In vitro silencing of RIP2 in naive CD4⁺ T cells from lupus-prone mice promotes pathogenic Th17 cell differentiation

This work aims to investigate whether RIP2 silencing in naive CD4⁺ T cells from lupus-prone mice impacts Th17 cell activity or differentiation in vitro.

Naive CD4⁺ T cells isolation from MRL/lpr mice's spleens. Three RNA interference target sequences of RIP2 were packaged with lentivirus and transfected into naive CD4⁺ T cells. The shRIP2 with the highest interference efficiency was selected and transfected into naive CD4⁺ T cells. Naive CD4⁺ T cells were cultured under conventional (TGF-β1 and IL-6) and pathogenic (IL-6, IL-23, IL-1β) differentiation environments, respectively. Then, RT-qPCR, Western blot or Flow Cytometry were used for measuring the amounts of RIP2 and IL-17 and the differentiation of Th17 cells in two settings.

Under the conventional Th17 (cTh17) cell differentiation environment (TGF-β1 and IL-6), RIP2 deficiency is linked to decreased IL-17A levels (1.00 ± 0.03 vs 0.80 ± 0.03) and attenuated cTh17 cell (2.46 ± 0.08 vs 0.78 ± 0.03) differentiation (all, P < 0.05). Under the pathogenic Th17 (pTh17) cell environment (IL-1β, IL-23, IL-6), RIP2 deficiency is linked to elevated IL-17A levels (1.03 ± 0.05 vs 1.63 ± 0.07) and enhanced pTh17 cell (3.69 ± 0.19 vs 5.49 ± 0.10) differentiation (all, P < 0.05).

Our data suggest that RIP2 inhibition induces preferential differentiation of naive CD4⁺ T cells to pathogenic Th17 cells, while being able to upregulate IL-17A levels in the context of pTh17 cell differentiation. Our study opens up new research areas to reveal the underlying mechanisms and potential therapeutic targets for the prevention and treatment of SLE patients.