anti human il 17a monoclonal antibody  (R&D Systems)

Journal: PLoS ONE

Article Title: CCL20 Secretion from the Nucleus Pulposus Improves the Recruitment of CCR6-Expressing Th17 Cells to Degenerated IVD Tissues

doi: 10.1371/journal.pone.0066286

Figure Lengend Snippet: Fifty IVD tissue samples (Group P, n = 20 and Group E, n = 30) from disc degeneration patients and 3 from scoliosis patients were analyzed. In Group P or the control group, there were few or no positive cells (data not shown). The representative results of group E are shown. (a), IL-17-producing cells were detected by a rabbit anti-IL-17 polyclonal antibody and a mouse anti-CD4 monoclonal Ab, followed by secondary staining with an Alexa 488-conjugated donkey anti-rabbit and an Alexa 568-conjugated donkey anti-mouse IgG. DAPI mounting medium was used for nuclear staining. (b), surface CCR6 expression on the cells was detected with a rabbit anti CD4 monoclonal Ab and a mouse anti-CCR6 monoclonal Ab, followed by secondary staining with an Alexa 488-conjugated donkey anti-rabbit and an Alexa 568-conjugated donkey anti-mouse IgG. DAPI mounting medium was used for nuclear staining. In the top panel, green and red represents the expression of IL-17 and CD4, respectively, and the double-stained cells represent the IL-17-producing cells. In the bottom panel, green and red represents the expression of CD4 and CCR6, respectively, and the double-stained cells demonstrate the surface expression of CCR6 on T lymphocytes.

Article Snippet: After surface staining, the cells were fixed and permeabilized (LiankeBio, LK-GAS003, FIX&PERM Kit) and stained with a PE-conjugated anti-human IL-17A monoclonal antibody (eBioscience, 12-7178-41).

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: CCL20 Secretion from the Nucleus Pulposus Improves the Recruitment of CCR6-Expressing Th17 Cells to Degenerated IVD Tissues

doi: 10.1371/journal.pone.0066286

Figure Lengend Snippet: Eight IVD tissue samples from disc degeneration patients were used as the primary cultures. NP cells were stimulated with the indicated concentrations of (a) IL-17A or (b) TNF-α for 48 hours. The cells were treated with the optimal concentrations of (c) IL-17A, TNF-α or both for 0, 24, 48 and 72 hours. The cell-free supernatants were harvested and the expression levels of CCL20 protein were quantified by ELISA. The data are representative of at least three separate experiments, performed in duplicate, and the mean protein levels and standard deviations (SD) are shown. Significant differences were detected by a least significance difference analysis (LSD) (P<0.05). There was statistically significant differences between each concentration of IL-17 when compared to the control; however, statistical significance was verified only for TNF-α concentrations higher than 10 ng/ml (IL-17 treatment: *, P<0.01, vs. control; +, P<0.001, vs. 10 ng/ml; # , p<0.05, vs. 1000 ng/ml; TNF-α treatment: *, p<0.001, vs. control; +, p<0.001, vs. 10 ng/ml).

Article Snippet: After surface staining, the cells were fixed and permeabilized (LiankeBio, LK-GAS003, FIX&PERM Kit) and stained with a PE-conjugated anti-human IL-17A monoclonal antibody (eBioscience, 12-7178-41).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: PLoS ONE

Article Title: CCL20 Secretion from the Nucleus Pulposus Improves the Recruitment of CCR6-Expressing Th17 Cells to Degenerated IVD Tissues

doi: 10.1371/journal.pone.0066286

Figure Lengend Snippet: Eight IVD tissue samples from disc degeneration patients were used as the primary cultures. The cells were treated with the indicated concentrations of (a) IL-17A or (b)TNF-α for 48 hours. The expression levels of CCL20 mRNA were detected and the CCL20 transcripts were quantified by real-time RT-PCR analyses in all experiments. The data are representative of at least two separate experiments, performed in duplicate, and the mean increases in the mRNA expression were recorded, after which we evaluated the standard deviations (SD). Significant differences were detected by a least significance difference analysis (LSD) (P<0.05). The results correspond with the findings obtained in the ELISA tests. (IL-17 treatment: *, P<0.001, vs. control; +, P<0.05, vs. 10 ng/ml; # , p<0.01, vs. 1000 ng/ml; TNF-α treatment: *, p<0.001, vs. control; +, p<0.01, vs. 10 ng/ml).

