Study On The Functions Of WDFY4 In Survival And Differentiation Of T Cells Using Knockout Mice

Autoimmune diseases(AIDs)are chronic diseases caused by the loss of immunologic tolerance to self-antigens and represent a heterogeneous group of disorders that affect specific target organs.AIDs have various clinical phenotypes because of different target cells and affected organs.However,the dysfunction of immune system,e.g.the production of autoantibodies and high level of cytotoxic cytokines,is the symptom shared by all the AID patients.Immune cells damage tissues directly by killing cells or indirectly by releasing cytotoxic cytokines,prostaglandins,reactive nitrogen or oxygen intermediates.The autoreactive responses of T cells reacting against autoantigens and the production of autoantibodies by B cells are the main pathogenesis of AIDs.Excessive immune responses of helper T cells,Th1 and Th17 cells,are important driver of inflammatory processes in tissue-specific autoimmunity.Follicular helper T(Tfh)cells have been reported to increase and correlate with disease activity and autoantibody production in human AIDs.Regulatory T(Treg)cells mainly exist in peripheral immune tissues and secrete anti-inflammatory cytokines to suppress Thl and Th17 immune responses,resulting in the reduction of inflammation.Therefore,Treg cells are considered as an important regulatory factor in autoimmune responses.AIDs may occur if environmental factors,e.g.infections or toxins,disrupt the regulation of autoantibody production and autoreactive T cells by Treg cells.The factors that lead to AIDs are complex and unclear.However,genetic factors were reported to be important in AIDs.Genome-wide association study(GWAS)is a way to discover genes or loci associated with human complex diseases.It can find genetic variations in the range of the whole genome,utilizes them as markers and screens genetic variations associated with complex diseases by conducting case-control study.Hundreds of genes,to date,have been identified as susceptibility genes of AIDs.In 2010,WDFY4(WDFY family member 4)was reported to be associated with systemic lupus erythematosus(SLE)in Asian populations and the result was verified in various ethnic populations.Subsequently,increasing studies have revealed the association between WDFY4 and AIDs,including rheumatoid arthritis,clinically amyopathic dermatomyositis and juvenile idiopathic arthritis.WDFY4 is encoded by WDFY4 which is located on chromosome 10q11.23.WDFY4 is the fourth member of WDFY family.WDFY4 is a huge gene containing approximately 300,000 bases and has several transcripts,of which can encode a protein with 3184 amino acid residues.WDFY4 is highly conserved in various species.The N-terminal of WDFY4 is an Armadillo-type fold domain and the C-terminal of WDFY4 consists of a Pleckstrin homology(PH)domain,a Beige and chediak-higashi(BEACH)domain and five WD repeat domains.Two recent studies showed WDFY4 was required for maintenance of B cells and cross-presentation of cell-associated antigens,but the way WDFY4 affects the development,survival and differentiation of T cells is still unclear.To resolve these questions,three parts of studies have been carried out.Part ?Generation and phenotype analysis of Wdfy4 knockout miceThe expression of Wdfy4/WDFY4 in mouse tissues and human cell lines commonly used in laboratory was detected by real-time quantitative PCR.The results showed WDFY4 was highly expressed in immune tissues and cell lines.To study the effects of WDFY4 on T cells,Wdfy4 knockout mice based on LoxP-Cre system were constructed.After acquiring homozygous Wdfy4fl/fl mice,we crossed them to Sox2-Cre+/-mice and obtained Wdfy4fl/-,Sox2-Cre+/-mice(KO mice).No antibody is available for detecting the protein level of WDFY4 in mice,so we confirmed knockout efficiency by DNA sequencing and real-time quantitative PCR.The results showed that Wdfy4 was effectively deleted in thymus and spleen of KO mice.Wdfy4fl/fl mice(WT mice),which matched knockout mice in gender and age,were referred as control for subsequent experiments.Phenotype analysis of WT mice and KO mice was performed to study how WDFY4 influences the development,survival and differentiation of T cells.Flow cytometry was performed to detect the proportions of double negative,double positive and single positive thymocytes,in order to confirm