anti il 17  (R&D Systems)

 
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    Name:
    Human Primate IL 17 IL 17A Antibody
    Description:
    The Human Primate IL 17 IL 17A Antibody from R D Systems is a mouse monoclonal antibody to IL 17 IL 17A This antibody reacts with human primate The Human Primate IL 17 IL 17A Antibody has been validated for the following applications Immunoprecipitation Neutralization ELISA Capture Matched Antibody Pair
    Catalog Number:
    MAB317-500
    Price:
    729
    Category:
    Primary Antibodies
    Applications:
    Immunoprecipitation, Neutralization, ELISA Capture (Matched Antibody Pair)
    Purity:
    Protein A or G purified from ascites
    Conjugate:
    Unconjugated
    Immunogen:
    E. coli-derived recombinant human IL-17, Ile20-Ala155, Accession # Q16552
    Size:
    500 ug
    Host:
    Mouse
    Isotype:
    IgG2b
    Buy from Supplier


    Structured Review

    R&D Systems anti il 17
    Human Primate IL 17 IL 17A Antibody
    The Human Primate IL 17 IL 17A Antibody from R D Systems is a mouse monoclonal antibody to IL 17 IL 17A This antibody reacts with human primate The Human Primate IL 17 IL 17A Antibody has been validated for the following applications Immunoprecipitation Neutralization ELISA Capture Matched Antibody Pair
    https://www.bioz.com/result/anti il 17/product/R&D Systems
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti il 17 - by Bioz Stars, 2021-09
    93/100 stars

    Images

    1) Product Images from "The Ratio of Circulating Regulatory T Cells (Tregs)/Th17 Cells Is Associated with Acute Allograft Rejection in Liver Transplantation"

    Article Title: The Ratio of Circulating Regulatory T Cells (Tregs)/Th17 Cells Is Associated with Acute Allograft Rejection in Liver Transplantation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0112135

    The distribution of Tregs and Th17 cells in the peripheral blood and in the grafts of patients with acute allograft rejection. (A) Representative profiles of Tregs and Th17 cells in peripheral blood collected using fluorescence-activated cell sorter (FACS). (B) The frequency of Tregs and Tregs/Th17 ratio were significantly higher in Gr-SF than in Gr-AR. On the contrary, the frequency of Th17 cells was significantly lower in Gr-SF than in Gr-AR. In addition, the frequency of IL-17 + IFN-γ + cells was lower in Gr-SF than in Gr-AR. (C) To evaluate the distribution pattern of Tregs and Th17 cells in allografts with acute rejection, we examined the infiltration of Tregs and Th17 cells using immunohistochemical staining. Anti-FoxP3 + and anti-IL-17 antibodies were used on paraffin embedded biopsy samples which were obtained from allograft with acute rejection. The results showed extensive infiltration of Tregs (red) and Th17 cells (red). Original magnification, ×400. (D) One representative patient with acute allograft rejection was followed-up for 12 months after liver transplantation. The dynamics of Tregs and Th17 cell frequencies were depicted during the follow-up period (the black line represents Th17 cells frequency; the blue line the Tregs frequency; and the red line Tregs/Th17 ratio). ARS: Acute rejection subsided.
    Figure Legend Snippet: The distribution of Tregs and Th17 cells in the peripheral blood and in the grafts of patients with acute allograft rejection. (A) Representative profiles of Tregs and Th17 cells in peripheral blood collected using fluorescence-activated cell sorter (FACS). (B) The frequency of Tregs and Tregs/Th17 ratio were significantly higher in Gr-SF than in Gr-AR. On the contrary, the frequency of Th17 cells was significantly lower in Gr-SF than in Gr-AR. In addition, the frequency of IL-17 + IFN-γ + cells was lower in Gr-SF than in Gr-AR. (C) To evaluate the distribution pattern of Tregs and Th17 cells in allografts with acute rejection, we examined the infiltration of Tregs and Th17 cells using immunohistochemical staining. Anti-FoxP3 + and anti-IL-17 antibodies were used on paraffin embedded biopsy samples which were obtained from allograft with acute rejection. The results showed extensive infiltration of Tregs (red) and Th17 cells (red). Original magnification, ×400. (D) One representative patient with acute allograft rejection was followed-up for 12 months after liver transplantation. The dynamics of Tregs and Th17 cell frequencies were depicted during the follow-up period (the black line represents Th17 cells frequency; the blue line the Tregs frequency; and the red line Tregs/Th17 ratio). ARS: Acute rejection subsided.

    Techniques Used: Fluorescence, FACS, Immunohistochemistry, Staining, Transplantation Assay

    2) Product Images from "Development of tibulizumab, a tetravalent bispecific antibody targeting BAFF and IL-17A for the treatment of autoimmune disease"

    Article Title: Development of tibulizumab, a tetravalent bispecific antibody targeting BAFF and IL-17A for the treatment of autoimmune disease

    Journal: mAbs

    doi: 10.1080/19420862.2019.1624463

    Serum concentrations of total BAFF or total IL-17 in male cynomolgus monkeys following a single IV bolus administration of 0.3, 1, 5 or 20 mg/kg of LY3090106. Data are the mean ± standard deviation (n = 3–4/group).
    Figure Legend Snippet: Serum concentrations of total BAFF or total IL-17 in male cynomolgus monkeys following a single IV bolus administration of 0.3, 1, 5 or 20 mg/kg of LY3090106. Data are the mean ± standard deviation (n = 3–4/group).

    Techniques Used: Standard Deviation

    Structural comparison of ixekizumab Fab and H44-L100 anti-IL-17 scFv. Cartoon depiction of ixekizumab HC in magenta and LC in blue, scFv V H in green and V k in cyan. The H44-L100 scFv disulfide is shown in sticks (inset). The AB loop of the V H domain of both molecules is indicated on the bottom right. The most significant structural rearrangement occurred immediately N-terminal to the V H Cys44 (inset).
    Figure Legend Snippet: Structural comparison of ixekizumab Fab and H44-L100 anti-IL-17 scFv. Cartoon depiction of ixekizumab HC in magenta and LC in blue, scFv V H in green and V k in cyan. The H44-L100 scFv disulfide is shown in sticks (inset). The AB loop of the V H domain of both molecules is indicated on the bottom right. The most significant structural rearrangement occurred immediately N-terminal to the V H Cys44 (inset).

    Techniques Used:

    Serum concentrations of LY3090106 in male cynomolgus monkeys following a single IV bolus administration of 0.3, 1, 5 or 20 mg/kg as measured by ELISA. The total IgG, BAFF antigen capture, and IL-17 antigen capture curves are nearly superimposable indicating LY3090106 remained functionally intact in vivo . Data are the mean ± standard deviation (n = 3–4/group).
    Figure Legend Snippet: Serum concentrations of LY3090106 in male cynomolgus monkeys following a single IV bolus administration of 0.3, 1, 5 or 20 mg/kg as measured by ELISA. The total IgG, BAFF antigen capture, and IL-17 antigen capture curves are nearly superimposable indicating LY3090106 remained functionally intact in vivo . Data are the mean ± standard deviation (n = 3–4/group).

    Techniques Used: Enzyme-linked Immunosorbent Assay, In Vivo, Standard Deviation

    In vitro neutralization of IL-17 and BAFF. A) LY3090106 (circles) dose-dependently inhibited IL-17-induced CXCL1 production by HT-29 cells in the absence (closed symbols) or presence (open symbols) of BAFF. Positive control (triangles) and negative isotype control (squares) is shown. B) LY3090106 (circles) dose-dependently inhibited proliferation of T1165 cells induced by BAFF in the absence (closed symbols) or presence (open symbols) of IL-17. Positive control (triangles) and negative isotype control (squares) is shown. All results are mean ± SEM.
    Figure Legend Snippet: In vitro neutralization of IL-17 and BAFF. A) LY3090106 (circles) dose-dependently inhibited IL-17-induced CXCL1 production by HT-29 cells in the absence (closed symbols) or presence (open symbols) of BAFF. Positive control (triangles) and negative isotype control (squares) is shown. B) LY3090106 (circles) dose-dependently inhibited proliferation of T1165 cells induced by BAFF in the absence (closed symbols) or presence (open symbols) of IL-17. Positive control (triangles) and negative isotype control (squares) is shown. All results are mean ± SEM.

    Techniques Used: In Vitro, Neutralization, Positive Control

    Biacore surface plasmon resonance demonstration of simultaneous target engagement by LY3090106. The IgG-scFv, or the parental antibodies, were captured by protein A immobilized onto a Biacore CM4 chip, followed by injections of either 10 nM IL-17, 50 nM BAFF, or both targets sequentially in either order. Binding stoichiometries were estimated based on the maximum signal changes from the binding of each target, relative to the signal response of the captured antibody (scaled by the relative molecular weights of antibody and target; the calculated stoichiometry values assume that IL-17 is predominantly dimeric, and that BAFF is predominantly trimeric).
    Figure Legend Snippet: Biacore surface plasmon resonance demonstration of simultaneous target engagement by LY3090106. The IgG-scFv, or the parental antibodies, were captured by protein A immobilized onto a Biacore CM4 chip, followed by injections of either 10 nM IL-17, 50 nM BAFF, or both targets sequentially in either order. Binding stoichiometries were estimated based on the maximum signal changes from the binding of each target, relative to the signal response of the captured antibody (scaled by the relative molecular weights of antibody and target; the calculated stoichiometry values assume that IL-17 is predominantly dimeric, and that BAFF is predominantly trimeric).

    Techniques Used: SPR Assay, Chromatin Immunoprecipitation, Binding Assay

    In vivo neutralization of IL-17 and BAFF. A) LY3090106 inhibited IL-17-induced CXCL1 production in mice. Each circle represents an individual animal; horizontal line represents the group mean. Positive control and LY3090106 have significantly lower CXCL1 levels compared to negative isotype control. B) LY3090106 significantly reduced the number of splenic B cells in huBAFF-transgenic mice (compared to negative control antibody) similar to the positive control. Each circle represents an individual animal; horizontal line represents the group mean.
    Figure Legend Snippet: In vivo neutralization of IL-17 and BAFF. A) LY3090106 inhibited IL-17-induced CXCL1 production in mice. Each circle represents an individual animal; horizontal line represents the group mean. Positive control and LY3090106 have significantly lower CXCL1 levels compared to negative isotype control. B) LY3090106 significantly reduced the number of splenic B cells in huBAFF-transgenic mice (compared to negative control antibody) similar to the positive control. Each circle represents an individual animal; horizontal line represents the group mean.

    Techniques Used: In Vivo, Neutralization, Mouse Assay, Positive Control, Transgenic Assay, Negative Control

    ANS binding to anti-IL-17 scFv (left panel) and H44-L100 anti-IL-17 scFv (right panel) as monitored by ANS fluorescence emission. Excitation wavelength at 360 nm. Emission scan from 400 to 700 nM. One nanometer excitation and emission slit widths.
    Figure Legend Snippet: ANS binding to anti-IL-17 scFv (left panel) and H44-L100 anti-IL-17 scFv (right panel) as monitored by ANS fluorescence emission. Excitation wavelength at 360 nm. Emission scan from 400 to 700 nM. One nanometer excitation and emission slit widths.

    Techniques Used: Binding Assay, Fluorescence

    3) Product Images from "Sera from Remitting and Secondary Progressive Multiple Sclerosis Patients Disrupt the Blood-Brain Barrier"

    Article Title: Sera from Remitting and Secondary Progressive Multiple Sclerosis Patients Disrupt the Blood-Brain Barrier

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092872

    The BBB disruption was restored after adding GM6001 or a neutralizing anti-VEGF antibody to the sera from relapsing MS patients. (A)–(J) The effects of TNF-α, IFN-γ, IL-17 or VEGF neutralizing antibodies or a matrix- metalloproteinases (MMPs) inhibitor, GM6001, on the amount of tight junction proteins in TY09 cells after exposure to the sera from SPMS or RRMS-R patients was determined by a Western blot analysis. (K)–(N) Each bar graph reflects the combined densitometry data from each independent experiment (mean±SEM, SPMS n = 6, RRMS-R n = 4). (M) In patients with RRMS-R, preincubation with a VEGF neutralizing antibody or GM6001 increased the amount of claudin-5 protein in TY09 cells. Part 2 (O) The TEER value of the TY09 cells significantly increased after incubation with the sera from RRMS-R patients that had been pretreated with an anti-VEGF neutralizing antibody or GM 6001 (mean±SEM, n = 6). SPMS: conditioned medium containing a 10% concentration of serum from a SPMS patient diluted with non-conditioned DMEM containing 10% FBS; SPMS+TNF-α Ab: conditioned medium with 10% SPMS sera pretreated with a TNF-α neutralizing antibody; SPMS+IFN-γ Ab: conditioned medium with 10% SPMS sera pretreated with an IFN-γ neutralizing antibody; SPMS+IL-17 Ab: conditioned medium with 10% SPMS sera pretreated with an IL-17 neutralizing antibody; SPMS+VEGF Ab: conditioned medium with 10% SPMS sera pretreated with a VEGF neutralizing antibody; SPMS+GM6001: conditioned medium with 10% SPMS sera pretreated with GM6001; RRMS-R: conditioned medium containing a 10% concentration of serum from a RRMS-R patient diluted with non-conditioned DMEM containing 10% FBS; RRMS-R+TNF-α Ab: conditioned medium with 10% RRMS-R sera pretreated with a TNF-α neutralizing antibody; RRMS-R+IFN-γ Ab: conditioned medium with 10% RRMS-R sera pretreated with an IFN-γ neutralizing antibody; RRMS-R+IL-17 Ab: conditioned medium with 10% RRMS-R sera pretreated with an IL-17 neutralizing antibody; RRMS-R+VEGF Ab: conditioned medium with 10% RRMS-R sera pretreated with a VEGF neutralizing antibody; RRMS-R+GM6001: conditioned medium with 10% RRMS-R sera pretreated with GM6001.
    Figure Legend Snippet: The BBB disruption was restored after adding GM6001 or a neutralizing anti-VEGF antibody to the sera from relapsing MS patients. (A)–(J) The effects of TNF-α, IFN-γ, IL-17 or VEGF neutralizing antibodies or a matrix- metalloproteinases (MMPs) inhibitor, GM6001, on the amount of tight junction proteins in TY09 cells after exposure to the sera from SPMS or RRMS-R patients was determined by a Western blot analysis. (K)–(N) Each bar graph reflects the combined densitometry data from each independent experiment (mean±SEM, SPMS n = 6, RRMS-R n = 4). (M) In patients with RRMS-R, preincubation with a VEGF neutralizing antibody or GM6001 increased the amount of claudin-5 protein in TY09 cells. Part 2 (O) The TEER value of the TY09 cells significantly increased after incubation with the sera from RRMS-R patients that had been pretreated with an anti-VEGF neutralizing antibody or GM 6001 (mean±SEM, n = 6). SPMS: conditioned medium containing a 10% concentration of serum from a SPMS patient diluted with non-conditioned DMEM containing 10% FBS; SPMS+TNF-α Ab: conditioned medium with 10% SPMS sera pretreated with a TNF-α neutralizing antibody; SPMS+IFN-γ Ab: conditioned medium with 10% SPMS sera pretreated with an IFN-γ neutralizing antibody; SPMS+IL-17 Ab: conditioned medium with 10% SPMS sera pretreated with an IL-17 neutralizing antibody; SPMS+VEGF Ab: conditioned medium with 10% SPMS sera pretreated with a VEGF neutralizing antibody; SPMS+GM6001: conditioned medium with 10% SPMS sera pretreated with GM6001; RRMS-R: conditioned medium containing a 10% concentration of serum from a RRMS-R patient diluted with non-conditioned DMEM containing 10% FBS; RRMS-R+TNF-α Ab: conditioned medium with 10% RRMS-R sera pretreated with a TNF-α neutralizing antibody; RRMS-R+IFN-γ Ab: conditioned medium with 10% RRMS-R sera pretreated with an IFN-γ neutralizing antibody; RRMS-R+IL-17 Ab: conditioned medium with 10% RRMS-R sera pretreated with an IL-17 neutralizing antibody; RRMS-R+VEGF Ab: conditioned medium with 10% RRMS-R sera pretreated with a VEGF neutralizing antibody; RRMS-R+GM6001: conditioned medium with 10% RRMS-R sera pretreated with GM6001.

    Techniques Used: Mass Spectrometry, Western Blot, Incubation, Concentration Assay

    4) Product Images from "5-Fluorouracil targets thymidylate synthase in the selective suppression of TH17 cell differentiation"

    Article Title: 5-Fluorouracil targets thymidylate synthase in the selective suppression of TH17 cell differentiation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8344

    5-FU inhibition is rescued by thymidine A. Naïve CD4 + T cells from C57BL/6 mice were differentiated under T H 17 polarizing conditions in the presence of 5-FU (1.0 μM) and thymidine (1.0 μM) for 3 days and then were re-stimulated with PMA/ionomycin for 5 hours, stained for intracellular IL-17, and analyzed by flow cytometry as shown. Supernatants from the cells were saved and tested via ELISA. The cells cultured for 48 hours and mRNA expression of the indicated genes was determined by qPCR. B. Repetition of (A) using T H 1 polarizing conditions. C. Repetition of (A) and (B), except using T H 2 polarizing conditions. D. Repetition of (A), (B), and (C) with Treg polarizing conditions instead. ** p
    Figure Legend Snippet: 5-FU inhibition is rescued by thymidine A. Naïve CD4 + T cells from C57BL/6 mice were differentiated under T H 17 polarizing conditions in the presence of 5-FU (1.0 μM) and thymidine (1.0 μM) for 3 days and then were re-stimulated with PMA/ionomycin for 5 hours, stained for intracellular IL-17, and analyzed by flow cytometry as shown. Supernatants from the cells were saved and tested via ELISA. The cells cultured for 48 hours and mRNA expression of the indicated genes was determined by qPCR. B. Repetition of (A) using T H 1 polarizing conditions. C. Repetition of (A) and (B), except using T H 2 polarizing conditions. D. Repetition of (A), (B), and (C) with Treg polarizing conditions instead. ** p

    Techniques Used: Inhibition, Mouse Assay, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, Real-time Polymerase Chain Reaction

    5-FU alters STAT3 DNA binding activity in T H 17 cells A. Naïve CD4 + T cells from C57BL/B6 mice cultured in T H 17 polarizing conditions in the presence of 5-FU (1.0 μM) for 3 days prior to western blotting for RORγt and other indicated proteins. B. The same cells were cultured for 60 hours, followed by ChIP assay. 3 μg of anti- RORγt antibody or isotype-matched IgG control antibody were used in the immunoprecipitation step. qPCR was used to quantify the amount of precipitated DNA with primers flanking the RORγt binding the CNS2 of the IL-17 promoter region. C. 293T cells transfected with STAT3 plasmid for 40 hours in the presence of 5-FU (1 μM). The cytosolic fraction and nuclear fraction proteins were analyzed via western blotting. Cells were cultured as in (A), with western blotting for STAT3 protein phosphorylation and STAT3 protein expression at the times indicated. Cells treated as in (A) analyzed via ChIP assay. 3 μg of anti-STAT3 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. qPCR was used to quantify the amount of precipitated DNA with primers flanking the STAT3 binding site of the RORγt D. and the CNS2 of the IL-17 (E) promoter region. * p
    Figure Legend Snippet: 5-FU alters STAT3 DNA binding activity in T H 17 cells A. Naïve CD4 + T cells from C57BL/B6 mice cultured in T H 17 polarizing conditions in the presence of 5-FU (1.0 μM) for 3 days prior to western blotting for RORγt and other indicated proteins. B. The same cells were cultured for 60 hours, followed by ChIP assay. 3 μg of anti- RORγt antibody or isotype-matched IgG control antibody were used in the immunoprecipitation step. qPCR was used to quantify the amount of precipitated DNA with primers flanking the RORγt binding the CNS2 of the IL-17 promoter region. C. 293T cells transfected with STAT3 plasmid for 40 hours in the presence of 5-FU (1 μM). The cytosolic fraction and nuclear fraction proteins were analyzed via western blotting. Cells were cultured as in (A), with western blotting for STAT3 protein phosphorylation and STAT3 protein expression at the times indicated. Cells treated as in (A) analyzed via ChIP assay. 3 μg of anti-STAT3 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. qPCR was used to quantify the amount of precipitated DNA with primers flanking the STAT3 binding site of the RORγt D. and the CNS2 of the IL-17 (E) promoter region. * p

    Techniques Used: Binding Assay, Activity Assay, Mouse Assay, Cell Culture, Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Expressing

    Knockdown of the TS suppresses T H 17 and T H 1 cell differentiation A. Naïve CD4 + T cells from C57BL/6 mice were transfected with TS siRNA or control siRNA and differentiated under T H 17 polarizing conditions for 72 hours. Cell lysates were analyzed by western blotting. The cells were cultured for 72 hours and then were re-stimulated with PMA/ionomycin for 5 hours, stained for intracellular IL-17, and analyzed by flow cytometry, and the supernatants were analyzed for IL-17 by ELISA. *** p
    Figure Legend Snippet: Knockdown of the TS suppresses T H 17 and T H 1 cell differentiation A. Naïve CD4 + T cells from C57BL/6 mice were transfected with TS siRNA or control siRNA and differentiated under T H 17 polarizing conditions for 72 hours. Cell lysates were analyzed by western blotting. The cells were cultured for 72 hours and then were re-stimulated with PMA/ionomycin for 5 hours, stained for intracellular IL-17, and analyzed by flow cytometry, and the supernatants were analyzed for IL-17 by ELISA. *** p

    Techniques Used: Cell Differentiation, Mouse Assay, Transfection, Western Blot, Cell Culture, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    5) Product Images from "Prostaglandin mediates IL-23/IL-17-induced neutrophil migration in inflammation by inhibiting IL-12 and IFN? production"

    Article Title: Prostaglandin mediates IL-23/IL-17-induced neutrophil migration in inflammation by inhibiting IL-12 and IFN? production

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

    doi: 10.1073/pnas.0812782106

    Schematic representation of IL-23-induced neutrophil migration in antigen (mBSA)-induced arthritis. Lines terminating in blunt ends indicate inhibition. During antigen-induced neutrophil migration to the joints, IL-23 is released early and, together with several cytokines, stimulates IL-17 production that induces the release of TNFα, the CXC chemokines (CXCL1 and CXCL5), and LTB 4 , which together contribute to neutrophil recruitment by inducing locomotion and the expression of adhesion molecules. IL-23 elicited during antigen challenge also can induce COX2 expression leading to the production of PGE 2 ), thus providing an IL-23-COX2-PGE-IL-23 amplification circuit, perpetuating neutrophil migration and inflammation.
    Figure Legend Snippet: Schematic representation of IL-23-induced neutrophil migration in antigen (mBSA)-induced arthritis. Lines terminating in blunt ends indicate inhibition. During antigen-induced neutrophil migration to the joints, IL-23 is released early and, together with several cytokines, stimulates IL-17 production that induces the release of TNFα, the CXC chemokines (CXCL1 and CXCL5), and LTB 4 , which together contribute to neutrophil recruitment by inducing locomotion and the expression of adhesion molecules. IL-23 elicited during antigen challenge also can induce COX2 expression leading to the production of PGE 2 ), thus providing an IL-23-COX2-PGE-IL-23 amplification circuit, perpetuating neutrophil migration and inflammation.

