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
    Category:
    Proteins and Enzymes
    Source:
    E. coli-derived Recombinant Canine IL-17A Protein
    Applications:
    Bioactivity
    Purity:
    >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
    Conjugate:
    Unconjugated
    Size:
    25 ug
    Buy from Supplier


    Structured Review

    R&D Systems il 17a
    Neutrophils are reduced in the joints of Il17ra −/− mice and are unresponsive to direct stimulation with <t>IL-17A.</t> 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
    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 1 article reviews
    Price from $9.99 to $1999.99
    il 17a - by Bioz Stars, 2021-07
    99/100 stars

    Images

    1) 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

    2) Product Images from "Th17 micro-milieu regulates NLRP1-dependent caspase-5 activity in skin autoinflammation"

    Article Title: Th17 micro-milieu regulates NLRP1-dependent caspase-5 activity in skin autoinflammation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0175153

    The antimicrobial peptide psoriasin (S100A7) mediates cytokine-dependent caspase regulation and IL-1β release by epidermal keratinocytes. A, E, Keratinocytes stimulated with IFNγ, IL-17A, transfected with dsDNA and indicated siRNA, and the psoriasin-dependent IL-1β release was analyzed by ELISA. Data represent mean + SEM, **, p
    Figure Legend Snippet: The antimicrobial peptide psoriasin (S100A7) mediates cytokine-dependent caspase regulation and IL-1β release by epidermal keratinocytes. A, E, Keratinocytes stimulated with IFNγ, IL-17A, transfected with dsDNA and indicated siRNA, and the psoriasin-dependent IL-1β release was analyzed by ELISA. Data represent mean + SEM, **, p

    Techniques Used: Transfection, Enzyme-linked Immunosorbent Assay

    Vitamin D interferes with IL-1β release by epidermal keratinocytes and suppresses caspase-5 expression in epidermal keratinocytes in psoriasis. A, dsDNA-transfected keratinocytes stimulated with IFNγ, IL-17A, and the vitamin D-dependent IL-1β release was analyzed by ELISA, n = 4. B, Keratinocytes stimulated with IFNγ, IL-17A and vitamin D-dependent caspase-5 levels were detected by immunoblotting, n = 3. C, Vitamin D-dependent regulation of caspase-5 in keratinocytes stimulated with IFNγ, IL-17A and analyzed by RTqPCR and normalized to β-actin. Data represent mean + SEM *, p
    Figure Legend Snippet: Vitamin D interferes with IL-1β release by epidermal keratinocytes and suppresses caspase-5 expression in epidermal keratinocytes in psoriasis. A, dsDNA-transfected keratinocytes stimulated with IFNγ, IL-17A, and the vitamin D-dependent IL-1β release was analyzed by ELISA, n = 4. B, Keratinocytes stimulated with IFNγ, IL-17A and vitamin D-dependent caspase-5 levels were detected by immunoblotting, n = 3. C, Vitamin D-dependent regulation of caspase-5 in keratinocytes stimulated with IFNγ, IL-17A and analyzed by RTqPCR and normalized to β-actin. Data represent mean + SEM *, p

    Techniques Used: Expressing, Transfection, Enzyme-linked Immunosorbent Assay

    IL-17A amplifies caspase-5 induction and controls NLRP1-mediated IL-1β release by epidermal keratinocytes. A-D, Regulation of IL-1β, caspase-1, caspase-5, NLRP1in IFNγ- and IL-17A -stimulated keratinocytes analyzed by RTqPCR and normalized to β-actin. Data represent mean + SEM of three independent experiments performed in triplicates *, p
    Figure Legend Snippet: IL-17A amplifies caspase-5 induction and controls NLRP1-mediated IL-1β release by epidermal keratinocytes. A-D, Regulation of IL-1β, caspase-1, caspase-5, NLRP1in IFNγ- and IL-17A -stimulated keratinocytes analyzed by RTqPCR and normalized to β-actin. Data represent mean + SEM of three independent experiments performed in triplicates *, p

    Techniques Used:

    3) Product Images from "Interleukin 17A promotes gallbladder cancer invasiveness via ERK/NF-κB signal pathway mediated epithelial-to-mesenchymal transition"

    Article Title: Interleukin 17A promotes gallbladder cancer invasiveness via ERK/NF-κB signal pathway mediated epithelial-to-mesenchymal transition

    Journal: Journal of Cancer

    doi: 10.7150/jca.40656

    IL-17A activates ERK signal pathway in GBC cells. (A)The protein level of p-ERK and ERK was analysed with Western blotting analysis in GBC cells treated with IL-17A (50 ng/ml) at different time points. (B) Phosphorylation densities of ERK were digitally scanned. All data are presented as the mean±standard deviation. * p
    Figure Legend Snippet: IL-17A activates ERK signal pathway in GBC cells. (A)The protein level of p-ERK and ERK was analysed with Western blotting analysis in GBC cells treated with IL-17A (50 ng/ml) at different time points. (B) Phosphorylation densities of ERK were digitally scanned. All data are presented as the mean±standard deviation. * p

    Techniques Used: Western Blot, Standard Deviation

    The pro-metastasis effect of IL-17A in GBC. (A) IL-17A has no effect on the proliferation of GBC cells. (B) IL-17A has no effect on the apoptosis of GBC cells. (C and D) The effect of IL-17A on cell migration was detected by using wound healing assays. IL-17A enhanced the motility of GBC cells. (E) The pro-invasion effect of IL-17A on GBC cells was assay via transwell assay. (F) The invasion cells per field were calculated. All data are presented as the mean±standard deviation. * p
    Figure Legend Snippet: The pro-metastasis effect of IL-17A in GBC. (A) IL-17A has no effect on the proliferation of GBC cells. (B) IL-17A has no effect on the apoptosis of GBC cells. (C and D) The effect of IL-17A on cell migration was detected by using wound healing assays. IL-17A enhanced the motility of GBC cells. (E) The pro-invasion effect of IL-17A on GBC cells was assay via transwell assay. (F) The invasion cells per field were calculated. All data are presented as the mean±standard deviation. * p

    Techniques Used: Migration, Transwell Assay, Standard Deviation

    IL-17A induces EMT of GBC cells. (A) Protein levels of Snail, E-cadherin, N-cadherin, Vimentin and Slug in GBC cells treated with IL-17A (50 ng/ml) for 24 h or not were detected with western blot. (B) The expression levels of E-cadherin, Vimentin, N-cadherin, Snail and Slug were quantified as percentage of control. (C) The effect of IL-17A on protein expressions of NF-κB/p50 and p65. (D) Quantification of the expressions of NF-κB/p50 and p65. All data are presented as the mean±standard deviation. * p
    Figure Legend Snippet: IL-17A induces EMT of GBC cells. (A) Protein levels of Snail, E-cadherin, N-cadherin, Vimentin and Slug in GBC cells treated with IL-17A (50 ng/ml) for 24 h or not were detected with western blot. (B) The expression levels of E-cadherin, Vimentin, N-cadherin, Snail and Slug were quantified as percentage of control. (C) The effect of IL-17A on protein expressions of NF-κB/p50 and p65. (D) Quantification of the expressions of NF-κB/p50 and p65. All data are presented as the mean±standard deviation. * p

    Techniques Used: Western Blot, Expressing, Standard Deviation

    Effects of the ERK signal pathway inhibitor and IL-17A on cell invasion and EMT in GBC cells. (A, B) The pro-invasion effect of IL-17A in GBC cells could been partially reversed by U0126. (C, D) After treated with an ERK inhibitor (U0126; 10 μM) for 30 min, GBC cells were cultured with IL-17A or not. Pretreated with U0126 (10 μM), the expression levels of Vimentin were significantly decreased, the expression levels of E-cadherin were significantly increased. (E, F) Pretreated with U0126 could block the nuclear translocation of NF-κB/p50 and p65 in GBC cells. All data are presented as the mean±standard deviation. * p
    Figure Legend Snippet: Effects of the ERK signal pathway inhibitor and IL-17A on cell invasion and EMT in GBC cells. (A, B) The pro-invasion effect of IL-17A in GBC cells could been partially reversed by U0126. (C, D) After treated with an ERK inhibitor (U0126; 10 μM) for 30 min, GBC cells were cultured with IL-17A or not. Pretreated with U0126 (10 μM), the expression levels of Vimentin were significantly decreased, the expression levels of E-cadherin were significantly increased. (E, F) Pretreated with U0126 could block the nuclear translocation of NF-κB/p50 and p65 in GBC cells. All data are presented as the mean±standard deviation. * p

    Techniques Used: Cell Culture, Expressing, Blocking Assay, Translocation Assay, Standard Deviation

    4) 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

    5) 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

    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 "Epicutaneous Exposure to Staphylococcal Superantigen Enterotoxin B Enhances Allergic Lung Inflammation via an IL-17A Dependent Mechanism"

    Article Title: Epicutaneous Exposure to Staphylococcal Superantigen Enterotoxin B Enhances Allergic Lung Inflammation via an IL-17A Dependent Mechanism

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039032

    SEB stimulated lymphocyte IL-17A production. IL-17A, IL-4 and IFN-γ produced by lymphocytes from cervical, axillary, inguinal draining lymph nodes (DLNs) or from spleen after SEB stimulation (a-f). Data are representative of two experiments with similar results. Columns and error bars represent Mean ± SEM (n = 5–7 per group; *p≤0.05, **p≤0.01).
    Figure Legend Snippet: SEB stimulated lymphocyte IL-17A production. IL-17A, IL-4 and IFN-γ produced by lymphocytes from cervical, axillary, inguinal draining lymph nodes (DLNs) or from spleen after SEB stimulation (a-f). Data are representative of two experiments with similar results. Columns and error bars represent Mean ± SEM (n = 5–7 per group; *p≤0.05, **p≤0.01).

    Techniques Used: Produced

    SEB enhanced IL-17A production and Langerhans cell accumulation in the skin. Mice were epicutaneously sensitized with PBS, SEB, Ova, or Ova + SEB and airway challenged with Ova. Skin samples were prepared for proteins and histology. (a) IL-17A was measured by ELISA of the skin protein extracts (n = 7–8 for each group; *p
    Figure Legend Snippet: SEB enhanced IL-17A production and Langerhans cell accumulation in the skin. Mice were epicutaneously sensitized with PBS, SEB, Ova, or Ova + SEB and airway challenged with Ova. Skin samples were prepared for proteins and histology. (a) IL-17A was measured by ELISA of the skin protein extracts (n = 7–8 for each group; *p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Effects of IL-17A gene disruption on SEB enhanced Ova-induced pulmonary inflammation, cytokine production and immunoglobulin production. Measurements were performed after EC-sensitization with PBS, SEB, Ova, or Ova + SEB and Ova airway challenge in IL-17A KO mice and compared with wild type mice. (a) H E staining of lung sections examined at magnification ×20 (representatives of at least 5 samples each group). (b, c) Total and differential cell counts in the BAL and (d, e) IL-4 and IFN-γ levels in the BAL. (f, g) SEB-specific IgG1 and Ova-specific IgG1. Data shown are Mean ± SEM (n = 5 for each group; *p
    Figure Legend Snippet: Effects of IL-17A gene disruption on SEB enhanced Ova-induced pulmonary inflammation, cytokine production and immunoglobulin production. Measurements were performed after EC-sensitization with PBS, SEB, Ova, or Ova + SEB and Ova airway challenge in IL-17A KO mice and compared with wild type mice. (a) H E staining of lung sections examined at magnification ×20 (representatives of at least 5 samples each group). (b, c) Total and differential cell counts in the BAL and (d, e) IL-4 and IFN-γ levels in the BAL. (f, g) SEB-specific IgG1 and Ova-specific IgG1. Data shown are Mean ± SEM (n = 5 for each group; *p

    Techniques Used: Mouse Assay, Staining

    SEB synergistically enhanced Ova stimulated IL-17A production by lymphocytes and exaggerated systemic Ova-specific IgE production. (a–f) Ova stimulated production of IL-17A, IL-4, and IFN-γ by lymphocytes from DLN or spleen of mice sensitized with PBS, SEB, Ova and Ova + SEB (n = 5–7 per group; *p≤0.05, **p≤0.01). (g) SEB-specific IgG1 and (h) Ova-specific IgE in the serum. Data are Mean ± SEM (n = 5–7 per group; *p≤0.05, **p≤0.01).
    Figure Legend Snippet: SEB synergistically enhanced Ova stimulated IL-17A production by lymphocytes and exaggerated systemic Ova-specific IgE production. (a–f) Ova stimulated production of IL-17A, IL-4, and IFN-γ by lymphocytes from DLN or spleen of mice sensitized with PBS, SEB, Ova and Ova + SEB (n = 5–7 per group; *p≤0.05, **p≤0.01). (g) SEB-specific IgG1 and (h) Ova-specific IgE in the serum. Data are Mean ± SEM (n = 5–7 per group; *p≤0.05, **p≤0.01).

    Techniques Used: Mouse Assay

    Deletion of IL-17A gene reduced SEB enhanced Ova-induced changes in lung physiology. (a) Airway resistance in response to methacholine after epicutaneous sensitization and Ova airway challenge. The columns and error bars represent Mean ± SEM (shown are combined data from two separate experiments; n = 4–5 per group; * p
    Figure Legend Snippet: Deletion of IL-17A gene reduced SEB enhanced Ova-induced changes in lung physiology. (a) Airway resistance in response to methacholine after epicutaneous sensitization and Ova airway challenge. The columns and error bars represent Mean ± SEM (shown are combined data from two separate experiments; n = 4–5 per group; * p

    Techniques Used:

    8) Product Images from "Self-reactive CD4+ IL-3+ T cells amplify autoimmune inflammation in myocarditis by inciting monocyte chemotaxis"

    Article Title: Self-reactive CD4+ IL-3+ T cells amplify autoimmune inflammation in myocarditis by inciting monocyte chemotaxis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20180722

    T cell–derived IL-3 is essential to cardiac inflammation in myocarditis. (A) Il3 mRNA levels in the heart (HT), BM, spleen (Sp), draining LN, thymus (TH), and lung (LG) before and 8, 14, and 21 d after the first immunization ( n = 6–9 per group representing two independent experiments). nd, not detected. (B) Representative flow dot plots of heart tissue cell suspensions to identify IL-3 + cells on day 21. (C) Further flow cytometric characterization of IL-3–producing CD4 + T cells by costaining for IFN-γ, IL-17A, and IL-4 in the inflamed heart. (D) T cells were isolated by draining LNs of either WT or Il3 −/− immunized mice on day 14 and culturing with WT BMDCs in the presence or absence of the indicated peptide (10 µg/ml) for 72 h. Culture supernatants were collected, and IL-3 levels were measured by ELISA. MOG, myelin oligodendrocyte glycoprotein. (E) Schematic diagram of T cell adoptive transfer–induced EAM. (F) Quantification of total leukocyte numbers in the hearts of recipient Scid mice ( n = 6–7 per group of two independent experiments). (G and H) WT mice were lethally irradiated and reconstituted with a mixture of BM cells obtained from Rag1 −/− and either WT or Il3 −/− mice at 1:1 ratio to generate Rag1 −/− /WT and Rag1 −/− / Il3 −/− BM mixed chimeras (G). After 6–7 wk to allow for reconstitution, the mice were subjected to EAM induction, and leukocyte subsets in the heart were evaluated by flow cytometry on day 21 (H; n = 7–8 per group of two independent experiments). *, P
    Figure Legend Snippet: T cell–derived IL-3 is essential to cardiac inflammation in myocarditis. (A) Il3 mRNA levels in the heart (HT), BM, spleen (Sp), draining LN, thymus (TH), and lung (LG) before and 8, 14, and 21 d after the first immunization ( n = 6–9 per group representing two independent experiments). nd, not detected. (B) Representative flow dot plots of heart tissue cell suspensions to identify IL-3 + cells on day 21. (C) Further flow cytometric characterization of IL-3–producing CD4 + T cells by costaining for IFN-γ, IL-17A, and IL-4 in the inflamed heart. (D) T cells were isolated by draining LNs of either WT or Il3 −/− immunized mice on day 14 and culturing with WT BMDCs in the presence or absence of the indicated peptide (10 µg/ml) for 72 h. Culture supernatants were collected, and IL-3 levels were measured by ELISA. MOG, myelin oligodendrocyte glycoprotein. (E) Schematic diagram of T cell adoptive transfer–induced EAM. (F) Quantification of total leukocyte numbers in the hearts of recipient Scid mice ( n = 6–7 per group of two independent experiments). (G and H) WT mice were lethally irradiated and reconstituted with a mixture of BM cells obtained from Rag1 −/− and either WT or Il3 −/− mice at 1:1 ratio to generate Rag1 −/− /WT and Rag1 −/− / Il3 −/− BM mixed chimeras (G). After 6–7 wk to allow for reconstitution, the mice were subjected to EAM induction, and leukocyte subsets in the heart were evaluated by flow cytometry on day 21 (H; n = 7–8 per group of two independent experiments). *, P

    Techniques Used: Derivative Assay, Flow Cytometry, Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Adoptive Transfer Assay, Irradiation, Cytometry

    IL-3 is dispensable for T cell sensitization. (A) In vivo T cell proliferation in the draining LNs was measured by BrdU incorporation before and 10 and 21 d after the first immunization ( n = 4–8 per group of two independent experiments). BrdU was injected intraperitoneally 2 h before the sacrifice. (B) In vitro T cell proliferation was assessed by a cell tracer dye, Cell Trace Violet. CD4 + T cells obtained from LNs of immunized WT or Il3 −/− mice were stained with Cell Trace Violet and cultured at indicated conditions for 72 h ( n = 4–8 per group of three independent experiments). (C) Enumeration of CD4 + T cell numbers in the draining LNs before and 10 d after the first immunization ( n = 4–8 per group of two independent experiments). (D) Representative flow dot plots to identify DC subsets in the draining LNs. (E) Quantification of migratory cDCs, resident cDCs, and moDCs in WT and Il3 −/− draining LNs on days 0 and 10 ( n = 4 per group of two independent experiments). (F) Production of IFN-γ and IL-17A by WT and Il3 −/− CD4 + T cells in the draining LNs on day 21. (G) Percentage of IFN-γ + or IL-17A + CD4 + T cells in the draining LNs on day 21 ( n = 4–8 per group of two independent experiments). (H) CD4 + T cells collected from draining LNs of immunized WT or Il3 −/− mice were cultured with BMDC in the presence of 10 µg/ml αMHC for 3 d, and indicated cytokines were measured in the supernatants ( n = 4–8 per group of two independent experiments). (I) Schematic diagram of the experimental design for BMDC-induced EAM. (J) Flow cytometry–based quantification of indicated cells in the hearts of WT and Il3 −/− mice 10 d after the first BMDC injection ( n = 4–14 per group grouped from at least two independent experiments). *, P
    Figure Legend Snippet: IL-3 is dispensable for T cell sensitization. (A) In vivo T cell proliferation in the draining LNs was measured by BrdU incorporation before and 10 and 21 d after the first immunization ( n = 4–8 per group of two independent experiments). BrdU was injected intraperitoneally 2 h before the sacrifice. (B) In vitro T cell proliferation was assessed by a cell tracer dye, Cell Trace Violet. CD4 + T cells obtained from LNs of immunized WT or Il3 −/− mice were stained with Cell Trace Violet and cultured at indicated conditions for 72 h ( n = 4–8 per group of three independent experiments). (C) Enumeration of CD4 + T cell numbers in the draining LNs before and 10 d after the first immunization ( n = 4–8 per group of two independent experiments). (D) Representative flow dot plots to identify DC subsets in the draining LNs. (E) Quantification of migratory cDCs, resident cDCs, and moDCs in WT and Il3 −/− draining LNs on days 0 and 10 ( n = 4 per group of two independent experiments). (F) Production of IFN-γ and IL-17A by WT and Il3 −/− CD4 + T cells in the draining LNs on day 21. (G) Percentage of IFN-γ + or IL-17A + CD4 + T cells in the draining LNs on day 21 ( n = 4–8 per group of two independent experiments). (H) CD4 + T cells collected from draining LNs of immunized WT or Il3 −/− mice were cultured with BMDC in the presence of 10 µg/ml αMHC for 3 d, and indicated cytokines were measured in the supernatants ( n = 4–8 per group of two independent experiments). (I) Schematic diagram of the experimental design for BMDC-induced EAM. (J) Flow cytometry–based quantification of indicated cells in the hearts of WT and Il3 −/− mice 10 d after the first BMDC injection ( n = 4–14 per group grouped from at least two independent experiments). *, P

    Techniques Used: In Vivo, BrdU Incorporation Assay, Injection, In Vitro, Mouse Assay, Staining, Cell Culture, Flow Cytometry, Cytometry

    Monocyte-derived APCs promote local T cell proliferation in the inflamed heart. (A) CD4 + T cell proliferation was measured by BrdU incorporation in the inflamed hearts (HT) and blood of WT and Il3 −/− mice 21 d after the first immunization. BrdU was injected 2 h before the sacrifice. (B) Percentage of BrdU + cells in cardiac and blood (BL) CD4 + T cells of WT and Il3 −/− mice at the indicated time points ( n = 4–8 per group of two independent experiments). (C) Representative flow cytometric dot plots of activated caspase-3 expression in WT and Il3 −/− CD4 + T cells in the heart 21 d after the first immunization. (D) Percentage of activated caspase-3 + cells shown in C ( n = 4 per group of two independent experiments). (E) Quantification of IFN-γ + , IL-17A + , and GM-CSF + CD4 + T cells in the hearts on days 0 and 21 ( n = 4–8 per group of two independent experiments). (F) 5 × 10 4 of autoreactive T cells and 10 4 of each of the sorted cardiac populations were cultured at indicated conditions for 3 d. The percentage of proliferating T cells was evaluated by Cell Trace Violet dye and normalized to T cells cultured with moDCs in the presence of αMHC peptide ( n = 4–7 per group of three independent experiments). (G) T cells sorted from day 14 draining LNs were stained with Cell Trace Violet and cultured with sorted cardiac moDCs at the indicated ratio. T cell proliferation was assessed 3 d later ( n = 4 per group of two independent experiments). (H) IL-3 protein levels were measured by ELISA in the supernatant of the culture as in G ( n = 4 per group of two independent experiments). (I) Correlation between T cell proliferation and the number of moDCs or MHCII + macrophages in WT hearts at peak of inflammation. *, P
    Figure Legend Snippet: Monocyte-derived APCs promote local T cell proliferation in the inflamed heart. (A) CD4 + T cell proliferation was measured by BrdU incorporation in the inflamed hearts (HT) and blood of WT and Il3 −/− mice 21 d after the first immunization. BrdU was injected 2 h before the sacrifice. (B) Percentage of BrdU + cells in cardiac and blood (BL) CD4 + T cells of WT and Il3 −/− mice at the indicated time points ( n = 4–8 per group of two independent experiments). (C) Representative flow cytometric dot plots of activated caspase-3 expression in WT and Il3 −/− CD4 + T cells in the heart 21 d after the first immunization. (D) Percentage of activated caspase-3 + cells shown in C ( n = 4 per group of two independent experiments). (E) Quantification of IFN-γ + , IL-17A + , and GM-CSF + CD4 + T cells in the hearts on days 0 and 21 ( n = 4–8 per group of two independent experiments). (F) 5 × 10 4 of autoreactive T cells and 10 4 of each of the sorted cardiac populations were cultured at indicated conditions for 3 d. The percentage of proliferating T cells was evaluated by Cell Trace Violet dye and normalized to T cells cultured with moDCs in the presence of αMHC peptide ( n = 4–7 per group of three independent experiments). (G) T cells sorted from day 14 draining LNs were stained with Cell Trace Violet and cultured with sorted cardiac moDCs at the indicated ratio. T cell proliferation was assessed 3 d later ( n = 4 per group of two independent experiments). (H) IL-3 protein levels were measured by ELISA in the supernatant of the culture as in G ( n = 4 per group of two independent experiments). (I) Correlation between T cell proliferation and the number of moDCs or MHCII + macrophages in WT hearts at peak of inflammation. *, P