Article Snippet: After surface staining, the cells were fixed and permeabilized (LiankeBio, LK-GAS003, FIX&PERM Kit) and stained with a PE-conjugated anti-human IL-17A monoclonal antibody (eBioscience, 12-7178-41).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: CCL20 Secretion from the Nucleus Pulposus Improves the Recruitment of CCR6-Expressing Th17 Cells to Degenerated IVD Tissues

doi: 10.1371/journal.pone.0066286

Figure Lengend Snippet: Heparinized peripheral whole blood cells from 20 patients and 15 healthy controls were stimulated with phorbol myristate acetate (PMA), ionomycin, and monensin for 4 h and subsequently stained with fluorochrome-labeled antibodies as described in Materials and Methods. A(a) Lymphocytes were gated by flow cytometry. A(b) CD3 + T subsets were gated by flow cytometry; the plots in the inset box represent the CD3 + T cells. A(c) Representative IL-17 expression levels in the CD3 + CD8 − T subsets (CD4 + T subsets) from each group are shown. The percentages of positive cells are shown in the upper left panels. A(d) Representative surface CCR6 expression levels on the CD3 + CD8 − IL-17 + subsets from each group are shown. The percentages of positive cells are shown in the right panel. (B) The percentage of circulating Th17 cells was significantly higher in IVD degenerated patients (2.973±0.689%) than in the control group (1.039±0.156%; *, p<0.001, significantly different from the control). (C) The rate of surface CCR6 expression on circulating Th17 cells was significantly higher in patients with degenerated IVD (59.69±4.48%) than the control group (28.75±2.09%; *, p<0.001, significantly different from the control).

Article Snippet: After surface staining, the cells were fixed and permeabilized (LiankeBio, LK-GAS003, FIX&PERM Kit) and stained with a PE-conjugated anti-human IL-17A monoclonal antibody (eBioscience, 12-7178-41).

Techniques: Staining, Labeling, Flow Cytometry, Expressing

Journal: Journal of Inflammation Research

Article Title: MBL Binding with AhR Controls Th17 Immunity in Silicosis-Associated Lung Inflammation and Fibrosis

doi: 10.2147/JIR.S357453

Figure Lengend Snippet: List of Primer Sequences Used for RT-qPCR Analysis in This Study

Article Snippet: Antibodies involved in this subsection were listed below: mouse anti-human/mouse CD4 monoclonal antibody (11–0049-42, Thermo Fisher Scientific, Inc), mouse anti-human IL-17a monoclonal antibody (12–7178-42, Thermo Fisher Scientific, Inc), mouse anti-human Foxp3 monoclonal antibody (17–4777-42, Thermo Fisher Scientific, Inc), mouse anti-human CD4/CD25 antibody cocktail (22–0425-71, Thermo Fisher Scientific, Inc), rat anti-human/mouse RORγ monoclonal antibody (12–6988-82, Thermo Fisher Scientific, Inc), rat anti-human/mouse IL-17a monoclonal antibody (12–7177-81, Thermo Fisher Scientific, Inc), rat anti-mouse CD25 monoclonal antibody (56–0251-82, Thermo Fisher Scientific, Inc), mouse- anti-human/mouse phosphor-STAT3 (TYR705)(48–9033-42, Thermo Fisher Scientific, Inc), mouse anti-human/mouse AhR (67785-1-Ig, Proteintech, Chicago, USA), Alexa Fluor ® 488 goat anti-mouse IgG antibody (405319, Biolegend, San Diego, USA).

