STING Couples With PI3K To Regulate Actin Reorganization During BCR Activation

Objective: Interferon gene stimulating factor(STING,Interferon Genes,STING),which can also be MITA,MPYS or ERIS,is an adaptor protein that is highly expressed in peripheral blood lymphocytes.STING interacts with IRF3 to activate the type I interferon production in response to foreign DNA from a variety of intracellular pathogens,which plays an important role in the innate immune response.However,its role in adaptive immunity as well as its underlying mechanism is not completely.The expression of STING is low both in SLE patients and MRL/lpr mice,of which the MRL/lpr mice have lower expression of tyrosine phosphotases,p SHP1 and p SHP2,however,SLE B cells have highly activated BCR mediated signaling.STING has been shown to regulate the tyrosine phosphatase,SHP.But whether it can regulate SHIP during BCR activation still remains elusive.BCR activation is vital for the function of B cells,BCR activation leads to actin reorganization,which offers feedback to the BCR signaling by modulating the spatiotemporal organization of BCRs.Whether STING regulates BCR signaling to orchestrate actin reorganization is still unknown.In this study,we used Sting KO mice and a patient's mutated STING cells to study the effect of STING deficiency on B cell development,differentiation,BCR signaling,and humoral immune response.Methods: The effects of STING deficiency on bone marrow and peripheral B cell development and differentiation were analyzed by flow cytometry.A bone marrow chimeric model was constructed to eliminate the effects of environmental factors lacking STING on cell development and differentiation.The effect of STING deficiency on BCR activation and BCR signal pathway in spleen B cells was analyzed by confocal.The effect of STING deficiency on early activation of splenic B cells was analyzed by TIRFm.The effect of STING deficiency on BCR signal was analyzed by Western blot and phosphorylation flow cytometry.Finally,the effect of STING deficiency on the immune function of B cells was analyzed by a mouse immune model.Then,the effects of STING mutations on the development and differentiation of B cells in patients were also analyzed by flow cytometry.The effect of STING mutation on BCR signal in B cells of patients was analyzed by Western blot.Results: 1.The deficiency of STING alters the homeostasis of peripheral B cells,but not the developmental subsets in the bone marrow.The percentage and number of MZ and GC B cells were significantly increased in Sting KO mice,but that of bone marrow B cell subsets and FO,T1 and T2 showed no changes.The percentage of CD45.2+ Sting KO MZ and GC B cells was increased compared with CD45.2+ WT MZ and GC B cells after bone marrow reconstitution.The enlarged and darker staining of the GCs and larger sizes of GCs in Sting KO spleens compared with that of WT in HE staining and immunofluorescence.2.STING deficiency does not affect development of thymus and extended T cell subsets.The percentage and number of CD4+ T,CD8+ T,CD4+CD8+ T,Treg,IFN-?+ T,IL-4+ T and IL-17A+ T cells were not altered in the thymus,spleen and LN of Sting KO mice.3.STING is involved in BCR activation and regulates B-cell peripheral tolerance.The correlation coefficient increased gradually up to 10 min and dropped afterwards in WT B cells.The staining pattern of the ER was the same for Sting KO B cells and WT B cells,as well as the colocalization of ER with BCR.3D microscopy results showed that both ER and STING colocalized with the BCR cap at 10 min upon antigenic stimulation.In addition,the level of anti-double-stranded DNA antibodies in the serum of Sting KO mice was significantly increased,the levels of IgG and complement in glomeruli were also significantly increased,and lymphocyte infiltration in the spleen,kidney,lung,and colon was significantly increased as well.4.STING deficiency upregulates the proximal positive BCR signaling.The colocalization between pY/p Btk/pCD19 and BCRs and the levels of pY and the ratio of p Syk/Syk and p Btk/Btk were significantly enhanced in Sting KO B cells compared with that of WT B cells.5.STING deficiency downregulates the activation of the negative BCR signaling molecule-SHIP.The correlation coefficient between BCR and p SHIP and the ratio of p SHIP/SHIP were markedly decreased in Sting KO B cells compared with that of WT B cells.6.STING deficiency causes an abnormal accumulation of F-actin via WASP.The correlation coefficient between pWASP and BCRs was significantly increased in Sting KO B cells.The Phosflow cytometry showed that,the expression level of pWASP in Sting KO B cells was increased when the antigen stimulated for 5 min and 10 min,and the depolymerization ability of F-actin was weakened at this stage compared with that of WT B cells,resulting in Actin aggregation increased.7.STING deficiency causes an abnormal accumulation of F-actin via PI3K mediated signaling.The BCR accumulation in the contact zone was significantly decreased,but the contact area and the accumulation of pY and F-actin in the contact zone of Sting KO B cells was significantly increased.The ratio of pPI3K/PI3K,pAkt/Akt,pFoxO-1/FoxO-1 and p S6/S6 were all clearly increased in Sting KO B cells.The level of pPI3K was higher during the resting and activated state in the peripheral B cells of the STING mutant patient cells compared with hat of the HC.The treatment with PI3K inhibitors largely reduced the colocalization between the BCR and STING compared with the treatment of Lyn and Syk inhibitors.8.STING deficiency causes a reduced humoral immune response.The number and percentage of MZ and GC B cells were both decreased in KO mice compared with WT mice after immunization.The titers of NP-specific IgG1 and IgM were both decreased in KO mice compared with that of WT mice after immunization.No difference in the frequency and number was observed for MBC,PC,switched,unswitched B cells and Tfh cells between WT and KO mice after immunization.Conclusion: STING uses PI3K mediated by the CD19-Btk axis as a central hub for controling the actin remodeling that,in turn,offers feedback to BCR signaling.Overall,our study provids a new mechanism of how STING regulates BCR signaling via feedback from actin reorganization.