Figure 5. IL-17C supports aortic chemokine expression and supports the recruitment of IL-17A⁺ Th17 cells to the aorta.

A) 12 week WD Apoe^−/− aortas were sterilely explanted and untreated (black bars) or cultured with 100 ng/ml IL-17C (grey bars) for 24 hours before being collected and processed for RT-PCR. Fold induction of Ccl2, Ccl7, Cxc3cl1, Cxcl1, Cxcl2, Cxcl5, Ccl20, Il6, Il23a, and Il1b expression. n=9 Apoe^−/− mice, 3 independent experiments. (B) 12 week WD-fed Apoe^−/− and Il17c^−/−Apoe^−/− aortic smooth muscle cells (CD45⁻CD31⁻CD29⁻ aortic cells) were sterilely FACS sorted and cultured for an hour in vitro to collect cell supernatants. CD4⁺ T cells were isolated in parallel from 40 week CD Il17a^icre/icre R26R^{tdTomato/tdTomato} Apoe^−/− mice. Il17a^icre/icreR26R^{tdTomato/tdTomato}Apoe^−/− CD4⁺ T cells migrated towards either a migration media (vehicle control), or 1000 ng/ml rCCL20, or Apoe^−/− or Il17c^−/−Apoe^−/− aortic smooth muscle cell supernatants for 2 hrs. The transmigrated cells were collected and assessed for IL-17A^tdTomato+ Th17 cells by flow cytometry and normalized to the percentage of Th17 cells in the starting population. n= 5 independent experiments, all assays were performed in triplicate. (C–F) 30×10⁶ 12 week WD Apoe^−/− splenocytes were labeled with cell trace violet (CTV) and adoptively transferred to 12 week WD Apoe^−/− and Il17c^−/−Apoe^−/− mice for 72 hours. The migration of CTV⁺CD4⁺ T cells, and CTV⁺ Th17 cells within the recipients was assessed by flow cytometry. Representative flow cytometry plots for CTV⁺ splenocytes, and CD4⁺ Th17 cell migration to recipient spleens (C) and aortas (E) are shown. Quantification of the percentage and number of donor CD4⁺ T cells and Th17 cells within the spleens (D) and aortas (F) of recipient 12 week WD Apoe^−/− (blue) and Il17c^−/−Apoe^−/− (red) recipients. n= 5 recipients/genotype, 4 independent experiments. Bars depict means±SEM. * - p<0.05, ** - p<0.01, *** - p<0.001.