Techniques:

Journal: Journal of Inflammation Research

Article Title: MBL Binding with AhR Controls Th17 Immunity in Silicosis-Associated Lung Inflammation and Fibrosis

doi: 10.2147/JIR.S357453

Figure Lengend Snippet: MBL inhibited Th17 cells polarization in vitro. ( A and C ) Naïve CD4 + T cells were cultured under Th17 differentiation conditions in the presence or absence of MBL protein (5μg/mL). ( A ) The expression of IL-17a and RORγt in CD4 + T cells were determined by FCM analysis. ( B ) The levels of IL-17a and IL-21 in the culture medium were evaluated by ELISA assay. ( C ) The mRNA levels of RORγt, IL-17a, and IL-21 were detected by RT-qPCR analysis. ( D – F ) Naïve CD4 + T cells were cultured under Treg differentiation conditions with or without MBL protein (5μg/mL). ( D ) The expression of Foxp3 in CD4 + T cells was determined by FCM analysis. ( E ) The levels of TGF-β and IL-10 in the culture medium were evaluated by ELISA assay. ( F ) The mRNA transcription levels of Foxp3, TGF-β, and IL-10 were detected by RT-qPCR analysis. * p < 0.05, ** p < 0.01. The data represent three independent experiments with similar results.

Article Snippet: Antibodies involved in this subsection were listed below: mouse anti-human/mouse CD4 monoclonal antibody (11–0049-42, Thermo Fisher Scientific, Inc), mouse anti-human IL-17a monoclonal antibody (12–7178-42, Thermo Fisher Scientific, Inc), mouse anti-human Foxp3 monoclonal antibody (17–4777-42, Thermo Fisher Scientific, Inc), mouse anti-human CD4/CD25 antibody cocktail (22–0425-71, Thermo Fisher Scientific, Inc), rat anti-human/mouse RORγ monoclonal antibody (12–6988-82, Thermo Fisher Scientific, Inc), rat anti-human/mouse IL-17a monoclonal antibody (12–7177-81, Thermo Fisher Scientific, Inc), rat anti-mouse CD25 monoclonal antibody (56–0251-82, Thermo Fisher Scientific, Inc), mouse- anti-human/mouse phosphor-STAT3 (TYR705)(48–9033-42, Thermo Fisher Scientific, Inc), mouse anti-human/mouse AhR (67785-1-Ig, Proteintech, Chicago, USA), Alexa Fluor ® 488 goat anti-mouse IgG antibody (405319, Biolegend, San Diego, USA).

Techniques: In Vitro, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Journal of Inflammation Research

Article Title: MBL Binding with AhR Controls Th17 Immunity in Silicosis-Associated Lung Inflammation and Fibrosis

doi: 10.2147/JIR.S357453

Figure Lengend Snippet: MBL limited AhR expression and its nuclear translocation in CD4 + T lymphocytes. ( A and C ) Naïve CD4 + T cells were cultured under Th17 differentiation conditions with or without MBL protein (5μg/mL). ( A ) The expression of AhR in CD4+ T cells was determined by FCM analysis. ( B ) The protein levels of AhR were determined by Western blot. ( C ) The level of phosphorylated STAT3 was assessed by FCM analysis. ( D – E ) Naïve CD4 + T cells were pretreated with 10μM Sc144 for 2h, then cultured under Th17 differentiation conditions with or without MBL protein (5μg/mL). ( D ) The mRNA expression of AhR in CD4 + T cells was determined by RT-qPCR analysis. ( E ) The protein level of AhR was assessed by Western blot analysis. ( F – H ) CD4 + T cells were treated with 200nM FICZ for 6h in the presence or absence of 5μg/mL MBL. ( F ) The fraction of cytoplasm and nucleus protein were separated, and the expression of AhR was determined by Western blot. ( G ) The mRNA levels of CYP1A1, CYP1A2, and CYP1B1 were detected by RT-qPCR analysis. ( H ) The mRNA levels of RORγt and IL-17 were detected by RT-qPCR analysis. * p < 0.05, ** p < 0.01. The data represent three independent experiments with similar results.

Article Snippet: Antibodies involved in this subsection were listed below: mouse anti-human/mouse CD4 monoclonal antibody (11–0049-42, Thermo Fisher Scientific, Inc), mouse anti-human IL-17a monoclonal antibody (12–7178-42, Thermo Fisher Scientific, Inc), mouse anti-human Foxp3 monoclonal antibody (17–4777-42, Thermo Fisher Scientific, Inc), mouse anti-human CD4/CD25 antibody cocktail (22–0425-71, Thermo Fisher Scientific, Inc), rat anti-human/mouse RORγ monoclonal antibody (12–6988-82, Thermo Fisher Scientific, Inc), rat anti-human/mouse IL-17a monoclonal antibody (12–7177-81, Thermo Fisher Scientific, Inc), rat anti-mouse CD25 monoclonal antibody (56–0251-82, Thermo Fisher Scientific, Inc), mouse- anti-human/mouse phosphor-STAT3 (TYR705)(48–9033-42, Thermo Fisher Scientific, Inc), mouse anti-human/mouse AhR (67785-1-Ig, Proteintech, Chicago, USA), Alexa Fluor ® 488 goat anti-mouse IgG antibody (405319, Biolegend, San Diego, USA).