    Techniques Used: Migration, Inhibition, Expressing, Amplification

    IL-17-mediated neutrophil chemotaxis depends on CXC chemokines released by these cells. ( A ) Neutrophils were harvested from bone marrow of mice (IL-12p40 −/− , IFNγ −/− and their control C57BL/6, or 5-LO −/− and their control 129S1/SvImJ [129]), treated for 30 min with indomethacin (Indo, 50 μg/ml). Chemotaxis was determined in a microwell chamber initiated by stimulation for 1 h with IL-17 (1–100 ng/ml), MIP-2 (positive control, 20 ng/ml) or RPMI (vehicle). ( B ) Neutrophils were harvested from bone marrow of C57BL/6 mice and treated for 30 min with IgG control (α-Ctl, 30 μg/ml), anti-CXCR2 antibodies (30 μg/ml), or repertaxin (RPTX, 30 μg/ml). Chemotaxis was determined in a microwell chamber initiated by stimulation for 1 h with IL-17 (50 ng/ml) or RPMI (vehicle). ( C ) The levels of CXCL1 in culture supernatants of neutrophils harvested from bone marrow of C57BL/6 mice stimulated or not with IL-17 (50 ng/ml) for 1 h. * P
    Figure Legend Snippet: IL-17-mediated neutrophil chemotaxis depends on CXC chemokines released by these cells. ( A ) Neutrophils were harvested from bone marrow of mice (IL-12p40 −/− , IFNγ −/− and their control C57BL/6, or 5-LO −/− and their control 129S1/SvImJ [129]), treated for 30 min with indomethacin (Indo, 50 μg/ml). Chemotaxis was determined in a microwell chamber initiated by stimulation for 1 h with IL-17 (1–100 ng/ml), MIP-2 (positive control, 20 ng/ml) or RPMI (vehicle). ( B ) Neutrophils were harvested from bone marrow of C57BL/6 mice and treated for 30 min with IgG control (α-Ctl, 30 μg/ml), anti-CXCR2 antibodies (30 μg/ml), or repertaxin (RPTX, 30 μg/ml). Chemotaxis was determined in a microwell chamber initiated by stimulation for 1 h with IL-17 (50 ng/ml) or RPMI (vehicle). ( C ) The levels of CXCL1 in culture supernatants of neutrophils harvested from bone marrow of C57BL/6 mice stimulated or not with IL-17 (50 ng/ml) for 1 h. * P

    Techniques Used: Chemotaxis Assay, Mouse Assay, Positive Control, CTL Assay

    The IL-23/IL-17 axis is essential for the neutrophil migration in antigen-induced arthritis. ( A ) Neutrophils harvested from articular cavity 24 h after intra-articular injection of mBSA (10 μg/cavity) or its vehicle (saline, Sal) in immunized (mBSA Im, closed bars ) or sham-immunized (Sham-Im, open bars ) mice treated with a co-injection of IgG control (α-CTL), α-IL-23 (700 ng/cavity), or α-IL-17 (700 ng/cavity) antibodies. ( B ) The levels of IL-23 and IL-17 in joint homogenate were determined 3 h after challenge with saline or mBSA (10 μg/cavity) in immunized mice. ( C and D ) Neutrophils harvested from articular cavity 6 h after intra-articular injection of IL-23 (1–10 ng/cavity, closed bars ) or saline (Sal, open bar ) in mice treated with a co-injection of IgG control (α-CTL), anti-IL-23 (5 μg/cavity), or anti-IL-17 (10 μg/cavity) antibodies. * P
    Figure Legend Snippet: The IL-23/IL-17 axis is essential for the neutrophil migration in antigen-induced arthritis. ( A ) Neutrophils harvested from articular cavity 24 h after intra-articular injection of mBSA (10 μg/cavity) or its vehicle (saline, Sal) in immunized (mBSA Im, closed bars ) or sham-immunized (Sham-Im, open bars ) mice treated with a co-injection of IgG control (α-CTL), α-IL-23 (700 ng/cavity), or α-IL-17 (700 ng/cavity) antibodies. ( B ) The levels of IL-23 and IL-17 in joint homogenate were determined 3 h after challenge with saline or mBSA (10 μg/cavity) in immunized mice. ( C and D ) Neutrophils harvested from articular cavity 6 h after intra-articular injection of IL-23 (1–10 ng/cavity, closed bars ) or saline (Sal, open bar ) in mice treated with a co-injection of IgG control (α-CTL), anti-IL-23 (5 μg/cavity), or anti-IL-17 (10 μg/cavity) antibodies. * P

    Techniques Used: Migration, Injection, Mouse Assay, CTL Assay

    PGE 2 enhances IL-23-induced neutrophil recruitment by increasing IL-17 synthesis via suppressing the IL-12/IFNγ axis. ( A ) Neutrophils harvested from the articular cavity 24 h after intra-articular injection of mBSA (1 or 10 μg/cavity) or saline (Sal) in immunized (mBSA-Im, closed bars ) or mBSA (10 μg/cavity) in sham-immunized (Sham-Im, open bar ) mice treated with a co-injection of PGE 2 (30 pg/cavity), IL-12 (10 pg/cavity), IFNγ (100 pg/cavity), or α-IFNγ antibody (700 ng/cavity). Some mice were pretreated 30 min earlier with indomethacin (Indo, 5 mg/kg, s.c.), as indicated. ( B and C ) Neutrophils harvested from articular cavity 6 h after intra-articular injection (1 or 10 ng/cavity) or saline ( open bar ) in wild-type, IL-12p40 −/− , or IFNγ −/− mice. Some mice were treated with a co-injection of PGE 2 (1 pg/cavity), IL-12 (0.1 ng/cavity), or IFNγ (1 ng/cavity) or were pretreated with indomethacin (Indo, 5 mg/kg, s.c. 30 min earlier). ( D and E ) Lymph node cells harvested from immunized mice were cultured with or without mBSA (100 μg/ml), indomethacin (Indo, 50 μg/ml), etoricoxib (Etori, 50 μg/ml), PGE 2 (1 μM), α-IFNγ (10 μg/ml), or α-IL-23 (100 ng/ml) for 36 h. IL-17 and IFNγ concentrations in culture supernatants were determined by ELISA. * P
    Figure Legend Snippet: PGE 2 enhances IL-23-induced neutrophil recruitment by increasing IL-17 synthesis via suppressing the IL-12/IFNγ axis. ( A ) Neutrophils harvested from the articular cavity 24 h after intra-articular injection of mBSA (1 or 10 μg/cavity) or saline (Sal) in immunized (mBSA-Im, closed bars ) or mBSA (10 μg/cavity) in sham-immunized (Sham-Im, open bar ) mice treated with a co-injection of PGE 2 (30 pg/cavity), IL-12 (10 pg/cavity), IFNγ (100 pg/cavity), or α-IFNγ antibody (700 ng/cavity). Some mice were pretreated 30 min earlier with indomethacin (Indo, 5 mg/kg, s.c.), as indicated. ( B and C ) Neutrophils harvested from articular cavity 6 h after intra-articular injection (1 or 10 ng/cavity) or saline ( open bar ) in wild-type, IL-12p40 −/− , or IFNγ −/− mice. Some mice were treated with a co-injection of PGE 2 (1 pg/cavity), IL-12 (0.1 ng/cavity), or IFNγ (1 ng/cavity) or were pretreated with indomethacin (Indo, 5 mg/kg, s.c. 30 min earlier). ( D and E ) Lymph node cells harvested from immunized mice were cultured with or without mBSA (100 μg/ml), indomethacin (Indo, 50 μg/ml), etoricoxib (Etori, 50 μg/ml), PGE 2 (1 μM), α-IFNγ (10 μg/ml), or α-IL-23 (100 ng/ml) for 36 h. IL-17 and IFNγ concentrations in culture supernatants were determined by ELISA. * P

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

    IL-17 mediates neutrophil migration via TNFα, leukotrienes, and CXC chemokines (CXCL1 and CXCL-5). ( A and C ) Neutrophils harvested from articular cavity 6 h after intra-articular injection of IL-17 (1–30 ng/joint), IL-23 (10 ng/cavity), or saline (Sal, open bars ) in mice treated with a co-injection of α-TNFα serum (5 μl/cavity), α-CXCL1 (700 ng/cavity), or α-CXCL5 (700 ng/cavity) antibodies. Some mice also were treated 30 min before with repertaxin (RPTX, 30 mg/kg, s.c.) or 1 h before with MK886 (1 mg/kg, by gavage). ( B ) Neutrophils harvested from articular cavity 24 h after intra-articular injection of mBSA (10 μg/cavity) or saline in immunized mice (mBSA Im, closed bars ) or mBSA (10 μg/cavity) in sham-immunized (Sham-Im, open bar ) mice treated with a co-injection of IgG control (α-CTL), α-TNFα serum (5 μl/cavity), α-CXCL1 (700 ng/cavity), or α-CXCL5 (700 ng/cavity) antibodies or 1 h before with MK886 (1 mg/kg, by gavage). * P
    Figure Legend Snippet: IL-17 mediates neutrophil migration via TNFα, leukotrienes, and CXC chemokines (CXCL1 and CXCL-5). ( A and C ) Neutrophils harvested from articular cavity 6 h after intra-articular injection of IL-17 (1–30 ng/joint), IL-23 (10 ng/cavity), or saline (Sal, open bars ) in mice treated with a co-injection of α-TNFα serum (5 μl/cavity), α-CXCL1 (700 ng/cavity), or α-CXCL5 (700 ng/cavity) antibodies. Some mice also were treated 30 min before with repertaxin (RPTX, 30 mg/kg, s.c.) or 1 h before with MK886 (1 mg/kg, by gavage). ( B ) Neutrophils harvested from articular cavity 24 h after intra-articular injection of mBSA (10 μg/cavity) or saline in immunized mice (mBSA Im, closed bars ) or mBSA (10 μg/cavity) in sham-immunized (Sham-Im, open bar ) mice treated with a co-injection of IgG control (α-CTL), α-TNFα serum (5 μl/cavity), α-CXCL1 (700 ng/cavity), or α-CXCL5 (700 ng/cavity) antibodies or 1 h before with MK886 (1 mg/kg, by gavage). * P

    Techniques Used: Migration, Injection, Mouse Assay, CTL Assay

    6) Product Images from "Ets-1 is a negative regulator of Th17 differentiation"

    Article Title: Ets-1 is a negative regulator of Th17 differentiation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20070994

    Increased IL-17 production by Ets-1 KO Th cells is reversible, and is associated with increased expression of Th17 markers. (A) Total Th cells from WT and Ets-1 KO mice were infected 2 d after activation under Th17 skewing conditions with either empty virus (GFP-rv) or virus expressing full-length Ets-1 isoform (Ets-1-rv). The panels are gated on GFP-positive cells. The production of IL-17 by the transduced (GFP-positive) cells was examined by ICS. Mean fluorescence intensity (MFI) of the IL-17-positive cells is indicated. Results are representative of three independent experiments. (B) After 5 d in culture in the indicated conditions, differentiated WT and Ets-1 KO Th cells were restimulated for 4 h with PMA/ionomycin, and the transcript levels of IL-17F, IL-22, IL-23R, and RORγt were measured by real-time PCR. Data are presented as the mean ± the SD and are representative of three independent experiments. *, P
    Figure Legend Snippet: Increased IL-17 production by Ets-1 KO Th cells is reversible, and is associated with increased expression of Th17 markers. (A) Total Th cells from WT and Ets-1 KO mice were infected 2 d after activation under Th17 skewing conditions with either empty virus (GFP-rv) or virus expressing full-length Ets-1 isoform (Ets-1-rv). The panels are gated on GFP-positive cells. The production of IL-17 by the transduced (GFP-positive) cells was examined by ICS. Mean fluorescence intensity (MFI) of the IL-17-positive cells is indicated. Results are representative of three independent experiments. (B) After 5 d in culture in the indicated conditions, differentiated WT and Ets-1 KO Th cells were restimulated for 4 h with PMA/ionomycin, and the transcript levels of IL-17F, IL-22, IL-23R, and RORγt were measured by real-time PCR. Data are presented as the mean ± the SD and are representative of three independent experiments. *, P

    Techniques Used: Expressing, Mouse Assay, Infection, Activation Assay, Fluorescence, Real-time Polymerase Chain Reaction

    In vivo evidence of increased Th17 differentiation in Ets-1 KO mice. Total RNA of indicated tissues from three WT and Ets-1 KO mice was extracted, and the transcript levels of IL-17 (A), IL-17F (B), IL-22 (C), and IFNγ (D) were quantified by real-time PCR. (E) Expression of various chemokines was analyzed in the lungs of WT and Ets-1 KO mice. Data are presented as the mean ± the SD. *, P
    Figure Legend Snippet: In vivo evidence of increased Th17 differentiation in Ets-1 KO mice. Total RNA of indicated tissues from three WT and Ets-1 KO mice was extracted, and the transcript levels of IL-17 (A), IL-17F (B), IL-22 (C), and IFNγ (D) were quantified by real-time PCR. (E) Expression of various chemokines was analyzed in the lungs of WT and Ets-1 KO mice. Data are presented as the mean ± the SD. *, P

    Techniques Used: In Vivo, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing

    Ets-1 does not bind to the IL-17 promoter or interfere with early signaling events during Th17 differentiation. (A) CHIP was performed on WT Th17 cells using anti–Ets-1 antibody. Binding to conserved Ets sites in the IL-17 and IFN-γ genetic region was analyzed by real-time PCR. Relative binding was calculated as described in the Materials and methods. (B) Total WT and Ets-1 KO Th cells were plated on anti-CD3–coated plates and given anti-CD28, but in the absence of APC, and skewed to the Th17 lineage with IL-6 and TGFβ1 for 5 d. The production of indicated cytokines by the differentiated Th cells was measured by ICS. (C) Freshly isolated total WT and Ets-1 KO Th cells were stimulated with TGFβ1 and IL-6 for the indicated number of minutes. The levels of phospho-Smad3, total Smad2/3, phospho-STAT3, and total STAT3 were examined with Western blotting. (D) Naive Th cells were cultured under Th17 conditions, and RNA was harvested on day 2 or 4 and analyzed for expression of IL-17 and RORγt by real-time PCR. Data are representative of three independent experiments and are presented as the mean ± the SD. *, P
    Figure Legend Snippet: Ets-1 does not bind to the IL-17 promoter or interfere with early signaling events during Th17 differentiation. (A) CHIP was performed on WT Th17 cells using anti–Ets-1 antibody. Binding to conserved Ets sites in the IL-17 and IFN-γ genetic region was analyzed by real-time PCR. Relative binding was calculated as described in the Materials and methods. (B) Total WT and Ets-1 KO Th cells were plated on anti-CD3–coated plates and given anti-CD28, but in the absence of APC, and skewed to the Th17 lineage with IL-6 and TGFβ1 for 5 d. The production of indicated cytokines by the differentiated Th cells was measured by ICS. (C) Freshly isolated total WT and Ets-1 KO Th cells were stimulated with TGFβ1 and IL-6 for the indicated number of minutes. The levels of phospho-Smad3, total Smad2/3, phospho-STAT3, and total STAT3 were examined with Western blotting. (D) Naive Th cells were cultured under Th17 conditions, and RNA was harvested on day 2 or 4 and analyzed for expression of IL-17 and RORγt by real-time PCR. Data are representative of three independent experiments and are presented as the mean ± the SD. *, P

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Isolation, Western Blot, Cell Culture, Expressing

    Ets-1 KO Th cells produce increased levels of IL-17. Total Th cells derived from WT or Ets-1 KO mice were cultured as described in the Materials and methods. (A) After 5 d in culture under Th0, Th1, Th2, or Th17 conditions, cells were restimulated with PMA/ionomycin for 10 min and lysed. Whole-cell extract was analyzed by Western blotting using antibodies against Ets-1 or phospho-T38 Ets-1. An antibody against Hsp90 was used as loading control. (B) Total Th cells from WT and Ets-1 KO mice were cultured under Th17 conditions for 5 d and restimulated with PMA/ionomycin. Cytokine production was assessed by ICS. The numbers represent the percentages of cells stained positive for the indicated cytokines. (C) Total Ets-1 KO and WT Th cells were differentiated for 5 d in the presence of WT irradiated splenocytes and indicated cytokines, and the production of IL-17 in response to PMA/ionomycin stimulation was analyzed by ICS. The percentages of IL-17–positive cells from five independent experiments are shown. (D) Some of the differentiated Th cells (at 1 million cells/ml) generated in C were restimulated with 0.1 μg/ml of plate-bound anti-CD3 for 24 h, and the level of IL-17 protein in the supernatant was measured by ELISA. (E) A fraction of the PMA/ionomycin-restimulated cells from C were analyzed for IL-17 expression by real-time PCR analysis. (F) Naive WT and Ets-1 KO Th cells were differentiated under Th17 conditions, and the production of IL-17 by the differentiated cells was analyzed by ICS after 5 d. Cumulative results of six independent experiments are shown. Data are presented as the mean ± the SD. *, P
    Figure Legend Snippet: Ets-1 KO Th cells produce increased levels of IL-17. Total Th cells derived from WT or Ets-1 KO mice were cultured as described in the Materials and methods. (A) After 5 d in culture under Th0, Th1, Th2, or Th17 conditions, cells were restimulated with PMA/ionomycin for 10 min and lysed. Whole-cell extract was analyzed by Western blotting using antibodies against Ets-1 or phospho-T38 Ets-1. An antibody against Hsp90 was used as loading control. (B) Total Th cells from WT and Ets-1 KO mice were cultured under Th17 conditions for 5 d and restimulated with PMA/ionomycin. Cytokine production was assessed by ICS. The numbers represent the percentages of cells stained positive for the indicated cytokines. (C) Total Ets-1 KO and WT Th cells were differentiated for 5 d in the presence of WT irradiated splenocytes and indicated cytokines, and the production of IL-17 in response to PMA/ionomycin stimulation was analyzed by ICS. The percentages of IL-17–positive cells from five independent experiments are shown. (D) Some of the differentiated Th cells (at 1 million cells/ml) generated in C were restimulated with 0.1 μg/ml of plate-bound anti-CD3 for 24 h, and the level of IL-17 protein in the supernatant was measured by ELISA. (E) A fraction of the PMA/ionomycin-restimulated cells from C were analyzed for IL-17 expression by real-time PCR analysis. (F) Naive WT and Ets-1 KO Th cells were differentiated under Th17 conditions, and the production of IL-17 by the differentiated cells was analyzed by ICS after 5 d. Cumulative results of six independent experiments are shown. Data are presented as the mean ± the SD. *, P

    Techniques Used: Derivative Assay, Mouse Assay, Cell Culture, Western Blot, Staining, Irradiation, Generated, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction

    IL-17–dependent mucus overproduction by Ets-1 KO airway epithelial cells. Histological sections of inflated lungs from WT and Ets-1 KO mice were stained for hematoxylin and eosin (A) and PAS (B). Bars, 1 mm. The arrow in B points out PAS-positive epithelial cells. The percentages of PAS-positive epithelial cells observed in WT and KO mice ( n = 3–4) at the indicated ages are shown in C. 2-mo-old Ets-1 KO mice were injected intraperitoneally with anti–IL-17 or control IgG every other day for 2 wk (6 injections in total) before killing. The histological sections of inflated lungs were stained with PAS (D). The percentages of PAS-positive epithelial cells are shown in F. Data are presented as the mean ± the SD. *, P
    Figure Legend Snippet: IL-17–dependent mucus overproduction by Ets-1 KO airway epithelial cells. Histological sections of inflated lungs from WT and Ets-1 KO mice were stained for hematoxylin and eosin (A) and PAS (B). Bars, 1 mm. The arrow in B points out PAS-positive epithelial cells. The percentages of PAS-positive epithelial cells observed in WT and KO mice ( n = 3–4) at the indicated ages are shown in C. 2-mo-old Ets-1 KO mice were injected intraperitoneally with anti–IL-17 or control IgG every other day for 2 wk (6 injections in total) before killing. The histological sections of inflated lungs were stained with PAS (D). The percentages of PAS-positive epithelial cells are shown in F. Data are presented as the mean ± the SD. *, P

    Techniques Used: Mouse Assay, Staining, Injection

    7) Product Images from "The role of Toll-like receptor 9 in a murine model of Cryptococcus gattii infection"

    Article Title: The role of Toll-like receptor 9 in a murine model of Cryptococcus gattii infection

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-80959-5

    Levels of IFN-γ and IL-17 in total lung homogenates of C. gattii -infected mice. Measurement of IFN-γ and IL-17 levels by ELISA of total lung homogenates of WT and TLR9 −/− mice, infected with C. gattii or given PBS, after 21 days. ( A ) Dosage of IFN-γ and ( B ) IL-17. The graphs show a representative result of two similar and independent experiments. Sham WT (n = 4), Sham TLR9 −/− (n = 4), infected WT (n = 6), infected TLR9 −/− (n = 7). **P
    Figure Legend Snippet: Levels of IFN-γ and IL-17 in total lung homogenates of C. gattii -infected mice. Measurement of IFN-γ and IL-17 levels by ELISA of total lung homogenates of WT and TLR9 −/− mice, infected with C. gattii or given PBS, after 21 days. ( A ) Dosage of IFN-γ and ( B ) IL-17. The graphs show a representative result of two similar and independent experiments. Sham WT (n = 4), Sham TLR9 −/− (n = 4), infected WT (n = 6), infected TLR9 −/− (n = 7). **P

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

    8) Product Images from "Association of Pathogenic Th17 Cells with the Disease Severity and Its Potential Implication for Biological Treatment Selection in Psoriasis Patients"

    Article Title: Association of Pathogenic Th17 Cells with the Disease Severity and Its Potential Implication for Biological Treatment Selection in Psoriasis Patients

    Journal: Mediators of Inflammation

    doi: 10.1155/2020/8065147

    Peripheral Th17 cell pool from psoriasis patients has different cytokine production potential. PBMC from psoriasis patients were stimulated with anti-CD3/CD28 (72 hours) or with anti-CD3/CD28 plus PMA/ionomycin (P/I) (6 hours before the end of the stimulation). IL-17 and IFN- γ production by Th17 cells was analyzed through flow cytometry. Representative plots of cytokine pattern expression in the Th17 cell (a) dynamic pool ( n = 3) or (b) stable pool ( n = 3). Numbers indicate the percentage of events in each plot quadrant.
    Figure Legend Snippet: Peripheral Th17 cell pool from psoriasis patients has different cytokine production potential. PBMC from psoriasis patients were stimulated with anti-CD3/CD28 (72 hours) or with anti-CD3/CD28 plus PMA/ionomycin (P/I) (6 hours before the end of the stimulation). IL-17 and IFN- γ production by Th17 cells was analyzed through flow cytometry. Representative plots of cytokine pattern expression in the Th17 cell (a) dynamic pool ( n = 3) or (b) stable pool ( n = 3). Numbers indicate the percentage of events in each plot quadrant.

