IgE在肥大细胞与2型固有淋巴样细胞活化及其相互影响介导过敏性哮喘中的作用

Allergic asthma is an IgE-mediated, mast cell (MC)-involved type 2 immune response. MC communicates with other cells through numerous surface molecules (such as IgE receptors: FcεRI), active mediators, and exosomes. The cross talk between MC and ILC2 has drawn recent attention, but little is known about the manner and nature of their interactions. Our group found that ILC2 from allergic asthmatic mice expressed IgE receptors. Normal allergic asthmatic mice produce significantly more Th2 cytokines than FcεRI-/- mice do. Therefore, it is speculated that ILC2 could be sensitized by IgE through FcεRI and that it interact with MC, thus enhance allergic inflammation. The subject is based on a mouse model of dust mite allergic asthma (wild-type and FcεRI-/-as well as ST2-/- mice as control); the objective is to①analyze mice and the cells of mouse bone marrow-derived MC and lung ILC2 response to IgE/non-IgE/IL-33, including asthma symptoms, tissue inflammation, degranulation, cytokine secretion, proliferation, chemotaxis, molecule phosphorylation of signal pass way, redox state and other changes.②observe and analyze the impact and manner of interaction between MC and ILC2. The aim is to clarify the role of IgE in the activation of MC and ILC2 and their interaction, and deepen the understanding of regional immune response to allergic asthma, hence providing evidence for disease treatment that uses IgE and key signaling molecules as target.

过敏性哮喘是IgE介导、肥大细胞（mast cell，MC）通过IgE受体（FcεRI）和其它表面分子、活性介质或外泌体与其他细胞相互作用所致的2型免疫反应。新近MC与ILC2的相互作用受到关注，但作用方式与性质所知甚少。课题组发现，过敏哮喘鼠ILC2表达FcεRI、2型细胞因子分泌较FcεRI-/-鼠增多。故推测，ILC2通过FcεRI接受IgE致敏、与MC相互作用，增强过敏炎症。本课题拟建立尘螨过敏哮喘鼠模型（FcεRI-/-和ST2-/-鼠对照）①分析小鼠及其骨髓MC和肺ILC2对IgE/非IgE/IL-33作用的应答：哮喘症状、组织炎症，脱颗粒、活性因子分泌、增殖、趋化、信号分子磷酸化和氧化还原状态等的变化；②观察分析MC与ILC2相互作用及其方式。以期明确IgE在MC和ILC2活化及其相互作用中的地位，深化对过敏哮喘区域免疫应答的认识，为以IgE和关键信号分子为靶点的治疗提供依据

方法：1.体外诱导小鼠骨髓获得肥大细胞（BMMC），差速离心提取胞外囊泡（MC-Evs），构建并分析静息和活化MC、MC-Evs的蛋白组与全转录组及其调控网络。.2.以上分析发现调控分子ORMDL3和ELK4,敲减ORMDL3和敲除ELK4探讨对BMMC脱颗粒和CKs产生的影响。3.IgE/Ag或C48/80刺激BMMC分析其连续应答能力。流式检测嗜碱性粒细胞膜分子，建立嗜碱粒细胞活化检测（BST）体系，提供过敏的功能检测方法。4.与尘螨过敏原（DfE）共育获得 BMMC-EVs-DfE。将其鼻腔免疫DfE过敏哮喘小鼠，肺组织HE和免疫组化、支气管肺泡灌洗液（BALF）细胞计数、检测细胞因子（CK）和血清抗体了解疾病变化及其机制5. 分析人银屑病（PS）和溃疡性结肠炎（UC）组织MC数量与疾病分期、分级的相关性。建立皮损和结肠炎模型，观察FcεR1α缺陷和野生型小鼠病变程度，明确肥大细胞的作用。.结果：1.活化前后MC差异表达mRNA 1655个（上、下调657、998个）。6444个蛋白中活化组47个高表达，36个低表达。MC-EVs转录组起调控作用的lncRNA 11个、circRNA 2个。2.IgE/Ag刺激MC活化后失去了再次应答能力。3.BMMC-EVs-DfE 免疫可减少哮喘小鼠BALF中巨噬、嗜酸细胞和细胞总数，显着降低肺内杯状细胞数量和MUC5AC、DfE-IgE和组胺水平，通过增加IFN-γ和降低IL-4表达调节Th1/Th2平衡。4.ORMDL3过表达可上调 ERS-UPR（SERCA2b、ATF6）和自噬（Beclin 1 和 LC3BII）相关因子表达以及脱颗粒，下调CK/趋化因子表达，后者可被 ATF6 的敲低和自噬抑制而逆转。 敲除Elk4可抑制细胞增殖并阻碍BMMC的细胞周期，降低CK和趋化因子的转录激活，但MC脱颗粒增强。5.人PS和UC的组织MC数量显著增多，与病情正相关。FcεR1α缺陷鼠银屑病和结肠炎模型的病变程度较野生鼠轻。.结论及意义：1.提供了MC与MC-EVs激活前后差异表达的蛋白组、转录组数据库，2.证实IgE和MC在自身免疫病中的作用，发现了MC重要调控因子ORMDL3和ELK4及其机制，为干预MC相关疾病提供了潜在靶点。3.MC-EVs-DeF的免疫耐受诱导，有望成为过敏原疫苗，4.BAT提供了过敏的功能检测方法