    Techniques Used: Derivative Assay, BrdU Incorporation Assay, Mouse Assay, Injection, Flow Cytometry, Expressing, Cell Culture, Staining, Enzyme-linked Immunosorbent Assay

    9) Product Images from "Early tissue responses in psoriasis to the anti-TNF-α biologic etanercept suggest reduced IL-17R expression and signalling"

    Article Title: Early tissue responses in psoriasis to the anti-TNF-α biologic etanercept suggest reduced IL-17R expression and signalling

    Journal: The British journal of dermatology

    doi: 10.1111/bjd.12937

    Enbrel treatment leads to suppression of IL-17-induced genes and its effect overlaps with that of the anti-IL-17A biologic LY2439821 (Ixekizumab)
    Figure Legend Snippet: Enbrel treatment leads to suppression of IL-17-induced genes and its effect overlaps with that of the anti-IL-17A biologic LY2439821 (Ixekizumab)

    Techniques Used:

    TNF-α induces IL-17R expression by keratinocytes and shRNA knock-down of IL17R expression abolishes synergistic TNF-α – IL-17A gene induction
    Figure Legend Snippet: TNF-α induces IL-17R expression by keratinocytes and shRNA knock-down of IL17R expression abolishes synergistic TNF-α – IL-17A gene induction

    Techniques Used: Expressing, shRNA

    10) Product Images from "Th17 cytokines differentiate obesity from obesity-associated type 2 diabetes and promote TNFα production"

    Article Title: Th17 cytokines differentiate obesity from obesity-associated type 2 diabetes and promote TNFα production

    Journal: Obesity (Silver Spring, Md.)

    doi: 10.1002/oby.21243

    IL-17 blockade decreases T cell TNFα production in T2DM (A) Percent intracellular TNFα-positive cells with surface marker expression as indicated in the absence (top panels) or presence (bottom panels) of IL-17 blocking antibody, following stimulation of PBMCs with αCD3/αCD28 (stim). Average percentage and SE of (B) CD4 + T cells (C) CD8 + T cells (D) CD11b + myeloid cells or (E) CD19 + B cells that stain for TNFα in the presence of stim +/− blocking antibodies as indicated. (F) Representative and (G) average (+/−SE) percentage of IL-17A and IL-17F staining in CD4 + T cells from panel B. “Non-stim” indicates PBMCs cultured in the absence of stimuli. “*” indicates significant differences ( p
    Figure Legend Snippet: IL-17 blockade decreases T cell TNFα production in T2DM (A) Percent intracellular TNFα-positive cells with surface marker expression as indicated in the absence (top panels) or presence (bottom panels) of IL-17 blocking antibody, following stimulation of PBMCs with αCD3/αCD28 (stim). Average percentage and SE of (B) CD4 + T cells (C) CD8 + T cells (D) CD11b + myeloid cells or (E) CD19 + B cells that stain for TNFα in the presence of stim +/− blocking antibodies as indicated. (F) Representative and (G) average (+/−SE) percentage of IL-17A and IL-17F staining in CD4 + T cells from panel B. “Non-stim” indicates PBMCs cultured in the absence of stimuli. “*” indicates significant differences ( p

    Techniques Used: Marker, Expressing, Blocking Assay, Staining, Cell Culture

    11) Product Images from "Inhibitory effects of the JAK inhibitor CP690,550 on human CD4+ T lymphocyte cytokine production"

    Article Title: Inhibitory effects of the JAK inhibitor CP690,550 on human CD4+ T lymphocyte cytokine production

    Journal: BMC Immunology

    doi: 10.1186/1471-2172-12-51

    Effects of CP690, 550 on cytokine genes expression in activated CD4 + T cells . CD4 + T cells were stimulated with CD3 monoclonal antibodies in the presence or absence of CP690,550. After 8-hr or 24-hr stimulation, total RNA was extracted and IL-2 (A), IFN-γ (B), IL-4 (C) and IL-17A (D) mRNA expression was measured by real-time PCR. * p
    Figure Legend Snippet: Effects of CP690, 550 on cytokine genes expression in activated CD4 + T cells . CD4 + T cells were stimulated with CD3 monoclonal antibodies in the presence or absence of CP690,550. After 8-hr or 24-hr stimulation, total RNA was extracted and IL-2 (A), IFN-γ (B), IL-4 (C) and IL-17A (D) mRNA expression was measured by real-time PCR. * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Effects of CP690, 550 on cytokine genes expression in Con-A activated CD4 + T cells . CD4 + T cells were stimulated with concanavalin A in the presence or absence of CP690,550. After 24-hr stimulation, total RNA was extracted and IL-2, IFN-γ, IL-4, IL-17A and IL-22 mRNA expression was measured by real-time PCR. The results show a representative result from three independent experiments.
    Figure Legend Snippet: Effects of CP690, 550 on cytokine genes expression in Con-A activated CD4 + T cells . CD4 + T cells were stimulated with concanavalin A in the presence or absence of CP690,550. After 24-hr stimulation, total RNA was extracted and IL-2, IFN-γ, IL-4, IL-17A and IL-22 mRNA expression was measured by real-time PCR. The results show a representative result from three independent experiments.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Effects of CP690, 550 activated CD4 + T cell cytokine production . CD4 + T cells were stimulated with CD3 monoclonal antibodies in the presence or absence of CP690,550 for 48 hr. Supernatants were collected and the levels of IL-2, IL-4 (A), IFN-γ (B), IL-17A (C), IL-22 (D) and IL-2 (E) were measured by ELISA. The figure shows the means ± SD of the three independent experiments performed in triplicate. * p
    Figure Legend Snippet: Effects of CP690, 550 activated CD4 + T cell cytokine production . CD4 + T cells were stimulated with CD3 monoclonal antibodies in the presence or absence of CP690,550 for 48 hr. Supernatants were collected and the levels of IL-2, IL-4 (A), IFN-γ (B), IL-17A (C), IL-22 (D) and IL-2 (E) were measured by ELISA. The figure shows the means ± SD of the three independent experiments performed in triplicate. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    12) Product Images from "Early tissue responses in psoriasis to the anti-TNF-α biologic etanercept suggest reduced IL-17R expression and signalling"

    Article Title: Early tissue responses in psoriasis to the anti-TNF-α biologic etanercept suggest reduced IL-17R expression and signalling

    Journal: The British journal of dermatology

    doi: 10.1111/bjd.12937

    Enbrel treatment leads to suppression of IL-17-induced genes and its effect overlaps with that of the anti-IL-17A biologic LY2439821 (Ixekizumab)
    Figure Legend Snippet: Enbrel treatment leads to suppression of IL-17-induced genes and its effect overlaps with that of the anti-IL-17A biologic LY2439821 (Ixekizumab)

    Techniques Used:

    TNF-α induces IL-17R expression by keratinocytes and shRNA knock-down of IL17R expression abolishes synergistic TNF-α – IL-17A gene induction
    Figure Legend Snippet: TNF-α induces IL-17R expression by keratinocytes and shRNA knock-down of IL17R expression abolishes synergistic TNF-α – IL-17A gene induction

    Techniques Used: Expressing, shRNA

    13) Product Images from "Th17/Treg homeostasis, but not Th1/Th2 homeostasis, is implicated in exacerbation of human bronchial asthma"

    Article Title: Th17/Treg homeostasis, but not Th1/Th2 homeostasis, is implicated in exacerbation of human bronchial asthma

    Journal: Therapeutics and Clinical Risk Management

    doi: 10.2147/TCRM.S172262

    The cytokine levels in plasma. Notes: ( A ) Cytokine IFN-γ, IL-4, IL-17A, IL-10, and IL-13 expression levels in the three groups. ( B ) The ratio of IL-4/IFN-γ and IL-17A/IL-10 in plasma. ( * P
    Figure Legend Snippet: The cytokine levels in plasma. Notes: ( A ) Cytokine IFN-γ, IL-4, IL-17A, IL-10, and IL-13 expression levels in the three groups. ( B ) The ratio of IL-4/IFN-γ and IL-17A/IL-10 in plasma. ( * P

    Techniques Used: Expressing

    Spearman’s correlation analysis between Th17/Treg and Th2/Th1 homeostasis in asthmatic patients. Notes: Correlation analysis between IL-17A/IL-10 and IL-4/IFN-γ ( A , r = −0.201, P = 0.278) and RORγt/Foxp3 and GATA-3/T-bet in exacerbation group ( B , r = −0.250, P = 0.173) and between IL-17A/IL-10 and IL-4/IFN-γ ( C , r = 0.876, P = 0.054) and RORγt/Foxp3 and GATA-3/T-bet ( D , r = −0.231, P = 0.338) in non-exacerbation group. P -value less than 0.05 means significant correlation. Abbreviations: Foxp3, forkhead/winged helix transcription factor 3; GATA-3, GATA binding protein 3; IFN-γ, interferon-γ; IL, interleukin; RORγt, retinoic acid–related orphan receptor gamma T; T-bet, T-box expressed in T cells.
    Figure Legend Snippet: Spearman’s correlation analysis between Th17/Treg and Th2/Th1 homeostasis in asthmatic patients. Notes: Correlation analysis between IL-17A/IL-10 and IL-4/IFN-γ ( A , r = −0.201, P = 0.278) and RORγt/Foxp3 and GATA-3/T-bet in exacerbation group ( B , r = −0.250, P = 0.173) and between IL-17A/IL-10 and IL-4/IFN-γ ( C , r = 0.876, P = 0.054) and RORγt/Foxp3 and GATA-3/T-bet ( D , r = −0.231, P = 0.338) in non-exacerbation group. P -value less than 0.05 means significant correlation. Abbreviations: Foxp3, forkhead/winged helix transcription factor 3; GATA-3, GATA binding protein 3; IFN-γ, interferon-γ; IL, interleukin; RORγt, retinoic acid–related orphan receptor gamma T; T-bet, T-box expressed in T cells.

    Techniques Used: Binding Assay

    Spearman’s correlation analysis between cytokine expression ratios and disease severity in exacerbation group. Notes: Correlation analysis between IL-17A/IL-10 and FEV 1 /FEV 1pred ( A , r = −0.769, P = 0.001) and ACT scores ( B , r = −0.487, P = 0.016) and between IL-4/IFN-γ and FEV 1 /FEV 1pred ( C , r = 0.307, P = 0.093) and ACT scores ( D , r = 0.415, P = 0.075). P -value less than 0.05 means significant correlation. Abbreviations: ACT, Asthma Control Test Questionnaire; FEV 1 , forced expiratory volume in the first second; IFN-γ, interferon-γ; IL, interleukin.
    Figure Legend Snippet: Spearman’s correlation analysis between cytokine expression ratios and disease severity in exacerbation group. Notes: Correlation analysis between IL-17A/IL-10 and FEV 1 /FEV 1pred ( A , r = −0.769, P = 0.001) and ACT scores ( B , r = −0.487, P = 0.016) and between IL-4/IFN-γ and FEV 1 /FEV 1pred ( C , r = 0.307, P = 0.093) and ACT scores ( D , r = 0.415, P = 0.075). P -value less than 0.05 means significant correlation. Abbreviations: ACT, Asthma Control Test Questionnaire; FEV 1 , forced expiratory volume in the first second; IFN-γ, interferon-γ; IL, interleukin.

    Techniques Used: Expressing, Activated Clotting Time Assay

    Spearman’s correlation analysis between cytokine expression ratios and disease severity in non-exacerbation group. Notes: Correlation analysis between IL-17A/IL-10 and FEV 1 /FEV 1pred ( A , r = −0.965, P = 0.003) and ACT scores ( B , r = −0.667, P = 0.029) and between IL-4/IFN-γ and FEV 1 /FEV 1pred ( C , r = −0.842, P = 0.004) and ACT scores ( D , r = −0.467, P = 0.044). P -value less than 0.05 means significant correlation. Abbreviations: ACT, Asthma Control Test Questionnaire; FEV 1 , forced expiratory volume in the first second; IFN-γ, interferon-γ; IL, interleukin.
    Figure Legend Snippet: Spearman’s correlation analysis between cytokine expression ratios and disease severity in non-exacerbation group. Notes: Correlation analysis between IL-17A/IL-10 and FEV 1 /FEV 1pred ( A , r = −0.965, P = 0.003) and ACT scores ( B , r = −0.667, P = 0.029) and between IL-4/IFN-γ and FEV 1 /FEV 1pred ( C , r = −0.842, P = 0.004) and ACT scores ( D , r = −0.467, P = 0.044). P -value less than 0.05 means significant correlation. Abbreviations: ACT, Asthma Control Test Questionnaire; FEV 1 , forced expiratory volume in the first second; IFN-γ, interferon-γ; IL, interleukin.

    Techniques Used: Expressing, Activated Clotting Time Assay

    14) Product Images from "Interleukin 17A Promotes Gastric Cancer Invasiveness via NF-?B Mediated Matrix Metalloproteinases 2 and 9 Expression"

    Article Title: Interleukin 17A Promotes Gastric Cancer Invasiveness via NF-?B Mediated Matrix Metalloproteinases 2 and 9 Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096678

    IL-17A promotes gastric cancer cell migration and invasion. (A) IL-17A treated GC cells (AGS, BGC-823 and SGC-7901) showed higher motility in a wound-healing assay, compared with cells without IL-17A treatment. (B) The percent migration rate is expressed as a percentage of the beginning area. (C) Effect of IL-17A on cell invasion was detected by transwell assay. Representative pictures of cells migrated through Matrigel-coated transwell were shown. (D) Total invasive cell number in each chamber was summarized as a percentage of control. Values represent the means ± SD of three independent experiments performed in triplicate. * p
    Figure Legend Snippet: IL-17A promotes gastric cancer cell migration and invasion. (A) IL-17A treated GC cells (AGS, BGC-823 and SGC-7901) showed higher motility in a wound-healing assay, compared with cells without IL-17A treatment. (B) The percent migration rate is expressed as a percentage of the beginning area. (C) Effect of IL-17A on cell invasion was detected by transwell assay. Representative pictures of cells migrated through Matrigel-coated transwell were shown. (D) Total invasive cell number in each chamber was summarized as a percentage of control. Values represent the means ± SD of three independent experiments performed in triplicate. * p

    Techniques Used: Migration, Wound Healing Assay, Transwell Assay

    IL-17A promotes the expressions and activities of MMP-2 and MMP-9 and suppresses the expressions of TIMP-1 and TIMP-2 in gastric cancer cells. (A) Expressions of MMPs in GC cells (AGS and BGC-823) were compared by western blotting between cells treated with and without IL-17A (50 ng/ml) for 24 h. (B) Quantification of the protein levels of MMP-2 and MMP-9. (C) Effects of IL-17A on the activities of MMP-2 and MMP-9. (D) Quantification of the activities of MMP-2 and MMP-9. Values represent the means ± SD of three independent experiments performed in triplicate. * p
    Figure Legend Snippet: IL-17A promotes the expressions and activities of MMP-2 and MMP-9 and suppresses the expressions of TIMP-1 and TIMP-2 in gastric cancer cells. (A) Expressions of MMPs in GC cells (AGS and BGC-823) were compared by western blotting between cells treated with and without IL-17A (50 ng/ml) for 24 h. (B) Quantification of the protein levels of MMP-2 and MMP-9. (C) Effects of IL-17A on the activities of MMP-2 and MMP-9. (D) Quantification of the activities of MMP-2 and MMP-9. Values represent the means ± SD of three independent experiments performed in triplicate. * p

    Techniques Used: Western Blot

    Effects of the NF-κB inhibitor and IL-17A on cell invasion and the expressions of MMP-2 and MMP-9 in AGS cells. (A) 1×10 6 AGS cells were pretreated with helenalin (5 µM) for 30 min and then incubated in the presence or absence of IL-17A (50 ng/ml) for 24 h. Cellular invasiveness was measured using the transwell invasion assay. (B) The percent invasion rate was expressed as a percentage of control. (C, D) AGS cells were treated and then subjected to western blotting to analyze the protein levels of MMP-2 and MMP-9. Values represent the means ± SD of three independent experiments performed in triplicate. * p
    Figure Legend Snippet: Effects of the NF-κB inhibitor and IL-17A on cell invasion and the expressions of MMP-2 and MMP-9 in AGS cells. (A) 1×10 6 AGS cells were pretreated with helenalin (5 µM) for 30 min and then incubated in the presence or absence of IL-17A (50 ng/ml) for 24 h. Cellular invasiveness was measured using the transwell invasion assay. (B) The percent invasion rate was expressed as a percentage of control. (C, D) AGS cells were treated and then subjected to western blotting to analyze the protein levels of MMP-2 and MMP-9. Values represent the means ± SD of three independent experiments performed in triplicate. * p

    Techniques Used: Incubation, Transwell Invasion Assay, Western Blot

    IL-17A activates NF-κB pathway in AGS cells. (A) Western blotting analysis was used to detect overall p50, p65, p52, c-Rel and RelB expression in AGS cells treated with IL-17A (50 ng/ml) at indicated time points. (B) Quantification of the protein levels of overall p50, p65, p52, c-Rel and RelB. (C) Western blotting analysis was used to detect nuclear p50, p65, p52, c-Rel and RelB expression in AGS cells treated with IL-17A (50 ng/mL) at indicated time points. (D) Quantification of the protein levels of nuclear p50, p65, p52, c-Rel and RelB. (E) The relative ratio of nuclear to overall fraction of p50, p65, p52, c-Rel and RelB. Values represent the means ± SD of three independent experiments performed in triplicate. ** p
    Figure Legend Snippet: IL-17A activates NF-κB pathway in AGS cells. (A) Western blotting analysis was used to detect overall p50, p65, p52, c-Rel and RelB expression in AGS cells treated with IL-17A (50 ng/ml) at indicated time points. (B) Quantification of the protein levels of overall p50, p65, p52, c-Rel and RelB. (C) Western blotting analysis was used to detect nuclear p50, p65, p52, c-Rel and RelB expression in AGS cells treated with IL-17A (50 ng/mL) at indicated time points. (D) Quantification of the protein levels of nuclear p50, p65, p52, c-Rel and RelB. (E) The relative ratio of nuclear to overall fraction of p50, p65, p52, c-Rel and RelB. Values represent the means ± SD of three independent experiments performed in triplicate. ** p

    Techniques Used: Western Blot, Expressing

    15) Product Images from "Targeting IL-17A in Multiple Myeloma: A Potential Novel Therapeutic Approach in Myeloma"

    Article Title: Targeting IL-17A in Multiple Myeloma: A Potential Novel Therapeutic Approach in Myeloma

    Journal: Leukemia

    doi: 10.1038/leu.2015.228

    Inhibition of tumor growth and prevention of bone resorption by AIN-457, IL-17A antibody in SCID human myeloma model A) SCID mice were transplanted with human fetal bones, and after four weeks, myeloma cells were injected into the bones. One group was treated with vehicle with isotype control antibody and another group of mice were treated subcutaneously with AIN-457 (10µg/ml-/mouse/injection) for four weeks following first detection of tumor by measuring human soluble IL-6R in the serum. Serum samples were collected weekly and level of humans IL-6R was measured by ELISA. Baseline values before treatment were not significantly different among groups. Representative of two experiments is shown (p
    Figure Legend Snippet: Inhibition of tumor growth and prevention of bone resorption by AIN-457, IL-17A antibody in SCID human myeloma model A) SCID mice were transplanted with human fetal bones, and after four weeks, myeloma cells were injected into the bones. One group was treated with vehicle with isotype control antibody and another group of mice were treated subcutaneously with AIN-457 (10µg/ml-/mouse/injection) for four weeks following first detection of tumor by measuring human soluble IL-6R in the serum. Serum samples were collected weekly and level of humans IL-6R was measured by ELISA. Baseline values before treatment were not significantly different among groups. Representative of two experiments is shown (p

    Techniques Used: Inhibition, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

    IL-17A knockdown decreases myeloma cell-number and their ability to produce IL-6 in a co-culture with bone-marrow stromal cells Myeloma cell-line (RPMI 8226) cells were used to transduce human IL-17A siRNA according to manufactures recommendations to determine the influence on myeloma cell-proliferation and their ability to produce IL-6 when co-cultured with bone-marrow stromal cells. Cell lysates were analyzed using immuno-blots to assess decrease in intra-cellular protein expression of IL-17A (A). Cell-proliferation was analyzed by counting live cells following transfection. Representative study results of three different experiments were shown. MM cell-line (U266) was used to transduce human IL-17A shRNA according to manufactures recommendations to determine the influence on myeloma cell-expression and –proliferation (B). Representative study results of three different experiments were shown. Colony formation was evaluated using MethoCult agar plates following transfection with siRNA or shRNA (C). Representative study results of five different experiments were shown. IL-6 production was measured with standard ELISA following co-culturing myeloma cells with or without transfected MM cells with BMSC (D). Representative study results of three different experiments were shown.
    Figure Legend Snippet: IL-17A knockdown decreases myeloma cell-number and their ability to produce IL-6 in a co-culture with bone-marrow stromal cells Myeloma cell-line (RPMI 8226) cells were used to transduce human IL-17A siRNA according to manufactures recommendations to determine the influence on myeloma cell-proliferation and their ability to produce IL-6 when co-cultured with bone-marrow stromal cells. Cell lysates were analyzed using immuno-blots to assess decrease in intra-cellular protein expression of IL-17A (A). Cell-proliferation was analyzed by counting live cells following transfection. Representative study results of three different experiments were shown. MM cell-line (U266) was used to transduce human IL-17A shRNA according to manufactures recommendations to determine the influence on myeloma cell-expression and –proliferation (B). Representative study results of three different experiments were shown. Colony formation was evaluated using MethoCult agar plates following transfection with siRNA or shRNA (C). Representative study results of five different experiments were shown. IL-6 production was measured with standard ELISA following co-culturing myeloma cells with or without transfected MM cells with BMSC (D). Representative study results of three different experiments were shown.