    Techniques Used: Flow Cytometry, Expressing

    Evaluation of the Th17 cell phenotype is helpful to select the biological treatment of a psoriasis patient. (a) Photography of psoriasis patient P91 (pretreatment, PASI 6.3) shows the most representative skin lesion. LS immunofluorescence staining for Th17 cells using anti-CD4 (green), anti-IFN- γ (cyan), anti-IL-17 or anti-CD161 (red), and nuclei (blue). PBMC were stimulated with anti-CD3/CD28. CD161 and ROR γ t were evaluated in CD4 T cells by flow cytometry as well as the production of IL-17 and IFN- γ in Th17 cells (bottom panels). (b) Photography of a psoriasis patient after secukinumab treatment (PASI 0) taken in the same anatomical site (a). Flow cytometry plot of IL-17 and IFN- γ expression in Th17 cells. Scale bar = 20 μ m.
    Figure Legend Snippet: Evaluation of the Th17 cell phenotype is helpful to select the biological treatment of a psoriasis patient. (a) Photography of psoriasis patient P91 (pretreatment, PASI 6.3) shows the most representative skin lesion. LS immunofluorescence staining for Th17 cells using anti-CD4 (green), anti-IFN- γ (cyan), anti-IL-17 or anti-CD161 (red), and nuclei (blue). PBMC were stimulated with anti-CD3/CD28. CD161 and ROR γ t were evaluated in CD4 T cells by flow cytometry as well as the production of IL-17 and IFN- γ in Th17 cells (bottom panels). (b) Photography of a psoriasis patient after secukinumab treatment (PASI 0) taken in the same anatomical site (a). Flow cytometry plot of IL-17 and IFN- γ expression in Th17 cells. Scale bar = 20 μ m.

    Techniques Used: Immunofluorescence, Staining, Flow Cytometry, Expressing

    Th17 cells with the pathogenic phenotype are infiltrating the lesional skin of psoriasis patients. Pathogenic Th17 cell identification in healthy (HS), nonlesional (NLS), and lesional (LS) skin biopsies was performed using antibodies anti-CD4 (green) and (a) anti-IL-17 (red) or (d) anti-CD161 (red) along with an anti-IFN- γ (cyan). Nuclei counterstaining was performed with Hoechst (blue). Confocal representative images were obtained (a, d). (b) Percentage of pathogenic Th17 lymphocytes (CD4 + IL-17 + IFN- γ + ) in HS ( n = 10), NLS ( n = 12), and LS ( n = 25), Mann-Whitney U test. (c) Spearman's correlation between the percentage of pathogenic Th17 cells (CD4 + IL-17 + IFN- γ + ) and the PASI score ( n = 25). (e) Quantification of pathogenic Th17 lymphocytes (CD4 + CD161 + IFN- γ + ) in HS ( n = 10), NLS ( n = 11), and LS ( n = 21), Mann-Whitney U test. (f) Spearman's correlation between the pathogenic Th17 cell (CD4 + CD161 + IFN- γ + ) percentage and the PASI score ( n = 21). ∗∗∗ P
    Figure Legend Snippet: Th17 cells with the pathogenic phenotype are infiltrating the lesional skin of psoriasis patients. Pathogenic Th17 cell identification in healthy (HS), nonlesional (NLS), and lesional (LS) skin biopsies was performed using antibodies anti-CD4 (green) and (a) anti-IL-17 (red) or (d) anti-CD161 (red) along with an anti-IFN- γ (cyan). Nuclei counterstaining was performed with Hoechst (blue). Confocal representative images were obtained (a, d). (b) Percentage of pathogenic Th17 lymphocytes (CD4 + IL-17 + IFN- γ + ) in HS ( n = 10), NLS ( n = 12), and LS ( n = 25), Mann-Whitney U test. (c) Spearman's correlation between the percentage of pathogenic Th17 cells (CD4 + IL-17 + IFN- γ + ) and the PASI score ( n = 25). (e) Quantification of pathogenic Th17 lymphocytes (CD4 + CD161 + IFN- γ + ) in HS ( n = 10), NLS ( n = 11), and LS ( n = 21), Mann-Whitney U test. (f) Spearman's correlation between the pathogenic Th17 cell (CD4 + CD161 + IFN- γ + ) percentage and the PASI score ( n = 21). ∗∗∗ P

    Techniques Used: MANN-WHITNEY

    Determination of the pathogenic phenotype of Th17 cells in a psoriasis patient. Consecutive immunologic surveillance is relevant to choose the biological therapy. Integral analysis of Th17 cells IL-17 and IFN- γ expression in the lesional skin shown in immunofluorescence panels (CD4: green; IFN- γ : cyan; IL-17 or CD161: red; and nuclei: blue) and in peripheral blood (cytometry plots) from patient P21. (a) After infliximab treatment for 14 weeks, the patient presented a PASI score of 3.4 and displayed psoriatic skin lesions (photography). (b) After infliximab treatment (18 weeks), the PASI score was 2.4. Photography shows the lesional skin. (c) A psoriasis patient following infliximab dose adjustment (PASI 0). Picture illustrates the same anatomical site shown in (a–c). Scale bar = 20 μ m.
    Figure Legend Snippet: Determination of the pathogenic phenotype of Th17 cells in a psoriasis patient. Consecutive immunologic surveillance is relevant to choose the biological therapy. Integral analysis of Th17 cells IL-17 and IFN- γ expression in the lesional skin shown in immunofluorescence panels (CD4: green; IFN- γ : cyan; IL-17 or CD161: red; and nuclei: blue) and in peripheral blood (cytometry plots) from patient P21. (a) After infliximab treatment for 14 weeks, the patient presented a PASI score of 3.4 and displayed psoriatic skin lesions (photography). (b) After infliximab treatment (18 weeks), the PASI score was 2.4. Photography shows the lesional skin. (c) A psoriasis patient following infliximab dose adjustment (PASI 0). Picture illustrates the same anatomical site shown in (a–c). Scale bar = 20 μ m.

    Techniques Used: Expressing, Immunofluorescence, Cytometry

    Th17 cells in peripheral blood of psoriasis patients could display conventional or pathogenic phenotype and correlate with the PASI score. PBMC from healthy and psoriasis subjects were stimulated for 72 h with anti-CD3 and anti-CD28 antibodies and analyzed by flow cytometry. (a) Representative plots for Th17 cell (CD4 + ROR γ t + ) identification. (b) Quantification of the percentage of Th17 cells (CD4 + ROR γ t + ) in healthy ( n = 9) and psoriasis subjects ( n = 25), Mann-Whitney U test. (c) Representative plots of IL-17 and IFN- γ expression by Th17 cells in a healthy subject and three psoriasis patients. (d) Percentage of pathogenic Th17 cells (CD4 + ROR γ t + IFN- γ + ) in healthy donors ( n = 9) and psoriasis patients ( n = 25), Mann-Whitney U test. (e) Spearman's correlation between the percentage of pathogenic Th17 cells (CD4 + ROR γ t + IFN- γ + ) and the PASI score ( n = 25). (f) Quantification of pathogenic Th17 lymphocytes (CD4 + CD161 + ROR γ t + IFN- γ + ) in healthy ( n = 10) and psoriasis ( n = 12) subjects, Mann-Whitney U test. (g) Spearman's correlation between the percentage of pathogenic Th17 cells (CD4 + CD161 + ROR γ t + IFN- γ + ) and the PASI score ( n = 12). ∗∗ P
    Figure Legend Snippet: Th17 cells in peripheral blood of psoriasis patients could display conventional or pathogenic phenotype and correlate with the PASI score. PBMC from healthy and psoriasis subjects were stimulated for 72 h with anti-CD3 and anti-CD28 antibodies and analyzed by flow cytometry. (a) Representative plots for Th17 cell (CD4 + ROR γ t + ) identification. (b) Quantification of the percentage of Th17 cells (CD4 + ROR γ t + ) in healthy ( n = 9) and psoriasis subjects ( n = 25), Mann-Whitney U test. (c) Representative plots of IL-17 and IFN- γ expression by Th17 cells in a healthy subject and three psoriasis patients. (d) Percentage of pathogenic Th17 cells (CD4 + ROR γ t + IFN- γ + ) in healthy donors ( n = 9) and psoriasis patients ( n = 25), Mann-Whitney U test. (e) Spearman's correlation between the percentage of pathogenic Th17 cells (CD4 + ROR γ t + IFN- γ + ) and the PASI score ( n = 25). (f) Quantification of pathogenic Th17 lymphocytes (CD4 + CD161 + ROR γ t + IFN- γ + ) in healthy ( n = 10) and psoriasis ( n = 12) subjects, Mann-Whitney U test. (g) Spearman's correlation between the percentage of pathogenic Th17 cells (CD4 + CD161 + ROR γ t + IFN- γ + ) and the PASI score ( n = 12). ∗∗ P

    Techniques Used: Flow Cytometry, MANN-WHITNEY, Expressing

    Determination of the pathogenic phenotype of Th17 cells in a psoriasis patient is useful to choose the biological treatment. (a) Prior to treatment, an integral analysis of Th17 cells was performed in patient P52 (PASI 18.3). The photograph shows the most significant psoriatic lesion. Microscopy images show the evaluation of Th17 cells in the lesional skin using antibodies anti-CD4 (green), anti-IFN- γ (cyan), anti-IL-17 or anti-CD161 (red), and nuclei (blue). Flow cytometry analyses of CD161 and ROR γ t expression in CD4 cells and production of IL-17 and IFN- γ in Th17 cells after stimulation. (b) Psoriasis patient photograph after adalimumab administration (PASI 0). Evaluation of IL-17 and IFN- γ expression in Th17 cells by flow cytometry (bottom plot). Scale bar = 20 μ m.
    Figure Legend Snippet: Determination of the pathogenic phenotype of Th17 cells in a psoriasis patient is useful to choose the biological treatment. (a) Prior to treatment, an integral analysis of Th17 cells was performed in patient P52 (PASI 18.3). The photograph shows the most significant psoriatic lesion. Microscopy images show the evaluation of Th17 cells in the lesional skin using antibodies anti-CD4 (green), anti-IFN- γ (cyan), anti-IL-17 or anti-CD161 (red), and nuclei (blue). Flow cytometry analyses of CD161 and ROR γ t expression in CD4 cells and production of IL-17 and IFN- γ in Th17 cells after stimulation. (b) Psoriasis patient photograph after adalimumab administration (PASI 0). Evaluation of IL-17 and IFN- γ expression in Th17 cells by flow cytometry (bottom plot). Scale bar = 20 μ m.

    Techniques Used: Microscopy, Flow Cytometry, Expressing

    9) Product Images from "Complementary action of granulocyte macrophage colony-stimulating factor and interleukin-17A induces interleukin-23, receptor activator of nuclear factor-κB ligand, and matrix metalloproteinases and drives bone and cartilage pathology in experimental arthritis: rationale for combination therapy in rheumatoid arthritis"

    Article Title: Complementary action of granulocyte macrophage colony-stimulating factor and interleukin-17A induces interleukin-23, receptor activator of nuclear factor-κB ligand, and matrix metalloproteinases and drives bone and cartilage pathology in experimental arthritis: rationale for combination therapy in rheumatoid arthritis

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-015-0683-5

    Cytokines in synovial washouts and serum and flow cytometric analysis of synovium and serum immunoglobulin G (IgG) after 14 days of interleukin (IL)-17 and granulocyte macrophage colony-stimulating factor (GM-CSF) blockade. a Luminex analysis of cytokines and chemokines in joint washouts ( n = 6 mice per group). b Flow cytometric analysis of phorbol 12-myristate 13-acetate/ionomycin-stimulated synovial tissue stained with monoclonal antibodies for CD4 and CD8 after 14 days of treatment. The gate depicts the percentage of CD4 + cells of single synoviocytes. Plots shown are representative of n = 6 joints/group. Mean ± standard error of the mean (SEM) for each group is given in the fluorescence-activated cell sorting plot. c Summary graph for the flow cytometric analysis of synovial tissue. Box depicts 25th to 75th percentiles. Line depicts median. Whiskers depict minimal to maximal values. n = 6 mice per group. d IL-6 levels in serum measured by Luminex assay. n = 10 mice/group. Mean ± SEM. e Total collagen-specific immunoglobulin G (IgG) in serum. n = 7–10 mice/group. * p
    Figure Legend Snippet: Cytokines in synovial washouts and serum and flow cytometric analysis of synovium and serum immunoglobulin G (IgG) after 14 days of interleukin (IL)-17 and granulocyte macrophage colony-stimulating factor (GM-CSF) blockade. a Luminex analysis of cytokines and chemokines in joint washouts ( n = 6 mice per group). b Flow cytometric analysis of phorbol 12-myristate 13-acetate/ionomycin-stimulated synovial tissue stained with monoclonal antibodies for CD4 and CD8 after 14 days of treatment. The gate depicts the percentage of CD4 + cells of single synoviocytes. Plots shown are representative of n = 6 joints/group. Mean ± standard error of the mean (SEM) for each group is given in the fluorescence-activated cell sorting plot. c Summary graph for the flow cytometric analysis of synovial tissue. Box depicts 25th to 75th percentiles. Line depicts median. Whiskers depict minimal to maximal values. n = 6 mice per group. d IL-6 levels in serum measured by Luminex assay. n = 10 mice/group. Mean ± SEM. e Total collagen-specific immunoglobulin G (IgG) in serum. n = 7–10 mice/group. * p

    Techniques Used: Flow Cytometry, Luminex, Mouse Assay, Staining, Fluorescence, FACS

    Quantitative PCR analysis of inflammatory and anti-inflammatory mediators in synovial tissue after adenoviral transfer into the knee joint. a Interleukin (IL)-17, granulocyte macrophage colony-stimulating factor (GM-CSF), RAR-related orphan receptor γt (RORγt) and IL-23 expression in synovial tissue determined at 4 h, day 1 and day 4 after adenoviral transfer. b Proinflammatory mediators IL-1β, receptor activator of nuclear factor κB ligand (RANKL) and S100A8 expression in synovial tissue determined at 4 h, day 1 and day 4 after adenoviral transfer. c Matrix metalloproteinase (MMP) expression in synovial tissue determined at 4 h, day 1 and day 4 after adenoviral transfer. d Expression of MMP inhibitors in synovial tissue determined at 4 h, day 1 and day 4 after adenoviral transfer. n = 6 joints/group. Mean ± standard error of the mean. * p
    Figure Legend Snippet: Quantitative PCR analysis of inflammatory and anti-inflammatory mediators in synovial tissue after adenoviral transfer into the knee joint. a Interleukin (IL)-17, granulocyte macrophage colony-stimulating factor (GM-CSF), RAR-related orphan receptor γt (RORγt) and IL-23 expression in synovial tissue determined at 4 h, day 1 and day 4 after adenoviral transfer. b Proinflammatory mediators IL-1β, receptor activator of nuclear factor κB ligand (RANKL) and S100A8 expression in synovial tissue determined at 4 h, day 1 and day 4 after adenoviral transfer. c Matrix metalloproteinase (MMP) expression in synovial tissue determined at 4 h, day 1 and day 4 after adenoviral transfer. d Expression of MMP inhibitors in synovial tissue determined at 4 h, day 1 and day 4 after adenoviral transfer. n = 6 joints/group. Mean ± standard error of the mean. * p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    Histological analysis of joint damage after intraarticular injection of adenoviral vectors for interleukin (IL)-17 and granulocyte macrophage colony-stimulating factor (GM-CSF). a Haematoxylin and eosin (H E)- and Safranin O (SafO)-stained sections of knee joints on days 4 and 7 after adenoviral transfer. E exudate, S synovitis, CD cartilage damage, B bone erosion, PG proteoglycan depletion. Representative sections are shown from n = 6 joints/group. b Individual and c total histological scores for day 4 after adenoviral transfer. d Individual and e total histological scores for day 7 after adenoviral transfer. Mean ± standard error of the mean for n = 6 joints per group. * p
    Figure Legend Snippet: Histological analysis of joint damage after intraarticular injection of adenoviral vectors for interleukin (IL)-17 and granulocyte macrophage colony-stimulating factor (GM-CSF). a Haematoxylin and eosin (H E)- and Safranin O (SafO)-stained sections of knee joints on days 4 and 7 after adenoviral transfer. E exudate, S synovitis, CD cartilage damage, B bone erosion, PG proteoglycan depletion. Representative sections are shown from n = 6 joints/group. b Individual and c total histological scores for day 4 after adenoviral transfer. d Individual and e total histological scores for day 7 after adenoviral transfer. Mean ± standard error of the mean for n = 6 joints per group. * p

    Techniques Used: Injection, Staining

    Neutralisation of interleukin (IL)-17 and granulocyte macrophage colony-stimulating factor (GM-CSF) during established collagen-induced arthritis (CIA). a Macroscopic disease scores followed over time from day 21 (day 0 of treatment). n = 10 mice/group. Arrows indicate times of antibody treatment. p values were calculated by two-way analysis of variance (ANOVA) after calculation of the area under the curve. b Endpoint X-ray analysis of ankle joints after CIA. Representative ankle joint shown for the isotype control and the anti-IL-17 + anti-GM-CSF groups. c Pooled endpoint X-ray analysis of ankle joints scored for bone damage [ n = 20 joints/group; mean ± standard error of the mean (SEM)]. d Histological analysis of ankle joints. Original magnification, ×50. e Detailed histological scores of the ankle joints after 14 days of treatment. n = 20 joints/group. f Total histological scores. n = 20 joints/group. Mean ± SEM. * p
    Figure Legend Snippet: Neutralisation of interleukin (IL)-17 and granulocyte macrophage colony-stimulating factor (GM-CSF) during established collagen-induced arthritis (CIA). a Macroscopic disease scores followed over time from day 21 (day 0 of treatment). n = 10 mice/group. Arrows indicate times of antibody treatment. p values were calculated by two-way analysis of variance (ANOVA) after calculation of the area under the curve. b Endpoint X-ray analysis of ankle joints after CIA. Representative ankle joint shown for the isotype control and the anti-IL-17 + anti-GM-CSF groups. c Pooled endpoint X-ray analysis of ankle joints scored for bone damage [ n = 20 joints/group; mean ± standard error of the mean (SEM)]. d Histological analysis of ankle joints. Original magnification, ×50. e Detailed histological scores of the ankle joints after 14 days of treatment. n = 20 joints/group. f Total histological scores. n = 20 joints/group. Mean ± SEM. * p

    Techniques Used: Mouse Assay

    Cytokine and chemokine analysis of joint washouts after adenoviral transfer into the knee joint . Luminex analysis for cytokines and chemokines at 4 h, 1 day and 4 days after adenoviral transfer. a Interleukin (IL)-17. b Granulocyte macrophage colony-stimulating factor (GM-CSF). c IL-1β. d IL-6. e CC chemokine ligand (CCL2). f CCL3. n = 6 mice per group. Mean ± standard error of the mean. ND not detected. * p
    Figure Legend Snippet: Cytokine and chemokine analysis of joint washouts after adenoviral transfer into the knee joint . Luminex analysis for cytokines and chemokines at 4 h, 1 day and 4 days after adenoviral transfer. a Interleukin (IL)-17. b Granulocyte macrophage colony-stimulating factor (GM-CSF). c IL-1β. d IL-6. e CC chemokine ligand (CCL2). f CCL3. n = 6 mice per group. Mean ± standard error of the mean. ND not detected. * p

    Techniques Used: Luminex, Mouse Assay

    10) Product Images from "Interleukin (IL)‐9/IL‐9R axis drives γδ T cells activation in psoriatic arthritis patients"

