The Role And Mechanism Of Eosinophil Extracellular Traps In Mediating Abnormal T Cell Differentiation In Bullous Pemphigoid

Background:Bullous pemphigoid(BP)is a common autoimmune skin disease in the elderly,characterized by widespread itchy blisters.The pathogenesis of this disease involves a series of complex immune regulatory processes,where the abnormal activation and imbalance of the adaptive immune response are widely considered to be the core driving factors.Pathologically,BP is characterized by the formation of blisters under the epidermis,accompanied by significant inflammatory cell infiltration,and the linear deposition of autoantibodies and complement components along the basement membrane.Notably,the number of eosinophils in the peripheral blood of BP patients often increases abnormally,and their abnormal accumulation at the lesion site is a typical pathological feature of BP.Although eosinophils are recognized to play a key role in the immunopathological process of BP,current understanding of their core pathological mechanisms is still limited,mainly focusing on their role in destroying the dermal-epidermal junction through the release of inflammatory mediators in the late stages of inflammation,further leading to the formation of subepidermal blisters.However,recent studies have revealed that eosinophils also have the ability to directly regulate T cell functions,becoming an important link between innate and adaptive immune responses.Therefore,whether eosinophils participate in the regulation of adaptive immune response cell functions in the early stages of BP disease,thereby affecting the activation of the adaptive immune response,has become an important scientific question worth in-depth study.Eosinophils,as key players in the immune system,their functions far exceed the traditional release of granule proteins and proteases.They can form eosinophil extracellular traps(EETs),which are complexes composed of DNA as the skeleton,with guanidylated histones and granule proteins attached to the surface.The latest research shows that in a mouse model of bacterial infection,eosinophils promote the generation of memory CD8~+T cells by secreting IL-4 to inhibit the apoptosis of CD8~+T cells.In addition,eosinophils activate neuroendocrine cells through EETs,integrating immune and neural signals to promote the allergic immune response of tissues.By revealing the complex role of eosinophils and their produced EETs in intercellular signaling and immune responses,these studies not only deepen our understanding of the functional diversity of eosinophils but also challenge and expand our traditional view of EETs as a single pro-inflammatory mediator.In comparison,although neutrophil extracellular traps have been proven to directly promote the abnormal differentiation of B cells and antibody production,and their histone components play a role in the abnormal differentiation of Th17 cells,the role of EETs in autoimmune responses and their impact on the function of adaptive immune response cells still require further in-depth study to reveal their potential mechanisms.In this research context,we propose the following hypothesis:In BP patients,the abnormally increased number of eosinophils and their produced EETs directly act on activated Na(?)ve CD4~+T cells.This process promotes the differentiation of initial CD4~+T cells into mature Th cell subsets,further assisting the activation of B cells and the production of pathogenic autoantibodies,thereby advancing the onset and development of BP.Objectives:1.Analyze the pathological characteristics of EETs in BPand their relationship with disease activity;2.Investigate the role and mechanism of EETs in the aberrant differentiation of na(?)ve CD4~+T cells;3.Explore new strategies for treating BP by targeting components of EETs or their downstream molecular targets.Methods:1.EETs in Peripheral Blood and Skin Lesions of BP Patients Correlate Positively with Disease Severity:BP patients exhibited significantly higher counts of eosinophils in peripheral blood compared to healthy controls,closely related to the titers of anti-BP180 and anti-BP230 antibodies.These counts could return to normal levels post-treatment.Specific eosinophils expressing EETs were identified in BP patients’blood via flow cytometry.Electron microscopy revealed irregular net-like structures on the surface of blister cells from BP patients,which were further confirmed to contain molecules marked with ECP,cit-H3,and Cytox Green through immunofluorescence analysis,with a significant presence of EETs in the lesioned skin of patients.Additionally,ELISA results showed elevated levels of ECP-DNA complexes in the blood and blister fluid of patients,significantly correlating with anti-BP180 antibody titers,and returning to normal upon treatment.These findings suggest a potential role of EETs in the pathogenesis of BP.2.Exploring the Role of EETs in the Differentiation of Human Na(?)ve CD4~+T Cells:The study employed flow cytometry to assess the proportions of T follicular helper cells(Tfh)and plasma cells in the peripheral blood of BP patients and healthy controls,analyzing their correlation with serum levels of EETs.Further,peripheral blood mononuclear cells(PBMCs)were collected from healthy volunteers and na(?)ve CD4~+T cells were isolated using magnetic bead sorting techniques.These cells were then allocated to various experimental groups for RNA sequencing to analyze gene expression differences under various stimuli.Through functional clustering,pathway analysis,quantitative real-time