【作者】 王亚楠；

【导师】 郑鹏远；

【作者基本信息】 郑州大学 ， 内科学， 2024， 博士

【摘要】 背景食物过敏(Foodallergy,FA)是一种由免疫系统对特定蛋白抗原异常反应引起的疾病,不仅严重影响患者的日常生活,还可能危及生命。近年来,FA的发病率逐年上升,尤其在儿童和青少年中更为常见。治疗过敏反应的一线药物是肾上腺素,非一线治疗药物包括H1-抗组胺药、糖皮质激素、口服免疫疗法、微生物治疗等。然而,每种治疗方案存在一定的局限性,因此亟需研究更安全、更高效的治疗方案。免疫球蛋白E(Immunoglobulin E,Ig E)介导的FA主要机制是Th2细胞(T helper 2 cells)极化,其特征为Th2细胞在肠黏膜固有层聚集,并分泌促炎细胞因子,导致肠粘膜嗜酸性粒细胞和肥大细胞的扩增,最终促进Ig E抗体的产生。Th2细胞极化可由CD4+T细胞凋亡机制障碍或Th2细胞分化机制异常所致,其具体分子机制有待进一步阐明。活化诱导的细胞死亡(Activation-induced cell death,AICD)是维持外周免疫耐受和T细胞免疫稳态的重要机制,其主要的分子机制是Fas信号通路和线粒体凋亡通路介导的细胞凋亡。然而,在过敏性疾病中,CD4+T细胞对AICD呈抵抗状态,进而导致其大量增殖活化,引起严重的过敏反应。然而,在FA中,导致CD4+T细胞发生AICD功能障碍的机制研究尚不足。调节性T细胞(Regulatory T cells,Treg)是机体重要的免疫调节细胞,可调控Th2细胞的免疫活性及分化。其中,1型调节性T细胞(Type 1 regulatory T cells,Tr1 cells)作为Treg细胞的一个重要亚群,通过分泌IL-10(Interleukin-10)诱导免疫耐受,与Foxp3+Treg细胞不同,其诱导不依赖于Foxp3(Forkheadbox P3)的表达。在FA中,Tr1细胞的免疫抑制功能存在障碍,但其分子机制研究尚不充分。内质网应激(Endoplasmic reticulum stress,ER stress)是错误折叠或未折叠蛋白质的积累所致,可破坏免疫系统的稳态。机体在应对过激的ER stress时,启动未折叠蛋白反应(Unfolded protein response,UPR)。有研究发现,在FA中,皮质醇和ER stress可通过上调Foxp3表达协同抑制Foxp3+Treg细胞的免疫调节功能。然而,ER stress对不依赖Foxp3的Tr1细胞的调控作用及分子机制均尚不完全清楚。本课题组前期使用人类全基因组寡核苷酸芯片对FA患者和健康对照志愿者(Health control,HC)的外周血单个核细胞(Peripheral blood mononuclear cells,PBMCs)进行差异基因表达检测发现,在FA中,Semaphorin 3A(Sema3A)的表达显著下调。Sema3A是Semaphorins家族中的重要成员,主要通过与Plexins A1-A4和神经纤毛蛋白-1(Neuropilin-1,Nrp1)形成复合体,发挥免疫调节功能。在过敏性疾病中,Sema 3A表达下调,其蛋白可改善哮喘模型小鼠临床症状并调节Th1/Th2细胞比例,但其具体机制不明确。在FA中,Sema 3A可以通过抑制Pak1的表达和RAS的激活,恢复肥大细胞对凋亡诱导的抵抗。然而,Sema 3A是否可诱导CD4+T细胞和Th2细胞发生AICD尚不清楚。既往研究发现,Sema 3A可通过上调CD4+T细胞中Foxp3的表达,促进Treg细胞分化,而对不依赖Foxp3的Tr1细胞的免疫调节功能的影响及分子机制未见报道。为深入研究Sema 3A在FA中对Th2细胞极化的调控机制,我们还通过实验验证Sema 3A的上游调节因素。既往研究发现,姜黄素可以通过抑制Th2细胞分化调节免疫反应。Treg细胞及其细胞因子TGF-β可通过Fas信号通路分别促进树突状细胞及血管内皮母细胞凋亡。所以,我们猜测姜黄素、Treg细胞及TGF-β可能作为Sema 3A的上游调节分子,参与Sema 3A对Th2细胞极化的调节。综上所述,本课题主要从三个部分表征在FA中,Sema 3A在Th2细胞极化中的作用机制。第一部分,研究Sema 3A抑制na(?)ve CD4+T细胞向Th2细胞分化的功能及分子机制。第二部分,探索Sema 3A促进CD4+T及Th2细胞发生AICD的作用及分子机制。第三部分,探讨Sema3A通过调控内质网应激来调节Tr1细胞的免疫抑制功能的作用及分子机制。本研究为Sema 3A调控Th2细胞极化的功能提供有力依据,为FA的潜在治疗靶点和新的研究方向提供了研究基础。第一部分 Semaphorin 3A抑制Na(?)ve CD4+T细胞向Th2细胞分化的分子机制目的探讨Seam 3A通过抑制na(?)ve CD4+T细胞向Th2细胞分化,从而抑制Th2细胞极化的分子机制,并进一步验证Sema3A是否可作为姜黄素的作用靶点。方法1.用卵白蛋白(Ovalbumin,OVA)及明矾佐剂对野生型(wild-type,WT)小鼠构建FA模型,用Western Blot及RT-qPCR检测小鼠肠道组织中Sema 3A表达。2.构建条件性敲除CD4+T细胞中SEMA 3A基因的小鼠(Conditional knockout,CKO),用CKO和WT小鼠构建FA模型,用ELISA检测肠道组织及血清中过敏相关免疫指标。3.利用磁性细胞分选技术(Magnetic activated cell sorting,MACS)从WT小鼠脾脏中分选na(?)ve CD4+T细胞,用重组IL-4细胞因子诱导Th2细胞分化,并且实验组用外源性重组小鼠Sema 3A蛋白(rSema 3A)干预。用流式细胞术、ELISA、RT-qPCR分别检测Th2细胞比例、Th2及Th1细胞因子表达量;用Western Blot检测IL-4转录因子GATA3及其磷酸化水平;通过分子对接及Co-IP实验验证Sema 3A与GATA3的结合。4.从CKO及WT小鼠脾脏中分别分选Sema 3A-/-na(?)ve CD4+T及na(?)ve CD4+T细胞,培养72 h。用流式细胞术、ELISA、RT-qPCR分别检测Th2细胞比例、Th2及Th1细胞因子的表达量;用Western Blot检测两组细胞中IL-4的转录因子GATA3及其磷酸化水平。用rSema 3A干预Sema 3A-/-na(?)ve CD4+T细胞,然后用Western Blot及RT-qPCR检测IL-4 的表达量。5.用分子对接方法验证姜黄素与Sema 3A的结合。然后,分选na(?)ve CD4+T细胞,实验组用姜黄素干预。用流式细胞术、Western Blot、RT-qPCR及ELISA分别检测Th2细胞比例、na(?)ve CD4+T细胞及Th2细胞中Sema3A、Th2细胞因子表达量。接下来,将细胞分为四组:姜黄素+Sema 3A/-na(?)ve CD4+T、姜黄素+na(?)ve CD4+T、Sema 3A-/-na(?)ve CD+T、na(?)ve CD4+T细胞,用 RT-qPCR检测细胞中IL-4 mRNA表达量。结果1.FA小鼠血清中OVA-specific IgE,及肠道组织中Th2细胞因子(IL-4、IL-5、IL-13)表达较control组上调,提示建模成功。FA小鼠肠道组织中Sema3A表达量较control组下调。2.与WT+FA组相比,CKO+FA组小鼠腹泻次数、维持时间,及粪便含水量增多,并且肠道组织中Th2细胞因子、及嗜酸性粒细胞过氧化物酶(Eosinophilic peroxidase,EPX)表达上调,血清中OVA-specific IgE、肥大细胞蛋白酶1(Mast cell proteinase 1,Mcpt1)表达上调,而肠道组织中Th1细胞因子(IFN-γ)表达下调。3.与na(?)ve CD4+T组相比,rSema 3A+na(?)ve CD4+T组Th2 细胞比例、Th2 细胞因子、GATA3的磷酸化水平均下调,IFN-γ表达上调。分子对接及Co-IP结果提示,GATA3蛋白与Sema 3A蛋白可相互结合。4.