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. 2021 Jul 8;11:14145. doi: 10.1038/s41598-021-93599-6

Figure 3.

Figure 3

Decreased levels of CCL20 in joints of myostatin-deficient arthritic mice and regulation of Th17 cell transmigration by FLS-secreted CCL20. Immunohistochemical staining of CCL20 in tarsal joint sections of arthritic mice and CCL20-mediated transmigration of Th cells in vitro. (a) Representative images of bright field (left) and fluorescence-stained hind paw sections from hTNFtg and Mstn−/− hTNFtg mice (nuclear staining: DAPI—blue, CCL20: Alexa 546—red) and a higher magnification of stained areas are depicted. Scale bars = 200 μm. (b) Fluorescence intensity of CCL20 stainings within the ROI (normalised to IgG control-stained sections) of hTNFtg and Mstn−/− hTNFtg mice. Data represent means ± SEM (hTNFtg: n = 3, Mstn−/− hTNFtg n = 5, Mann–Whitney U Test, *p ≤ 0.05). (c) CCL20 serum levels of hTNFtg and Mstn−/− hTNFtg mice. Data represent means ± SEM (hTNFtg: n = 12, Mstn−/− hTNFtg n = 10, Mann–Whitney U Test). (d) Proportion of CCR6-expressing cells in LN-derived CD4+ Th cells (%). (e) CD4+ Th cell transmigration rate towards 20% conditioned culture medium (CCM) of myostatin-stimulated, IL-17A-stimulated, or simultaneously stimulated FLS either with or without anti-CCL20 antibody treatment. Data represent means ± SEM (n = 4, two-way ANOVA and multiple comparison test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). (f) Specificity of CCL20 inhibition on CD4+ Th cell transmigration by the anti-CCL20 antibody. Data represent means ± SEM (n = 4, Wilcoxon Test). (g) Effect of CCM of hTNFtg and Mstn−/− hTNFtg FLS stimulated with or without IL-17A. Data represent means ± SEM (hTNFtg and Mstn−/− hTNFtg n = 4, each, Mann–Whitney U Test, *p ≤ 0.05). TH17 medium (TH17 M) without CCM served as a negative control.