Study On The Mechanism Of MiR-23b-3p/TLR4/STAT1 Axis In Immunoglobulin A Vasculitis

Objective:Immunoglobulin A associated vasculitis(IgAV),also known as Henoch-Schonlein purpura(HSP),is an immunoglobulin A mediated systemic small vasculitis common in children,often involving the skin,joints,gastrointestinal tract,and kidneys.About 3%of patients with IgAV vasculitis with nephritis(IgAVN)progressed to end-stage renal disease.Currently,long-term glucocorticoids combined with immunosuppressants are the primary treatment for IgAVN patients.However,the hormone often produces serious side effects on developing children and has significant effects on the physical and mental health of patients.Therefore,it is very important to study the pathogenesis of IgAVN and provide theoretical basis for finding effective treatment.Studies have found that infection,allergy,genetic susceptibility and immune response disorders are closely related to the development of the disease.IgAV immune response disorders involve innate and adaptive immune abnormalities,mainly manifested as excessive activation of B cells,resulting in a large number of IgA antibodies.Follicular helper T cells(Tfh)mainly regulate the activation,proliferation,antibody affinity maturation and isotype conversion of B cells in the germinal center(GC),and are regarded as a kind of CD4~+T cells that are particularly important in assisting the production of immunoglobulin by B cells and promoting the occurrence of inflammation.Abnormal differentiation and proliferation of Tfh cells can lead to autoimmune diseases.The proportion of Tfh cells in peripheral blood of IgAV patients was significantly increased.Therefore,targeted inhibition of abnormal differentiation and proliferation of Tfh cells may reduce IgA secretion by B cells.Therefore,it is necessary to further study the mechanism of Tfh cell differentiation.Tfh differentiation involves a variety of signaling factors,including a variety of cytokines,receptors and transcription factors.Many cytokines influence the differentiation of Tfh cells through signal transducer and activator of transcription(STAT)family signaling pathways.Activated STAT1 promotes the expression of Bcl6 and is a key transcription factor in Tfh cell differentiation.A variety of cytokines can lead to increased expression of STAT1.It has been reported that IL-6 and IL-12 are significantly increased in IgAV patients,and IL-6 and IL-12 can play an important role in Tfh cell differentiation by regulating STAT1,so we considered that these two factors are mediated.In addition,infection is an important cause of IgAV.During prophase infection,the pathogen activates the innate immune response,which produces immune effector molecules.Toll-like receptors(TLRs)are a group of important pattern recognition receptors involved in the innate immune system.They recognize pathogen-associated molecular patterns in pathogenic microorganisms to activate intracellular signaling pathways,leading to the release of a series of inflammatory cytokines and the initiation of adaptive immune responses.Infection-induced activation of TLRs may play an important role in initiating abnormal IgAV immune responses.TLR4 is a transmembrane,non-catalytic receptor that is usually expressed in dendritic cells(DCs).TLR4 expressed on DC can recognize pathogen-related molecular patterns and initiate signaling pathways to induce DC maturation and cytokine secretion.The initial CD4~+T cells recognize the antigens presented by MHC Class II molecules on the DC and are activated into antigen-specific helper T cells,which differentiate into Tfh cells under the induction of cytokines secreted by the DC(IL-6 or IL-12).Therefore,DC may play an important role in regulating Tfh cell differentiation.Micro RNAs are a group of small non-coding RNAs that are evolutionarily conserved and endogenous.miRNA inhibits the translation of target genes by binding to the 3’-terminal untranslated region(3’UTR)of m RNA.Several miRNAs are involved in DC maturation and function.Since DC recognizes pathogens through TLR4 and initiates intracellular signaling pathways,TLR4 plays an important role in DC maturation and function.We speculate that miRNA can regulate the maturation and function of DC by targeting TLR4 gene expression.However,the role of miRNA in the occurrence and development of IgAV is rarely studied,and the exact molecular mechanism of miRNA remains unclear.Methods:PartⅠ:First,an IgAV-related m RNA sequencing dataset,GSE102114,was downloaded from GEO,a public database,to analyze differential gene expression between IgAV patients and healthy control children.Then the protein-protein interaction(PPI)network of differential genes was constructed using STRING database.Key hub genes were identified by cyto Hubba plugin for functional enrichment analysis.Then,the abundance of 24 immune cells was measured by Immu Cell AI(Immune cell abundance identifier)to assess the proportion of immune cells in IgAV.Finally,qRT-PCR and the proportion of immune cells were verified based on patient samples.PartⅡ:1.Previous studies have shown that TLR4 and STAT1 are involved in the occurrence and development of IgAV.In order to further study the specific mechanism of action of TLR4 and STAT1,the potential miRNA binding sites of TLR4 and STAT1 can be predicted by using Target Scan and miRWalk website.Comprehensive analysis of the predicted results from the above sites suggested that both TLR4 and STAT1 may be regulated by miR-23b-3p.Subsequently,male SD rats aged 5-6 weeks were selected to establish IgAV rat model.A series of histopathological experiments verified the successful establishment of the rat model.The proportion of Tfh cells and the expression levels of TLR4 and STAT1 in IgAV model rats were detected.2.In the cell experiment,spleen cells of IgAV model rats were isolated and cultured,miR-23b-3p was knocked down/overexpressed,and the proportion of Tfh cells was detected by flow cytometry.3.DC and CD4~+T cells were isolated and cultured in IgAV model rats.DC was pretreated and co-cultured with CD4~+T cells to observe its effect on Tfh cell differentiation.Dual luciferase reporter assay was used to confirm the targeting binding sites of miR-23b-3p and TLR4.After knockdown/overexpression of miR-23b-3p in DC cells,the changes of TLR4,DC maturation and the effects of co-culture with CD4~+T cells on downstream STAT1 and Tfh cell differentiation were observed.The design of functional recovery experiments confirmed that miR-23b-3p affected the phosphorylation level of STAT1 by targeting the inhibition of TLR4 gene expression,thus regulating the differentiation of CD4~+T into Tfh cells.4.CD4~+T cells were isolated and cultured in IgAV model rats,and the CD4~+T cells were treated with miR-23b-3p intervention to observe its effect on Tfh cell differentiation.Dual luciferase reporter assay was used to confirm the targeting binding sites of miR-23b-3p and STAT1.After knockdown/overexpression of miR-23b-3p in CD4~+T cells,the changes of the target gene STAT1 and its effect on Tfh cell differentiation were observed.Designed functional recovery experiments confirmed that miR-23b-3p regulates CD4~+T differentiation into Tfh cells by targeting the inhibition of STAT1 gene expression.PartⅢ:Effect of miR-23b-3p on pathogenesis in IgAV rats.After injecting miR-23b-3p-NC and lentivirus overexpressing miR-23b-3p into the tail vein of IgAV model rats,the clinical symptoms of IgAV rats were observed.The changes of feeding status,activity and rash of rats in two groups were observed.IgA deposition in kidney was observed by immunofluorescence.HE staining was used to observe the pathological changes of skin and gastrointestinal tract.The changes of mesangial hyperplasia were observed by HE and PAS staining.The changes of spleen appearance size and spleen index were observed.Results:PartⅠ:A total of 4200 DEGs were screened for IgAV patients compared with healthy control children.Among the top 10 hub genes in the PPI network,STAT1,TLR4,PTEN,UBB,HSPA8,ATP5B,UBA52 and CDC42 were significantly upregulated in IgAV patients.KEGG and GSEA enrichment analysis showed that differential genes were mainly concentrated in Th1,Th2,Th17,Tfh,TLR and JAK/STAT signaling pathways.Finally,we found that excessive differentiation of Th2,Th17 and Tfh cells in IgAV may be involved in the occurrence and development of IgAVN.PartⅡ:1.The IgAV model rats showed hemorrhagic rash,IgA deposition in the kidney,significant enlargement of spleen,mesangial hyperplasia in the kidney tissue,obvious vasodilation,bleeding and perivascular inflammatory cell infiltration in the skin and gastrointestinal tract,suggesting that the IgAV model was successfully constructed.In IgAV rats,the expressions of TLR4 and STAT1 were significantly up-regulated,and the expressions of miR-23b-3p were significantly down-regulated,and the proportion of Tfh cells in IgAV rats was significantly increased.2.Flow cytometry showed that inhibition of miR-23b-3p expression could promote Tfh cell differentiation in whole spleen cells,and overexpression could inhibit Tfh cell differentiation.These results suggest that miR-23b-3p plays a role in the differentiation of Tfh cells.We speculated that miR-23b-3p may have a regulatory effect on both DC and CD4~+T cells.Therefore,we separately sorted DC and CD4~+T cells for further study.3.Double luciferase assay confirmed that miR-23b-3p could directly target and inhibit the expression of TLR4 gene.Knockdown/overexpression of miR-23b-3p in DC can promote/inhibit TLR4 gene expression and DC maturation,as well as significantly increase/decrease STAT1 phosphorylation level and promote/inhibit CD4~+T cell differentiation into Tfh cells after co-culture with CD4~+T cells.In addition,inhibition of TLR4 gene in DC partially reversed increased phosphorylation of STAT1 and increased differentiation of CD4~+T cells into Tfh cells in co-culture system caused by knockdown of miR-23b-3p.These results suggest that the miR-23b-3p/TLR4 axis may affect Tfh cell differentiation by regulating STAT1 phosphorylation and play an important regulatory role in the development of IgAV.4.Knockdown/overexpression of miR-23b-3p in CD4~+T cells can promote/inhibit the differentiation of CD4~+T cells into Tfh cells.In addition,STAT1 knockdown could reverse the increased differentiation of CD4~+T cells into Tfh cells caused by miR-23b-3p knockdown.These results suggest that the knockdown of miR-23b-3p expression promotes the phosphorylation of STAT1,thereby inducing the differentiation of CD4~+T cells into Tfh cells.PartⅢ:An IgAV rat model has demonstrated that overexpression of miR-23b-3p lentivirus in vivo can effectively relieve clinical symptoms in IgAV rats.Compared with the IgAV group that was injected with miR-23b-3p-NC through the tail vein,the IgAV group that was injected with miR-23b-3p lentivirus overexpressed showed less hemorrhagic rash and IgA deposition in the kidney.Renal mesangial hyperplasia decreased;Cutaneous gastrointestinal vascular dilatation,bleeding and perivascular inflammatory cell infiltration were significantly reduced.Conclusions:1.Screen out key genes(TLR4 and STAT1),signaling pathways(TLR and JAK/STAT signaling pathways)and dysregulated immune cells(Th2,Th17 and Tfh cells)related to the pathogenesis of IgAV.2.The low expression of miR-23b-3p can not only target and regulate the high expression of TLR4,promote the maturation of DC,increase the secretion of IL-6 and IL-12,cause the phosphorylation of STAT1,and thus improve the differentiation of CD4~+T to Tfh cells,which may induce the activation effect of IgAV;In addition,the low expression of miR-23b-3p targets to regulate the high expression of STAT1 gene,thereby promoting the differentiation of CD4~+T into Tfh cells,which is closely related to the occurrence and development of IgAV,and may be an important reason for the recurrence and progression of the disease.3.Overexpression of miR-23b-3p in vivo can indirectly or directly inhibit pathological expansion of Tfh cells and serum IgA levels by targeting the expression of TLR4 or STAT1,thus alleviating the occurrence and development of IgAV disease.