【作者】 李妍；

【导师】 刘奇迹；

【作者基本信息】 山东大学 ， 遗传学， 2021， 博士

【摘要】 自身免疫性疾病(Autoimmune disease,AID)是一类累及特定靶器官或多器官的异质性慢性疾病,免疫系统对自身抗原失去耐受性是导致疾病发生的原因。因靶细胞和受累器官不同,AID的临床表型也各不相同,但患者的免疫系统均存在功能失调,表现出多种异常表型,如高水平的细胞因子和自身性体的产生。在AID中,免疫细胞可通过直接杀伤细胞或释放细胞毒性细胞因子、前列腺素、活性氮或氧中间产物造成组织损伤。T细胞对自身抗原的免疫应答以及B细胞分泌的自身抗体是AID的主要致病机制。在组织特异性自身免疫中,辅助性T细胞(Th1细胞和Th17细胞)过度的免疫应答是导致炎症的重要驱动力。在人类AID中,滤泡辅助性T(Follicular helper T,Tfh)细胞数量增多,并与自身抗体的产生及疾病进程有关。调节性T(Regulatory T,Treg)细胞主要存在于机体的外周免疫组织中,可以通过分泌抗炎性细胞因子抑制Th1细胞和Th17细胞的免疫应答,减轻炎症,因而在自身免疫应答中极为重要。如果Treg细胞对自身抗体的产生和自身反应性T细胞的调控被环境因素(如感染或毒素)破坏,则可导致AID的发生。全基因组关联分析(Genome-wide association study,GWAS)是一种在全基因组序列内找出遗传多态性位点并将其作为标记,通过病例-对照研究从中找出与复杂性疾病有关的基因或位点的方法。当前,人们已利用GWAS找出了数百个与AID有关的基因或位点。2010年的一项GWAS报道,WDFY4(WDFY family member 4)是亚洲人群系统性红斑狼疮(Systemic lupus erythematosus,SLE)的一个易感基因,这是关于WDFY4的首篇报道。随即又有多篇GWAS相继报道,WDFY4是类风湿性关节炎、临床性无肌炎性皮肌炎和幼年特发性关节炎等AID的易感基因。人类WDFY4基因定位于10号染色体,其编码的蛋白WDFY4是WDFY家族的第四个成员。WDFY4基因是一个含有约30万个碱基的巨大基因,WDFY4蛋白是一个含有3184个氨基酸残基的巨大蛋白。WDFY4蛋白在多个物种间具有高度的保守性,其N端是Armadillo-type fold结构域,C端则由一个PH(Pleckstrin homology)样结构域、一个 BEACH(Beige and chediak-higashi)结构域和五个WD repeat结构域组成。近期发表的两项研究分别证实了 WDFY4在维持B细胞存活及细胞相关抗原交叉递呈中具有重要作用。然而,WDFY4是否会影响T细胞的发育、存活及分化呢?其机制为何?WDFY4又是否能通过影响T细胞介入到疾病的进程中呢?为了解答以上问题,我们进行了以下三部分的研究。第一部分Wdfy4敲除小鼠模型构建及表型分析对WDFY4基因在野生型小鼠各组织及实验室常用工具细胞系中的表达情况进行检测发现,WDFY4在免疫组织及免疫细胞系中具有高水平的表达。接下来,我们构建了Wdfy4基因全身性以及T细胞条件性敲除小鼠模型。首先通过雌雄交配的方法获得了 LoxP纯合的Wdfy4fl/fl小鼠,并将其与Sox2-Cre+/-小鼠进行杂交,最终获得Wdfy4fl/-,Sox2-Cre+/-小鼠。Sox2-Cre广泛表达于胚内各组织,因而Wdfy4fl/-,Sox2-Cre+/-小鼠胚内各组织中Wdfy4基因被全部敲除,将该小鼠记为KO小鼠。由于目前尚未有针对小鼠WDFY4的蛋白抗体,我们利用PCR实验对小鼠基因型进行了鉴定,并通过实时荧光定量PCR的方法对基因敲除效率进行了检测,结果证明Wdfy4基因在敲除小鼠的胸腺和脾脏中具有较好的敲除效率。与此同时,将性别、年龄相匹配的Wdfy4fl/fl小鼠(记为WT小鼠)作为对照小鼠,进行后续研究。为了研究WDFY4对T细胞发育、存活及分化的影响,我们对KO小鼠与WT小鼠的相关表型进行了检测。利用流式细胞术检测两组小鼠胸腺中各时期胸腺细胞的比例,以期研究敲除Wdfy4是否会影响T细胞的发育,然而结果显示两组小鼠发育各期胸腺细胞比例并无差异。我们又利用流式细胞术检测了 KO小鼠与WT小鼠外周免疫器官脾脏及淋巴结中T细胞、各亚型T细胞以及活化的T细胞的比例,结果也未发现两组小鼠在以上方面存在差异。随后,利用流式细胞术检测了与AID发生有关的Treg细胞及Tfh细胞在两组小鼠外周免疫器官中的比例,结果发现二者并无差异。