PDPK1-SGK1 Inhibitor Improves Rheumatoid Arthritis By Regulating Th17/Treg Balance And Its Underlying Mechanism

Objective:Rheumatoid arthritis（RA）is a common autoimmune disease.Abnormal activation of T helper cell 17（Th17）and the decreased functional competency of regulatory T cell（Treg）are the key features underlying RA immunopathology.The 3’-phosphoinositide dependent kinase 1（PDPK1）and serum-and glucocorticoid-regulated kinase 1（SGK1）are involved in the regulation of growth,proliferation and functional capacity of immune cells.Of note,the PDPK1-SGK1 signaling pathway is substantially activated upon the high salt stimulation.High salt diet has been identified as a risk factor for the progression of RA.Nonetheless,the specific mechanisms through which high salt diet participates in RA pathogenesis remains unclear.Based on these lines of evidence,our study aims to explore the potential involvement of PDPK1-SGK1 signaling and high salt diet in RA progression through the regulatory effect on Th17/Treg homeostasis.Methods:In the study,clinical samples were collected to determine the phosphorylation levels of PDPK1 and SGK1 in peripheral blood CD4 T cells,the correlation between the phosphorylation levels of PDPK1 and SGK1 and disease activity of RA patients,as well as the effects of PDPK1-SGK1 inhibitor and high salt stimulation on Th17/Treg balance of RA patients.The effects of PDPK1-SGK1 inhibitor and high salt stimulation（in vitro）or high salt diet（in vivo）on Th17/Treg balance was further confirmed by in vitro T cell polarization assay,adoptive transfer experiment and Treg lineage tracing mice(Foxp3^{Cre-e GFP};Rosa26^{flox-stop-flox-td Red}).Collagen-induced arthritis（CIA）model was constructed to explore the effects of PDPK1-SGK1 inhibitor and high salt diet on arthritic DBA/1 mice.The specific mechanisms of PDPK1-SGK1 inhibitor and high salt stimulation in modulating Th17/Treg balance were explored through glucose uptake test,Seahorse glycolysis rate measurement,western blotting and series of rescue assays.Results:Compared with healthy controls,the phosphorylation levels of PDPK1 and SGK1in peripheral blood CD4 T cells of RA patients were significantly increased,and the phosphorylation level of PDPK1 or SGK1 was positively correlated with the disease activity of RA.The proportion of Th17 cells was markedly increased while Treg cells were decreased in CD4 T cells from RA patients after the treatment of high salt.However,PDPK1-SGK1 inhibitor reduced the proportion of Th17 cells,increased the proportion of Treg cells,and corrected the Th17/Treg imbalance induced by high salt stimulation.In vitro T cell polarization showed that PDPK1-SGK1 inhibitor hindered the differentiation of Th17cells,promoted the differentiation of Treg cells,enhanced the stability of Treg cells and rectified the stimulatory effect of high salt.Treg lineage tracing mice and the adoptive transfer model further verified the effects of PDPK1-SGK1 inhibitor and high salt diet on Th17/Treg balance in vivo.Additionally,the adoptive transfer model suggested that high salt diet not only affected the immune status during the feeding period,but the impact could be long-lasting after the high salt diet withdrawal.High salt diet aggravated arthritis in mice by promoting Th17/Treg imbalance and inflammatory cytokine secretion,while PDPK1-SGK1 inhibitor alleviated arthritis by restoring Th17/Treg balance and restraining inflammatory cytokine secretion.Mechanistic studies demonstrated that PDPK1-SGK1inhibitor elevated the expression of forkhead box protein O1（Fox O1）,inhibited the expression of key glycolytic enzymes,and repressed glycolytic metabolism,thus restoring Th17/Treg balance and alleviating joint inflammation.Conclusions:Our study supports that PDPK1-SGK1 inhibitor could restore Th17/Treg balance by Fox O1 reprogramming glycolytic metabolism,which not only alleviates joint inflammation but reverse the adverse effects of high salt diet on RA prognosis.Targeted inhibition on PDPK1-SGK1 axis paves a new way for RA immunotherapy.