The Regulation Of AIM2 In T_FH Cell Differentiation And Its Molecular Mechanism In The Development Of Systemic Lupus Erythematosus

Systemic lupus erythematosus（SLE）is a classic chronic autoimmune disease,which can involve multiple tissues and organs.SLE patients have the salient feature of immune system dysfunction and impaired body immune tolerance,resulting in autoimmune response and damage in their body tissues.The etiology of SLE is not clear,early studies have shown that CD4⁺T cells play a key role in the pathogenesis of lupus^[1].In recent years,more and more studies have found that follicular helper T cells(T_FH)can promote the over activation of immune complexes and the response of autoimmune T and B cells,contributing to the development of SLE^[2].Absent in melanoma 2（human AIM2/mouse Aim2）is a DNA sensor,which is mainly involved in the assembly of AIM2 inflammatory bodies to resist the pathogen related molecular pattern（PAMP）in innate immunity^[3].Previous studies have focused on the role of inflammatory bodies in innate immune AIM2.However,the latest research showed that AIM2 can contribute to the regulation of adaptive immunity,especially in the development of tumors and autoimmune diseases^[4,5].In our research,we hypothesize:IL-21 of T_FHcell activation pathway recruit DNA hydroxyltransferase TET2,leading to demethylation of AIM2 gene promoter in T_FHcells and increased AIM2levels,promoting the interaction of AIM2 and c-MAF and the differentiation of T_FHand GC B cells,which then promotes the production of autoantibodies and IL-21 and further results in the onset of SLE.Our results revealed the IL-21-TET2-AIM2-c-MAF signaling pathway in the activation and differentiation of T_FHcells,which provides new insights for the molecular mechanism and target therapy of SLE.Objectives:To study the role of AIM2 in the regulation of T_FHcells,and its effect and molecular mechanism in the development of SLE.Methods:1.Normal human Na?ve CD4⁺T cells were isolated and differentiated to T_FHcells.AIM2 expression of in vitro-differentiated T_FHcells and human tonsil T_FHcells were detected by flow cytometry;m RNA sequencing,RT-PCR,Western blot and confocal immunofluorescence staining were used to detect the AIM2 expression levels during the in vitro T_FHcell differentiation;At the same time,AIM2 expression in T_FH-like cells of SLE patients was detected by flow cytometry,Western blot and confocal immunofluorescence staining.2.The frequencies of CD4⁺T,CD8⁺T,effector T and no-effector T cells in CD4^creAim2^fl/fland Aim2^fl/flmice was detected by flow cytometry at steady state;Proliferated CFSE⁺CD4⁺T cells were detected by flow cytometry after anti-CD3 and anti-CD28 stimulation.The whole genome expressions of spleen CD4⁺T cells in KLH stimulated mice were detected by m RNA-seq,the frequencies of T_FHand GC B cells were detected by flow cytometry,and the production of KLH antigen-specific antibody was detected by ELISA;Effect of Aim2 deficiency on murine in vitro-differentiation of T_FHcells was detected by flow cytometry.3.Lupus like symptoms were accessed:urinary protein level was detected by urinary protein test paper,anti-autoantibody titer was detected by ELISA,and renal Ig G and C3 deposition were detected by immunofluorescence staining;The frequencies of T_FHand GC B cells in mice was detected by flow cytometry.4.AIM2 antisense oligonucleotides（ASO）was transfected into Na?ve CD4⁺T cells for T_FHcell differentiation,following T_FHcell purity and IL-21/c-MAF m RNA were detected by flow cytometry and RT-PCR.The interaction between AIM2 and c-MAF was verified by protein interaction region prediction and CO-IP;Gene methylation sequencing and bisulfite sequencing（BSP）were used to detect the methylation level of AIM2 gene promoter in T_FHcells and SLE CD4⁺T cells;The effects of TET2 deficiency on AIM2 protein level and frequencies of T_FHand GC B cells were detected by western blot and flow cytometry;Chromatin immunoprecipitation（Ch IP）was used to verify TET2 binding to the AIM2 gene promoter in T_FHcells,which was regulated by upstream IL-21.Results:1.AIM2 expression level of human tonsil T_FHcells was higher compared with Na?ve T,T_H1 and T_H2 cells;Compared with T_H1,T_H2,T_H17 and Treg cells,AIM2 expression of in vitro-differentiated T_FHcells was higher;The levels of AIM2 m RNA,AIM2 protein and AIM2immunofluorescence intensity in vitro-differentiated T_FHcells were significantly higher than those of Na?ve CD4⁺T