Background/Purpose: Recent evidence indicates that IL-17A plays a key role in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE). SLE patients have a higher serum level of IL-17A which correlates with disease activity. However, the exact mechanisms of increased IL-17A level remain uncertain. Ten-eleven translocation (TET) family of dioxygenases catalyze the conversion of 5-methylcytosine into 5-hydroxymethylcytosine, and  regulate DNA methylation and gene expression dynamicly. Our previous results showed that both TET1 and TET2, particularly TET2 were increased in SLE CD4⁺T cells, which suggests TET2 may play a role in the pathogenesis of SLE. In this study, we aim to explore the effect of TET2 on IL-17A expression and the underlying mechanisms in SLE CD4⁺T cells.

Methods: Fifteen SLE patients and fifteen healthy controls were recruited. All patients fulfilled at least 4 of the SLE classification criteria of the American College of Rheumatology. Naive T cells or CD4⁺T cells were isolated by Ficoll-Hypaque density gradient centrifugation and magnetic sorting. The IL-17A levels in serum or culture supernatant were measured by ELISA kits. Bisulfite sequencing was done to assess the methylation status of the IL-17A promoter. Th17 cells were induced from Naive T cells of healthy donors in vitro. TET2-siRNA or TET2-expressing plasmid was transfected into CD4⁺T cells by transient electroporation. The mRNA levels of IL-17A and TET2 were examined by real-time PCR.The protein levels of IL-17A and TET2 were measured by flow cytometric analysis or western blot, respectively. The enrichment of TET2 in the promoter region of IL-17A gene was investigated by chromatin immunoprecipitation and real-time PCR.

Results: Compared to controls, both the serum IL-17A levels and IL-17A mRNA levels in CD4⁺T cells were elevated in SLE patients (p<0.05; p<0.05), the levels of Tet2 mRNA and protein were also increased in lupus CD4⁺T cells (p<0.05; p<0.05). The IL-17A promoter region in SLE CD4⁺T cells were found to be demethylated, which negatively correlated with the increased IL-17A mRNA expression (r=0.725; p<0.05). TET2 enrichment  at IL-17A promoter was increased in SLE CD4⁺T cells (p <0.05).Both IL-17A mRNA and protein levels were increased under the Th17 induction treatment on naive CD4⁺T cells (p<0.05; p<0.05). TET2 mRNA and protein levels were also increased in a time-dependent manner during the process of Th17 differentiation(p<0.05; p<0.05). IL-17A mRNA and protein levels were decreased in SLE CD4⁺T cells transfected with TET2-siRNA (p<0.05; p<0.05), while increased in normal CD4⁺T cells overexpressed  TET2 (p<0.05; p<0.05). The methylation levels of IL-17A promoter were up-regulated in SLE CD4⁺T cells transfected with TET2-siRNA (p<0.05), while down-regulated in normal CD4⁺T cells overexpressed TET2 (p<0.05). TET2 enrichment at IL-17A promoter was reduced in SLE CD4⁺T cells transfected with TET2-siRNA (p<0.05), while elevated in normal CD4⁺T cells overexpressed  TET2 (p<0.05).

Conclusion: Our results indicate that TET2 promotes IL-17A expression through demethylation of its promoter in SLE CD4⁺T cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ten-eleven-translocation-2-protein-down-regulates-dna-methylation-of-interleukin-17a-promoter-and-induces-its-expression-in-cd4t-cells-of-patients-with-systemic-lupus-erythematosus/