then harvested for RNA extraction to evaluate IL-17A (A) , IL-17F (B) , IL-17RC (C) , IL-17RA (D) , and SOCS3 (F) mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization (mean ± SEM, n = 4). Asterisks stand for significant differences as compared to untreated cells: * P

    Techniques Used: Expressing, Incubation, RNA Extraction, Real-time Polymerase Chain Reaction

    Lack of IL-17A and IL-17F production by human neutrophils activated by IL-6 plus IL-23 in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 and then cultured for three more hours in the presence or not of inactivated A. fumigatus conidia and hyphae (used at 1:5 and 1:1, respectively). After incubation, IL-17A (A) and CXCL8 (B) levels were determined in cell-free supernatants and in corresponding cell pellets by specific ELISA. Values are depicted as the mean ± SD or as not detected (nd) when values were under the detection limit ( n = 3). Asterisks stand for significant differences as compared to untreated cells: * P
    Figure Legend Snippet: Lack of IL-17A and IL-17F production by human neutrophils activated by IL-6 plus IL-23 in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 and then cultured for three more hours in the presence or not of inactivated A. fumigatus conidia and hyphae (used at 1:5 and 1:1, respectively). After incubation, IL-17A (A) and CXCL8 (B) levels were determined in cell-free supernatants and in corresponding cell pellets by specific ELISA. Values are depicted as the mean ± SD or as not detected (nd) when values were under the detection limit ( n = 3). Asterisks stand for significant differences as compared to untreated cells: * P

    Techniques Used: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

    Expression of surface IL-17RA and IL-17RC in neutrophils activated under various experimental conditions. Expression of surface IL-17RA (left panel) and IL-17RC (right panel) was evaluated by flow cytometry in neutrophils either freshly isolated or cultured for 3 h without or with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848 (A) , 20 µg/ml IL-6 plus 2 µg/ml IL-23 alone or in the presence of inactivated Aspergillus fumigatus conidia, hyphae or 500 ng/ml rIL-17A (B) . Graphs depict a representative experiment out of three independent ones with similar results. Histograms show staining by specific and isotype control Abs, respectively, for each stimulatory condition.
    Figure Legend Snippet: Expression of surface IL-17RA and IL-17RC in neutrophils activated under various experimental conditions. Expression of surface IL-17RA (left panel) and IL-17RC (right panel) was evaluated by flow cytometry in neutrophils either freshly isolated or cultured for 3 h without or with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848 (A) , 20 µg/ml IL-6 plus 2 µg/ml IL-23 alone or in the presence of inactivated Aspergillus fumigatus conidia, hyphae or 500 ng/ml rIL-17A (B) . Graphs depict a representative experiment out of three independent ones with similar results. Histograms show staining by specific and isotype control Abs, respectively, for each stimulatory condition.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Isolation, Cell Culture, Staining

    H3K4me1 or H3K27Ac levels at the IL-17A, IL-17F, and SOCS3 genomic loci of Th17 cell lines and resting/IL-6 plus IL-23-activated neutrophils. Enrichment levels of H3K4me1 (left panels) and H3K27Ac (right panels) at the IL-17A (A) , IL-17F (B) , and SOCS3 (C) genomic loci by chromatin immunoprecipitation (ChIP) analysis in human Th17 cell lines and neutrophils incubated for 1 h with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23. (A–C) Schemes illustrating the positions of the designed primer pairs amplifying promoter and potential enhancer regions of IL-17A, IL-17F, and SOCS3 for ChIP analysis are depicted at the top of each panel. Coimmunoprecipitated DNA samples were expressed as percent of the total input. Panels in (A–C) depict a representative experiment out of two independent ones with similar results. Error bars represent SEs calculated from triplicate qPCR reactions.
    Figure Legend Snippet: H3K4me1 or H3K27Ac levels at the IL-17A, IL-17F, and SOCS3 genomic loci of Th17 cell lines and resting/IL-6 plus IL-23-activated neutrophils. Enrichment levels of H3K4me1 (left panels) and H3K27Ac (right panels) at the IL-17A (A) , IL-17F (B) , and SOCS3 (C) genomic loci by chromatin immunoprecipitation (ChIP) analysis in human Th17 cell lines and neutrophils incubated for 1 h with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23. (A–C) Schemes illustrating the positions of the designed primer pairs amplifying promoter and potential enhancer regions of IL-17A, IL-17F, and SOCS3 for ChIP analysis are depicted at the top of each panel. Coimmunoprecipitated DNA samples were expressed as percent of the total input. Panels in (A–C) depict a representative experiment out of two independent ones with similar results. Error bars represent SEs calculated from triplicate qPCR reactions.

    Techniques Used: Chromatin Immunoprecipitation, Incubation, Real-time Polymerase Chain Reaction

    IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression levels in human neutrophils activated by a variety of stimuli. Human neutrophils were cultured at 5 × 10 6 /ml for up to 20 h with (A) 100 U/ml IFNγ and/or 100 ng/ml LPS; (B) 1,000 U/ml IFNα and/or 5 µM R848; (C) 10 ng/ml GM-CSF or 100 nM fMLF; (D) 1,000 U/ml G-CSF or 5 ng/ml TNFα. IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression was evaluated by reverse transcription quantitative real-time PCR (RT-qPCR) and data depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. The experiments depicted in each panels (A–D) are representative of at least three ones with similar results. Error bars stand for SEs calculated from triplicate qPCR reactions.
    Figure Legend Snippet: IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression levels in human neutrophils activated by a variety of stimuli. Human neutrophils were cultured at 5 × 10 6 /ml for up to 20 h with (A) 100 U/ml IFNγ and/or 100 ng/ml LPS; (B) 1,000 U/ml IFNα and/or 5 µM R848; (C) 10 ng/ml GM-CSF or 100 nM fMLF; (D) 1,000 U/ml G-CSF or 5 ng/ml TNFα. IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression was evaluated by reverse transcription quantitative real-time PCR (RT-qPCR) and data depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. The experiments depicted in each panels (A–D) are representative of at least three ones with similar results. Error bars stand for SEs calculated from triplicate qPCR reactions.

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Staining human neutrophils by anti-IL-17B (AF1248) antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) staining of two FFPE cases of human pustular psoriasis using anti-IL-17B (AF1248) and CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils incubated without (top panel) or with 5 µM R848 (bottom panel) for 3 h. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF1248 immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17B (rhIL-17B) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.
    Figure Legend Snippet: Staining human neutrophils by anti-IL-17B (AF1248) antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) staining of two FFPE cases of human pustular psoriasis using anti-IL-17B (AF1248) and CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils incubated without (top panel) or with 5 µM R848 (bottom panel) for 3 h. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF1248 immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17B (rhIL-17B) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.

    Techniques Used: Staining, Immunofluorescence, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Labeling, Marker, Incubation, Isolation, Recombinant, Positive Control

    26) Product Images from "A Reappraisal on the Potential Ability of Human Neutrophils to Express and Produce IL-17 Family Members In Vitro: Failure to Reproducibly Detect It"

    Article Title: A Reappraisal on the Potential Ability of Human Neutrophils to Express and Produce IL-17 Family Members In Vitro: Failure to Reproducibly Detect It

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00795

    Staining human neutrophils by anti-IL-17A (AF-317-NA) polyclonal antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) stainings of two FFPE cases of human pustular psoriasis using anti-IL-17A (AF-317-NA) and anti-CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils, either untreated (top panels) or treated with 5 µM R848 (bottom panels) for 3 h, were stained with anti-IL-17A (AF-317-NA, left panels) and anti-CXCL8 (right panels) Abs. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF-317-NA immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17A (rhIL-17A) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.
    Figure Legend Snippet: Staining human neutrophils by anti-IL-17A (AF-317-NA) polyclonal antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) stainings of two FFPE cases of human pustular psoriasis using anti-IL-17A (AF-317-NA) and anti-CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils, either untreated (top panels) or treated with 5 µM R848 (bottom panels) for 3 h, were stained with anti-IL-17A (AF-317-NA, left panels) and anti-CXCL8 (right panels) Abs. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF-317-NA immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17A (rhIL-17A) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.

    Techniques Used: Staining, Immunofluorescence, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Labeling, Marker, Isolation, Incubation, Recombinant, Positive Control

    IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression, as well as IL-17R surface expression, in neutrophils from patients with psoriasis. (A) Neutrophils isolated from healthy donors (HDs) ( n = 3) or psoriatic patients ( n = 3) were cultured for 20 h with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848, or 500 ng/ml IL-17A to evaluate IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. (B) Surface IL-17RA and IL-17RC expression evaluated by flow cytometry in human neutrophils from HDs or psoriatic patients. Values represent the mean ± SEM ( n = 3). For the data of panels (A,B) no significant differences between HDs or psoriatic patients were observed by two-way ANOVA followed by Bonferroni’s post-test.
    Figure Legend Snippet: IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression, as well as IL-17R surface expression, in neutrophils from patients with psoriasis. (A) Neutrophils isolated from healthy donors (HDs) ( n = 3) or psoriatic patients ( n = 3) were cultured for 20 h with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848, or 500 ng/ml IL-17A to evaluate IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. (B) Surface IL-17RA and IL-17RC expression evaluated by flow cytometry in human neutrophils from HDs or psoriatic patients. Values represent the mean ± SEM ( n = 3). For the data of panels (A,B) no significant differences between HDs or psoriatic patients were observed by two-way ANOVA followed by Bonferroni’s post-test.

    Techniques Used: Expressing, Isolation, Cell Culture, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry

    Levels of IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, IL-17RA, azurocidin, neutrophil elastase, and myeloperoxidase (MPO) mRNA expression in neutrophils at different stages of maturation. mRNA expression data derive from Gene Expression Omnibus database (accession number GSE42519) ( 65 ). (A) IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, and IL-17RA or (B) azurocidin (AZU1), neutrophil elastase (ELANE), and MPO mRNA expression levels were measured in the following cell types: hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), early and late promyelocytes (PMs), myelocytes (MYs), metamyelocytes (MMs), band cells (BCs), and bone marrow polymorphonuclear neutrophil granulocytes. Values represent the mean ± SEM as calculated from data of the biological replicates present in the database.
    Figure Legend Snippet: Levels of IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, IL-17RA, azurocidin, neutrophil elastase, and myeloperoxidase (MPO) mRNA expression in neutrophils at different stages of maturation. mRNA expression data derive from Gene Expression Omnibus database (accession number GSE42519) ( 65 ). (A) IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, and IL-17RA or (B) azurocidin (AZU1), neutrophil elastase (ELANE), and MPO mRNA expression levels were measured in the following cell types: hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), early and late promyelocytes (PMs), myelocytes (MYs), metamyelocytes (MMs), band cells (BCs), and bone marrow polymorphonuclear neutrophil granulocytes. Values represent the mean ± SEM as calculated from data of the biological replicates present in the database.

    Techniques Used: Expressing

    No induction of IL-17A, IL-17F, and IL-17RC mRNA expression in neutrophils incubated with IL-6 plus IL-23, in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated either with 100 ng/ml rIL-17A for 2 h or with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 for 1 h, prior to adding, or not, inactivated A. fumigatus conidia (1:5 neutrophils/conidia ratio) and hyphae (1:1 neutrophils/hyphae ratio) for additional 1 h. Neutrophils were then harvested for RNA extraction to evaluate IL-17A (A) , IL-17F (B) , IL-17RC (C) , IL-17RA (D) , and SOCS3 (F) mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization (mean ± SEM, n = 4). Asterisks stand for significant differences as compared to untreated cells: * P
    Figure Legend Snippet: No induction of IL-17A, IL-17F, and IL-17RC mRNA expression in neutrophils incubated with IL-6 plus IL-23, in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated either with 100 ng/ml rIL-17A for 2 h or with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 for 1 h, prior to adding, or not, inactivated A. fumigatus conidia (1:5 neutrophils/conidia ratio) and hyphae (1:1 neutrophils/hyphae ratio) for additional 1 h. Neutrophils were then harvested for RNA extraction to evaluate IL-17A (A) , IL-17F (B) , IL-17RC (C) , IL-17RA (D) , and SOCS3 (F) mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization (mean ± SEM, n = 4). Asterisks stand for significant differences as compared to untreated cells: * P

    Techniques Used: Expressing, Incubation, RNA Extraction, Real-time Polymerase Chain Reaction

    Lack of IL-17A and IL-17F production by human neutrophils activated by IL-6 plus IL-23 in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 and then cultured for three more hours in the presence or not of inactivated A. fumigatus conidia and hyphae (used at 1:5 and 1:1, respectively). After incubation, IL-17A (A) and CXCL8 (B) levels were determined in cell-free supernatants and in corresponding cell pellets by specific ELISA. Values are depicted as the mean ± SD or as not detected (nd) when values were under the detection limit ( n = 3). Asterisks stand for significant differences as compared to untreated cells: * P
    Figure Legend Snippet: Lack of IL-17A and IL-17F production by human neutrophils activated by IL-6 plus IL-23 in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 and then cultured for three more hours in the presence or not of inactivated A. fumigatus conidia and hyphae (used at 1:5 and 1:1, respectively). After incubation, IL-17A (A) and CXCL8 (B) levels were determined in cell-free supernatants and in corresponding cell pellets by specific ELISA. Values are depicted as the mean ± SD or as not detected (nd) when values were under the detection limit ( n = 3). Asterisks stand for significant differences as compared to untreated cells: * P

    Techniques Used: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

    Expression of surface IL-17RA and IL-17RC in neutrophils activated under various experimental conditions. Expression of surface IL-17RA (left panel) and IL-17RC (right panel) was evaluated by flow cytometry in neutrophils either freshly isolated or cultured for 3 h without or with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848 (A) , 20 µg/ml IL-6 plus 2 µg/ml IL-23 alone or in the presence of inactivated Aspergillus fumigatus conidia, hyphae or 500 ng/ml rIL-17A (B) . Graphs depict a representative experiment out of three independent ones with similar results. Histograms show staining by specific and isotype control Abs, respectively, for each stimulatory condition.
    Figure Legend Snippet: Expression of surface IL-17RA and IL-17RC in neutrophils activated under various experimental conditions. Expression of surface IL-17RA (left panel) and IL-17RC (right panel) was evaluated by flow cytometry in neutrophils either freshly isolated or cultured for 3 h without or with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848 (A) , 20 µg/ml IL-6 plus 2 µg/ml IL-23 alone or in the presence of inactivated Aspergillus fumigatus conidia, hyphae or 500 ng/ml rIL-17A (B) . Graphs depict a representative experiment out of three independent ones with similar results. Histograms show staining by specific and isotype control Abs, respectively, for each stimulatory condition.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Isolation, Cell Culture, Staining

    H3K4me1 or H3K27Ac levels at the IL-17A, IL-17F, and SOCS3 genomic loci of Th17 cell lines and resting/IL-6 plus IL-23-activated neutrophils. Enrichment levels of H3K4me1 (left panels) and H3K27Ac (right panels) at the IL-17A (A) , IL-17F (B) , and SOCS3 (C) genomic loci by chromatin immunoprecipitation (ChIP) analysis in human Th17 cell lines and neutrophils incubated for 1 h with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23. (A–C) Schemes illustrating the positions of the designed primer pairs amplifying promoter and potential enhancer regions of IL-17A, IL-17F, and SOCS3 for ChIP analysis are depicted at the top of each panel. Coimmunoprecipitated DNA samples were expressed as percent of the total input. Panels in (A–C) depict a representative experiment out of two independent ones with similar results. Error bars represent SEs calculated from triplicate qPCR reactions.
    Figure Legend Snippet: H3K4me1 or H3K27Ac levels at the IL-17A, IL-17F, and SOCS3 genomic loci of Th17 cell lines and resting/IL-6 plus IL-23-activated neutrophils. Enrichment levels of H3K4me1 (left panels) and H3K27Ac (right panels) at the IL-17A (A) , IL-17F (B) , and SOCS3 (C) genomic loci by chromatin immunoprecipitation (ChIP) analysis in human Th17 cell lines and neutrophils incubated for 1 h with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23. (A–C) Schemes illustrating the positions of the designed primer pairs amplifying promoter and potential enhancer regions of IL-17A, IL-17F, and SOCS3 for ChIP analysis are depicted at the top of each panel. Coimmunoprecipitated DNA samples were expressed as percent of the total input. Panels in (A–C) depict a representative experiment out of two independent ones with similar results. Error bars represent SEs calculated from triplicate qPCR reactions.

    Techniques Used: Chromatin Immunoprecipitation, Incubation, Real-time Polymerase Chain Reaction

    IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression levels in human neutrophils activated by a variety of stimuli. Human neutrophils were cultured at 5 × 10 6 /ml for up to 20 h with (A) 100 U/ml IFNγ and/or 100 ng/ml LPS; (B) 1,000 U/ml IFNα and/or 5 µM R848; (C) 10 ng/ml GM-CSF or 100 nM fMLF; (D) 1,000 U/ml G-CSF or 5 ng/ml TNFα. IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression was evaluated by reverse transcription quantitative real-time PCR (RT-qPCR) and data depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. The experiments depicted in each panels (A–D) are representative of at least three ones with similar results. Error bars stand for SEs calculated from triplicate qPCR reactions.
    Figure Legend Snippet: IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression levels in human neutrophils activated by a variety of stimuli. Human neutrophils were cultured at 5 × 10 6 /ml for up to 20 h with (A) 100 U/ml IFNγ and/or 100 ng/ml LPS; (B) 1,000 U/ml IFNα and/or 5 µM R848; (C) 10 ng/ml GM-CSF or 100 nM fMLF; (D) 1,000 U/ml G-CSF or 5 ng/ml TNFα. IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression was evaluated by reverse transcription quantitative real-time PCR (RT-qPCR) and data depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. The experiments depicted in each panels (A–D) are representative of at least three ones with similar results. Error bars stand for SEs calculated from triplicate qPCR reactions.

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Staining human neutrophils by anti-IL-17B (AF1248) antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) staining of two FFPE cases of human pustular psoriasis using anti-IL-17B (AF1248) and CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils incubated without (top panel) or with 5 µM R848 (bottom panel) for 3 h. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF1248 immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17B (rhIL-17B) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.
    Figure Legend Snippet: Staining human neutrophils by anti-IL-17B (AF1248) antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) staining of two FFPE cases of human pustular psoriasis using anti-IL-17B (AF1248) and CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils incubated without (top panel) or with 5 µM R848 (bottom panel) for 3 h. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF1248 immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17B (rhIL-17B) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.

    Techniques Used: Staining, Immunofluorescence, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Labeling, Marker, Incubation, Isolation, Recombinant, Positive Control

    27) Product Images from "A Reappraisal on the Potential Ability of Human Neutrophils to Express and Produce IL-17 Family Members In Vitro: Failure to Reproducibly Detect It"

    Article Title: A Reappraisal on the Potential Ability of Human Neutrophils to Express and Produce IL-17 Family Members In Vitro: Failure to Reproducibly Detect It

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00795

    Staining human neutrophils by anti-IL-17A (AF-317-NA) polyclonal antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) stainings of two FFPE cases of human pustular psoriasis using anti-IL-17A (AF-317-NA) and anti-CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils, either untreated (top panels) or treated with 5 µM R848 (bottom panels) for 3 h, were stained with anti-IL-17A (AF-317-NA, left panels) and anti-CXCL8 (right panels) Abs. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF-317-NA immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17A (rhIL-17A) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.
    Figure Legend Snippet: Staining human neutrophils by anti-IL-17A (AF-317-NA) polyclonal antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) stainings of two FFPE cases of human pustular psoriasis using anti-IL-17A (AF-317-NA) and anti-CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils, either untreated (top panels) or treated with 5 µM R848 (bottom panels) for 3 h, were stained with anti-IL-17A (AF-317-NA, left panels) and anti-CXCL8 (right panels) Abs. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF-317-NA immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17A (rhIL-17A) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.

    Techniques Used: Staining, Immunofluorescence, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Labeling, Marker, Isolation, Incubation, Recombinant, Positive Control

    IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression, as well as IL-17R surface expression, in neutrophils from patients with psoriasis. (A) Neutrophils isolated from healthy donors (HDs) ( n = 3) or psoriatic patients ( n = 3) were cultured for 20 h with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848, or 500 ng/ml IL-17A to evaluate IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. (B) Surface IL-17RA and IL-17RC expression evaluated by flow cytometry in human neutrophils from HDs or psoriatic patients. Values represent the mean ± SEM ( n = 3). For the data of panels (A,B) no significant differences between HDs or psoriatic patients were observed by two-way ANOVA followed by Bonferroni’s post-test.
    Figure Legend Snippet: IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression, as well as IL-17R surface expression, in neutrophils from patients with psoriasis. (A) Neutrophils isolated from healthy donors (HDs) ( n = 3) or psoriatic patients ( n = 3) were cultured for 20 h with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848, or 500 ng/ml IL-17A to evaluate IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. (B) Surface IL-17RA and IL-17RC expression evaluated by flow cytometry in human neutrophils from HDs or psoriatic patients. Values represent the mean ± SEM ( n = 3). For the data of panels (A,B) no significant differences between HDs or psoriatic patients were observed by two-way ANOVA followed by Bonferroni’s post-test.

