IL17 dependent angiocrine signaling drives inflammation in alcohol associated hepatitis

项目摘要 酒精性肝炎(AH)的特征是在过量饮酒的背景下发生严重的肝脏炎症和损伤 摄取。细胞因子和趋化因子的上调导致免疫细胞的渗透，并推动炎症反应。 肝窦内皮细胞(LSEC)是肝脏趋化因子表达的重要来源 参与旁分泌信号以吸引免疫细胞，这一过程被称为“血管分泌信号”。路径 对AH肝RNA测序的分析表明，IL17在介导LSEC血管分泌信号中具有潜在的作用。 IL17是一种参与多种自身免疫性和炎症性疾病的细胞因子。我们的初步数据显示， IL-17与肿瘤坏死因子协同刺激CXCL趋化因子的产生，但其机制尚不清楚。 T细胞产生IL17的调控也需要进一步研究。DNA监管是超级增强剂 与靶基因启动子复合以驱动基因表达的元件。我们之前的工作强调了 超级增强剂在急性肝炎炎症反应中的作用。在这里，我们假设LSEC增加 炎性趋化因子的表达与促进免疫细胞向肝实质的迁移 通过超级增强子的激活反应T细胞IL17的上调。为了检验我们的假设，我们将使用 补充细胞生物学和体内方法研究以下特定目的：目的1.LSECs增强 白细胞介素17对依赖于CXCL1的中性粒细胞经内皮细胞迁移的反应；目的2.治疗 靶向T细胞中的超级增强子可下调IL17并缓解急性肝炎的炎症反应。在AIM 1，我们将探讨IκB𝜁，一个先前参与IL17信号转导的转录因子在介导 白介素17和肿瘤坏死因子信号通路的相互作用。我们还将评估IL17的转录变化 测序结果显示，肿瘤坏死因子刺激和IκB𝜁沉默作用于LSEC。利用微流控装置进行模拟 肝脏微血管循环，我们将直接观察IL-17/肿瘤坏死因子刺激和I-κ-B𝜁抑制的效果 关于中性粒细胞的趋化作用。在目标2中，我们将通过染色体构象来鉴定IL17超级增强子 捕获化验。我们将评估CRISPR介导的序列特异性抑制IL17的可行性 超级增强剂。我们将使用依赖Cre的dCas9-KRAb敲门小鼠与AAV6病毒传递单一指南 在体内靶向IL17超级增强子的RNA。我们的目标是实现T细胞特异性抑制IL17的表达 并在小鼠模型上评估其对急性胰腺炎炎症反应的影响。事实上，体内CRISPR基因编辑已经 已经被临床应用于治疗遗传性疾病，突出了精确度的翻译承诺 基因组靶向疗法。更好地了解IL17介导的血管分泌信号转导过程和IL17 超增强子调控可能为治疗AH提供新的治疗靶点。因此，我们的总体目标是 和方法与NIAAA进一步了解和治疗酒精的使命一致- 相关的肝病。

Project Abstract Alcoholic hepatitis (AH) is characterized by intense liver inflammation and injury in the setting of excess alcohol ingestion. Cytokine and chemokine upregulation leads to immune cell infiltration and drives inflammation in AH. Liver sinusoidal endothelial cells (LSEC) are an important source of chemokine expression in the liver and participate in paracrine signaling to attract immune cells in a process termed “angiocrine signaling”. Pathway analysis of AH liver RNA-sequencing suggests a potential role of IL17 in mediating LSEC angiocrine signaling. IL17 is a cytokine involved in many autoimmune and inflammatory disorders. Our preliminary data shows that IL17 synergically stimulate CXCL chemokine production with TNF, but the underlying mechanism is not clear. The regulation of IL17 production from T cells also requires further study. Super enhancers are DNA regulatory elements that complex with target gene promoters to drive gene expression. Our previous work has highlighted the role of super enhancers in AH inflammation response. Here, we hypothesize that LSECs increase inflammatory chemokine expression and enhance immune cell transmigration into the liver parenchyma in response to T cell IL17 upregulation by super enhancer activation. To test our hypothesis, we will employ complementary cell biologic and in vivo approaches to study the following specific aims: Aim 1. LSECs enhance CXCL1-dependent neutrophil transendothelial migration in response to IL17; Aim 2. Therapeutic targeting of a super enhancer in T cells downregulates IL17 and ameliorates inflammation in AH. In Aim 1, we will explore the role of IκB𝜁, a transcription factor previously implicated in IL17 signaling, in mediating the interaction between IL17 and TNF signaling pathways. We will also assess the transcriptomic changes of IL17 and TNF stimulation and IκB𝜁 silencing on LSECs by RNA-sequencing. Using microfluidic devices to simulate liver microvascular circulation, we will directly observe the effects of IL17/TNF stimulation and IκB𝜁 inhibition on neutrophil chemotaxis. In Aim 2, we will identify the IL17 super enhancer by chromosome conformation capture assays. We will assess the feasibility of CRISPR-mediated sequence-specific suppression of the IL17 super enhancer. We will use a Cre dependent dCas9-KRAB knockin mice with AAV6 viral delivery of single guide RNA to target the IL17 super enhancer in vivo. We aim to achieve T cell-specific suppression of IL17 expression and assess its effect on AH inflammatory response in a murine model. Indeed, in vivo CRISPR gene-editing has already been applied clinically to treat hereditary disorders, highlighting the translational promise of precision genome-targeting therapies. Better understanding of the IL17 mediated angiocrine signaling process and IL17 super enhancer regulation may reveal novel therapeutic targets for treatment of AH. Therefore, our overall aims and approaches are aligned with the mission of NIAAA to further understanding and treatment of alcohol- associated liver diseases.