Fig. 10: Direct recognition of TLR4 ligands present in synovial fluid drives IL-17 production, independently of antigen recognition.

a–c Correlation between synovial fluid tenascin-C levels and a DMARD duration (n = 6 RA patients), b frequency of circulating (PB) TLR4⁺ T cells (n = 7 RA patients), and c frequency of synovial fluid (SF) TLR4⁺ T cells (n = 7 RA patients). d–g FACS-purified CD3^highCD4^high T cells from peripheral blood were cultured for 18 hours in the presence of medium (Med), synovial fluid (SF), or TLR4 signaling inhibitor (CLI-095). Frequency of d IL-17, e IL-10, f TNF-α, and g IL-21 production by TLR4⁺ T cells (n = 5 RA patients). h–k Ex vivo production of h IL-17 (n = 6 RA patients), i IL-10 (n = 5 RA patients), j TNF-α (n = 5 RA patients) and k IL-21 (n = 5 RA patients) by TLR4⁺ T cells in freshly obtained peripheral blood (PB) and synovial fluid (SF) donor paired samples. ΔMFI was calculated by subtracting the fluorescence intensity minus one (FMO) from median fluorescence intensity (MFI) for each given marker. FMOs were calculated independently for blood and synovial fluid FACS analysis. Data are presented as mean ± SD, for parametric statistical tests, or median ± IQR, for non-parametric statistical tests. Sample normality distribution was tested by using D’Agostino & Pearson normality test (n > 6) or Shapiro–Wilk normality test (n ≤ 6). P values ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 were determined by (a–c) Pearson correlation; (d, f, g) Friedman test with posttest Dunn’s multiple comparisons when significant results were obtained and (e) repeated measures ANOVA with posttest Tukey’s multiple comparisons; (h–j) Paired t test and (k) Wilcoxon matched-pairs rank test; the p values are adjusted for multiple comparisons. Effect size measures ⁺⁺⁺high, ⁺⁺medium, ⁺small were determined by (a–c) r_p – Pearson’s correlation coefficient; (d, f, g) W – Kendall’s W; (e) η_p² – partial eta-squared; (h–j) d – Cohen’s d and (k) r – correlation coefficient r.