    Techniques Used: Expressing, Isolation, Cell Culture, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry

    Levels of IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, IL-17RA, azurocidin, neutrophil elastase, and myeloperoxidase (MPO) mRNA expression in neutrophils at different stages of maturation. mRNA expression data derive from Gene Expression Omnibus database (accession number GSE42519) ( 65 ). (A) IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, and IL-17RA or (B) azurocidin (AZU1), neutrophil elastase (ELANE), and MPO mRNA expression levels were measured in the following cell types: hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), early and late promyelocytes (PMs), myelocytes (MYs), metamyelocytes (MMs), band cells (BCs), and bone marrow polymorphonuclear neutrophil granulocytes. Values represent the mean ± SEM as calculated from data of the biological replicates present in the database.
    Figure Legend Snippet: Levels of IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, IL-17RA, azurocidin, neutrophil elastase, and myeloperoxidase (MPO) mRNA expression in neutrophils at different stages of maturation. mRNA expression data derive from Gene Expression Omnibus database (accession number GSE42519) ( 65 ). (A) IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, and IL-17RA or (B) azurocidin (AZU1), neutrophil elastase (ELANE), and MPO mRNA expression levels were measured in the following cell types: hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), early and late promyelocytes (PMs), myelocytes (MYs), metamyelocytes (MMs), band cells (BCs), and bone marrow polymorphonuclear neutrophil granulocytes. Values represent the mean ± SEM as calculated from data of the biological replicates present in the database.

    Techniques Used: Expressing

    No induction of IL-17A, IL-17F, and IL-17RC mRNA expression in neutrophils incubated with IL-6 plus IL-23, in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated either with 100 ng/ml rIL-17A for 2 h or with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 for 1 h, prior to adding, or not, inactivated A. fumigatus conidia (1:5 neutrophils/conidia ratio) and hyphae (1:1 neutrophils/hyphae ratio) for additional 1 h. Neutrophils were then harvested for RNA extraction to evaluate IL-17A (A) , IL-17F (B) , IL-17RC (C) , IL-17RA (D) , and SOCS3 (F) mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization (mean ± SEM, n = 4). Asterisks stand for significant differences as compared to untreated cells: * P
    Figure Legend Snippet: No induction of IL-17A, IL-17F, and IL-17RC mRNA expression in neutrophils incubated with IL-6 plus IL-23, in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated either with 100 ng/ml rIL-17A for 2 h or with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 for 1 h, prior to adding, or not, inactivated A. fumigatus conidia (1:5 neutrophils/conidia ratio) and hyphae (1:1 neutrophils/hyphae ratio) for additional 1 h. Neutrophils were then harvested for RNA extraction to evaluate IL-17A (A) , IL-17F (B) , IL-17RC (C) , IL-17RA (D) , and SOCS3 (F) mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization (mean ± SEM, n = 4). Asterisks stand for significant differences as compared to untreated cells: * P

    Techniques Used: Expressing, Incubation, RNA Extraction, Real-time Polymerase Chain Reaction

    Lack of IL-17A and IL-17F production by human neutrophils activated by IL-6 plus IL-23 in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 and then cultured for three more hours in the presence or not of inactivated A. fumigatus conidia and hyphae (used at 1:5 and 1:1, respectively). After incubation, IL-17A (A) and CXCL8 (B) levels were determined in cell-free supernatants and in corresponding cell pellets by specific ELISA. Values are depicted as the mean ± SD or as not detected (nd) when values were under the detection limit ( n = 3). Asterisks stand for significant differences as compared to untreated cells: * P
    Figure Legend Snippet: Lack of IL-17A and IL-17F production by human neutrophils activated by IL-6 plus IL-23 in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 and then cultured for three more hours in the presence or not of inactivated A. fumigatus conidia and hyphae (used at 1:5 and 1:1, respectively). After incubation, IL-17A (A) and CXCL8 (B) levels were determined in cell-free supernatants and in corresponding cell pellets by specific ELISA. Values are depicted as the mean ± SD or as not detected (nd) when values were under the detection limit ( n = 3). Asterisks stand for significant differences as compared to untreated cells: * P

    Techniques Used: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

    Expression of surface IL-17RA and IL-17RC in neutrophils activated under various experimental conditions. Expression of surface IL-17RA (left panel) and IL-17RC (right panel) was evaluated by flow cytometry in neutrophils either freshly isolated or cultured for 3 h without or with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848 (A) , 20 µg/ml IL-6 plus 2 µg/ml IL-23 alone or in the presence of inactivated Aspergillus fumigatus conidia, hyphae or 500 ng/ml rIL-17A (B) . Graphs depict a representative experiment out of three independent ones with similar results. Histograms show staining by specific and isotype control Abs, respectively, for each stimulatory condition.
    Figure Legend Snippet: Expression of surface IL-17RA and IL-17RC in neutrophils activated under various experimental conditions. Expression of surface IL-17RA (left panel) and IL-17RC (right panel) was evaluated by flow cytometry in neutrophils either freshly isolated or cultured for 3 h without or with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848 (A) , 20 µg/ml IL-6 plus 2 µg/ml IL-23 alone or in the presence of inactivated Aspergillus fumigatus conidia, hyphae or 500 ng/ml rIL-17A (B) . Graphs depict a representative experiment out of three independent ones with similar results. Histograms show staining by specific and isotype control Abs, respectively, for each stimulatory condition.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Isolation, Cell Culture, Staining

    H3K4me1 or H3K27Ac levels at the IL-17A, IL-17F, and SOCS3 genomic loci of Th17 cell lines and resting/IL-6 plus IL-23-activated neutrophils. Enrichment levels of H3K4me1 (left panels) and H3K27Ac (right panels) at the IL-17A (A) , IL-17F (B) , and SOCS3 (C) genomic loci by chromatin immunoprecipitation (ChIP) analysis in human Th17 cell lines and neutrophils incubated for 1 h with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23. (A–C) Schemes illustrating the positions of the designed primer pairs amplifying promoter and potential enhancer regions of IL-17A, IL-17F, and SOCS3 for ChIP analysis are depicted at the top of each panel. Coimmunoprecipitated DNA samples were expressed as percent of the total input. Panels in (A–C) depict a representative experiment out of two independent ones with similar results. Error bars represent SEs calculated from triplicate qPCR reactions.
    Figure Legend Snippet: H3K4me1 or H3K27Ac levels at the IL-17A, IL-17F, and SOCS3 genomic loci of Th17 cell lines and resting/IL-6 plus IL-23-activated neutrophils. Enrichment levels of H3K4me1 (left panels) and H3K27Ac (right panels) at the IL-17A (A) , IL-17F (B) , and SOCS3 (C) genomic loci by chromatin immunoprecipitation (ChIP) analysis in human Th17 cell lines and neutrophils incubated for 1 h with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23. (A–C) Schemes illustrating the positions of the designed primer pairs amplifying promoter and potential enhancer regions of IL-17A, IL-17F, and SOCS3 for ChIP analysis are depicted at the top of each panel. Coimmunoprecipitated DNA samples were expressed as percent of the total input. Panels in (A–C) depict a representative experiment out of two independent ones with similar results. Error bars represent SEs calculated from triplicate qPCR reactions.

    Techniques Used: Chromatin Immunoprecipitation, Incubation, Real-time Polymerase Chain Reaction

    IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression levels in human neutrophils activated by a variety of stimuli. Human neutrophils were cultured at 5 × 10 6 /ml for up to 20 h with (A) 100 U/ml IFNγ and/or 100 ng/ml LPS; (B) 1,000 U/ml IFNα and/or 5 µM R848; (C) 10 ng/ml GM-CSF or 100 nM fMLF; (D) 1,000 U/ml G-CSF or 5 ng/ml TNFα. IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression was evaluated by reverse transcription quantitative real-time PCR (RT-qPCR) and data depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. The experiments depicted in each panels (A–D) are representative of at least three ones with similar results. Error bars stand for SEs calculated from triplicate qPCR reactions.
    Figure Legend Snippet: IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression levels in human neutrophils activated by a variety of stimuli. Human neutrophils were cultured at 5 × 10 6 /ml for up to 20 h with (A) 100 U/ml IFNγ and/or 100 ng/ml LPS; (B) 1,000 U/ml IFNα and/or 5 µM R848; (C) 10 ng/ml GM-CSF or 100 nM fMLF; (D) 1,000 U/ml G-CSF or 5 ng/ml TNFα. IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression was evaluated by reverse transcription quantitative real-time PCR (RT-qPCR) and data depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. The experiments depicted in each panels (A–D) are representative of at least three ones with similar results. Error bars stand for SEs calculated from triplicate qPCR reactions.

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Staining human neutrophils by anti-IL-17B (AF1248) antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) staining of two FFPE cases of human pustular psoriasis using anti-IL-17B (AF1248) and CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils incubated without (top panel) or with 5 µM R848 (bottom panel) for 3 h. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF1248 immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17B (rhIL-17B) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.
    Figure Legend Snippet: Staining human neutrophils by anti-IL-17B (AF1248) antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) staining of two FFPE cases of human pustular psoriasis using anti-IL-17B (AF1248) and CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils incubated without (top panel) or with 5 µM R848 (bottom panel) for 3 h. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF1248 immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17B (rhIL-17B) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.

    Techniques Used: Staining, Immunofluorescence, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Labeling, Marker, Incubation, Isolation, Recombinant, Positive Control

    28) Product Images from "A Reappraisal on the Potential Ability of Human Neutrophils to Express and Produce IL-17 Family Members In Vitro: Failure to Reproducibly Detect It"

    Article Title: A Reappraisal on the Potential Ability of Human Neutrophils to Express and Produce IL-17 Family Members In Vitro: Failure to Reproducibly Detect It

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00795

    Staining human neutrophils by anti-IL-17A (AF-317-NA) polyclonal antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) stainings of two FFPE cases of human pustular psoriasis using anti-IL-17A (AF-317-NA) and anti-CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils, either untreated (top panels) or treated with 5 µM R848 (bottom panels) for 3 h, were stained with anti-IL-17A (AF-317-NA, left panels) and anti-CXCL8 (right panels) Abs. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF-317-NA immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17A (rhIL-17A) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.
    Figure Legend Snippet: Staining human neutrophils by anti-IL-17A (AF-317-NA) polyclonal antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) stainings of two FFPE cases of human pustular psoriasis using anti-IL-17A (AF-317-NA) and anti-CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils, either untreated (top panels) or treated with 5 µM R848 (bottom panels) for 3 h, were stained with anti-IL-17A (AF-317-NA, left panels) and anti-CXCL8 (right panels) Abs. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF-317-NA immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17A (rhIL-17A) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.

    Techniques Used: Staining, Immunofluorescence, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Labeling, Marker, Isolation, Incubation, Recombinant, Positive Control

    IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression, as well as IL-17R surface expression, in neutrophils from patients with psoriasis. (A) Neutrophils isolated from healthy donors (HDs) ( n = 3) or psoriatic patients ( n = 3) were cultured for 20 h with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848, or 500 ng/ml IL-17A to evaluate IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. (B) Surface IL-17RA and IL-17RC expression evaluated by flow cytometry in human neutrophils from HDs or psoriatic patients. Values represent the mean ± SEM ( n = 3). For the data of panels (A,B) no significant differences between HDs or psoriatic patients were observed by two-way ANOVA followed by Bonferroni’s post-test.
    Figure Legend Snippet: IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression, as well as IL-17R surface expression, in neutrophils from patients with psoriasis. (A) Neutrophils isolated from healthy donors (HDs) ( n = 3) or psoriatic patients ( n = 3) were cultured for 20 h with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848, or 500 ng/ml IL-17A to evaluate IL-17A, IL-17F, IL-17RA, IL-17RC, CXCL8, TNFα, and SOCS3 mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. (B) Surface IL-17RA and IL-17RC expression evaluated by flow cytometry in human neutrophils from HDs or psoriatic patients. Values represent the mean ± SEM ( n = 3). For the data of panels (A,B) no significant differences between HDs or psoriatic patients were observed by two-way ANOVA followed by Bonferroni’s post-test.

    Techniques Used: Expressing, Isolation, Cell Culture, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry

    Levels of IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, IL-17RA, azurocidin, neutrophil elastase, and myeloperoxidase (MPO) mRNA expression in neutrophils at different stages of maturation. mRNA expression data derive from Gene Expression Omnibus database (accession number GSE42519) ( 65 ). (A) IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, and IL-17RA or (B) azurocidin (AZU1), neutrophil elastase (ELANE), and MPO mRNA expression levels were measured in the following cell types: hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), early and late promyelocytes (PMs), myelocytes (MYs), metamyelocytes (MMs), band cells (BCs), and bone marrow polymorphonuclear neutrophil granulocytes. Values represent the mean ± SEM as calculated from data of the biological replicates present in the database.
    Figure Legend Snippet: Levels of IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, IL-17RA, azurocidin, neutrophil elastase, and myeloperoxidase (MPO) mRNA expression in neutrophils at different stages of maturation. mRNA expression data derive from Gene Expression Omnibus database (accession number GSE42519) ( 65 ). (A) IL-17A, IL-17B, IL-17F, IL-10, IL-17RC, and IL-17RA or (B) azurocidin (AZU1), neutrophil elastase (ELANE), and MPO mRNA expression levels were measured in the following cell types: hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), early and late promyelocytes (PMs), myelocytes (MYs), metamyelocytes (MMs), band cells (BCs), and bone marrow polymorphonuclear neutrophil granulocytes. Values represent the mean ± SEM as calculated from data of the biological replicates present in the database.

    Techniques Used: Expressing

    No induction of IL-17A, IL-17F, and IL-17RC mRNA expression in neutrophils incubated with IL-6 plus IL-23, in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated either with 100 ng/ml rIL-17A for 2 h or with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 for 1 h, prior to adding, or not, inactivated A. fumigatus conidia (1:5 neutrophils/conidia ratio) and hyphae (1:1 neutrophils/hyphae ratio) for additional 1 h. Neutrophils were then harvested for RNA extraction to evaluate IL-17A (A) , IL-17F (B) , IL-17RC (C) , IL-17RA (D) , and SOCS3 (F) mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization (mean ± SEM, n = 4). Asterisks stand for significant differences as compared to untreated cells: * P
    Figure Legend Snippet: No induction of IL-17A, IL-17F, and IL-17RC mRNA expression in neutrophils incubated with IL-6 plus IL-23, in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated either with 100 ng/ml rIL-17A for 2 h or with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 for 1 h, prior to adding, or not, inactivated A. fumigatus conidia (1:5 neutrophils/conidia ratio) and hyphae (1:1 neutrophils/hyphae ratio) for additional 1 h. Neutrophils were then harvested for RNA extraction to evaluate IL-17A (A) , IL-17F (B) , IL-17RC (C) , IL-17RA (D) , and SOCS3 (F) mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization (mean ± SEM, n = 4). Asterisks stand for significant differences as compared to untreated cells: * P

    Techniques Used: Expressing, Incubation, RNA Extraction, Real-time Polymerase Chain Reaction

    Lack of IL-17A and IL-17F production by human neutrophils activated by IL-6 plus IL-23 in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 and then cultured for three more hours in the presence or not of inactivated A. fumigatus conidia and hyphae (used at 1:5 and 1:1, respectively). After incubation, IL-17A (A) and CXCL8 (B) levels were determined in cell-free supernatants and in corresponding cell pellets by specific ELISA. Values are depicted as the mean ± SD or as not detected (nd) when values were under the detection limit ( n = 3). Asterisks stand for significant differences as compared to untreated cells: * P
    Figure Legend Snippet: Lack of IL-17A and IL-17F production by human neutrophils activated by IL-6 plus IL-23 in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 10 6 /ml) were incubated with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 and then cultured for three more hours in the presence or not of inactivated A. fumigatus conidia and hyphae (used at 1:5 and 1:1, respectively). After incubation, IL-17A (A) and CXCL8 (B) levels were determined in cell-free supernatants and in corresponding cell pellets by specific ELISA. Values are depicted as the mean ± SD or as not detected (nd) when values were under the detection limit ( n = 3). Asterisks stand for significant differences as compared to untreated cells: * P

    Techniques Used: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

    Expression of surface IL-17RA and IL-17RC in neutrophils activated under various experimental conditions. Expression of surface IL-17RA (left panel) and IL-17RC (right panel) was evaluated by flow cytometry in neutrophils either freshly isolated or cultured for 3 h without or with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848 (A) , 20 µg/ml IL-6 plus 2 µg/ml IL-23 alone or in the presence of inactivated Aspergillus fumigatus conidia, hyphae or 500 ng/ml rIL-17A (B) . Graphs depict a representative experiment out of three independent ones with similar results. Histograms show staining by specific and isotype control Abs, respectively, for each stimulatory condition.
    Figure Legend Snippet: Expression of surface IL-17RA and IL-17RC in neutrophils activated under various experimental conditions. Expression of surface IL-17RA (left panel) and IL-17RC (right panel) was evaluated by flow cytometry in neutrophils either freshly isolated or cultured for 3 h without or with 100 U/ml IFNγ plus 100 ng/ml LPS, 5 µM R848 (A) , 20 µg/ml IL-6 plus 2 µg/ml IL-23 alone or in the presence of inactivated Aspergillus fumigatus conidia, hyphae or 500 ng/ml rIL-17A (B) . Graphs depict a representative experiment out of three independent ones with similar results. Histograms show staining by specific and isotype control Abs, respectively, for each stimulatory condition.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Isolation, Cell Culture, Staining

    H3K4me1 or H3K27Ac levels at the IL-17A, IL-17F, and SOCS3 genomic loci of Th17 cell lines and resting/IL-6 plus IL-23-activated neutrophils. Enrichment levels of H3K4me1 (left panels) and H3K27Ac (right panels) at the IL-17A (A) , IL-17F (B) , and SOCS3 (C) genomic loci by chromatin immunoprecipitation (ChIP) analysis in human Th17 cell lines and neutrophils incubated for 1 h with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23. (A–C) Schemes illustrating the positions of the designed primer pairs amplifying promoter and potential enhancer regions of IL-17A, IL-17F, and SOCS3 for ChIP analysis are depicted at the top of each panel. Coimmunoprecipitated DNA samples were expressed as percent of the total input. Panels in (A–C) depict a representative experiment out of two independent ones with similar results. Error bars represent SEs calculated from triplicate qPCR reactions.
    Figure Legend Snippet: H3K4me1 or H3K27Ac levels at the IL-17A, IL-17F, and SOCS3 genomic loci of Th17 cell lines and resting/IL-6 plus IL-23-activated neutrophils. Enrichment levels of H3K4me1 (left panels) and H3K27Ac (right panels) at the IL-17A (A) , IL-17F (B) , and SOCS3 (C) genomic loci by chromatin immunoprecipitation (ChIP) analysis in human Th17 cell lines and neutrophils incubated for 1 h with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23. (A–C) Schemes illustrating the positions of the designed primer pairs amplifying promoter and potential enhancer regions of IL-17A, IL-17F, and SOCS3 for ChIP analysis are depicted at the top of each panel. Coimmunoprecipitated DNA samples were expressed as percent of the total input. Panels in (A–C) depict a representative experiment out of two independent ones with similar results. Error bars represent SEs calculated from triplicate qPCR reactions.

    Techniques Used: Chromatin Immunoprecipitation, Incubation, Real-time Polymerase Chain Reaction

    IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression levels in human neutrophils activated by a variety of stimuli. Human neutrophils were cultured at 5 × 10 6 /ml for up to 20 h with (A) 100 U/ml IFNγ and/or 100 ng/ml LPS; (B) 1,000 U/ml IFNα and/or 5 µM R848; (C) 10 ng/ml GM-CSF or 100 nM fMLF; (D) 1,000 U/ml G-CSF or 5 ng/ml TNFα. IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression was evaluated by reverse transcription quantitative real-time PCR (RT-qPCR) and data depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. The experiments depicted in each panels (A–D) are representative of at least three ones with similar results. Error bars stand for SEs calculated from triplicate qPCR reactions.
    Figure Legend Snippet: IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression levels in human neutrophils activated by a variety of stimuli. Human neutrophils were cultured at 5 × 10 6 /ml for up to 20 h with (A) 100 U/ml IFNγ and/or 100 ng/ml LPS; (B) 1,000 U/ml IFNα and/or 5 µM R848; (C) 10 ng/ml GM-CSF or 100 nM fMLF; (D) 1,000 U/ml G-CSF or 5 ng/ml TNFα. IL-17A, IL-17F, CXCL8, and IL-1ra mRNA expression was evaluated by reverse transcription quantitative real-time PCR (RT-qPCR) and data depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization. The experiments depicted in each panels (A–D) are representative of at least three ones with similar results. Error bars stand for SEs calculated from triplicate qPCR reactions.