由于遗传性代偿效应可能掩盖了 KO小鼠的表型。所以,我们又将Wdfy4fl/fl小鼠与Lck-Cre+/-小鼠进行杂交,最终获得Wdfy4fl/fl,Lck-Cre+/-小鼠,即T细胞条件性敲除小鼠,记为CKO小鼠。对CKO小鼠与WT小鼠的相关表型进行了检测发现,在免疫静息状态下,WDFY4并不影响小鼠外周Treg细胞与Tfh细胞的分化。但是,CKO小鼠外周T细胞及CD8+T细胞的比例明显低于WT小鼠,从而导致CKO小鼠外周CD4+T细胞/CD8+T细胞比值升高。进一步分析发现,CKO小鼠T细胞的发育并不存在异常,脾脏发育正常,脾脏细胞总数与WT小鼠相近,但CKO小鼠外周CD8+T细胞的比例以及数量均明显明显低于WT小鼠,而这种减少也影响了 CKO小鼠的抗肿瘤能力。对CKO小鼠及WT小鼠进行荷瘤实验,结果显示CKO小鼠的肿瘤生长速度明显快于WT小鼠,其肿瘤体积及重量均大于WT小鼠。在本部分中,我们成功构建了Wdfy4基因全身性以及T细胞条件性敲除小鼠模型。通过对小鼠表型进行比较分析发现,在T细胞中敲除Wdfy4可影响小鼠外周CD8+T细胞的存活,从而影响小鼠的抗肿瘤能力。第二部分WDFY4通过p53信号通路参与小鼠外周CD8+T细胞的凋亡研究发现,WDFY4蛋白C末端的BEACH-WD repeat结构域在细胞凋亡中具有极为重要的作用。我们首先利用流式分析了 WT小鼠与CKO小鼠外周免疫器官淋巴结中T细胞与CD8+T细胞的凋亡水平,结果显示CKO小鼠外周T细胞与CD8+T细胞的凋亡水平均远高于WT小鼠。为了对以上结果进行验证,我们构建了稳定低表达WDFY4的Jurkat细胞(人T淋巴细胞瘤细胞)与对照细胞。通过Western blot对两组细胞凋亡相关蛋白的表达水平进行检测,结果显示缺失WDFY4可上调Jurkat细胞凋亡相关蛋白的水平。为了探讨WDFY4参与凋亡的机制,我们利用磁珠分选出了 WT小鼠与CKO小鼠的CD8+T细胞。活性氧群(Reactive oxygen species,ROS)在诱导凋亡发生中饰演着重要的角色,我们发现CKO小鼠CD8+T细胞胞内ROS水平显著高于WT小鼠。利用Jurkat细胞进行实验,结果也显示缺失WDFY4可上调胞内ROS的水平。NADPH氧化酶复合物NOX是哺乳动物体内产生ROS的主要酶系统。实时荧光定量PCR结果显示,CKO小鼠CD8+T细胞和低表达WDFY4的Jurkat细胞中NADPH氧化酶的表达水平均高于对照。为了进一步证实WDFY4是通过调控ROS来参与凋亡过程的,我们对磁珠分选得到的小鼠CD8+T细胞进行了体外培养,并在培养期间向培养液中加入含巯基的抗氧化剂NAC以清除胞内ROS。Western blot结果显示,未加入NAC时CKO小鼠CD8+T细胞的凋亡水平明显高于WT小鼠。然而加入NAC后,CKO小鼠CD8+T细胞中cleaved PARP以及cleaved caspase-7的表达水平接近于WT小鼠。既往文献报道,ROS能通过p53信号通路参与凋亡的调控。利用Western blot对p53第15位丝氨酸的磷酸化水平进行检测,结果显示CKO小鼠CD8+T细胞中p53的磷酸化水平高于WT小鼠的CD8+T细胞。通过实时荧光定量PCR对p53下游的Noxα、Gadd45b以及Puma基因的表达水平进行检测,结果显示缺失WDFY4可明显上调p53下游参与凋亡的靶基因的表达。胞外调控蛋白激酶(Extracellular regulated protein kinase,ERK)也可看作是p53下游的一个靶分子,抑制ERK活性可促进凋亡的发生。Western blot结果显示,CKO小鼠CD8+T细胞中磷酸化的ERK水平低于WT小鼠CD8+T细胞;而在低表达WDFY4的Jurkat细胞中,我们也检测到了 ERK磷酸化水平的下调。ERK可使促凋亡蛋白Bim第69位丝氨酸发生磷酸化,从而介导Bim的降解。我们发现缺失WDFY4确实可以下调Bim第69位丝氨酸的磷酸化水平。课题组前期的研究发现,WDFY4可通过非经典自噬介导B细胞的死亡,从而影响小鼠SLE的疾病进程。在本研究中,我们利用Western blot检测了小鼠CD8+T细胞和Jurkat细胞中非经典自噬相关因子的表达水平,结果并未发现差异。对Jurkat细胞进行饥饿处理以诱导自噬,结果显示WDFY4确实不影响LC3的脂化。增殖受阻以及细胞周期停滞也可能是造成CKO小鼠外周CD8+T细胞数量减少的原因。因而,我们首先通过腹腔注射BrdU的方法对两组小鼠外周CD8+T细胞的增殖水平进行了检测。