Ets-2通过控制日本血吸虫感染所致的FceRI阳性细胞分化而在Th2应答及免疫病理中起重要作用

Increased number of cells positive for high affinity receptor for IgE Fc (FceRIa) have been found by us in several Th2 response-associated senarios. First, the number of FceRI-positive cells were greatly increased in the spleen from Schistosoma japonica infection, and these cells demonstrated great capacity of inducing Th2 cells which may contribute to the Th2-dominated liver granuloma and fibrosis seen in schistosomiasis. Second, in addition to IL-3, addition of Th2 antigens including schistosome egg antigens or heat inactivated papain to GM-CSF treated bone marrow cell cultures also induced significant number of FceRI-positive cells while decreased the number of cells positive for CD11c, a marker for conventional dendritic cells. Thus, we predicted that FceRI is a signature molecule for cells promoting Th2 polarization. Except Gata-2, it is not clear regarding to the transcriptional control on the expression of FceRI as well as the development of FceRI-positive cells like basophils and mast cells. Screened by RNASeq and then verified by qRT-PCR on both FcRI-positive cells as well as FceRI-negative dendritic cells isolated from 5 weeks Schistosoma japonica infected spleen, we found that transcriptional factor Ets-2 was expressed at higher levels in all FceRI-positive cell groups with Th2-promoting activities than in dendritic cells which only promotes Th1 cells. The expression levels of Ets-2 in FceRI-positive cells are higher than the levels of Gata-2, a known FceRIa-promoting transcriptional factor. Therefore, we proposed in this study to explore the required role played by Ets-2 in regulation of FceRIa expression as well as in development of FceRI-positive cells. Conditional Ets-2 knockout mice will be made to investigate if Ets-2 is required for the induction of FceRI-positive cells induced by IL-3 or by Th2 antigens. Meanwhile, mixed chimeric mice will be built by adoptively transfer bone marrow cells from both conditional Ets-2 knockout mice and conditional knockout of FceRI-positive cells using FcRI-DTR transgenic mice made by our laboratory at 1:1 ratio. After observing presence of FceRI-positive cells can rescue the reduced Th2 responses due to lack of Ets-2 in schistosoma infected mixed chimeric mice, the pathway that Ets-2 controls schistosome infection induced Th2 responses via induction of bone marrow cells to differentiate into FceRI-positive cells is established. This proposed study will provide the support at the molecular level to apply FceRI as a new target molecule to the treatment of schistosomiasis and other types of type II inflammation associated diseases.

日本血吸虫（Sj）感染鼠脾脏中FceRI+细胞在诱导Th2极化中起重要作用。IL-3及Th2类抗原从骨髓诱导的FceRI+细胞均可促Th2分化。但骨髓前体细胞分化为FceRI+细胞的转录调控机制不清楚。RNASeq筛选发现，除正向调控FceRIa表达的Gata2外，Ets-2在Sj感染脾脏的FceRI+细胞中高表达。qRT-PCR证实Ets-2在FceRI+细胞中的mRNA水平高于Gata2，是FceRI-树突状细胞的2到4倍。本研究拟通过构建条件性Ets-2基因敲除鼠，明确Ets-2在IL-3、Th2类抗原诱导FceRI+细胞产生中为必须；并通过构建来源于条件性Ets-2基因敲除鼠和条件性FceRI清除鼠的骨髓细胞共转输嵌合体，明确Ets-2通过控制骨髓细胞分化为FceRI+细胞，而在Sj感染的Th2应答和肝脏病理中起重要作用。为以FceRI为干预Th2型免疫病理的新靶标提供理论依据。

研究背景：. 作为IgE高亲和力受体的a亚基，FceRIa在 IgE 介导的过敏和其他 IgE 相关疾病的发病机制中起着关键作用。FceRIa通常仅在嗜碱性粒细胞和肥大细胞等有限范围的细胞中表达，但控制这些细胞中FcRI表达的机制尚不清楚。自2010年起。我一直得到国家自然科学基金资助，研究FceRIa阳性细胞群在负责抗蠕虫免疫，过敏反应及组织修复的Th2型免疫应答的启动和效应中是否起着不可或缺的作用。实施过程中，构建的2株FceRIa基因区的基因敲入鼠，均出现基因位点上的基因不表达。由此在这项研究中，我们揭示依赖于该位点序列信息调控FceRIa表达的新机制。发现FceRIa在表达时，其反义转录本FCERIA-AS也共表达，且FCERIA-AS正向调控FceRIa。.研究内容：. 首先构建条件性清除FcRI阳性细胞小鼠模型FceRIa-DTR-II鼠，发现和之前敲入构建的FceRIa-DTR-I一样（81273326），敲入基因仍然不表达。.进而研究FCERIA基因表达是否有反义链调节存在及其机制及体内对过敏应答的调控。.重要结果、关键数据及科学意义：. 在 IL-3 诱导的FceRIa表达细胞或在高FceRIa表达细胞系 MC/9 中，存在FceRIa的完全序列重叠的天然反义转录物FCER1A-AS，并与正义转录本FCER1A-S共表达。当在 MC/9 中通过 CRISPR/RfxCas13d (CasRx) 方法选择性敲除 FCER1A-AS 时，FCER1A-S 的 mRNA和蛋白质的表达也显著降低。此外，在 FCER1A 基因座实行两种不同基因靶向小鼠系中发现了 FCER1A-AS 的缺失伴随着 FCER1A-S 表达的抑制。更重要的是，FCER1A-AS 缺陷型纯合小鼠过敏反应的缺失与 FCER1A 基因敲除小鼠相似。因此我们的研究成果有2个意义，一是发现 FCER1A-S 的表达受共表达的反义转录产物的正向调节，为抗IgE依赖的过敏性疾病提供新药靶；二是提示基因敲入改造时，受反向链调控的基因改造需谨慎。