3型固有淋巴细胞介导中性粒细胞性哮喘的实验研究

Severe asthma or difficult-to-treat asthma is predominantly induced by neutrophilic airway inflammation, which has been reported to be associated with the type 3 cytokines including IL-17 secreted by type 17 helper T-cells (Th17). However the precise mechanisms under the pathogenesis of neutrophilic asthma are still unknown. Innate lymphoid cells (ILCs) are a newly recongized immune regulatory cells without T or B lymphocytes antigen-specific receptors dependent on Rag genes, of which ILC2 has been found to involve the development of eosinophilic asthma. ILC3 preserves similar morphological and functional properties with Th17, but researches of roles of ILC3 in asthma airway inflammation are scarce. Recent studies found that ILC3 induced airway inflammation in obesity patients with asthma via IL-17A expression suggesting potential roles of ILC3 in neutrophilic airway inflammation development. Based on the lack of evidence in ILC3 and neutrophilic asthma, this study will establish neutrophilic or eosinophilic asthmatic model in different genes deficiency mice with to 1) measure the distribution of ILC3 and other immune cell types as well as the relevant cytokines expression levels in different asthma airway inflammation phenotypes, 2) verify the extracellular (TLR—NFκB/SAA—NLRP3—Caspase-1) and intracellular (IL-1βR—JAK/STAT—AhR—IL-17) signal pathways in ILC3 activation and neutrophilic airway inflammation induction, and 3) investigate the effects of ILC3 on triggering neutrophil related chemokines secretion by airway epithelial cells.The study results may,with reasonable possibilities, identify novel treatment targets in severe asthma.

重度哮喘或难治性哮喘常常表现为中性粒细胞性气道炎症，现有研究发现与Th17细胞所介导的获得性免疫反应关系密切，但具体发生机制尚不够明了。固有淋巴样细胞（ ILCs）是新近发现的一种非T非B细胞，已发现ILC2参与嗜酸粒细胞性哮喘的调控。ILC3与哮喘的相关研究不多，近期有研究显示在肥胖哮喘患者ILC3可分泌IL-17A，诱导中性粒细胞气道炎症。本课题拟采用野生型及各种免疫缺陷型小鼠，制作中性粒细胞性哮喘动物模型，测定ILC3及其他免疫细胞和相关细胞因子在不同哮喘气道炎症表型中的表达水平，以明确ILC3在中性粒细胞性哮喘的作用；通过体内外实验研究ILC3的活化及介导中性粒细胞性气道炎症的细胞内外信号传导途径；观察ILC3与气道上皮细胞的相互作用及对中性粒细胞的趋化作用。本研究的结果有可能揭示3型固有淋巴样细胞在中性粒细胞性哮喘中的作用，为重度哮喘的治疗探索新的靶点。

固有淋巴细胞（Innate lymphoid cells，ILC）是近年来发现的一类非T、非B淋巴样细胞，目前大多数研究集中在2型固有淋巴细胞（ILC2），ILC2与Th2细胞共同调控嗜酸粒细胞性气道炎症。此外尚有约1/3的哮喘属于中性粒细胞炎症，多属于重度哮喘，关于中性粒细胞性哮喘的机制尚未充分阐明，既往认为主要与Th17及其分泌的IL-17A有关。近期有研究显示在肥胖哮喘患者ILC3可分泌IL-17A，诱导中性粒细胞气道炎症。本课题拟通过体内外实验，明确ILC3在中性粒细胞性哮喘中的调控作用。课题组在细胞实验中有以下发现（1）健康人外周血分选出的ILC3细胞经体外与IL-2共培养后主要表现为CD3-CD56+RORγt+ NKp44+亚型，故以该亚型为主要研究细胞。QPCR、ELISA检测及流式细胞内蛋白检测检测到ILC3主要表达IL-22，仅极微量表达IL-17A和IL-17F，在IL-1β+IL-23刺激下IL-22和IL-17F的表达量显著增加。（2）QPCR和ELISA检测发现：在转录和蛋白水平，IL-1β+IL-23刺激组CXCL1和CXCL8表达水平显著高于ILC3未刺激组。（3）ILC3通过IL-1β—NF-κB—p38/JNK MAPK-CXCL8 信号途径释放CXCL8。（4）采用浓缩ILC3细胞培养上清液干预气道上皮细胞株16HBE后CXCL1和CXCL8基因表达量显著增加。（5）ILC3细胞FGF2的基因和蛋白表达量显著高于ILC2，采用FGFR1抑制剂YTH17与ILC3细胞培养上清液共同孵育16HBE细胞，发现YTH17可显著抑制ILC3细胞培养上清液孵育对16HBE细胞的干预效应及中性粒细胞趋化活性。（6）FGF2主要通过FGFR1-JAK2-STAT3通路促进16HBE细胞分泌CXCL1和CXCL8。在动物实验中发现：（1）中性粒细胞性小鼠模型中ILC3比例及其相关细胞因子IL-17A、IL-17F、IL-22、TNFa显著升高。（2）适应性免疫缺陷Rag2-/-小鼠经OVA/LPS致敏-OVA激发造模，仍可出现气道炎性细胞浸润及气道粘液高分泌哮喘等特征。采用抗CD90.2单克隆抗体敲除Rag2-/-小鼠ILC细胞后气道炎症减弱，IL-17A、IL-17F、IL-22表达降低，提示ILC3可不依赖适应性免疫介导中性粒细胞气道炎症