    Article Title: Interleukin (IL)‐9/IL‐9R axis drives γδ T cells activation in psoriatic arthritis patients

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/cei.12853

    Phenotypical analysis and cytokine production of Vγ9Vδ2T cells from peripheral blood and synovial fluid of psoriatic arthritis (PsA) patients. (a) Percentages of total Vγ9Vδ2 T cells, their naive (T naive ) (CD45RA + CD27 + ), T central memory (T CM ) (CD45R − CD27 + ), T effector memory (T EM ) (CD45RA − CD27 − ), effector memory terminally differentiated (T EMRA ) (CD45RA + CD27 − ) subsets and correlation between percentage of Vγ9Vδ2 T EM subset and disease activity index (BASDAI and DAS28). (b) Mean percentage of interferon (IFN)‐γ‐ and interleukin (IL)‐17‐producing Vγ9Vδ2 T cells. (c) Dot‐plot analysis and gating strategy of one representative PsA patient and one control [healthy donor (HD)]. (d) Percentages of total synovial fluid (SF) Vγ9Vδ2 T cells and mean percentage of Vγ9Vδ2 T cells subsets from PsA patients and controls [osteoarthritis (OA)]. Data are expressed as mean [standard error of the mean (s.e.m.)]. * P ]
    Figure Legend Snippet: Phenotypical analysis and cytokine production of Vγ9Vδ2T cells from peripheral blood and synovial fluid of psoriatic arthritis (PsA) patients. (a) Percentages of total Vγ9Vδ2 T cells, their naive (T naive ) (CD45RA + CD27 + ), T central memory (T CM ) (CD45R − CD27 + ), T effector memory (T EM ) (CD45RA − CD27 − ), effector memory terminally differentiated (T EMRA ) (CD45RA + CD27 − ) subsets and correlation between percentage of Vγ9Vδ2 T EM subset and disease activity index (BASDAI and DAS28). (b) Mean percentage of interferon (IFN)‐γ‐ and interleukin (IL)‐17‐producing Vγ9Vδ2 T cells. (c) Dot‐plot analysis and gating strategy of one representative PsA patient and one control [healthy donor (HD)]. (d) Percentages of total synovial fluid (SF) Vγ9Vδ2 T cells and mean percentage of Vγ9Vδ2 T cells subsets from PsA patients and controls [osteoarthritis (OA)]. Data are expressed as mean [standard error of the mean (s.e.m.)]. * P ]

    Techniques Used: Activity Assay

    Cytokine production by Vγ9Vδ2 T cells from synovial fluid (SF) of psoriatic arthritis (PsA) patients and comparison of paired peripheral blood and synovial fluid (SF) samples from patients with PsA. Interleukin (IL)‐9R and IL‐23R expression and in‐vitro response to recombinant IL‐23 and IL‐9. (a) Mean percentages of interferon (IFN)‐γ, IL‐17‐producing Vγ9Vδ2 T cells in PsA and osteoarthritis (OA) patients. (b) Dot‐plot analysis of one representative PsA and OA patient. (c) Increased frequencies of IFN‐γ + Vγ9Vδ2 + and IL‐17 + Vγ9Vδ2 + T in the PsA SF compared to peripheral blood. (d) Reverse transcription–polymerase chain reaction (RT–PCR) of IL‐9R and IL‐23R gene expression on Vγ9Vδ2 T cells, either unstimulated or stimulated with isopentenyl pyrophosphate (IPP) for 6 h or 7 days. (e) Fluorescence activated cell sorter (FACS) analysis of IL‐9R and IL‐23R expression by Vγ9Vδ2 T cells of PsA patients and healthy donors (HD). Dot‐plot analysis of IFN‐γ/IL‐17 producing Vγ9Vδ2 T cells and Vγ9Vδ2 T cell expansion from one representative PsA patient after in‐vitro stimulation with rIL‐9 or rIL‐23. Mean percentage of IFN‐γ/IL‐17‐producing Vγ9Vδ2 T cells after in‐vitro stimulation with recombinant IL (rIL)‐9 or rIL‐23. Data represent the mean values ± standard error of the mean (s.e.m.). * P
    Figure Legend Snippet: Cytokine production by Vγ9Vδ2 T cells from synovial fluid (SF) of psoriatic arthritis (PsA) patients and comparison of paired peripheral blood and synovial fluid (SF) samples from patients with PsA. Interleukin (IL)‐9R and IL‐23R expression and in‐vitro response to recombinant IL‐23 and IL‐9. (a) Mean percentages of interferon (IFN)‐γ, IL‐17‐producing Vγ9Vδ2 T cells in PsA and osteoarthritis (OA) patients. (b) Dot‐plot analysis of one representative PsA and OA patient. (c) Increased frequencies of IFN‐γ + Vγ9Vδ2 + and IL‐17 + Vγ9Vδ2 + T in the PsA SF compared to peripheral blood. (d) Reverse transcription–polymerase chain reaction (RT–PCR) of IL‐9R and IL‐23R gene expression on Vγ9Vδ2 T cells, either unstimulated or stimulated with isopentenyl pyrophosphate (IPP) for 6 h or 7 days. (e) Fluorescence activated cell sorter (FACS) analysis of IL‐9R and IL‐23R expression by Vγ9Vδ2 T cells of PsA patients and healthy donors (HD). Dot‐plot analysis of IFN‐γ/IL‐17 producing Vγ9Vδ2 T cells and Vγ9Vδ2 T cell expansion from one representative PsA patient after in‐vitro stimulation with rIL‐9 or rIL‐23. Mean percentage of IFN‐γ/IL‐17‐producing Vγ9Vδ2 T cells after in‐vitro stimulation with recombinant IL (rIL)‐9 or rIL‐23. Data represent the mean values ± standard error of the mean (s.e.m.). * P

    Techniques Used: Expressing, In Vitro, Recombinant, Reverse Transcription Polymerase Chain Reaction, Fluorescence, FACS

    11) Product Images from "A novel pro-lymphangiogenic function for Th17/IL-17"

    Article Title: A novel pro-lymphangiogenic function for Th17/IL-17

    Journal: Blood

    doi: 10.1182/blood-2011-01-332049

    In vivo blockade of IL-17 inhibits inflammatory lymphangiogenesis in Th17-dominant autoimmune DED. a readout for clinical signs of dry eye inflammation, from day 0 to day 12, at which time corneas were harvested for immunohistochemical and real-time PCR analyses. (A) Corneas were immunostained with CD31 (green) and Lyve1 (red) antibodies. Digital micrographs using epifluorescence microscopy were captured and ImageJ software was used to (B) quantify the growth of blood (CD31 hi Lyve1 − ) and lymphatic (CD31 lo Lyve1 + ) vessels. (C) Real-time PCR analysis was performed to measure the expression levels of lymphangiogenic-specific VEGF-D and VEGF-C in untreated, isotype Ab-treated, and anti–IL-17 Ab-treated DED corneas. Expression levels of VEGF-D and VEGF-C were normalized to GAPDH levels as an internal control and then with their levels in normal corneas. (D) Corneal fluorescein staining scores showing severity of clinical disease in untreated, isotype, and anti–IL-17 Ab-treated groups. Each group consists of 6 mice. Data are mean ± SEM. * P
    Figure Legend Snippet: In vivo blockade of IL-17 inhibits inflammatory lymphangiogenesis in Th17-dominant autoimmune DED. a readout for clinical signs of dry eye inflammation, from day 0 to day 12, at which time corneas were harvested for immunohistochemical and real-time PCR analyses. (A) Corneas were immunostained with CD31 (green) and Lyve1 (red) antibodies. Digital micrographs using epifluorescence microscopy were captured and ImageJ software was used to (B) quantify the growth of blood (CD31 hi Lyve1 − ) and lymphatic (CD31 lo Lyve1 + ) vessels. (C) Real-time PCR analysis was performed to measure the expression levels of lymphangiogenic-specific VEGF-D and VEGF-C in untreated, isotype Ab-treated, and anti–IL-17 Ab-treated DED corneas. Expression levels of VEGF-D and VEGF-C were normalized to GAPDH levels as an internal control and then with their levels in normal corneas. (D) Corneal fluorescein staining scores showing severity of clinical disease in untreated, isotype, and anti–IL-17 Ab-treated groups. Each group consists of 6 mice. Data are mean ± SEM. * P

    Techniques Used: In Vivo, Immunohistochemistry, Real-time Polymerase Chain Reaction, Epifluorescence Microscopy, Software, Expressing, Staining, Mouse Assay

    IL-17 promotes lymphangiogenesis by inducing VEGF-D secretion, and proliferation of and tube formation by LECs. After 7 days, corneas were evaluated biomicroscopically and then harvested for immunostaining with CD31 (green) and Lyve1 (red). Digital micrographs using epifluorescence microscopy were captured, and ImageJ 1.34s software was used to quantify the growth of (B) blood (CD31 hi Lyve1 − ) and (C) lymphatic (CD31 lo Lyve1 + ) vessels. (D) Primary human corneal epithelial cells were cultured with 10 ng/mL concentration of IL-1β, IL-17, and IL-17 with IL-1β–blocking antibodies for 24 hours, and expression levels of VEGF-A, VEGF-C, and VEGF-D mRNA in cells, and protein in culture supernatants, were measured by real-time PCR and ELISA, respectively. (E) Primary human LECs were cultured with 10 ng/mL concentrations of IL-1β, IL-17, and IL-17 with blockade of IL-1β or VEGFR-3 for 24 hours, and then proliferation was measured using BrdU incorporation assay. (F) In a transwell Matrigel assay, in vitro polarized 2 × 10 5 Th17 cells (in transwell) were cocultured with 5 × 10 4 LECs (on Matrigel) in basal MEM with reduced serum (2% FBS). Positive controls consisted of LEC cultures on Matrigel in MEM supplemented with growth factors (5% FBS, VEGF, FGF, EGF). Negative controls consisted of LEC cultures on Matrigel in basal MEM only with reduced serum (2% FBS). In some wells of Th17-LEC coculture, soluble-IL-17receptor-Fc or soluble-VEGFR3-Fc was added in media. After 8- and 24-hour incubation at 37°C, wells were visualized under bright-field inverted microscope to study the LEC tube formations on Matrigel, and digital micrographs were then captured for quantitative analysis of tube length. Data are mean ± SEM values of 3 independent experiments. * P
    Figure Legend Snippet: IL-17 promotes lymphangiogenesis by inducing VEGF-D secretion, and proliferation of and tube formation by LECs. After 7 days, corneas were evaluated biomicroscopically and then harvested for immunostaining with CD31 (green) and Lyve1 (red). Digital micrographs using epifluorescence microscopy were captured, and ImageJ 1.34s software was used to quantify the growth of (B) blood (CD31 hi Lyve1 − ) and (C) lymphatic (CD31 lo Lyve1 + ) vessels. (D) Primary human corneal epithelial cells were cultured with 10 ng/mL concentration of IL-1β, IL-17, and IL-17 with IL-1β–blocking antibodies for 24 hours, and expression levels of VEGF-A, VEGF-C, and VEGF-D mRNA in cells, and protein in culture supernatants, were measured by real-time PCR and ELISA, respectively. (E) Primary human LECs were cultured with 10 ng/mL concentrations of IL-1β, IL-17, and IL-17 with blockade of IL-1β or VEGFR-3 for 24 hours, and then proliferation was measured using BrdU incorporation assay. (F) In a transwell Matrigel assay, in vitro polarized 2 × 10 5 Th17 cells (in transwell) were cocultured with 5 × 10 4 LECs (on Matrigel) in basal MEM with reduced serum (2% FBS). Positive controls consisted of LEC cultures on Matrigel in MEM supplemented with growth factors (5% FBS, VEGF, FGF, EGF). Negative controls consisted of LEC cultures on Matrigel in basal MEM only with reduced serum (2% FBS). In some wells of Th17-LEC coculture, soluble-IL-17receptor-Fc or soluble-VEGFR3-Fc was added in media. After 8- and 24-hour incubation at 37°C, wells were visualized under bright-field inverted microscope to study the LEC tube formations on Matrigel, and digital micrographs were then captured for quantitative analysis of tube length. Data are mean ± SEM values of 3 independent experiments. * P

    Techniques Used: Immunostaining, Epifluorescence Microscopy, Software, Cell Culture, Concentration Assay, Blocking Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, BrdU Incorporation Assay, Matrigel Assay, In Vitro, Incubation, Inverted Microscopy

    12) Product Images from "Interleukin 17 inhibits progenitor cells in rheumatoid arthritis cartilage"

    Article Title: Interleukin 17 inhibits progenitor cells in rheumatoid arthritis cartilage

    Journal: European Journal of Immunology

    doi: 10.1002/eji.201545910

    The effects of IL‐17 on RA‐CPCs: expression of IL‐17RA and IL‐17RC receptor mRNA (A), Western blots (B), and immuncytochemistry (C) of the IL‐17RA and IL‐17RC proteins. RA‐CPCs are also positive for IL‐6 (D, upper), as well as IL‐10 (D, lower). MMP3 mRNA was significantly upregulated by IL‐17 (E). TIMP1 (F) and TIMP3 (G) were significantly downregulated. However, IL‐6 was significantly upregulated by IL‐17 (H). In contrast, blocking IL‐17 (IL‐17AB) produced the opposite effects (E–H). Western blot analysis (I, J), secukinumab significantly reduced the amount of RUNX2 (K), as well as the amount of IL‐6 (L). Western blots of low molecular weight enriched medium from RA‐CPCs treated with adalimumab or secukinumab (M) exhibited significantly more IL‐10 than the controls (N). (O) Role of IL‐17 on RA‐CPCs, with IL‐17 promoting IL‐6, while antagonizing IL‐17 promotes IL‐10 and chondrogenesis. All figures are representative of at least three different experiments ( n = 3). Significant differences (* p ≤ 0.05); error bars denote the means ± SD.
    Figure Legend Snippet: The effects of IL‐17 on RA‐CPCs: expression of IL‐17RA and IL‐17RC receptor mRNA (A), Western blots (B), and immuncytochemistry (C) of the IL‐17RA and IL‐17RC proteins. RA‐CPCs are also positive for IL‐6 (D, upper), as well as IL‐10 (D, lower). MMP3 mRNA was significantly upregulated by IL‐17 (E). TIMP1 (F) and TIMP3 (G) were significantly downregulated. However, IL‐6 was significantly upregulated by IL‐17 (H). In contrast, blocking IL‐17 (IL‐17AB) produced the opposite effects (E–H). Western blot analysis (I, J), secukinumab significantly reduced the amount of RUNX2 (K), as well as the amount of IL‐6 (L). Western blots of low molecular weight enriched medium from RA‐CPCs treated with adalimumab or secukinumab (M) exhibited significantly more IL‐10 than the controls (N). (O) Role of IL‐17 on RA‐CPCs, with IL‐17 promoting IL‐6, while antagonizing IL‐17 promotes IL‐10 and chondrogenesis. All figures are representative of at least three different experiments ( n = 3). Significant differences (* p ≤ 0.05); error bars denote the means ± SD.

    Techniques Used: Expressing, Western Blot, Blocking Assay, Produced, Molecular Weight

    13) Product Images from "An Atlas of Human Regulatory T Helper-like Cells Reveals Features of Th2-like Tregs that Support a Tumorigenic Environment"

    Article Title: An Atlas of Human Regulatory T Helper-like Cells Reveals Features of Th2-like Tregs that Support a Tumorigenic Environment

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2017.06.079

    Th2-like Tregs Exhibit Higher Viability, Activation, and JAK-STAT Signaling Pathway Than Other Treg Subsets (A) Total percentages of live and blasting cells between Th-like Tregs 72 hr post-TCR activation in the absence of IL-2 (n = 10, mean ± SEM using independent values, RM one-way ANOVA with Tukey’s test). (B) Distribution of dead, apoptotic, and live cells between Th-like Tregs after TCR activation (n = 5). (C and D) The percentage of live and blasting cells was analyzed in Th-like Treg subsets activated in the presence of neutralizing antibodies for IL-2, IL-4, IFN-γ, and IL-17 (all at 10 μg/mL) (C) or 250 U/ml exogenous IL-2 (D) (n = 4, mean ± SEM using bar charts, RM two-way ANOVA with Tukey’s test). (E) STAT5 signaling and p53 expression was measured in Th-like Tregs 16 hr post-TCR activation in the presence or absence of IL-2 (250 U/mL) (n = 4, mean ± SEM using bar charts, RM two-way ANOVA with Sidak’s test). (F) Heatmap showing upregulation of JAK-STAT, TCR signaling pathway and pro and anti-apoptotic genes between Th-like Treg subsets using Partek software and the KEGG database. For all statistical tests, ∗∗∗∗ p
    Figure Legend Snippet: Th2-like Tregs Exhibit Higher Viability, Activation, and JAK-STAT Signaling Pathway Than Other Treg Subsets (A) Total percentages of live and blasting cells between Th-like Tregs 72 hr post-TCR activation in the absence of IL-2 (n = 10, mean ± SEM using independent values, RM one-way ANOVA with Tukey’s test). (B) Distribution of dead, apoptotic, and live cells between Th-like Tregs after TCR activation (n = 5). (C and D) The percentage of live and blasting cells was analyzed in Th-like Treg subsets activated in the presence of neutralizing antibodies for IL-2, IL-4, IFN-γ, and IL-17 (all at 10 μg/mL) (C) or 250 U/ml exogenous IL-2 (D) (n = 4, mean ± SEM using bar charts, RM two-way ANOVA with Tukey’s test). (E) STAT5 signaling and p53 expression was measured in Th-like Tregs 16 hr post-TCR activation in the presence or absence of IL-2 (250 U/mL) (n = 4, mean ± SEM using bar charts, RM two-way ANOVA with Sidak’s test). (F) Heatmap showing upregulation of JAK-STAT, TCR signaling pathway and pro and anti-apoptotic genes between Th-like Treg subsets using Partek software and the KEGG database. For all statistical tests, ∗∗∗∗ p

    Techniques Used: Activation Assay, Expressing, Software

    Th-like Tregs Suppress Cell Division of Th-like Teffs without Preference for Lineage Counterparts (A) Representative dot plots of Th-like Teffs. Th2, Th17, Th1, and Th1/17 were identified from memory Teff CCR4 + cells. (B) Representative histograms and total percentages of divided Cell Trace Violet + Th-like Teff subsets (1 × 10 5 ) stimulated with anti-CD3/CD28 beads at a 40:1 (cell/bead) ratio for 4 days (n = 5, mean ± SEM using bar chart and independent values, RM one-way ANOVA with Tukey’s multiple comparison test). (C and D) Representative histograms (C) and division (Div.) index (D) were obtained from suppression assays between memory Teff or Th-like Teff and Th-like Treg subsets. Teffs (1 × 10 5 ) alone or with autologous Tregs (0.5 × 10 5 ) were activated with anti-CD3/CD28 beads at a 40:1 (cell/bead) ratio for 4 days. The data are presented as division index obtained from FlowJo analysis (n = 6, mean ± SEM using bar charts, RM two-way ANOVA with Tukey’s test). (E) Absolute values of IL-4, IFN-γ, IL-17, and IL-10 obtained from supernatants after 4 days of suppression assays (n = 6, mean ± SD using bars, RM Two-way ANOVA with Tukey’s test). For all statistical tests, ∗∗∗∗ p
    Figure Legend Snippet: Th-like Tregs Suppress Cell Division of Th-like Teffs without Preference for Lineage Counterparts (A) Representative dot plots of Th-like Teffs. Th2, Th17, Th1, and Th1/17 were identified from memory Teff CCR4 + cells. (B) Representative histograms and total percentages of divided Cell Trace Violet + Th-like Teff subsets (1 × 10 5 ) stimulated with anti-CD3/CD28 beads at a 40:1 (cell/bead) ratio for 4 days (n = 5, mean ± SEM using bar chart and independent values, RM one-way ANOVA with Tukey’s multiple comparison test). (C and D) Representative histograms (C) and division (Div.) index (D) were obtained from suppression assays between memory Teff or Th-like Teff and Th-like Treg subsets. Teffs (1 × 10 5 ) alone or with autologous Tregs (0.5 × 10 5 ) were activated with anti-CD3/CD28 beads at a 40:1 (cell/bead) ratio for 4 days. The data are presented as division index obtained from FlowJo analysis (n = 6, mean ± SEM using bar charts, RM two-way ANOVA with Tukey’s test). (E) Absolute values of IL-4, IFN-γ, IL-17, and IL-10 obtained from supernatants after 4 days of suppression assays (n = 6, mean ± SD using bars, RM Two-way ANOVA with Tukey’s test). For all statistical tests, ∗∗∗∗ p

    Techniques Used:

    14) Product Images from "A specific role for TLR1 in protective TH17 immunity during mucosal infection"

    Article Title: A specific role for TLR1 in protective TH17 immunity during mucosal infection

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20112339

    TLR1 is important for inducing IL-17–mediated immunity during oral infection. (A and B) TLR1 −/− , TLR6 −/− , and littermates were infected orally (A) or i.v. (B) with 10 5 CFU Y. enterocolitica . 6 d after infection, MLNs (oral) or SPLs (i.v.) were harvested. Intracellular cytokine production was analyzed by flow cytometry; plots are representative of one out of six infected mice. Total cell counts were determined for naive (open circles) or infected (closed circles) mice. *, P
    Figure Legend Snippet: TLR1 is important for inducing IL-17–mediated immunity during oral infection. (A and B) TLR1 −/− , TLR6 −/− , and littermates were infected orally (A) or i.v. (B) with 10 5 CFU Y. enterocolitica . 6 d after infection, MLNs (oral) or SPLs (i.v.) were harvested. Intracellular cytokine production was analyzed by flow cytometry; plots are representative of one out of six infected mice. Total cell counts were determined for naive (open circles) or infected (closed circles) mice. *, P

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

    TLR1-dependent IL-6 is critical for inducing T H 17 cells during oral infection. (A) Levels of IFN-γ and IL-17 from co-cultures of naive SPL CD4 + T cells and MLN or SPL DCs from TLR1 +/− or TLR1 −/− mice stimulated with 10 µg/ml Y. enterocolitica lysate (Ye). Data shown are the mean ± SEM ( n = 5) from two individual experiments. *, P
    Figure Legend Snippet: TLR1-dependent IL-6 is critical for inducing T H 17 cells during oral infection. (A) Levels of IFN-γ and IL-17 from co-cultures of naive SPL CD4 + T cells and MLN or SPL DCs from TLR1 +/− or TLR1 −/− mice stimulated with 10 µg/ml Y. enterocolitica lysate (Ye). Data shown are the mean ± SEM ( n = 5) from two individual experiments. *, P

    Techniques Used: Infection, Mouse Assay

    15) Product Images from "Development of tibulizumab, a tetravalent bispecific antibody targeting BAFF and IL-17A for the treatment of autoimmune disease"

    Article Title: Development of tibulizumab, a tetravalent bispecific antibody targeting BAFF and IL-17A for the treatment of autoimmune disease

    Journal: mAbs

    doi: 10.1080/19420862.2019.1624463

    Serum concentrations of total BAFF or total IL-17 in male cynomolgus monkeys following a single IV bolus administration of 0.3, 1, 5 or 20 mg/kg of LY3090106. Data are the mean ± standard deviation (n = 3–4/group).
    Figure Legend Snippet: Serum concentrations of total BAFF or total IL-17 in male cynomolgus monkeys following a single IV bolus administration of 0.3, 1, 5 or 20 mg/kg of LY3090106. Data are the mean ± standard deviation (n = 3–4/group).