Techniques: Expressing, Translocation Assay, Cell Culture, Western Blot, Quantitative RT-PCR

Journal: Journal of Inflammation Research

Article Title: MBL Binding with AhR Controls Th17 Immunity in Silicosis-Associated Lung Inflammation and Fibrosis

doi: 10.2147/JIR.S357453

Figure Lengend Snippet: MBL interacted with AhR and regulated Th17 cell polarization through AhR signaling. ( A and B ) CD4 + T cells were stimulated with 200nM FICZ with 5μg/mL MBL for 6 h. ( A ) The association of AhR with MBL was analyzed by immunoprecipitation assay. ( B ) Colocalization of AhR and internalized MBL in T cells was determined by immunofluorescence analysis. ( C – E ) Naïve CD4 + T cells were cultured under Th17 differentiation conditions in the presence or absence of MBL protein (5μg/mL) combined with 1μM CH-223191 for 7d, t. ( C ) The expression of IL-17a and RORγt in CD4 + T cells was determined by FCM analysis. ( D ) The levels of IL-17a and IL-21 in the culture medium were evaluated by ELISA assay. ( E ) The mRNA levels of RORγt, IL-17a, and IL-21 were detected by RT-qPCR analysis. * p < 0.05, ** p < 0.01. The data represent three independent experiments with similar results.

Article Snippet: Antibodies involved in this subsection were listed below: mouse anti-human/mouse CD4 monoclonal antibody (11–0049-42, Thermo Fisher Scientific, Inc), mouse anti-human IL-17a monoclonal antibody (12–7178-42, Thermo Fisher Scientific, Inc), mouse anti-human Foxp3 monoclonal antibody (17–4777-42, Thermo Fisher Scientific, Inc), mouse anti-human CD4/CD25 antibody cocktail (22–0425-71, Thermo Fisher Scientific, Inc), rat anti-human/mouse RORγ monoclonal antibody (12–6988-82, Thermo Fisher Scientific, Inc), rat anti-human/mouse IL-17a monoclonal antibody (12–7177-81, Thermo Fisher Scientific, Inc), rat anti-mouse CD25 monoclonal antibody (56–0251-82, Thermo Fisher Scientific, Inc), mouse- anti-human/mouse phosphor-STAT3 (TYR705)(48–9033-42, Thermo Fisher Scientific, Inc), mouse anti-human/mouse AhR (67785-1-Ig, Proteintech, Chicago, USA), Alexa Fluor ® 488 goat anti-mouse IgG antibody (405319, Biolegend, San Diego, USA).

Techniques: Immunoprecipitation, Immunofluorescence, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Journal of Inflammation Research

Article Title: MBL Binding with AhR Controls Th17 Immunity in Silicosis-Associated Lung Inflammation and Fibrosis

doi: 10.2147/JIR.S357453

Figure Lengend Snippet: MBL deficiency aggravated silica-induced pulmonary inflammation in mice. ( A – G ) WT and MBL −/− mice were intratracheally injected with 50μL silica suspension containing 5mg silica for 3 d. The lung tissues and sera were collected at the end of the experiment. ( A ) Histological analysis of mouse lungs was determined by H&E staining. ( B ) Cell counts in BALF were calculated under the microscope. ( C ) Total protein level was detected in BALF by BCA analysis. ( D ) FCM experiments were performed to measure Th17 or Treg frequency in lung tissues, HLN, and BALF from silica-challenged mice. ( E ) The levels of IL-17a, IL-6, TNFα, TGF-β, and IL-10 levels in BALF were evaluated by ELISA analysis. ( F ) The mRNA expression levels of IL-17a, IL-6, TNFα, RORγt, TGF-β, IL-10, and Foxp3 in lung tissues were determined by RT-qPCR analysis. ( G ) AhR expression in CD4 + T cells was evaluated by FCM assay. * p < 0.05, ** p < 0.01. The data represent three independent experiments with similar results.

