Defining the roles of an enhancer long non-coding RNA eIncRNA-ID2 in rickettsial pathogenesis and immunity

项目总结 基因表达的精确和动态变化是调节宿主免疫的关键决定因素 微生物病原体。斑点热组中的致病性立克次体引起一些最严重的 人类感染性疾病，以微血管炎症和功能障碍为特征 内皮细胞播散性感染和血管通透性增加导致肺/脑 浮肿。≥200核苷酸的长非编码(Lnc)rna通过 一系列机制及其表达水平的变化现在与这种决定错综复杂地联系在一起 先天免疫反应和细胞免疫反应。作为LncRNAs的一个重要子集，增强子LncRNAs 通过以顺式或反式方式增强蛋白质编码基因(PCG)来实现它们的调节作用。 我们对肺部进行了rna测序，肺是易感疾病的主要受影响的靶器官之一。 感染康氏杆菌的小鼠鉴定179个lncRNAs的上调。通过跟踪分析来实现差异化 启动子相关(Plnc)RNA的增强子(Elnc)基于 组蛋白3位于赖氨酸4(H3K4me1：H3K4me3)和其他基于POLR2A，P300， DNase I超敏位点、CTCF和Hi-3C芯片-序列数据集，我们进一步确定显著更高 活性elncRNA013718及其靶向DNA结合抑制因子2(ID2)在小鼠体内的表达 康氏立克次体感染时的肺、脾和CD8 T细胞。我们的初步发现进一步表明 ElncRNA013718正向调节E蛋白转录的蛋白拮抗剂ID2的表达 免疫系统中T细胞的因子和调节器。因此，我们将elncRNA013718称为elncRNA- ID2和假说elncRNA-ID2：ID2在调节保护性宿主中的作用 立克次体感染期间的免疫力。我们建议通过两个独立但有主题的假设来检验这个假设 相互关联的具体目标。目标1将区分elncRNA-1的细胞类型特异性表达和功能作用 康诺里弧菌和澳大利亚弧菌感染小鼠的宿主肺和脾中的ID2和ID2。 在目标2中，我们将确定全局和细胞特异性干扰对elncRNA- ID2对宿主免疫反应和疾病进展/结果的影响。鉴于细胞免疫的复杂性 ，我们将使用两个独立的和已建立的体内感染模型来密切模拟 人类立克次体病的病理生理学及细胞微生物学和免疫学的前沿研究 确定elncRNA-ID2作为一种新的elncRNA在潜在的分子电路中的调节潜力 调节宿主免疫力。所获得的洞察力将增强我们对特定背景的生理学知识 ElncRNA-ID2在确定宿主对致病性立克次体应答中的作用 新策略的独特切入点，可增强宿主对细胞内微生物感染的免疫力。

PROJECT SUMMARY Precise and dynamic alterations in gene expression are critical determinants of the regulation of host immunity to microbial pathogens. Pathogenic rickettsiae in the spotted fever group cause some of the most severe infectious diseases in humans, characterized by microvascular inflammation and dysfunction attributed to disseminated infection of endothelial cells and increased vascular permeability resulting in pulmonary/cerebral edema. Long non-coding (lnc) RNAs of ≥ 200 nucleotides regulate a panoply of biological responses through an array of mechanisms and changes in their expression levels are now intricately linked to the determination of innate as well as cell-mediated immune responses. As an important subset of lncRNAs, enhancer lncRNAs implement their regulatory roles by enhancing protein coding genes (PCGs) in a cis- or trans-acting manner. We performed RNA-sequencing on the lungs as one of the predominantly affected target organs of susceptible mice infected with R. conorii to identify up-regulation of 179 lncRNAs. Via follow-up analysis to differentiate enhancer (elnc) from promoter-associated (plnc) RNAs based on the ratio of single- versus tri-methylation of histone 3 at lysine 4 (H3K4Me1:H3K4Me3) and other active enhancer signatures based on POLR2A, p300, DNase I hypersensitivity sites, CTCF, and Hi-3C ChIP-Seq datasets, we further determined significantly higher expression of an active elncRNA013718 and its target PCG Inhibitor of DNA binding 2 (ID2) in the mouse lungs, spleen, and CD8+ T-cells during Rickettsia conorii infection. Our preliminary findings further suggest that elncRNA013718 positively regulates the expression of ID2, a protein antagonist of E protein transcription factors and a regulator of T cells in the immune system. Accordingly, we refer to elncRNA013718 as elncRNA- ID2 and hypothesize novel contributory roles for elncRNA-ID2:Id2 interplay in the regulation of protective host immunity during rickettsial infections. We propose to test this hypothesis via two independent yet thematically interlinked specific aims. Aim 1 will distinguish cell type-specific expression and functional roles of elncRNA- ID2 and ID2 in the host lungs and spleen in experimental murine models of R. conorii and R. australis infection. In Aim 2, we will determine the modulatory effects of both global and cell-specific interference with elncRNA- ID2 on host immune responses and disease progression/outcome. Given the complexity of cellular immune responses, we will employ two independent and established in vivo models of infection closely mimicking the pathophysiology of human rickettsioses and cutting-edge approaches of cellular microbiology and immunology to determine the regulatory potential of elncRNA-ID2 as a novel elncRNA in the molecular circuitry underlying regulation of host immunity. The acquired insights will enhance our knowledge of context-specific physiological roles of elncRNA-ID2 in the determination of host responses to pathogenic rickettsiae and reveal potentially unique entry points for novel strategies to enhance host immunity against intracellular microbial infections.