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Staining human neutrophils by anti-IL-17B (AF1248) antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) staining of two FFPE cases of human pustular psoriasis using anti-IL-17B (AF1248) and CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils incubated without (top panel) or with 5 µM R848 (bottom panel) for 3 h. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF1248 immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17B (rhIL-17B) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.
    Figure Legend Snippet: Staining human neutrophils by anti-IL-17B (AF1248) antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) staining of two FFPE cases of human pustular psoriasis using anti-IL-17B (AF1248) and CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils incubated without (top panel) or with 5 µM R848 (bottom panel) for 3 h. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF1248 immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17B (rhIL-17B) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.

    Techniques Used: Staining, Immunofluorescence, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Labeling, Marker, Incubation, Isolation, Recombinant, Positive Control

    29) Product Images from "Type I Interferon signaling constrains IL-17A/F secretion by ?? T cells during bacterial infections"

    Article Title: Type I Interferon signaling constrains IL-17A/F secretion by ?? T cells during bacterial infections

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0902065

    Type I IFN negatively regulate IL-17A secretion by γδ T cells during Listeria infection Results from 2 independent experiments (A, E, G) or from 1 experiment representative of 2 independent experiments (B, C, D, F) are shown. (A) Bacterial burden was determined at 48h PI in the spleen following tail vein injection of 1.4 10 4 cfu of L. monocytogenes . Each circle represents the cfu in one mouse, geometric mean is shown. IL-17A (B) and IFN-γ (C) transcript levels in the spleen were determined by qRT-PCR at 48h and expressed as fold increase over WT uninfected. (D) Splenocytes from WT or IFNAR1 −/− mice infected for 48h with L. monocytogenes were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (left panels) or the isotype control (right panel) in singlets, live, CD19 − , CD3ε + , CD4 − , CD8 − , TCRαβ − , TCRγδ + cells versus the expression of TCR γδ is shown. Numbers indicate the percentage of IL-17A + cells of the total γδ T cells. FACS plots corresponding to five mice, are shown. (E) Percentage of IL-17A + cells over the total of γδ T cells is shown. Each circle corresponds to the value in one mouse, geometric mean is shown. Total numbers of γδ T cell (F) and neutrophil (G) per spleen are shown. 1: p
    Figure Legend Snippet: Type I IFN negatively regulate IL-17A secretion by γδ T cells during Listeria infection Results from 2 independent experiments (A, E, G) or from 1 experiment representative of 2 independent experiments (B, C, D, F) are shown. (A) Bacterial burden was determined at 48h PI in the spleen following tail vein injection of 1.4 10 4 cfu of L. monocytogenes . Each circle represents the cfu in one mouse, geometric mean is shown. IL-17A (B) and IFN-γ (C) transcript levels in the spleen were determined by qRT-PCR at 48h and expressed as fold increase over WT uninfected. (D) Splenocytes from WT or IFNAR1 −/− mice infected for 48h with L. monocytogenes were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (left panels) or the isotype control (right panel) in singlets, live, CD19 − , CD3ε + , CD4 − , CD8 − , TCRαβ − , TCRγδ + cells versus the expression of TCR γδ is shown. Numbers indicate the percentage of IL-17A + cells of the total γδ T cells. FACS plots corresponding to five mice, are shown. (E) Percentage of IL-17A + cells over the total of γδ T cells is shown. Each circle corresponds to the value in one mouse, geometric mean is shown. Total numbers of γδ T cell (F) and neutrophil (G) per spleen are shown. 1: p

    Techniques Used: Infection, Injection, Quantitative RT-PCR, Mouse Assay, Isolation, Cell Culture, Ex Vivo, Staining, FACS, Expressing

    Type I IFN signaling contrains IL-17A secretion by γδ T cells during i.n. infection with 50 cfu F. novicida (A) Type I IFN signaling is detrimental in a survival experiment during during i.n. infection with 50 cfu F. novicida . WT (n=10) and IFNAR1 −/− (n=10) C57BL/6J mice were inoculated with 50 F. novicida cfu by the intranasal route. Survival was monitored twice daily during 10 days (1: p=0.0053). (B) Type I IFN signaling is detrimental to control the bacterial burden during during i.n. infection with 50 cfu F. novicida . Bacterial colonization in the spleen was determined at 72h and 96h PI (at this latter time point, 2 WT mice had died as indicated †) (2: p=0.0286; 3: p=0.0286). (C) type I IFN signaling negatively controls IL-17A transcript level in the spleen (3: p=0.0286). (D) type I IFN signaling negatively controls IL-17A production by γδ T cells in the spleen of infected mice. Splenocytes from infected mice were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A or the isotype control (as indicated) in singlets, live, CD19 − , CD3ε + , TCR αβ − , TCR γδ + cells is shown. Numbers indicate the percentage of IL-17A + cells (or isotype + cells) in total γδ T cells. FACS plots corresponding to four mice are shown. (E) The number of IL-17A-producing γδ T cells was determined in each mouse and expressed as the percentage of total γδ T cells. Each circle represents the value in a single mouse. Geometric mean is shown. 4: p=0.0286. (E) The number of splenic neutrophil per mouse was determined. Each circle corresponds to a single mouse, geometric mean is shown. 5: p=0.0286.
    Figure Legend Snippet: Type I IFN signaling contrains IL-17A secretion by γδ T cells during i.n. infection with 50 cfu F. novicida (A) Type I IFN signaling is detrimental in a survival experiment during during i.n. infection with 50 cfu F. novicida . WT (n=10) and IFNAR1 −/− (n=10) C57BL/6J mice were inoculated with 50 F. novicida cfu by the intranasal route. Survival was monitored twice daily during 10 days (1: p=0.0053). (B) Type I IFN signaling is detrimental to control the bacterial burden during during i.n. infection with 50 cfu F. novicida . Bacterial colonization in the spleen was determined at 72h and 96h PI (at this latter time point, 2 WT mice had died as indicated †) (2: p=0.0286; 3: p=0.0286). (C) type I IFN signaling negatively controls IL-17A transcript level in the spleen (3: p=0.0286). (D) type I IFN signaling negatively controls IL-17A production by γδ T cells in the spleen of infected mice. Splenocytes from infected mice were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A or the isotype control (as indicated) in singlets, live, CD19 − , CD3ε + , TCR αβ − , TCR γδ + cells is shown. Numbers indicate the percentage of IL-17A + cells (or isotype + cells) in total γδ T cells. FACS plots corresponding to four mice are shown. (E) The number of IL-17A-producing γδ T cells was determined in each mouse and expressed as the percentage of total γδ T cells. Each circle represents the value in a single mouse. Geometric mean is shown. 4: p=0.0286. (E) The number of splenic neutrophil per mouse was determined. Each circle corresponds to a single mouse, geometric mean is shown. 5: p=0.0286.

    Techniques Used: Infection, Mouse Assay, Isolation, Cell Culture, Ex Vivo, Staining, FACS

    Type I IFN negatively regulate IL-17A secretion during intranasal inoculation with highly virulen t F. tularensis SchuS4 strain Results from one experiment are shown. Bacterial burden was determined at 48h and 96h PI in the spleen (A) and the lung (B) following intranasal inoculation of BALB/cJ mice with 25 F. tularensis SchuS4 cfu. Each circle represents the cfu in one mouse, geometric mean is shown. IFN-β (C, D) and IL-17A (E, F) transcripts levels in the spleen (C, E) and the lung (D, F) were determined by qRT-PCR at 96h PI, normalized to GAPDH transcript levels and expressed as fold increase over WT uninfected. IL-17A (G, J), IFN-γ (H) and TNF-α (I) protein levels in spleen (G, H, I) and lung (J) lysates at 96h PI were determined by ELISA and expressed as mg per organ. 1: p=0.0022; 2: p=0.0022; 3: p=0.0022; 4: p=0.026; 5: p=0.0087.
    Figure Legend Snippet: Type I IFN negatively regulate IL-17A secretion during intranasal inoculation with highly virulen t F. tularensis SchuS4 strain Results from one experiment are shown. Bacterial burden was determined at 48h and 96h PI in the spleen (A) and the lung (B) following intranasal inoculation of BALB/cJ mice with 25 F. tularensis SchuS4 cfu. Each circle represents the cfu in one mouse, geometric mean is shown. IFN-β (C, D) and IL-17A (E, F) transcripts levels in the spleen (C, E) and the lung (D, F) were determined by qRT-PCR at 96h PI, normalized to GAPDH transcript levels and expressed as fold increase over WT uninfected. IL-17A (G, J), IFN-γ (H) and TNF-α (I) protein levels in spleen (G, H, I) and lung (J) lysates at 96h PI were determined by ELISA and expressed as mg per organ. 1: p=0.0022; 2: p=0.0022; 3: p=0.0022; 4: p=0.026; 5: p=0.0087.

    Techniques Used: Mouse Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Increase in IL-17A in IFNAR1 −/− mice is associated with an increase in splenic neutrophil numbers (A) Numbers of Monocytes (Mono.), Neutrophils (Neutro.), Macrophages (Macro.), T and B cells in the spleen were determined by FACS, the averages from 3 (uninfected samples) to 7 (infected samples) animals from one experiment representative of 3 independent experiments are shown. Neutrophil number was statistically higher in infected IFNAR1 −/− mice than in WT infected mice (1, p=0.0013). (B, C) IFNAR1-deficiency and IL-17A deficiency are associated with neutrophilia and neutropenia respectively. Each dot represents the percentage of splenic neutrophils from one individual mouse. Geometric mean is shown. Results from three independent experiments, one including IFNAR1 −/− mice, are shown. 2: p=0.0159, 3: p
    Figure Legend Snippet: Increase in IL-17A in IFNAR1 −/− mice is associated with an increase in splenic neutrophil numbers (A) Numbers of Monocytes (Mono.), Neutrophils (Neutro.), Macrophages (Macro.), T and B cells in the spleen were determined by FACS, the averages from 3 (uninfected samples) to 7 (infected samples) animals from one experiment representative of 3 independent experiments are shown. Neutrophil number was statistically higher in infected IFNAR1 −/− mice than in WT infected mice (1, p=0.0013). (B, C) IFNAR1-deficiency and IL-17A deficiency are associated with neutrophilia and neutropenia respectively. Each dot represents the percentage of splenic neutrophils from one individual mouse. Geometric mean is shown. Results from three independent experiments, one including IFNAR1 −/− mice, are shown. 2: p=0.0159, 3: p

    Techniques Used: Mouse Assay, FACS, Infection

    Type I IFN negatively regulate IL-17A and IL-17F production as soon as 24h PI independently of the bacterial burden Mice were injected with 10 5 F. novicida cfu i.d., IL-17A (A, D), IL-17F (B, E), IFN-γ (C) transcript levels in the spleen were determined at the indicated time points by qRT-PCR and expressed as fold increase over the corresponding transcript levels in the spleen of uninfected mice. Each dot represents one mouse. The geometric means from a single experiment are shown and are representative of three independent experiments. ns: not significant, 1: p=0.0023, 2: p=0.0012, 3: p=0.0023, 4: p=0.0012. (D, E) Splenic bacterial burden was determined for each mouse at 48h PI and correlated to IL-17A (D) or IL-17F (E) transcript levels. (F) Splenocytes from either WT or IFNAR1 −/− mice PBS-injected or infected for 48h, were cultured ex vivo for 24h, the concentration of IL-17A released in the supernatant was determined by Elisa. ND: not detectable.
    Figure Legend Snippet: Type I IFN negatively regulate IL-17A and IL-17F production as soon as 24h PI independently of the bacterial burden Mice were injected with 10 5 F. novicida cfu i.d., IL-17A (A, D), IL-17F (B, E), IFN-γ (C) transcript levels in the spleen were determined at the indicated time points by qRT-PCR and expressed as fold increase over the corresponding transcript levels in the spleen of uninfected mice. Each dot represents one mouse. The geometric means from a single experiment are shown and are representative of three independent experiments. ns: not significant, 1: p=0.0023, 2: p=0.0012, 3: p=0.0023, 4: p=0.0012. (D, E) Splenic bacterial burden was determined for each mouse at 48h PI and correlated to IL-17A (D) or IL-17F (E) transcript levels. (F) Splenocytes from either WT or IFNAR1 −/− mice PBS-injected or infected for 48h, were cultured ex vivo for 24h, the concentration of IL-17A released in the supernatant was determined by Elisa. ND: not detectable.

    Techniques Used: Mouse Assay, Injection, Quantitative RT-PCR, Infection, Cell Culture, Ex Vivo, Concentration Assay, Enzyme-linked Immunosorbent Assay

    CD27 − γδ T cells are the main population of IL-17A-producing cells early on during infection (A) Splenocytes from two IFNAR1 −/− mice infected for 48h with U112 were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (top panels) or the isotype control (bottom panels) in singlets, live, CD19 − , CD3ε + cells versus the expression of TCR αβ (left panels) or TCR γδ (right panels) is shown. Numbers indicate the percentage of positive cells in each gate, numbers in brackets indicate the percentage of IL-17A + cells in total TCRαβ + T cells and total TCRγδ + T cells (left and right panels respectively). (B) CD27 staining intensity in total γδ T cells (white histogram) and IL-17A + γδ T cells (grey histogram) from 5 infected IFNAR1 −/− is shown. Percentage of CD27 − cells in the corresponding population is indicated within brackets (C) Splenocytes from WT or IFNAR1 −/− mice uninfected or infected for 48h with F. novicida were isolated, cultured ex-vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (left panels) or the isotype control (right panels) in singlets, live, CD19 − , CD3ε + CD4 − CD8 − cells versus the expression of TCR γδ is shown. Numbers indicate the percentage of positive cells in each gate, numbers in brackets indicate the percentage of IL-17A + cells in total TCRγδ + T cells. FACS plots corresponding to 3 (uninfected) to 6 (infected) mice are shown. (D) The number of IL-17A-producing γδ T cells was determined in each mouse and expressed as the percentage of total γδ T cells. Each circle represents the value in a single mouse. Geometric mean is shown. 1: p=0.003, 2: p=0.0043, 3: p=0.0011, 4: p=0.0001. (E) Total splenocytes (10 6 per well) or MACS-purified γδ T cells (10 5 per well) isolated from WT mice at 24h PI were restimulated for 20h on anti-CD3ε antibody coated plates in the presence of rIFN-β at the indicated concentrations. IL-17A concentration in the supernatant was determined by ELISA. (F) The number of γδ T cells per mouse spleen was determined. Each circle corresponds to a single mouse, geometric mean is shown. One experiment, representative of three independent experiments, is shown.
    Figure Legend Snippet: CD27 − γδ T cells are the main population of IL-17A-producing cells early on during infection (A) Splenocytes from two IFNAR1 −/− mice infected for 48h with U112 were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (top panels) or the isotype control (bottom panels) in singlets, live, CD19 − , CD3ε + cells versus the expression of TCR αβ (left panels) or TCR γδ (right panels) is shown. Numbers indicate the percentage of positive cells in each gate, numbers in brackets indicate the percentage of IL-17A + cells in total TCRαβ + T cells and total TCRγδ + T cells (left and right panels respectively). (B) CD27 staining intensity in total γδ T cells (white histogram) and IL-17A + γδ T cells (grey histogram) from 5 infected IFNAR1 −/− is shown. Percentage of CD27 − cells in the corresponding population is indicated within brackets (C) Splenocytes from WT or IFNAR1 −/− mice uninfected or infected for 48h with F. novicida were isolated, cultured ex-vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (left panels) or the isotype control (right panels) in singlets, live, CD19 − , CD3ε + CD4 − CD8 − cells versus the expression of TCR γδ is shown. Numbers indicate the percentage of positive cells in each gate, numbers in brackets indicate the percentage of IL-17A + cells in total TCRγδ + T cells. FACS plots corresponding to 3 (uninfected) to 6 (infected) mice are shown. (D) The number of IL-17A-producing γδ T cells was determined in each mouse and expressed as the percentage of total γδ T cells. Each circle represents the value in a single mouse. Geometric mean is shown. 1: p=0.003, 2: p=0.0043, 3: p=0.0011, 4: p=0.0001. (E) Total splenocytes (10 6 per well) or MACS-purified γδ T cells (10 5 per well) isolated from WT mice at 24h PI were restimulated for 20h on anti-CD3ε antibody coated plates in the presence of rIFN-β at the indicated concentrations. IL-17A concentration in the supernatant was determined by ELISA. (F) The number of γδ T cells per mouse spleen was determined. Each circle corresponds to a single mouse, geometric mean is shown. One experiment, representative of three independent experiments, is shown.

    Techniques Used: Infection, Mouse Assay, Isolation, Cell Culture, Ex Vivo, Staining, FACS, Expressing, Magnetic Cell Separation, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Bone marrow transplants experiment suggests a dominant effect of type I IFN signaling in controlling IL-17A production by γδ T cells Recipient mice (B6.SJL-Ptprc a Pepc b /BoyJ) which harbor the CD45.1 (Ptprc a ) marker were irradiated twice with 500 rad and reconstituted with 2.10 6 bone-marrow cells from WT C57BL/6J mice (left panel, WT CD45.2 mice in WT CD45.1), from IFNAR1 −/− mice (right panel, IFNAR1 −/− CD45.2 mice in WT CD45.1) or with 1 10 6 bone-marrow cells from WT B6.SJL-Ptprc a Pepc b /BoyJ mice and 1 10 6 bone-marrow cells from IFNAR1 −/− mice (center panel, chimera WT (CD45.1) + IFNAR1 −/− (CD45.2) mice in WT CD45.1). 8 weeks post transplant, mice were injected i.d. with 1 10 5 F. novicida cfu. At 48h PI, splenocytes were isolated, cultured ex-vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A or the isotype control (as indicated) in singlets, live, CD19 − , CD3ε + , TCR αβ − , TCR γδ + cells is shown. Numbers indicate the percentage of IL-17A + cells (or isotype + cells) in each gate. FACS plots corresponding 5 mice are shown.
    Figure Legend Snippet: Bone marrow transplants experiment suggests a dominant effect of type I IFN signaling in controlling IL-17A production by γδ T cells Recipient mice (B6.SJL-Ptprc a Pepc b /BoyJ) which harbor the CD45.1 (Ptprc a ) marker were irradiated twice with 500 rad and reconstituted with 2.10 6 bone-marrow cells from WT C57BL/6J mice (left panel, WT CD45.2 mice in WT CD45.1), from IFNAR1 −/− mice (right panel, IFNAR1 −/− CD45.2 mice in WT CD45.1) or with 1 10 6 bone-marrow cells from WT B6.SJL-Ptprc a Pepc b /BoyJ mice and 1 10 6 bone-marrow cells from IFNAR1 −/− mice (center panel, chimera WT (CD45.1) + IFNAR1 −/− (CD45.2) mice in WT CD45.1). 8 weeks post transplant, mice were injected i.d. with 1 10 5 F. novicida cfu. At 48h PI, splenocytes were isolated, cultured ex-vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A or the isotype control (as indicated) in singlets, live, CD19 − , CD3ε + , TCR αβ − , TCR γδ + cells is shown. Numbers indicate the percentage of IL-17A + cells (or isotype + cells) in each gate. FACS plots corresponding 5 mice are shown.

    Techniques Used: Mouse Assay, Marker, Irradiation, Injection, Isolation, Cell Culture, Ex Vivo, Staining, FACS

    In an in vitro assay, type I IFN signaling in macrophages and T cells contributes to the inhibition of IL-17A production (A, B, C) 5 10 4 bone marrow-derived macrophages (BMM) from WT or IFNAR1 −/− mice were infected with F. novicida (U112) at a MOI of 10:1. 10 6 splenocytes from uninfected WT or IFNAR1 −/− mice were added at 1h PI, IL-17A released in the supernatant was determined by ELISA at 24h PI. In A, the in vitro system reconstitutes spleen environment from WT and IFNAR1 −/− mice. In B, the in vitro system tests the role of type I IFN signaling in macrophages (WT splenocytes added in both cases). In C, the in vitro system tests the role of type I IFN signaling in splenocytes (WT macrophages added in both cases). One experiment, representative of three independent experiments, is shown. (D) 5 10 4 IFNAR1 −/− BMM infected with U112 at a MOI of 10:1 were incubated with 10 5 MACS-purified splenocytes from either WT or IFNAR1 −/− mice that had been infected for 24h with F. novicida . 10 5 CD19 + and Gr1 + enriched cells; CD19 − , Gr1 − , γδ-depleted cells or γδ-enriched cells from either WT or IFNAR1 −/− mice infected for 24h were added to IFNAR1 −/− BMM at 1h PI. Concentration of the IL-17A released in the supernatant at 24h PI was determined by ELISA. Results from one experiment are shown.
    Figure Legend Snippet: In an in vitro assay, type I IFN signaling in macrophages and T cells contributes to the inhibition of IL-17A production (A, B, C) 5 10 4 bone marrow-derived macrophages (BMM) from WT or IFNAR1 −/− mice were infected with F. novicida (U112) at a MOI of 10:1. 10 6 splenocytes from uninfected WT or IFNAR1 −/− mice were added at 1h PI, IL-17A released in the supernatant was determined by ELISA at 24h PI. In A, the in vitro system reconstitutes spleen environment from WT and IFNAR1 −/− mice. In B, the in vitro system tests the role of type I IFN signaling in macrophages (WT splenocytes added in both cases). In C, the in vitro system tests the role of type I IFN signaling in splenocytes (WT macrophages added in both cases). One experiment, representative of three independent experiments, is shown. (D) 5 10 4 IFNAR1 −/− BMM infected with U112 at a MOI of 10:1 were incubated with 10 5 MACS-purified splenocytes from either WT or IFNAR1 −/− mice that had been infected for 24h with F. novicida . 10 5 CD19 + and Gr1 + enriched cells; CD19 − , Gr1 − , γδ-depleted cells or γδ-enriched cells from either WT or IFNAR1 −/− mice infected for 24h were added to IFNAR1 −/− BMM at 1h PI. Concentration of the IL-17A released in the supernatant at 24h PI was determined by ELISA. Results from one experiment are shown.