与na(?)ve CD4+T组相比,Sema 3A-/-na(?)ve CD4+T组Th2 细胞、GATA3 磷酸化水平、Th2细胞因子表达均上调,IFN-γ表达下调。rSema 3A+Sema 3A-/-na(?)ve CD4+T 组 IL-4 表达量较 Sema 3A-/na(?)ve CD4+T 组下调。5.分子对接结果提示姜黄素可与Sema 3A结合。与na(?)ve CD4+T相比,姜黄素+na(?)ve CD4+T组Th2细胞比例、Th2细胞因子表达均下调,而na(?)ve CD4+T细胞及Th2细胞中Sema 3A表达上调。姜黄素+Sema 3A-/-na(?)ve CD4+T组IL-4 mRNA表达较姜黄素+na(?)ve CD4+T组上调,同时较Sema 3A-/-na(?)ve CD4+T组下调。小结1.Sema 3A在FA患者及FA模型小鼠中表达水平下调。2.CKO+FA模型小鼠在肠道组织中表现出Th2细胞偏向性炎症。3.Sema 3A可通过抑制转录因子GATA3磷酸化,或通过与转录因子GATA 3结合,从而抑制IL-4蛋白合成,进而抑制Th2细胞分化,最终抑制Th2细胞极化。4.Sema3A可作为姜黄素的作用靶点发挥功能。第二部分 Semaphorin 3A促进CD4+T细胞及Th2细胞发生活化诱导的细胞死亡的分子机制目的探讨Sema 3A通过促进CD4+T细胞及Th2细胞发生AICD,从而抑制Th2细胞极化的机制,并进一步探讨Sema 3A是否可作为Treg细胞及其细胞因子TGF-β的作用靶点。方法1.分选CD4+T、Sema 3A-/-CD4+T细胞,用anti-CD3/28单克隆抗体诱导细胞发生AICD,实验组用rSema 3A干预。用流式细胞术、Western Blot及RT-qPCR分别检测细胞凋亡水平、Fas信号通路及线粒体凋亡通路中明星分子的表达量。2.双荧光素酶报告基因实验验证Sema3A对Fas启动子的活性的调节。3.分选na(?)ve CD4+T、Sema 3A-/-na(?)ve CD4+T细胞,用rSema 3A干预Sema 3A-/-na(?)ve CD4+T细胞。用流式细胞术检测Th2细胞中Fas的表达量。4.分选CD4+T细胞,用Caspase 8抑制剂Z-IETD-FMK阻断Fas信号通路。用流式细胞术检测CD4+T细胞的凋亡比例。5.用Transwell共培养CD4+CD25+Treg细胞与CD4+CD25-Teff细胞。用流式细胞术、Western Blot及RT-qPCR分别检测Teff细胞及Th2细胞中Sema 3A的表达量。6.分选CD4+T、Sema 3A-/-CD4+T细胞,用TGF-β1干预细胞。用流式细胞术检测细胞凋亡比例、CD4+T细胞及Th2细胞中Sema 3A及Fas表达量,并用Western Blot及RT-qPCR检测Sema 3A、Fas信号通路及线粒体凋亡信号通路相关蛋白表达。结果1.与CD4+T组相比,rSema 3A+CD4+T组细胞凋亡比例、Fas信号通路中相关蛋白(Fas、Cleaved-Caspase 3/8)、FAS mRNA水平上调;线粒体凋亡通路相关蛋白Bax表达上调、抗凋亡蛋白Bcl-2表达下调。与CD4+T组相比,Sema 3A-/-CD4+T组细胞凋亡比例、Fas表达均下调,Bcl-2表达上调。与Sema 3A-/-CD4+T组相比,rSema 3A+Sema 3A-/-CD4+T组细胞凋亡比例、Fas表达上调,Bcl-2表达下调。2.双荧光素酶报告基因实验结果提示,转染Sema 3A过表达质粒组Fas启动子活性较转染对照质粒组上调。3.与na(?)ve CD4+T组相比,rSema 3A+na(?)ve CD4+T组Th2 细胞中Fas表达水平上调,Sema 3A-/-na(?)ve CD4+T组Th2细胞中Fas表达水平下调。4.与CD4+T组相比,Z-IETD-FMK+CD4+T组细胞凋亡比例下调;rSema 3A+Z-IETD-FMK+CD4+T组细胞凋亡比例较rSema 3A+CD4+T组下调,同时较Z-IETD-FMK+CD4+T 组上调。5.Treg+Teff组CD4+T细胞、Th2细胞中Sema 3A表达较Teff组上调。6.与CD4+T组相比,TGF-β1+CD4+T细胞凋亡比例、Fas信号通路相关蛋白、CD4+T及Th2细胞中Sema 3A及Fas表达均上调,Bcl-2表达下调。TGF-β1+Sema 3A-/-CD4+T组细胞凋亡比例、Fas表达量较Sema 3A-/-CD4+T组上调,较TGF-β1+CD4+T组下调。小结1.Sema 3A可通过激活Fas信号通路及线粒体凋亡通路,促进CD4+T细胞发生AICD。2.Sema 3A可通过激活Fas信号通路,促进Th2细胞发生AICD。3.Sema 3A可作为Treg细胞及其细胞因子TGF-β的作用靶点发挥功能。第三部分Semaphorin 3A通过调节内质网应激来调节1型调节性T细胞免疫抑制功能的分子机制目的在FA中,检测Tr1细胞免疫抑制功能水平及其内质网应激水平,并进一步探讨Sema 3A通过调节内质网应激来恢复Tr1细胞的免疫抑制功能的分子机制。方法1.收集FA患者及HC志愿者的PBMCs,用流式细胞术分选CD4+CD25+LAG3+CD49b+Tr1细胞,用RNA-seq进行测序及生物信息学分析、RT-qPCR、Western Blot分别检测Tr1细胞中内质网应激相关基因、IL-10及其相关基因表达水平,并分析XBP1及IL-10两基因的相关性。2.用电穿孔法转染构建IL-10过表达质粒至EL-4细胞,构建表达IL-10的细胞模型(IL 10-EL-4细胞)。在IL10-EL-4细胞中过表达XBP1s蛋白后,用RNA-seq测序分析其内质网应激相关基因、IL-0及其相关基因表达水平以及相关性,并用RT-qPCR检测其内质网应激相关基因mRNA表达水平。3.用非特异性细胞激活剂PMA(Phorbol myristate acetate)刺激IL10-EL-4细胞,用RT-qPCR检测各组细胞中IL-10 mRNA表达水平。用双荧光素酶报告基因实验验证XBP1s对IL-10启动子活性的调节作用。4.用RNAi方法敲低IL10-EL-4细胞中eIF2α的表达(EIF2ARNAi)。用ELISA、Western Blot、ChIP分别检测IL-10蛋白、CMIP蛋白及IL-10启动子部位c-Maf表达量。5.用RT-qPCR检测FA患者及HC志愿者Tr1细胞中NRP1及EIF2A mRNA水平,并分析两者的相关性。rSema 3A干预FA患者Tr1细胞(FA.Sema 3A),用RT-qPCR检测NRP1及EIF2A mRNA表达量,Western Blot检测eIF2α蛋白水平变化。6.流式细胞分别分选FA患者及HC志愿者PBMCs中Tr1细胞及Teff细胞。用CFSE染料对Teff细胞进行染色处理,将经rSema 3A干预或未经rSema 3A干预的Tr1细胞与Teff细胞(比例1:5)共孵育3天,流式细胞术检测Teff细胞增殖水平。7.构建特异性敲除CD4+T细胞中IL-10的IL10f/f Cd4-Cre小鼠(CKO)。用WT及CKO小鼠建立FA小鼠模型,从建模第9天开始使用rSema 3A干预。检测小鼠腹泻,核心体温,并用ELISA检测FA相关免疫指标表达量。结果1.与HC组相比,FA组内质网应激相关分子IRE1、iXBP1、ATF6、PERK、GRP78、EIF2A mRNA表达上调,XBP1s蛋白表达上调;IL-10及其相关基因MAF、CMIP mRNA表达下调,并且IL-10与XBP1基因mRNA水平呈负相关性。2.与IL10-EL-4组相比,XBP1s过表达组,内质网应激相关基因GRP78、GRP94、PERK和EIF2A表达上调,而IL10、CMIP、MAF和STA3表达下调,且EIF2A与IL-10、AF、CMIP、STAT3均有相关性。3.与Control.plasmid组相比,XBP1.plasmid组IL-10 mRNA表达水平及IL-10启动子活性均下调。4.XBP1sOE组培养液中IL-10表达水平较Control组下调,XBP1sOE.EIF2A RNAi组IL-10表达水平较XBP1sOE组上调,并且CMIP蛋白及IL-10启动子部位c-Maf蛋白表达有上述相同趋势。5.与HC组相比,FA组Tr1细胞中NRP1 mRNA表达下调,EIF2A mRNA表达上调,并且两者呈负相关性。与FA组相比,FA.rSema 3A组NRP1 mRNA水平上调,eIF2α蛋白及mRNA水平下调。6.与FA组相比,rSema 3A+FA组Teff细胞的增殖能力下调。7.与NC组相比,FA组小鼠表现出腹泻、核心温度降低、Serum OVA-sIg E、肠道灌洗液中过敏介质、Th2细胞因子表达上调,提示建模成功。与FA组相比,FA.Sema3A组小鼠过敏症状减轻,上述免疫指标均下调。与FA.Sema3A组相比,FA.Sema 3A.CKO组小鼠的过敏症状加重,上述免疫指标均上调。小结1.FA中,Tr1细胞免疫抑制功能受到抑制并存在过激的内质网应激反应。2.FA中,内质网应激相关蛋白XBP1s可通过eIF2α蛋白抑制IL-10的表达水平。3.Sema 3A通过上调并结合其受体Nrp1,抑制内质网应激反应,从而上调IL-10表达水平,进而上调Tr1的免疫抑制功能,抑制FA反应。全文结论在Food allergy中,Sema 3A可通过以下三个机制抑制Th2细胞极化:1.Sema 3A可通过抑制转录因子GATA3磷酸化,或通过与转录因子GATA3紧密结合,抑制IL-4蛋白合成,最终抑制Th2细胞分化。2.Sema 3A可通过激活Fas信号通路及线粒体凋亡通路,促进CD4+T细胞及Th2细胞发生AICD。3.Sema 3A可通过上调并结合其受体Nrp1,抑制内质网应激,从而上调IL-10表达,进而上调Tr1细胞的免疫抑制功能。更多还原

【Abstract】 BackgroundFood allergy(FA)is a disease caused by an abnormal response of the immune system to specific protein antigens.It not only seriously affects the patient’s daily life but may also be life-threatening.In recent years,the incidence of FA has increased year by year,especially in children and adolescents.The first-line drug for treating allergic reactions is epinephrine,and non-first-line treatment drugs include H1antihistamines,glucocorticoids,oral immunotherapy,microbial therapy,etc.However,each treatment option has certain limitations,so there is an urgent need to develop safer and more efficient treatment options.The main mechanism of immunoglobulin E(Ig E)-mediated FA is the polarisation of Th2 cells(T helper 2 cells),which is manifested by the aggregation of Th2 cells in the lamina propria of the intestinal mucosa and promotes the secretion of pro-inflammatory cytokines,which leads to the expansion of intestinal mucosal eosinophils and mast cells,and ultimately promotes the production of Ig E antibodies.Th2 cell polarisation can be caused by impaired CD4+T cell apoptosis or abnormal Th2 cell differentiation mechanisms,and its specific molecular mechanisms need to be further elucidated.Activation-induced cell death(AICD)is one of the important mechanisms for building peripheral immune tolerance and T-cell immune homeostasis,and its main molecular mechanism is apoptosis mediated by Fas signalling pathway and mitochondrial apoptosis pathway.In allergic diseases,CD4+T cells are resistant to AICD,which in turn leads to their massive proliferation and activation,causing severe allergic reactions.However,the mechanisms leading to AICD dysfunction in CD4+T cells are understudied in FA.Regulatory T cells(Treg)are one of the important immunoregulatory cells in the organism,regulating the immunoreactivity and differentiation of Th2 cells.Type 1 regulatory T cells(Trl cells),an important subpopulation of Treg cells,induce immune tolerance to antigens in the organism by secreting IL-10(Interleukin-10)to induce organismal immune tolerance to antigens,and unlike Foxp3+Treg cells,its induction does not depend on the expression of Foxp3(Forkhead box P3).In FA,the immunosuppressive function of Trl cells is impaired,but the molecular mechanisms are not well studied.Endoplasmic reticulum stress(ER stress)results from the accumulation of misfolded or unfolded proteins and can disrupt the homeostasis of the immune system.The organism initiates the unfolded protein response(UPR)in response to excessive ER stress.It has been found that in FA,cortisol and ER stress synergistically inhibit the immunomodulatory function of Foxp3+Treg cells by upregulating Foxp3 expression.However,both the regulatory effects of ER stress on Foxp3-independent Trl cells and the molecular mechanisms are not fully understood.Our research group previously used a human whole-genome oligonucleotide microarray to detect differential gene expression in peripheral blood mononuclear cells(PBMCs)of FA patients and healthy control volunteers.We found that the expression of Semaphorin 3A(Sema 3A)was significantly down-regulated in FA patients.Sema 3A is an important member of the Semaphorins family and mainly exerts its immune regulatory function by forming a complex with Plexins A1-A4 and Neuropilin-1(Nrp1).In allergic diseases,the downregulation of Sema 3A expression has been observed,and Sema 3A protein treatment can improve the clinical symptoms of asthma model mice and the Th1/Th2 cell ratio in their intestinal tissues,but the specific mechanism is unclear.Additionally,in FA,Sema 3A can restore the resistance of mast cells to apoptosis induction by inhibiting the expression of Pak1 and the activation of RAS.However,it is unclear whether Sema 3A can induce AICD in CD4+T cells and Th2 cells.Previous studies have found that Sema 3A can promote the differentiation of Treg cells by upregulating the expression of Foxp3 in CD4+T cells,but the impact of Sema 3A on the immune regulatory function of Foxp3independed Trl and the underlying molecular mechanisms have not been reported.To further investigate the regulatory mechanisms of Sema 3 A in the polarization of Th2 cells in FA,we also experimentally validated the upstream mechanism of Sema 3A.Previous studies have found that curcumin can regulate the immune response by inhibiting the differentiation of Th2 cells.Treg cells and their cytokine TGF-β can promote apoptosis of dendritic cells and vascular endothelial cells through the Fas signaling pathway,respectively.Therefore,we have proposed the scientific hypothesis that curcumin,Treg cells,and TGF-β may act as upstream regulatory factors of Sema 3A,participating in the regulation of Sema 3A on the polarization of Th2 cells.In summary,this study primarily studied the mechanisms of action of Sema 3A in the polarization of Th2 cells in FA from three aspects.In the first part,we investigate the inhibitory function and molecular mechanism of Sema 3A in suppressing the differentiation of na(?)ve CD4+T cells into Th2 cells.In the second part,we explore the role and molecular mechanism of Sema 3A in promoting AICD in CD4+T cells and Th2 cells.In the third part,we examine the role and molecular mechanism of Sema 3A in regulating the immune inhibitory function of Tr1 cells by modulating endoplasmic reticulum stress.These studies provide strong evidence for the regulatory function of Sema 3 A in the polarization of Th2 cells in FA,laying the foundation for potential therapeutic targets and new research directions in FA.Part Ⅰ Semaphorin 3A Inhibits the Differentiation of Na(?)ve CD4+T Cells into Th2 CellsObjectivesTo investigate the molecular mechanism by which Seam 3A inhibits Th2 cell polarization by inhibiting na(?)ve CD4+T cell differentiation to Th2 cells,and to further verify whether Seam 3A could be a target for curcumin.Methods1.FA model was established in wild-type(WT)mice using ovalbumin(OVA)and alum adjuvant.The expression of Sema 3A in the intestinal tissues of these mice was detected using Western Blot and RT-qPCR.2.A conditional knockout(CKO)mouse model with Sema 3A deleted in CD4+T cells was constructed,then we established the FA model using CKO and WT mice.Allergy-related immune indicators were measured in the intestinal tissues and serum using ELISA.3.Na(?)ve CD4+T cells were isolated from the spleens of WT mice using magnetic-activated cell sorting(MACS).Then Th2 cell differentiation was induced using recombinant IL-4 cytokine.The experimental group was treated with exogenous recombinant mouse Sema 3A protein(rSema 3A).The proportion of Th2 cells and the expression of Th2 and Thl cytokines were detected using flow cytometry,ELISA,and RT-qPCR.The expression and phosphorylation levels of the Th2 transcription factor GATA3 were examined using Western Blot.The binding of Sema 3A to GATA3 was verified using molecular docking and coimmunoprecipitation(Co-IP)experiments.4.Sema 3A-/-na(?)ve CD4+T cells and na(?)ve CD4+T cells were isolated from the spleens of CKO and WT mice,respectively.The proportion of Th2 cells and the expression of Th2 and Th1 cytokines were detected using flow cytometry,ELISA,and RT-qPCR.The expression and phosphorylation levels of the Th2 transcription factor GATA3 were examined using Western Blot.The Sema 3A-/-na(?)ve CD4+T cells were treated with rSema 3A and the expression of IL-4 was detected using Western Blot and RT-qPCR.5.Na(?)ve CD4+T cells were treated with curcumin and the proportion of Th2 cells,the expression of Sema 3A in na(?)ve CD4+T cells and Th2 cells,and the expression of Th2 cytokines were detected using flow cytometry,Western Blot,RT-qPCR,and ELISA.Then the cells were divided into four groups:curcumin+Sema 3A-/-na(?)ve CD4+T,curcumin+na(?)ve CD4+T,Sema 3A-/-na(?)ve CD4+T,and na(?)ve CD4+T cells,and the mRNA expression of IL-4 was detected using RT-qPCR.Results1.The serum OVA-specific Ig E and Th2 cytokines(IL-4,IL-5,IL-13)in the intestinal tissues of the FA mice were upregulated compared to the control group,indicating successful model establishment.The expression of Sema 3A in the intestinal tissues of the FA mice was down-regulated compared to the control group.2.Compared with the WT+FA group,mice in the CKO+FA group had an increased number of diarrhea episodes,duration of diarrhea,and fecal water content,as well as an up-regulation of Th2 cytokine and eosinophilic peroxidase(EPX)in intestinal tissues,and an up-regulation of OVA-specific IgE,mast cell proteinase 1(Mcpt1)in serum,while the expression of Th1 cytokine(IFN-y)downregulated in intestinal tissues.3.The rSema 3A+na(?)ve CD4+T group showed a decreased proportion of Th2 cells,Th2 cytokine,and GATA3 phosphorylation,while IFN-y was up-regulated comparing to the na(?)ve CD4+T group.The molecular docking and Co-IP results suggested a tight binding between the GATA3 and Sema 3 A proteins.4.In contrast to the na(?)ve CD4+T group,the Sema 3A-/-na(?)ve CD4+T group exhibited an increased proportion of Th2 cells,GATA3 phosphorylation,and Th2 cytokine,while IFN-y was down-regulated.The rSema 3A+Sema 3A-/-na(?)ve CD4+T group showed a decreased IL-4 compared to the Sema 3A-/-na(?)ve CD4+T group.5.Compared with the na(?)ve CD4+T group,the curcumin+na(?)ve CD4+T group showed a decreased proportion of Th2 cells and Th2 cytokine,while the expression of Sema 3A in na(?)ve CD4+T cells and Th2 cells was up-regulated.The curcumin+Sema 3A-/-na(?)ve CD4+T group showed a higher IL-4 mRNA expression than the curcumin+naive CD4+T group,but lower than the Sema 3A-/-A naive CD4+T group.Summary1.The expression level of Sema 3A decreased in patients with food allergy and in mouse models of food allergy.2.The CKO+FA model mice exhibit a Th2 cell-biased inflammation in the intestinal tissues.3.Sema 3A can inhibit IL-4 protein synthesis and ultimately suppress Th2 cell differentiation by inhibiting the phosphorylation of the transcription factor GATA3 or by tightly binding to the transcription factor GATA3.4.Sema 3A can function as a target of curcumin.Part Ⅱ Semaphorin 3A Promotes Activation-Induced Cell Death in CD4+T Cells and Th2 CellsObjectivesTo investigate the mechanism by which Sema 3A inhibits Th2 cell polarization by promoting AICD in CD4+T cells and Th2 cells,and to futher explore whether Sema 3 A may serve as a target for Treg cells and their cytokine TGF-β.Methods1.CD4+T and Sema 3A-/-CD4+T cells were sorted and cells were induced to undergo AICD with anti-CD3/28 monoclonal antibody,and the experimental group was intervened with rSema 3A.Flow cytometry,Western Blot,and RT-qPCR methods were used to detect the level of cell apoptosis,expression of key molecules in the Fas signaling pathway,and mitochondrial apoptosis pathway,respectively.2.Dual luciferase reporter gene assay was used to validate the regulation of Fas promoter activity by Sema3 A.3.Sema 3A-/-na(?)ve CD4+T and na(?)ve CD4+T cells were isolated and Sema 3A-/na(?)ve CD4+T cells were interfered with rSema 3A.Flow cytometry was used to detect the expression of Fas in Th2 cells.4.CD4+T cells were sorted,and the Fas signalling pathway was blocked with the caspase 8 inhibitor Z-IETD-FMK.The percentage of apoptotic CD4+T cells was detected by flow cytometry.5.CD4+CD25+Treg cells and CD4+CD25-Teff cells were co-cultured with Transwell.The expression of Sema 3A in Teff cells and Th2 cells was detected by flow cytometry,Western Blot and RT-qPCR,respectively.6.Sema 3A-/-CD4+T and CD4+T cells were isolated,and the experimental group was intervened with TGF-β1.Flow cytometry was used to detect the apoptosis ratio,as well as the expression of Sema 3A and Fas in CD4+T cells and Th2 cells.Western Blot and RT-qPCR were used to detect the expression of Sema 3A,proteins of Fas signaling pathway,and mitochondrial apoptosis pathway.Results1.Compared with CD4+T group,rSema 3A+CD4+T group showed upregulation of apoptosis proportion,related proteins(Fas,Cleaved-Caspase 3/8)in Fas signalling pathway,and FAS mRNA level;mitochondrial apoptosis pathway related protein Bax was up-regulated,and anti-apoptosis protein Bcl-2 was down-regulated.Compared to the CD4+T group,the apoptosis ratio and Fas were down-regulated and Bcl-2 was up-regulated in the Sema 3A-/-CD4+T group.Compared with Sema 3A-/CD4+T group,apoptosis proportion and Fas were up-regulated and Bcl-2 was downregulated in rSema 3A+Sema 3A-/-CD4+T group.2.The results of the dual-luciferase reporter gene experiment suggested that transfection of the Sema 3A overexpression plasmid upregulated the activity of the Fas promoter compared to the transfection of the control plasmid.3.In contrast to the na(?)ve CD4+T group,the rSema 3A+ na(?)ve CD4+T group exhibited an upregulation of Fas in Th2 cells.The Sema 3A-/-na(?)ve CD4+T group showed a downregulation of Fas in Th2 cells compared to the na(?)ve CD4+T group.4.The Z-IETD-FMK+CD4+T group showed a downregulation of cell apoptosis ratio than the CD4+T group.The rSema 3A+Z-IETD-FMK+CD4+T group exhibited a lower cell apoptosis ratio compared to the rSema 3A+CD4+T group,and a higher cell apoptosis ratio compared to the Z-IETD-FMK+CD4+T group.5.The expression of Sema 3 A in CD4+T cells and Th2 cells was upregulated in the Treg+Teff group compared to the Teff group.6.In comparison to the CD4+T group,the TGF-β1+CD4+T group exhibited an upregulation of cell apoptosis ratio,Fas signaling pathway-related proteins,and the expression of Sema 3A and Fas in CD4+T and Th2 cells,with a downregulation of Bcl-2 expression.The TGF-β1+Sema 3A-/-CD4+T group showed an upregulation of cell apoptosis ratio and Fas expression compared to the Sema 3A-/-CD4+T group,and a downregulation compared to the TGF-β1+CD4+T group.Summary1.Sema 3 A can promote activation-induced cell death(AICD)in CD4 +T cells by activating the Fas signaling pathway and the mitochondrial apoptosis pathway.2.Sema 3A can promote AICD in Th2 cells by activating the Fas signaling pathway.3.Sema 3A can function as a target of Treg cells and their cytokine TGF-β.Part Ⅲ Semaphorin 3A Regulates the Immunosuppressive Function of Type 1 Regulatory T Cells by Regulating Endoplasmic Reticulum StressObjectivesThe immunosuppressive function level of Tr1 cells and their endoplasmic reticulum stress level were examined in FA,and the molecular mechanism by which Sema 3A restores the immunosuppressive function of Tr1 cells by regulating endoplasmic reticulum stress was further explored.Methods1.PBMCs from FA patients and HC volunteers(n=20)were collected,and CD4+CD25+LAG3+CD49b+Tr1 cells were sorted by flow cytometry,and the expression levels of endoplasmic reticulum stress-related genes,IL-10 and its related genes were detected in Tr1 cells by sequencing and bioinformatic analysis with RNAseq,RT-qPCR,and Western Blot,respectively,and the correlation between XBP1 and IL-10 genes was analyzed.2.EL-4 cells were transfected with IL-10 overexpression plasmid by electroporation to construct a cell model expressing IL-10(IL 10-EL-4 cells).After overexpression of XBP1s protein in IL10-EL-4 cells,the expression levels of endoplasmic reticulum stress-related genes,IL-10 and its related genes as well as their correlations were analysed by RNA-seq sequencing,and the expression levels of endoplasmic reticulum stress-related genes mRNA were detected by RT-qPCR.3.IL10-EL-4 cells were stimulated with the non-specific cell activator PMA(Phorbol myristate acetate),and IL-10 mRNA expression levels were detected by RTqPCR in each group.A dual luciferase reporter gene assay was used to verify the regulatory effect of XBP1s on IL-10 promoter activity.4.eIF2α was knocked down in IL10-EL-4 cells using RNAi(EIF2ARNAi).ELISA,Western Blot,and ChIP were used to respectively detect the expression of IL10,CMIP,and the c-Maf binding site of the IL-10 promoter.5.RT-qPCR was used to detect the mRNA levels of NRP1 and EIF2A in Tr1 cells from patients with FA and HC volunteers and analyzed their correlation.Tr1 cells from patients with FA(FA.Sema 3A)were intervened with rSema 3 A,then the expression of NRP1 and EIF2A mRNA was detected using RT-qPCR,and Western Blot was used to detect changes in eIF2α protein.6.Tr1 cells and Teff cells from PBMCs of patients with FA and HC volunteers were isolated using flow cytometry.Teff cells were stained with CFSE dye.Tr1 cells treated with or without rSema 3A were co-culture with Teff cells(at a ratio of 1:5)3days,and flow cytometry was used to detect the proliferation of Teff cells.7.Specific knockout IL-10 in CD4+T cells in IL-10f/f Cd4-Cre mice(CKO)were generated.FA mouse model was established using WT and CKO mice and rSema 3 A intervention was started from the 9th day of modeling.Measure mouse diarrhea,core body temperature was tested,and ELISA was used to detect the expression of FArelated immune indicators.Results1.Compared with the HC group,the expression of endoplasmic reticulum stressrelated molecules IRE1,XBP1,ATF6,PERK,GRP78,and EIF2A mRNA was upregulated in the FA group,and the expression of XBP1s protein was up-regulated;the expression of IL-10 and its related genes MAF and CMIP mRNA was down-regulated,and there was a negative correlation between the mRNA levels of IL-10 and XBP1 gene.2.The XBP1s overexpression group exhibited upregulation of endoplasmic reticulum stress-related genes GRP78,GRP94,PERK,and EIF2A,and downregulation of IL10,CMIP,MAF,and STAT3 comparing to the IL10-EL-4 group.Additionally,there was a correlation between EIF2A and IL-10,MAF,CMIP,and STAT3.3.The XBP1.plasmid group showed a downregulation of IL-10 mRNA expression compared to the Control.plasmid group.Results from the dual-luciferase reporter gene experiment indicated a downregulation of IL-10 promoter activity in the XBP1.plasmid group compared to the Control.plasmid group.4.The XBP1sOE group exhibited a decreased expression of IL-10 in the culture medium than the Control group,while the XBP1sOE.EIF2A RNAi group showed an increased expression of IL-10 compared to the XBP1sOE group.Additionally,the expression of CMIP protein and the c-Maf protein binding site of the IL-10 promoter showed a similar trend.5.The FA group showed downregulation of NRP1 mRNA expression and upregulation of EIF2A mRNA expression in Tr1 cells comparing to the HC group,and with a negative correlation between the two genes.In comparison to the FA group,the FA.rSema 3A group exhibited upregulation of NRP1 mRNA levels and downregulation of eIF2α protein and mRNA levels.6.The proliferation ability of Teff cells reduced in the rSema 3A+FA group than the FA group.7.FA mice exhibited diarrhea,decreased core body temperature,elevated Serum OVA-sIg E,allergic mediators in intestinal lavage,and increased expression of Th2 cytokines comparing to the NC group,indicating successful modeling.Compared to the FA group,the FA.Sema 3A group of mice showed reduced allergic symptoms and downregulation of the immune indicators.In contrast,compared to the FA.Sema 3A group,the FA.Sema 3A.CKO group of mice showed aggravated allergic symptoms and upregulation of the immune indicators.Summary1.In FA,the immunosuppressive function of Tr1 cells is inhibited,and there is excessive ER stress.2.In FA,the ER stress-related protein XBP1s suppress the expression level of IL-10 through the eIF2α protein.3.Sema 3A can upregulate and bind to its receptor Nrp1,thereby suppressing ER stress and upregulating the expression level of IL-10,which in turn enhances the immunosuppressive function of Tr1 cells and inhibits the FA response.ConclusionsIn Food allergy,Sema 3A can inhibit Th2 cell polarization through the following three mechanisms:1.Sema 3 A can inhibit the phosphorylation of the transcription factor GATA3,or closely bind to the transcription factor GATA3,to suppress IL-4 protein synthesis,ultimately inhibiting Th2 cell differentiation.2.Sema 3A can promote AICD in CD4+T cells and Th2 cells by activating the Fas signaling pathway and the mitochondrial apoptotic pathway.3.Sema 3A can inhibit Endoplasmic reticulum stress by up-regulating and binding to its receptor Nrp1,thereby up-regulating IL-10 expression and consequently the immunosuppressive function of Tr1 cells.更多还原