    Techniques Used: Co-Culture Assay, Transduction, Cell Culture, Western Blot, Expressing, Transfection, shRNA, Enzyme-linked Immunosorbent Assay

    Down regulation of BMSC-mediated MM cell-growth by anti-IL-17A antibody via blockade of IL-6 production (A) MM cell lines (N=7) were cultured with BMSCs in the presence of isotype control antibody or anti-IL-17A mAb and proliferation measured by 3 H-thymidine incorporation after 3 days and presented as percentage of inhibition in proliferation with isotype control antibody. (B) BMSC was cultured for three days in the presence or absence of IL-17A (100ng/ml), IL-21, IL-22, IL-23, IL-27 and LPS at 10ng/ml concentration. IL-6 production was measured by standard ELISA (R D Systems, Minneapolis, MN). Bar graph represents mean ± SEM of the data (N=4) calculated as percent of increase or decrease in IL-6 levels in culture supernatants compared to IL-6 levels (as endogenous IL-6 production) obtained from BMSC alone without any stimulation. (C) BMSC in the presence (right or absence (left) of MM cell-lines was cultured with isotype control antibody, IL-17A, or anti-IL-17A mAb or in combination with IL-17A + anti-IL-17A mAb and IL-6 levels were measured in culture supernatants by ELISA. Bar graph represents mean ± SEM of the data calculated as percent of increase or decrease in IL-6 levels in culture supernatants compared to IL-6 levels (as endogenous IL-6 production) obtained from BMSC alone with isotype control antibody without any stimulation. (D) Serum-starved MM cells were labeled with calcein AM, washed, and added to BMSC-coated plates in the presence of isotype control antibody or anti-IL-17A mAb for 4 hours and non-adherent cells were removed by washing. Adhesion was measured by measuring the absorbance using 492/520 nm filter set with a fluorescence plate reader. Results represent mean ± SEM of 3 independent experiments performed in triplicate. The absorbance values obtained with isotype control antibody was considered as 100% and percentage of inhibition was calculated for anti-IL-17A treatment. (E) The fetal bone chips with MM cells were incubated in the presence of isotype control antibody or anti-IL-17A mAb for 2 days for ex-vivo for the evaluation of IL-6 production that is measured by ELISA (R D Systems). Bar graph represents mean ± SEM of the data calculated as percent of IL-6 levels (as endogenous IL-6 production) in culture supernatants from BMSC alone with isotype control antibody. (F) The immunohistochemical analysis of bones was performed by staining them with anti-CD138 antibody. Arrows indicates presence bright CD138+cells. (G) Myeloma cells were co-cultured with BMSC in the presence of IL-17A with or without cell-signaling inhibitors (JAK2, STAT3, JNK, MEK, NFkB and PI3 inhibitors) and IL-6 production was measured with standard ELISA. *P
    Figure Legend Snippet: Down regulation of BMSC-mediated MM cell-growth by anti-IL-17A antibody via blockade of IL-6 production (A) MM cell lines (N=7) were cultured with BMSCs in the presence of isotype control antibody or anti-IL-17A mAb and proliferation measured by 3 H-thymidine incorporation after 3 days and presented as percentage of inhibition in proliferation with isotype control antibody. (B) BMSC was cultured for three days in the presence or absence of IL-17A (100ng/ml), IL-21, IL-22, IL-23, IL-27 and LPS at 10ng/ml concentration. IL-6 production was measured by standard ELISA (R D Systems, Minneapolis, MN). Bar graph represents mean ± SEM of the data (N=4) calculated as percent of increase or decrease in IL-6 levels in culture supernatants compared to IL-6 levels (as endogenous IL-6 production) obtained from BMSC alone without any stimulation. (C) BMSC in the presence (right or absence (left) of MM cell-lines was cultured with isotype control antibody, IL-17A, or anti-IL-17A mAb or in combination with IL-17A + anti-IL-17A mAb and IL-6 levels were measured in culture supernatants by ELISA. Bar graph represents mean ± SEM of the data calculated as percent of increase or decrease in IL-6 levels in culture supernatants compared to IL-6 levels (as endogenous IL-6 production) obtained from BMSC alone with isotype control antibody without any stimulation. (D) Serum-starved MM cells were labeled with calcein AM, washed, and added to BMSC-coated plates in the presence of isotype control antibody or anti-IL-17A mAb for 4 hours and non-adherent cells were removed by washing. Adhesion was measured by measuring the absorbance using 492/520 nm filter set with a fluorescence plate reader. Results represent mean ± SEM of 3 independent experiments performed in triplicate. The absorbance values obtained with isotype control antibody was considered as 100% and percentage of inhibition was calculated for anti-IL-17A treatment. (E) The fetal bone chips with MM cells were incubated in the presence of isotype control antibody or anti-IL-17A mAb for 2 days for ex-vivo for the evaluation of IL-6 production that is measured by ELISA (R D Systems). Bar graph represents mean ± SEM of the data calculated as percent of IL-6 levels (as endogenous IL-6 production) in culture supernatants from BMSC alone with isotype control antibody. (F) The immunohistochemical analysis of bones was performed by staining them with anti-CD138 antibody. Arrows indicates presence bright CD138+cells. (G) Myeloma cells were co-cultured with BMSC in the presence of IL-17A with or without cell-signaling inhibitors (JAK2, STAT3, JNK, MEK, NFkB and PI3 inhibitors) and IL-6 production was measured with standard ELISA. *P

    Techniques Used: Cell Culture, Inhibition, Concentration Assay, Enzyme-linked Immunosorbent Assay, Labeling, Fluorescence, Incubation, Ex Vivo, Immunohistochemistry, Staining

    Reduced osteoclast cell numbers by the antibody Normal BM cells were cultured for three weeks in osteoclast supporting-medium (consisting 25ng/ml of macrophage colony-stimulating factor and 25 ng/ml of receptor activator of nuclear factor kappa-B ligand) with isotype control antibody (as positive control) or anti-IL-17A antibody and the tartrate-resistant acid phosphatage (TRAP+) multinucleated osteoclast cells were stained and cell-numbers were counted. Cells were cultured without osteoclast-supporting-medium as negative control. Images were obtained at 10 magnifications with microscope (Eclipse TS100, Nikon instruments, Melville, NY, USA) with spot insight camera. TRAP-positive cells containing 5 or more nuclei/cell were enumerated using image J 1.45 software (NIH, Bethesda, MD, USA). A representative image was depicted (Figure A) and composite results from three experiments were shown in bar graph Figure B). Star indicates statistical signifies (p
    Figure Legend Snippet: Reduced osteoclast cell numbers by the antibody Normal BM cells were cultured for three weeks in osteoclast supporting-medium (consisting 25ng/ml of macrophage colony-stimulating factor and 25 ng/ml of receptor activator of nuclear factor kappa-B ligand) with isotype control antibody (as positive control) or anti-IL-17A antibody and the tartrate-resistant acid phosphatage (TRAP+) multinucleated osteoclast cells were stained and cell-numbers were counted. Cells were cultured without osteoclast-supporting-medium as negative control. Images were obtained at 10 magnifications with microscope (Eclipse TS100, Nikon instruments, Melville, NY, USA) with spot insight camera. TRAP-positive cells containing 5 or more nuclei/cell were enumerated using image J 1.45 software (NIH, Bethesda, MD, USA). A representative image was depicted (Figure A) and composite results from three experiments were shown in bar graph Figure B). Star indicates statistical signifies (p

    Techniques Used: Cell Culture, Positive Control, Staining, Negative Control, Microscopy, Software

    Effect of AIN-457 humanized antibody on myeloma growth using three mouse models without tumor microenvironment (A) SCID mice were injected with myeloma cells by sc route. Mice were injected in 2 groups (N=3). One group was injected with myeloma cells with isotype control antibody; and second of mice injected with MM cells in anti-IL-17A mAb (10µg/ml). Tumor volume was measured at indicated time intervals from the MM cell-injections. Representative of two experiments is shown (p
    Figure Legend Snippet: Effect of AIN-457 humanized antibody on myeloma growth using three mouse models without tumor microenvironment (A) SCID mice were injected with myeloma cells by sc route. Mice were injected in 2 groups (N=3). One group was injected with myeloma cells with isotype control antibody; and second of mice injected with MM cells in anti-IL-17A mAb (10µg/ml). Tumor volume was measured at indicated time intervals from the MM cell-injections. Representative of two experiments is shown (p

    Techniques Used: Mouse Assay, Injection

    Analysis of IL-17A expression in myeloma cells The MM patient samples were collected after informed consent in accordance with the Declaration of Helsinki and approved by the institutional review board (IRB) from Dana-Farber Cancer Institute. Healthy donor bone marrow samples were obtained from AllCells (Emeryville, CA). MM primary cells were purified as described earlier5. RNA was isolated from MM cell-lines (A) and purified MM primary cells (B). Quantitative PCR was performed for IL-17A using 7900HT from Applied Biosystems. Representative experimental results from three different experiments were presented as relative expression value in comparison with GAPDH. For immuno-blot experiments, total cell lysates were prepared from MM cell-lines (C) and purified CD138+ MM primary cells (D), and separated by electrophoresis on 5% to 20% polyacrylamide gradient gels. Samples were probed with anti-sera to IL-17A and GAPDH as indicated. Representative immunoblot of three different experiments was shown. Myeloma cell lines were stained with isotype control antibody or anti–IL-17A antibody and analyzed by confocal microscopy. One representative cell line of 4 experiments was shown at 640 magnifications (E).
    Figure Legend Snippet: Analysis of IL-17A expression in myeloma cells The MM patient samples were collected after informed consent in accordance with the Declaration of Helsinki and approved by the institutional review board (IRB) from Dana-Farber Cancer Institute. Healthy donor bone marrow samples were obtained from AllCells (Emeryville, CA). MM primary cells were purified as described earlier5. RNA was isolated from MM cell-lines (A) and purified MM primary cells (B). Quantitative PCR was performed for IL-17A using 7900HT from Applied Biosystems. Representative experimental results from three different experiments were presented as relative expression value in comparison with GAPDH. For immuno-blot experiments, total cell lysates were prepared from MM cell-lines (C) and purified CD138+ MM primary cells (D), and separated by electrophoresis on 5% to 20% polyacrylamide gradient gels. Samples were probed with anti-sera to IL-17A and GAPDH as indicated. Representative immunoblot of three different experiments was shown. Myeloma cell lines were stained with isotype control antibody or anti–IL-17A antibody and analyzed by confocal microscopy. One representative cell line of 4 experiments was shown at 640 magnifications (E).

    Techniques Used: Expressing, Purification, Isolation, Real-time Polymerase Chain Reaction, Electrophoresis, Staining, Confocal Microscopy

    Inhibitory activity of anti-IL-17A antibody on myeloma cell proliferation (A) Myeloma cell lines alone (N=8) were incubated with isotype control antibody or anti-IL-17A mAb to measure proliferation by 3 H-thymidine incorporation after 3 days. Data is presented as percentage inhibition in proliferation in presence of anti-IL-17A mAb compared with isotype control antibody and showed as mean ± SEM. (B) Myeloma cell lines alone (N=5) were incubated with isotype control antibody or anti-IL-17A mAb to measure metabolic activity by MTT assay after 3 days. Data is presented as percentage inhibition in proliferation in presence of anti-IL-17A mAb compared with isotype control antibody and showed as mean ± SEM. (C) Myeloma cell lines (U266) were cultured in methocult agar plates in the presence of isotype control antibody or anti-IL-17A mAb. Representative photomicrograph and results (N=3) are presented. Photographs were obtained using a Nikon TE200 microscope (40× objective) with attached camera (Nikon) at room temperature (total magnification 200) and analyzed with Metafluor software (Molecular Devices). The number of colonies were counted in unit area and presented as mean ± SEM. (D) Primary MM cells (N=4) were cultured in methocult agar plates in the presence of isotype control antibody or anti-IL-17A mAb. The number of colonies were counted in unit area and presented as mean ± SEM. *P
    Figure Legend Snippet: Inhibitory activity of anti-IL-17A antibody on myeloma cell proliferation (A) Myeloma cell lines alone (N=8) were incubated with isotype control antibody or anti-IL-17A mAb to measure proliferation by 3 H-thymidine incorporation after 3 days. Data is presented as percentage inhibition in proliferation in presence of anti-IL-17A mAb compared with isotype control antibody and showed as mean ± SEM. (B) Myeloma cell lines alone (N=5) were incubated with isotype control antibody or anti-IL-17A mAb to measure metabolic activity by MTT assay after 3 days. Data is presented as percentage inhibition in proliferation in presence of anti-IL-17A mAb compared with isotype control antibody and showed as mean ± SEM. (C) Myeloma cell lines (U266) were cultured in methocult agar plates in the presence of isotype control antibody or anti-IL-17A mAb. Representative photomicrograph and results (N=3) are presented. Photographs were obtained using a Nikon TE200 microscope (40× objective) with attached camera (Nikon) at room temperature (total magnification 200) and analyzed with Metafluor software (Molecular Devices). The number of colonies were counted in unit area and presented as mean ± SEM. (D) Primary MM cells (N=4) were cultured in methocult agar plates in the presence of isotype control antibody or anti-IL-17A mAb. The number of colonies were counted in unit area and presented as mean ± SEM. *P

    Techniques Used: Activity Assay, Incubation, Inhibition, MTT Assay, Cell Culture, Microscopy, Software

    16) Product Images from "Blockade of Interplay between IL-17A and Endoplasmic Reticulum Stress Attenuates LPS-Induced Lung Injury"

    Article Title: Blockade of Interplay between IL-17A and Endoplasmic Reticulum Stress Attenuates LPS-Induced Lung Injury

    Journal: Theranostics

    doi: 10.7150/thno.11685

    Schematic diagram for the role of the interplay between ER stress and IL-17A in LPS-induced lung injury.
    Figure Legend Snippet: Schematic diagram for the role of the interplay between ER stress and IL-17A in LPS-induced lung injury.

    Techniques Used:

    Levels of IL-17A, GRP78, and CHOP in lung tissues from LPS-instilled mice and LPS-stimulated NHBE cells. Representative immunoblots of IL-17A (A) , GRP78 (C) , and CHOP (E) in lung tissues from SV, LV, and LPS-instilled mice given intravenous injections of TAK-242 of 200 μg/mouse (LT) and densitometric analysis of IL-17A (B) , GRP78 (D) , and CHOP (F) . Sampling was performed at 48 hours after the instillation of LPS. Bars represent mean ± SEM from 5 mice/group. # P
    Figure Legend Snippet: Levels of IL-17A, GRP78, and CHOP in lung tissues from LPS-instilled mice and LPS-stimulated NHBE cells. Representative immunoblots of IL-17A (A) , GRP78 (C) , and CHOP (E) in lung tissues from SV, LV, and LPS-instilled mice given intravenous injections of TAK-242 of 200 μg/mouse (LT) and densitometric analysis of IL-17A (B) , GRP78 (D) , and CHOP (F) . Sampling was performed at 48 hours after the instillation of LPS. Bars represent mean ± SEM from 5 mice/group. # P

    Techniques Used: Mouse Assay, Western Blot, Sampling

    Effects of anti-IL-17A antibody and 4-PBA on the expression of TLR4 and NF-κB in airway epithelial cells, macrophages, neutrophils, and dendritic cells from LPS-instilled mice. (A) Representative histogram of the expression of TLR4 in various cells of lung from LV-instilled mice. (B-E) Fluorescence intensity of TLR4 in airway epithelial cells (B) , macrophage (C) , neutrophils (D) , and dendritic cells (E) is presented as the ratio of the levels of TLR4 in each group relative to those in SV mice. (F) Representative histogram of the expression of NF-κB in nuclear extracts of various cells of the lung from LV-instilled mice. (G-J) Fluorescence intensity of NF-κB in nuclear extracts of airway epithelial cells (G) , macrophage (H) , neutrophils (I) , and dendritic cells (J) is presented as the ratio of the levels of NF-κB in each group relative to those in SV mice. Sampling was performed at 48 hours after the instillation of LPS. Bars represent mean ± SEM from 5 mice/group. # P
    Figure Legend Snippet: Effects of anti-IL-17A antibody and 4-PBA on the expression of TLR4 and NF-κB in airway epithelial cells, macrophages, neutrophils, and dendritic cells from LPS-instilled mice. (A) Representative histogram of the expression of TLR4 in various cells of lung from LV-instilled mice. (B-E) Fluorescence intensity of TLR4 in airway epithelial cells (B) , macrophage (C) , neutrophils (D) , and dendritic cells (E) is presented as the ratio of the levels of TLR4 in each group relative to those in SV mice. (F) Representative histogram of the expression of NF-κB in nuclear extracts of various cells of the lung from LV-instilled mice. (G-J) Fluorescence intensity of NF-κB in nuclear extracts of airway epithelial cells (G) , macrophage (H) , neutrophils (I) , and dendritic cells (J) is presented as the ratio of the levels of NF-κB in each group relative to those in SV mice. Sampling was performed at 48 hours after the instillation of LPS. Bars represent mean ± SEM from 5 mice/group. # P

    Techniques Used: Expressing, Mouse Assay, Fluorescence, Sampling

    Levels of pro-inflammatory mediators and time-kinetics of IL-17A in lung tissues of LPS-instilled mice. Representative immunoblots of IL-4 (A) , ICAM-1 (C) , VEGF (E), IL-17 (G) and KC (I) in lung tissues and densitometric analysis of IL-4 (B) , ICAM-1 (D) , VEGF (F) , IL-17 (H) and KC (J) . Sampling was performed at 48 hours after the instillation of LPS. (K and L) Kinetics of IL-17A in lung tissues over time; One, 6, 12, 24, 48, and 72 hours are the time periods of the sampling after the instillation of LPS. Pre, 1 hour before the instillation of LPS. Bars represent mean ± SEM from 5 or 6 mice/group. # P
    Figure Legend Snippet: Levels of pro-inflammatory mediators and time-kinetics of IL-17A in lung tissues of LPS-instilled mice. Representative immunoblots of IL-4 (A) , ICAM-1 (C) , VEGF (E), IL-17 (G) and KC (I) in lung tissues and densitometric analysis of IL-4 (B) , ICAM-1 (D) , VEGF (F) , IL-17 (H) and KC (J) . Sampling was performed at 48 hours after the instillation of LPS. (K and L) Kinetics of IL-17A in lung tissues over time; One, 6, 12, 24, 48, and 72 hours are the time periods of the sampling after the instillation of LPS. Pre, 1 hour before the instillation of LPS. Bars represent mean ± SEM from 5 or 6 mice/group. # P

    Techniques Used: Mouse Assay, Western Blot, Sampling

    Effect of anti-IL-17A antibody or 4-PBA on levels of TLR4 and infiltration of CD11b + CD11c + MHC II + cells in lung tissues of LPS-instilled mice. (A and B) Representative immunoblot of TLR4 in lung tissues (A) and densitometric analysis of TLR4 (B) . (C) Dot plot analysis of CD11b + CD11c + MHC II + cells that infiltrated lung tissues in SV, LV, LP, LIL17-Ab, and LCON-Ab mice. (D and E) Numbers of infiltrated DCs. (F) Mean fluorescence intensity for MHCII + cells. Sampling was performed at 48 hours after the instillation of LPS. Bars represent mean±SEM from 6 mice/group. # P
    Figure Legend Snippet: Effect of anti-IL-17A antibody or 4-PBA on levels of TLR4 and infiltration of CD11b + CD11c + MHC II + cells in lung tissues of LPS-instilled mice. (A and B) Representative immunoblot of TLR4 in lung tissues (A) and densitometric analysis of TLR4 (B) . (C) Dot plot analysis of CD11b + CD11c + MHC II + cells that infiltrated lung tissues in SV, LV, LP, LIL17-Ab, and LCON-Ab mice. (D and E) Numbers of infiltrated DCs. (F) Mean fluorescence intensity for MHCII + cells. Sampling was performed at 48 hours after the instillation of LPS. Bars represent mean±SEM from 6 mice/group. # P

    Techniques Used: Mouse Assay, Fluorescence, Sampling

    Effects of anti-IL-17A antibody or 4-PBA on ER stress markers and UPR-related proteins. (A and B) Representative confocal laser immunofluorescence photomicrographs of lung tissues (A) and BAL cells (B) from saline-instilled mice given injections of vehicle (SV), LPS-instilled mice given injections of vehicle (LV), LPS-instilled mice given intraperitoneal injections of 4-PBA of 200 mg/kg (LP), LPS-instilled mice given intravenous injections of anti-IL-17 antibody of 5 mg/kg (LIL17-Ab), or LPS-instilled mice given intravenous injections of isotype control monoclonal antibody (LCON-Ab). Sampling was performed at 48 hours after the instillation of LPS. Bars indicate 50 μm. DIC means 'Differential interference contrast'. (C and D) Representative RT-PCR and semi-quantative analyses for mRNA of GRP78 and CHOP. (E-N) Representative immunoblots of GRP78 (E) , CHOP (E) , XBP-1 (G) , ATF-4 (I) , ATF-6 (K) and p-eIF2α (M) in lung tissues and densitometric analyses of GRP78 (F) , CHOP (F) , XBP-1 (H) , ATF-4 (J) , ATF-6 (L) , and p-eIF2α (M) . Bars represent mean ± SEM from 5 or 6 mice/group. # P
    Figure Legend Snippet: Effects of anti-IL-17A antibody or 4-PBA on ER stress markers and UPR-related proteins. (A and B) Representative confocal laser immunofluorescence photomicrographs of lung tissues (A) and BAL cells (B) from saline-instilled mice given injections of vehicle (SV), LPS-instilled mice given injections of vehicle (LV), LPS-instilled mice given intraperitoneal injections of 4-PBA of 200 mg/kg (LP), LPS-instilled mice given intravenous injections of anti-IL-17 antibody of 5 mg/kg (LIL17-Ab), or LPS-instilled mice given intravenous injections of isotype control monoclonal antibody (LCON-Ab). Sampling was performed at 48 hours after the instillation of LPS. Bars indicate 50 μm. DIC means 'Differential interference contrast'. (C and D) Representative RT-PCR and semi-quantative analyses for mRNA of GRP78 and CHOP. (E-N) Representative immunoblots of GRP78 (E) , CHOP (E) , XBP-1 (G) , ATF-4 (I) , ATF-6 (K) and p-eIF2α (M) in lung tissues and densitometric analyses of GRP78 (F) , CHOP (F) , XBP-1 (H) , ATF-4 (J) , ATF-6 (L) , and p-eIF2α (M) . Bars represent mean ± SEM from 5 or 6 mice/group. # P

    Techniques Used: Immunofluorescence, Mouse Assay, Sampling, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Levels of IL-17A, GRP78 and NF-κB p65 in LPS-stimulated NHBE cells. (A) Representative confocal laser immunofluorescence photomicrographs of NHBE cells with no stimulation (Control), LPS stimulation (LPS), pre-treatment of vehicle and LPS stimulation (LV), pre-treatment with 10 mmol/L 4-PBA for 1 hour and LPS stimulation (100 μg/ml) (LP), pre-treatment with 2 μg/ml of anti-IL-17 antibody and LPS stimulation (100 μg/ml) (LIL17-Ab), or pre-treatment with 2 μg/ml of isotype control monoclonal antibody and LPS stimulation (100 μg/ml) (LCON-Ab). Bars indicate 20 μm. (B and C) Representative RT-PCR of IL-17A mRNA and semi-quantitative analyses of IL-17A mRNA. (D and E) Representative immunoblots of GRP78 in NHBE cells and densitometric analyses of GRP78. (F-I) Representative immunoblots of NF-κB p65 in NHBE cells and densitometric analyses of NF-κB p65. Data are from 3 independent experiments. NHBE cells were stimulated with 100 μg/ml of LPS and/or 100 ng/ml of IL-17A. Pre-treatment with 10 mmol/L 4-PBA or 2 μg/ml of a human anti-IL-17A antibody (IL-17Ab) or an isotype control monoclonal antibody (CON-Ab) was performed at 1 hour before LPS stimulation. Cells were harvested at 12 hours after LPS stimulation. # P
    Figure Legend Snippet: Levels of IL-17A, GRP78 and NF-κB p65 in LPS-stimulated NHBE cells. (A) Representative confocal laser immunofluorescence photomicrographs of NHBE cells with no stimulation (Control), LPS stimulation (LPS), pre-treatment of vehicle and LPS stimulation (LV), pre-treatment with 10 mmol/L 4-PBA for 1 hour and LPS stimulation (100 μg/ml) (LP), pre-treatment with 2 μg/ml of anti-IL-17 antibody and LPS stimulation (100 μg/ml) (LIL17-Ab), or pre-treatment with 2 μg/ml of isotype control monoclonal antibody and LPS stimulation (100 μg/ml) (LCON-Ab). Bars indicate 20 μm. (B and C) Representative RT-PCR of IL-17A mRNA and semi-quantitative analyses of IL-17A mRNA. (D and E) Representative immunoblots of GRP78 in NHBE cells and densitometric analyses of GRP78. (F-I) Representative immunoblots of NF-κB p65 in NHBE cells and densitometric analyses of NF-κB p65. Data are from 3 independent experiments. NHBE cells were stimulated with 100 μg/ml of LPS and/or 100 ng/ml of IL-17A. Pre-treatment with 10 mmol/L 4-PBA or 2 μg/ml of a human anti-IL-17A antibody (IL-17Ab) or an isotype control monoclonal antibody (CON-Ab) was performed at 1 hour before LPS stimulation. Cells were harvested at 12 hours after LPS stimulation. # P