流式细胞术结果显示,CKO小鼠外周CD8+T细胞中BrdU+细胞明显少于WT小鼠,提示CKO小鼠CD8+T细胞增殖受阻。此外,利用流式细胞术对两组小鼠的细胞周期进行检测,发现WDFY4的缺失并不影响CKO小鼠脾脏细胞的细胞周期。对低表达WDFY4的Jurkat细胞进行Western blot检测发现,WDFY4的缺失并不影响细胞周期相关蛋白的表达。在本部分中我们发现,缺失WDFY4可通过激活p53通路、抑制ERK活性来参与ROS介导的凋亡。此外,敲除Wdfy4可导致CKO小鼠外周CD8+T细胞的增殖受损。第三部分敲除Wdfy4促进Th2细胞分化并加重Th2细胞介导的哮喘表型WDFY4已被证实是多种人群SLE的易感基因,之前认为SLE是由Th2细胞驱动的疾病。在本部分中,我们通过磁珠分选得到两组小鼠脾脏中的初始CD4+T细胞并在体外对其进行诱导活化,从而研究WDFY4在Th细胞分化,尤其是Th2细胞分化中的作用。我们首先以两组小鼠的全脾细胞为研究对象,在体外对其进行诱导活化。将两组小鼠的全脾细胞分别分为两组,一组给予PBS作为对照,一组给予anti-CD3抗体与anti-CD28抗体以在体外活化小鼠T细胞,使其不定向地向各T细胞亚群分化。流式细胞术结果表明,与PBS组相比,无论是来源于WT小鼠还是来源于CKO小鼠的T细胞在接受免疫刺激活化后各Th细胞亚群的比例均有所升高,说明此法可刺激小鼠T细胞活化。进一步分析发现,敲除Wdfy4的T细胞在接受刺激活化后,Th2细胞的比例要高于来源于WT小鼠的T细胞,提示敲除Wdfy4基因可导致T细胞倾向于向Th2方向分化。我们又分选出了两组小鼠脾脏中的初始CD4+T细胞,并以其为研究对象重复上述实验,得到了相同的结果。为了明确WDFY4在Th2细胞分化中的作用,我们对两组小鼠的初始CD4+T细胞进行了体外定向诱导分化,使其特异性地向Th2细胞分化。实时荧光定量PCR和Western blot的结果显示,敲除Wdfy 4的Th2细胞表达更高水平的GATA3与c-Maf。实时荧光定量PCR和ELISA结果则表明,敲除Wdfy4的Th2细胞表达更高水平的Th2型细胞因子。Th2细胞在慢性炎症(如哮喘)及过敏性疾病(如过敏性腹泻)中扮演着举足轻重的角色。为了明确WDFY4是否能参与Th2细胞介导的疾病,我们利用卵清蛋白OVA构建了慢性哮喘小鼠模型。实时荧光定量PCR和ELISA结果表明,CKO哮喘小鼠肺组织中表达更高水平的Th2型细胞因子与转录因子。对两组哮喘小鼠的肺组织切片进行组织病理学分析,我们发现CKO哮喘小鼠存在更为严重的气道炎症,表现为更多的嗜中性粒细胞浸润、更为明显与严重的杯状细胞增生以及黏蛋白分泌增多。实时荧光定量PCR结果也显示,与WT哮喘小鼠相比,CKO哮喘小鼠肺组织中表达更高水平的黏蛋白MUC5AC。对小鼠支气管肺泡灌注液中的细胞进行分析,结果显示CKO哮喘小鼠肺泡灌注液中含有更高比例及数量的嗜中性粒细胞。此外,两组哮喘小鼠均表现出一定程度的气道重塑,相较于WT哮喘小鼠,敲除Wdfy4可明显加重哮喘小鼠的胶原沉积。实时荧光定量PCR结果也显示,CKO哮喘小鼠表达更高水平的I型胶原蛋白与α-SMA。在本部分中,我们通过体外诱导小鼠T细胞分化发现,敲除Wdfy4可促进Th2细胞的分化、上调Th2细胞相关因子的表达水平,并因此加重哮喘小鼠的疾病表型。通过本研究我们发现,WDFY4可以参与细胞增殖并通过p53和ERK信号通路参与ROS介导的凋亡,从而导致小鼠外周CD8+T细胞比例及数量的减少,CKO小鼠的抗肿瘤免疫能力因此受损。敲除Wdfy4可促进Th2细胞分化并加重Th2细胞介导的哮喘表型。我们的研究结果阐明了 WDFY4在细胞凋亡及Th2细胞分化中的生物学作用,并建立了WDFY4基因与T细胞之间的联系,从而为解释WDFY4基因在人类AID中的易感性提供了理论依据。更多还原

【Abstract】 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 ⅠGeneration 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 Ⅰ/Ⅱ 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-Ⅱ/LC3-Ⅰ 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 α-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.更多还原