Article Snippet: Antibodies involved in this subsection were listed below: mouse anti-human/mouse CD4 monoclonal antibody (11–0049-42, Thermo Fisher Scientific, Inc), mouse anti-human IL-17a monoclonal antibody (12–7178-42, Thermo Fisher Scientific, Inc), mouse anti-human Foxp3 monoclonal antibody (17–4777-42, Thermo Fisher Scientific, Inc), mouse anti-human CD4/CD25 antibody cocktail (22–0425-71, Thermo Fisher Scientific, Inc), rat anti-human/mouse RORγ monoclonal antibody (12–6988-82, Thermo Fisher Scientific, Inc), rat anti-human/mouse IL-17a monoclonal antibody (12–7177-81, Thermo Fisher Scientific, Inc), rat anti-mouse CD25 monoclonal antibody (56–0251-82, Thermo Fisher Scientific, Inc), mouse- anti-human/mouse phosphor-STAT3 (TYR705)(48–9033-42, Thermo Fisher Scientific, Inc), mouse anti-human/mouse AhR (67785-1-Ig, Proteintech, Chicago, USA), Alexa Fluor ® 488 goat anti-mouse IgG antibody (405319, Biolegend, San Diego, USA).

Techniques: Injection, Staining, Microscopy, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

Journal: Journal of Inflammation Research

Article Title: MBL Binding with AhR Controls Th17 Immunity in Silicosis-Associated Lung Inflammation and Fibrosis

doi: 10.2147/JIR.S357453

Figure Lengend Snippet: MBL-deficient mice exhibited aggravated silica-induced pulmonary fibrosis. ( A – G ) Mice were intratracheally injected with silica suspension (5mg silica in 50μL sterile PBS) for 35 d. The lung tissues and sera were collected at the end of the experiment. ( A ) Histological analysis of lung tissues was detected by H&E staining. ( B ) Ashcroft scores of lung tissues were determined under a microscope. ( C ) Hydroxyproline levels in lung tissues were tested. ( D ) Th17 or Treg frequency in lung tissues, HLN, and BALF were assessed by FCM analysis. ( E ) ELISA analysis was performed to evaluate IL-17a, IL-6, TNFα, TGF-β, and IL-10 levels in BALF collected from silica-treated mice at the last day. ( F ) The mRNA expression levels of IL-17a, IL-6, TNFα, RORγt, TGF-β, IL-10, and Foxp3 in lung tissues were determined by RT-qPCR analysis ( G ) Expression of AhR in CD4 + T cells was assessed by FCM assay. * p < 0.05, ** p < 0.01. The data represent three independent experiments with similar results.

Article Snippet: Antibodies involved in this subsection were listed below: mouse anti-human/mouse CD4 monoclonal antibody (11–0049-42, Thermo Fisher Scientific, Inc), mouse anti-human IL-17a monoclonal antibody (12–7178-42, Thermo Fisher Scientific, Inc), mouse anti-human Foxp3 monoclonal antibody (17–4777-42, Thermo Fisher Scientific, Inc), mouse anti-human CD4/CD25 antibody cocktail (22–0425-71, Thermo Fisher Scientific, Inc), rat anti-human/mouse RORγ monoclonal antibody (12–6988-82, Thermo Fisher Scientific, Inc), rat anti-human/mouse IL-17a monoclonal antibody (12–7177-81, Thermo Fisher Scientific, Inc), rat anti-mouse CD25 monoclonal antibody (56–0251-82, Thermo Fisher Scientific, Inc), mouse- anti-human/mouse phosphor-STAT3 (TYR705)(48–9033-42, Thermo Fisher Scientific, Inc), mouse anti-human/mouse AhR (67785-1-Ig, Proteintech, Chicago, USA), Alexa Fluor ® 488 goat anti-mouse IgG antibody (405319, Biolegend, San Diego, USA).

Techniques: Injection, Staining, Microscopy, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR