LIGHT-HVEM/LTβR通路对哮喘CD4+T细胞-气道平滑肌细胞交叉串扰的调控作用及机制研究

Asthma is characterized by airway inflammation and airway remodeling,and the crosstalk between CD4+T cells and airway smooth muscle(ASM) cells plays a critical role in regulating these two features. However, the mechanisms underlying this crosstalk are not clear. Recently, the central roles for LIGHT-HVEM/LTβR pathway regulating heterotypic cells proliferation and differentiation are emerging, highlighting the pathway may be a key bridging that controls the CD4+T cell-ASM cell interactions in asthma. So we plan to carry out this research to explore the mechanisms of LIGHT-HVEM/LTβR pathway regulating the crosstalk between CD4+T cells and ASM cells in asthma. Firstly, we perform several experiments on the asthma model of mice both in vitro and in vivo assays. These experiments are carried out from cellular, protein and genetic levels, to verify our scientific hypotheses that LIGHT-HVEM/LTβR pathway controls the crosstalk between CD4+T cells and ASM cells in asthma. Secondly, many biotechnologies are used to study the effects of LIGHT-HVEM/LTβR pathway on the cell proliferation and differentiation of CD4+T cells and ASM cells, and to explore the signal transduction mechanisms underlying the effects of the pathway. These assays rely on a suite of technologies including co-culture of CD4+T cels and ASM cells, protein chip, and immuno-electrophysiology, etc. Lastly, the exogenous recombinant lentivirus vectors are constructed to block or promote the gene expression of LIGHT in vivo assays. Then the changes of airway inflammation and airway remodeling are collected to investigate the molecular mechanisms of targeted gene intervention in asthma. All the above experiments will demonstrate that LIGHT-HVEM/LTβR pathway plays a key role in regulating the crosstalk between CD4+T cels and ASM cells in asthma, and suggest that the gene of LIGHT may be a potent novel target for the future development of genetic intervention of asthma.

CD4+T细胞-气道平滑肌(ASM)细胞的交叉串扰是导致哮喘病理特征- - 气道炎症及气道重塑的重要原因，但机制不清。鉴于LIGHT-HVEM/LTβR通路在调控多种细胞、特别是炎症细胞增殖、分化过程中的重要地位，可能是CD4+T细胞与ASM细胞相互作用的关键桥接。我们拟在前期研究基础上，对哮喘小鼠模型进行在体、离体研究，从细胞、蛋白、基因不同水平探讨LIGHT-HVEM/LTβR通路与CD4+T细胞-ASM细胞交叉串扰的关系；在此基础上采用细胞共培养、蛋白质芯片、免疫电生理等技术，探讨LIGHT-HVEM/LTβR通路对哮喘CD4+T细胞分化、ASMC增殖及凋亡的作用及其信号调控机制；利用外源性重组慢病毒载体经气道对哮喘小鼠模型进行体内LIGHT基因靶向干预，运用动物活体成像等方法观察该基因沉默或过表达时气道炎症及气道重塑的变化，探讨基因靶向干预的分子机制，为哮喘基因靶向治疗提供实验依据。

CD4+T细胞与气道平滑肌(ASM)细胞的交叉串扰是导致哮喘气道炎症及重塑的重要原因，但其机制尚不明确。TNF超家族新成员LIGHT（TNFSF14）能激活共刺激途径、诱导炎症应答；并在气道重塑中起作用。因此，探讨LIGHT在CD4+T细胞-ASM细胞交叉串扰中的作用对哮喘的诊治研究有重要意义。.本项目分别从动物和临床实验两方面开展研究。我们首先构建了小鼠慢性哮喘模型，比较了哮喘鼠与对照鼠间气道内LIGHT、HVEM及LTβR的蛋白及mRNA表达差异，证实以上三种分子在哮喘鼠气道内均表达升高。同时，布地奈德雾化干预能显著降低哮喘气道的LIGHT及HVEM水平，并与肺泡灌洗液中IL-6浓度呈正相关。因此，LIGHT/HVEM/LTβR通路可能在哮喘气道炎症中起重要作用。随后，我们从哮喘小鼠的气道中提取了气道平滑肌细胞，从脾脏中分离出T淋巴细胞，分别进行分离纯化后，进行体外共培养（直接或间接接触），分别加入LIGHT单抗或LIGHT单抗+HVEM单抗干预72小时。随后对比了不同干预情况下，干预孔与对照孔间HVEM、LTβR、NF-κB、JNK、c-JUN蛋白、基因表达差异；ASM细胞增殖、凋亡、迁移的差异；CD4+T细胞分化为Th1、Th2、Th17、Treg比例的变化；多种细胞因子释放浓度的差异。结果证实，拮抗LIGHT表达能通过HVEM-NF-κB/c-JUN通路抑制ASM细胞增殖、促进细胞凋亡、延缓细胞迁移；抑制CD4+T细胞向Th1、Th2、Th17分化；并能减少多种细胞因子释放（IL-13、TGF-β及IFN-γ），进而影响ASM细胞增殖、CD4+T细胞分化。同时，靶向沉默LIGHT的shRNA重组载体雾化干预，能降低哮喘鼠的气道炎症水平及ASM厚度。本项目还进行了临床研究的扩展，包括FeNO在不同严重程度哮喘患者中的浓度变化；FeNO在老年ACOS及其他慢性气道炎症疾病的差异；哮喘患者FeNO水平与外周血LIGHT浓度的相关性分析等。结果证实，LIGHT浓度与哮喘嗜酸性粒细胞性炎症呈正相关。因此，LIGHT是哮喘CD4+T细胞与ASM细胞交叉串扰的关键环节，可作为气道炎症及重塑的有效靶点，进而为后续哮喘基因治疗研究提供坚实的实验基础和理论依据。本项目已有2篇中文论著在核心期刊发表，3篇英文论著送SCI收录期刊外审，1项实用新型专利获批，并培养硕士研究生3人。