cells;Compared with normal controls,AIM2 expression levels of T_FH-like cells in peripheral blood in SLE patients were increased;compared with normal controls,the expression levels of AIM2 protein of CD4⁺T cells by western blot and immunofluorescence were increased in SLE patients;Compared with normal controls,patients with lupus erythematosus(including discoid lupus erythematosus（DLE）,acute cutaneous lupus erythematosus（ACLE）and subacute cutaneous lupus erythematosus（SCLE）had higher m RNA levels of AIM2,BCL6,IL-21,and STAT3 in skin lesions;compared with patients with psoriasis and normal controls,expression levels of AIM2,co-localization of AIM2 and T_FH-like cells in skin lesions by immunofluorescence were increased in patients with DLE and SLE.2.The frequency of CD4⁺T cells in DLN of CD4^creAim2^fl/flmice was lower than that of Aim2^fl/flmice.The frequency of proliferated CFSE⁺CD4⁺T cells was lower in CD4^creAim2^fl/flmice than that in Aim2^fl/flmice after the treatment of CD3 and CD28;The m RNA expressions of T_FHsignature genes such as IL4,IL21,PD1,CXCR5,ICOS,STAT4 and ASCL2 were significantly lower in spleen CD4⁺T cells of CD4^creAim2^fl/flmice than that of Aim2^fl/flmice;The frequencies of T_FHand GC B cells in CD4^creAim2^fl/flmice were lower than those in Aim2^fl/flmice after KLH stimulation;The serum anti-NP2 Ig G production of CD4^creAim2^fl/flmice was lower than that of Aim2^fl/flmice;While anti-NP2 Ig M levels of CD4^creAim2^fl/flmice was higher than that of Aim2^fl/flmice.3.Compared with CD4^creAim2^fl/flmice,the levels of urinary protein,anti-ds DNA and ANA titers in Aim2^fl/flmice were in were significantly increased;compared with CD4^creAim2^fl/flmice,more severe renal damage by H&E staining,more Ig G and C3 precipitation in kidney by IHC staining were showed in Aim2^fl/flmice;Compared with CD4^creAim2^fl/flmice,the frequencies of T_FHcells and GC B cells in Aim2^fl/flmice was significantly increased.4.AIM2 silence led to the reduced T_FHcell differentiation,decreased m RNA levels of IL21 and c-MAF in ASO group;The m RNA levels of AIM2,IL-21 and c-MAF were higher in peripheral blood CD4⁺T cells of SLE patients than those of normal controls;Moreover,AIM2 m RNA level was positively correlated with IL-21 m RNA level and c-MAF m RNA level,respectively;Prediction software（Py MOL v2.0 and Cluspro v2.0）of protein interaction,immunoprecipitation（Co-IP）confirmed that AIM2 can directedly bind c-MAF in T_FHcells;Methylation sequencing and BSP sequencing confirmed that the methylation level of AIM2 gene promoter region in T_FHcells was lower than that in Na?ve CD4⁺T cells.Ch IP confirmed that DNA hydroxyltransferase TET2 could bind to the AIM2 gene promoter in T_FHcells,resulting in the decreased methylation level of AIM2 promoter;BSP sequencing and Ch IP assays confirmed that the AIM2 methylation level in CD4⁺T cells of SLE patients was lower than that of normal controls,which resulted of TET2 binding to AIM2 gene promoter in SLE patients;Flow cytometry and western blot results showed that TET2 deficiency reduced the frequencies of T_FHand GC B cells in CD4^creTET2^fl/flmice than that of TET2^fl/flmice after KLH stimulation.RT-PCR and western blot results showed that IL-21 was an upstream regulation of AIM2.Chromatin immunoprecipitation（Ch IP）confirmed that TET2 could bind to the promoter sequence of AIM2 in CD4⁺T cells after IL-21 stimuli.Conclusions:1.T_FHcells expressed the highest level of AIM2 in all CD4⁺T cell subsets.AIM2 expression levels of T_FHlike cells in peripheral blood and skin lesions of SLE patients were abnormally increased.2.Aim2 may regulate the differentiation of CD4⁺T cells and T_FHcells.3.Aim2 deficiency alleviated the clinical symptoms and reduce the differentiation levels of T_FHand GC B cells in CD4^creAim2^fl/flmice in pristane induced lupus model.4.AIM2 may regulate the in vitro-differentiation of T_FHcells;The AIM2 expression in SLE patients was positively related with the expression levels of IL-21 and c-MAF;AIM2 directly interacts with c-MAF and thus involved in IL-21-c-MAF signaling pathway in T_FHcell differentiation;TET2 can bind to the AIM2 gene promoter sequence in T_FHcells,resulting in the decreased methylation level of AIM2 promoter and increased frequencies of T_FHand GC B cells,which were regulated by the upstream IL-21;IL-21 can promote TET2 binding to the AIM2gene promoter sequence and increase the expression level of AIM2 in T_FHcells;IL-21 may regulate AIM2 mediated by TET2.