    Techniques Used: Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Western Blot

    17) Product Images from "Linker Length Matters, Fynomer-Fc Fusion with an Optimized Linker Displaying Picomolar IL-17A Inhibition Potency *"

    Article Title: Linker Length Matters, Fynomer-Fc Fusion with an Optimized Linker Displaying Picomolar IL-17A Inhibition Potency *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.534578

    IL-17A Inhibition Cell Assays
    Figure Legend Snippet: IL-17A Inhibition Cell Assays

    Techniques Used: Inhibition

    IL-17A inhibition in vivo by 2C1 L3 Fc. Intravenously injected 2C1 L3 Fc completely inhibited IL-17A-induced KC up-regulation in mice. Mean KC levels of five mice per group are shown (± S.D.). ***, p
    Figure Legend Snippet: IL-17A inhibition in vivo by 2C1 L3 Fc. Intravenously injected 2C1 L3 Fc completely inhibited IL-17A-induced KC up-regulation in mice. Mean KC levels of five mice per group are shown (± S.D.). ***, p

    Techniques Used: Inhibition, In Vivo, Injection, Mouse Assay

    The Fynomer 2C1 exhibits excellent biophysical properties and a high affinity and specificity to its target protein IL-17A. A , the monomeric Fynomer was expressed in E. coli and purified via a His 6 tag affinity chromatography. The resulting protein was 95% pure and monomeric ( M ), as determined by SEC and SDS-PAGE ( B ). C , real-time interaction analysis on a BIAcore chip coated with IL-17A was used to determine the dissociation constant ( K D ). Different concentrations of Fynomer 2C1 (highest concentration, 100 n m and subsequent 3-fold dilution series) were injected revealing a K D value of 1.8 n m at the antigen surface density used. D , to investigate binding specificity of the monomeric Fynomer 2C1, indicated proteins were coated onto an ELISA plate. 2C1 bound human and cynomolgus ( cyno ) IL-17A in a specific manner and did not bind to any of the other IL-17 family members or IL-17A cytokines from other species nor to unrelated proteins. Binding to IL17-C was determined in a separate experiment, indicated by the dashed line. neg ctrl , negative control; EDB , extradomain B of fibronectin. E , 2C1 specifically inhibited IL-17A in a dose-dependent manner. As a positive control for IL-17A inhibition, commercially available IL-17R-Fc fusion was used.
    Figure Legend Snippet: The Fynomer 2C1 exhibits excellent biophysical properties and a high affinity and specificity to its target protein IL-17A. A , the monomeric Fynomer was expressed in E. coli and purified via a His 6 tag affinity chromatography. The resulting protein was 95% pure and monomeric ( M ), as determined by SEC and SDS-PAGE ( B ). C , real-time interaction analysis on a BIAcore chip coated with IL-17A was used to determine the dissociation constant ( K D ). Different concentrations of Fynomer 2C1 (highest concentration, 100 n m and subsequent 3-fold dilution series) were injected revealing a K D value of 1.8 n m at the antigen surface density used. D , to investigate binding specificity of the monomeric Fynomer 2C1, indicated proteins were coated onto an ELISA plate. 2C1 bound human and cynomolgus ( cyno ) IL-17A in a specific manner and did not bind to any of the other IL-17 family members or IL-17A cytokines from other species nor to unrelated proteins. Binding to IL17-C was determined in a separate experiment, indicated by the dashed line. neg ctrl , negative control; EDB , extradomain B of fibronectin. E , 2C1 specifically inhibited IL-17A in a dose-dependent manner. As a positive control for IL-17A inhibition, commercially available IL-17R-Fc fusion was used.

    Techniques Used: Purification, Affinity Chromatography, Size-exclusion Chromatography, SDS Page, Chromatin Immunoprecipitation, Concentration Assay, Injection, Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Positive Control, Inhibition

    2C1-Fc fusion molecules show linker-length dependent IL-17A inhibition in vitro . 2C1 was genetically fused to the Fc-part of a human IgG1 using four linkers of variable length (L0–L3, see Fig. 1 C ). The Fc-fusion molecules were tested in a cell assay after stimulation with IL-17A and TNF. The Fynomer-Fc fusion proteins with longer and more flexible linkers, 2C1 L2 Fc and 2C1 L3 Fc, showed most potent inhibition of IL-17A. log conc , log concentration.
    Figure Legend Snippet: 2C1-Fc fusion molecules show linker-length dependent IL-17A inhibition in vitro . 2C1 was genetically fused to the Fc-part of a human IgG1 using four linkers of variable length (L0–L3, see Fig. 1 C ). The Fc-fusion molecules were tested in a cell assay after stimulation with IL-17A and TNF. The Fynomer-Fc fusion proteins with longer and more flexible linkers, 2C1 L2 Fc and 2C1 L3 Fc, showed most potent inhibition of IL-17A. log conc , log concentration.

    Techniques Used: Inhibition, In Vitro, Concentration Assay

    18) 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

    19) 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

    20) Product Images from "Identification of Protective Pneumococcal TH17 Antigens from the Soluble Fraction of a Killed Whole Cell Vaccine"

    Article Title: Identification of Protective Pneumococcal TH17 Antigens from the Soluble Fraction of a Killed Whole Cell Vaccine

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043445

    Size separation of fractions and stimulation of splenocytes. A. SDS-PAGE of fractions was performed and gel was silver-stained; shown here are the electrophoretic patterns of three fractions eluted into the same chamber during different transverse elutions. B. Results from stimulation of splenocytes from WCC immunized mice (n = 6) with equal concentrations of each fraction. Supernatants were collected after 3 days of incubation and IL-17A concentration in the supernatant was measured by ELISA. IL-17A values are shown here, normalized to the DMEM-stimulated response of each animal. Bars represent medians with interquartile range. WCC: chloroform-inactivated pneumococcal whole cell antigen, 10 µg protein/ml; WCCsup: soluble fraction of WCC, 7 µg protein/ml.
    Figure Legend Snippet: Size separation of fractions and stimulation of splenocytes. A. SDS-PAGE of fractions was performed and gel was silver-stained; shown here are the electrophoretic patterns of three fractions eluted into the same chamber during different transverse elutions. B. Results from stimulation of splenocytes from WCC immunized mice (n = 6) with equal concentrations of each fraction. Supernatants were collected after 3 days of incubation and IL-17A concentration in the supernatant was measured by ELISA. IL-17A values are shown here, normalized to the DMEM-stimulated response of each animal. Bars represent medians with interquartile range. WCC: chloroform-inactivated pneumococcal whole cell antigen, 10 µg protein/ml; WCCsup: soluble fraction of WCC, 7 µg protein/ml.

    Techniques Used: SDS Page, Staining, Mouse Assay, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Stimulation of splenocytes from immune mice with purified recombinant proteins. Mice (n = 7–10) were intranasally immunized with WCC and cholera toxin as described. Splenocytes from immunized mice were stimulated with 10 μg/ml of the indicated recombinant protein for 3 days, after which supernatants were harvested and assayed for IL-17A concentration. Values are normalized to the DMEM stimulated response for each animal. Bars represent medians with interquartile ranges.
    Figure Legend Snippet: Stimulation of splenocytes from immune mice with purified recombinant proteins. Mice (n = 7–10) were intranasally immunized with WCC and cholera toxin as described. Splenocytes from immunized mice were stimulated with 10 μg/ml of the indicated recombinant protein for 3 days, after which supernatants were harvested and assayed for IL-17A concentration. Values are normalized to the DMEM stimulated response for each animal. Bars represent medians with interquartile ranges.

    Techniques Used: Mouse Assay, Purification, Recombinant, Concentration Assay

    Protection against colonization by intranasal immunization with a mixture of proteins. Mice were intranasally immunized with a mixture (Protein mix) containing 4 μg of SP0435, SP1534, and SP2070 and 1 μg of cholera toxin (CT) as adjuvant; control mice received CT alone or WCC with CT (WCV). Blood was obtained 3 weeks after last immunization and mice were challenged one week later with strain 0603. Density of colonization was determined one week after challenge by quantifying pneumococcal carriage from nasal washes. (A) IL-17A production was determined in vitro from whole blood stimulated with pneumococcal whole-cell antigen. (B) Mice immunized with the WCV or the protein mixture were significantly protected against colonization compared to mice that received CT alone. Bars indicate median values and P values were determined by Mann-Whitney U test compared to mice immunized with CT alone.
    Figure Legend Snippet: Protection against colonization by intranasal immunization with a mixture of proteins. Mice were intranasally immunized with a mixture (Protein mix) containing 4 μg of SP0435, SP1534, and SP2070 and 1 μg of cholera toxin (CT) as adjuvant; control mice received CT alone or WCC with CT (WCV). Blood was obtained 3 weeks after last immunization and mice were challenged one week later with strain 0603. Density of colonization was determined one week after challenge by quantifying pneumococcal carriage from nasal washes. (A) IL-17A production was determined in vitro from whole blood stimulated with pneumococcal whole-cell antigen. (B) Mice immunized with the WCV or the protein mixture were significantly protected against colonization compared to mice that received CT alone. Bars indicate median values and P values were determined by Mann-Whitney U test compared to mice immunized with CT alone.

    Techniques Used: Mouse Assay, In Vitro, MANN-WHITNEY

    21) 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

    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

    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 repres ent 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 repres ent 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

    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

    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

    22) Product Images from "Effects of 1,25(OH)2D3 in immune response regulation of systemic lupus erithematosus (SLE) patient with hypovitamin D"

    Article Title: Effects of 1,25(OH)2D3 in immune response regulation of systemic lupus erithematosus (SLE) patient with hypovitamin D

    Journal: International Journal of Clinical and Experimental Medicine

    doi:

    The difference of IL-17A secretion level in treatment groups of 1,25(OH) 2 D 3 in CD4 T cells. There was a decrease in IL-17A level in groups of P1, P2, dan P3 compared with P0 (control). Group P2 was the lowest (p=0.047). *Significant (p
    Figure Legend Snippet: The difference of IL-17A secretion level in treatment groups of 1,25(OH) 2 D 3 in CD4 T cells. There was a decrease in IL-17A level in groups of P1, P2, dan P3 compared with P0 (control). Group P2 was the lowest (p=0.047). *Significant (p

    Techniques Used:

    23) 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

    24) 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

    25) Product Images from "Lung epithelial cells coordinate innate lymphocytes and immunity against pulmonary fungal infection"

    Article Title: Lung epithelial cells coordinate innate lymphocytes and immunity against pulmonary fungal infection

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2018.02.011

    Collaborative killing of yeast by alveolar MØ, DCs, and neutrophils is dependent on LEC NFκB, IL-17A and GM-CSF (A–B) IKK2 fl/fl and IKK2 ΔLEC mice were infected i.t. with DsRed yeast stained with Uvitex. The proportion and number of live (red) and dead (blue) yeast among total yeasts (red+blue) were quantified by flow cytometry. Two pooled experiments depicted. (C) Proportion of yeast-associated (uvitex + ) alveolar MØ (CD11c + Siglec F + ), DCs (Siglec F − CD11c + MHCII + ) and neutrophils (Siglec F − CD11b + Ly6G + ) that were DsRed- (associated with dead yeast). Two pooled experiments depicted. (D) WT mice were infected i.t. with DsRed yeast and the proportion of dead yeasts was calculated by flow cytometry. IL-17A and GM-CSF were neutralized as noted in Methods. Data representative of 3 experiments. One-way ANOVA with Tukey’s multiple comparison. (E) IKK2 fl/fl and IKK2 ΔLEC were infected with DsRed yeast. rIL-17A and rGM-CSF were given i.t. together with the inoculum. 48 h.p.i lung cell suspensions were analyzed by flow cytometry to quantify the proportion of dead yeast (DeRed − Uvitex + ) in total lung homogenate, and proportoin of phagocytes associated with dead yeast. One-Way ANOVA with Tukey’s multiple comparison.
    Figure Legend Snippet: Collaborative killing of yeast by alveolar MØ, DCs, and neutrophils is dependent on LEC NFκB, IL-17A and GM-CSF (A–B) IKK2 fl/fl and IKK2 ΔLEC mice were infected i.t. with DsRed yeast stained with Uvitex. The proportion and number of live (red) and dead (blue) yeast among total yeasts (red+blue) were quantified by flow cytometry. Two pooled experiments depicted. (C) Proportion of yeast-associated (uvitex + ) alveolar MØ (CD11c + Siglec F + ), DCs (Siglec F − CD11c + MHCII + ) and neutrophils (Siglec F − CD11b + Ly6G + ) that were DsRed- (associated with dead yeast). Two pooled experiments depicted. (D) WT mice were infected i.t. with DsRed yeast and the proportion of dead yeasts was calculated by flow cytometry. IL-17A and GM-CSF were neutralized as noted in Methods. Data representative of 3 experiments. One-way ANOVA with Tukey’s multiple comparison. (E) IKK2 fl/fl and IKK2 ΔLEC were infected with DsRed yeast. rIL-17A and rGM-CSF were given i.t. together with the inoculum. 48 h.p.i lung cell suspensions were analyzed by flow cytometry to quantify the proportion of dead yeast (DeRed − Uvitex + ) in total lung homogenate, and proportoin of phagocytes associated with dead yeast. One-Way ANOVA with Tukey’s multiple comparison.

    Techniques Used: Mouse Assay, Infection, Staining, Flow Cytometry, Cytometry

    ) (A) WT mice were infected i.t. with yeast. IL-17A and GM-CSF were neutralized as noted in Methods. At 48 h.p.i., lung CFU was enumerated. Each panel represents a separate experiment (2 pooled experiments depicted in left panel); each symbol denotes one mouse. Mann-Whitney test (48 h.p.i.). (B) IKK2 fl/fl and IKK2 ΔLEC mice were infected i.t. with yeast and IL-17A content was analyzed by ELISA in BALF at 48 h.p.i. A representative of 4 experiments is depicted. Mann-Whitney test. (C) IKK2 fl/fl and IKK2 ΔLEC mice were infected i.t. with yeast. At 48 h.p.i., lung cell suspensions were analyzed by flow cytometry for intracellular IL-17A and GM-CSF in lymphocytes (CD90.2 + CD44 hi ). Two pooled experiments depicted. Mann-Whitney test. (D) WT, IL-17A −/− and GM-CSF −/− were infected with 5×10 5 yeasts; at 48 h.p.i., lungs were analyzed by confocal imaging. Club cells depicted in green (CC10), nuclei in blue (DAPI) and GM-CSF in red. (E) Procedure was done as in A with one further injection of antibody at 48 h.p.i. At 96 h.p.i., lung CFU were quantified. Data representative of 3 experiments. Kruskal-Wallis with Dunn’s multiple comparison.
    Figure Legend Snippet: ) (A) WT mice were infected i.t. with yeast. IL-17A and GM-CSF were neutralized as noted in Methods. At 48 h.p.i., lung CFU was enumerated. Each panel represents a separate experiment (2 pooled experiments depicted in left panel); each symbol denotes one mouse. Mann-Whitney test (48 h.p.i.). (B) IKK2 fl/fl and IKK2 ΔLEC mice were infected i.t. with yeast and IL-17A content was analyzed by ELISA in BALF at 48 h.p.i. A representative of 4 experiments is depicted. Mann-Whitney test. (C) IKK2 fl/fl and IKK2 ΔLEC mice were infected i.t. with yeast. At 48 h.p.i., lung cell suspensions were analyzed by flow cytometry for intracellular IL-17A and GM-CSF in lymphocytes (CD90.2 + CD44 hi ). Two pooled experiments depicted. Mann-Whitney test. (D) WT, IL-17A −/− and GM-CSF −/− were infected with 5×10 5 yeasts; at 48 h.p.i., lungs were analyzed by confocal imaging. Club cells depicted in green (CC10), nuclei in blue (DAPI) and GM-CSF in red. (E) Procedure was done as in A with one further injection of antibody at 48 h.p.i. At 96 h.p.i., lung CFU were quantified. Data representative of 3 experiments. Kruskal-Wallis with Dunn’s multiple comparison.

    Techniques Used: Mouse Assay, Infection, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Imaging, Injection

    ) (A–B) IL-17 cre Rosa26R eYFP mice were infected i.t. with yeast. Lung cell suspensions were analyzed by flow cytometry at 48 h.p.i. to identify IL-17A + innate lymphocytes. Cells were gated as CD90.2 + IL-17 + and the proportion (A) and number (B) of nTh17, TCRβ + CD4 − , TCRγδ, and ILCs quantified. Total lymphocytes (B) means IL-17 + cells within the total CD90.2 + population. A representative of 3 experiments depicted; n=5/group. Mann-Whitney test (B). (C–D) IKK2 fl/fl and IKK2 ΔLEC mice were infected i.t. with yeast and the proportion (C) and number (D) of indicated lymphocyte populations quantified by flow cytometry at 48 h.p.i. The TCRβ + CD4 − gate includes CD8 + T cells. Concatenated plots depicted in C (n=11 in IKK2 fl/fl , n=9 in IKK2 ΔLEC ). Two pooled experiments depicted in D; each symbol is one mouse. Mann-Whitney test.
    Figure Legend Snippet: ) (A–B) IL-17 cre Rosa26R eYFP mice were infected i.t. with yeast. Lung cell suspensions were analyzed by flow cytometry at 48 h.p.i. to identify IL-17A + innate lymphocytes. Cells were gated as CD90.2 + IL-17 + and the proportion (A) and number (B) of nTh17, TCRβ + CD4 − , TCRγδ, and ILCs quantified. Total lymphocytes (B) means IL-17 + cells within the total CD90.2 + population. A representative of 3 experiments depicted; n=5/group. Mann-Whitney test (B). (C–D) IKK2 fl/fl and IKK2 ΔLEC mice were infected i.t. with yeast and the proportion (C) and number (D) of indicated lymphocyte populations quantified by flow cytometry at 48 h.p.i. The TCRβ + CD4 − gate includes CD8 + T cells. Concatenated plots depicted in C (n=11 in IKK2 fl/fl , n=9 in IKK2 ΔLEC ). Two pooled experiments depicted in D; each symbol is one mouse. Mann-Whitney test.

    Techniques Used: Mouse Assay, Infection, Flow Cytometry, Cytometry, MANN-WHITNEY

    ) (A) Bone marrow chimeric mice were infected i.t. with yeast and lung CFU counted at 48 h.p.i. A representative of 2 experiments is shown. One-way ANOVA with Bonferroni’s correction. (B) WT mice were infected i.t. with yeast and lung CFU counted at 48 h.p.i. IL-1α and IL-1β were neutralized at the time of infection. Two pooled experiments depicted. One-way ANOVA with Bonferroni’s correction. (C–D) IL-17A and GM-CSF production by innate lymphocytes in WT and IL-1R −/− mice were analyzed by intracellular cytokine staining. Proportion (C) and number (D) of cytokine-producing cells are depicted. Concatenated plots depicted in C (n=5 WT and 5 IL-1R −/− ). A representative experiment of two is shown. Mann Whitney test. (E) IL-1R fl/fl and IL-1R ΔLEC mice were infected with yeast and CFU counted at 48 h.p.i. Mann Whitney test.
    Figure Legend Snippet: ) (A) Bone marrow chimeric mice were infected i.t. with yeast and lung CFU counted at 48 h.p.i. A representative of 2 experiments is shown. One-way ANOVA with Bonferroni’s correction. (B) WT mice were infected i.t. with yeast and lung CFU counted at 48 h.p.i. IL-1α and IL-1β were neutralized at the time of infection. Two pooled experiments depicted. One-way ANOVA with Bonferroni’s correction. (C–D) IL-17A and GM-CSF production by innate lymphocytes in WT and IL-1R −/− mice were analyzed by intracellular cytokine staining. Proportion (C) and number (D) of cytokine-producing cells are depicted. Concatenated plots depicted in C (n=5 WT and 5 IL-1R −/− ). A representative experiment of two is shown. Mann Whitney test. (E) IL-1R fl/fl and IL-1R ΔLEC mice were infected with yeast and CFU counted at 48 h.p.i. Mann Whitney test.