    Techniques Used: Standard Deviation

    Structural comparison of ixekizumab Fab and H44-L100 anti-IL-17 scFv. Cartoon depiction of ixekizumab HC in magenta and LC in blue, scFv V H in green and V k in cyan. The H44-L100 scFv disulfide is shown in sticks (inset). The AB loop of the V H domain of both molecules is indicated on the bottom right. The most significant structural rearrangement occurred immediately N-terminal to the V H Cys44 (inset).
    Figure Legend Snippet: Structural comparison of ixekizumab Fab and H44-L100 anti-IL-17 scFv. Cartoon depiction of ixekizumab HC in magenta and LC in blue, scFv V H in green and V k in cyan. The H44-L100 scFv disulfide is shown in sticks (inset). The AB loop of the V H domain of both molecules is indicated on the bottom right. The most significant structural rearrangement occurred immediately N-terminal to the V H Cys44 (inset).

    Techniques Used:

    Serum concentrations of LY3090106 in male cynomolgus monkeys following a single IV bolus administration of 0.3, 1, 5 or 20 mg/kg as measured by ELISA. The total IgG, BAFF antigen capture, and IL-17 antigen capture curves are nearly superimposable indicating LY3090106 remained functionally intact in vivo . Data are the mean ± standard deviation (n = 3–4/group).
    Figure Legend Snippet: Serum concentrations of LY3090106 in male cynomolgus monkeys following a single IV bolus administration of 0.3, 1, 5 or 20 mg/kg as measured by ELISA. The total IgG, BAFF antigen capture, and IL-17 antigen capture curves are nearly superimposable indicating LY3090106 remained functionally intact in vivo . Data are the mean ± standard deviation (n = 3–4/group).

    Techniques Used: Enzyme-linked Immunosorbent Assay, In Vivo, Standard Deviation

    In vitro neutralization of IL-17 and BAFF. A) LY3090106 (circles) dose-dependently inhibited IL-17-induced CXCL1 production by HT-29 cells in the absence (closed symbols) or presence (open symbols) of BAFF. Positive control (triangles) and negative isotype control (squares) is shown. B) LY3090106 (circles) dose-dependently inhibited proliferation of T1165 cells induced by BAFF in the absence (closed symbols) or presence (open symbols) of IL-17. Positive control (triangles) and negative isotype control (squares) is shown. All results are mean ± SEM.
    Figure Legend Snippet: In vitro neutralization of IL-17 and BAFF. A) LY3090106 (circles) dose-dependently inhibited IL-17-induced CXCL1 production by HT-29 cells in the absence (closed symbols) or presence (open symbols) of BAFF. Positive control (triangles) and negative isotype control (squares) is shown. B) LY3090106 (circles) dose-dependently inhibited proliferation of T1165 cells induced by BAFF in the absence (closed symbols) or presence (open symbols) of IL-17. Positive control (triangles) and negative isotype control (squares) is shown. All results are mean ± SEM.

    Techniques Used: In Vitro, Neutralization, Positive Control

    Biacore surface plasmon resonance demonstration of simultaneous target engagement by LY3090106. The IgG-scFv, or the parental antibodies, were captured by protein A immobilized onto a Biacore CM4 chip, followed by injections of either 10 nM IL-17, 50 nM BAFF, or both targets sequentially in either order. Binding stoichiometries were estimated based on the maximum signal changes from the binding of each target, relative to the signal response of the captured antibody (scaled by the relative molecular weights of antibody and target; the calculated stoichiometry values assume that IL-17 is predominantly dimeric, and that BAFF is predominantly trimeric).
    Figure Legend Snippet: Biacore surface plasmon resonance demonstration of simultaneous target engagement by LY3090106. The IgG-scFv, or the parental antibodies, were captured by protein A immobilized onto a Biacore CM4 chip, followed by injections of either 10 nM IL-17, 50 nM BAFF, or both targets sequentially in either order. Binding stoichiometries were estimated based on the maximum signal changes from the binding of each target, relative to the signal response of the captured antibody (scaled by the relative molecular weights of antibody and target; the calculated stoichiometry values assume that IL-17 is predominantly dimeric, and that BAFF is predominantly trimeric).

    Techniques Used: SPR Assay, Chromatin Immunoprecipitation, Binding Assay

    In vivo neutralization of IL-17 and BAFF. A) LY3090106 inhibited IL-17-induced CXCL1 production in mice. Each circle represents an individual animal; horizontal line represents the group mean. Positive control and LY3090106 have significantly lower CXCL1 levels compared to negative isotype control. B) LY3090106 significantly reduced the number of splenic B cells in huBAFF-transgenic mice (compared to negative control antibody) similar to the positive control. Each circle represents an individual animal; horizontal line represents the group mean.
    Figure Legend Snippet: In vivo neutralization of IL-17 and BAFF. A) LY3090106 inhibited IL-17-induced CXCL1 production in mice. Each circle represents an individual animal; horizontal line represents the group mean. Positive control and LY3090106 have significantly lower CXCL1 levels compared to negative isotype control. B) LY3090106 significantly reduced the number of splenic B cells in huBAFF-transgenic mice (compared to negative control antibody) similar to the positive control. Each circle represents an individual animal; horizontal line represents the group mean.

    Techniques Used: In Vivo, Neutralization, Mouse Assay, Positive Control, Transgenic Assay, Negative Control

    ANS binding to anti-IL-17 scFv (left panel) and H44-L100 anti-IL-17 scFv (right panel) as monitored by ANS fluorescence emission. Excitation wavelength at 360 nm. Emission scan from 400 to 700 nM. One nanometer excitation and emission slit widths.
    Figure Legend Snippet: ANS binding to anti-IL-17 scFv (left panel) and H44-L100 anti-IL-17 scFv (right panel) as monitored by ANS fluorescence emission. Excitation wavelength at 360 nm. Emission scan from 400 to 700 nM. One nanometer excitation and emission slit widths.

    Techniques Used: Binding Assay, Fluorescence

    16) Product Images from "Transcription factor IRF8 directs a silencing programme for TH17 cell differentiation"

    Article Title: Transcription factor IRF8 directs a silencing programme for TH17 cell differentiation

    Journal: Nature Communications

    doi: 10.1038/ncomms1311

    IRF8 inhibits the expression of T H 17-associated genes. Naive CD4 + T cells from wild-type and Irf8 –/– mice were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of IL-6 (10 ng ml −1 ) plus differing concentrations of TGF-β (0.1, 1, 5 ng ml −1 ) ( a ), or TGF-β (5 ng ml −1 ), TGF-β (5 ng ml −1 )/IL-6 (10 ng ml −1 ) with or without retinoic acid (RA, 100 nM) ( b ). After 4 days of stimulation, IL-17 and FOXP3 intracellular staining was performed and analysed by flow cytometry. ( c ) The cells prepared in a and b except for the presence of TGF-β (5 ng ml −1 ) plus various concentrations of IL-6 (5, 10, 50, 100 ng ml −1 ) were re-stimulated with PMA/ionomycin for 5 h and stained for intracellular IL-17 and IFN-γ and analysed by flow cytometry. ( d ) Naive CD4 + T cells from wild-type and Irf8 –/– mice were stimulated with IL-6, IL-23, TGF-β or different combinations of these cytokines for various intervals. Total RNA was extracted and analysed by RT–PCR for mRNA expression of IL-17 (top panel) and IL-17F (bottom panel). Data are from one experiment representative of three independent experiments. Error bars, s.d. ( e ) Naive CD4 + T cells from WT and Irf8 –/– mice were stimulated with the indicated cytokines for 4 days. Cells were re-stimulated with PMA/ionomycin for 5 h and stained for intracellular IL-17 and IFN-γ and analysed by flow cytometry. The results are representative of three independent experiments. ( f ) Cells were prepared as in e and the culture supernatants were collected after 4 days of stimulation. IL-17 protein secretion was analysed by ELISA. ( g ) Naive CD4 + T cells from WT and Irf8 –/– mice were prepared and stimulated with TGF-β and IL-6 in the presence of neutralizing anti-IFN-γ (10 μg ml −1 ) and anti-IL-4 (10 μg ml −1 ) antibodies. At 72 h after the stimulation, IL-17 protein secretion in culture supernatants was analysed by ELISA. Data are the mean±s.d. of triplicate cultures. ( h ) Naive CD4 + T cells from wild-type or Irf8 –/– mice were stimulated with the indicated cytokine combinations for 48 h. Total RNA was extracted and analysed by qPCR for mRNA expression of the indicated genes. Data are from one experiment representative of three independent experiments. Error bars, s.d.
    Figure Legend Snippet: IRF8 inhibits the expression of T H 17-associated genes. Naive CD4 + T cells from wild-type and Irf8 –/– mice were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of IL-6 (10 ng ml −1 ) plus differing concentrations of TGF-β (0.1, 1, 5 ng ml −1 ) ( a ), or TGF-β (5 ng ml −1 ), TGF-β (5 ng ml −1 )/IL-6 (10 ng ml −1 ) with or without retinoic acid (RA, 100 nM) ( b ). After 4 days of stimulation, IL-17 and FOXP3 intracellular staining was performed and analysed by flow cytometry. ( c ) The cells prepared in a and b except for the presence of TGF-β (5 ng ml −1 ) plus various concentrations of IL-6 (5, 10, 50, 100 ng ml −1 ) were re-stimulated with PMA/ionomycin for 5 h and stained for intracellular IL-17 and IFN-γ and analysed by flow cytometry. ( d ) Naive CD4 + T cells from wild-type and Irf8 –/– mice were stimulated with IL-6, IL-23, TGF-β or different combinations of these cytokines for various intervals. Total RNA was extracted and analysed by RT–PCR for mRNA expression of IL-17 (top panel) and IL-17F (bottom panel). Data are from one experiment representative of three independent experiments. Error bars, s.d. ( e ) Naive CD4 + T cells from WT and Irf8 –/– mice were stimulated with the indicated cytokines for 4 days. Cells were re-stimulated with PMA/ionomycin for 5 h and stained for intracellular IL-17 and IFN-γ and analysed by flow cytometry. The results are representative of three independent experiments. ( f ) Cells were prepared as in e and the culture supernatants were collected after 4 days of stimulation. IL-17 protein secretion was analysed by ELISA. ( g ) Naive CD4 + T cells from WT and Irf8 –/– mice were prepared and stimulated with TGF-β and IL-6 in the presence of neutralizing anti-IFN-γ (10 μg ml −1 ) and anti-IL-4 (10 μg ml −1 ) antibodies. At 72 h after the stimulation, IL-17 protein secretion in culture supernatants was analysed by ELISA. Data are the mean±s.d. of triplicate cultures. ( h ) Naive CD4 + T cells from wild-type or Irf8 –/– mice were stimulated with the indicated cytokine combinations for 48 h. Total RNA was extracted and analysed by qPCR for mRNA expression of the indicated genes. Data are from one experiment representative of three independent experiments. Error bars, s.d.

    Techniques Used: Expressing, Mouse Assay, Staining, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

    Transduction of IRF8 inhibits T H 17-associated genes. ( a ) Naive CD4 + T cells from C57BL/6 mice were infected with retrovirus encoding IRF8 or empty vector and were activated under T H 17-inducing conditions for 4 days. The cells were re-stimulated with PMA/ionomycin for 5 h and stained for intracellular IL-17 and RORγt and analysed by flow cytometry. ( b ) EL4 cells were transduced with retroviruses encoding IRF8 (black column) or GFP (white column) for 48 h and the transduced cells were then stimulated with PMA/ionomycin for various times as indicated. Total RNA was extracted and the transcript levels of T H 17-associated genes were analysed by qPCR as indicated. The results are normalized to ubiquitin levels. Data are from one experiment representative of three independent experiments. Error bars, s.d.
    Figure Legend Snippet: Transduction of IRF8 inhibits T H 17-associated genes. ( a ) Naive CD4 + T cells from C57BL/6 mice were infected with retrovirus encoding IRF8 or empty vector and were activated under T H 17-inducing conditions for 4 days. The cells were re-stimulated with PMA/ionomycin for 5 h and stained for intracellular IL-17 and RORγt and analysed by flow cytometry. ( b ) EL4 cells were transduced with retroviruses encoding IRF8 (black column) or GFP (white column) for 48 h and the transduced cells were then stimulated with PMA/ionomycin for various times as indicated. Total RNA was extracted and the transcript levels of T H 17-associated genes were analysed by qPCR as indicated. The results are normalized to ubiquitin levels. Data are from one experiment representative of three independent experiments. Error bars, s.d.

    Techniques Used: Transduction, Mouse Assay, Infection, Plasmid Preparation, Staining, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction

    IL-21 signalling and IL-10 production during T H 17-cell differentiation in Irf8 –/– mice. Naive CD4 + T cells from WT and Irf8 –/– mice were prepared, and the cells were stimulated with anti-CD3 and anti-CD28 antibodies in the presence or absence of TGF-β plus IL-21 or IL-6 for 72 h. Intracellular staining for IL-17 and IFN-γ expression was performed and analysed by flow cytometry ( a ). The secretion of IL-21 protein in culture supernatants was analysed by ELISA ( b ). Data are from one experiment representative of three independent experiments. Error bars, s.d. Naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of TGF-β plus IL-6 or IL-21. At 96 h after stimulation, intracellular staining for IL-17 and IL-10 was performed and analysed after re-stimulation with PMA/ionomycin by flow cytometry ( c ). Naive CD4 + T cells from spleens and lymph nodes of WT and Irf8 –/– mice were prepared and the cells were stimulated under T H 0, T H 1, T H 2 and T H 17 polarizing conditions for 4 days. Then the cells were re-stimulated with PMA/ionomycin for 12 h and IL-10 protein secretion was analysed by ELISA ( d ). Data are from one experiment representative of three independent experiments. Error bars, s.d. Naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of TGF-β plus IL-6 or IL-21 for 72 h. IL-10 expression was analysed by RT–PCR ( e ). Data are from one experiment representative of three independent experiments. Error bars, s.d.
    Figure Legend Snippet: IL-21 signalling and IL-10 production during T H 17-cell differentiation in Irf8 –/– mice. Naive CD4 + T cells from WT and Irf8 –/– mice were prepared, and the cells were stimulated with anti-CD3 and anti-CD28 antibodies in the presence or absence of TGF-β plus IL-21 or IL-6 for 72 h. Intracellular staining for IL-17 and IFN-γ expression was performed and analysed by flow cytometry ( a ). The secretion of IL-21 protein in culture supernatants was analysed by ELISA ( b ). Data are from one experiment representative of three independent experiments. Error bars, s.d. Naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of TGF-β plus IL-6 or IL-21. At 96 h after stimulation, intracellular staining for IL-17 and IL-10 was performed and analysed after re-stimulation with PMA/ionomycin by flow cytometry ( c ). Naive CD4 + T cells from spleens and lymph nodes of WT and Irf8 –/– mice were prepared and the cells were stimulated under T H 0, T H 1, T H 2 and T H 17 polarizing conditions for 4 days. Then the cells were re-stimulated with PMA/ionomycin for 12 h and IL-10 protein secretion was analysed by ELISA ( d ). Data are from one experiment representative of three independent experiments. Error bars, s.d. Naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of TGF-β plus IL-6 or IL-21 for 72 h. IL-10 expression was analysed by RT–PCR ( e ). Data are from one experiment representative of three independent experiments. Error bars, s.d.

    Techniques Used: Cell Differentiation, Mouse Assay, Staining, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

    IRF-8 represses IL-17 transcription. ( a ) IRF8-expressing EL4 cells (black column) or control cells (white column) were transiently transfected with a RORγt plasmid for 24 h, and the cells were treated with plate-bound anti-CD3 and anti-CD28 antibodies in the presence or absence of the indicated cytokines for 8 h. qPCR analyses of transcripts of the indicated genes were performed and the results were normalized to the levels of ubiquitin transcripts. ** P
    Figure Legend Snippet: IRF-8 represses IL-17 transcription. ( a ) IRF8-expressing EL4 cells (black column) or control cells (white column) were transiently transfected with a RORγt plasmid for 24 h, and the cells were treated with plate-bound anti-CD3 and anti-CD28 antibodies in the presence or absence of the indicated cytokines for 8 h. qPCR analyses of transcripts of the indicated genes were performed and the results were normalized to the levels of ubiquitin transcripts. ** P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction

    Increased IL-17 production in IRF8 deficient T helper cells. ( a ) Naive CD4 + T cells from wild-type (WT) or Irf8 –/– mice were differentiated under T H 0 and T H 17 polarizing conditions for 4 days. Cells were then re-stimulated with PMA/ionomycin for 5 h, stained for intracellular IL-17 and IFN-γ, and analysed by flow cytometry. Representative fluorescence-activated cell sorting (FACS) dot plots gated on CD4 + cells and the percentages of IL-17-producing CD4 + cells are shown. Data are from one experiment representative of three independent experiments. Error bars, s.d. ( b ) The cells prepared in a , T H 1 and T H 2 polarizing conditions were re-stimulated with PMA/ionomycin for 24 h and the supernatants were analysed for IL-17 by ELISA. Data are from one experiment representative of three independent experiments. Error bars, s.d. ( c ) Wild-type or Irf8 –/– lamina propria lymphocytes (LPL) were differentiated under T H 17 conditions for 4 days. Cells were then re-stimulated with PMA/ionomycin for 5 h, stained for intracellular IL-17 and analysed by flow cytometry. ( d ) Naive CD4 + T cells from Lck-Cre + Irf8 wt/wt and Lck-Cre + Irf8 fl/fl mice were differentiated under T H 0 and T H 17 polarizing conditions for 4 days and the cells were re-stimulated with PMA/ionomycin for 5 h for staining of IL-17, IFN-γ and FOXP3. Representative FACS dot plots gated on CD4 + cells are shown. ( e ) The cells prepared in d were re-stimulated with PMA/ionomycin for 24 h and the supernatants were analysed for IL-17 by ELISA. Data are from one experiment representative of two independent experiments. Error bars, s.d. ( f ) Naive CD4 + T cells from C57BL/6 mice were differentiated under T H 0 and T H 17 conditions for 4 days. The cells were re-stimulated with PMA/ionomycin for 12 h and IRF8 expression was analysed by western blot. ( g ) The cells prepared in a were re-stimulated with PMA/ionomycin for 5 h and mRNA expression of indicated genes was determined by qPCR. The data shown were normalized to levels of ubiquitin expression as analysed by qPCR. The results are representative of three independent experiments. Error bars, s.d.
    Figure Legend Snippet: Increased IL-17 production in IRF8 deficient T helper cells. ( a ) Naive CD4 + T cells from wild-type (WT) or Irf8 –/– mice were differentiated under T H 0 and T H 17 polarizing conditions for 4 days. Cells were then re-stimulated with PMA/ionomycin for 5 h, stained for intracellular IL-17 and IFN-γ, and analysed by flow cytometry. Representative fluorescence-activated cell sorting (FACS) dot plots gated on CD4 + cells and the percentages of IL-17-producing CD4 + cells are shown. Data are from one experiment representative of three independent experiments. Error bars, s.d. ( b ) The cells prepared in a , T H 1 and T H 2 polarizing conditions were re-stimulated with PMA/ionomycin for 24 h and the supernatants were analysed for IL-17 by ELISA. Data are from one experiment representative of three independent experiments. Error bars, s.d. ( c ) Wild-type or Irf8 –/– lamina propria lymphocytes (LPL) were differentiated under T H 17 conditions for 4 days. Cells were then re-stimulated with PMA/ionomycin for 5 h, stained for intracellular IL-17 and analysed by flow cytometry. ( d ) Naive CD4 + T cells from Lck-Cre + Irf8 wt/wt and Lck-Cre + Irf8 fl/fl mice were differentiated under T H 0 and T H 17 polarizing conditions for 4 days and the cells were re-stimulated with PMA/ionomycin for 5 h for staining of IL-17, IFN-γ and FOXP3. Representative FACS dot plots gated on CD4 + cells are shown. ( e ) The cells prepared in d were re-stimulated with PMA/ionomycin for 24 h and the supernatants were analysed for IL-17 by ELISA. Data are from one experiment representative of two independent experiments. Error bars, s.d. ( f ) Naive CD4 + T cells from C57BL/6 mice were differentiated under T H 0 and T H 17 conditions for 4 days. The cells were re-stimulated with PMA/ionomycin for 12 h and IRF8 expression was analysed by western blot. ( g ) The cells prepared in a were re-stimulated with PMA/ionomycin for 5 h and mRNA expression of indicated genes was determined by qPCR. The data shown were normalized to levels of ubiquitin expression as analysed by qPCR. The results are representative of three independent experiments. Error bars, s.d.

    Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry, Fluorescence, FACS, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    IRF8 interacts physically with RORγ. ( a ) 293T cells were transfected with HA-tagged IRF8 and T7-tagged RORγt overexpression plasmids for 40 h and cell lysates were prepared in the presence or absence of DNase I or ethidium bromide. 500 μg of cell lysates were immunoprecipitated with an anti-T7 antibody and immunoblotted with specific antibodies as indicated. Data are representative of three independent experiments. ( b ) NIH3T3 cells were transiently transfected with GFP-IRF8 and T7-RORγt for 40 h, and cells were fixed and stained red for RORγt followed by confocal microscopic analysis. Scale bar, 50 μm. ( c ) Naive CD4 + T cells from WT mice were cultured under T H 17-polarizing conditions for 72 h and the expression of RORγt and IRF8 was analysed by flow cytometry. The cells were gated on CD4 + T cells. Data are representative of three independent experiments. ( d ) Naive CD4 + T cells from wild-type or Irf8 –/– mice were cultured under T H 17-polarizing conditions for 60 h and the cell lysates were then immunoprecipitated with an anti-IRF8 antibody and western blotted (WB) with anti-RORγt and anti-IRF8 antibodies. Data represent three independent experiments. ( e ) Diagrams of IRF8 protein domains. ( f ) 293T cells were co-transfected with plasmids containing Flag-tagged full-length IRF8, IRF8 fragments (1–230, 1–190, 1–154) and T7-tagged RORγt plasmid for 40 h, and co-immunoprecipitation using anti-Flag antibody from the cell extracts was performed and immunoblotted with anti-T7 antibody. Data are representative of three independent experiments. ( g ) 293T cells were co-transfected with an IL-17 promoter reporter construct containing the 6-kbp promoter, a RORγt plasmid and either a full-length IRF8 or the IRF8 truncation mutant (1–114) construct for 30 h. Luciferase assays were performed as described in b . Data indicate mean±s.d. of triplicate cultures and are representative of three independent experiments. * P
    Figure Legend Snippet: IRF8 interacts physically with RORγ. ( a ) 293T cells were transfected with HA-tagged IRF8 and T7-tagged RORγt overexpression plasmids for 40 h and cell lysates were prepared in the presence or absence of DNase I or ethidium bromide. 500 μg of cell lysates were immunoprecipitated with an anti-T7 antibody and immunoblotted with specific antibodies as indicated. Data are representative of three independent experiments. ( b ) NIH3T3 cells were transiently transfected with GFP-IRF8 and T7-RORγt for 40 h, and cells were fixed and stained red for RORγt followed by confocal microscopic analysis. Scale bar, 50 μm. ( c ) Naive CD4 + T cells from WT mice were cultured under T H 17-polarizing conditions for 72 h and the expression of RORγt and IRF8 was analysed by flow cytometry. The cells were gated on CD4 + T cells. Data are representative of three independent experiments. ( d ) Naive CD4 + T cells from wild-type or Irf8 –/– mice were cultured under T H 17-polarizing conditions for 60 h and the cell lysates were then immunoprecipitated with an anti-IRF8 antibody and western blotted (WB) with anti-RORγt and anti-IRF8 antibodies. Data represent three independent experiments. ( e ) Diagrams of IRF8 protein domains. ( f ) 293T cells were co-transfected with plasmids containing Flag-tagged full-length IRF8, IRF8 fragments (1–230, 1–190, 1–154) and T7-tagged RORγt plasmid for 40 h, and co-immunoprecipitation using anti-Flag antibody from the cell extracts was performed and immunoblotted with anti-T7 antibody. Data are representative of three independent experiments. ( g ) 293T cells were co-transfected with an IL-17 promoter reporter construct containing the 6-kbp promoter, a RORγt plasmid and either a full-length IRF8 or the IRF8 truncation mutant (1–114) construct for 30 h. Luciferase assays were performed as described in b . Data indicate mean±s.d. of triplicate cultures and are representative of three independent experiments. * P

    Techniques Used: Transfection, Over Expression, Immunoprecipitation, Staining, Mouse Assay, Cell Culture, Expressing, Flow Cytometry, Cytometry, Western Blot, Plasmid Preparation, Construct, Mutagenesis, Luciferase

    17) Product Images from "Successful Regulatory T Cell-Based Therapy Relies on Inhibition of T Cell Effector Function and Enrichment of FOXP3+ Cells in a Humanized Mouse Model of Skin Inflammation"

    Article Title: Successful Regulatory T Cell-Based Therapy Relies on Inhibition of T Cell Effector Function and Enrichment of FOXP3+ Cells in a Humanized Mouse Model of Skin Inflammation

    Journal: Journal of Immunology Research

    doi: 10.1155/2020/7680131

    Treg infusion inhibits skin-infiltrating T cells and downregulates local IL-17 production while promoting FOXP3+ Treg enrichment. Representative histology at 10x magnification and quantitative analysis in skin grafts from SCID beige mice at 28 days after cell injection. Expression of human (a) CD4+ and (b) CD8+ T cells. (c) Expression of IL-17-secreting cells and (d) expression of CD3+ (blue) and FOXP3 (red). Mean ± SEM is shown for (a–c) ( n = 3). Statistical significance was analysed by the Mann–Whitney U test. ∗ p value
    Figure Legend Snippet: Treg infusion inhibits skin-infiltrating T cells and downregulates local IL-17 production while promoting FOXP3+ Treg enrichment. Representative histology at 10x magnification and quantitative analysis in skin grafts from SCID beige mice at 28 days after cell injection. Expression of human (a) CD4+ and (b) CD8+ T cells. (c) Expression of IL-17-secreting cells and (d) expression of CD3+ (blue) and FOXP3 (red). Mean ± SEM is shown for (a–c) ( n = 3). Statistical significance was analysed by the Mann–Whitney U test. ∗ p value

    Techniques Used: Mouse Assay, Injection, Expressing, MANN-WHITNEY

    Treg infusion affects systemic proinflammatory cytokine production by T cells. Representative flow cytometry pictures and quantitative analysis of systemic human CD45+ and CD3+ T cells harvested from the mouse spleen of SCID beige mice infused with PBMC with or without Treg. (a) Percentage of human CD45 cells. (b) Percentage of human CD4+ and CD8+ cells within human CD45+ cells. (c) Representative example of the percentages of dividing (Ki67+) CD4+ and CD8+ cells ( n = 3). (d) Percentage of human IFN γ and IL-17A-secreting T cells. (e) Frequency of CD4+CD25+FOXP3+ cells within CD45+ cells and Treg : CD4 ratio analysis. Mean ± SEM is shown for (a, b, d) ( n = 3‐8). Statistical significance was analysed by the Mann–Whitney U test. ∗ p value
    Figure Legend Snippet: Treg infusion affects systemic proinflammatory cytokine production by T cells. Representative flow cytometry pictures and quantitative analysis of systemic human CD45+ and CD3+ T cells harvested from the mouse spleen of SCID beige mice infused with PBMC with or without Treg. (a) Percentage of human CD45 cells. (b) Percentage of human CD4+ and CD8+ cells within human CD45+ cells. (c) Representative example of the percentages of dividing (Ki67+) CD4+ and CD8+ cells ( n = 3). (d) Percentage of human IFN γ and IL-17A-secreting T cells. (e) Frequency of CD4+CD25+FOXP3+ cells within CD45+ cells and Treg : CD4 ratio analysis. Mean ± SEM is shown for (a, b, d) ( n = 3‐8). Statistical significance was analysed by the Mann–Whitney U test. ∗ p value

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

    18) Product Images from "Th17-related cytokines contribute to recall-like expansion/effector function of HMBPP-specific Vγ2Vδ2 T cells after Mycobacterium tuberculosis infection or vaccination"

    Article Title: Th17-related cytokines contribute to recall-like expansion/effector function of HMBPP-specific Vγ2Vδ2 T cells after Mycobacterium tuberculosis infection or vaccination

    Journal: European journal of immunology

    doi: 10.1002/eji.201444635

    Primary Mtb infection of macaques promotes the ability of IL-23 or IL-17/IL-22 to expand HMBPP-stimulated Vγ2Vδ2 T cells, and IL-23-driven expansion is reduced by blockade of IL-23 signaling. (A) Flow cytometry was performed to assess
    Figure Legend Snippet: Primary Mtb infection of macaques promotes the ability of IL-23 or IL-17/IL-22 to expand HMBPP-stimulated Vγ2Vδ2 T cells, and IL-23-driven expansion is reduced by blockade of IL-23 signaling. (A) Flow cytometry was performed to assess

    Techniques Used: Infection, Flow Cytometry, Cytometry

    HMBPP/IL-23-expanded Vγ2Vδ2 T cells from macaques infected with Mtb or vaccinated with BCG or r Listeria Δ actA prfA *- ESAT6/Ag85B can produce IL-17, IL-22, IL-2, and IFN-γ. (A) Flow cytometry was used to assess the ability
    Figure Legend Snippet: HMBPP/IL-23-expanded Vγ2Vδ2 T cells from macaques infected with Mtb or vaccinated with BCG or r Listeria Δ actA prfA *- ESAT6/Ag85B can produce IL-17, IL-22, IL-2, and IFN-γ. (A) Flow cytometry was used to assess the ability

    Techniques Used: Infection, Flow Cytometry, Cytometry

    19) Product Images from "Neutralization of IL-17 ameliorates uveitis but damages photoreceptors in a murine model of spondyloarthritis"

    Article Title: Neutralization of IL-17 ameliorates uveitis but damages photoreceptors in a murine model of spondyloarthritis

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar3697

    Cellular trafficking responses exacerbated in the iris by IFNγ deficiency areabrogated by IL-17A blockade . ( A ) Weekly i.p. injections of anti-IL-17A blocking antibody or isotype-matched control antibody (IC) were administered into PG-immunized GKO/TCR-Tg mice. Adjuvant-immunized control mice were also administered the anti-IL-17A blocking antibody and IC. The leukocyte trafficking response within the iris was assessed by intravital microscopy at three weeks following PG-immunization. ** P
    Figure Legend Snippet: Cellular trafficking responses exacerbated in the iris by IFNγ deficiency areabrogated by IL-17A blockade . ( A ) Weekly i.p. injections of anti-IL-17A blocking antibody or isotype-matched control antibody (IC) were administered into PG-immunized GKO/TCR-Tg mice. Adjuvant-immunized control mice were also administered the anti-IL-17A blocking antibody and IC. The leukocyte trafficking response within the iris was assessed by intravital microscopy at three weeks following PG-immunization. ** P

    Techniques Used: Blocking Assay, Mouse Assay, Intravital Microscopy

    IFNγ-deficiency results in an altered cytokine profile including exacerbated IL-17A production . Antigen specific T cell cytokine production was measured in splenocyte cultures from TCR-Tg mice with intact IFNγ (WT) or TCR-Tg mice lacking IFNγ (IFNγ KO) that were stimulated with the arthritogenic G1 peptide carrying the TCR-specific epitiope. Cytokine levels in the supernatants were quantified by multiplex-ELISA (Luminex) at 48 h post stimulation. Data are the mean ± SEM of values combined from three independent experiments. * P
    Figure Legend Snippet: IFNγ-deficiency results in an altered cytokine profile including exacerbated IL-17A production . Antigen specific T cell cytokine production was measured in splenocyte cultures from TCR-Tg mice with intact IFNγ (WT) or TCR-Tg mice lacking IFNγ (IFNγ KO) that were stimulated with the arthritogenic G1 peptide carrying the TCR-specific epitiope. Cytokine levels in the supernatants were quantified by multiplex-ELISA (Luminex) at 48 h post stimulation. Data are the mean ± SEM of values combined from three independent experiments. * P

    Techniques Used: Mouse Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Luminex

    IL-17 inhibition prevents uveitis but results in photoreceptor toxicity . ( A ) PG-immunized GKO/TCR-Tg mice were administered weekly i.p. injections of anti-IL-17A blocking antibody or isotype-matched control antibody (IC). Control mice were immunized with adjuvant and the anti-IL-17A blocking antibody or IC. Histological assessment of uveitis was performed at three weeks post PG-immunization. Representative images of the anterior and posterior eye segments are shown. Increased leukocyte infiltration in IC control mice is indicated by arrows (two left panels) (H E stain). The brackets on the far right panel (anti-IL-17 treated group) indicate the space where the ONL and photoreceptor layers would normally be present, but which are absent. Original magnification: 200X. AC, anterior chamber; Co, cornea; CB, ciliary body, Ir, iris; Li, limbus; Vi, vitreous, GCL, ganglion cell layer, INL, inner nuclear layer, ONL, outer nuclear layer, PRL, photoreceptor layer. ( B ) The infiltrating cells present within the aqueous humor of the anterior eye segment or vitreous body of the posterior eye segment were quantified. ( C ) Structural changes within the retina were scored in both eyes of each mouse. Data points represent individual eyes from each mouse. * P
    Figure Legend Snippet: IL-17 inhibition prevents uveitis but results in photoreceptor toxicity . ( A ) PG-immunized GKO/TCR-Tg mice were administered weekly i.p. injections of anti-IL-17A blocking antibody or isotype-matched control antibody (IC). Control mice were immunized with adjuvant and the anti-IL-17A blocking antibody or IC. Histological assessment of uveitis was performed at three weeks post PG-immunization. Representative images of the anterior and posterior eye segments are shown. Increased leukocyte infiltration in IC control mice is indicated by arrows (two left panels) (H E stain). The brackets on the far right panel (anti-IL-17 treated group) indicate the space where the ONL and photoreceptor layers would normally be present, but which are absent. Original magnification: 200X. AC, anterior chamber; Co, cornea; CB, ciliary body, Ir, iris; Li, limbus; Vi, vitreous, GCL, ganglion cell layer, INL, inner nuclear layer, ONL, outer nuclear layer, PRL, photoreceptor layer. ( B ) The infiltrating cells present within the aqueous humor of the anterior eye segment or vitreous body of the posterior eye segment were quantified. ( C ) Structural changes within the retina were scored in both eyes of each mouse. Data points represent individual eyes from each mouse. * P

    Techniques Used: Inhibition, Mouse Assay, Blocking Assay, H&E Stain

    IFNγ regulates the requirement for IL-17 in PG-induced arthritis . PG-immunized GKO/TCR-Tg mice were administered weekly i.p. injections of anti-IL-17A blocking antibody or isotype-matched control antibody (IC). Mice were also immunized with adjuvant along with anti-IL-17A blocking antibody and IC. The peripheral arthritis was assessed as a function of time. ( A ) Clinical arthritic scores in GKO/TCR-Tg mice treated with anti-IL-17A or IC; ** P
    Figure Legend Snippet: IFNγ regulates the requirement for IL-17 in PG-induced arthritis . PG-immunized GKO/TCR-Tg mice were administered weekly i.p. injections of anti-IL-17A blocking antibody or isotype-matched control antibody (IC). Mice were also immunized with adjuvant along with anti-IL-17A blocking antibody and IC. The peripheral arthritis was assessed as a function of time. ( A ) Clinical arthritic scores in GKO/TCR-Tg mice treated with anti-IL-17A or IC; ** P

    Techniques Used: Mouse Assay, Blocking Assay

    20) Product Images from "The role of Toll-like receptor 9 in a murine model of Cryptococcus gattii infection"

    Article Title: The role of Toll-like receptor 9 in a murine model of Cryptococcus gattii infection

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-80959-5

    Levels of IFN-γ and IL-17 in total lung homogenates of C. gattii -infected mice. Measurement of IFN-γ and IL-17 levels by ELISA of total lung homogenates of WT and TLR9 −/− mice, infected with C. gattii or given PBS, after 21 days. ( A ) Dosage of IFN-γ and ( B ) IL-17. The graphs show a representative result of two similar and independent experiments. Sham WT (n = 4), Sham TLR9 −/− (n = 4), infected WT (n = 6), infected TLR9 −/− (n = 7). **P
    Figure Legend Snippet: Levels of IFN-γ and IL-17 in total lung homogenates of C. gattii -infected mice. Measurement of IFN-γ and IL-17 levels by ELISA of total lung homogenates of WT and TLR9 −/− mice, infected with C. gattii or given PBS, after 21 days. ( A ) Dosage of IFN-γ and ( B ) IL-17. The graphs show a representative result of two similar and independent experiments. Sham WT (n = 4), Sham TLR9 −/− (n = 4), infected WT (n = 6), infected TLR9 −/− (n = 7). **P

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

    21) Product Images from "Propionibacterium acnes induces an interleukin-17 response in acne vulgaris that is regulated by vitamin A and vitamin D"

    Article Title: Propionibacterium acnes induces an interleukin-17 response in acne vulgaris that is regulated by vitamin A and vitamin D

    Journal: The Journal of investigative dermatology

    doi: 10.1038/jid.2013.334

    P. acnes stimulate production of IL-17A and IFN-γbut not IL-4 in PBMCs PBMCs were cultured (2–5 × 10 6 /ml) in the presence of P. acnes sonicate (2 µg/ml) or P. acnes clinical isolates for 7 days. a) Levels of IL-17, IL-4 and IFN-γaccumulated in culture supernatants were measured using ELISA. Experiments were performed at least five times using PBMCs from five different donors with similar results. b) PBMCs (2–5 × 10 6 /ml) were cultured either in the presence or absence of seven P. acnes clinical isolates (C1–C7). Levels of IL-17, IL-4, and IFN-γaccumulated in culture supernatants were measured using ELISA. Experiments were performed at least three times using PBMCs from three different donors with similar results. The overall group effect was statistically significant (p
    Figure Legend Snippet: P. acnes stimulate production of IL-17A and IFN-γbut not IL-4 in PBMCs PBMCs were cultured (2–5 × 10 6 /ml) in the presence of P. acnes sonicate (2 µg/ml) or P. acnes clinical isolates for 7 days. a) Levels of IL-17, IL-4 and IFN-γaccumulated in culture supernatants were measured using ELISA. Experiments were performed at least five times using PBMCs from five different donors with similar results. b) PBMCs (2–5 × 10 6 /ml) were cultured either in the presence or absence of seven P. acnes clinical isolates (C1–C7). Levels of IL-17, IL-4, and IFN-γaccumulated in culture supernatants were measured using ELISA. Experiments were performed at least three times using PBMCs from three different donors with similar results. The overall group effect was statistically significant (p

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    Immunohistochemistry of IL-17 in acne lesion Frozen skin sections were obtained from patients with acne lesion (a) and (b) and nomal control skin (c) . Immunohistochemical staining was carried out using anti-human IL-17 antibody (a) and (c) and an isotype control (b) . IL-17 expressing lymphoid cells (brown) can be seen scattered around the inflammation surrounding the pilosebaceous follicle (a) . Data is representative of three independent experiments from three different lesions. Scale bars represent 75 Zm and 30 Zm respectively.
    Figure Legend Snippet: Immunohistochemistry of IL-17 in acne lesion Frozen skin sections were obtained from patients with acne lesion (a) and (b) and nomal control skin (c) . Immunohistochemical staining was carried out using anti-human IL-17 antibody (a) and (c) and an isotype control (b) . IL-17 expressing lymphoid cells (brown) can be seen scattered around the inflammation surrounding the pilosebaceous follicle (a) . Data is representative of three independent experiments from three different lesions. Scale bars represent 75 Zm and 30 Zm respectively.

