Fig 2: miR-21-5p overexpression may protect melanocytes via regulating T effector/regulatory T cell balance through STAT3. (A) Apoptosis rates of melanocytes in the control, MC+NC, M+NC, MC+STAT3 and M+STAT3 groups were measured via flow cytometry. Protein levels of (B) IL-17A and (C) IL-22 in the lower chamber of Transwell inserts after co-culture of melanocytes and Th17-polarized CD4+ T cells in the control, MC+NC, M+NC, MC+STAT3 and M+STAT3 groups was measured via enzyme-linked immunosorbent assay. (D) Tyrosinase activity and (E) relative melanin content in melanocytes in the control, MC+NC, M+NC, MC+STAT3 and M+STAT3 groups were measured using a microplate reader. *P<0.05, **P<0.01 and ***P<0.001 vs. MC+NC; ^P<0.05 and ^^P<0.01 vs. MC+STAT3; ##P<0.01 and ###P<0.001 vs. M+NC. Th, T helper; IL, interleukin; miR, microRNA; M, miR-21-5p mimic; MC, mimic control; NC, negative control.

Fig 3: IL-17–producing cells infiltrate musculoskeletal tissues in RRV disease.A) Gating strategy used to quantify infiltrating cells in the feet in RRV-infected IL-17GFP mice. Live singlet cells were gated for CD45+ and subsequently excluded from the CD11b+ MHC-II+ myeloid lineage (Lin) gate. CD3+ cell and subsequent CD4+ and CD8+ cell populations gated on IL-17A+ (or IL-17F+), and ?dTCR-positive T cells (?d T cells) were gated from the CD3+?dTCR+ cell population. B) IL-17-expressing CD8+ and CD4+ T cells in the feet and C) quadriceps muscle of RRV-infected IL-17GFP mice at 7, 10, 14 and 30 dpi. Data shown (mean +/- SEM) as number of LIVE CD45+Lin-CD3+CD8+IL-17AGFP+ or LIVE CD45+Lin-CD3+CD4+IL-17AGFP+ T cells, per paired feet or per gram of muscle tissue. D) IL-17F+ CD8+ and CD4+ T cells in the feet of RRV-infected IL-17GFP mice at 7, 10 and 14 dpi. Data shown (mean +/- SEM) as number of LIVE CD45+ Lin-CD3+CD8+IL-17F+ or LIVE CD45+ Lin-CD3+CD4+IL-17F+ T cells, per paired feet. E) IL-17A+ ?d T cells in the feet (left) and quadriceps muscle (right) of RRV-infected IL-17GFP mice at 7, 10 and 14 dpi. Data shown (mean +/- SEM) as number of LIVE CD45+ Lin-CD3+CD4-CD8- ?dTCR+ T cells, per paired feet, or per gram of muscle tissue. IL-17F expression were assessed by intracellular cytokine staining. Data in B-E representative of 3 independent experiments (n = 5 mice per group) and statistically significant differences between groups for each time point determined by Mann-Whitney U Test. *, p < 0.05; **, p < 0.01; ns: not significant. F) Flow cytometry gating strategy for IL-17GFP+ CD45+ cells and corresponding histogram (normalized counts from concatenated samples) for IL-17GFP reporter expression in CD3+ T cells and CD11b+ myeloid cells in the joints of RRV-infected mice, at 10 dpi. Populations gated from GFP-ve (grey histogram) and GFP+ve (green histogram). Mean population frequencies shown in gates. Data representative of 3 independent experiments (n = 5 mice per group).

Fig 4: IL-17–expressing cells infiltrate joint tissues during RRV disease.A) Confocal microscopy of immunofluorescent staining of optically cleared 150µm-thick vibratome sections of feet from mock-infected or RRV-infected IL-17GFP mice, at 10 dpi. Scale bars = 50µm. Wholemount sections were stained for Type IV Collagen (red) and nuclei labelled with DAPI (blue). B) Immunofluorescent staining of CD4+, CD8+ T cells and Type IV Collagen in foot cryosections from mock-infected or RRV-infected IL-17GFP mice at 7 and 10 dpi. Scale bars = 100µm (left panels) and 15µm (right panels). C) Immunofluorescent staining of Ly6G+ neutrophils and Type IV Collagen in foot cryosections RRV-infected IL-17GFP mice, at 10 dpi. Arrows indicate GFP+ve cells. Nuclei were labelled with Hoechst 33258. Scale bars = 30µm (left panel) and 10µm (insets). Images acquired as z-stacks and shown as maximum intensity projections. Data representative of 3 independent experiments (n = 5 mice per group).

Fig 5: miR-21-5p overexpression regulates T effector/Treg cell balance via STAT3. Percentages of (A) Th17, (B) Th22 and (C) Treg cells in CD4+ T cells of the control, MC+NC, M+NC, MC+STAT3 and M+STAT3 groups were measured via flow cytometry. Protein levels of (D) IL-17A and (E) IL-22 of T cells in the control, MC+NC, M+NC, MC+STAT3 and M+STAT3 groups were measured via enzyme-linked immunosorbent assay. (F) Relative Foxp3 expression in the control, MC+NC, M+NC, MC+STAT3 and M+STAT3 groups were measured via reverse transcription-quantitative PCR. GAPDH was used as the internal control. *P<0.05, **P<0.01 and ***P<0.001 vs. MC+NC; ^P<0.05 and ^^P<0.01 vs. MC+STAT3; ##P<0.01 and ###P<0.001 vs. M+NC. Th, T helper; IL, interleukin; miR, microRNA; Treg cells, regulatory T cells; M, miR-21-5p mimic; MC, mimic control; NC, negative control.