    Techniques Used: Mouse Assay, Infection, Staining, MANN-WHITNEY

    26) Product Images from "LXR-Mediated Inhibition of CD4+ T Helper Cells"

    Article Title: LXR-Mediated Inhibition of CD4+ T Helper Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046615

    T09 inhibits IL-17 cytokine production. A , The effect of T09 (5 µM) on IL-17 cytokine expression during murine T H 17 cell differentiation. Left: Intracellular cytokine staining on splenocytes treated with T09 for the duration of the time course (4 days). Middle: Intracellular cytokine staining on splenocytes treated with T09 24 hours prior to the end of the time course. Right: Graphs summarizing the FACS plots. Data was normalized to DMSO controls. B , The effect of T09 treatment (5 µM, 24 hours) on IL-17 cytokine expression on differentiated human peripheral blood mononuclear cells (hPBMCs) using intracellular cytokine staining. C , The effect of T09 (5 µM) on IL-17 cytokine secretion during murine T H 17 cell differentiation. ELISA assay on splenocytes treated with T09 for the duration of the three day time course (T = 0, left) or treated with T09 24 hours prior to the end of the time course (T-24h, right). D , The effect of T09 (5 µM) on IL-17A/F cytokine secretion during murine T H 17 cell differentiation. ELISA assay on splenocytes treated with T09 for the duration of the three day time course (T = 0, left), or on splenocytes treated with T09 24 hours prior to the end of the time course (T-24h, right). ( n = 3 , * p
    Figure Legend Snippet: T09 inhibits IL-17 cytokine production. A , The effect of T09 (5 µM) on IL-17 cytokine expression during murine T H 17 cell differentiation. Left: Intracellular cytokine staining on splenocytes treated with T09 for the duration of the time course (4 days). Middle: Intracellular cytokine staining on splenocytes treated with T09 24 hours prior to the end of the time course. Right: Graphs summarizing the FACS plots. Data was normalized to DMSO controls. B , The effect of T09 treatment (5 µM, 24 hours) on IL-17 cytokine expression on differentiated human peripheral blood mononuclear cells (hPBMCs) using intracellular cytokine staining. C , The effect of T09 (5 µM) on IL-17 cytokine secretion during murine T H 17 cell differentiation. ELISA assay on splenocytes treated with T09 for the duration of the three day time course (T = 0, left) or treated with T09 24 hours prior to the end of the time course (T-24h, right). D , The effect of T09 (5 µM) on IL-17A/F cytokine secretion during murine T H 17 cell differentiation. ELISA assay on splenocytes treated with T09 for the duration of the three day time course (T = 0, left), or on splenocytes treated with T09 24 hours prior to the end of the time course (T-24h, right). ( n = 3 , * p

    Techniques Used: Expressing, Cell Differentiation, Staining, FACS, Enzyme-linked Immunosorbent Assay

    27) Product Images from "Interleukin 17A Promotes Gastric Cancer Invasiveness via NF-?B Mediated Matrix Metalloproteinases 2 and 9 Expression"

    Article Title: Interleukin 17A Promotes Gastric Cancer Invasiveness via NF-?B Mediated Matrix Metalloproteinases 2 and 9 Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096678

    IL-17A promotes gastric cancer cell migration and invasion. (A) IL-17A treated GC cells (AGS, BGC-823 and SGC-7901) showed higher motility in a wound-healing assay, compared with cells without IL-17A treatment. (B) The percent migration rate is expressed as a percentage of the beginning area. (C) Effect of IL-17A on cell invasion was detected by transwell assay. Representative pictures of cells migrated through Matrigel-coated transwell were shown. (D) Total invasive cell number in each chamber was summarized as a percentage of control. Values represent the means ± SD of three independent experiments performed in triplicate. * p
    Figure Legend Snippet: IL-17A promotes gastric cancer cell migration and invasion. (A) IL-17A treated GC cells (AGS, BGC-823 and SGC-7901) showed higher motility in a wound-healing assay, compared with cells without IL-17A treatment. (B) The percent migration rate is expressed as a percentage of the beginning area. (C) Effect of IL-17A on cell invasion was detected by transwell assay. Representative pictures of cells migrated through Matrigel-coated transwell were shown. (D) Total invasive cell number in each chamber was summarized as a percentage of control. Values represent the means ± SD of three independent experiments performed in triplicate. * p

    Techniques Used: Migration, Wound Healing Assay, Transwell Assay

    IL-17A promotes the expressions and activities of MMP-2 and MMP-9 and suppresses the expressions of TIMP-1 and TIMP-2 in gastric cancer cells. (A) Expressions of MMPs in GC cells (AGS and BGC-823) were compared by western blotting between cells treated with and without IL-17A (50 ng/ml) for 24 h. (B) Quantification of the protein levels of MMP-2 and MMP-9. (C) Effects of IL-17A on the activities of MMP-2 and MMP-9. (D) Quantification of the activities of MMP-2 and MMP-9. Values represent the means ± SD of three independent experiments performed in triplicate. * p
    Figure Legend Snippet: IL-17A promotes the expressions and activities of MMP-2 and MMP-9 and suppresses the expressions of TIMP-1 and TIMP-2 in gastric cancer cells. (A) Expressions of MMPs in GC cells (AGS and BGC-823) were compared by western blotting between cells treated with and without IL-17A (50 ng/ml) for 24 h. (B) Quantification of the protein levels of MMP-2 and MMP-9. (C) Effects of IL-17A on the activities of MMP-2 and MMP-9. (D) Quantification of the activities of MMP-2 and MMP-9. Values represent the means ± SD of three independent experiments performed in triplicate. * p

    Techniques Used: Western Blot

    Effects of the NF-κB inhibitor and IL-17A on cell invasion and the expressions of MMP-2 and MMP-9 in AGS cells. (A) 1×10 6 AGS cells were pretreated with helenalin (5 µM) for 30 min and then incubated in the presence or absence of IL-17A (50 ng/ml) for 24 h. Cellular invasiveness was measured using the transwell invasion assay. (B) The percent invasion rate was expressed as a percentage of control. (C, D) AGS cells were treated and then subjected to western blotting to analyze the protein levels of MMP-2 and MMP-9. Values represent the means ± SD of three independent experiments performed in triplicate. * p
    Figure Legend Snippet: Effects of the NF-κB inhibitor and IL-17A on cell invasion and the expressions of MMP-2 and MMP-9 in AGS cells. (A) 1×10 6 AGS cells were pretreated with helenalin (5 µM) for 30 min and then incubated in the presence or absence of IL-17A (50 ng/ml) for 24 h. Cellular invasiveness was measured using the transwell invasion assay. (B) The percent invasion rate was expressed as a percentage of control. (C, D) AGS cells were treated and then subjected to western blotting to analyze the protein levels of MMP-2 and MMP-9. Values represent the means ± SD of three independent experiments performed in triplicate. * p

    Techniques Used: Incubation, Transwell Invasion Assay, Western Blot

    IL-17A activates NF-κB pathway in AGS cells. (A) Western blotting analysis was used to detect overall p50, p65, p52, c-Rel and RelB expression in AGS cells treated with IL-17A (50 ng/ml) at indicated time points. (B) Quantification of the protein levels of overall p50, p65, p52, c-Rel and RelB. (C) Western blotting analysis was used to detect nuclear p50, p65, p52, c-Rel and RelB expression in AGS cells treated with IL-17A (50 ng/mL) at indicated time points. (D) Quantification of the protein levels of nuclear p50, p65, p52, c-Rel and RelB. (E) The relative ratio of nuclear to overall fraction of p50, p65, p52, c-Rel and RelB. Values represent the means ± SD of three independent experiments performed in triplicate. ** p
    Figure Legend Snippet: IL-17A activates NF-κB pathway in AGS cells. (A) Western blotting analysis was used to detect overall p50, p65, p52, c-Rel and RelB expression in AGS cells treated with IL-17A (50 ng/ml) at indicated time points. (B) Quantification of the protein levels of overall p50, p65, p52, c-Rel and RelB. (C) Western blotting analysis was used to detect nuclear p50, p65, p52, c-Rel and RelB expression in AGS cells treated with IL-17A (50 ng/mL) at indicated time points. (D) Quantification of the protein levels of nuclear p50, p65, p52, c-Rel and RelB. (E) The relative ratio of nuclear to overall fraction of p50, p65, p52, c-Rel and RelB. Values represent the means ± SD of three independent experiments performed in triplicate. ** p

    Techniques Used: Western Blot, Expressing

    28) Product Images from "Regulation of IL-17A Production Is Distinct from IL-17F in a Primary Human Cell Co-culture Model of T Cell-Mediated B Cell Activation"

    Article Title: Regulation of IL-17A Production Is Distinct from IL-17F in a Primary Human Cell Co-culture Model of T Cell-Mediated B Cell Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0058966

    Production of IL-17A and IL-17F protein in BT co-cultures requires B cell and CD4 T cell interaction. Measurement of IL-17A (A) and IL-17F (B) by ELISA in supernatants from 25,000 B cells/well co-cultured with 25,000 PBMC/well and stimulated with α-IgM and SAg for three days. Measurement of IL-17A (C) and IL-17F (D) by ELISA in supernatants from 25,000 B cells/well and 25,000 CD4 T cells/well cultured alone or 25,000 B cells cultured with increasing numbers of CD4 T cells in the presence of α-IgM and SAg or carrier (Control) for three days. Data are the means ± standard deviation of three B cell and CD4 T cell donor pools and two independent experiments.
    Figure Legend Snippet: Production of IL-17A and IL-17F protein in BT co-cultures requires B cell and CD4 T cell interaction. Measurement of IL-17A (A) and IL-17F (B) by ELISA in supernatants from 25,000 B cells/well co-cultured with 25,000 PBMC/well and stimulated with α-IgM and SAg for three days. Measurement of IL-17A (C) and IL-17F (D) by ELISA in supernatants from 25,000 B cells/well and 25,000 CD4 T cells/well cultured alone or 25,000 B cells cultured with increasing numbers of CD4 T cells in the presence of α-IgM and SAg or carrier (Control) for three days. Data are the means ± standard deviation of three B cell and CD4 T cell donor pools and two independent experiments.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Cell Culture, Standard Deviation

    Summary of pathways and targets identified in a screen of pharmacologic modulators that positively or negatively regulate IL-17A and/or IL-17F in stimulated BT co-cultures. Pathways and targets are listed next to arrows that indicate selectivity for IL-17A, IL-17A and IL-17F, or IL-17F.
    Figure Legend Snippet: Summary of pathways and targets identified in a screen of pharmacologic modulators that positively or negatively regulate IL-17A and/or IL-17F in stimulated BT co-cultures. Pathways and targets are listed next to arrows that indicate selectivity for IL-17A, IL-17A and IL-17F, or IL-17F.

    Techniques Used:

    PGE1 and PGE2 stimulate production of IL-17A and impair production of IL-17F. Profiles of PGE1 (0.37–3.33 μM; A) and PGE2 (0.37–3.33 μM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. PGE1 and PGE2 increased production of IL-17A and IL-6, but inhibited IL-17F, IL-2, and TNFα production. Data are presented as the Log 10 ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.
    Figure Legend Snippet: PGE1 and PGE2 stimulate production of IL-17A and impair production of IL-17F. Profiles of PGE1 (0.37–3.33 μM; A) and PGE2 (0.37–3.33 μM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. PGE1 and PGE2 increased production of IL-17A and IL-6, but inhibited IL-17F, IL-2, and TNFα production. Data are presented as the Log 10 ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.

    Techniques Used:

    Erythromycin and Wortmannin at select doses inhibit IL-17A and IL-17F without modulating other parameters. Profiles of erythromycin (3.3–90 μM; A) or wortmannin (1.5–13.7 nM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. Erythromycin inhibited IL-17A and IL-17F production but did not affect B cell proliferation, secreted IgG, IL-2, IL-6, or TNFα, whereas wortmannin selectively inhibited IL-17A and IL-17F only at lower doses. Data are presented as the Log 10 ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.
    Figure Legend Snippet: Erythromycin and Wortmannin at select doses inhibit IL-17A and IL-17F without modulating other parameters. Profiles of erythromycin (3.3–90 μM; A) or wortmannin (1.5–13.7 nM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. Erythromycin inhibited IL-17A and IL-17F production but did not affect B cell proliferation, secreted IgG, IL-2, IL-6, or TNFα, whereas wortmannin selectively inhibited IL-17A and IL-17F only at lower doses. Data are presented as the Log 10 ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.

    Techniques Used:

    Torin-1, CP-690,550, and Axitinib are examples of compounds that regulated production of IL-17A, IL-17F or both IL-17A and IL-17F. Profiles of Torin-1 (0.460–12.3 nM; A), CP-690,550 (0.041–1.11 μM; B), or Axitinib (0.333–9 μM; C) added to BT co-cultures stimulated with α-IgM and SAg for three days. Parameters measured are indicated along the x-axis. Torin-1 inhibits IL-17A more potently than IL-17F (A), whereas CP-690,550 inhibits IL-17F more potently than IL-17A (B). Similarly, Axitinib blocks IL-17F production but does not affect IL-17A (C). Data are presented as the Log 10 ratio of drug-treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.
    Figure Legend Snippet: Torin-1, CP-690,550, and Axitinib are examples of compounds that regulated production of IL-17A, IL-17F or both IL-17A and IL-17F. Profiles of Torin-1 (0.460–12.3 nM; A), CP-690,550 (0.041–1.11 μM; B), or Axitinib (0.333–9 μM; C) added to BT co-cultures stimulated with α-IgM and SAg for three days. Parameters measured are indicated along the x-axis. Torin-1 inhibits IL-17A more potently than IL-17F (A), whereas CP-690,550 inhibits IL-17F more potently than IL-17A (B). Similarly, Axitinib blocks IL-17F production but does not affect IL-17A (C). Data are presented as the Log 10 ratio of drug-treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.

    Techniques Used:

    IL-17A and IL-17F are predominantly expressed by CD4 T cells in a BT co-culture model of human B cell-dependent T cell responses. FACS analysis gating strategy for cell types present in BT co-cultures after stimulation for three days with α-IgM and SAg (A). Gated cell populations are listed above the FACS plots and the percentage of cells present in each gate relative to the parent population is shown. To detect intracellular cytokine expression, BT co-cultures were treated for 5 hours with PMA, ionomycin, and monensin, and then stained with antibodies specific to IL-17A (A), IL-17F (B), or with an isotype control antibody common to the isotype of α-IL-17A and α-IL-17F (C).
    Figure Legend Snippet: IL-17A and IL-17F are predominantly expressed by CD4 T cells in a BT co-culture model of human B cell-dependent T cell responses. FACS analysis gating strategy for cell types present in BT co-cultures after stimulation for three days with α-IgM and SAg (A). Gated cell populations are listed above the FACS plots and the percentage of cells present in each gate relative to the parent population is shown. To detect intracellular cytokine expression, BT co-cultures were treated for 5 hours with PMA, ionomycin, and monensin, and then stained with antibodies specific to IL-17A (A), IL-17F (B), or with an isotype control antibody common to the isotype of α-IL-17A and α-IL-17F (C).

    Techniques Used: Co-Culture Assay, FACS, Expressing, Staining

    Examples of agents that potentiated production of IL-17A and IL-17F in BT co-cultures. Profiles of BW 245C (0.37–10 μM; A), LPS (0.1–100 ng/ml; B), or HKLM (10–10,000 U/ml; C) added to BT co-cultures stimulated with α-IgM and SAg for three days. BW245C and LPS enhance IL-17A and IL-6 production, while LPS also stimulates IL-17F, secreted IgG, IL-6, and TNFα. HKLM stimulates IL-17F more potently than IL-17A. Data are presented as the log 10 ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.
    Figure Legend Snippet: Examples of agents that potentiated production of IL-17A and IL-17F in BT co-cultures. Profiles of BW 245C (0.37–10 μM; A), LPS (0.1–100 ng/ml; B), or HKLM (10–10,000 U/ml; C) added to BT co-cultures stimulated with α-IgM and SAg for three days. BW245C and LPS enhance IL-17A and IL-6 production, while LPS also stimulates IL-17F, secreted IgG, IL-6, and TNFα. HKLM stimulates IL-17F more potently than IL-17A. Data are presented as the log 10 ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.

    Techniques Used:

    SR2211, an RORγ inverse agonist, and Calcitriol, a Vitamin D3 receptor agonist, block production of IL-17A and IL-17F in BT co-cultures. Profiles of SR2211 (0.370–10 μM; A) or Calcitriol (0.15–4.1 nM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. Parameters measured (B cell Proliferation, PBMC Cytotoxicity, Secreted IgG, sIL-17A, sIL-17F, sIL-2, sIL-6, and sTNF-alpha) are indicated along the x-axis. Data are presented as the Log 10 ratio of drug-treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.
    Figure Legend Snippet: SR2211, an RORγ inverse agonist, and Calcitriol, a Vitamin D3 receptor agonist, block production of IL-17A and IL-17F in BT co-cultures. Profiles of SR2211 (0.370–10 μM; A) or Calcitriol (0.15–4.1 nM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. Parameters measured (B cell Proliferation, PBMC Cytotoxicity, Secreted IgG, sIL-17A, sIL-17F, sIL-2, sIL-6, and sTNF-alpha) are indicated along the x-axis. Data are presented as the Log 10 ratio of drug-treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.

    Techniques Used: Blocking Assay

    29) Product Images from "Contribution of IL-17 in Steroid Hyporesponsiveness in Obese Asthmatics Through Dysregulation of Glucocorticoid Receptors α and β"

    Article Title: Contribution of IL-17 in Steroid Hyporesponsiveness in Obese Asthmatics Through Dysregulation of Glucocorticoid Receptors α and β

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.01724

    Stimulation with IL-17A F and IL-17A alone induces changes in inflammatory cytokine profiles in adipocytes from lean and obese subjects. A multiplex assay was used to measure the levels of inflammatory cytokines: IL-8 (A) , IL-6 (B) , IL-5 (C) , IL-4 (D) , and IFN-γ (E) secreted by mature adipocytes after 48 h stimulation with IL-17A F combination or IL-17A alone. One independent experiment preformed per subject. n = 4 lean subjects, n = 3 obese subjects, One-way ANOVA, Mean ± SE; * P
    Figure Legend Snippet: Stimulation with IL-17A F and IL-17A alone induces changes in inflammatory cytokine profiles in adipocytes from lean and obese subjects. A multiplex assay was used to measure the levels of inflammatory cytokines: IL-8 (A) , IL-6 (B) , IL-5 (C) , IL-4 (D) , and IFN-γ (E) secreted by mature adipocytes after 48 h stimulation with IL-17A F combination or IL-17A alone. One independent experiment preformed per subject. n = 4 lean subjects, n = 3 obese subjects, One-way ANOVA, Mean ± SE; * P

    Techniques Used: Multiplex Assay

    IL-17A F and IL-17A alone induces changes in protein levels of GR-β. GR-α (A) and GR- β (B) protein expression in adipocytes from lean and obese subjects following 48 h stimulation with IL-17A F combination or IL-17A alone (C) Ratio of GR-α/GR-β. n = 4 lean subjects, n = 3 obese subjects, One-way ANOVA, Mean ± SE; * P
    Figure Legend Snippet: IL-17A F and IL-17A alone induces changes in protein levels of GR-β. GR-α (A) and GR- β (B) protein expression in adipocytes from lean and obese subjects following 48 h stimulation with IL-17A F combination or IL-17A alone (C) Ratio of GR-α/GR-β. n = 4 lean subjects, n = 3 obese subjects, One-way ANOVA, Mean ± SE; * P

    Techniques Used: Expressing

    Pre-treatment with Dexamethasone followed by IL-17A stimulation induces changes in GR-α/GR-β mRNA ratio. Adipocytes from lean and obese subjects were pre-treated with 500 ng/ml dexamethasone followed by a 48 h stimulation with 100 ng/ml of IL-17A. mRNA expression of GR-α and GR-β were measured by qRT-PCR in duplicates using TaqMan probes. One independent experiment preformed per subject: n = 4 lean subjects, n = 3 obese subjects, One-Way ANOVA, Mean ± SE; * P
    Figure Legend Snippet: Pre-treatment with Dexamethasone followed by IL-17A stimulation induces changes in GR-α/GR-β mRNA ratio. Adipocytes from lean and obese subjects were pre-treated with 500 ng/ml dexamethasone followed by a 48 h stimulation with 100 ng/ml of IL-17A. mRNA expression of GR-α and GR-β were measured by qRT-PCR in duplicates using TaqMan probes. One independent experiment preformed per subject: n = 4 lean subjects, n = 3 obese subjects, One-Way ANOVA, Mean ± SE; * P

    Techniques Used: Expressing, Quantitative RT-PCR

    Stimulation with IL-17A F and IL-17A alone induces changes in mRNA expression of inflammatory mediators in adipocytes from lean and obese subjects. qRT-PCR analysis of detected mRNA expression of inflammatory markers: IL-8 (A) , IL-6 (B) , TGF-β (C) , IL-1β (D) in mature adipocytes after 48 h stimulation with combination of IL-17A F or IL-17A alone. n = 4 lean subjects, n = 3 obese subjects, One-way ANOVA, Mean ± SE; * P
    Figure Legend Snippet: Stimulation with IL-17A F and IL-17A alone induces changes in mRNA expression of inflammatory mediators in adipocytes from lean and obese subjects. qRT-PCR analysis of detected mRNA expression of inflammatory markers: IL-8 (A) , IL-6 (B) , TGF-β (C) , IL-1β (D) in mature adipocytes after 48 h stimulation with combination of IL-17A F or IL-17A alone. n = 4 lean subjects, n = 3 obese subjects, One-way ANOVA, Mean ± SE; * P

    Techniques Used: Expressing, Quantitative RT-PCR

    Stimulation with IL-17A F and IL-17A alone induces changes in GR-α/GR-β mRNA ratio. Adipocytes from lean and obese subjects were stimulated with 100 ng/mL of IL-17A and IL-17F in combination or IL-17A alone for 48 h. Cells were collected and qRT-PCR analysis was performed in duplicate using TaqMan probes to assess GR-α and GR-β mRNA expression. One independent experiment performed per subject. n = 4 lean subjects, n = 3 obese subjects, One-Way ANOVA, Mean ± SE; * P
    Figure Legend Snippet: Stimulation with IL-17A F and IL-17A alone induces changes in GR-α/GR-β mRNA ratio. Adipocytes from lean and obese subjects were stimulated with 100 ng/mL of IL-17A and IL-17F in combination or IL-17A alone for 48 h. Cells were collected and qRT-PCR analysis was performed in duplicate using TaqMan probes to assess GR-α and GR-β mRNA expression. One independent experiment performed per subject. n = 4 lean subjects, n = 3 obese subjects, One-Way ANOVA, Mean ± SE; * P

    Techniques Used: Quantitative RT-PCR, Expressing

    30) Product Images from "IL-17A Influences Essential Functions of the Monocyte/Macrophage Lineage and Is Involved in Advanced Murine and Human Atherosclerosis"

    Article Title: IL-17A Influences Essential Functions of the Monocyte/Macrophage Lineage and Is Involved in Advanced Murine and Human Atherosclerosis

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1400181

    Cellular composition in atherosclerotic lesions of Apoe −/− mice. ( A ) Representative photomicrographs of immunohistochemistry stainings of T cells (CD3, original magnification ×40), Mac-2 + cells (original magnification ×40), CD206 + cells (original magnification ×40), and B cells (B220; original magnification ×40). ( B ) Quantitative analysis of plaque inflammation are presented in 42-wk-old Apoe −/− mice with ( n = 12–15) and without IL-17A mAb treatment ( n = 8–10) (as well as 26-wk-old baseline mice, n = 4 to 5). Results are presented as dot plots displaying mean.
    Figure Legend Snippet: Cellular composition in atherosclerotic lesions of Apoe −/− mice. ( A ) Representative photomicrographs of immunohistochemistry stainings of T cells (CD3, original magnification ×40), Mac-2 + cells (original magnification ×40), CD206 + cells (original magnification ×40), and B cells (B220; original magnification ×40). ( B ) Quantitative analysis of plaque inflammation are presented in 42-wk-old Apoe −/− mice with ( n = 12–15) and without IL-17A mAb treatment ( n = 8–10) (as well as 26-wk-old baseline mice, n = 4 to 5). Results are presented as dot plots displaying mean.

    Techniques Used: Mouse Assay, Immunohistochemistry

    Effects of IL-17A on monocytes and monocyte-derived cells. ( A and B ) Foam cell formation assay. (A) Representative photomicrographs of monocyte-derived macrophages incubated with oxLDL in addition to IL-17A in different concentrations are shown (original magnification ×20). Representative Oil Red O staining of different groups are shown: no oxLDL (control), oxLDL, and oxLDL plus IL-17A. (B) Data are given as mean ± SEM of five independent experiments. ( C ) oxLDL uptake assay. Flow cytometry analysis of DiI-labeled oxLDL uptake (in addition to IL-17A incubation) of monocyte-derived macrophages: solid green line (oxLDL) and dotted pink line (oxLDL+IL-17A). Histogram represents one out of three independent experiments. ( D ) For T cell activation assays, macrophages or DCs were incubated in 96-well round-bottom plates and stimulated with oxLDL (Sanbio), IL-17A (R D Systems), or oxLDL in combination with IL-17A. After 6-h preincubation, CD4 + T cells were added in a ratio of 1:5 and stimulated for 8 h. Expression of activation-associated cytokines and transcription factors in CD4 + for details). Representative analysis of T-box21 (T-bet), IL2 , and IFNγ are shown.
    Figure Legend Snippet: Effects of IL-17A on monocytes and monocyte-derived cells. ( A and B ) Foam cell formation assay. (A) Representative photomicrographs of monocyte-derived macrophages incubated with oxLDL in addition to IL-17A in different concentrations are shown (original magnification ×20). Representative Oil Red O staining of different groups are shown: no oxLDL (control), oxLDL, and oxLDL plus IL-17A. (B) Data are given as mean ± SEM of five independent experiments. ( C ) oxLDL uptake assay. Flow cytometry analysis of DiI-labeled oxLDL uptake (in addition to IL-17A incubation) of monocyte-derived macrophages: solid green line (oxLDL) and dotted pink line (oxLDL+IL-17A). Histogram represents one out of three independent experiments. ( D ) For T cell activation assays, macrophages or DCs were incubated in 96-well round-bottom plates and stimulated with oxLDL (Sanbio), IL-17A (R D Systems), or oxLDL in combination with IL-17A. After 6-h preincubation, CD4 + T cells were added in a ratio of 1:5 and stimulated for 8 h. Expression of activation-associated cytokines and transcription factors in CD4 + for details). Representative analysis of T-box21 (T-bet), IL2 , and IFNγ are shown.

    Techniques Used: Derivative Assay, Tube Formation Assay, Incubation, Staining, Flow Cytometry, Cytometry, Labeling, Activation Assay, Expressing

    Effects of IL-17A mAb treatment on atherosclerotic plaques in Apoe −/− mice. Representative Oil Red O immunohistostainings (original magnification ×4) from aortic root of control [ n = 10; ( A )]; IL-17A mAb-treated mice [ n = 15; ( B )] and baseline [ n = 5; ( C )] are shown. ( D – F ) Morphometric quantification of cross-sectional area (μm 2 ), fractional stenosis (%), and maximum stenosis (%) of late-stage atherosclerotic lesions were compared. Results are shown as dot plots displaying mean.
    Figure Legend Snippet: Effects of IL-17A mAb treatment on atherosclerotic plaques in Apoe −/− mice. Representative Oil Red O immunohistostainings (original magnification ×4) from aortic root of control [ n = 10; ( A )]; IL-17A mAb-treated mice [ n = 15; ( B )] and baseline [ n = 5; ( C )] are shown. ( D – F ) Morphometric quantification of cross-sectional area (μm 2 ), fractional stenosis (%), and maximum stenosis (%) of late-stage atherosclerotic lesions were compared. Results are shown as dot plots displaying mean.

    Techniques Used: Mouse Assay

    31) 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:

    32) Product Images from "Broad antibody and T cell reactivity induced by a pneumococcal whole-cell vaccine"

    Article Title: Broad antibody and T cell reactivity induced by a pneumococcal whole-cell vaccine

    Journal: Vaccine

    doi: 10.1016/j.vaccine.2012.01.034

    WCV-induced IL-17A response to isogenic pneumococcal strains bearing differing capsular types from splenocytes obtained from mice immunized with WCV
    Figure Legend Snippet: WCV-induced IL-17A response to isogenic pneumococcal strains bearing differing capsular types from splenocytes obtained from mice immunized with WCV

    Techniques Used: Mouse Assay

    33) 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

    34) Product Images from "Contribution of IL-17 in Steroid Hyporesponsiveness in Obese Asthmatics Through Dysregulation of Glucocorticoid Receptors α and β"

    Article Title: Contribution of IL-17 in Steroid Hyporesponsiveness in Obese Asthmatics Through Dysregulation of Glucocorticoid Receptors α and β

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.01724

    Stimulation with IL-17A F and IL-17A alone induces changes in inflammatory cytokine profiles in adipocytes from lean and obese subjects. A multiplex assay was used to measure the levels of inflammatory cytokines: IL-8 (A) , IL-6 (B) , IL-5 (C) , IL-4 (D) , and IFN-γ (E) secreted by mature adipocytes after 48 h stimulation with IL-17A F combination or IL-17A alone. One independent experiment preformed per subject. n = 4 lean subjects, n = 3 obese subjects, One-way ANOVA, Mean ± SE; * P
    Figure Legend Snippet: Stimulation with IL-17A F and IL-17A alone induces changes in inflammatory cytokine profiles in adipocytes from lean and obese subjects. A multiplex assay was used to measure the levels of inflammatory cytokines: IL-8 (A) , IL-6 (B) , IL-5 (C) , IL-4 (D) , and IFN-γ (E) secreted by mature adipocytes after 48 h stimulation with IL-17A F combination or IL-17A alone. One independent experiment preformed per subject. n = 4 lean subjects, n = 3 obese subjects, One-way ANOVA, Mean ± SE; * P

    Techniques Used: Multiplex Assay

    IL-17A F and IL-17A alone induces changes in protein levels of GR-β. GR-α (A) and GR- β (B) protein expression in adipocytes from lean and obese subjects following 48 h stimulation with IL-17A F combination or IL-17A alone (C) Ratio of GR-α/GR-β. n = 4 lean subjects, n = 3 obese subjects, One-way ANOVA, Mean ± SE; * P
    Figure Legend Snippet: IL-17A F and IL-17A alone induces changes in protein levels of GR-β. GR-α (A) and GR- β (B) protein expression in adipocytes from lean and obese subjects following 48 h stimulation with IL-17A F combination or IL-17A alone (C) Ratio of GR-α/GR-β. n = 4 lean subjects, n = 3 obese subjects, One-way ANOVA, Mean ± SE; * P

    Techniques Used: Expressing

    Pre-treatment with Dexamethasone followed by IL-17A stimulation induces changes in GR-α/GR-β mRNA ratio. Adipocytes from lean and obese subjects were pre-treated with 500 ng/ml dexamethasone followed by a 48 h stimulation with 100 ng/ml of IL-17A. mRNA expression of GR-α and GR-β were measured by qRT-PCR in duplicates using TaqMan probes. One independent experiment preformed per subject: n = 4 lean subjects, n = 3 obese subjects, One-Way ANOVA, Mean ± SE; * P
    Figure Legend Snippet: Pre-treatment with Dexamethasone followed by IL-17A stimulation induces changes in GR-α/GR-β mRNA ratio. Adipocytes from lean and obese subjects were pre-treated with 500 ng/ml dexamethasone followed by a 48 h stimulation with 100 ng/ml of IL-17A. mRNA expression of GR-α and GR-β were measured by qRT-PCR in duplicates using TaqMan probes. One independent experiment preformed per subject: n = 4 lean subjects, n = 3 obese subjects, One-Way ANOVA, Mean ± SE; * P

    Techniques Used: Expressing, Quantitative RT-PCR

    Stimulation with IL-17A F and IL-17A alone induces changes in mRNA expression of inflammatory mediators in adipocytes from lean and obese subjects. qRT-PCR analysis of detected mRNA expression of inflammatory markers: IL-8 (A) , IL-6 (B) , TGF-β (C) , IL-1β (D) in mature adipocytes after 48 h stimulation with combination of IL-17A F or IL-17A alone. n = 4 lean subjects, n = 3 obese subjects, One-way ANOVA, Mean ± SE; * P
    Figure Legend Snippet: Stimulation with IL-17A F and IL-17A alone induces changes in mRNA expression of inflammatory mediators in adipocytes from lean and obese subjects. qRT-PCR analysis of detected mRNA expression of inflammatory markers: IL-8 (A) , IL-6 (B) , TGF-β (C) , IL-1β (D) in mature adipocytes after 48 h stimulation with combination of IL-17A F or IL-17A alone. n = 4 lean subjects, n = 3 obese subjects, One-way ANOVA, Mean ± SE; * P

    Techniques Used: Expressing, Quantitative RT-PCR

    Stimulation with IL-17A F and IL-17A alone induces changes in GR-α/GR-β mRNA ratio. Adipocytes from lean and obese subjects were stimulated with 100 ng/mL of IL-17A and IL-17F in combination or IL-17A alone for 48 h. Cells were collected and qRT-PCR analysis was performed in duplicate using TaqMan probes to assess GR-α and GR-β mRNA expression. One independent experiment performed per subject. n = 4 lean subjects, n = 3 obese subjects, One-Way ANOVA, Mean ± SE; * P
    Figure Legend Snippet: Stimulation with IL-17A F and IL-17A alone induces changes in GR-α/GR-β mRNA ratio. Adipocytes from lean and obese subjects were stimulated with 100 ng/mL of IL-17A and IL-17F in combination or IL-17A alone for 48 h. Cells were collected and qRT-PCR analysis was performed in duplicate using TaqMan probes to assess GR-α and GR-β mRNA expression. One independent experiment performed per subject. n = 4 lean subjects, n = 3 obese subjects, One-Way ANOVA, Mean ± SE; * P

    Techniques Used: Quantitative RT-PCR, Expressing

    35) Product Images from "Bifidobacterium longum Alleviates Dextran Sulfate Sodium-Induced Colitis by Suppressing IL-17A Response: Involvement of Intestinal Epithelial Costimulatory Molecules"

    Article Title: Bifidobacterium longum Alleviates Dextran Sulfate Sodium-Induced Colitis by Suppressing IL-17A Response: Involvement of Intestinal Epithelial Costimulatory Molecules

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0079735

    IECs from DSS mice, but not B. longum JCM 1222 T (B.l) -treated mice, induces IL-17A response. IECs were co-cultured with pan T cells (a), CD4 + T cells (b), and CD8 + T cells (c) from control mice in the presence of anti-CD3 antibody. The cytokine concentration in the supernatant was measured by ELISA. The data are representative of four experiments. Results are expressed as means ± standard error (n = 4). ** p
    Figure Legend Snippet: IECs from DSS mice, but not B. longum JCM 1222 T (B.l) -treated mice, induces IL-17A response. IECs were co-cultured with pan T cells (a), CD4 + T cells (b), and CD8 + T cells (c) from control mice in the presence of anti-CD3 antibody. The cytokine concentration in the supernatant was measured by ELISA. The data are representative of four experiments. Results are expressed as means ± standard error (n = 4). ** p

    Techniques Used: Mouse Assay, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

    IECs from DSS mice induce IL-17A response via CD80/CD86 and CD40 dependent costimulation. (a) IECs from DSS mice were co-cultured with CD4 + T cells from control mice in the presence of anti-CD3 antibody. In the indicated groups, IECs were pretreated with CD80 or CD86 blocking antibodies, and CD4 + T cells were pretreated with CD40L blocking antibody before co-culturing. The data are representative of two experiments. Results are expressed as means ± standard error (n = 4). ** p
    Figure Legend Snippet: IECs from DSS mice induce IL-17A response via CD80/CD86 and CD40 dependent costimulation. (a) IECs from DSS mice were co-cultured with CD4 + T cells from control mice in the presence of anti-CD3 antibody. In the indicated groups, IECs were pretreated with CD80 or CD86 blocking antibodies, and CD4 + T cells were pretreated with CD40L blocking antibody before co-culturing. The data are representative of two experiments. Results are expressed as means ± standard error (n = 4). ** p

    Techniques Used: Mouse Assay, Cell Culture, Blocking Assay

    36) Product Images from "Differential Effects of IL-17A and TNF-α on Osteoblastic Differentiation of Isolated Synoviocytes and on Bone Explants from Arthritis Patients"

    Article Title: Differential Effects of IL-17A and TNF-α on Osteoblastic Differentiation of Isolated Synoviocytes and on Bone Explants from Arthritis Patients

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2015.00151

    Effects of IL-17A and/or TNF-α on alkaline phosphatase activity . (A) OA and (B) RA FLS were cultured at a density 5 × 10 3 cell/cm 2 in osteogenic medium in the absence (0) or presence of TNF-α 1 ng/ml and/or IL-17A 50 ng/ml for 14 days. (C) Bone samples were cultured in six well-plates for 7 days. Supernatants were collected for ALP measurements by fluorometry. Results were analyzed using the Wilcoxon test. ** p
    Figure Legend Snippet: Effects of IL-17A and/or TNF-α on alkaline phosphatase activity . (A) OA and (B) RA FLS were cultured at a density 5 × 10 3 cell/cm 2 in osteogenic medium in the absence (0) or presence of TNF-α 1 ng/ml and/or IL-17A 50 ng/ml for 14 days. (C) Bone samples were cultured in six well-plates for 7 days. Supernatants were collected for ALP measurements by fluorometry. Results were analyzed using the Wilcoxon test. ** p

    Techniques Used: Activity Assay, Cell Culture, ALP Assay

    Effects of IL-17A and/or TNF-α on BV/TV ratio . Bone samples were cultured for 7 days in six well-plates, in the presence or absence of IL-17A (50 ng/ml) and TNF-α (1 ng/ml) or both, then bone samples were used for histomorphometric analysis. Bone volume over tissue volume (BV/TV) was measured with an automatic image analyzer at low power field. (A) Results were analyzed using the Wilcoxon test. * p
    Figure Legend Snippet: Effects of IL-17A and/or TNF-α on BV/TV ratio . Bone samples were cultured for 7 days in six well-plates, in the presence or absence of IL-17A (50 ng/ml) and TNF-α (1 ng/ml) or both, then bone samples were used for histomorphometric analysis. Bone volume over tissue volume (BV/TV) was measured with an automatic image analyzer at low power field. (A) Results were analyzed using the Wilcoxon test. * p

    Techniques Used: Cell Culture

    Effects of IL-17A and/or TNF-α on extracellular matrix of synoviocytes . FLS were cultured at a density 5 × 10 3 cell/cm 2 . (A) In the absence (column 1, negative control as basic DMEM medium without any addition) or presence (column 2) of osteogenic factors. TNF-α 1 ng/ml (column 3) or IL-17A 50 ng/ml (column 4), or both (column 5) were added or not to cultures. After 7, 14, and 17 days, cells were stained with alizarin red and (B) calcium deposits were quantified and analyzed for each condition using the Wilcoxon test * p
    Figure Legend Snippet: Effects of IL-17A and/or TNF-α on extracellular matrix of synoviocytes . FLS were cultured at a density 5 × 10 3 cell/cm 2 . (A) In the absence (column 1, negative control as basic DMEM medium without any addition) or presence (column 2) of osteogenic factors. TNF-α 1 ng/ml (column 3) or IL-17A 50 ng/ml (column 4), or both (column 5) were added or not to cultures. After 7, 14, and 17 days, cells were stained with alizarin red and (B) calcium deposits were quantified and analyzed for each condition using the Wilcoxon test * p

    Techniques Used: Cell Culture, Negative Control, Staining

    Effects of TNF-α and IL-17A on osteogenic marker genes . FLS were cultured in osteogenic medium after 1 day of plating, in the presence or absence of TNF-α 1 ng/ml and/or IL-17A 50 ng/ml for the indicated times. Osteogenic gene expression of (A) BMP2, (B) Wnt5a, and (C) Runx2 was measured by q-RT-PCR at 6, 12, and 24 h. Results were analyzed using the Wilcoxon test. * p
    Figure Legend Snippet: Effects of TNF-α and IL-17A on osteogenic marker genes . FLS were cultured in osteogenic medium after 1 day of plating, in the presence or absence of TNF-α 1 ng/ml and/or IL-17A 50 ng/ml for the indicated times. Osteogenic gene expression of (A) BMP2, (B) Wnt5a, and (C) Runx2 was measured by q-RT-PCR at 6, 12, and 24 h. Results were analyzed using the Wilcoxon test. * p

    Techniques Used: Marker, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction

    Effects of TNF-α and IL-17A on IL-6 and IL-8 production . (A) OA and (B) RA FLS were cultured in osteogenic medium for 14 days and bone samples (C,D) for 7 days, in the presence or absence of TNF-α 1 ng/ml and/or IL-17A 50 ng/ml. IL-6 (C) and IL-8 (D) concentrations in supernatants were quantified by ELISA. Results were analyzed using the Wilcoxon test. * p
    Figure Legend Snippet: Effects of TNF-α and IL-17A on IL-6 and IL-8 production . (A) OA and (B) RA FLS were cultured in osteogenic medium for 14 days and bone samples (C,D) for 7 days, in the presence or absence of TNF-α 1 ng/ml and/or IL-17A 50 ng/ml. IL-6 (C) and IL-8 (D) concentrations in supernatants were quantified by ELISA. Results were analyzed using the Wilcoxon test. * p

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    37) Product Images from "Regulation of IL-17A Production Is Distinct from IL-17F in a Primary Human Cell Co-culture Model of T Cell-Mediated B Cell Activation"

    Article Title: Regulation of IL-17A Production Is Distinct from IL-17F in a Primary Human Cell Co-culture Model of T Cell-Mediated B Cell Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0058966

    Production of IL-17A and IL-17F protein in BT co-cultures requires B cell and CD4 T cell interaction. Measurement of IL-17A (A) and IL-17F (B) by ELISA in supernatants from 25,000 B cells/well co-cultured with 25,000 PBMC/well and stimulated with α-IgM and SAg for three days. Measurement of IL-17A (C) and IL-17F (D) by ELISA in supernatants from 25,000 B cells/well and 25,000 CD4 T cells/well cultured alone or 25,000 B cells cultured with increasing numbers of CD4 T cells in the presence of α-IgM and SAg or carrier (Control) for three days. Data are the means ± standard deviation of three B cell and CD4 T cell donor pools and two independent experiments.
    Figure Legend Snippet: Production of IL-17A and IL-17F protein in BT co-cultures requires B cell and CD4 T cell interaction. Measurement of IL-17A (A) and IL-17F (B) by ELISA in supernatants from 25,000 B cells/well co-cultured with 25,000 PBMC/well and stimulated with α-IgM and SAg for three days. Measurement of IL-17A (C) and IL-17F (D) by ELISA in supernatants from 25,000 B cells/well and 25,000 CD4 T cells/well cultured alone or 25,000 B cells cultured with increasing numbers of CD4 T cells in the presence of α-IgM and SAg or carrier (Control) for three days. Data are the means ± standard deviation of three B cell and CD4 T cell donor pools and two independent experiments.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Cell Culture, Standard Deviation

    Summary of pathways and targets identified in a screen of pharmacologic modulators that positively or negatively regulate IL-17A and/or IL-17F in stimulated BT co-cultures. Pathways and targets are listed next to arrows that indicate selectivity for IL-17A, IL-17A and IL-17F, or IL-17F.
    Figure Legend Snippet: Summary of pathways and targets identified in a screen of pharmacologic modulators that positively or negatively regulate IL-17A and/or IL-17F in stimulated BT co-cultures. Pathways and targets are listed next to arrows that indicate selectivity for IL-17A, IL-17A and IL-17F, or IL-17F.

    Techniques Used:

    PGE1 and PGE2 stimulate production of IL-17A and impair production of IL-17F. Profiles of PGE1 (0.37–3.33 μM; A) and PGE2 (0.37–3.33 μM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. PGE1 and PGE2 increased production of IL-17A and IL-6, but inhibited IL-17F, IL-2, and TNFα production. Data are presented as the Log 10 ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.
    Figure Legend Snippet: PGE1 and PGE2 stimulate production of IL-17A and impair production of IL-17F. Profiles of PGE1 (0.37–3.33 μM; A) and PGE2 (0.37–3.33 μM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. PGE1 and PGE2 increased production of IL-17A and IL-6, but inhibited IL-17F, IL-2, and TNFα production. Data are presented as the Log 10 ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.

    Techniques Used:

    Erythromycin and Wortmannin at select doses inhibit IL-17A and IL-17F without modulating other parameters. Profiles of erythromycin (3.3–90 μM; A) or wortmannin (1.5–13.7 nM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. Erythromycin inhibited IL-17A and IL-17F production but did not affect B cell proliferation, secreted IgG, IL-2, IL-6, or TNFα, whereas wortmannin selectively inhibited IL-17A and IL-17F only at lower doses. Data are presented as the Log 10 ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.
    Figure Legend Snippet: Erythromycin and Wortmannin at select doses inhibit IL-17A and IL-17F without modulating other parameters. Profiles of erythromycin (3.3–90 μM; A) or wortmannin (1.5–13.7 nM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. Erythromycin inhibited IL-17A and IL-17F production but did not affect B cell proliferation, secreted IgG, IL-2, IL-6, or TNFα, whereas wortmannin selectively inhibited IL-17A and IL-17F only at lower doses. Data are presented as the Log 10 ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.

    Techniques Used:

    Torin-1, CP-690,550, and Axitinib are examples of compounds that regulated production of IL-17A, IL-17F or both IL-17A and IL-17F. Profiles of Torin-1 (0.460–12.3 nM; A), CP-690,550 (0.041–1.11 μM; B), or Axitinib (0.333–9 μM; C) added to BT co-cultures stimulated with α-IgM and SAg for three days. Parameters measured are indicated along the x-axis. Torin-1 inhibits IL-17A more potently than IL-17F (A), whereas CP-690,550 inhibits IL-17F more potently than IL-17A (B). Similarly, Axitinib blocks IL-17F production but does not affect IL-17A (C). Data are presented as the Log 10 ratio of drug-treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.
    Figure Legend Snippet: Torin-1, CP-690,550, and Axitinib are examples of compounds that regulated production of IL-17A, IL-17F or both IL-17A and IL-17F. Profiles of Torin-1 (0.460–12.3 nM; A), CP-690,550 (0.041–1.11 μM; B), or Axitinib (0.333–9 μM; C) added to BT co-cultures stimulated with α-IgM and SAg for three days. Parameters measured are indicated along the x-axis. Torin-1 inhibits IL-17A more potently than IL-17F (A), whereas CP-690,550 inhibits IL-17F more potently than IL-17A (B). Similarly, Axitinib blocks IL-17F production but does not affect IL-17A (C). Data are presented as the Log 10 ratio of drug-treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.

    Techniques Used:

    IL-17A and IL-17F are predominantly expressed by CD4 T cells in a BT co-culture model of human B cell-dependent T cell responses. FACS analysis gating strategy for cell types present in BT co-cultures after stimulation for three days with α-IgM and SAg (A). Gated cell populations are listed above the FACS plots and the percentage of cells present in each gate relative to the parent population is shown. To detect intracellular cytokine expression, BT co-cultures were treated for 5 hours with PMA, ionomycin, and monensin, and then stained with antibodies specific to IL-17A (A), IL-17F (B), or with an isotype control antibody common to the isotype of α-IL-17A and α-IL-17F (C).
    Figure Legend Snippet: IL-17A and IL-17F are predominantly expressed by CD4 T cells in a BT co-culture model of human B cell-dependent T cell responses. FACS analysis gating strategy for cell types present in BT co-cultures after stimulation for three days with α-IgM and SAg (A). Gated cell populations are listed above the FACS plots and the percentage of cells present in each gate relative to the parent population is shown. To detect intracellular cytokine expression, BT co-cultures were treated for 5 hours with PMA, ionomycin, and monensin, and then stained with antibodies specific to IL-17A (A), IL-17F (B), or with an isotype control antibody common to the isotype of α-IL-17A and α-IL-17F (C).

    Techniques Used: Co-Culture Assay, FACS, Expressing, Staining

    Examples of agents that potentiated production of IL-17A and IL-17F in BT co-cultures. Profiles of BW 245C (0.37–10 μM; A), LPS (0.1–100 ng/ml; B), or HKLM (10–10,000 U/ml; C) added to BT co-cultures stimulated with α-IgM and SAg for three days. BW245C and LPS enhance IL-17A and IL-6 production, while LPS also stimulates IL-17F, secreted IgG, IL-6, and TNFα. HKLM stimulates IL-17F more potently than IL-17A. Data are presented as the log 10 ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.
    Figure Legend Snippet: Examples of agents that potentiated production of IL-17A and IL-17F in BT co-cultures. Profiles of BW 245C (0.37–10 μM; A), LPS (0.1–100 ng/ml; B), or HKLM (10–10,000 U/ml; C) added to BT co-cultures stimulated with α-IgM and SAg for three days. BW245C and LPS enhance IL-17A and IL-6 production, while LPS also stimulates IL-17F, secreted IgG, IL-6, and TNFα. HKLM stimulates IL-17F more potently than IL-17A. Data are presented as the log 10 ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.

    Techniques Used:

    SR2211, an RORγ inverse agonist, and Calcitriol, a Vitamin D3 receptor agonist, block production of IL-17A and IL-17F in BT co-cultures. Profiles of SR2211 (0.370–10 μM; A) or Calcitriol (0.15–4.1 nM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. Parameters measured (B cell Proliferation, PBMC Cytotoxicity, Secreted IgG, sIL-17A, sIL-17F, sIL-2, sIL-6, and sTNF-alpha) are indicated along the x-axis. Data are presented as the Log 10 ratio of drug-treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.
    Figure Legend Snippet: SR2211, an RORγ inverse agonist, and Calcitriol, a Vitamin D3 receptor agonist, block production of IL-17A and IL-17F in BT co-cultures. Profiles of SR2211 (0.370–10 μM; A) or Calcitriol (0.15–4.1 nM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. Parameters measured (B cell Proliferation, PBMC Cytotoxicity, Secreted IgG, sIL-17A, sIL-17F, sIL-2, sIL-6, and sTNF-alpha) are indicated along the x-axis. Data are presented as the Log 10 ratio of drug-treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.