the role of WDFY4 in the development of T cells.We found normal development of thymocytes in KO mice.Subsequently,flow cytometry was performed to detect the proportions of T cells,CD4+T cells,CD8+T cells and active T cells in peripheral immune organs.However,we found there were no differences between KO mice and WT mice.In addition,flow cytometry was also performed to detect the proportions of Treg cells and Tfh cells,which were associated with AIDs.The results showed similar proportions between two groups.The general knockout of gene may lead to genetic compensation response,thus hiding abnormal phenotypes.So we crossed Wdfy4fl/fl mice with Lck-Cre+/-mice and obtained conditional knockout mice(CKO mice),in which Wdfy4 was knocked out only in T cells.Phenotypes of CKO mice were analyzed as described above.We found CKO mice had similar proportions of Treg cells and Tfh cells when compared with WT mice.Significant reduction of T cells and CD8+T cells and increased ratio of CD4+/CD8+T cell were observed in periphery of CKO mice.Consistent with these results,the number of CD8+T cells in spleen of CKO mice was less than that of WT mice,though the numbers of thymic CD8+T cells and splenic cells were normal in CKO mice.As a consequence,tumor-bearing CKO mice showed faster growth rate and larger volume of tumors.In this part,Wdfy4 knockout mice were successfully constructed.We found that lack of Wdfy4 in T cells led to reduced numbers of T cells and CD8+T cells in the periphery,which resulted in uncontrolled tumor growth.Part II WDFY4 participates in CD8+T-cell apoptosis via p53 signalingIt has been reported that BEACH and WD repeat domains play an important role in apoptosis.Therefore,apoptosis levels of T cells and CD8+T cells in WT mice and CKO mice were detected by flow cytometry.We found that T cells and CD8+T cells of CKO mice showed higher apoptosis levels.To verify the above results,we used small hairpin RNA for stable knockdown of WDFY4 in Jurkat cells.Expression levels of proteins related to apoptosis were detected by Western blot and the results showed enhanced apoptosis in WDFY4-deficient cells.CD8+T cells of WT mice and CKO mice were isolated using magnetic beads to investigate the mechanism of high-level apoptosis in CKO mice.Reactive oxygen species(ROS)is an important mediator in apoptosis and we found that CD8+T cells from CKO mice showed significantly greater ROS level than those from WT mice.Jurkat cells showed the same results.The NADPH oxidase enzyme complex is main enzyme system dedicated to producing ROS.Real-time quantitative PCR showed upregulated expression of Nox in CKO CD8+T cells and WDFY4-deficient Jurkat cells when compared with controls.To further verify that enhanced apoptosis in WDFY4-deficient cells is caused by ROS,CD8+T cells of WT and CKO mice were treated with an ROS scavenger,NAC.Western blot showed that CKO CD8+T cells showed higher levels of cleaved PARP and cleaved caspase-7.However,treatment with NAC could eliminate the differences between WT CD8+T cells and CKO CD8+T cells.ROS has been reported to regulate apoptosis via p53 signaling.Expression of factors associated with p53 pathway was detected.Upregulation of phosphorylated p53 and the downstream targets of p53 were observed in CKO CD8+T cells by carrying out Western blot and real-time quantitative PCR.Extracellular regulated protein kinase(ERK)is another target of p53 and inhibition of ERK can enhance apoptosis.Phosphorylation of Bim by ERK1/2 on serine69 promotes its degradation via the proteasome pathway and regulates its proapoptotic function.Downregulation of phosphorylation of ERK and Bim was observed in WDFY4-deficient cells.WDFY4 can modulate B cell fate via noncanonical autophagy,thus affecting the progression of SLE.Western blot was performed to detect the levels of LC3 and PI3KC3.Nevertheless,we did not detect any differences between WT CD8+T cells and CKO CD8+T cells.Moreover,we did not detect any differences in LC3 ?/? and PI3KC3 level between WDFY4-deficient Jurkat cells and negative controls.To further confirm above results,Jurkat cells were cultured under starvation condition to induce autophagy.The ratio of LC3-?