PCR(q RT-PCR),and flow cytometry,the differentiation direction of na(?)ve CD4~+T cells was further validated.Specifically,by blocking the DNA and histone receptor components of EETs,flow cytometry was used to study changes in Th cell differentiation,identifying key components of EETs that mediate aberrant Th cell differentiation.Transcriptome sequencing was utilized to screen for receptors that influence the aberrant differentiation of Th cells mediated by key components of EETs.Furthermore,flow cytometry and immunofluorescence techniques were used to analyze differences in the expression of receptors for key components of EETs between BP patients and healthy controls.Finally,the impact of EET-mediated na(?)ve CD4~+T cell differentiation was assessed by interfering with the expression of receptors for key components of EETs through si RNA experiments.3.Unveiling the Mechanism of EET-Mediated Differentiation of Na(?)ve CD4~+T Cells into Tfh Cells:Utilizing Gene Set Enrichment Analysis(GSEA),the study identified key downstream pathways and employed colorimetric methods to measure differences in N6-methyladenosine(m6A)levels in na(?)ve CD4~+T cells under different stimulation conditions and in RNA from PBMCs of BP patients and healthy controls in clinical samples.The correlation between ECP-DNA complexes in the peripheral blood of BP patients and the overall m6A methylation levels of RNA from PBMCs was further validated and analyzed using clinical samples.Subsequently,to identify key genes playing roles in m6A modification and their downstream key transcription factors in this experiment,RNA sequencing data were reanalyzed.Then,predictive modification sites of transcription factors genes,which showed significant differences in Tfh cells,were predicted using online tools and validated through Methylated RNA Immunoprecipitation(Me RIP)experiments.After identifying the key genes involved in m6A modification and their downstream key transcription factors,si RNA interference was used on upstream key receptors to assess their impact on m6A modification of key transcription factors and Tfh cell differentiation.4.Assessing the Impact of Targeted EET Intervention on an Active Immunization Mouse Model of BP180:An active immunization mouse model using the BP180 protein was utilized to investigate the effects of EET synthesis inhibitors,DNA components,and histone receptor blockers on the autoimmune response.ELISA was employed to evaluate the impact of these treatments on the levels of EETs in mouse serum and the titers of anti-BP180 antibodies.Further,tissue immunofluorescence analysis was conducted to study the deposition patterns of Ig G and complement C3 in the skin lesions of mice from different treatment groups.Additionally,flow cytometry analysis revealed changes in the composition of immune cells in the peripheral blood,spleen,and lymph nodes of the treated mice.Lastly,tissue immunofluorescence analysis further explored the distribution patterns of EETs in the lymph nodes and spleens of mice from different treatment groups and the formation of germinal centers.Results:1.Large amounts of EETs are present in the peripheral blood and skin lesions of BP patients and correlate positively with disease severity:Retrospective analysis showed that the peripheral blood eosinophil count in BP patients was higher than in healthy controls and returned to normal after treatment.The eosinophil count was positively correlated with anti-BP180 and anti-BP230 antibody titers.Flow cytometry revealed eosinophils expressing EETs in the blood of BP patients.Electron microscopy observed irregular reticular structures on the surface of BP blister cells.Immunofluorescence analysis confirmed these structures were composed of molecules marked by ECP,cit-H3,and Cytox Green.A large amount of EETs was also detected in the skin lesion tissues of patients.The levels of ECP-DNA complexes in the blood and blister fluid of BP patients were high,related to anti-BP180 antibody titers,and returned to normal after treatment.2.EETs Mediate Differentiation of Na(?)ve CD4~+T Cells into Tfh Cells:Flow cytometry observations revealed a significant increase in the numbers of Tfh and plasma cells in the peripheral blood of BP patients compared to healthy controls.Further correlation analysis showed a positive relationship between serum levels of EETs and the proportion of Tfh cells in peripheral blood,with no significant correlation with plasma cell ratios.Our gene sequencing analysis identified extensive gene expression changes in the EETs group compared to the Na(?)ve and Th0 groups,primarily involving immune responses and Tfh cell differentiation.Functional clustering and pathway analysis linked these gene changes closely with immune responses,the STAT signaling pathway,and T cell differentiation.Deeper gene set enrichment analysis indicated that EETs promoted the expression of genes associated with Tfh cell differentiation.Cell experiments further confirmed that exposure to EETs significantly increased the expression of Tfh characteristic genes(Bcl6,IL21,CXCR5,PDCD1),suggesting a role for EETs in mediating the differentiation of na(?)ve CD4~+T cells into Tfh cells.3.EETs’DNA as the Principal Component Mediating Differentiation of Na(?)ve CD4~+T Cells into Tfh Cells:Flow cytometry results showed that EETs treated with Deoxyribonuclease I(DNAse I)significantly reduced Tfh cell differentiation,while Toll-like receptor 2/4 inhibitors had no significant effect.QRT-PCR further confirmed that DNAse I treatment suppressed the expression of characteristic Tfh cell genes Bcl6,IL-21,CXCR5,PDCD1.Additionally,ELISA tests revealed elevated levels of ECP-DNA complexes and anti-His-BP180 antibodies in the serum of mice immunized with mouse BP180 recombinant protein,which significantly decreased after treatment with Protein arginine deiminases 4(PAD4)inhibitor or DNAse I,unaffected by Toll-like receptor 2/4inhibitors.Tissue immunofluorescence and flow cytometry analysis of mice immunized with mouse BP180 recombinant protein showed linear deposition of Ig G and complement C3 at the skin basement membrane,significant infiltration of EETs~+eosinophils,increased germinal center numbers,and higher ratios of Tfh and GCB cells in lymph nodes and spleen.PAD4 inhibitor and DNAse I treatment significantly suppressed these immune characteristics.4.EETs-DNA Promotes Tfh Cell Differentiation via CD4~+T Cell Surface Nucleic Acid Sensor CCDC25:Activating na(?)ve CD4~+T cells with CD3/CD28 magnetic beads,we noted the most significant expression differential within the nucleic acid sensor gene family was coiled-coil domain containing 25(CCDC25).Flow cytometry and immunofluorescence analysis revealed that CD3/CD28 stimulation significantly increased the expression of CCDC25 protein on the surface of na(?)ve CD4~+T cells.In the peripheral blood of BP patients,the expression level of CCDC25 on CD4~+T cells was higher and returned to normal levels after treatment.Furthermore,the positive proportion of CCDC25correlated positively with the proportion of Tfh cells,emphasizing its importance in regulating Tfh cell differentiation.Tissue immunofluorescence analysis also identified CD4 and CCDC25 co-localized T cells in the lesions of patients,further validating the role of CCDC25 in the pathogenesis of BP.Finally,cell experiments confirmed that si-RNA interference of CCDC25 expression significantly inhibited the proportion of EETs-mediated Tfh cell differentiation and the expression of characteristic Tfh genes(Bcl6,IL21,CXCR5,PDCD1).5.EETs-DNA-CCDC25 Enhances m6A Methylation Levels in CD4~+T Cells via VIRMA:GSEA analysis revealed significant enrichment of differential genes between EETs and Th0 cells in m6A methylation-related gene sets.Colorimetric assays confirmed that the overall RNA m6A methylation levels in PBMCs from BP patients were higher than those in healthy controls and correlated positively with serum ECP-DNA complex concentrations.CD3/CD28 magnetic beads combined with EETs stimulation increased the overall RNA m6A methylation levels in na(?)ve CD4~+T cells,with Vir like m6A methyltransferase associated protein(VIRMA)showing significant expression differences among writer genes.Downregulation of CCDC25 could inhibit this process.Clinical samples also confirmed that the expression level of VIRMA in CD4~+T from BP patients’PBMCs was elevated,and the proportion of CD4~+T cells’surface CCDC25 expression in BP patients’peripheral blood PBMCs significantly correlated with the intracellular expression of VIRMA.Finally,cell experiments confirmed that si-RNA interference in VIRMA expression significantly inhibited the proportion of EETs-mediated Tfh cell differentiation and the expression of characteristic Tfh genes(Bcl6,IL21,CXCR5,PDCD1).6.EETs-CCDC25-VIRMA Enhances MAF’s m6A Methylation Levels to Mediate Tfh Cell Differentiation:In our pursuit to identify key transcription factors undergoing m6A modification and mediating Tfh cell differentiation,we re-analyzed RNA sequencing data.We discovered that under co-stimulation with CD3/CD28 and EETs,the transcription factor MAF showed the most significant expression difference among all transcription factors related to Tfh cell differentiation.Online predictive modeling indicated three high-confidence sites in MAF’s 3’UTR,and through Me RIP experiments,we identified an m6A modification site.Cell experiments further confirmed that co-stimulation with CD3/CD28 and EETs increased the m6A methylation modification level of MAF in na(?)ve CD4~+T cells,thus promoting the stability and expression of its m RNA.si-RNA inhibition of CCDC25 or VIRMA expression significantly reduced MAF’s m6A methylation modification level and m RNA expression.Finally,verification through clinical samples from BP patients showed high expression of VIRMA and MAF in PBMCs,with a positive correlation between the expression levels of both genes.In RNA samples from PBMCs of BP patients,the m6A modification of MAF was significantly higher than in healthy controls.Conclusion:This study delves into the intricate link between eosinophils,their production of EETs,and the severity of Bullous Pemphigoid(BP)in patients.We observed that the DNA component of EETs,by activating the CCDC25 receptor,significantly promotes the differentiation of na(?)ve CD4~+T cells into Tfh cells.In terms of therapeutic strategies,we found that using PAD4 inhibitors to prevent the formation of EETs,or employing DNase I to break down the DNA structure of EETs,effectively mitigated the abnormal differentiation of Tfh cells and the production of autoantibodies in a BP180-immunized mouse model,with similar effects observed in in vitro cellular experiments.Furthermore,our research unveiled a novel mechanism where EETs enhance the m6A methylation of transcription factor MAF through the DNA-CCDC25-VIRMA pathway,subsequently stabilizing MAF m RNA and promoting Tfh cell differentiation.This pivotal discovery offers new potential targets for the treatment of BP and other autoimmune diseases,laying the groundwork for the development of more precise targeted therapeutic strategies.