    Techniques Used: Blocking Assay

    38) Product Images from "Long-term PM2.5 exposure increases the risk of non-small cell lung cancer (NSCLC) progression by enhancing interleukin-17a (IL-17a)-regulated proliferation and metastasis"

    Article Title: Long-term PM2.5 exposure increases the risk of non-small cell lung cancer (NSCLC) progression by enhancing interleukin-17a (IL-17a)-regulated proliferation and metastasis

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.103319

    PM 2.5 enhances IL-17a expression in mice. ( A , B ) IL-17a contents in BALF and serum were measured by ELISA, respectively (n = 8). ( C ) RT-qPCR, ( D ) western blotting and ( E ) IHC analysis of IL-17a in lung tissues of mice challenged with PM 2.5 for the indicated time (n = 6). Scale bar, 100 μm. ( F , G ) IL-17a levels in the peripheral blood lymphocytes and in splenic lymphocytes were calculated using ELISA analysis (n = 8). All data are expressed as mean ± SEM. * p
    Figure Legend Snippet: PM 2.5 enhances IL-17a expression in mice. ( A , B ) IL-17a contents in BALF and serum were measured by ELISA, respectively (n = 8). ( C ) RT-qPCR, ( D ) western blotting and ( E ) IHC analysis of IL-17a in lung tissues of mice challenged with PM 2.5 for the indicated time (n = 6). Scale bar, 100 μm. ( F , G ) IL-17a levels in the peripheral blood lymphocytes and in splenic lymphocytes were calculated using ELISA analysis (n = 8). All data are expressed as mean ± SEM. * p

    Techniques Used: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Immunohistochemistry

    IL-17a knockout alleviates pulmonary injury and cancer stem cell properties in mice following PM 2.5 exposure for 3 months. ( A ) H E staining (up panel) and Masson trichrome staining (down panel) of lung sections from IL-17a +/+ and IL-17a -/- mice challenged with or without PM 2.5 for 3 months (n = 6). Scale bar, 100 μm. ( B ) Protein levels in BALF were measured (n = 8). ( C ) The total number of cells in BALF was assessed (n = 8). ( D ) The number of neutrophils in BALF was measured (n = 8). ( E ) Serum TNF-α and IL-6 levels in mice were measured by ELISA (n = 8). ( F ) TNF-α and IL-6 mRNA levels in the pulmonary samples were determined using RT-qPCR analysis (n = 4). ( G ) Fibrosis-associated genes as shown were tested using RT-qPCR analysis (n = 4). ( H ) Genes associated with lung cancer progression were calculated using RT-qPCR analysis (n = 4). ( I ) IHC staining of c-Myc and SOX2 in pulmonary sections from the indicated groups of mice (n = 6). Scale bar, 100 μm. All data are expressed as mean ± SEM. * p
    Figure Legend Snippet: IL-17a knockout alleviates pulmonary injury and cancer stem cell properties in mice following PM 2.5 exposure for 3 months. ( A ) H E staining (up panel) and Masson trichrome staining (down panel) of lung sections from IL-17a +/+ and IL-17a -/- mice challenged with or without PM 2.5 for 3 months (n = 6). Scale bar, 100 μm. ( B ) Protein levels in BALF were measured (n = 8). ( C ) The total number of cells in BALF was assessed (n = 8). ( D ) The number of neutrophils in BALF was measured (n = 8). ( E ) Serum TNF-α and IL-6 levels in mice were measured by ELISA (n = 8). ( F ) TNF-α and IL-6 mRNA levels in the pulmonary samples were determined using RT-qPCR analysis (n = 4). ( G ) Fibrosis-associated genes as shown were tested using RT-qPCR analysis (n = 4). ( H ) Genes associated with lung cancer progression were calculated using RT-qPCR analysis (n = 4). ( I ) IHC staining of c-Myc and SOX2 in pulmonary sections from the indicated groups of mice (n = 6). Scale bar, 100 μm. All data are expressed as mean ± SEM. * p

    Techniques Used: Knock-Out, Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Immunohistochemistry

    PM 2.5 -promoted tumor growth and metastasis are associated with IL-17a in xenograft mouse models. ( A ) Representative images of tumor samples isolated from each group of mice as indicated (n = 6). Scale bar, 1 cm. ( B ) Tumor volume was measured (n = 6). ( C ) Tumor weight was recorded (n = 6). ( D ) The body weight of mice was recorded (n = 6). ( E ) IHC staining was used to determine KI-67 and SOX2 expression levels in the tumor sections (n = 4). Scale bar, 100 μm. ( F ) H E staining of pulmonary tissues (n = 4; Scale bar, 100 μm) and ( G ) pictures of lung samples isolated from the indicated groups of mice to calculate the metastatic nodules on the surface of lungs (n = 6). ( H ) The number of lung metastatic nodules was quantified (n = 6). ( I ) RT-qPCR analysis was used to calculate the expression of metastasis-associated genes as displayed (n = 4). All data are expressed as mean ± SEM. * p
    Figure Legend Snippet: PM 2.5 -promoted tumor growth and metastasis are associated with IL-17a in xenograft mouse models. ( A ) Representative images of tumor samples isolated from each group of mice as indicated (n = 6). Scale bar, 1 cm. ( B ) Tumor volume was measured (n = 6). ( C ) Tumor weight was recorded (n = 6). ( D ) The body weight of mice was recorded (n = 6). ( E ) IHC staining was used to determine KI-67 and SOX2 expression levels in the tumor sections (n = 4). Scale bar, 100 μm. ( F ) H E staining of pulmonary tissues (n = 4; Scale bar, 100 μm) and ( G ) pictures of lung samples isolated from the indicated groups of mice to calculate the metastatic nodules on the surface of lungs (n = 6). ( H ) The number of lung metastatic nodules was quantified (n = 6). ( I ) RT-qPCR analysis was used to calculate the expression of metastasis-associated genes as displayed (n = 4). All data are expressed as mean ± SEM. * p

    Techniques Used: Isolation, Mouse Assay, Immunohistochemistry, Staining, Expressing, Quantitative RT-PCR

    PM 2.5 elevates the proliferation in NSCLC cells. ( A – E ) Th17 cells were treated with 100 μg/cm 2 of PM 2.5 for 24 h, and then all cells and supernatants were collected for the analysis. ( A ) TNF-α and IL-6 levels in the supernatants were measured using ELISA. ( B ) IL-17a contents in the collected supernatants were calculated using ELISA. ( C ) RT-qPCR and ( D ) western blot analysis were used to measure IL-17a expression levels in the harvested cells. ( E ) IF staining of IL-17a in the harvested cells. Scale bar, 20 μm. ( F ) Th17 cells were treated with 100 μg/cm 2 of PM 2.5 for 24 h, and then the conditional medium was collected, and mixed with fresh RPMI1640 absolute medium at 1:3. The composed culture medium was exposed to A549 and H1350 cells for 12, 24, 48 or 72 h. Then, the NSCLC cells were collected for cell proliferation analysis using CCK-8 analysis. ( G , H ) Th17 cells were incubated with 100 μg/cm 2 of PM 2.5 for 24 h, and then the conditional medium was collected, and mixed with fresh RPMI1640 absolute medium at 1:3. Then, the composed culture medium was subjected to A549 and H1350 cells for another 24 h. Subsequently, all cells were harvested to assess the cell proliferation using colony formation and EdU assays. Scale bar, 100 μm. Quantification of colony formation assay and EdU was exhibited. All data are expressed as mean ± SEM. * p
    Figure Legend Snippet: PM 2.5 elevates the proliferation in NSCLC cells. ( A – E ) Th17 cells were treated with 100 μg/cm 2 of PM 2.5 for 24 h, and then all cells and supernatants were collected for the analysis. ( A ) TNF-α and IL-6 levels in the supernatants were measured using ELISA. ( B ) IL-17a contents in the collected supernatants were calculated using ELISA. ( C ) RT-qPCR and ( D ) western blot analysis were used to measure IL-17a expression levels in the harvested cells. ( E ) IF staining of IL-17a in the harvested cells. Scale bar, 20 μm. ( F ) Th17 cells were treated with 100 μg/cm 2 of PM 2.5 for 24 h, and then the conditional medium was collected, and mixed with fresh RPMI1640 absolute medium at 1:3. The composed culture medium was exposed to A549 and H1350 cells for 12, 24, 48 or 72 h. Then, the NSCLC cells were collected for cell proliferation analysis using CCK-8 analysis. ( G , H ) Th17 cells were incubated with 100 μg/cm 2 of PM 2.5 for 24 h, and then the conditional medium was collected, and mixed with fresh RPMI1640 absolute medium at 1:3. Then, the composed culture medium was subjected to A549 and H1350 cells for another 24 h. Subsequently, all cells were harvested to assess the cell proliferation using colony formation and EdU assays. Scale bar, 100 μm. Quantification of colony formation assay and EdU was exhibited. All data are expressed as mean ± SEM. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Expressing, Staining, CCK-8 Assay, Incubation, Colony Assay

    IL-17a expression is up-regulated in patients with lung cancer. ( A ) Images showing IL-17a expression levels in tumor samples from patients with lung cancer using IHC. Scale bar, 100 μm. ( B ) IL-17a expression levels in the primary lung tumor specimens and the adjacent normal tissue samples by RT-qPCR (n = 48). ( C ) Western blot analysis of IL-17a expression levels in the primary lung tumor specimens and the paired lung alveolar tissue samples (n = 8). ( D ) Kaplan-Meier survival curves for overall survival (OS) in lung cancer patients according to the expression levels of IL-17a. All data are expressed as mean ± SEM. ** p
    Figure Legend Snippet: IL-17a expression is up-regulated in patients with lung cancer. ( A ) Images showing IL-17a expression levels in tumor samples from patients with lung cancer using IHC. Scale bar, 100 μm. ( B ) IL-17a expression levels in the primary lung tumor specimens and the adjacent normal tissue samples by RT-qPCR (n = 48). ( C ) Western blot analysis of IL-17a expression levels in the primary lung tumor specimens and the paired lung alveolar tissue samples (n = 8). ( D ) Kaplan-Meier survival curves for overall survival (OS) in lung cancer patients according to the expression levels of IL-17a. All data are expressed as mean ± SEM. ** p

    Techniques Used: Expressing, Immunohistochemistry, Quantitative RT-PCR, Western Blot

    IL-17a treatment promotes the proliferation and EMT in NSCLC cells. ( A – G ) A549 and H1350 cells were treated with or without IL-17a (100 ng/ml) for 24 h, and then were collected for the following studies. ( A ) CCK-8 analysis was used to determine the cell proliferative activity. ( B ) Colony formation and EdU assays were used to determine the cell proliferative condition. Scale bar, 100 μm. ( C ) Quantification of the number of cells by colony formation and EdU analysis. ( D ) Transwell analysis was used to calculate the number of cells in migration and invasion. Scale bar, 100 μm. ( E ) Quantification of the counts of the migrated and invaded cells. ( F ) RT-qPCR analysis was used to measure EMT-associated genes in A549 and H1350 cells. ( G ) IF staining of N-cadherin in A549 and H1350 cells. Scale bar, 50 μm. All data are expressed as mean ± SEM. * p
    Figure Legend Snippet: IL-17a treatment promotes the proliferation and EMT in NSCLC cells. ( A – G ) A549 and H1350 cells were treated with or without IL-17a (100 ng/ml) for 24 h, and then were collected for the following studies. ( A ) CCK-8 analysis was used to determine the cell proliferative activity. ( B ) Colony formation and EdU assays were used to determine the cell proliferative condition. Scale bar, 100 μm. ( C ) Quantification of the number of cells by colony formation and EdU analysis. ( D ) Transwell analysis was used to calculate the number of cells in migration and invasion. Scale bar, 100 μm. ( E ) Quantification of the counts of the migrated and invaded cells. ( F ) RT-qPCR analysis was used to measure EMT-associated genes in A549 and H1350 cells. ( G ) IF staining of N-cadherin in A549 and H1350 cells. Scale bar, 50 μm. All data are expressed as mean ± SEM. * p

    Techniques Used: CCK-8 Assay, Activity Assay, Migration, Quantitative RT-PCR, Staining

    39) Product Images from "Targeting IL-17A in Multiple Myeloma: A Potential Novel Therapeutic Approach in Myeloma"

    Article Title: Targeting IL-17A in Multiple Myeloma: A Potential Novel Therapeutic Approach in Myeloma

    Journal: Leukemia

    doi: 10.1038/leu.2015.228

    Inhibition of tumor growth and prevention of bone resorption by AIN-457, IL-17A antibody in SCID human myeloma model A) SCID mice were transplanted with human fetal bones, and after four weeks, myeloma cells were injected into the bones. One group was treated with vehicle with isotype control antibody and another group of mice were treated subcutaneously with AIN-457 (10µg/ml-/mouse/injection) for four weeks following first detection of tumor by measuring human soluble IL-6R in the serum. Serum samples were collected weekly and level of humans IL-6R was measured by ELISA. Baseline values before treatment were not significantly different among groups. Representative of two experiments is shown (p
    Figure Legend Snippet: Inhibition of tumor growth and prevention of bone resorption by AIN-457, IL-17A antibody in SCID human myeloma model A) SCID mice were transplanted with human fetal bones, and after four weeks, myeloma cells were injected into the bones. One group was treated with vehicle with isotype control antibody and another group of mice were treated subcutaneously with AIN-457 (10µg/ml-/mouse/injection) for four weeks following first detection of tumor by measuring human soluble IL-6R in the serum. Serum samples were collected weekly and level of humans IL-6R was measured by ELISA. Baseline values before treatment were not significantly different among groups. Representative of two experiments is shown (p

    Techniques Used: Inhibition, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

    IL-17A knockdown decreases myeloma cell-number and their ability to produce IL-6 in a co-culture with bone-marrow stromal cells Myeloma cell-line (RPMI 8226) cells were used to transduce human IL-17A siRNA according to manufactures recommendations to determine the influence on myeloma cell-proliferation and their ability to produce IL-6 when co-cultured with bone-marrow stromal cells. Cell lysates were analyzed using immuno-blots to assess decrease in intra-cellular protein expression of IL-17A (A). Cell-proliferation was analyzed by counting live cells following transfection. Representative study results of three different experiments were shown. MM cell-line (U266) was used to transduce human IL-17A shRNA according to manufactures recommendations to determine the influence on myeloma cell-expression and –proliferation (B). Representative study results of three different experiments were shown. Colony formation was evaluated using MethoCult agar plates following transfection with siRNA or shRNA (C). Representative study results of five different experiments were shown. IL-6 production was measured with standard ELISA following co-culturing myeloma cells with or without transfected MM cells with BMSC (D). Representative study results of three different experiments were shown.
    Figure Legend Snippet: IL-17A knockdown decreases myeloma cell-number and their ability to produce IL-6 in a co-culture with bone-marrow stromal cells Myeloma cell-line (RPMI 8226) cells were used to transduce human IL-17A siRNA according to manufactures recommendations to determine the influence on myeloma cell-proliferation and their ability to produce IL-6 when co-cultured with bone-marrow stromal cells. Cell lysates were analyzed using immuno-blots to assess decrease in intra-cellular protein expression of IL-17A (A). Cell-proliferation was analyzed by counting live cells following transfection. Representative study results of three different experiments were shown. MM cell-line (U266) was used to transduce human IL-17A shRNA according to manufactures recommendations to determine the influence on myeloma cell-expression and –proliferation (B). Representative study results of three different experiments were shown. Colony formation was evaluated using MethoCult agar plates following transfection with siRNA or shRNA (C). Representative study results of five different experiments were shown. IL-6 production was measured with standard ELISA following co-culturing myeloma cells with or without transfected MM cells with BMSC (D). Representative study results of three different experiments were shown.

    Techniques Used: Co-Culture Assay, Transduction, Cell Culture, Western Blot, Expressing, Transfection, shRNA, Enzyme-linked Immunosorbent Assay

    Down regulation of BMSC-mediated MM cell-growth by anti-IL-17A antibody via blockade of IL-6 production (A) MM cell lines (N=7) were cultured with BMSCs in the presence of isotype control antibody or anti-IL-17A mAb and proliferation measured by 3 H-thymidine incorporation after 3 days and presented as percentage of inhibition in proliferation with isotype control antibody. (B) BMSC was cultured for three days in the presence or absence of IL-17A (100ng/ml), IL-21, IL-22, IL-23, IL-27 and LPS at 10ng/ml concentration. IL-6 production was measured by standard ELISA (R D Systems, Minneapolis, MN). Bar graph represents mean ± SEM of the data (N=4) calculated as percent of increase or decrease in IL-6 levels in culture supernatants compared to IL-6 levels (as endogenous IL-6 production) obtained from BMSC alone without any stimulation. (C) BMSC in the presence (right or absence (left) of MM cell-lines was cultured with isotype control antibody, IL-17A, or anti-IL-17A mAb or in combination with IL-17A + anti-IL-17A mAb and IL-6 levels were measured in culture supernatants by ELISA. Bar graph represents mean ± SEM of the data calculated as percent of increase or decrease in IL-6 levels in culture supernatants compared to IL-6 levels (as endogenous IL-6 production) obtained from BMSC alone with isotype control antibody without any stimulation. (D) Serum-starved MM cells were labeled with calcein AM, washed, and added to BMSC-coated plates in the presence of isotype control antibody or anti-IL-17A mAb for 4 hours and non-adherent cells were removed by washing. Adhesion was measured by measuring the absorbance using 492/520 nm filter set with a fluorescence plate reader. Results represent mean ± SEM of 3 independent experiments performed in triplicate. The absorbance values obtained with isotype control antibody was considered as 100% and percentage of inhibition was calculated for anti-IL-17A treatment. (E) The fetal bone chips with MM cells were incubated in the presence of isotype control antibody or anti-IL-17A mAb for 2 days for ex-vivo for the evaluation of IL-6 production that is measured by ELISA (R D Systems). Bar graph represents mean ± SEM of the data calculated as percent of IL-6 levels (as endogenous IL-6 production) in culture supernatants from BMSC alone with isotype control antibody. (F) The immunohistochemical analysis of bones was performed by staining them with anti-CD138 antibody. Arrows indicates presence bright CD138+cells. (G) Myeloma cells were co-cultured with BMSC in the presence of IL-17A with or without cell-signaling inhibitors (JAK2, STAT3, JNK, MEK, NFkB and PI3 inhibitors) and IL-6 production was measured with standard ELISA. *P
    Figure Legend Snippet: Down regulation of BMSC-mediated MM cell-growth by anti-IL-17A antibody via blockade of IL-6 production (A) MM cell lines (N=7) were cultured with BMSCs in the presence of isotype control antibody or anti-IL-17A mAb and proliferation measured by 3 H-thymidine incorporation after 3 days and presented as percentage of inhibition in proliferation with isotype control antibody. (B) BMSC was cultured for three days in the presence or absence of IL-17A (100ng/ml), IL-21, IL-22, IL-23, IL-27 and LPS at 10ng/ml concentration. IL-6 production was measured by standard ELISA (R D Systems, Minneapolis, MN). Bar graph represents mean ± SEM of the data (N=4) calculated as percent of increase or decrease in IL-6 levels in culture supernatants compared to IL-6 levels (as endogenous IL-6 production) obtained from BMSC alone without any stimulation. (C) BMSC in the presence (right or absence (left) of MM cell-lines was cultured with isotype control antibody, IL-17A, or anti-IL-17A mAb or in combination with IL-17A + anti-IL-17A mAb and IL-6 levels were measured in culture supernatants by ELISA. Bar graph represents mean ± SEM of the data calculated as percent of increase or decrease in IL-6 levels in culture supernatants compared to IL-6 levels (as endogenous IL-6 production) obtained from BMSC alone with isotype control antibody without any stimulation. (D) Serum-starved MM cells were labeled with calcein AM, washed, and added to BMSC-coated plates in the presence of isotype control antibody or anti-IL-17A mAb for 4 hours and non-adherent cells were removed by washing. Adhesion was measured by measuring the absorbance using 492/520 nm filter set with a fluorescence plate reader. Results represent mean ± SEM of 3 independent experiments performed in triplicate. The absorbance values obtained with isotype control antibody was considered as 100% and percentage of inhibition was calculated for anti-IL-17A treatment. (E) The fetal bone chips with MM cells were incubated in the presence of isotype control antibody or anti-IL-17A mAb for 2 days for ex-vivo for the evaluation of IL-6 production that is measured by ELISA (R D Systems). Bar graph represents mean ± SEM of the data calculated as percent of IL-6 levels (as endogenous IL-6 production) in culture supernatants from BMSC alone with isotype control antibody. (F) The immunohistochemical analysis of bones was performed by staining them with anti-CD138 antibody. Arrows indicates presence bright CD138+cells. (G) Myeloma cells were co-cultured with BMSC in the presence of IL-17A with or without cell-signaling inhibitors (JAK2, STAT3, JNK, MEK, NFkB and PI3 inhibitors) and IL-6 production was measured with standard ELISA. *P

    Techniques Used: Cell Culture, Inhibition, Concentration Assay, Enzyme-linked Immunosorbent Assay, Labeling, Fluorescence, Incubation, Ex Vivo, Immunohistochemistry, Staining

    Reduced osteoclast cell numbers by the antibody Normal BM cells were cultured for three weeks in osteoclast supporting-medium (consisting 25ng/ml of macrophage colony-stimulating factor and 25 ng/ml of receptor activator of nuclear factor kappa-B ligand) with isotype control antibody (as positive control) or anti-IL-17A antibody and the tartrate-resistant acid phosphatage (TRAP+) multinucleated osteoclast cells were stained and cell-numbers were counted. Cells were cultured without osteoclast-supporting-medium as negative control. Images were obtained at 10 magnifications with microscope (Eclipse TS100, Nikon instruments, Melville, NY, USA) with spot insight camera. TRAP-positive cells containing 5 or more nuclei/cell were enumerated using image J 1.45 software (NIH, Bethesda, MD, USA). A representative image was depicted (Figure A) and composite results from three experiments were shown in bar graph Figure B). Star indicates statistical signifies (p
    Figure Legend Snippet: Reduced osteoclast cell numbers by the antibody Normal BM cells were cultured for three weeks in osteoclast supporting-medium (consisting 25ng/ml of macrophage colony-stimulating factor and 25 ng/ml of receptor activator of nuclear factor kappa-B ligand) with isotype control antibody (as positive control) or anti-IL-17A antibody and the tartrate-resistant acid phosphatage (TRAP+) multinucleated osteoclast cells were stained and cell-numbers were counted. Cells were cultured without osteoclast-supporting-medium as negative control. Images were obtained at 10 magnifications with microscope (Eclipse TS100, Nikon instruments, Melville, NY, USA) with spot insight camera. TRAP-positive cells containing 5 or more nuclei/cell were enumerated using image J 1.45 software (NIH, Bethesda, MD, USA). A representative image was depicted (Figure A) and composite results from three experiments were shown in bar graph Figure B). Star indicates statistical signifies (p

    Techniques Used: Cell Culture, Positive Control, Staining, Negative Control, Microscopy, Software

    Effect of AIN-457 humanized antibody on myeloma growth using three mouse models without tumor microenvironment (A) SCID mice were injected with myeloma cells by sc route. Mice were injected in 2 groups (N=3). One group was injected with myeloma cells with isotype control antibody; and second of mice injected with MM cells in anti-IL-17A mAb (10µg/ml). Tumor volume was measured at indicated time intervals from the MM cell-injections. Representative of two experiments is shown (p
    Figure Legend Snippet: Effect of AIN-457 humanized antibody on myeloma growth using three mouse models without tumor microenvironment (A) SCID mice were injected with myeloma cells by sc route. Mice were injected in 2 groups (N=3). One group was injected with myeloma cells with isotype control antibody; and second of mice injected with MM cells in anti-IL-17A mAb (10µg/ml). Tumor volume was measured at indicated time intervals from the MM cell-injections. Representative of two experiments is shown (p

    Techniques Used: Mouse Assay, Injection

    Analysis of IL-17A expression in myeloma cells The MM patient samples were collected after informed consent in accordance with the Declaration of Helsinki and approved by the institutional review board (IRB) from Dana-Farber Cancer Institute. Healthy donor bone marrow samples were obtained from AllCells (Emeryville, CA). MM primary cells were purified as described earlier5. RNA was isolated from MM cell-lines (A) and purified MM primary cells (B). Quantitative PCR was performed for IL-17A using 7900HT from Applied Biosystems. Representative experimental results from three different experiments were presented as relative expression value in comparison with GAPDH. For immuno-blot experiments, total cell lysates were prepared from MM cell-lines (C) and purified CD138+ MM primary cells (D), and separated by electrophoresis on 5% to 20% polyacrylamide gradient gels. Samples were probed with anti-sera to IL-17A and GAPDH as indicated. Representative immunoblot of three different experiments was shown. Myeloma cell lines were stained with isotype control antibody or anti–IL-17A antibody and analyzed by confocal microscopy. One representative cell line of 4 experiments was shown at 640 magnifications (E).
    Figure Legend Snippet: Analysis of IL-17A expression in myeloma cells The MM patient samples were collected after informed consent in accordance with the Declaration of Helsinki and approved by the institutional review board (IRB) from Dana-Farber Cancer Institute. Healthy donor bone marrow samples were obtained from AllCells (Emeryville, CA). MM primary cells were purified as described earlier5. RNA was isolated from MM cell-lines (A) and purified MM primary cells (B). Quantitative PCR was performed for IL-17A using 7900HT from Applied Biosystems. Representative experimental results from three different experiments were presented as relative expression value in comparison with GAPDH. For immuno-blot experiments, total cell lysates were prepared from MM cell-lines (C) and purified CD138+ MM primary cells (D), and separated by electrophoresis on 5% to 20% polyacrylamide gradient gels. Samples were probed with anti-sera to IL-17A and GAPDH as indicated. Representative immunoblot of three different experiments was shown. Myeloma cell lines were stained with isotype control antibody or anti–IL-17A antibody and analyzed by confocal microscopy. One representative cell line of 4 experiments was shown at 640 magnifications (E).