/LC3-? in WDFY4-deficient cells was equal to that in control cells,which suggest that the role of WDFY4 in autophagy depends on the cell type.Impaired proliferation and cell cycle arrest are also responsible to the reduction of cells.Consequently,proliferation of CD8+T cells was detected by BrdU incorporation assay.In vivo study revealed impaired proliferation of CD8+ T cells in CKO mice.In addition,flow cytometry was performed to analyze the cell cycle of CD8+ T cells in two group mice and no differences were observed.Immunoblot assay showed no differences in expression of proteins associated with cell cycle.In general,in vivo and in vitro studies showed lack of WDFY4 could activate p53 signaling and suppress ERK activity,thus inducing ROS-mediated apoptosis.Moreover,WDFY4-deficient CD8+T cells showed impaired proliferation.Part IIIDeficiency in WDFY4 promotes Th2 differentiation and aggravates Th2 cell-mediated diseaseWDFY4 has been identified as a susceptibility gene in SLE in various populations.SLE was previously thought to be a disease driven by Th2 cells.In this part,naive CD4+T cells of mice were isolated and induced to differentiation in vitro,so that the role of WDFY4 in T cell differentiation could be studied.Splenocytes of WT mice and CKO mice were respectively divided into two groups,of which one group was treated with PBS and the other one was treated with anti-CD3 antibody plus anti-CD28 antibody.In this way,cells treated with anti-CD3 antibody plus anti-CD28 antibody would differentiate to Th cells randomly.Flow cytometry was performed and the results showed that compared with cells treated with PBS,cells treated with anti-CD3 antibody plus anti-CD28 antibody were more likely to differentiate to Th cells,which indicated anti-CD3 antibody plus anti-CD28 antibody could active T cells.In group which was treated with anti-CD3 antibody plus anti-CD28 antibody,the proportion of Th2 cells from WDFY4-deficient T cells was higher than that from WT T cells,indicating WDFY4-deficient T cells were more likely to differentiate to Th2 cells.To confirm this result,experiments were repeated using naive CD4+T cells and the same results were obtained.Naive CD4+T cells isolated from two group mice were cultured under Th2 polarization condition to study the role of WDFY4 in Th2 differentiation.Real-time quantitative PCR and Western blot showed WDFY4-deficient Th2 cells expressed higher levels of transcription factors associated with Th2 differentiation.Real-time quantitative PCR and ELISA showed more production of Th2 cytokines in WDFY4-deficient Th2 cells.Th2 cells play an important role in chronic inflammation and allergic diseases.To determine whether WDFY4 is involved in Th2 cell-mediated diseases,a mouse model of asthma was established by sensitization with ovalbumin.Real-time quantitative PCR and ELISA were performed and we found that deficiency in WDFY4 promoted the production of Th2 cytokines in asthmatic mice.The histopathological analysis was carried out on the lung sections of asthmatic mice and CKO asthmatic mice showed aggravated airway inflammation,including more inflammatory cell infiltration,more severe goblet cell hyperplasia and more mucus production.Consistently,real-time quantitative PCR showed higher level of Muc5ac in CKO asthmatic mice.More neutrophils were observed in bronchoalveolar lavage fluid of CKO asthmatic mice,compared with WT asthmatic mice.In addition,Sirius red staining was performed and we found the collagen deposition of CKO asthmatic mice was more severe than that of WT asthmatic mice.Consistently,the results of real-time quantitative PCR showed upregulated expression of Collal and ?-SMA in CKO asthmatic mice.In summary,by inducing T cell differentiation in vitro,we found lack of WDFY4 could promote Th2 cell differentiation and the production of Th2 cytokines.As a consequence,excess production of Th2 cytokines could exacerbate Th2 cell-mediated asthma.In this study,we demonstrated that lack of WDFY4 in T cells could enhance ROS-induced apoptosis via p53 and ERK signaling pathways and retard cell proliferation,which was responsible for decreased number of CD8+ T cells and an impaired anti-tumor response.Deficiency in WDFY4 promoted Th2 cell differentiation and aggravated Th2 cell-mediated asthma.Our results elucidate the biological involvement of WDFY4 in apoptosis,proliferation and Th2 differentiation,and also establish the relationship between WDFY4 and T cells,which provides an explanation for the susceptibility of WDFY4 in human autoimmune diseases.