    Techniques Used: Immunohistochemistry, Staining, Expressing

    Induction of IL-17, IL-17RA , IL-17RC, RORc and RORa mRNAs expression in PBMCs stimulated with P. acnes PBMCs were cultured (2–5 × 10 6 /ml) with P. acnes sonicate (2 µg/ml). Real time PCR of IL-17 , IL-17RA , IL-17RC, RORc and RORa mRNA expression was analyzed 24 hours following P. acnes stimulation. Gene expression was normalized to the housekeeping genes GAPDH and quantified by the comparative method 2 −ΔΔCT . Data are representative of four independent experiments. Data represent mean ± SD (***p ≤ 0.001)
    Figure Legend Snippet: Induction of IL-17, IL-17RA , IL-17RC, RORc and RORa mRNAs expression in PBMCs stimulated with P. acnes PBMCs were cultured (2–5 × 10 6 /ml) with P. acnes sonicate (2 µg/ml). Real time PCR of IL-17 , IL-17RA , IL-17RC, RORc and RORa mRNA expression was analyzed 24 hours following P. acnes stimulation. Gene expression was normalized to the housekeeping genes GAPDH and quantified by the comparative method 2 −ΔΔCT . Data are representative of four independent experiments. Data represent mean ± SD (***p ≤ 0.001)

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

    P. acnes lab strain and clinical isolates stimulate production of IL-17 in human PBMCs a) PBMCs were cultured (2–5 × 10 6 /ml) in the presence of P. acnes sonicate (2 µg/ml), live P. acnes (0.5 muliplicity of infection), M. tuberculosis (5 µg/ml), M. leprae (5 µg/ml), and Staphylococcus enterotoxin B (SEB 2 µg/ml) for seven days. b) PBMCs (2–5 × 10 6 /ml) were cultured either in the presence or absence of seven P. acnes clinical isolates (C1–C7). Levels of IL-17 accumulated in culture supernatants were measured using ELISA. Experiments were performed at least five times using PBMCs from five different donors with similar results. (** p ≤ 0.05, ***p ≤ 0.001)
    Figure Legend Snippet: P. acnes lab strain and clinical isolates stimulate production of IL-17 in human PBMCs a) PBMCs were cultured (2–5 × 10 6 /ml) in the presence of P. acnes sonicate (2 µg/ml), live P. acnes (0.5 muliplicity of infection), M. tuberculosis (5 µg/ml), M. leprae (5 µg/ml), and Staphylococcus enterotoxin B (SEB 2 µg/ml) for seven days. b) PBMCs (2–5 × 10 6 /ml) were cultured either in the presence or absence of seven P. acnes clinical isolates (C1–C7). Levels of IL-17 accumulated in culture supernatants were measured using ELISA. Experiments were performed at least five times using PBMCs from five different donors with similar results. (** p ≤ 0.05, ***p ≤ 0.001)

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

    Supernatants from PBMCs treated with P. acnes differentiate naïve CD4 + T cells to IL-17 producing T cells a) PBMCs (2–5 × 10 6 /ml) were stimulated overnight with P. acnes sonicate (2 µg/ml). Culture supernatants were then collected and used to stimulate naive CD4 + CD45RA T cells for seven days in 96 well plates with plate bound anti-CD3 and soluble CD28. Cells were harvested and intracellular cytokine staining for IL-17 was performed. Each panel is representative of three independent experiments. b) PBMCs (2–5 × 10 6 /ml) were cultured with IL-17, IL-1β, IL-6, TGF-β, IL-23p19, IL-4 and IFN-γneutralizing antibodies for one hour followed by seven days of stimulation with P. acnes . IL-17 production was then measured using ELISA. Data are representative of four independent experiments. Data represent mean ± SD (** p ≤ 0.05, ***p ≤ 0.001)
    Figure Legend Snippet: Supernatants from PBMCs treated with P. acnes differentiate naïve CD4 + T cells to IL-17 producing T cells a) PBMCs (2–5 × 10 6 /ml) were stimulated overnight with P. acnes sonicate (2 µg/ml). Culture supernatants were then collected and used to stimulate naive CD4 + CD45RA T cells for seven days in 96 well plates with plate bound anti-CD3 and soluble CD28. Cells were harvested and intracellular cytokine staining for IL-17 was performed. Each panel is representative of three independent experiments. b) PBMCs (2–5 × 10 6 /ml) were cultured with IL-17, IL-1β, IL-6, TGF-β, IL-23p19, IL-4 and IFN-γneutralizing antibodies for one hour followed by seven days of stimulation with P. acnes . IL-17 production was then measured using ELISA. Data are representative of four independent experiments. Data represent mean ± SD (** p ≤ 0.05, ***p ≤ 0.001)

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

    Effects of 1,25D3, 25D3 and ATRA on Th17 differentiation Human PBMCs were stimulated with P. acnes sonicate (2 µg/ml) in the presence of 1, 25D3 (10 −7 M), 25D3 (10 −7 M) and ATRA (10 −7 M). IL-17 (a) , RORa (c) , RORc (d) , IL-17RA (e) and IL-17RC (f) mRNA expression was analyzed 24 hours following P. acnes stimulation. Gene expression was normalized to the housekeeping genes GAPDH and quantified by the comparative method 2 −ΔΔCT . Each panel is representative of three independent donors. b) PBMCs were cultured (2–5 × 10 6 /ml) with 1,25D3, 25D3 and ATRA for one hour followed by seven days of stimulation with P. acnes (sonicate or live), IL-17 production was then measured using ELISA. Data are representative of four independent experiments. Data represent mean ± SD (***p ≤ 0.001)
    Figure Legend Snippet: Effects of 1,25D3, 25D3 and ATRA on Th17 differentiation Human PBMCs were stimulated with P. acnes sonicate (2 µg/ml) in the presence of 1, 25D3 (10 −7 M), 25D3 (10 −7 M) and ATRA (10 −7 M). IL-17 (a) , RORa (c) , RORc (d) , IL-17RA (e) and IL-17RC (f) mRNA expression was analyzed 24 hours following P. acnes stimulation. Gene expression was normalized to the housekeeping genes GAPDH and quantified by the comparative method 2 −ΔΔCT . Each panel is representative of three independent donors. b) PBMCs were cultured (2–5 × 10 6 /ml) with 1,25D3, 25D3 and ATRA for one hour followed by seven days of stimulation with P. acnes (sonicate or live), IL-17 production was then measured using ELISA. Data are representative of four independent experiments. Data represent mean ± SD (***p ≤ 0.001)

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

    22) Product Images from "Angiogenesis in rheumatoid arthritis is directly fostered by TLR5 ligation and indirectly through IL-17 induction"

    Article Title: Angiogenesis in rheumatoid arthritis is directly fostered by TLR5 ligation and indirectly through IL-17 induction

    Journal: Arthritis and rheumatism

    doi: 10.1002/art.37992

    IL-17 and its associated factors are elevated in flagellin compared to control treatment in CIA mice
    Figure Legend Snippet: IL-17 and its associated factors are elevated in flagellin compared to control treatment in CIA mice

    Techniques Used: Mouse Assay

    Myeloid cell production of IL-6 and IL-1β is responsible for TLR5 mediated TH-17 cell differentiation and blockade of IL-17 pathway reduces flagellin mediated endothelial chemotaxis
    Figure Legend Snippet: Myeloid cell production of IL-6 and IL-1β is responsible for TLR5 mediated TH-17 cell differentiation and blockade of IL-17 pathway reduces flagellin mediated endothelial chemotaxis

    Techniques Used: Cell Differentiation, Chemotaxis Assay

    23) Product Images from "Regulation of Pathogenic Th17 Cell Differentiation by IL-10 in the Development of Glomerulonephritis"

    Article Title: Regulation of Pathogenic Th17 Cell Differentiation by IL-10 in the Development of Glomerulonephritis

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2013.05.001

    Enhanced immune responses in IL-10 −/− mice with GN. A : Splenocytes were prepared from WT or IL-10 −/− mice with GN induced by aGBM, and the cells were activated with SG for 24 hours. IL-17–producing CD4 + T cells
    Figure Legend Snippet: Enhanced immune responses in IL-10 −/− mice with GN. A : Splenocytes were prepared from WT or IL-10 −/− mice with GN induced by aGBM, and the cells were activated with SG for 24 hours. IL-17–producing CD4 + T cells

    Techniques Used: Mouse Assay

    24) Product Images from "IL-17 is required for Th1 immunity and host resistance to the intracellular pathogen Francisella tularensis LVS"

    Article Title: IL-17 is required for Th1 immunity and host resistance to the intracellular pathogen Francisella tularensis LVS

    Journal: Immunity

    doi: 10.1016/j.immuni.2009.08.025

    IL-17 can induce the polarization of naïve T cells into IFNγ-producing T cells
    Figure Legend Snippet: IL-17 can induce the polarization of naïve T cells into IFNγ-producing T cells

    Techniques Used:

    Cellular sources of IL-17 in the lung following LVS infection
    Figure Legend Snippet: Cellular sources of IL-17 in the lung following LVS infection

    Techniques Used: Infection

    IL-23 dependent IL-17 is required for protection against pulmonary tularemia
    Figure Legend Snippet: IL-23 dependent IL-17 is required for protection against pulmonary tularemia

    Techniques Used:

    IL-17 is required for induction of IFNγ responses during pulmonary tularemia
    Figure Legend Snippet: IL-17 is required for induction of IFNγ responses during pulmonary tularemia

    Techniques Used:

    IL-17 but not IL-17F or IL-22 can induce IL-12 from BMDCs
    Figure Legend Snippet: IL-17 but not IL-17F or IL-22 can induce IL-12 from BMDCs

    Techniques Used:

    IL-17 induces IFNγ and IL-12 from macrophages and enhances bacterial clearance
    Figure Legend Snippet: IL-17 induces IFNγ and IL-12 from macrophages and enhances bacterial clearance

    Techniques Used:

    25) Product Images from "Protective effect of 1α,25-dihydroxyvitamin D3 on effector CD4+ T cell induced injury in human renal proximal tubular epithelial cells"

    Article Title: Protective effect of 1α,25-dihydroxyvitamin D3 on effector CD4+ T cell induced injury in human renal proximal tubular epithelial cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0172536

    Effect of 1,25(OH) 2 D3 on the production of KIM-1 and fibronectin 1 from HRPTEpiC induced by recombinant human IL-17 (rhIL-17), human TNF- α or TGF-beta. (A) The expression of KIM-1 by HRPTEpiC was pretreated with 1,25(OH) 2 D3 (10 nM) as indicated, and then cultured for 24 hours with TNF-α (50 ng/mL) and/or IL-17 (50ng/mL) (n = 3). The expression of KIM-1 was measured by real-time PCR. Note that addition of 1,25(OH) 2 D3 significantly decrease KIM-1 expression which was increased by TNF-α and IL-17. *P
    Figure Legend Snippet: Effect of 1,25(OH) 2 D3 on the production of KIM-1 and fibronectin 1 from HRPTEpiC induced by recombinant human IL-17 (rhIL-17), human TNF- α or TGF-beta. (A) The expression of KIM-1 by HRPTEpiC was pretreated with 1,25(OH) 2 D3 (10 nM) as indicated, and then cultured for 24 hours with TNF-α (50 ng/mL) and/or IL-17 (50ng/mL) (n = 3). The expression of KIM-1 was measured by real-time PCR. Note that addition of 1,25(OH) 2 D3 significantly decrease KIM-1 expression which was increased by TNF-α and IL-17. *P

    Techniques Used: Recombinant, Expressing, Cell Culture, Real-time Polymerase Chain Reaction

    Effects of 1,25(OH) 2 D3 on the expression of mTOR and STAT3 proteins in HRPTEpiC. (A) Immunoblotting of VDR, p -mTOR, mTOR, p- Akt, Akt, p- s6k, p- STAT3(705), STAT3, p -ERK, ERK and REDD1 in HRPTEpiC pretreated with or without 1,25(OH) 2 D3 (10 nM) and then cultured with recombinant IL-17 for 1 hour. (B) Stimulation of HRPTEpiC with recombinant IL-17 activated the phosphorylation of mTOR, Akt, STAT3,ERK and s6k as detected by Western blotting and shown by the ratio of phosphorylated to total proteins. Note that combined use of 1,25(OH) 2 D3 resulted in the most inhibitory effect on the expression of VDR, mTOR, Akt, STAT3, ERK and s6k. Bars show the mean ±SD results in 3 patients, in 1 of 3 independent experiments. **P
    Figure Legend Snippet: Effects of 1,25(OH) 2 D3 on the expression of mTOR and STAT3 proteins in HRPTEpiC. (A) Immunoblotting of VDR, p -mTOR, mTOR, p- Akt, Akt, p- s6k, p- STAT3(705), STAT3, p -ERK, ERK and REDD1 in HRPTEpiC pretreated with or without 1,25(OH) 2 D3 (10 nM) and then cultured with recombinant IL-17 for 1 hour. (B) Stimulation of HRPTEpiC with recombinant IL-17 activated the phosphorylation of mTOR, Akt, STAT3,ERK and s6k as detected by Western blotting and shown by the ratio of phosphorylated to total proteins. Note that combined use of 1,25(OH) 2 D3 resulted in the most inhibitory effect on the expression of VDR, mTOR, Akt, STAT3, ERK and s6k. Bars show the mean ±SD results in 3 patients, in 1 of 3 independent experiments. **P

    Techniques Used: Expressing, Cell Culture, Recombinant, Western Blot

    Effect of 1,25(OH) 2 D3 on the production of inflammatory cytokines by HRPTEpiC, induced by recombinant human IL-17 (rhIL-17) or human TNF-α. HRPTEpiC were cultured with rhIL-17 (0, 1, 10, 50, or 100 ng/mL) or TNF-α (0, 1, 10, or 50 ng/mL) for 48 hours, and the production of (A) IL-6 and (B) IL-8 was measured (n = 3). Note that IL-6 and IL-8 levels were significantly increased by IL-17 or TNF-α in a dose-dependent manner. *P
    Figure Legend Snippet: Effect of 1,25(OH) 2 D3 on the production of inflammatory cytokines by HRPTEpiC, induced by recombinant human IL-17 (rhIL-17) or human TNF-α. HRPTEpiC were cultured with rhIL-17 (0, 1, 10, 50, or 100 ng/mL) or TNF-α (0, 1, 10, or 50 ng/mL) for 48 hours, and the production of (A) IL-6 and (B) IL-8 was measured (n = 3). Note that IL-6 and IL-8 levels were significantly increased by IL-17 or TNF-α in a dose-dependent manner. *P

    Techniques Used: Recombinant, Cell Culture

    Effect of 1,25(OH) 2 D3 on CD4 + T cells isolated from the PBMCs of healthy donors and cultured under Th0-polarizing conditions. Human PBMCs (n = 3) were isolated from healthy subjects and pre-incubated with 1,25(OH) 2 D3 (1, 10, 100 nM) as indicated. They were then cultured under Th0-polarizing conditions (anti-CD3, 1 μg/mL and anti-CD28, 1 μg/mL) for 48 h. (A) PBMCs were stained with anti-CD4 PE-cy7, anti-CD25 APC, anti-IFN-γ FITC, anti-IL-17 PE, anti-IL-4 APC and anti-Foxp3 FITC. CD4 + T cells were gated for further analysis. Then, the percentage of (B) IFN-γ + /CD4 + T cells, (C) IL-17 + /CD4 + T cells, (D) IL-4 + /CD4 + T cells, and (E) CD25 + FOXP3 + /CD4 + T cells was measured by flow cytometry. The production of (F) IFN-γ, (G) IL-17, (H) IL-22 and (I) IL-23 by Th0-polarizing CD4 + T cells and secretion into the culture supernatant by ELISA. Bars represent the mean 0± SD. * P
    Figure Legend Snippet: Effect of 1,25(OH) 2 D3 on CD4 + T cells isolated from the PBMCs of healthy donors and cultured under Th0-polarizing conditions. Human PBMCs (n = 3) were isolated from healthy subjects and pre-incubated with 1,25(OH) 2 D3 (1, 10, 100 nM) as indicated. They were then cultured under Th0-polarizing conditions (anti-CD3, 1 μg/mL and anti-CD28, 1 μg/mL) for 48 h. (A) PBMCs were stained with anti-CD4 PE-cy7, anti-CD25 APC, anti-IFN-γ FITC, anti-IL-17 PE, anti-IL-4 APC and anti-Foxp3 FITC. CD4 + T cells were gated for further analysis. Then, the percentage of (B) IFN-γ + /CD4 + T cells, (C) IL-17 + /CD4 + T cells, (D) IL-4 + /CD4 + T cells, and (E) CD25 + FOXP3 + /CD4 + T cells was measured by flow cytometry. The production of (F) IFN-γ, (G) IL-17, (H) IL-22 and (I) IL-23 by Th0-polarizing CD4 + T cells and secretion into the culture supernatant by ELISA. Bars represent the mean 0± SD. * P

    Techniques Used: Isolation, Cell Culture, Incubation, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    26) Product Images from "Serum Amyloid A Promotes Lung Neutrophilia by Increasing IL-17A Levels in the Mucosa and \u03b3\u03b4 T Cells"

    Article Title: Serum Amyloid A Promotes Lung Neutrophilia by Increasing IL-17A Levels in the Mucosa and \u03b3\u03b4 T Cells

    Journal: American Journal of Respiratory and Critical Care Medicine

    doi: 10.1164/rccm.201211-2139OC

    Enzyme-linked immunospot assay of the cellular source of IL-17A in response to serum amyloid A (SAA). Mice were intranasally treated with SAA and γδT (CD45 + , γδTCR + ), and CD4 + T (TCR + , CD4 + TCR) cells were sorted. All cell
    Figure Legend Snippet: Enzyme-linked immunospot assay of the cellular source of IL-17A in response to serum amyloid A (SAA). Mice were intranasally treated with SAA and γδT (CD45 + , γδTCR + ), and CD4 + T (TCR + , CD4 + TCR) cells were sorted. All cell

    Techniques Used: Enzyme-linked Immunospot, Mouse Assay

    Serum amyloid A (SAA)–induced IL-17A from multiple cellular sources. ( A ) Total bronchoalveolar lavage (BAL) neutrophils as determined by fluorescence-activated cell sorter in Balb/c and NOD.SCID mice treated with saline or SAA. ( B ) IL-17A gene
    Figure Legend Snippet: Serum amyloid A (SAA)–induced IL-17A from multiple cellular sources. ( A ) Total bronchoalveolar lavage (BAL) neutrophils as determined by fluorescence-activated cell sorter in Balb/c and NOD.SCID mice treated with saline or SAA. ( B ) IL-17A gene

    Techniques Used: Fluorescence, Mouse Assay

    Serum amyloid A (SAA)–mediated expression of IL-17A and its related cytokines is dependent on ALX-FPR2. Mice were simultaneously challenged with SAA (2 μg) and 15-epi-LXA 4 (4 μg) or vehicle by intranasal administration and gene
    Figure Legend Snippet: Serum amyloid A (SAA)–mediated expression of IL-17A and its related cytokines is dependent on ALX-FPR2. Mice were simultaneously challenged with SAA (2 μg) and 15-epi-LXA 4 (4 μg) or vehicle by intranasal administration and gene

    Techniques Used: Expressing, Mouse Assay

    Serum amyloid A (SAA)–mediated bronchoalveolar lavage (BAL) neutrophil recruitment is associated with expression of IL-17–related cytokine members in the lung. Mice were given a single dose of saline or SAA (2 μg) intranasally.
    Figure Legend Snippet: Serum amyloid A (SAA)–mediated bronchoalveolar lavage (BAL) neutrophil recruitment is associated with expression of IL-17–related cytokine members in the lung. Mice were given a single dose of saline or SAA (2 μg) intranasally.

    Techniques Used: Expressing, Mouse Assay

    Serum amyloid A (SAA) predominantly promotes protein secretion of the IL-17A–regulating cytokine, IL-6. Mice were treated intranasally with saline or SAA (2 μg) and bronchoalveolar lavage fluid collected at the indicated time points (hours).
    Figure Legend Snippet: Serum amyloid A (SAA) predominantly promotes protein secretion of the IL-17A–regulating cytokine, IL-6. Mice were treated intranasally with saline or SAA (2 μg) and bronchoalveolar lavage fluid collected at the indicated time points (hours).

    Techniques Used: Mouse Assay

    Blocking IL-17A inhibits serum amyloid A (SAA)–induced neutrophil recruitment. Mice were pretreated with anti–IL-17A or rat IgG2 A (isotype) antibody (50 μg) intranasally 1 hour before saline or SAA (2 μg) administration.
    Figure Legend Snippet: Blocking IL-17A inhibits serum amyloid A (SAA)–induced neutrophil recruitment. Mice were pretreated with anti–IL-17A or rat IgG2 A (isotype) antibody (50 μg) intranasally 1 hour before saline or SAA (2 μg) administration.

    Techniques Used: Blocking Assay, Mouse Assay

    27) Product Images from "Brown adipose tissue ameliorates autoimmune arthritis via inhibition of Th17 cells"

    Article Title: Brown adipose tissue ameliorates autoimmune arthritis via inhibition of Th17 cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-68749-x

    BAT inhibits Th17 differentiation and induces IL-10 expression in vitro. ( A ) IL-17 and IL-10 levels were measured by ELISA in BAT and eWAT. ( B ) Levels of IL-17 and IL-10 in BAT and eWAT were detected by ELISA under Th17-inducing conditions for 72 h. ( C ) IL-17 and IL-10 levels of normal BAT and CIA BAT were measured by ELISA. ( D , E ) The population of Th17 cells and Treg cells in BAT co-culture condition were measured. *P
    Figure Legend Snippet: BAT inhibits Th17 differentiation and induces IL-10 expression in vitro. ( A ) IL-17 and IL-10 levels were measured by ELISA in BAT and eWAT. ( B ) Levels of IL-17 and IL-10 in BAT and eWAT were detected by ELISA under Th17-inducing conditions for 72 h. ( C ) IL-17 and IL-10 levels of normal BAT and CIA BAT were measured by ELISA. ( D , E ) The population of Th17 cells and Treg cells in BAT co-culture condition were measured. *P

    Techniques Used: Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

    Transplantation of normal brown adipose tissue reduces secretion of proinflammatory cytokines. Immunohistochemistry images are shown with tissues stained by anti-IL-12, anti-IL-17, anti-IL-6, anti-TNF-α, and anti-IL-10 antibodies. *P
    Figure Legend Snippet: Transplantation of normal brown adipose tissue reduces secretion of proinflammatory cytokines. Immunohistochemistry images are shown with tissues stained by anti-IL-12, anti-IL-17, anti-IL-6, anti-TNF-α, and anti-IL-10 antibodies. *P

    Techniques Used: Transplantation Assay, Immunohistochemistry, Staining

    28) Product Images from "Interleukin-17 Promotes Pseudomonas aeruginosa Keratitis in C57BL/6 Mouse Corneas"

    Article Title: Interleukin-17 Promotes Pseudomonas aeruginosa Keratitis in C57BL/6 Mouse Corneas

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

    doi: 10.4049/jimmunol.1900736

    PA infection increases IL23/17-axis signaling in B6 mouse cornea. (A) Mouse corneas were scratched with a needle and inoculated with 1.0 × 10 4 CFU PA . Whole corneas were collected at 1dpi for quantitative real-time PCR or semiquantitative RT-PCR analysis of IL-23, IL-17A, IL-17RA, IL-17RC. (N=3) (B) Western-blot analysis of IL-23, IL-23R, IL-17A, IL17-RC in cell lysates of whole corneas infected with PA at 1dpi. β-actin serves as the loading control. The results are presented as a relative increase (fold) to those of naive corneas, set as 1. Data are representative of three independent experiments (A, mean ± SEM). *p
    Figure Legend Snippet: PA infection increases IL23/17-axis signaling in B6 mouse cornea. (A) Mouse corneas were scratched with a needle and inoculated with 1.0 × 10 4 CFU PA . Whole corneas were collected at 1dpi for quantitative real-time PCR or semiquantitative RT-PCR analysis of IL-23, IL-17A, IL-17RA, IL-17RC. (N=3) (B) Western-blot analysis of IL-23, IL-23R, IL-17A, IL17-RC in cell lysates of whole corneas infected with PA at 1dpi. β-actin serves as the loading control. The results are presented as a relative increase (fold) to those of naive corneas, set as 1. Data are representative of three independent experiments (A, mean ± SEM). *p

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Blockade of IL-17A alteres gene expression in B6 mouse corneas in response to PA infection. Mouse corneas were treated with anti-IL17A antibody or control IgG and inoculated with 1.0 × 10 4 CFU PA . Corneal epithelial cells were collected at 6 hpi and analyzed by real-time PCR. The results are presented as a relative increase (fold) to those of naive corneas, set as 1. Data are representative of three independent experiments with three corneas per group (mean ± SEM). (N=3) *p
    Figure Legend Snippet: Blockade of IL-17A alteres gene expression in B6 mouse corneas in response to PA infection. Mouse corneas were treated with anti-IL17A antibody or control IgG and inoculated with 1.0 × 10 4 CFU PA . Corneal epithelial cells were collected at 6 hpi and analyzed by real-time PCR. The results are presented as a relative increase (fold) to those of naive corneas, set as 1. Data are representative of three independent experiments with three corneas per group (mean ± SEM). (N=3) *p

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

    IL-17A regulated OPG expression in B6 mouse corneas in response to PA infection. Mouse Corneas were treated with anti-IL17A antibody or rmIL-17A and inoculated with 1.0 × 10 4 CFU PA . Whole corneas were collected at 1dpi. ( A ) Protein array analysis revealed the effect of IL-17A on cytokine expression. Selected images for Osteoprotegerin was shown. ( B ) q-PCR analysis of Osteoprotegerin in whole corneas infected with P. aeruginosa at 1dpi. (N=3) ( C ) Western-blot analysis of Osteoprotegerin in cell lysate of whole corneas infected with P. aeruginosa at 1dpi. β-actin serves as the loading control. Data are representative of three independent experiments with three corneas per group (B, mean ± SEM). *p
    Figure Legend Snippet: IL-17A regulated OPG expression in B6 mouse corneas in response to PA infection. Mouse Corneas were treated with anti-IL17A antibody or rmIL-17A and inoculated with 1.0 × 10 4 CFU PA . Whole corneas were collected at 1dpi. ( A ) Protein array analysis revealed the effect of IL-17A on cytokine expression. Selected images for Osteoprotegerin was shown. ( B ) q-PCR analysis of Osteoprotegerin in whole corneas infected with P. aeruginosa at 1dpi. (N=3) ( C ) Western-blot analysis of Osteoprotegerin in cell lysate of whole corneas infected with P. aeruginosa at 1dpi. β-actin serves as the loading control. Data are representative of three independent experiments with three corneas per group (B, mean ± SEM). *p

    Techniques Used: Expressing, Infection, Protein Array, Polymerase Chain Reaction, Western Blot

    IL-17A neutralizing antibody and rmIL-17 have opposing effects on the outcome of PA infection in B6 mouse corneas. Mice were subconjunctivally injected with IL-17A neutralizing antibody (250ng/5μl) or recombinant mouse IL-17A (200ng/5μl in 0.1% BSA) 4h before the inoculation with 1.0 × 10 4 CFU PA . Mouse IgG or 0.1% BSA serves as control. ( A D ) Mouse corneas were monitored and photographed (original magnification × 10) at 1dpi. The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. ( B E C F ) At 1 dpi, the corneas were excised and subjected to bacterial plate counting (CFU per cornea) and MPO unit determination. Data are representative of three independent experiments with five corneas per group (B, C, mean ± SEM). (N=5) *p
    Figure Legend Snippet: IL-17A neutralizing antibody and rmIL-17 have opposing effects on the outcome of PA infection in B6 mouse corneas. Mice were subconjunctivally injected with IL-17A neutralizing antibody (250ng/5μl) or recombinant mouse IL-17A (200ng/5μl in 0.1% BSA) 4h before the inoculation with 1.0 × 10 4 CFU PA . Mouse IgG or 0.1% BSA serves as control. ( A D ) Mouse corneas were monitored and photographed (original magnification × 10) at 1dpi. The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. ( B E C F ) At 1 dpi, the corneas were excised and subjected to bacterial plate counting (CFU per cornea) and MPO unit determination. Data are representative of three independent experiments with five corneas per group (B, C, mean ± SEM). (N=5) *p

    Techniques Used: Infection, Mouse Assay, Injection, Recombinant

    OPG upregulation is partially responsible for IL-17-worsened outcome of PA keratitis in B6 mice. Mouse corneas were treated with rmIL-17A or rmIL-17A+Anti-OPG antibody and inoculated with 1.0 × 10 4 CFU PA , 0.1% BSA serves as control. ( A ) Mouse corneas were monitored and photographed (original magnification×10) at 1dpi. The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. ( B C ) At 1 dpi, the corneas were excised and subjected to bacterial plate counting (CFU per cornea) and MPO unit determination. Data are representative of three independent experiments with five corneas per group (B, C, mean ± SEM). (N=5) *p
    Figure Legend Snippet: OPG upregulation is partially responsible for IL-17-worsened outcome of PA keratitis in B6 mice. Mouse corneas were treated with rmIL-17A or rmIL-17A+Anti-OPG antibody and inoculated with 1.0 × 10 4 CFU PA , 0.1% BSA serves as control. ( A ) Mouse corneas were monitored and photographed (original magnification×10) at 1dpi. The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. ( B C ) At 1 dpi, the corneas were excised and subjected to bacterial plate counting (CFU per cornea) and MPO unit determination. Data are representative of three independent experiments with five corneas per group (B, C, mean ± SEM). (N=5) *p

    Techniques Used: Mouse Assay

    Concurrent topical application of IL-17A neutralizing antibody and ciprofloxacin eradicates PA -infection associated inflammation in B6 mice. C57BL/6 mouse corneas were inoculated with 1.0 × 10 4 CFU PA . Topical solution containing ciprofloxacin (Cip) was used to dissolve IL-17A neutralizing antibody. Topical antibiotic with or without anti-IL17A Ab was then applied, starting 16 hours after infection, at 2-hour intervals during the first and second days of treatment and at 4-hour intervals on the third day of treatment. The infected corneas were ( A ) photographed and ( B ) scored, and ( C ) myeloperoxidase (MPO) activity assay was performed at the end of experiment. The results are representative of 3 independent experiments. Data are representative of three independent experiments with five corneas per group (B, C, mean ± SEM). (N=5) *p
    Figure Legend Snippet: Concurrent topical application of IL-17A neutralizing antibody and ciprofloxacin eradicates PA -infection associated inflammation in B6 mice. C57BL/6 mouse corneas were inoculated with 1.0 × 10 4 CFU PA . Topical solution containing ciprofloxacin (Cip) was used to dissolve IL-17A neutralizing antibody. Topical antibiotic with or without anti-IL17A Ab was then applied, starting 16 hours after infection, at 2-hour intervals during the first and second days of treatment and at 4-hour intervals on the third day of treatment. The infected corneas were ( A ) photographed and ( B ) scored, and ( C ) myeloperoxidase (MPO) activity assay was performed at the end of experiment. The results are representative of 3 independent experiments. Data are representative of three independent experiments with five corneas per group (B, C, mean ± SEM). (N=5) *p

    Techniques Used: Infection, Mouse Assay, Activity Assay

    Blockade of either IL-17A or OPG attenuates the severity of PA keratitis in B6 mouse cornea at 3 dpi. Mice were subconjunctivally injected with either IL-17A neutralizing or OPG neutralizing antibody 4h before the inoculation with 1.0 × 10 4 CFU PA . Mouse IgG serves as control. ( A ) Mouse corneas were monitored and photographed (original magnification×10) at 3 dpi. The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. ( B C ) At 3 dpi, the corneas were excised and subjected to bacterial plate counting (CFU per cornea) and MPO unit determination. (N=5) *p
    Figure Legend Snippet: Blockade of either IL-17A or OPG attenuates the severity of PA keratitis in B6 mouse cornea at 3 dpi. Mice were subconjunctivally injected with either IL-17A neutralizing or OPG neutralizing antibody 4h before the inoculation with 1.0 × 10 4 CFU PA . Mouse IgG serves as control. ( A ) Mouse corneas were monitored and photographed (original magnification×10) at 3 dpi. The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. ( B C ) At 3 dpi, the corneas were excised and subjected to bacterial plate counting (CFU per cornea) and MPO unit determination. (N=5) *p

    Techniques Used: Mouse Assay, Injection

    Related Articles

    Mouse Assay:

    Article Title: Type IIb Heat Labile Enterotoxin B Subunit as a Mucosal Adjuvant to Enhance Protective Immunity against H5N1 Avian Influenza Viruses
    Article Snippet: .. For IL-17A depletion, two groups of BALB/c mice immunized with 10 μg HA + 5 μg LTIIb-B5 + PELC were intraperitoneally injected with 0.1 mg of anti-IL-17A monoclonal antibody (mAb) (R & D SYSTEMS) or IgG2A isotype control mAb (R & D SYSTEMS) at 1 day before and 1, 3, 5 days after viral challenges. ..

    Injection:

    Article Title: Type IIb Heat Labile Enterotoxin B Subunit as a Mucosal Adjuvant to Enhance Protective Immunity against H5N1 Avian Influenza Viruses
    Article Snippet: .. For IL-17A depletion, two groups of BALB/c mice immunized with 10 μg HA + 5 μg LTIIb-B5 + PELC were intraperitoneally injected with 0.1 mg of anti-IL-17A monoclonal antibody (mAb) (R & D SYSTEMS) or IgG2A isotype control mAb (R & D SYSTEMS) at 1 day before and 1, 3, 5 days after viral challenges. ..

    Concentration Assay:

    Article Title: Early kinetics of serum Interleukine-17A and infarct size in patients with reperfused acute ST-elevated myocardial infarction
    Article Snippet: .. HUVEC were then exposed to 10% of patients’ sera (diluted in EGM™ -2 medium) with or without addition of anti-IL-17A antibody at the concentration of 10 μg/mL (R & D Systems, Minneapolis MN, USA). ..

    Staining:

    Article Title: Association of Pathogenic Th17 Cells with the Disease Severity and Its Potential Implication for Biological Treatment Selection in Psoriasis Patients
    Article Snippet: .. According to the staining, permeabilized tissue was incubated overnight with primary antibodies anti-CD4 (Sigma Aldrich), anti-IL-17 (RD Systems), and anti-IFN-γ (BioLegend). ..

    Incubation:

    Article Title: Association of Pathogenic Th17 Cells with the Disease Severity and Its Potential Implication for Biological Treatment Selection in Psoriasis Patients
    Article Snippet: .. According to the staining, permeabilized tissue was incubated overnight with primary antibodies anti-CD4 (Sigma Aldrich), anti-IL-17 (RD Systems), and anti-IFN-γ (BioLegend). ..

    Article Title: Third-party regulatory T cells prevent murine acute graft-versus-host disease
    Article Snippet: .. Anti-mouse IL-4, IL-21, IL-10, IFN-γ, IL-6, or IL-17 monoclonal antibodies (R & D Systems, Minneapolis, MN, USA) were added to a 96-well plate (Nunc, Roskilde, Denmark) and incubated overnight at 4°C. ..

    Neutralization:

    Article Title: Blockade of Store-Operated Calcium Entry Reduces IL-17/TNF Cytokine-Induced Inflammatory Response in Human Myoblasts
    Article Snippet: .. For the IL-17 and TNFα neutralization assays, PBMCs activated or not with PHA for 24 h were exposed to an anti-IL-17 antibody (R & D Systems) and/or the anti-TNFα antibody infliximab (Merck, Kenilworth, USA) at 10 μg/mL for 3 h before being added to the HepaRG cells. ..

    Enzyme-linked Immunospot:

    Article Title: Pharmacological inhibition of RORγt suppresses the Th17 pathway and alleviates arthritis in vivo
    Article Snippet: .. In brief, inguinal draining lymph node cells (1 × 105 /well) from compound or vehicle treated rats used in the AiA study were added to 96-well polyvinylidene difluoride (PVDF) plates (Millipore), pre-coated with anti-IL-17 capture Ab (ELISPOT development module, SEL421, R & D Systems). ..

    Blocking Assay:

    Article Title: Synergistic Interaction Between High Bioactive IL-17A and Joint Destruction for the Occurrence of Cardiovascular Events in Rheumatoid Arthritis
    Article Snippet: .. To measure the specific contribution of IL-17A in IL-8 production, plasma samples (diluted at 10%) are first pre-incubated with or without a blocking anti-IL-17A monoclonal antibody (10 μg/ml, R & D Systems, Paris, France) and then added to HUVEC for 48 h. The difference between IL-8 production by ELISA with and without anti-IL-17A antibody represents the contribution of the bioactive fraction of IL-17A ( ). ..

    Enzyme-linked Immunosorbent Assay:

    Article Title: Synergistic Interaction Between High Bioactive IL-17A and Joint Destruction for the Occurrence of Cardiovascular Events in Rheumatoid Arthritis
    Article Snippet: .. To measure the specific contribution of IL-17A in IL-8 production, plasma samples (diluted at 10%) are first pre-incubated with or without a blocking anti-IL-17A monoclonal antibody (10 μg/ml, R & D Systems, Paris, France) and then added to HUVEC for 48 h. The difference between IL-8 production by ELISA with and without anti-IL-17A antibody represents the contribution of the bioactive fraction of IL-17A ( ). ..

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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 anti il 17a antibody
    Schematic diagram for the role of the interplay between ER stress and <t>IL-17A</t> in LPS-induced lung injury.
    Anti Il 17a Antibody, 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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    Percentage of <t>IL-17A</t> + and IL-17F + cells in airway submucosal cells of COPD patients . (A) Submucosal cells in airways of COPD patients. (B) Percentage of IL-17A + and IL-17F + cells in airway submucosal cells of COPD patients. Results are expressed as median (range), n = 15 and 16 subjects for controls and COPD patients respectively. ** P
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    Image Search Results


    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

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

    Journal: Theranostics

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

    doi: 10.7150/thno.11685

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

    Article Snippet: Anti-IL-17A antibody or 4-PBA reduced the increases of GRP78 and CHOP expression in BAL cells and lung tissues of LV mice.

    Techniques:

    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

    Journal: Theranostics

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

    doi: 10.7150/thno.11685

    Figure Lengend 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

    Article Snippet: Anti-IL-17A antibody or 4-PBA reduced the increases of GRP78 and CHOP expression in BAL cells and lung tissues of LV mice.

    Techniques: 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

    Journal: Theranostics

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

    doi: 10.7150/thno.11685

    Figure Lengend 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

    Article Snippet: Anti-IL-17A antibody or 4-PBA reduced the increases of GRP78 and CHOP expression in BAL cells and lung tissues of LV mice.

    Techniques: 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

    Journal: Theranostics

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

    doi: 10.7150/thno.11685

    Figure Lengend 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

    Article Snippet: Anti-IL-17A antibody or 4-PBA reduced the increases of GRP78 and CHOP expression in BAL cells and lung tissues of LV mice.

    Techniques: 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

    Journal: Theranostics

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

    doi: 10.7150/thno.11685

    Figure Lengend 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

    Article Snippet: Anti-IL-17A antibody or 4-PBA reduced the increases of GRP78 and CHOP expression in BAL cells and lung tissues of LV mice.

    Techniques: 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

    Journal: Theranostics

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

    doi: 10.7150/thno.11685

    Figure Lengend 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

    Article Snippet: Anti-IL-17A antibody or 4-PBA reduced the increases of GRP78 and CHOP expression in BAL cells and lung tissues of LV mice.

    Techniques: 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

    Journal: Theranostics

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

    doi: 10.7150/thno.11685

    Figure Lengend 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

    Article Snippet: Anti-IL-17A antibody or 4-PBA reduced the increases of GRP78 and CHOP expression in BAL cells and lung tissues of LV mice.

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

    Percentage of IL-17A + and IL-17F + cells in airway submucosal cells of COPD patients . (A) Submucosal cells in airways of COPD patients. (B) Percentage of IL-17A + and IL-17F + cells in airway submucosal cells of COPD patients. Results are expressed as median (range), n = 15 and 16 subjects for controls and COPD patients respectively. ** P

    Journal: Respiratory Research

    Article Title: CD8 positive T cells express IL-17 in patients with chronic obstructive pulmonary disease

    doi: 10.1186/1465-9921-12-43

    Figure Lengend Snippet: Percentage of IL-17A + and IL-17F + cells in airway submucosal cells of COPD patients . (A) Submucosal cells in airways of COPD patients. (B) Percentage of IL-17A + and IL-17F + cells in airway submucosal cells of COPD patients. Results are expressed as median (range), n = 15 and 16 subjects for controls and COPD patients respectively. ** P

    Article Snippet: The sections for double labeling with IL-17A or IL-17F paired with CD4 and CD8 respectively were incubated with diluted goat anti-human IL-17A antibody (1:100) or IL-17F antibody (1:200) (R & D Systems) paired with mouse anti-human CD4 antibody (1:40) (VP-C319, Vector), CD8 antibody (1:120) (M7103, Dako) or relevant isotype controls (MAB002, R & D Systems) for overnight at 4°C.

    Techniques:

    IL-17A and IL-17F expression in COPD patients . (A) Immunohistochemistry, positive staining appears brown color. Magnification, 100 ×. Scale bar = 50 μm. (B) IL-17F expression in epithelium of airways of COPD patients. IL-17F positive area in epithelium was measured as outlined in text. (C) IL-17A and IL-17F expression in submucosa of airways of COPD patients. Absolute IL-17A + and IL-17F + positive cells in submucosa were counted. Results are expressed as median (range), n = 15 and 16 subjects for controls and COPD patients respectively. *** P

    Journal: Respiratory Research

    Article Title: CD8 positive T cells express IL-17 in patients with chronic obstructive pulmonary disease

    doi: 10.1186/1465-9921-12-43

    Figure Lengend Snippet: IL-17A and IL-17F expression in COPD patients . (A) Immunohistochemistry, positive staining appears brown color. Magnification, 100 ×. Scale bar = 50 μm. (B) IL-17F expression in epithelium of airways of COPD patients. IL-17F positive area in epithelium was measured as outlined in text. (C) IL-17A and IL-17F expression in submucosa of airways of COPD patients. Absolute IL-17A + and IL-17F + positive cells in submucosa were counted. Results are expressed as median (range), n = 15 and 16 subjects for controls and COPD patients respectively. *** P

    Article Snippet: The sections for double labeling with IL-17A or IL-17F paired with CD4 and CD8 respectively were incubated with diluted goat anti-human IL-17A antibody (1:100) or IL-17F antibody (1:200) (R & D Systems) paired with mouse anti-human CD4 antibody (1:40) (VP-C319, Vector), CD8 antibody (1:120) (M7103, Dako) or relevant isotype controls (MAB002, R & D Systems) for overnight at 4°C.

    Techniques: Expressing, Immunohistochemistry, Staining

    IL-17A and IL-17F mRNA expression in airways of COPD patients . (A) Quantitative RT-PCR was performed from frozen airways sections of COPD patients. One representative example from 7 subjects with similar results is shown. (B) Quantification of IL-17A and IL-17F mRNA expression in airways of control subjects and COPD patients. Results are expressed as means ± SEM. N = 7 for both control subjects and COPD patients.

    Journal: Respiratory Research

    Article Title: CD8 positive T cells express IL-17 in patients with chronic obstructive pulmonary disease

    doi: 10.1186/1465-9921-12-43

    Figure Lengend Snippet: IL-17A and IL-17F mRNA expression in airways of COPD patients . (A) Quantitative RT-PCR was performed from frozen airways sections of COPD patients. One representative example from 7 subjects with similar results is shown. (B) Quantification of IL-17A and IL-17F mRNA expression in airways of control subjects and COPD patients. Results are expressed as means ± SEM. N = 7 for both control subjects and COPD patients.

    Article Snippet: The sections for double labeling with IL-17A or IL-17F paired with CD4 and CD8 respectively were incubated with diluted goat anti-human IL-17A antibody (1:100) or IL-17F antibody (1:200) (R & D Systems) paired with mouse anti-human CD4 antibody (1:40) (VP-C319, Vector), CD8 antibody (1:120) (M7103, Dako) or relevant isotype controls (MAB002, R & D Systems) for overnight at 4°C.

    Techniques: Expressing, Quantitative RT-PCR

    Double immunofluorescence staining for detection of IL-17A and IL-17F expression in CD4 + and CD8 + T cells in airways of COPD patients . (A) Double immunofluorescence staining was performed. Scale bar = 5 μm. (B) Percentage of CD4 + and CD8 + T cells that express IL-17A and IL-17F. Results are expressed as means ± SEM. (C) Percentage of CD4 + and CD8 + T cells that express IL-17A and IL-17F in total IL-17A + and IL-17F + cells. Results are expressed as means ± SEM. *P

    Journal: Respiratory Research

    Article Title: CD8 positive T cells express IL-17 in patients with chronic obstructive pulmonary disease

    doi: 10.1186/1465-9921-12-43

    Figure Lengend Snippet: Double immunofluorescence staining for detection of IL-17A and IL-17F expression in CD4 + and CD8 + T cells in airways of COPD patients . (A) Double immunofluorescence staining was performed. Scale bar = 5 μm. (B) Percentage of CD4 + and CD8 + T cells that express IL-17A and IL-17F. Results are expressed as means ± SEM. (C) Percentage of CD4 + and CD8 + T cells that express IL-17A and IL-17F in total IL-17A + and IL-17F + cells. Results are expressed as means ± SEM. *P

    Article Snippet: The sections for double labeling with IL-17A or IL-17F paired with CD4 and CD8 respectively were incubated with diluted goat anti-human IL-17A antibody (1:100) or IL-17F antibody (1:200) (R & D Systems) paired with mouse anti-human CD4 antibody (1:40) (VP-C319, Vector), CD8 antibody (1:120) (M7103, Dako) or relevant isotype controls (MAB002, R & D Systems) for overnight at 4°C.

    Techniques: Double Immunofluorescence Staining, Expressing