    Techniques Used: Expressing, Purification, Isolation, Real-time Polymerase Chain Reaction, Electrophoresis, Staining, Confocal Microscopy

    Inhibitory activity of anti-IL-17A antibody on myeloma cell proliferation (A) Myeloma cell lines alone (N=8) were incubated with isotype control antibody or anti-IL-17A mAb to measure proliferation by 3 H-thymidine incorporation after 3 days. Data is presented as percentage inhibition in proliferation in presence of anti-IL-17A mAb compared with isotype control antibody and showed as mean ± SEM. (B) Myeloma cell lines alone (N=5) were incubated with isotype control antibody or anti-IL-17A mAb to measure metabolic activity by MTT assay after 3 days. Data is presented as percentage inhibition in proliferation in presence of anti-IL-17A mAb compared with isotype control antibody and showed as mean ± SEM. (C) Myeloma cell lines (U266) were cultured in methocult agar plates in the presence of isotype control antibody or anti-IL-17A mAb. Representative photomicrograph and results (N=3) are presented. Photographs were obtained using a Nikon TE200 microscope (40× objective) with attached camera (Nikon) at room temperature (total magnification 200) and analyzed with Metafluor software (Molecular Devices). The number of colonies were counted in unit area and presented as mean ± SEM. (D) Primary MM cells (N=4) were cultured in methocult agar plates in the presence of isotype control antibody or anti-IL-17A mAb. The number of colonies were counted in unit area and presented as mean ± SEM. *P
    Figure Legend Snippet: Inhibitory activity of anti-IL-17A antibody on myeloma cell proliferation (A) Myeloma cell lines alone (N=8) were incubated with isotype control antibody or anti-IL-17A mAb to measure proliferation by 3 H-thymidine incorporation after 3 days. Data is presented as percentage inhibition in proliferation in presence of anti-IL-17A mAb compared with isotype control antibody and showed as mean ± SEM. (B) Myeloma cell lines alone (N=5) were incubated with isotype control antibody or anti-IL-17A mAb to measure metabolic activity by MTT assay after 3 days. Data is presented as percentage inhibition in proliferation in presence of anti-IL-17A mAb compared with isotype control antibody and showed as mean ± SEM. (C) Myeloma cell lines (U266) were cultured in methocult agar plates in the presence of isotype control antibody or anti-IL-17A mAb. Representative photomicrograph and results (N=3) are presented. Photographs were obtained using a Nikon TE200 microscope (40× objective) with attached camera (Nikon) at room temperature (total magnification 200) and analyzed with Metafluor software (Molecular Devices). The number of colonies were counted in unit area and presented as mean ± SEM. (D) Primary MM cells (N=4) were cultured in methocult agar plates in the presence of isotype control antibody or anti-IL-17A mAb. The number of colonies were counted in unit area and presented as mean ± SEM. *P

    Techniques Used: Activity Assay, Incubation, Inhibition, MTT Assay, Cell Culture, Microscopy, Software

    40) Product Images from "IL-17A Influences Essential Functions of the Monocyte/Macrophage Lineage and Is Involved in Advanced Murine and Human Atherosclerosis"

    Article Title: IL-17A Influences Essential Functions of the Monocyte/Macrophage Lineage and Is Involved in Advanced Murine and Human Atherosclerosis

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1400181

    Cellular composition in atherosclerotic lesions of Apoe −/− mice. ( A ) Representative photomicrographs of immunohistochemistry stainings of T cells (CD3, original magnification ×40), Mac-2 + cells (original magnification ×40), CD206 + cells (original magnification ×40), and B cells (B220; original magnification ×40). ( B ) Quantitative analysis of plaque inflammation are presented in 42-wk-old Apoe −/− mice with ( n = 12–15) and without IL-17A mAb treatment ( n = 8–10) (as well as 26-wk-old baseline mice, n = 4 to 5). Results are presented as dot plots displaying mean.
    Figure Legend Snippet: Cellular composition in atherosclerotic lesions of Apoe −/− mice. ( A ) Representative photomicrographs of immunohistochemistry stainings of T cells (CD3, original magnification ×40), Mac-2 + cells (original magnification ×40), CD206 + cells (original magnification ×40), and B cells (B220; original magnification ×40). ( B ) Quantitative analysis of plaque inflammation are presented in 42-wk-old Apoe −/− mice with ( n = 12–15) and without IL-17A mAb treatment ( n = 8–10) (as well as 26-wk-old baseline mice, n = 4 to 5). Results are presented as dot plots displaying mean.

    Techniques Used: Mouse Assay, Immunohistochemistry

    Effects of IL-17A on monocytes and monocyte-derived cells. ( A and B ) Foam cell formation assay. (A) Representative photomicrographs of monocyte-derived macrophages incubated with oxLDL in addition to IL-17A in different concentrations are shown (original magnification ×20). Representative Oil Red O staining of different groups are shown: no oxLDL (control), oxLDL, and oxLDL plus IL-17A. (B) Data are given as mean ± SEM of five independent experiments. ( C ) oxLDL uptake assay. Flow cytometry analysis of DiI-labeled oxLDL uptake (in addition to IL-17A incubation) of monocyte-derived macrophages: solid green line (oxLDL) and dotted pink line (oxLDL+IL-17A). Histogram represents one out of three independent experiments. ( D ) For T cell activation assays, macrophages or DCs were incubated in 96-well round-bottom plates and stimulated with oxLDL (Sanbio), IL-17A (R D Systems), or oxLDL in combination with IL-17A. After 6-h preincubation, CD4 + T cells were added in a ratio of 1:5 and stimulated for 8 h. Expression of activation-associated cytokines and transcription factors in CD4 + for details). Representative analysis of T-box21 (T-bet), IL2 , and IFNγ are shown.
    Figure Legend Snippet: Effects of IL-17A on monocytes and monocyte-derived cells. ( A and B ) Foam cell formation assay. (A) Representative photomicrographs of monocyte-derived macrophages incubated with oxLDL in addition to IL-17A in different concentrations are shown (original magnification ×20). Representative Oil Red O staining of different groups are shown: no oxLDL (control), oxLDL, and oxLDL plus IL-17A. (B) Data are given as mean ± SEM of five independent experiments. ( C ) oxLDL uptake assay. Flow cytometry analysis of DiI-labeled oxLDL uptake (in addition to IL-17A incubation) of monocyte-derived macrophages: solid green line (oxLDL) and dotted pink line (oxLDL+IL-17A). Histogram represents one out of three independent experiments. ( D ) For T cell activation assays, macrophages or DCs were incubated in 96-well round-bottom plates and stimulated with oxLDL (Sanbio), IL-17A (R D Systems), or oxLDL in combination with IL-17A. After 6-h preincubation, CD4 + T cells were added in a ratio of 1:5 and stimulated for 8 h. Expression of activation-associated cytokines and transcription factors in CD4 + for details). Representative analysis of T-box21 (T-bet), IL2 , and IFNγ are shown.

    Techniques Used: Derivative Assay, Tube Formation Assay, Incubation, Staining, Flow Cytometry, Cytometry, Labeling, Activation Assay, Expressing

    Effects of IL-17A mAb treatment on atherosclerotic plaques in Apoe −/− mice. Representative Oil Red O immunohistostainings (original magnification ×4) from aortic root of control [ n = 10; ( A )]; IL-17A mAb-treated mice [ n = 15; ( B )] and baseline [ n = 5; ( C )] are shown. ( D – F ) Morphometric quantification of cross-sectional area (μm 2 ), fractional stenosis (%), and maximum stenosis (%) of late-stage atherosclerotic lesions were compared. Results are shown as dot plots displaying mean.
    Figure Legend Snippet: Effects of IL-17A mAb treatment on atherosclerotic plaques in Apoe −/− mice. Representative Oil Red O immunohistostainings (original magnification ×4) from aortic root of control [ n = 10; ( A )]; IL-17A mAb-treated mice [ n = 15; ( B )] and baseline [ n = 5; ( C )] are shown. ( D – F ) Morphometric quantification of cross-sectional area (μm 2 ), fractional stenosis (%), and maximum stenosis (%) of late-stage atherosclerotic lesions were compared. Results are shown as dot plots displaying mean.

    Techniques Used: Mouse Assay

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    Mouse Assay:

    Article Title: IL-17RA Signaling Amplifies Antibody-Induced Arthritis
    Article Snippet: IL-17A induces neutrophil-active chemokines and MMPs in FLS in vitro Synovial cells play a crucial active role in the development of RA, and IL-17A has previously been shown to directly activate synovial cells in vitro , . .. In order to determine which of the genes downregulated in the ankles of Il17ra−/− mice might be directly regulated by IL-17RA signaling, murine fibroblast-like synoviocytes (FLS) were either left unstimulated or stimulated with 100 ng/ml IL-17A for 16 h. RNA was then isolated and gene expression assessed by qPCR. .. IL-17RA signaling in FLS induced a number of genes, which were down-regulated in the joints of Il17ra−/− mice, suggesting that IL-17A directly regulated these genes in vivo .

    Isolation:

    Article Title: IL-17RA Signaling Amplifies Antibody-Induced Arthritis
    Article Snippet: IL-17A induces neutrophil-active chemokines and MMPs in FLS in vitro Synovial cells play a crucial active role in the development of RA, and IL-17A has previously been shown to directly activate synovial cells in vitro , . .. In order to determine which of the genes downregulated in the ankles of Il17ra−/− mice might be directly regulated by IL-17RA signaling, murine fibroblast-like synoviocytes (FLS) were either left unstimulated or stimulated with 100 ng/ml IL-17A for 16 h. RNA was then isolated and gene expression assessed by qPCR. .. IL-17RA signaling in FLS induced a number of genes, which were down-regulated in the joints of Il17ra−/− mice, suggesting that IL-17A directly regulated these genes in vivo .

    Expressing:

    Article Title: IL-17RA Signaling Amplifies Antibody-Induced Arthritis
    Article Snippet: IL-17A induces neutrophil-active chemokines and MMPs in FLS in vitro Synovial cells play a crucial active role in the development of RA, and IL-17A has previously been shown to directly activate synovial cells in vitro , . .. In order to determine which of the genes downregulated in the ankles of Il17ra−/− mice might be directly regulated by IL-17RA signaling, murine fibroblast-like synoviocytes (FLS) were either left unstimulated or stimulated with 100 ng/ml IL-17A for 16 h. RNA was then isolated and gene expression assessed by qPCR. .. IL-17RA signaling in FLS induced a number of genes, which were down-regulated in the joints of Il17ra−/− mice, suggesting that IL-17A directly regulated these genes in vivo .

    Real-time Polymerase Chain Reaction:

    Article Title: IL-17RA Signaling Amplifies Antibody-Induced Arthritis
    Article Snippet: IL-17A induces neutrophil-active chemokines and MMPs in FLS in vitro Synovial cells play a crucial active role in the development of RA, and IL-17A has previously been shown to directly activate synovial cells in vitro , . .. In order to determine which of the genes downregulated in the ankles of Il17ra−/− mice might be directly regulated by IL-17RA signaling, murine fibroblast-like synoviocytes (FLS) were either left unstimulated or stimulated with 100 ng/ml IL-17A for 16 h. RNA was then isolated and gene expression assessed by qPCR. .. IL-17RA signaling in FLS induced a number of genes, which were down-regulated in the joints of Il17ra−/− mice, suggesting that IL-17A directly regulated these genes in vivo .

    Enzyme-linked Immunosorbent Assay:

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  • 93
    R&D Systems mouse recombinant il 17a
    DI inhibits IL-17-mediated IκBζ induction in keratinocytes and ameliorates psoriatic pathology a , b , Western blot of IκBζ expression in DI-treated, <t>IL-17A-stimulated</t> primary mouse ( a ) and human ( b . c , d , mRNA expression in DI-treated, IL-17A-stimulated (4 h) primary mouse ( c ) and human ( d ) keratinocytes, mean ± s.e.m., n = 3 mice or donors. e , Schematic of DI administration in a psoriasis model. f , Ear histology of control mice (left), IMQ-treated mice (middle) and mice treated with both IMQ and DI (right). Scale bars, 100 µm. Representative of six mice in two experiments. g , Quantification of the histology results in f , showing the relative change in ear thickness. Mean ± s.e.m., n = 6 mice. h , mRNA expression to show the induction of the indicated IκBζ target genes in ear tissue, mean ± s.e.m., n = 3 mice. Statistical tests used were two-tailed t -tests.
    Mouse Recombinant Il 17a, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems il 17a
    Neutrophils are reduced in the joints of Il17ra −/− mice and are unresponsive to direct stimulation with <t>IL-17A.</t> 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
    Il 17a, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems goat anti mouse il 17
    Tbet deficiency prevents induction of transplantation tolerance by combined co-stimulation blockade with persistent T17/Th2 skewing of alloantigen specific cytokine profile, and PMN, CD4, and <t>IL-17-producing</t> CD8 T cell infiltration. ( A ) Kaplan-Meier survival
    Goat Anti Mouse Il 17, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems il 17f
    Immunostaining for chemoattractant receptor CXCR1/IL-8RA and <t>IL-17F</t> in representative samples of prostates from C57BL/6J mice. IL-1 β (10 μg/kg) was i.v. injected in the tail vein. To proof pro-inflammatory ability of IL-1 β along
    Il 17f, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DI inhibits IL-17-mediated IκBζ induction in keratinocytes and ameliorates psoriatic pathology a , b , Western blot of IκBζ expression in DI-treated, IL-17A-stimulated primary mouse ( a ) and human ( b . c , d , mRNA expression in DI-treated, IL-17A-stimulated (4 h) primary mouse ( c ) and human ( d ) keratinocytes, mean ± s.e.m., n = 3 mice or donors. e , Schematic of DI administration in a psoriasis model. f , Ear histology of control mice (left), IMQ-treated mice (middle) and mice treated with both IMQ and DI (right). Scale bars, 100 µm. Representative of six mice in two experiments. g , Quantification of the histology results in f , showing the relative change in ear thickness. Mean ± s.e.m., n = 6 mice. h , mRNA expression to show the induction of the indicated IκBζ target genes in ear tissue, mean ± s.e.m., n = 3 mice. Statistical tests used were two-tailed t -tests.

    Journal: Nature

    Article Title: Electrophilic properties of itaconate and derivatives regulate the IκBζ–ATF3 inflammatory axis

    doi: 10.1038/s41586-018-0052-z

    Figure Lengend Snippet: DI inhibits IL-17-mediated IκBζ induction in keratinocytes and ameliorates psoriatic pathology a , b , Western blot of IκBζ expression in DI-treated, IL-17A-stimulated primary mouse ( a ) and human ( b . c , d , mRNA expression in DI-treated, IL-17A-stimulated (4 h) primary mouse ( c ) and human ( d ) keratinocytes, mean ± s.e.m., n = 3 mice or donors. e , Schematic of DI administration in a psoriasis model. f , Ear histology of control mice (left), IMQ-treated mice (middle) and mice treated with both IMQ and DI (right). Scale bars, 100 µm. Representative of six mice in two experiments. g , Quantification of the histology results in f , showing the relative change in ear thickness. Mean ± s.e.m., n = 6 mice. h , mRNA expression to show the induction of the indicated IκBζ target genes in ear tissue, mean ± s.e.m., n = 3 mice. Statistical tests used were two-tailed t -tests.

    Article Snippet: After 2–3 days of cultivation, mouse or human cells were treated with DI for 12 h and then stimulated with mouse recombinant IL-17A (100 ng ml−1 ; cat. no. 421-ML, R & D Systems) or human recombinant IL-17A (100 ng ml−1 ; 7955-IL, R & D Systems) for 4 h or as indicated.

    Techniques: Western Blot, Expressing, Mouse Assay, Two Tailed Test

    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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0026342

    Figure Lengend 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

    Article Snippet: In order to determine which of the genes downregulated in the ankles of Il17ra−/− mice might be directly regulated by IL-17RA signaling, murine fibroblast-like synoviocytes (FLS) were either left unstimulated or stimulated with 100 ng/ml IL-17A for 16 h. RNA was then isolated and gene expression assessed by qPCR.

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

    Tbet deficiency prevents induction of transplantation tolerance by combined co-stimulation blockade with persistent T17/Th2 skewing of alloantigen specific cytokine profile, and PMN, CD4, and IL-17-producing CD8 T cell infiltration. ( A ) Kaplan-Meier survival

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

    Article Title: Targeting Tim-1 to overcome resistance to transplantation tolerance mediated by CD8 T17 cells

    doi: 10.1073/pnas.0812538106

    Figure Lengend Snippet: Tbet deficiency prevents induction of transplantation tolerance by combined co-stimulation blockade with persistent T17/Th2 skewing of alloantigen specific cytokine profile, and PMN, CD4, and IL-17-producing CD8 T cell infiltration. ( A ) Kaplan-Meier survival

    Article Snippet: Frozen sections were used for immunofluorescence staining using goat anti-mouse IL-17 (R & D Systems), rat anti-Mouse CD4 and CD8 (both from BioExpress) as primary antibodies.

    Techniques: Transplantation Assay

    In vivo IL-17 neutralization inhibits rejection and facilitates allograft survival with combined co-stimulation blockade in Tbet KO recipients. ( A ) Fully mismatched cardiac allograft survival in Tbet KO recipients treated with CTLA4Ig+MR1 and anti-IL-17

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

    Article Title: Targeting Tim-1 to overcome resistance to transplantation tolerance mediated by CD8 T17 cells

    doi: 10.1073/pnas.0812538106

    Figure Lengend Snippet: In vivo IL-17 neutralization inhibits rejection and facilitates allograft survival with combined co-stimulation blockade in Tbet KO recipients. ( A ) Fully mismatched cardiac allograft survival in Tbet KO recipients treated with CTLA4Ig+MR1 and anti-IL-17

    Article Snippet: Frozen sections were used for immunofluorescence staining using goat anti-mouse IL-17 (R & D Systems), rat anti-Mouse CD4 and CD8 (both from BioExpress) as primary antibodies.

    Techniques: In Vivo, Neutralization

    Immunostaining for chemoattractant receptor CXCR1/IL-8RA and IL-17F in representative samples of prostates from C57BL/6J mice. IL-1 β (10 μg/kg) was i.v. injected in the tail vein. To proof pro-inflammatory ability of IL-1 β along

    Journal:

    Article Title: IL-1?-INDUCED POST-TRANSITION EFFECT OF NF-KAPPAB PROVIDES TIME-DEPENDENT WAVE OF SIGNALS FOR INITIAL PHASE OF INTRAPOSTATIC INFLAMMATION

    doi: 10.1002/pros.20916

    Figure Lengend Snippet: Immunostaining for chemoattractant receptor CXCR1/IL-8RA and IL-17F in representative samples of prostates from C57BL/6J mice. IL-1 β (10 μg/kg) was i.v. injected in the tail vein. To proof pro-inflammatory ability of IL-1 β along

    Article Snippet: The intraprostatic expression of chemokine receptors, adhesion molecules, surface cell markers, and interleukin in formalin-fixed paraffin-embedded prostate tissue was evaluated using the following markers: for chemokine receptors - CXCR1/IL-8RA (Santa Cruz Biotechnology, Santa Cruz, CA) and CXCR4 (R & D System, Minneapolis, MN); for adhesion molecules - VCAM1 (Santa Cruz); for neutrophils - NIMP-R14 (Santa Cruz); for mononuclear cells (MNC) - CD45 (R & D System); and for IL-17F - (R & D System).

    Techniques: Immunostaining, Mouse Assay, Injection

    Systemic administration of IL-1β induces multitype time-dependent behavior of intraprostatic chemoattractant receptors and IL-17F

    Journal:

    Article Title: IL-1?-INDUCED POST-TRANSITION EFFECT OF NF-KAPPAB PROVIDES TIME-DEPENDENT WAVE OF SIGNALS FOR INITIAL PHASE OF INTRAPOSTATIC INFLAMMATION

    doi: 10.1002/pros.20916

    Figure Lengend Snippet: Systemic administration of IL-1β induces multitype time-dependent behavior of intraprostatic chemoattractant receptors and IL-17F

    Article Snippet: The intraprostatic expression of chemokine receptors, adhesion molecules, surface cell markers, and interleukin in formalin-fixed paraffin-embedded prostate tissue was evaluated using the following markers: for chemokine receptors - CXCR1/IL-8RA (Santa Cruz Biotechnology, Santa Cruz, CA) and CXCR4 (R & D System, Minneapolis, MN); for adhesion molecules - VCAM1 (Santa Cruz); for neutrophils - NIMP-R14 (Santa Cruz); for mononuclear cells (MNC) - CD45 (R & D System); and for IL-17F - (R & D System).

    Techniques: