TNFα Promotes Th17 Cell Differentiation through IL-6 and IL-1β Produced by Monocytes in Rheumatoid Arthritis

Figure 4

Role of TNFα in the Th17 cell differentiation through IL-6 and IL-1β in CD14⁺ monocytes. (a) CD4⁺ T cells purified from PBMC of healthy individuals were cultured with RA SF (dilution 1 : 8) in the presence or absence of the indicated antibodies in presence of anti-CD3 and anti-CD28 for 5 days. Percentage of Th1 and Th17 cells was analyzed in CD4 subset by flow cytometry. (b) FACS-sorted CD4⁺CD45RA⁺CD25⁻ naïve T cells from healthy individuals were stimulated with the indicated conditions for 5 days and intracellular staining of IL-17 was analyzed by flow cytometry (up row) and the supernatant was collected for IL-17 detection by ELISA (down row). (c) CD14⁺ monocytes purified from PBMC of healthy individuals were cultured overnight with RA SF in the presence or absence of the indicated antibodies. The resulting cells were cocultured with CD4⁺ T cells derived from the same donor in presence of anti-CD3 and anti-CD28 for 5 days. Percentage of Th17 cells was analyzed in CD4 subset by flow cytometry. (d) TNFRI and TNFRII expression levels were detected by flow cytometry gated on the CD14⁺ monocytes from the healthy individuals. Bars show the mean and SEM. (e) Purified CD4⁺ T cells derived from healthy individuals were cocultured with CD14⁺ monocytes with different ratios in the presence of TNFα (50 ng/mL) for 5 days and percentage of Th17 cells was analyzed in CD4 subset by flow cytometry. (f) Purified CD4⁺ T cells derived from healthy individuals were cocultured with CD14⁺ monocytes in the presence of TNFα and the indicated antibodies for 5 days. Percentage of Th17 cells was analyzed in CD4 subset by flow cytometry. (g) Purified CD4⁺ T cells and CD14⁺ monocytes from healthy individual were cocultured with TNFα or not. After being cultured for 5 days, percentage of IL-6 or IL-1β producing cells was analyzed in CD14⁺ cells by flow cytometry. (h) In the same experiment setting as in (g), STAT3 phosphorylation in the CD4⁺ T cell population was analyzed by flow cytometry (left panel) after 24 h culture and mRNA abundance of RORC was determined by real-time PCR (right panel) after 18 h culture. Bars show the mean ± SEM. versus medium control.

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