    Techniques Used: In Vitro, Inhibition, Derivative Assay, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Incubation, Magnetic Cell Separation, Purification, Concentration Assay

    30) Product Images from "Type I Interferon signaling constrains IL-17A/F secretion by ?? T cells during bacterial infections"

    Article Title: Type I Interferon signaling constrains IL-17A/F secretion by ?? T cells during bacterial infections

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0902065

    Type I IFN negatively regulate IL-17A secretion by γδ T cells during Listeria infection Results from 2 independent experiments (A, E, G) or from 1 experiment representative of 2 independent experiments (B, C, D, F) are shown. (A) Bacterial burden was determined at 48h PI in the spleen following tail vein injection of 1.4 10 4 cfu of L. monocytogenes . Each circle represents the cfu in one mouse, geometric mean is shown. IL-17A (B) and IFN-γ (C) transcript levels in the spleen were determined by qRT-PCR at 48h and expressed as fold increase over WT uninfected. (D) Splenocytes from WT or IFNAR1 −/− mice infected for 48h with L. monocytogenes were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (left panels) or the isotype control (right panel) in singlets, live, CD19 − , CD3ε + , CD4 − , CD8 − , TCRαβ − , TCRγδ + cells versus the expression of TCR γδ is shown. Numbers indicate the percentage of IL-17A + cells of the total γδ T cells. FACS plots corresponding to five mice, are shown. (E) Percentage of IL-17A + cells over the total of γδ T cells is shown. Each circle corresponds to the value in one mouse, geometric mean is shown. Total numbers of γδ T cell (F) and neutrophil (G) per spleen are shown. 1: p
    Figure Legend Snippet: Type I IFN negatively regulate IL-17A secretion by γδ T cells during Listeria infection Results from 2 independent experiments (A, E, G) or from 1 experiment representative of 2 independent experiments (B, C, D, F) are shown. (A) Bacterial burden was determined at 48h PI in the spleen following tail vein injection of 1.4 10 4 cfu of L. monocytogenes . Each circle represents the cfu in one mouse, geometric mean is shown. IL-17A (B) and IFN-γ (C) transcript levels in the spleen were determined by qRT-PCR at 48h and expressed as fold increase over WT uninfected. (D) Splenocytes from WT or IFNAR1 −/− mice infected for 48h with L. monocytogenes were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (left panels) or the isotype control (right panel) in singlets, live, CD19 − , CD3ε + , CD4 − , CD8 − , TCRαβ − , TCRγδ + cells versus the expression of TCR γδ is shown. Numbers indicate the percentage of IL-17A + cells of the total γδ T cells. FACS plots corresponding to five mice, are shown. (E) Percentage of IL-17A + cells over the total of γδ T cells is shown. Each circle corresponds to the value in one mouse, geometric mean is shown. Total numbers of γδ T cell (F) and neutrophil (G) per spleen are shown. 1: p

    Techniques Used: Infection, Injection, Quantitative RT-PCR, Mouse Assay, Isolation, Cell Culture, Ex Vivo, Staining, FACS, Expressing

    Type I IFN signaling contrains IL-17A secretion by γδ T cells during i.n. infection with 50 cfu F. novicida (A) Type I IFN signaling is detrimental in a survival experiment during during i.n. infection with 50 cfu F. novicida . WT (n=10) and IFNAR1 −/− (n=10) C57BL/6J mice were inoculated with 50 F. novicida cfu by the intranasal route. Survival was monitored twice daily during 10 days (1: p=0.0053). (B) Type I IFN signaling is detrimental to control the bacterial burden during during i.n. infection with 50 cfu F. novicida . Bacterial colonization in the spleen was determined at 72h and 96h PI (at this latter time point, 2 WT mice had died as indicated †) (2: p=0.0286; 3: p=0.0286). (C) type I IFN signaling negatively controls IL-17A transcript level in the spleen (3: p=0.0286). (D) type I IFN signaling negatively controls IL-17A production by γδ T cells in the spleen of infected mice. Splenocytes from infected mice were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A or the isotype control (as indicated) in singlets, live, CD19 − , CD3ε + , TCR αβ − , TCR γδ + cells is shown. Numbers indicate the percentage of IL-17A + cells (or isotype + cells) in total γδ T cells. FACS plots corresponding to four mice are shown. (E) The number of IL-17A-producing γδ T cells was determined in each mouse and expressed as the percentage of total γδ T cells. Each circle represents the value in a single mouse. Geometric mean is shown. 4: p=0.0286. (E) The number of splenic neutrophil per mouse was determined. Each circle corresponds to a single mouse, geometric mean is shown. 5: p=0.0286.
    Figure Legend Snippet: Type I IFN signaling contrains IL-17A secretion by γδ T cells during i.n. infection with 50 cfu F. novicida (A) Type I IFN signaling is detrimental in a survival experiment during during i.n. infection with 50 cfu F. novicida . WT (n=10) and IFNAR1 −/− (n=10) C57BL/6J mice were inoculated with 50 F. novicida cfu by the intranasal route. Survival was monitored twice daily during 10 days (1: p=0.0053). (B) Type I IFN signaling is detrimental to control the bacterial burden during during i.n. infection with 50 cfu F. novicida . Bacterial colonization in the spleen was determined at 72h and 96h PI (at this latter time point, 2 WT mice had died as indicated †) (2: p=0.0286; 3: p=0.0286). (C) type I IFN signaling negatively controls IL-17A transcript level in the spleen (3: p=0.0286). (D) type I IFN signaling negatively controls IL-17A production by γδ T cells in the spleen of infected mice. Splenocytes from infected mice were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A or the isotype control (as indicated) in singlets, live, CD19 − , CD3ε + , TCR αβ − , TCR γδ + cells is shown. Numbers indicate the percentage of IL-17A + cells (or isotype + cells) in total γδ T cells. FACS plots corresponding to four mice are shown. (E) The number of IL-17A-producing γδ T cells was determined in each mouse and expressed as the percentage of total γδ T cells. Each circle represents the value in a single mouse. Geometric mean is shown. 4: p=0.0286. (E) The number of splenic neutrophil per mouse was determined. Each circle corresponds to a single mouse, geometric mean is shown. 5: p=0.0286.

    Techniques Used: Infection, Mouse Assay, Isolation, Cell Culture, Ex Vivo, Staining, FACS

    Type I IFN negatively regulate IL-17A secretion during intranasal inoculation with highly virulen t F. tularensis SchuS4 strain Results from one experiment are shown. Bacterial burden was determined at 48h and 96h PI in the spleen (A) and the lung (B) following intranasal inoculation of BALB/cJ mice with 25 F. tularensis SchuS4 cfu. Each circle represents the cfu in one mouse, geometric mean is shown. IFN-β (C, D) and IL-17A (E, F) transcripts levels in the spleen (C, E) and the lung (D, F) were determined by qRT-PCR at 96h PI, normalized to GAPDH transcript levels and expressed as fold increase over WT uninfected. IL-17A (G, J), IFN-γ (H) and TNF-α (I) protein levels in spleen (G, H, I) and lung (J) lysates at 96h PI were determined by ELISA and expressed as mg per organ. 1: p=0.0022; 2: p=0.0022; 3: p=0.0022; 4: p=0.026; 5: p=0.0087.
    Figure Legend Snippet: Type I IFN negatively regulate IL-17A secretion during intranasal inoculation with highly virulen t F. tularensis SchuS4 strain Results from one experiment are shown. Bacterial burden was determined at 48h and 96h PI in the spleen (A) and the lung (B) following intranasal inoculation of BALB/cJ mice with 25 F. tularensis SchuS4 cfu. Each circle represents the cfu in one mouse, geometric mean is shown. IFN-β (C, D) and IL-17A (E, F) transcripts levels in the spleen (C, E) and the lung (D, F) were determined by qRT-PCR at 96h PI, normalized to GAPDH transcript levels and expressed as fold increase over WT uninfected. IL-17A (G, J), IFN-γ (H) and TNF-α (I) protein levels in spleen (G, H, I) and lung (J) lysates at 96h PI were determined by ELISA and expressed as mg per organ. 1: p=0.0022; 2: p=0.0022; 3: p=0.0022; 4: p=0.026; 5: p=0.0087.

    Techniques Used: Mouse Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Increase in IL-17A in IFNAR1 −/− mice is associated with an increase in splenic neutrophil numbers (A) Numbers of Monocytes (Mono.), Neutrophils (Neutro.), Macrophages (Macro.), T and B cells in the spleen were determined by FACS, the averages from 3 (uninfected samples) to 7 (infected samples) animals from one experiment representative of 3 independent experiments are shown. Neutrophil number was statistically higher in infected IFNAR1 −/− mice than in WT infected mice (1, p=0.0013). (B, C) IFNAR1-deficiency and IL-17A deficiency are associated with neutrophilia and neutropenia respectively. Each dot represents the percentage of splenic neutrophils from one individual mouse. Geometric mean is shown. Results from three independent experiments, one including IFNAR1 −/− mice, are shown. 2: p=0.0159, 3: p
    Figure Legend Snippet: Increase in IL-17A in IFNAR1 −/− mice is associated with an increase in splenic neutrophil numbers (A) Numbers of Monocytes (Mono.), Neutrophils (Neutro.), Macrophages (Macro.), T and B cells in the spleen were determined by FACS, the averages from 3 (uninfected samples) to 7 (infected samples) animals from one experiment representative of 3 independent experiments are shown. Neutrophil number was statistically higher in infected IFNAR1 −/− mice than in WT infected mice (1, p=0.0013). (B, C) IFNAR1-deficiency and IL-17A deficiency are associated with neutrophilia and neutropenia respectively. Each dot represents the percentage of splenic neutrophils from one individual mouse. Geometric mean is shown. Results from three independent experiments, one including IFNAR1 −/− mice, are shown. 2: p=0.0159, 3: p

    Techniques Used: Mouse Assay, FACS, Infection

    Type I IFN negatively regulate IL-17A and IL-17F production as soon as 24h PI independently of the bacterial burden Mice were injected with 10 5 F. novicida cfu i.d., IL-17A (A, D), IL-17F (B, E), IFN-γ (C) transcript levels in the spleen were determined at the indicated time points by qRT-PCR and expressed as fold increase over the corresponding transcript levels in the spleen of uninfected mice. Each dot represents one mouse. The geometric means from a single experiment are shown and are representative of three independent experiments. ns: not significant, 1: p=0.0023, 2: p=0.0012, 3: p=0.0023, 4: p=0.0012. (D, E) Splenic bacterial burden was determined for each mouse at 48h PI and correlated to IL-17A (D) or IL-17F (E) transcript levels. (F) Splenocytes from either WT or IFNAR1 −/− mice PBS-injected or infected for 48h, were cultured ex vivo for 24h, the concentration of IL-17A released in the supernatant was determined by Elisa. ND: not detectable.
    Figure Legend Snippet: Type I IFN negatively regulate IL-17A and IL-17F production as soon as 24h PI independently of the bacterial burden Mice were injected with 10 5 F. novicida cfu i.d., IL-17A (A, D), IL-17F (B, E), IFN-γ (C) transcript levels in the spleen were determined at the indicated time points by qRT-PCR and expressed as fold increase over the corresponding transcript levels in the spleen of uninfected mice. Each dot represents one mouse. The geometric means from a single experiment are shown and are representative of three independent experiments. ns: not significant, 1: p=0.0023, 2: p=0.0012, 3: p=0.0023, 4: p=0.0012. (D, E) Splenic bacterial burden was determined for each mouse at 48h PI and correlated to IL-17A (D) or IL-17F (E) transcript levels. (F) Splenocytes from either WT or IFNAR1 −/− mice PBS-injected or infected for 48h, were cultured ex vivo for 24h, the concentration of IL-17A released in the supernatant was determined by Elisa. ND: not detectable.

    Techniques Used: Mouse Assay, Injection, Quantitative RT-PCR, Infection, Cell Culture, Ex Vivo, Concentration Assay, Enzyme-linked Immunosorbent Assay

    CD27 − γδ T cells are the main population of IL-17A-producing cells early on during infection (A) Splenocytes from two IFNAR1 −/− mice infected for 48h with U112 were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (top panels) or the isotype control (bottom panels) in singlets, live, CD19 − , CD3ε + cells versus the expression of TCR αβ (left panels) or TCR γδ (right panels) is shown. Numbers indicate the percentage of positive cells in each gate, numbers in brackets indicate the percentage of IL-17A + cells in total TCRαβ + T cells and total TCRγδ + T cells (left and right panels respectively). (B) CD27 staining intensity in total γδ T cells (white histogram) and IL-17A + γδ T cells (grey histogram) from 5 infected IFNAR1 −/− is shown. Percentage of CD27 − cells in the corresponding population is indicated within brackets (C) Splenocytes from WT or IFNAR1 −/− mice uninfected or infected for 48h with F. novicida were isolated, cultured ex-vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (left panels) or the isotype control (right panels) in singlets, live, CD19 − , CD3ε + CD4 − CD8 − cells versus the expression of TCR γδ is shown. Numbers indicate the percentage of positive cells in each gate, numbers in brackets indicate the percentage of IL-17A + cells in total TCRγδ + T cells. FACS plots corresponding to 3 (uninfected) to 6 (infected) mice are shown. (D) The number of IL-17A-producing γδ T cells was determined in each mouse and expressed as the percentage of total γδ T cells. Each circle represents the value in a single mouse. Geometric mean is shown. 1: p=0.003, 2: p=0.0043, 3: p=0.0011, 4: p=0.0001. (E) Total splenocytes (10 6 per well) or MACS-purified γδ T cells (10 5 per well) isolated from WT mice at 24h PI were restimulated for 20h on anti-CD3ε antibody coated plates in the presence of rIFN-β at the indicated concentrations. IL-17A concentration in the supernatant was determined by ELISA. (F) The number of γδ T cells per mouse spleen was determined. Each circle corresponds to a single mouse, geometric mean is shown. One experiment, representative of three independent experiments, is shown.
    Figure Legend Snippet: CD27 − γδ T cells are the main population of IL-17A-producing cells early on during infection (A) Splenocytes from two IFNAR1 −/− mice infected for 48h with U112 were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (top panels) or the isotype control (bottom panels) in singlets, live, CD19 − , CD3ε + cells versus the expression of TCR αβ (left panels) or TCR γδ (right panels) is shown. Numbers indicate the percentage of positive cells in each gate, numbers in brackets indicate the percentage of IL-17A + cells in total TCRαβ + T cells and total TCRγδ + T cells (left and right panels respectively). (B) CD27 staining intensity in total γδ T cells (white histogram) and IL-17A + γδ T cells (grey histogram) from 5 infected IFNAR1 −/− is shown. Percentage of CD27 − cells in the corresponding population is indicated within brackets (C) Splenocytes from WT or IFNAR1 −/− mice uninfected or infected for 48h with F. novicida were isolated, cultured ex-vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (left panels) or the isotype control (right panels) in singlets, live, CD19 − , CD3ε + CD4 − CD8 − cells versus the expression of TCR γδ is shown. Numbers indicate the percentage of positive cells in each gate, numbers in brackets indicate the percentage of IL-17A + cells in total TCRγδ + T cells. FACS plots corresponding to 3 (uninfected) to 6 (infected) mice are shown. (D) The number of IL-17A-producing γδ T cells was determined in each mouse and expressed as the percentage of total γδ T cells. Each circle represents the value in a single mouse. Geometric mean is shown. 1: p=0.003, 2: p=0.0043, 3: p=0.0011, 4: p=0.0001. (E) Total splenocytes (10 6 per well) or MACS-purified γδ T cells (10 5 per well) isolated from WT mice at 24h PI were restimulated for 20h on anti-CD3ε antibody coated plates in the presence of rIFN-β at the indicated concentrations. IL-17A concentration in the supernatant was determined by ELISA. (F) The number of γδ T cells per mouse spleen was determined. Each circle corresponds to a single mouse, geometric mean is shown. One experiment, representative of three independent experiments, is shown.

    Techniques Used: Infection, Mouse Assay, Isolation, Cell Culture, Ex Vivo, Staining, FACS, Expressing, Magnetic Cell Separation, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Bone marrow transplants experiment suggests a dominant effect of type I IFN signaling in controlling IL-17A production by γδ T cells Recipient mice (B6.SJL-Ptprc a Pepc b /BoyJ) which harbor the CD45.1 (Ptprc a ) marker were irradiated twice with 500 rad and reconstituted with 2.10 6 bone-marrow cells from WT C57BL/6J mice (left panel, WT CD45.2 mice in WT CD45.1), from IFNAR1 −/− mice (right panel, IFNAR1 −/− CD45.2 mice in WT CD45.1) or with 1 10 6 bone-marrow cells from WT B6.SJL-Ptprc a Pepc b /BoyJ mice and 1 10 6 bone-marrow cells from IFNAR1 −/− mice (center panel, chimera WT (CD45.1) + IFNAR1 −/− (CD45.2) mice in WT CD45.1). 8 weeks post transplant, mice were injected i.d. with 1 10 5 F. novicida cfu. At 48h PI, splenocytes were isolated, cultured ex-vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A or the isotype control (as indicated) in singlets, live, CD19 − , CD3ε + , TCR αβ − , TCR γδ + cells is shown. Numbers indicate the percentage of IL-17A + cells (or isotype + cells) in each gate. FACS plots corresponding 5 mice are shown.
    Figure Legend Snippet: Bone marrow transplants experiment suggests a dominant effect of type I IFN signaling in controlling IL-17A production by γδ T cells Recipient mice (B6.SJL-Ptprc a Pepc b /BoyJ) which harbor the CD45.1 (Ptprc a ) marker were irradiated twice with 500 rad and reconstituted with 2.10 6 bone-marrow cells from WT C57BL/6J mice (left panel, WT CD45.2 mice in WT CD45.1), from IFNAR1 −/− mice (right panel, IFNAR1 −/− CD45.2 mice in WT CD45.1) or with 1 10 6 bone-marrow cells from WT B6.SJL-Ptprc a Pepc b /BoyJ mice and 1 10 6 bone-marrow cells from IFNAR1 −/− mice (center panel, chimera WT (CD45.1) + IFNAR1 −/− (CD45.2) mice in WT CD45.1). 8 weeks post transplant, mice were injected i.d. with 1 10 5 F. novicida cfu. At 48h PI, splenocytes were isolated, cultured ex-vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A or the isotype control (as indicated) in singlets, live, CD19 − , CD3ε + , TCR αβ − , TCR γδ + cells is shown. Numbers indicate the percentage of IL-17A + cells (or isotype + cells) in each gate. FACS plots corresponding 5 mice are shown.

    Techniques Used: Mouse Assay, Marker, Irradiation, Injection, Isolation, Cell Culture, Ex Vivo, Staining, FACS

    In an in vitro assay, type I IFN signaling in macrophages and T cells contributes to the inhibition of IL-17A production (A, B, C) 5 10 4 bone marrow-derived macrophages (BMM) from WT or IFNAR1 −/− mice were infected with F. novicida (U112) at a MOI of 10:1. 10 6 splenocytes from uninfected WT or IFNAR1 −/− mice were added at 1h PI, IL-17A released in the supernatant was determined by ELISA at 24h PI. In A, the in vitro system reconstitutes spleen environment from WT and IFNAR1 −/− mice. In B, the in vitro system tests the role of type I IFN signaling in macrophages (WT splenocytes added in both cases). In C, the in vitro system tests the role of type I IFN signaling in splenocytes (WT macrophages added in both cases). One experiment, representative of three independent experiments, is shown. (D) 5 10 4 IFNAR1 −/− BMM infected with U112 at a MOI of 10:1 were incubated with 10 5 MACS-purified splenocytes from either WT or IFNAR1 −/− mice that had been infected for 24h with F. novicida . 10 5 CD19 + and Gr1 + enriched cells; CD19 − , Gr1 − , γδ-depleted cells or γδ-enriched cells from either WT or IFNAR1 −/− mice infected for 24h were added to IFNAR1 −/− BMM at 1h PI. Concentration of the IL-17A released in the supernatant at 24h PI was determined by ELISA. Results from one experiment are shown.
    Figure Legend Snippet: In an in vitro assay, type I IFN signaling in macrophages and T cells contributes to the inhibition of IL-17A production (A, B, C) 5 10 4 bone marrow-derived macrophages (BMM) from WT or IFNAR1 −/− mice were infected with F. novicida (U112) at a MOI of 10:1. 10 6 splenocytes from uninfected WT or IFNAR1 −/− mice were added at 1h PI, IL-17A released in the supernatant was determined by ELISA at 24h PI. In A, the in vitro system reconstitutes spleen environment from WT and IFNAR1 −/− mice. In B, the in vitro system tests the role of type I IFN signaling in macrophages (WT splenocytes added in both cases). In C, the in vitro system tests the role of type I IFN signaling in splenocytes (WT macrophages added in both cases). One experiment, representative of three independent experiments, is shown. (D) 5 10 4 IFNAR1 −/− BMM infected with U112 at a MOI of 10:1 were incubated with 10 5 MACS-purified splenocytes from either WT or IFNAR1 −/− mice that had been infected for 24h with F. novicida . 10 5 CD19 + and Gr1 + enriched cells; CD19 − , Gr1 − , γδ-depleted cells or γδ-enriched cells from either WT or IFNAR1 −/− mice infected for 24h were added to IFNAR1 −/− BMM at 1h PI. Concentration of the IL-17A released in the supernatant at 24h PI was determined by ELISA. Results from one experiment are shown.

    Techniques Used: In Vitro, Inhibition, Derivative Assay, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Incubation, Magnetic Cell Separation, Purification, Concentration Assay

    31) Product Images from "Type I Interferon signaling constrains IL-17A/F secretion by ?? T cells during bacterial infections"

    Article Title: Type I Interferon signaling constrains IL-17A/F secretion by ?? T cells during bacterial infections

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0902065

    Type I IFN negatively regulate IL-17A secretion by γδ T cells during Listeria infection Results from 2 independent experiments (A, E, G) or from 1 experiment representative of 2 independent experiments (B, C, D, F) are shown. (A) Bacterial burden was determined at 48h PI in the spleen following tail vein injection of 1.4 10 4 cfu of L. monocytogenes . Each circle represents the cfu in one mouse, geometric mean is shown. IL-17A (B) and IFN-γ (C) transcript levels in the spleen were determined by qRT-PCR at 48h and expressed as fold increase over WT uninfected. (D) Splenocytes from WT or IFNAR1 −/− mice infected for 48h with L. monocytogenes were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (left panels) or the isotype control (right panel) in singlets, live, CD19 − , CD3ε + , CD4 − , CD8 − , TCRαβ − , TCRγδ + cells versus the expression of TCR γδ is shown. Numbers indicate the percentage of IL-17A + cells of the total γδ T cells. FACS plots corresponding to five mice, are shown. (E) Percentage of IL-17A + cells over the total of γδ T cells is shown. Each circle corresponds to the value in one mouse, geometric mean is shown. Total numbers of γδ T cell (F) and neutrophil (G) per spleen are shown. 1: p
    Figure Legend Snippet: Type I IFN negatively regulate IL-17A secretion by γδ T cells during Listeria infection Results from 2 independent experiments (A, E, G) or from 1 experiment representative of 2 independent experiments (B, C, D, F) are shown. (A) Bacterial burden was determined at 48h PI in the spleen following tail vein injection of 1.4 10 4 cfu of L. monocytogenes . Each circle represents the cfu in one mouse, geometric mean is shown. IL-17A (B) and IFN-γ (C) transcript levels in the spleen were determined by qRT-PCR at 48h and expressed as fold increase over WT uninfected. (D) Splenocytes from WT or IFNAR1 −/− mice infected for 48h with L. monocytogenes were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (left panels) or the isotype control (right panel) in singlets, live, CD19 − , CD3ε + , CD4 − , CD8 − , TCRαβ − , TCRγδ + cells versus the expression of TCR γδ is shown. Numbers indicate the percentage of IL-17A + cells of the total γδ T cells. FACS plots corresponding to five mice, are shown. (E) Percentage of IL-17A + cells over the total of γδ T cells is shown. Each circle corresponds to the value in one mouse, geometric mean is shown. Total numbers of γδ T cell (F) and neutrophil (G) per spleen are shown. 1: p

    Techniques Used: Infection, Injection, Quantitative RT-PCR, Mouse Assay, Isolation, Cell Culture, Ex Vivo, Staining, FACS, Expressing

    Type I IFN signaling contrains IL-17A secretion by γδ T cells during i.n. infection with 50 cfu F. novicida (A) Type I IFN signaling is detrimental in a survival experiment during during i.n. infection with 50 cfu F. novicida . WT (n=10) and IFNAR1 −/− (n=10) C57BL/6J mice were inoculated with 50 F. novicida cfu by the intranasal route. Survival was monitored twice daily during 10 days (1: p=0.0053). (B) Type I IFN signaling is detrimental to control the bacterial burden during during i.n. infection with 50 cfu F. novicida . Bacterial colonization in the spleen was determined at 72h and 96h PI (at this latter time point, 2 WT mice had died as indicated †) (2: p=0.0286; 3: p=0.0286). (C) type I IFN signaling negatively controls IL-17A transcript level in the spleen (3: p=0.0286). (D) type I IFN signaling negatively controls IL-17A production by γδ T cells in the spleen of infected mice. Splenocytes from infected mice were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A or the isotype control (as indicated) in singlets, live, CD19 − , CD3ε + , TCR αβ − , TCR γδ + cells is shown. Numbers indicate the percentage of IL-17A + cells (or isotype + cells) in total γδ T cells. FACS plots corresponding to four mice are shown. (E) The number of IL-17A-producing γδ T cells was determined in each mouse and expressed as the percentage of total γδ T cells. Each circle represents the value in a single mouse. Geometric mean is shown. 4: p=0.0286. (E) The number of splenic neutrophil per mouse was determined. Each circle corresponds to a single mouse, geometric mean is shown. 5: p=0.0286.
    Figure Legend Snippet: Type I IFN signaling contrains IL-17A secretion by γδ T cells during i.n. infection with 50 cfu F. novicida (A) Type I IFN signaling is detrimental in a survival experiment during during i.n. infection with 50 cfu F. novicida . WT (n=10) and IFNAR1 −/− (n=10) C57BL/6J mice were inoculated with 50 F. novicida cfu by the intranasal route. Survival was monitored twice daily during 10 days (1: p=0.0053). (B) Type I IFN signaling is detrimental to control the bacterial burden during during i.n. infection with 50 cfu F. novicida . Bacterial colonization in the spleen was determined at 72h and 96h PI (at this latter time point, 2 WT mice had died as indicated †) (2: p=0.0286; 3: p=0.0286). (C) type I IFN signaling negatively controls IL-17A transcript level in the spleen (3: p=0.0286). (D) type I IFN signaling negatively controls IL-17A production by γδ T cells in the spleen of infected mice. Splenocytes from infected mice were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A or the isotype control (as indicated) in singlets, live, CD19 − , CD3ε + , TCR αβ − , TCR γδ + cells is shown. Numbers indicate the percentage of IL-17A + cells (or isotype + cells) in total γδ T cells. FACS plots corresponding to four mice are shown. (E) The number of IL-17A-producing γδ T cells was determined in each mouse and expressed as the percentage of total γδ T cells. Each circle represents the value in a single mouse. Geometric mean is shown. 4: p=0.0286. (E) The number of splenic neutrophil per mouse was determined. Each circle corresponds to a single mouse, geometric mean is shown. 5: p=0.0286.

    Techniques Used: Infection, Mouse Assay, Isolation, Cell Culture, Ex Vivo, Staining, FACS

    Type I IFN negatively regulate IL-17A secretion during intranasal inoculation with highly virulen t F. tularensis SchuS4 strain Results from one experiment are shown. Bacterial burden was determined at 48h and 96h PI in the spleen (A) and the lung (B) following intranasal inoculation of BALB/cJ mice with 25 F. tularensis SchuS4 cfu. Each circle represents the cfu in one mouse, geometric mean is shown. IFN-β (C, D) and IL-17A (E, F) transcripts levels in the spleen (C, E) and the lung (D, F) were determined by qRT-PCR at 96h PI, normalized to GAPDH transcript levels and expressed as fold increase over WT uninfected. IL-17A (G, J), IFN-γ (H) and TNF-α (I) protein levels in spleen (G, H, I) and lung (J) lysates at 96h PI were determined by ELISA and expressed as mg per organ. 1: p=0.0022; 2: p=0.0022; 3: p=0.0022; 4: p=0.026; 5: p=0.0087.
    Figure Legend Snippet: Type I IFN negatively regulate IL-17A secretion during intranasal inoculation with highly virulen t F. tularensis SchuS4 strain Results from one experiment are shown. Bacterial burden was determined at 48h and 96h PI in the spleen (A) and the lung (B) following intranasal inoculation of BALB/cJ mice with 25 F. tularensis SchuS4 cfu. Each circle represents the cfu in one mouse, geometric mean is shown. IFN-β (C, D) and IL-17A (E, F) transcripts levels in the spleen (C, E) and the lung (D, F) were determined by qRT-PCR at 96h PI, normalized to GAPDH transcript levels and expressed as fold increase over WT uninfected. IL-17A (G, J), IFN-γ (H) and TNF-α (I) protein levels in spleen (G, H, I) and lung (J) lysates at 96h PI were determined by ELISA and expressed as mg per organ. 1: p=0.0022; 2: p=0.0022; 3: p=0.0022; 4: p=0.026; 5: p=0.0087.

    Techniques Used: Mouse Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Increase in IL-17A in IFNAR1 −/− mice is associated with an increase in splenic neutrophil numbers (A) Numbers of Monocytes (Mono.), Neutrophils (Neutro.), Macrophages (Macro.), T and B cells in the spleen were determined by FACS, the averages from 3 (uninfected samples) to 7 (infected samples) animals from one experiment representative of 3 independent experiments are shown. Neutrophil number was statistically higher in infected IFNAR1 −/− mice than in WT infected mice (1, p=0.0013). (B, C) IFNAR1-deficiency and IL-17A deficiency are associated with neutrophilia and neutropenia respectively. Each dot represents the percentage of splenic neutrophils from one individual mouse. Geometric mean is shown. Results from three independent experiments, one including IFNAR1 −/− mice, are shown. 2: p=0.0159, 3: p
    Figure Legend Snippet: Increase in IL-17A in IFNAR1 −/− mice is associated with an increase in splenic neutrophil numbers (A) Numbers of Monocytes (Mono.), Neutrophils (Neutro.), Macrophages (Macro.), T and B cells in the spleen were determined by FACS, the averages from 3 (uninfected samples) to 7 (infected samples) animals from one experiment representative of 3 independent experiments are shown. Neutrophil number was statistically higher in infected IFNAR1 −/− mice than in WT infected mice (1, p=0.0013). (B, C) IFNAR1-deficiency and IL-17A deficiency are associated with neutrophilia and neutropenia respectively. Each dot represents the percentage of splenic neutrophils from one individual mouse. Geometric mean is shown. Results from three independent experiments, one including IFNAR1 −/− mice, are shown. 2: p=0.0159, 3: p

    Techniques Used: Mouse Assay, FACS, Infection

    Type I IFN negatively regulate IL-17A and IL-17F production as soon as 24h PI independently of the bacterial burden Mice were injected with 10 5 F. novicida cfu i.d., IL-17A (A, D), IL-17F (B, E), IFN-γ (C) transcript levels in the spleen were determined at the indicated time points by qRT-PCR and expressed as fold increase over the corresponding transcript levels in the spleen of uninfected mice. Each dot represents one mouse. The geometric means from a single experiment are shown and are representative of three independent experiments. ns: not significant, 1: p=0.0023, 2: p=0.0012, 3: p=0.0023, 4: p=0.0012. (D, E) Splenic bacterial burden was determined for each mouse at 48h PI and correlated to IL-17A (D) or IL-17F (E) transcript levels. (F) Splenocytes from either WT or IFNAR1 −/− mice PBS-injected or infected for 48h, were cultured ex vivo for 24h, the concentration of IL-17A released in the supernatant was determined by Elisa. ND: not detectable.
    Figure Legend Snippet: Type I IFN negatively regulate IL-17A and IL-17F production as soon as 24h PI independently of the bacterial burden Mice were injected with 10 5 F. novicida cfu i.d., IL-17A (A, D), IL-17F (B, E), IFN-γ (C) transcript levels in the spleen were determined at the indicated time points by qRT-PCR and expressed as fold increase over the corresponding transcript levels in the spleen of uninfected mice. Each dot represents one mouse. The geometric means from a single experiment are shown and are representative of three independent experiments. ns: not significant, 1: p=0.0023, 2: p=0.0012, 3: p=0.0023, 4: p=0.0012. (D, E) Splenic bacterial burden was determined for each mouse at 48h PI and correlated to IL-17A (D) or IL-17F (E) transcript levels. (F) Splenocytes from either WT or IFNAR1 −/− mice PBS-injected or infected for 48h, were cultured ex vivo for 24h, the concentration of IL-17A released in the supernatant was determined by Elisa. ND: not detectable.

    Techniques Used: Mouse Assay, Injection, Quantitative RT-PCR, Infection, Cell Culture, Ex Vivo, Concentration Assay, Enzyme-linked Immunosorbent Assay

    CD27 − γδ T cells are the main population of IL-17A-producing cells early on during infection (A) Splenocytes from two IFNAR1 −/− mice infected for 48h with U112 were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (top panels) or the isotype control (bottom panels) in singlets, live, CD19 − , CD3ε + cells versus the expression of TCR αβ (left panels) or TCR γδ (right panels) is shown. Numbers indicate the percentage of positive cells in each gate, numbers in brackets indicate the percentage of IL-17A + cells in total TCRαβ + T cells and total TCRγδ + T cells (left and right panels respectively). (B) CD27 staining intensity in total γδ T cells (white histogram) and IL-17A + γδ T cells (grey histogram) from 5 infected IFNAR1 −/− is shown. Percentage of CD27 − cells in the corresponding population is indicated within brackets (C) Splenocytes from WT or IFNAR1 −/− mice uninfected or infected for 48h with F. novicida were isolated, cultured ex-vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (left panels) or the isotype control (right panels) in singlets, live, CD19 − , CD3ε + CD4 − CD8 − cells versus the expression of TCR γδ is shown. Numbers indicate the percentage of positive cells in each gate, numbers in brackets indicate the percentage of IL-17A + cells in total TCRγδ + T cells. FACS plots corresponding to 3 (uninfected) to 6 (infected) mice are shown. (D) The number of IL-17A-producing γδ T cells was determined in each mouse and expressed as the percentage of total γδ T cells. Each circle represents the value in a single mouse. Geometric mean is shown. 1: p=0.003, 2: p=0.0043, 3: p=0.0011, 4: p=0.0001. (E) Total splenocytes (10 6 per well) or MACS-purified γδ T cells (10 5 per well) isolated from WT mice at 24h PI were restimulated for 20h on anti-CD3ε antibody coated plates in the presence of rIFN-β at the indicated concentrations. IL-17A concentration in the supernatant was determined by ELISA. (F) The number of γδ T cells per mouse spleen was determined. Each circle corresponds to a single mouse, geometric mean is shown. One experiment, representative of three independent experiments, is shown.
    Figure Legend Snippet: CD27 − γδ T cells are the main population of IL-17A-producing cells early on during infection (A) Splenocytes from two IFNAR1 −/− mice infected for 48h with U112 were isolated, cultured ex vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (top panels) or the isotype control (bottom panels) in singlets, live, CD19 − , CD3ε + cells versus the expression of TCR αβ (left panels) or TCR γδ (right panels) is shown. Numbers indicate the percentage of positive cells in each gate, numbers in brackets indicate the percentage of IL-17A + cells in total TCRαβ + T cells and total TCRγδ + T cells (left and right panels respectively). (B) CD27 staining intensity in total γδ T cells (white histogram) and IL-17A + γδ T cells (grey histogram) from 5 infected IFNAR1 −/− is shown. Percentage of CD27 − cells in the corresponding population is indicated within brackets (C) Splenocytes from WT or IFNAR1 −/− mice uninfected or infected for 48h with F. novicida were isolated, cultured ex-vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A (left panels) or the isotype control (right panels) in singlets, live, CD19 − , CD3ε + CD4 − CD8 − cells versus the expression of TCR γδ is shown. Numbers indicate the percentage of positive cells in each gate, numbers in brackets indicate the percentage of IL-17A + cells in total TCRγδ + T cells. FACS plots corresponding to 3 (uninfected) to 6 (infected) mice are shown. (D) The number of IL-17A-producing γδ T cells was determined in each mouse and expressed as the percentage of total γδ T cells. Each circle represents the value in a single mouse. Geometric mean is shown. 1: p=0.003, 2: p=0.0043, 3: p=0.0011, 4: p=0.0001. (E) Total splenocytes (10 6 per well) or MACS-purified γδ T cells (10 5 per well) isolated from WT mice at 24h PI were restimulated for 20h on anti-CD3ε antibody coated plates in the presence of rIFN-β at the indicated concentrations. IL-17A concentration in the supernatant was determined by ELISA. (F) The number of γδ T cells per mouse spleen was determined. Each circle corresponds to a single mouse, geometric mean is shown. One experiment, representative of three independent experiments, is shown.

    Techniques Used: Infection, Mouse Assay, Isolation, Cell Culture, Ex Vivo, Staining, FACS, Expressing, Magnetic Cell Separation, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Bone marrow transplants experiment suggests a dominant effect of type I IFN signaling in controlling IL-17A production by γδ T cells Recipient mice (B6.SJL-Ptprc a Pepc b /BoyJ) which harbor the CD45.1 (Ptprc a ) marker were irradiated twice with 500 rad and reconstituted with 2.10 6 bone-marrow cells from WT C57BL/6J mice (left panel, WT CD45.2 mice in WT CD45.1), from IFNAR1 −/− mice (right panel, IFNAR1 −/− CD45.2 mice in WT CD45.1) or with 1 10 6 bone-marrow cells from WT B6.SJL-Ptprc a Pepc b /BoyJ mice and 1 10 6 bone-marrow cells from IFNAR1 −/− mice (center panel, chimera WT (CD45.1) + IFNAR1 −/− (CD45.2) mice in WT CD45.1). 8 weeks post transplant, mice were injected i.d. with 1 10 5 F. novicida cfu. At 48h PI, splenocytes were isolated, cultured ex-vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A or the isotype control (as indicated) in singlets, live, CD19 − , CD3ε + , TCR αβ − , TCR γδ + cells is shown. Numbers indicate the percentage of IL-17A + cells (or isotype + cells) in each gate. FACS plots corresponding 5 mice are shown.
    Figure Legend Snippet: Bone marrow transplants experiment suggests a dominant effect of type I IFN signaling in controlling IL-17A production by γδ T cells Recipient mice (B6.SJL-Ptprc a Pepc b /BoyJ) which harbor the CD45.1 (Ptprc a ) marker were irradiated twice with 500 rad and reconstituted with 2.10 6 bone-marrow cells from WT C57BL/6J mice (left panel, WT CD45.2 mice in WT CD45.1), from IFNAR1 −/− mice (right panel, IFNAR1 −/− CD45.2 mice in WT CD45.1) or with 1 10 6 bone-marrow cells from WT B6.SJL-Ptprc a Pepc b /BoyJ mice and 1 10 6 bone-marrow cells from IFNAR1 −/− mice (center panel, chimera WT (CD45.1) + IFNAR1 −/− (CD45.2) mice in WT CD45.1). 8 weeks post transplant, mice were injected i.d. with 1 10 5 F. novicida cfu. At 48h PI, splenocytes were isolated, cultured ex-vivo for 5h in the presence of monensin, stained and analyzed by FACS. Intracellular detection of IL-17A or the isotype control (as indicated) in singlets, live, CD19 − , CD3ε + , TCR αβ − , TCR γδ + cells is shown. Numbers indicate the percentage of IL-17A + cells (or isotype + cells) in each gate. FACS plots corresponding 5 mice are shown.

    Techniques Used: Mouse Assay, Marker, Irradiation, Injection, Isolation, Cell Culture, Ex Vivo, Staining, FACS

    In an in vitro assay, type I IFN signaling in macrophages and T cells contributes to the inhibition of IL-17A production (A, B, C) 5 10 4 bone marrow-derived macrophages (BMM) from WT or IFNAR1 −/− mice were infected with F. novicida (U112) at a MOI of 10:1. 10 6 splenocytes from uninfected WT or IFNAR1 −/− mice were added at 1h PI, IL-17A released in the supernatant was determined by ELISA at 24h PI. In A, the in vitro system reconstitutes spleen environment from WT and IFNAR1 −/− mice. In B, the in vitro system tests the role of type I IFN signaling in macrophages (WT splenocytes added in both cases). In C, the in vitro system tests the role of type I IFN signaling in splenocytes (WT macrophages added in both cases). One experiment, representative of three independent experiments, is shown. (D) 5 10 4 IFNAR1 −/− BMM infected with U112 at a MOI of 10:1 were incubated with 10 5 MACS-purified splenocytes from either WT or IFNAR1 −/− mice that had been infected for 24h with F. novicida . 10 5 CD19 + and Gr1 + enriched cells; CD19 − , Gr1 − , γδ-depleted cells or γδ-enriched cells from either WT or IFNAR1 −/− mice infected for 24h were added to IFNAR1 −/− BMM at 1h PI. Concentration of the IL-17A released in the supernatant at 24h PI was determined by ELISA. Results from one experiment are shown.
    Figure Legend Snippet: In an in vitro assay, type I IFN signaling in macrophages and T cells contributes to the inhibition of IL-17A production (A, B, C) 5 10 4 bone marrow-derived macrophages (BMM) from WT or IFNAR1 −/− mice were infected with F. novicida (U112) at a MOI of 10:1. 10 6 splenocytes from uninfected WT or IFNAR1 −/− mice were added at 1h PI, IL-17A released in the supernatant was determined by ELISA at 24h PI. In A, the in vitro system reconstitutes spleen environment from WT and IFNAR1 −/− mice. In B, the in vitro system tests the role of type I IFN signaling in macrophages (WT splenocytes added in both cases). In C, the in vitro system tests the role of type I IFN signaling in splenocytes (WT macrophages added in both cases). One experiment, representative of three independent experiments, is shown. (D) 5 10 4 IFNAR1 −/− BMM infected with U112 at a MOI of 10:1 were incubated with 10 5 MACS-purified splenocytes from either WT or IFNAR1 −/− mice that had been infected for 24h with F. novicida . 10 5 CD19 + and Gr1 + enriched cells; CD19 − , Gr1 − , γδ-depleted cells or γδ-enriched cells from either WT or IFNAR1 −/− mice infected for 24h were added to IFNAR1 −/− BMM at 1h PI. Concentration of the IL-17A released in the supernatant at 24h PI was determined by ELISA. Results from one experiment are shown.

    Techniques Used: In Vitro, Inhibition, Derivative Assay, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Incubation, Magnetic Cell Separation, Purification, Concentration Assay

    32) Product Images from "IL-17 induces type V collagen overexpression and EMT via TGF-?-dependent pathways in obliterative bronchiolitis"

    Article Title: IL-17 induces type V collagen overexpression and EMT via TGF-?-dependent pathways in obliterative bronchiolitis

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00080.2012

    Neutralization of IL-17 bioactivity suppresses IL-17A-induced TGF-β in murine OB model of orthotopic lung transplant. A : qPCR analyses of TGF-β and SMAD7 mRNA expression in response to IL-17 in RLE-6TN cells. Values represent means ±
    Figure Legend Snippet: Neutralization of IL-17 bioactivity suppresses IL-17A-induced TGF-β in murine OB model of orthotopic lung transplant. A : qPCR analyses of TGF-β and SMAD7 mRNA expression in response to IL-17 in RLE-6TN cells. Values represent means ±

    Techniques Used: Neutralization, Real-time Polymerase Chain Reaction, Expressing

    IL-17A induces EMT in airway epithelial cells located in clinical OB lesions and in a murine model of orthotopic lung transplant. A : OB lesions were immunostained for epithelial marker, zona occludens [ZO-1-rhodamine (red)] and mesenchymal marker, α-SMA-FITC
    Figure Legend Snippet: IL-17A induces EMT in airway epithelial cells located in clinical OB lesions and in a murine model of orthotopic lung transplant. A : OB lesions were immunostained for epithelial marker, zona occludens [ZO-1-rhodamine (red)] and mesenchymal marker, α-SMA-FITC

    Techniques Used: Marker

    IL-17A is expressed in clinical OB lesions after lung transplant. A : immunohistochemical staining showing expression of IL-17A in a representative human subject with OB and normal lungs. Hematoxylin and eosin staining reveals the respective lung architecture.
    Figure Legend Snippet: IL-17A is expressed in clinical OB lesions after lung transplant. A : immunohistochemical staining showing expression of IL-17A in a representative human subject with OB and normal lungs. Hematoxylin and eosin staining reveals the respective lung architecture.

    Techniques Used: Immunohistochemistry, Staining, Expressing

    IL-17A induces epithelial mesenchymal transition (EMT) in airway epithelial cells. A : E-cadherin (E-CAD) protein expression as determined by Western blotting. B : densitometry normalized to GAPDH in a dose-response treatment of normal human small airway
    Figure Legend Snippet: IL-17A induces epithelial mesenchymal transition (EMT) in airway epithelial cells. A : E-cadherin (E-CAD) protein expression as determined by Western blotting. B : densitometry normalized to GAPDH in a dose-response treatment of normal human small airway

    Techniques Used: Expressing, Western Blot

    33) Product Images from "IL-17 induces type V collagen overexpression and EMT via TGF-?-dependent pathways in obliterative bronchiolitis"

    Article Title: IL-17 induces type V collagen overexpression and EMT via TGF-?-dependent pathways in obliterative bronchiolitis

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00080.2012

    Neutralization of IL-17 bioactivity suppresses IL-17A-induced TGF-β in murine OB model of orthotopic lung transplant. A : qPCR analyses of TGF-β and SMAD7 mRNA expression in response to IL-17 in RLE-6TN cells. Values represent means ±
    Figure Legend Snippet: Neutralization of IL-17 bioactivity suppresses IL-17A-induced TGF-β in murine OB model of orthotopic lung transplant. A : qPCR analyses of TGF-β and SMAD7 mRNA expression in response to IL-17 in RLE-6TN cells. Values represent means ±

    Techniques Used: Neutralization, Real-time Polymerase Chain Reaction, Expressing

    IL-17A induces EMT in airway epithelial cells located in clinical OB lesions and in a murine model of orthotopic lung transplant. A : OB lesions were immunostained for epithelial marker, zona occludens [ZO-1-rhodamine (red)] and mesenchymal marker, α-SMA-FITC
    Figure Legend Snippet: IL-17A induces EMT in airway epithelial cells located in clinical OB lesions and in a murine model of orthotopic lung transplant. A : OB lesions were immunostained for epithelial marker, zona occludens [ZO-1-rhodamine (red)] and mesenchymal marker, α-SMA-FITC

    Techniques Used: Marker

    IL-17A is expressed in clinical OB lesions after lung transplant. A : immunohistochemical staining showing expression of IL-17A in a representative human subject with OB and normal lungs. Hematoxylin and eosin staining reveals the respective lung architecture.
    Figure Legend Snippet: IL-17A is expressed in clinical OB lesions after lung transplant. A : immunohistochemical staining showing expression of IL-17A in a representative human subject with OB and normal lungs. Hematoxylin and eosin staining reveals the respective lung architecture.

    Techniques Used: Immunohistochemistry, Staining, Expressing

    IL-17A induces epithelial mesenchymal transition (EMT) in airway epithelial cells. A : E-cadherin (E-CAD) protein expression as determined by Western blotting. B : densitometry normalized to GAPDH in a dose-response treatment of normal human small airway
    Figure Legend Snippet: IL-17A induces epithelial mesenchymal transition (EMT) in airway epithelial cells. A : E-cadherin (E-CAD) protein expression as determined by Western blotting. B : densitometry normalized to GAPDH in a dose-response treatment of normal human small airway

    Techniques Used: Expressing, Western Blot

    34) Product Images from "IL-17 induces type V collagen overexpression and EMT via TGF-?-dependent pathways in obliterative bronchiolitis"

    Article Title: IL-17 induces type V collagen overexpression and EMT via TGF-?-dependent pathways in obliterative bronchiolitis

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00080.2012

    Neutralization of IL-17 bioactivity suppresses IL-17A-induced TGF-β in murine OB model of orthotopic lung transplant. A : qPCR analyses of TGF-β and SMAD7 mRNA expression in response to IL-17 in RLE-6TN cells. Values represent means ±
    Figure Legend Snippet: Neutralization of IL-17 bioactivity suppresses IL-17A-induced TGF-β in murine OB model of orthotopic lung transplant. A : qPCR analyses of TGF-β and SMAD7 mRNA expression in response to IL-17 in RLE-6TN cells. Values represent means ±

    Techniques Used: Neutralization, Real-time Polymerase Chain Reaction, Expressing

    IL-17A induces EMT in airway epithelial cells located in clinical OB lesions and in a murine model of orthotopic lung transplant. A : OB lesions were immunostained for epithelial marker, zona occludens [ZO-1-rhodamine (red)] and mesenchymal marker, α-SMA-FITC
    Figure Legend Snippet: IL-17A induces EMT in airway epithelial cells located in clinical OB lesions and in a murine model of orthotopic lung transplant. A : OB lesions were immunostained for epithelial marker, zona occludens [ZO-1-rhodamine (red)] and mesenchymal marker, α-SMA-FITC

    Techniques Used: Marker

    IL-17A is expressed in clinical OB lesions after lung transplant. A : immunohistochemical staining showing expression of IL-17A in a representative human subject with OB and normal lungs. Hematoxylin and eosin staining reveals the respective lung architecture.
    Figure Legend Snippet: IL-17A is expressed in clinical OB lesions after lung transplant. A : immunohistochemical staining showing expression of IL-17A in a representative human subject with OB and normal lungs. Hematoxylin and eosin staining reveals the respective lung architecture.

    Techniques Used: Immunohistochemistry, Staining, Expressing

    IL-17A induces epithelial mesenchymal transition (EMT) in airway epithelial cells. A : E-cadherin (E-CAD) protein expression as determined by Western blotting. B : densitometry normalized to GAPDH in a dose-response treatment of normal human small airway
    Figure Legend Snippet: IL-17A induces epithelial mesenchymal transition (EMT) in airway epithelial cells. A : E-cadherin (E-CAD) protein expression as determined by Western blotting. B : densitometry normalized to GAPDH in a dose-response treatment of normal human small airway

    Techniques Used: Expressing, Western Blot

    35) Product Images from "Composition of the Schistosoma mansoni worm secretome: Identification of immune modulatory Cyclophilin A"

    Article Title: Composition of the Schistosoma mansoni worm secretome: Identification of immune modulatory Cyclophilin A

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006012

    Modulation of CD4 + T cells by SmCypA-treated BMDC. ( A ) Representative histograms from flow cytometric analysis of CFSE labeled TCR OVA CD4 + T cell cultures with OVA and LPS treated or LPS and rSmCypA co-treated BMDC as APC. ( B ) Flow cytometric analysis for the identification of Treg (FOXP3 + ), Th17 (RORc + ), Th2 (GATA3 + ) and Th1 (TBET + ) CD4 + T cells following culture with OVA and BMDC as APC, activated under the outlined conditions, n = 4 per group. ( C ) Heatmap of ELISA for the detection of IL-10, IL-17A, IFN-γ and IL-4 in the supernatant of TCR OVA CD4 + T cells and BMDC co-cultures. BMDC were activated, or not, under the indicated conditions prior to their co-culture with the TCR OVA CD4 + T cells ± antigen (OVA), n = 6 per group. Data are presented as mean and SEM and statistical difference between groups was determined using Student's t test.
    Figure Legend Snippet: Modulation of CD4 + T cells by SmCypA-treated BMDC. ( A ) Representative histograms from flow cytometric analysis of CFSE labeled TCR OVA CD4 + T cell cultures with OVA and LPS treated or LPS and rSmCypA co-treated BMDC as APC. ( B ) Flow cytometric analysis for the identification of Treg (FOXP3 + ), Th17 (RORc + ), Th2 (GATA3 + ) and Th1 (TBET + ) CD4 + T cells following culture with OVA and BMDC as APC, activated under the outlined conditions, n = 4 per group. ( C ) Heatmap of ELISA for the detection of IL-10, IL-17A, IFN-γ and IL-4 in the supernatant of TCR OVA CD4 + T cells and BMDC co-cultures. BMDC were activated, or not, under the indicated conditions prior to their co-culture with the TCR OVA CD4 + T cells ± antigen (OVA), n = 6 per group. Data are presented as mean and SEM and statistical difference between groups was determined using Student's t test.

    Techniques Used: Flow Cytometry, Labeling, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

    36) Product Images from "Bowman-Birk Inhibitor attenuates experimental autoimmune encephalomyelitis by delaying infiltration of inflammatory cells into the CNS."

    Article Title: Bowman-Birk Inhibitor attenuates experimental autoimmune encephalomyelitis by delaying infiltration of inflammatory cells into the CNS.

    Journal: Immunologic research

    doi: 10.1007/s12026-011-8254-6

    BBI reduces infiltration of the CNS by inflammatory cells. C57BL/6 mice were immunized with MOG 35–55 and treated daily with 1 mg/mouse BBI or PBS starting on the day of immunization. On day 7, 14, 21, and 30 p.i., mice were sacrificed, their spinal cords pooled and cells from the CNS isolated. a Total numbers of cells isolated from the CNS, and total numbers of CD4 + , CD4 + INF-γ + , and CD4 + IL-17A + cells. b Mononuclear cells isolated from the CNS were analyzed by flow cytometry for proportion of CD4 + IFN-γ + IL-17A + cells. Data shown are of three independent experiments.
    Figure Legend Snippet: BBI reduces infiltration of the CNS by inflammatory cells. C57BL/6 mice were immunized with MOG 35–55 and treated daily with 1 mg/mouse BBI or PBS starting on the day of immunization. On day 7, 14, 21, and 30 p.i., mice were sacrificed, their spinal cords pooled and cells from the CNS isolated. a Total numbers of cells isolated from the CNS, and total numbers of CD4 + , CD4 + INF-γ + , and CD4 + IL-17A + cells. b Mononuclear cells isolated from the CNS were analyzed by flow cytometry for proportion of CD4 + IFN-γ + IL-17A + cells. Data shown are of three independent experiments.

    Techniques Used: Mouse Assay, Isolation, Flow Cytometry, Cytometry

    BBI treatment results in enhanced cytokine production by peripheral cells at later stage of EAE development. C57BL/6 mice were immunized with MOG 35–55 and treated daily by oral gavage with 1 mg/mouse BBI or PBS starting on the day of immunization. On days 7, 14, and 21 p.i., mice were sacrificed, splenocytes, and LN cells were stimulated with MOG 35–55 for 3 days, and GM-CSF, IL-17A, and IFN-γ concentrations in the supernatants measured by ELISA. Data are representative of three independent experiments.
    Figure Legend Snippet: BBI treatment results in enhanced cytokine production by peripheral cells at later stage of EAE development. C57BL/6 mice were immunized with MOG 35–55 and treated daily by oral gavage with 1 mg/mouse BBI or PBS starting on the day of immunization. On days 7, 14, and 21 p.i., mice were sacrificed, splenocytes, and LN cells were stimulated with MOG 35–55 for 3 days, and GM-CSF, IL-17A, and IFN-γ concentrations in the supernatants measured by ELISA. Data are representative of three independent experiments.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    BBI-treated mice initially have reduced numbers of Th1 and Th17 cells, followed by an increase. C57BL/6 mice were immunized with MOG 35–55 and treated daily by oral gavage with 1 mg/mouse BBI or PBS starting on the day of immunization. On days 7, 14, and 21 p.i., mice were sacrificed, and splenocytes and LN cells were analyzed by flow cytometry for the proportions of IL-17A + and IFN-γ + cells among CD4 + T cells. Data are representative of three independent experiments.
    Figure Legend Snippet: BBI-treated mice initially have reduced numbers of Th1 and Th17 cells, followed by an increase. C57BL/6 mice were immunized with MOG 35–55 and treated daily by oral gavage with 1 mg/mouse BBI or PBS starting on the day of immunization. On days 7, 14, and 21 p.i., mice were sacrificed, and splenocytes and LN cells were analyzed by flow cytometry for the proportions of IL-17A + and IFN-γ + cells among CD4 + T cells. Data are representative of three independent experiments.

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

    37) Product Images from "The Kinase Inhibitory Region of SOCS-1 is Sufficient to Inhibit T-helper 17 and other Immune Functions in Experimental Allergic Encephalomyelitis"

    Article Title: The Kinase Inhibitory Region of SOCS-1 is Sufficient to Inhibit T-helper 17 and other Immune Functions in Experimental Allergic Encephalomyelitis

    Journal: Journal of neuroimmunology

    doi: 10.1016/j.jneuroim.2010.10.018

    IL-17A production by splenocytes is inhibited in EAE by treatment with SOCS1- KIR A) Splenocytes were isolated from SJL/J mice 36 days after immunization with MBP. Prior to harvesting the spleens, the mice had been receiving i.p. injections of 100 μL PBS, SOCS1-KIR (60 μg/mouse), or SOCS1-KIR2A (60 μg/mouse) every other day beginning on day 12 post-immunization, and were scored EAE stage 1. Cells were seeded at 5 × 10 6 cells/well in RPMI (10% FBS) and incubated 24 hours at 37°C, 5%CO 2 . Supernatants were collected and analyzed for IL-17A using the IL-17A Ready-Set-Go ELISA kit (eBioscience, San Diego, CA). B) IL-17A production by splenocytes in response to MBP stimulation is inhibited in mice treated with SOCS1-KIR. Splenocytes were isolated from SJL/J mice 27 days after immunization with MBP. Prior to harvesting the spleens, the mice had been treated as described in part A. Cells were seeded as described above and treated with or without 25 μg/ml MBP and incubated 24 hours. Supernatants were collected and analyzed for IL-17A as in part A. *P
    Figure Legend Snippet: IL-17A production by splenocytes is inhibited in EAE by treatment with SOCS1- KIR A) Splenocytes were isolated from SJL/J mice 36 days after immunization with MBP. Prior to harvesting the spleens, the mice had been receiving i.p. injections of 100 μL PBS, SOCS1-KIR (60 μg/mouse), or SOCS1-KIR2A (60 μg/mouse) every other day beginning on day 12 post-immunization, and were scored EAE stage 1. Cells were seeded at 5 × 10 6 cells/well in RPMI (10% FBS) and incubated 24 hours at 37°C, 5%CO 2 . Supernatants were collected and analyzed for IL-17A using the IL-17A Ready-Set-Go ELISA kit (eBioscience, San Diego, CA). B) IL-17A production by splenocytes in response to MBP stimulation is inhibited in mice treated with SOCS1-KIR. Splenocytes were isolated from SJL/J mice 27 days after immunization with MBP. Prior to harvesting the spleens, the mice had been treated as described in part A. Cells were seeded as described above and treated with or without 25 μg/ml MBP and incubated 24 hours. Supernatants were collected and analyzed for IL-17A as in part A. *P

    Techniques Used: Isolation, Mouse Assay, Incubation, Enzyme-linked Immunosorbent Assay

    Induction of IL-17A and IFNã in MBP-sensitized splenocytes by IL-23 is inhibited by SOCS1-KIR A) IL-17A production by splenocytes in response to IL-23 is inhibited by SOCS1- KIR. Splenocytes were isolated from SJL/J mice 37 days after immunization with MBP. Prior to harvesting the spleen, the mouse was scored at EAE stage one. Cells were seeded at 5 × 10 6 cells/well in RPMI (10% FBS) in a 96-well plate. Peptides were added at the above concentrations and cells were incubated at 37°C, 5% CO 2 , for 2 hours. IL-23 (10 ng/ml) was added to each well and the cells were incubated an additional 48 hours. Supernatants were collected and analyzed for IL-17A production using the IL-17A Ready-Set-Go ELISA kit (eBioscience, San Diego, CA). *P
    Figure Legend Snippet: Induction of IL-17A and IFNã in MBP-sensitized splenocytes by IL-23 is inhibited by SOCS1-KIR A) IL-17A production by splenocytes in response to IL-23 is inhibited by SOCS1- KIR. Splenocytes were isolated from SJL/J mice 37 days after immunization with MBP. Prior to harvesting the spleen, the mouse was scored at EAE stage one. Cells were seeded at 5 × 10 6 cells/well in RPMI (10% FBS) in a 96-well plate. Peptides were added at the above concentrations and cells were incubated at 37°C, 5% CO 2 , for 2 hours. IL-23 (10 ng/ml) was added to each well and the cells were incubated an additional 48 hours. Supernatants were collected and analyzed for IL-17A production using the IL-17A Ready-Set-Go ELISA kit (eBioscience, San Diego, CA). *P

    Techniques Used: Isolation, Mouse Assay, Incubation, Enzyme-linked Immunosorbent Assay

    SOCS1-KIR inhibits enhanced STAT3 activation by IL-23 and IL-17A A) SOCS1- KIR inhibits IL-23-enhanced STAT3 phosphorylation. Splenocytes isolated from MBP-immunized mice experiencing EAE (stage 1, five weeks after immunization), were treated with the above peptides for 2 hours, followed by incubation with IL-23 (10 ng/ml) for 10 minutes. The cells were lysed and protein concentration was determined by the standard BCA assay. The cell lysates were resolved using SDS-PAGE and proteins were transferred onto a nitrocellulose membrane. The membranes were probed for pSTAT3 (top) or STAT3 (bottom). Relative intensity of the bands was measured with ImageJ software (NIH) and is shown below the pSTAT3 blot. B) SOCS1-KIR inhibits STAT3 phosphorylation in splenocytes treated with IL-17. Splenocytes isolated from MBPimmunized mice were treated with the above peptides for 2 hours, followed by incubation with IL-17 (100 ng/ml) for 2 hours. Western blots were performed and analyzed as described for part A; however, longer exposure times were necessary. The experiments were performed twice with similar results.
    Figure Legend Snippet: SOCS1-KIR inhibits enhanced STAT3 activation by IL-23 and IL-17A A) SOCS1- KIR inhibits IL-23-enhanced STAT3 phosphorylation. Splenocytes isolated from MBP-immunized mice experiencing EAE (stage 1, five weeks after immunization), were treated with the above peptides for 2 hours, followed by incubation with IL-23 (10 ng/ml) for 10 minutes. The cells were lysed and protein concentration was determined by the standard BCA assay. The cell lysates were resolved using SDS-PAGE and proteins were transferred onto a nitrocellulose membrane. The membranes were probed for pSTAT3 (top) or STAT3 (bottom). Relative intensity of the bands was measured with ImageJ software (NIH) and is shown below the pSTAT3 blot. B) SOCS1-KIR inhibits STAT3 phosphorylation in splenocytes treated with IL-17. Splenocytes isolated from MBPimmunized mice were treated with the above peptides for 2 hours, followed by incubation with IL-17 (100 ng/ml) for 2 hours. Western blots were performed and analyzed as described for part A; however, longer exposure times were necessary. The experiments were performed twice with similar results.

    Techniques Used: Activation Assay, Isolation, Mouse Assay, Incubation, Protein Concentration, BIA-KA, SDS Page, Software, Western Blot

    38) Product Images from "The Kinase Inhibitory Region of SOCS-1 is Sufficient to Inhibit T-helper 17 and other Immune Functions in Experimental Allergic Encephalomyelitis"

    Article Title: The Kinase Inhibitory Region of SOCS-1 is Sufficient to Inhibit T-helper 17 and other Immune Functions in Experimental Allergic Encephalomyelitis

    Journal: Journal of neuroimmunology

    doi: 10.1016/j.jneuroim.2010.10.018

    IL-17A production by splenocytes is inhibited in EAE by treatment with SOCS1- KIR A) Splenocytes were isolated from SJL/J mice 36 days after immunization with MBP. Prior to harvesting the spleens, the mice had been receiving i.p. injections of 100 μL PBS, SOCS1-KIR (60 μg/mouse), or SOCS1-KIR2A (60 μg/mouse) every other day beginning on day 12 post-immunization, and were scored EAE stage 1. Cells were seeded at 5 × 10 6 cells/well in RPMI (10% FBS) and incubated 24 hours at 37°C, 5%CO 2 . Supernatants were collected and analyzed for IL-17A using the IL-17A Ready-Set-Go ELISA kit (eBioscience, San Diego, CA). B) IL-17A production by splenocytes in response to MBP stimulation is inhibited in mice treated with SOCS1-KIR. Splenocytes were isolated from SJL/J mice 27 days after immunization with MBP. Prior to harvesting the spleens, the mice had been treated as described in part A. Cells were seeded as described above and treated with or without 25 μg/ml MBP and incubated 24 hours. Supernatants were collected and analyzed for IL-17A as in part A. *P
    Figure Legend Snippet: IL-17A production by splenocytes is inhibited in EAE by treatment with SOCS1- KIR A) Splenocytes were isolated from SJL/J mice 36 days after immunization with MBP. Prior to harvesting the spleens, the mice had been receiving i.p. injections of 100 μL PBS, SOCS1-KIR (60 μg/mouse), or SOCS1-KIR2A (60 μg/mouse) every other day beginning on day 12 post-immunization, and were scored EAE stage 1. Cells were seeded at 5 × 10 6 cells/well in RPMI (10% FBS) and incubated 24 hours at 37°C, 5%CO 2 . Supernatants were collected and analyzed for IL-17A using the IL-17A Ready-Set-Go ELISA kit (eBioscience, San Diego, CA). B) IL-17A production by splenocytes in response to MBP stimulation is inhibited in mice treated with SOCS1-KIR. Splenocytes were isolated from SJL/J mice 27 days after immunization with MBP. Prior to harvesting the spleens, the mice had been treated as described in part A. Cells were seeded as described above and treated with or without 25 μg/ml MBP and incubated 24 hours. Supernatants were collected and analyzed for IL-17A as in part A. *P

    Techniques Used: Isolation, Mouse Assay, Incubation, Enzyme-linked Immunosorbent Assay

    Induction of IL-17A and IFNã in MBP-sensitized splenocytes by IL-23 is inhibited by SOCS1-KIR A) IL-17A production by splenocytes in response to IL-23 is inhibited by SOCS1- KIR. Splenocytes were isolated from SJL/J mice 37 days after immunization with MBP. Prior to harvesting the spleen, the mouse was scored at EAE stage one. Cells were seeded at 5 × 10 6 cells/well in RPMI (10% FBS) in a 96-well plate. Peptides were added at the above concentrations and cells were incubated at 37°C, 5% CO 2 , for 2 hours. IL-23 (10 ng/ml) was added to each well and the cells were incubated an additional 48 hours. Supernatants were collected and analyzed for IL-17A production using the IL-17A Ready-Set-Go ELISA kit (eBioscience, San Diego, CA). *P
    Figure Legend Snippet: Induction of IL-17A and IFNã in MBP-sensitized splenocytes by IL-23 is inhibited by SOCS1-KIR A) IL-17A production by splenocytes in response to IL-23 is inhibited by SOCS1- KIR. Splenocytes were isolated from SJL/J mice 37 days after immunization with MBP. Prior to harvesting the spleen, the mouse was scored at EAE stage one. Cells were seeded at 5 × 10 6 cells/well in RPMI (10% FBS) in a 96-well plate. Peptides were added at the above concentrations and cells were incubated at 37°C, 5% CO 2 , for 2 hours. IL-23 (10 ng/ml) was added to each well and the cells were incubated an additional 48 hours. Supernatants were collected and analyzed for IL-17A production using the IL-17A Ready-Set-Go ELISA kit (eBioscience, San Diego, CA). *P

    Techniques Used: Isolation, Mouse Assay, Incubation, Enzyme-linked Immunosorbent Assay

    SOCS1-KIR inhibits enhanced STAT3 activation by IL-23 and IL-17A A) SOCS1- KIR inhibits IL-23-enhanced STAT3 phosphorylation. Splenocytes isolated from MBP-immunized mice experiencing EAE (stage 1, five weeks after immunization), were treated with the above peptides for 2 hours, followed by incubation with IL-23 (10 ng/ml) for 10 minutes. The cells were lysed and protein concentration was determined by the standard BCA assay. The cell lysates were resolved using SDS-PAGE and proteins were transferred onto a nitrocellulose membrane. The membranes were probed for pSTAT3 (top) or STAT3 (bottom). Relative intensity of the bands was measured with ImageJ software (NIH) and is shown below the pSTAT3 blot. B) SOCS1-KIR inhibits STAT3 phosphorylation in splenocytes treated with IL-17. Splenocytes isolated from MBPimmunized mice were treated with the above peptides for 2 hours, followed by incubation with IL-17 (100 ng/ml) for 2 hours. Western blots were performed and analyzed as described for part A; however, longer exposure times were necessary. The experiments were performed twice with similar results.
    Figure Legend Snippet: SOCS1-KIR inhibits enhanced STAT3 activation by IL-23 and IL-17A A) SOCS1- KIR inhibits IL-23-enhanced STAT3 phosphorylation. Splenocytes isolated from MBP-immunized mice experiencing EAE (stage 1, five weeks after immunization), were treated with the above peptides for 2 hours, followed by incubation with IL-23 (10 ng/ml) for 10 minutes. The cells were lysed and protein concentration was determined by the standard BCA assay. The cell lysates were resolved using SDS-PAGE and proteins were transferred onto a nitrocellulose membrane. The membranes were probed for pSTAT3 (top) or STAT3 (bottom). Relative intensity of the bands was measured with ImageJ software (NIH) and is shown below the pSTAT3 blot. B) SOCS1-KIR inhibits STAT3 phosphorylation in splenocytes treated with IL-17. Splenocytes isolated from MBPimmunized mice were treated with the above peptides for 2 hours, followed by incubation with IL-17 (100 ng/ml) for 2 hours. Western blots were performed and analyzed as described for part A; however, longer exposure times were necessary. The experiments were performed twice with similar results.

    Techniques Used: Activation Assay, Isolation, Mouse Assay, Incubation, Protein Concentration, BIA-KA, SDS Page, Software, Western Blot

    39) Product Images from "IL-17RA Signaling Amplifies Antibody-Induced Arthritis"

    Article Title: IL-17RA Signaling Amplifies Antibody-Induced Arthritis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026342

    Neutrophils are reduced in the joints of Il17ra −/− mice and are unresponsive to direct stimulation with IL-17A. A, Number of neutrophils in the ankle joints of WT and Il17ra −/− mice on day 12 was determined by FACS analysis counting Ly6G + cells in relation to counting beads. Data are presented as mean ± SEM (n = 3 mice per group). One of three independent experiments is shown. B, Chemotaxis of freshly isolated murine bone marrow-derived neutrophils towards 100 nM LTB 4 and IL-17A (10 and 100 ng/ml) assessed using 24-well transwell assays. Data represent numbers of migrated neutrophils (n = 3 independently performed experiments). C, Chemotaxis of freshly isolated murine bone marrow-derived neutrophils towards LTB 4 (100 nM) MIP-2 (100 nM) and IL-17A (1, 10, 100, 1000 ng/ml) as well as their corresponding chemokinesis controls assessed using 96-well ChemoTx assays. Data are presented as chemotactic index (number of cells migrating to chemoattractant/number of cell migrating to medium control). Data shown are mean ± SEM (n = 4 independently performed experiments). D, Levels of IL-17RA and IL-17RC mRNA determined by qPCR on RNA isolated from murine FLS and freshly isolated bone marrow-derived neutrophils (n = 3 independently performed experiments). Data were compared by unpaired two-tailed Student's t test, ** = p
    Figure Legend Snippet: Neutrophils are reduced in the joints of Il17ra −/− mice and are unresponsive to direct stimulation with IL-17A. A, Number of neutrophils in the ankle joints of WT and Il17ra −/− mice on day 12 was determined by FACS analysis counting Ly6G + cells in relation to counting beads. Data are presented as mean ± SEM (n = 3 mice per group). One of three independent experiments is shown. B, Chemotaxis of freshly isolated murine bone marrow-derived neutrophils towards 100 nM LTB 4 and IL-17A (10 and 100 ng/ml) assessed using 24-well transwell assays. Data represent numbers of migrated neutrophils (n = 3 independently performed experiments). C, Chemotaxis of freshly isolated murine bone marrow-derived neutrophils towards LTB 4 (100 nM) MIP-2 (100 nM) and IL-17A (1, 10, 100, 1000 ng/ml) as well as their corresponding chemokinesis controls assessed using 96-well ChemoTx assays. Data are presented as chemotactic index (number of cells migrating to chemoattractant/number of cell migrating to medium control). Data shown are mean ± SEM (n = 4 independently performed experiments). D, Levels of IL-17RA and IL-17RC mRNA determined by qPCR on RNA isolated from murine FLS and freshly isolated bone marrow-derived neutrophils (n = 3 independently performed experiments). Data were compared by unpaired two-tailed Student's t test, ** = p

    Techniques Used: Mouse Assay, FACS, Chemotaxis Assay, Isolation, Derivative Assay, Real-time Polymerase Chain Reaction, Two Tailed Test

    40) Product Images from "IL-17RA Signaling Amplifies Antibody-Induced Arthritis"

    Article Title: IL-17RA Signaling Amplifies Antibody-Induced Arthritis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026342

    Neutrophils are reduced in the joints of Il17ra −/− mice and are unresponsive to direct stimulation with IL-17A. A, Number of neutrophils in the ankle joints of WT and Il17ra −/− mice on day 12 was determined by FACS analysis counting Ly6G + cells in relation to counting beads. Data are presented as mean ± SEM (n = 3 mice per group). One of three independent experiments is shown. B, Chemotaxis of freshly isolated murine bone marrow-derived neutrophils towards 100 nM LTB 4 and IL-17A (10 and 100 ng/ml) assessed using 24-well transwell assays. Data represent numbers of migrated neutrophils (n = 3 independently performed experiments). C, Chemotaxis of freshly isolated murine bone marrow-derived neutrophils towards LTB 4 (100 nM) MIP-2 (100 nM) and IL-17A (1, 10, 100, 1000 ng/ml) as well as their corresponding chemokinesis controls assessed using 96-well ChemoTx assays. Data are presented as chemotactic index (number of cells migrating to chemoattractant/number of cell migrating to medium control). Data shown are mean ± SEM (n = 4 independently performed experiments). D, Levels of IL-17RA and IL-17RC mRNA determined by qPCR on RNA isolated from murine FLS and freshly isolated bone marrow-derived neutrophils (n = 3 independently performed experiments). Data were compared by unpaired two-tailed Student's t test, ** = p
    Figure Legend Snippet: Neutrophils are reduced in the joints of Il17ra −/− mice and are unresponsive to direct stimulation with IL-17A. A, Number of neutrophils in the ankle joints of WT and Il17ra −/− mice on day 12 was determined by FACS analysis counting Ly6G + cells in relation to counting beads. Data are presented as mean ± SEM (n = 3 mice per group). One of three independent experiments is shown. B, Chemotaxis of freshly isolated murine bone marrow-derived neutrophils towards 100 nM LTB 4 and IL-17A (10 and 100 ng/ml) assessed using 24-well transwell assays. Data represent numbers of migrated neutrophils (n = 3 independently performed experiments). C, Chemotaxis of freshly isolated murine bone marrow-derived neutrophils towards LTB 4 (100 nM) MIP-2 (100 nM) and IL-17A (1, 10, 100, 1000 ng/ml) as well as their corresponding chemokinesis controls assessed using 96-well ChemoTx assays. Data are presented as chemotactic index (number of cells migrating to chemoattractant/number of cell migrating to medium control). Data shown are mean ± SEM (n = 4 independently performed experiments). D, Levels of IL-17RA and IL-17RC mRNA determined by qPCR on RNA isolated from murine FLS and freshly isolated bone marrow-derived neutrophils (n = 3 independently performed experiments). Data were compared by unpaired two-tailed Student's t test, ** = p

    Techniques Used: Mouse Assay, FACS, Chemotaxis Assay, Isolation, Derivative Assay, Real-time Polymerase Chain Reaction, Two Tailed Test

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    ALP Assay:

    Article Title: IL-17A Inhibits Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Wnt Signaling Pathway
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    Inhibition:

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    Expressing:

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    R&D Systems tnfα neutralization assays
    Inhibition of SOCE reduces IL-6 production induced by the <t>IL-17/TNFα</t> combination. Myoblasts were stimulated with IL-17 and/or TNFα in presence or not of the SOCE inhibitor 2-APB (10, 25, and 50 μM) or BTP2 (10 and 20 μM) for 48 h. CCL20 and IL-6 secretion by myoblasts was quantified by ELISA (A,B) . Data are the mean of 7 independent experiments ± SEM; ** p
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    <t>IL-17</t> promotes, but IFN-γ suppresses, neutrophilic airway inflammation . Mice were treated with 50 μg of OVA or PBS intranasally for 3 days (once per day) (A-C) or 10 μg of LPS intranasally (D). Cells in BALF were quantified 24 hours after the last OVA, PBS, or LPS inhalation. (A) OVA or PBS-challenged IFN-γ +/+ OTII mice (PBS, n = 16; OVA, n = 21), and IFN-γ −/− OTII mice (PBS, n = 6; OVA, n = 14). (B) OVA- or PBS-challenged IL-17 +/+ OTII mice (PBS, n = 12; OVA, n = 14), and IL-17 −/− OTII mice (PBS, n = 7; OVA, n = 14). (C) Cytokine levels in BALF obtained 24 hours after the last OVA or PBS inhalation in mice shown in panels A and B. (D) LPS-challenged wild-type mice (n = 15), OTII mice (n = 7), IL-17 −/− mice (n = 15), IFN-γ −/− mice (n = 12), and Rag-1 −/− mice (n = 7). * P
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    R&D Systems il 17f cytokine levels
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    Inhibition of SOCE reduces IL-6 production induced by the IL-17/TNFα combination. Myoblasts were stimulated with IL-17 and/or TNFα in presence or not of the SOCE inhibitor 2-APB (10, 25, and 50 μM) or BTP2 (10 and 20 μM) for 48 h. CCL20 and IL-6 secretion by myoblasts was quantified by ELISA (A,B) . Data are the mean of 7 independent experiments ± SEM; ** p

    Journal: Frontiers in Immunology

    Article Title: Blockade of Store-Operated Calcium Entry Reduces IL-17/TNF Cytokine-Induced Inflammatory Response in Human Myoblasts

    doi: 10.3389/fimmu.2018.03170

    Figure Lengend Snippet: Inhibition of SOCE reduces IL-6 production induced by the IL-17/TNFα combination. Myoblasts were stimulated with IL-17 and/or TNFα in presence or not of the SOCE inhibitor 2-APB (10, 25, and 50 μM) or BTP2 (10 and 20 μM) for 48 h. CCL20 and IL-6 secretion by myoblasts was quantified by ELISA (A,B) . Data are the mean of 7 independent experiments ± SEM; ** p

    Article Snippet: For the IL-17 and TNFα neutralization assays, PBMCs activated or not with PHA for 24 h were exposed to an anti-IL-17 antibody (R & D Systems) and/or the anti-TNFα antibody infliximab (Merck, Kenilworth, USA) at 10 μg/mL for 3 h before being added to the HepaRG cells.

    Techniques: Inhibition, Enzyme-linked Immunosorbent Assay

    IL-17 and TNFα increase ER stress and mitochondrial ROS in myoblasts. Myoblasts were treated with IL-17 (50 ng/mL) and/or TNFα (1 ng/mL) for 24 h. Expression of BiP/Grp78 protein was measured by western-blot and the band density was normalized with tubulin expression (A,B) . Mitochondrial oxidative stress measurements (ROS) of human myoblasts was measured with the fluorescence intensity of CellRox Dye, using 40x objective of a confocal microscope Nikon A1r, scale bar 70 μm (C,D) . Data are the mean of 4 to 7 independent experiments ± SEM, *** p

    Journal: Frontiers in Immunology

    Article Title: Blockade of Store-Operated Calcium Entry Reduces IL-17/TNF Cytokine-Induced Inflammatory Response in Human Myoblasts

    doi: 10.3389/fimmu.2018.03170

    Figure Lengend Snippet: IL-17 and TNFα increase ER stress and mitochondrial ROS in myoblasts. Myoblasts were treated with IL-17 (50 ng/mL) and/or TNFα (1 ng/mL) for 24 h. Expression of BiP/Grp78 protein was measured by western-blot and the band density was normalized with tubulin expression (A,B) . Mitochondrial oxidative stress measurements (ROS) of human myoblasts was measured with the fluorescence intensity of CellRox Dye, using 40x objective of a confocal microscope Nikon A1r, scale bar 70 μm (C,D) . Data are the mean of 4 to 7 independent experiments ± SEM, *** p

    Article Snippet: For the IL-17 and TNFα neutralization assays, PBMCs activated or not with PHA for 24 h were exposed to an anti-IL-17 antibody (R & D Systems) and/or the anti-TNFα antibody infliximab (Merck, Kenilworth, USA) at 10 μg/mL for 3 h before being added to the HepaRG cells.

    Techniques: Expressing, Western Blot, Fluorescence, Microscopy

    PBMC-myoblast interaction induces a strong production CCL20 and IL-6. PBMC and myoblasts were cultured alone or in co-culture at a ratio of 5 PBMCs for 1 myoblast for 48 h in the presence or not of PHA (5 μg/mL). CCL20 and IL-6 secretion by myoblasts was quantified by ELISA (A–F) . The contribution of direct cell-cell contact was investigated with a cell culture permeable insert (C,D) . PBMCs were pre-incubated for 24 h in presence or not of PHA and then exposed or not to an anti-IL-17 antibody and/or an anti-TNFα antibody for 3 h before being added to the myoblast cultures. Data are expressed as CCL20 and IL-6 supernatant level percentages compared to the non-activated pre-incubated PBMC—myoblast co-cultures (E,F) . Data are the mean of 6–14 independent experiments ± SEM; * p

    Journal: Frontiers in Immunology

    Article Title: Blockade of Store-Operated Calcium Entry Reduces IL-17/TNF Cytokine-Induced Inflammatory Response in Human Myoblasts

    doi: 10.3389/fimmu.2018.03170

    Figure Lengend Snippet: PBMC-myoblast interaction induces a strong production CCL20 and IL-6. PBMC and myoblasts were cultured alone or in co-culture at a ratio of 5 PBMCs for 1 myoblast for 48 h in the presence or not of PHA (5 μg/mL). CCL20 and IL-6 secretion by myoblasts was quantified by ELISA (A–F) . The contribution of direct cell-cell contact was investigated with a cell culture permeable insert (C,D) . PBMCs were pre-incubated for 24 h in presence or not of PHA and then exposed or not to an anti-IL-17 antibody and/or an anti-TNFα antibody for 3 h before being added to the myoblast cultures. Data are expressed as CCL20 and IL-6 supernatant level percentages compared to the non-activated pre-incubated PBMC—myoblast co-cultures (E,F) . Data are the mean of 6–14 independent experiments ± SEM; * p

    Article Snippet: For the IL-17 and TNFα neutralization assays, PBMCs activated or not with PHA for 24 h were exposed to an anti-IL-17 antibody (R & D Systems) and/or the anti-TNFα antibody infliximab (Merck, Kenilworth, USA) at 10 μg/mL for 3 h before being added to the HepaRG cells.

    Techniques: Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Incubation

    IL-17 and TNFα increase store-operated calcium entry. Myoblasts were treated with IL-17 (50 ng/mL) and/or TNFα (1 ng/mL). mRNA levels of STIM1 and ORAI1 at 12 h was expressed as fold changes compared to control (A,B) . ORAI1 and STIM1 protein was measured by western-blot and the band density was normalized with the tubulin expression. (C–F) Representative image of STIM1 puncta in human myoblast treated with IL-17 (50 ng/mL) and TNFα (1 ng/mL) for 24 h. Image Correlation Spectroscopy (ICS) analysis of STIM1 puncta (left inset) mean density of puncta (μm2). (right inset) mean surface of puncta (puncta/μm2). Data are the mean of 3 independent experiments with cells from 3 different donors ± SEM; * p

    Journal: Frontiers in Immunology

    Article Title: Blockade of Store-Operated Calcium Entry Reduces IL-17/TNF Cytokine-Induced Inflammatory Response in Human Myoblasts

    doi: 10.3389/fimmu.2018.03170

    Figure Lengend Snippet: IL-17 and TNFα increase store-operated calcium entry. Myoblasts were treated with IL-17 (50 ng/mL) and/or TNFα (1 ng/mL). mRNA levels of STIM1 and ORAI1 at 12 h was expressed as fold changes compared to control (A,B) . ORAI1 and STIM1 protein was measured by western-blot and the band density was normalized with the tubulin expression. (C–F) Representative image of STIM1 puncta in human myoblast treated with IL-17 (50 ng/mL) and TNFα (1 ng/mL) for 24 h. Image Correlation Spectroscopy (ICS) analysis of STIM1 puncta (left inset) mean density of puncta (μm2). (right inset) mean surface of puncta (puncta/μm2). Data are the mean of 3 independent experiments with cells from 3 different donors ± SEM; * p

    Article Snippet: For the IL-17 and TNFα neutralization assays, PBMCs activated or not with PHA for 24 h were exposed to an anti-IL-17 antibody (R & D Systems) and/or the anti-TNFα antibody infliximab (Merck, Kenilworth, USA) at 10 μg/mL for 3 h before being added to the HepaRG cells.

    Techniques: Western Blot, Expressing, Spectroscopy

    IL-17 and TNFα mediate muscle damage and weakness through immune and non-immune pathways in myoblasts. The immune cell infiltration in IIM constitutes a local source of cytokines and promotes the cell-cell interactions. IL-17 mainly produced by Th17 cells, and TNFα act in synergy on myoblasts to increase IL-6 and CCL20 secretion. Because IL-6 is involved in the Th17 cell differentiation and CCL20 in dendritic and Th17 cell recruitment, IL-6 and CCL20 mediate a positive feedback loop promoting local IL-17 production. IL-17 and TNFα induce also non-immune pathways with ROS production, ER stress and SOCE activation. The IL-17/TNFα effect of mitochondrial dysfunction, ER stress, and SOCE activation are probably closely linked. SOCE and calcium dysregulation contribute to the IL-6 release induced by IL-17/TNFα. CCL20, chemokine (C-C motif) ligand 20; DC, dendritic cells; ER, endoplasmic reticulum; IL, interleukin; ROS, reactive oxygen species; STIM, stromal interacting molecule; TNFα, tumor necrosis factor-α.

    Journal: Frontiers in Immunology

    Article Title: Blockade of Store-Operated Calcium Entry Reduces IL-17/TNF Cytokine-Induced Inflammatory Response in Human Myoblasts

    doi: 10.3389/fimmu.2018.03170

    Figure Lengend Snippet: IL-17 and TNFα mediate muscle damage and weakness through immune and non-immune pathways in myoblasts. The immune cell infiltration in IIM constitutes a local source of cytokines and promotes the cell-cell interactions. IL-17 mainly produced by Th17 cells, and TNFα act in synergy on myoblasts to increase IL-6 and CCL20 secretion. Because IL-6 is involved in the Th17 cell differentiation and CCL20 in dendritic and Th17 cell recruitment, IL-6 and CCL20 mediate a positive feedback loop promoting local IL-17 production. IL-17 and TNFα induce also non-immune pathways with ROS production, ER stress and SOCE activation. The IL-17/TNFα effect of mitochondrial dysfunction, ER stress, and SOCE activation are probably closely linked. SOCE and calcium dysregulation contribute to the IL-6 release induced by IL-17/TNFα. CCL20, chemokine (C-C motif) ligand 20; DC, dendritic cells; ER, endoplasmic reticulum; IL, interleukin; ROS, reactive oxygen species; STIM, stromal interacting molecule; TNFα, tumor necrosis factor-α.

    Article Snippet: For the IL-17 and TNFα neutralization assays, PBMCs activated or not with PHA for 24 h were exposed to an anti-IL-17 antibody (R & D Systems) and/or the anti-TNFα antibody infliximab (Merck, Kenilworth, USA) at 10 μg/mL for 3 h before being added to the HepaRG cells.

    Techniques: Produced, Activated Clotting Time Assay, Cell Differentiation, Activation Assay

    Synergistic effect of IL-17 and TNFα on the CCL20 and IL-6 production by myoblasts. Myoblasts were treated with IL-17 (50 ng/mL) and/or TNFα (1 ng/mL) for 48 h. IL-6 and CCL20 secretion by myoblasts was quantified by ELISA (A,B) . Data are the mean of 5–8 independent experiments ± SEM; * p

    Journal: Frontiers in Immunology

    Article Title: Blockade of Store-Operated Calcium Entry Reduces IL-17/TNF Cytokine-Induced Inflammatory Response in Human Myoblasts

    doi: 10.3389/fimmu.2018.03170

    Figure Lengend Snippet: Synergistic effect of IL-17 and TNFα on the CCL20 and IL-6 production by myoblasts. Myoblasts were treated with IL-17 (50 ng/mL) and/or TNFα (1 ng/mL) for 48 h. IL-6 and CCL20 secretion by myoblasts was quantified by ELISA (A,B) . Data are the mean of 5–8 independent experiments ± SEM; * p

    Article Snippet: For the IL-17 and TNFα neutralization assays, PBMCs activated or not with PHA for 24 h were exposed to an anti-IL-17 antibody (R & D Systems) and/or the anti-TNFα antibody infliximab (Merck, Kenilworth, USA) at 10 μg/mL for 3 h before being added to the HepaRG cells.

    Techniques: Enzyme-linked Immunosorbent Assay

    IL-17 promotes, but IFN-γ suppresses, neutrophilic airway inflammation . Mice were treated with 50 μg of OVA or PBS intranasally for 3 days (once per day) (A-C) or 10 μg of LPS intranasally (D). Cells in BALF were quantified 24 hours after the last OVA, PBS, or LPS inhalation. (A) OVA or PBS-challenged IFN-γ +/+ OTII mice (PBS, n = 16; OVA, n = 21), and IFN-γ −/− OTII mice (PBS, n = 6; OVA, n = 14). (B) OVA- or PBS-challenged IL-17 +/+ OTII mice (PBS, n = 12; OVA, n = 14), and IL-17 −/− OTII mice (PBS, n = 7; OVA, n = 14). (C) Cytokine levels in BALF obtained 24 hours after the last OVA or PBS inhalation in mice shown in panels A and B. (D) LPS-challenged wild-type mice (n = 15), OTII mice (n = 7), IL-17 −/− mice (n = 15), IFN-γ −/− mice (n = 12), and Rag-1 −/− mice (n = 7). * P

    Journal: Blood

    Article Title: Mast cell-derived TNF can promote Th17 cell-dependent neutrophil recruitment in ovalbumin-challenged OTII mice

    doi: 10.1182/blood-2006-09-046128

    Figure Lengend Snippet: IL-17 promotes, but IFN-γ suppresses, neutrophilic airway inflammation . Mice were treated with 50 μg of OVA or PBS intranasally for 3 days (once per day) (A-C) or 10 μg of LPS intranasally (D). Cells in BALF were quantified 24 hours after the last OVA, PBS, or LPS inhalation. (A) OVA or PBS-challenged IFN-γ +/+ OTII mice (PBS, n = 16; OVA, n = 21), and IFN-γ −/− OTII mice (PBS, n = 6; OVA, n = 14). (B) OVA- or PBS-challenged IL-17 +/+ OTII mice (PBS, n = 12; OVA, n = 14), and IL-17 −/− OTII mice (PBS, n = 7; OVA, n = 14). (C) Cytokine levels in BALF obtained 24 hours after the last OVA or PBS inhalation in mice shown in panels A and B. (D) LPS-challenged wild-type mice (n = 15), OTII mice (n = 7), IL-17 −/− mice (n = 15), IFN-γ −/− mice (n = 12), and Rag-1 −/− mice (n = 7). * P

    Article Snippet: For IL-17 inhalation, mice were treated intranasally with 10 μg recombinant mouse IL-17 (rmIL-17; R & D Systems, Minneapolis MN) and BAL cells were collected 6 hours later.

    Techniques: Mouse Assay

    TNF is required for IL-17–induced neutrophil influx in the lungs . (A) Cells in BALF were quantified 8 hours after intranasal treatment of mice with PBS or rmIL-17 (10 μg). Wild-type mice (PBS, n = 5; IL-17, n = 4) and TNF −/− mice (PBS, n = 5; IL-17, n = 4). Data are the average + SD of results pooled from 2 independent experiments, each of which gave similar results. * P

    Journal: Blood

    Article Title: Mast cell-derived TNF can promote Th17 cell-dependent neutrophil recruitment in ovalbumin-challenged OTII mice

    doi: 10.1182/blood-2006-09-046128

    Figure Lengend Snippet: TNF is required for IL-17–induced neutrophil influx in the lungs . (A) Cells in BALF were quantified 8 hours after intranasal treatment of mice with PBS or rmIL-17 (10 μg). Wild-type mice (PBS, n = 5; IL-17, n = 4) and TNF −/− mice (PBS, n = 5; IL-17, n = 4). Data are the average + SD of results pooled from 2 independent experiments, each of which gave similar results. * P

    Article Snippet: For IL-17 inhalation, mice were treated intranasally with 10 μg recombinant mouse IL-17 (rmIL-17; R & D Systems, Minneapolis MN) and BAL cells were collected 6 hours later.

    Techniques: Mouse Assay

    IL-18-mediated resistance is dependent on endogenous IL-12. (A) SCID mice were infected with T. gondii and treated with IL-18 or PBS alone or in combination with anti-IL-12 or rat IgG. The treatment with anti-IL-12 resulted in complete abrogation of serum levels of IL-12p40 on days 3, 5, and 7 postinfection. The serum levels of IFN-γ on day 5 postinfection were measured by ELISA (A), and the percentage of infected PECs on day 7 postinfection calculated (B) as described in Materials and Methods. The data shown are the means ± the SD of the pooled data from three experiments with three to five mice per group. (C) Effect of depletion of endogenous IL-12 on the survival of SCID mice treated with IL-18. Similar results were observed in a repeat experiment with four mice per group.

    Journal: Infection and Immunity

    Article Title: Interleukin-18 (IL-18) Enhances Innate IL-12-Mediated Resistance to Toxoplasma gondii

    doi:

    Figure Lengend Snippet: IL-18-mediated resistance is dependent on endogenous IL-12. (A) SCID mice were infected with T. gondii and treated with IL-18 or PBS alone or in combination with anti-IL-12 or rat IgG. The treatment with anti-IL-12 resulted in complete abrogation of serum levels of IL-12p40 on days 3, 5, and 7 postinfection. The serum levels of IFN-γ on day 5 postinfection were measured by ELISA (A), and the percentage of infected PECs on day 7 postinfection calculated (B) as described in Materials and Methods. The data shown are the means ± the SD of the pooled data from three experiments with three to five mice per group. (C) Effect of depletion of endogenous IL-12 on the survival of SCID mice treated with IL-18. Similar results were observed in a repeat experiment with four mice per group.

    Article Snippet: Cultures were stimulated with cytokines and/or RH strain tachyzoite lysate antigen (TLA) or cytokine(s) with or without antibody for 24 h. For the depletion of cytokines in the cultures, 30 μg of anti-IL-12 (C17.8), 20 μg of rat anti-IL-18 MAb (R & D Systems, Inc.), or 30 μg of rabbit anti-IL-18 polyclonal antibody per ml was added to the culture.

    Techniques: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay

    Expression of IL-18 during toxoplasmosis. (A) Total RNA from the spleens of uninfected SCID mice or SCID mice infected for 7 days was extracted, and RPA analysis was performed as described in Materials and Methods. Similar results were observed in two experiments. (B) Densitometric analysis of RNA levels and expression relative to the housekeeping L32 gene. The results shown are the means ± the SD of the data presented. (C) Levels of IL-18 and IL-12 were measured by using ELISA in the serum from uninfected mice or mice infected for 7 days. The data shown are the means ± the SD from four independent experiments with three to six mice per group.

    Journal: Infection and Immunity

    Article Title: Interleukin-18 (IL-18) Enhances Innate IL-12-Mediated Resistance to Toxoplasma gondii

    doi:

    Figure Lengend Snippet: Expression of IL-18 during toxoplasmosis. (A) Total RNA from the spleens of uninfected SCID mice or SCID mice infected for 7 days was extracted, and RPA analysis was performed as described in Materials and Methods. Similar results were observed in two experiments. (B) Densitometric analysis of RNA levels and expression relative to the housekeeping L32 gene. The results shown are the means ± the SD of the data presented. (C) Levels of IL-18 and IL-12 were measured by using ELISA in the serum from uninfected mice or mice infected for 7 days. The data shown are the means ± the SD from four independent experiments with three to six mice per group.

    Article Snippet: Cultures were stimulated with cytokines and/or RH strain tachyzoite lysate antigen (TLA) or cytokine(s) with or without antibody for 24 h. For the depletion of cytokines in the cultures, 30 μg of anti-IL-12 (C17.8), 20 μg of rat anti-IL-18 MAb (R & D Systems, Inc.), or 30 μg of rabbit anti-IL-18 polyclonal antibody per ml was added to the culture.

    Techniques: Expressing, Mouse Assay, Infection, Recombinase Polymerase Amplification, Enzyme-linked Immunosorbent Assay

    IL-18 induced production of IFN-γ by splenocytes from infected SCID mice is dependent on IL-12. Splenocytes from SCID mice infected for 7 days were stimulated with 30 μg of TLA, 10 ng of IL-12, or 10 ng of IL-18 per ml in combination with 30 μg of rat IgG or anti-IL-12 (C17.8) per ml for 24 h, and the production of IFN-γ in the supernatants was measured by ELISA. The data shown are the means ± the SD from a single experiment done in triplicate. Similar results were observed in two additional experiments.

    Journal: Infection and Immunity

    Article Title: Interleukin-18 (IL-18) Enhances Innate IL-12-Mediated Resistance to Toxoplasma gondii

    doi:

    Figure Lengend Snippet: IL-18 induced production of IFN-γ by splenocytes from infected SCID mice is dependent on IL-12. Splenocytes from SCID mice infected for 7 days were stimulated with 30 μg of TLA, 10 ng of IL-12, or 10 ng of IL-18 per ml in combination with 30 μg of rat IgG or anti-IL-12 (C17.8) per ml for 24 h, and the production of IFN-γ in the supernatants was measured by ELISA. The data shown are the means ± the SD from a single experiment done in triplicate. Similar results were observed in two additional experiments.

    Article Snippet: Cultures were stimulated with cytokines and/or RH strain tachyzoite lysate antigen (TLA) or cytokine(s) with or without antibody for 24 h. For the depletion of cytokines in the cultures, 30 μg of anti-IL-12 (C17.8), 20 μg of rat anti-IL-18 MAb (R & D Systems, Inc.), or 30 μg of rabbit anti-IL-18 polyclonal antibody per ml was added to the culture.

    Techniques: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay

    IL23R A/Glu381 allele reduces IL-23 responsiveness in human memory Th17 cells and interrupts the IL-23/th17 axis in the skin of heterozygous psoriatic patients. ( a ) Memory CD4+ T cell supernatants from psoriatic patients homozygous GG (green dots) and heterozygous GA (red dots), cultured with or without IL-23 for 72 hours, were assayed for IL-17A, IL-17F, IFN-γ, and IL-22 secretion. Data are shown as differential cytokine production (Δ) in cells cultured with versus without IL-23. Each symbol represents one individual donor. Dotted line denotes Δ=0. Horizontal bars represent medians. Mann–Whitney test was performed; * P

    Journal: The Journal of Investigative Dermatology

    Article Title: The IL23R A/Gln381 Allele Promotes IL-23 Unresponsiveness in Human Memory T-Helper 17 Cells and Impairs Th17 Responses in Psoriasis Patients

    doi: 10.1038/jid.2013.170

    Figure Lengend Snippet: IL23R A/Glu381 allele reduces IL-23 responsiveness in human memory Th17 cells and interrupts the IL-23/th17 axis in the skin of heterozygous psoriatic patients. ( a ) Memory CD4+ T cell supernatants from psoriatic patients homozygous GG (green dots) and heterozygous GA (red dots), cultured with or without IL-23 for 72 hours, were assayed for IL-17A, IL-17F, IFN-γ, and IL-22 secretion. Data are shown as differential cytokine production (Δ) in cells cultured with versus without IL-23. Each symbol represents one individual donor. Dotted line denotes Δ=0. Horizontal bars represent medians. Mann–Whitney test was performed; * P

    Article Snippet: IL-22 and IL-17F cytokine levels were assayed using commercially available ELISA kits (R & D Systems).

    Techniques: Cell Culture, MANN-WHITNEY

    IL-23R A/Glu381 allele promotes IL-23 unresponsiveness in human memory Th17 cells. Supernatants of memory CD4+ T cells from healthy individuals homozygous for the common G allele (GG, green dots), heterozygous GA (red dots), or homozygous for the protective A allele (AA, blue dots), cultured with or without IL-23 for 72 hours, were assayed for IL-17A ( a ), IL-17F ( b ), IFN-γ ( c ), and IL-22 ( d ) secretion. Data are shown as differential cytokine production (Δ) in cells cultured with versus without IL-23. Each symbol represents one individual donor. Dotted line denotes Δ=0. Horizontal bars represent means ( a ) or medians ( b , c , d ). One-way analysis of variance, followed by Bonferroni post test ( a ) or Kruskal–Wallis test and Dunn's multiple-comparison test ( b , c , d ), was performed. * P

    Journal: The Journal of Investigative Dermatology

    Article Title: The IL23R A/Gln381 Allele Promotes IL-23 Unresponsiveness in Human Memory T-Helper 17 Cells and Impairs Th17 Responses in Psoriasis Patients

    doi: 10.1038/jid.2013.170

    Figure Lengend Snippet: IL-23R A/Glu381 allele promotes IL-23 unresponsiveness in human memory Th17 cells. Supernatants of memory CD4+ T cells from healthy individuals homozygous for the common G allele (GG, green dots), heterozygous GA (red dots), or homozygous for the protective A allele (AA, blue dots), cultured with or without IL-23 for 72 hours, were assayed for IL-17A ( a ), IL-17F ( b ), IFN-γ ( c ), and IL-22 ( d ) secretion. Data are shown as differential cytokine production (Δ) in cells cultured with versus without IL-23. Each symbol represents one individual donor. Dotted line denotes Δ=0. Horizontal bars represent means ( a ) or medians ( b , c , d ). One-way analysis of variance, followed by Bonferroni post test ( a ) or Kruskal–Wallis test and Dunn's multiple-comparison test ( b , c , d ), was performed. * P

    Article Snippet: IL-22 and IL-17F cytokine levels were assayed using commercially available ELISA kits (R & D Systems).

    Techniques: Cell Culture