Distinguishing inflammatory Th17 subsets through using an autoimmune Th17-selective inhibitor

项目摘要 Th17细胞是产生IL-17的辅助性T细胞CD4T细胞的一个亚群，在非霍奇金淋巴瘤的致病过程中起着关键作用。 各种自身免疫性疾病，这些疾病正在影响着2300多万无法治愈的美国人。了解以下内容 Th17细胞的调控机制可能为治疗这些疾病提供新的机会。基于 需要IL-23的发育，目前Th17细胞处于非IL-23的稳态状态 和IL-23依赖的炎性Th17细胞，后者执行生理(清除 细胞外病原体感染)和致病(引发自身免疫)功能。已经做出了重大努力 被用来定义区分稳态和炎症性Th17细胞的基因调控网络。 然而，如何解剖炎性Th17细胞亚群在很大程度上是未知的。这一知识是 非常需要，因为对炎性Th17细胞的非选择性抑制，就像新批准的IL- 23基于单抗的自身免疫性疾病治疗不可避免地增加了对真菌的易感性 和细菌感染。在此，自身免疫和抗感染的Th17亚群对 小分子氯法齐明：在抗感染的同时抑制Th17亚群的自身免疫效应 Th17亚群功能未受影响。我们在这项应用中的总体目标是阐明基因 表达和代谢程序区分自身免疫和抗感染Th17亚群。中环 假设自身免疫和抗感染的Th17亚群是可区分的，SHMT1是一个检查点 自身免疫Th17细胞内丝氨酸水平的变化。这个项目的基本原理是确定 自身免疫和抗感染Th17亚群之间的差异可能提供一个强有力的科学框架 因此，自身免疫性疾病治疗的新策略大大增加了选择性，减少了 可能会产生不良影响。核心假设将通过追求两个具体目标来检验：目标1) 鉴定体内抗感染和自身免疫Th17亚群的基因表达特征；并目的2) 确定SHMT1在自身免疫性Th17细胞内丝氨酸调节中的作用。在目标1下，单一- 细胞RNA-seq分析将利用基于Cas9的基因敲除分析进行验证。对于目标2， 自身免疫Th17细胞的致病性及其细胞内丝氨酸水平将在存在 SHMT1的调节：药物抑制、基因敲除和失活酶的点突变 活动。本申请中提出的研究具有创新性，因为它关注的是监管网络 区分抗感染和自身免疫的Th17亚群，这是一种迄今未被研究的机制。这个 提出的研究具有重要意义，因为它有望为发展自身免疫提供新的机会 TH17-自身免疫性疾病的选择性治疗。这对患者来说非常重要， 已感染或接触某些病原体，如结核病，因为现有的Th17抑制剂不能 由于降低患者对感染的控制的风险而使用。

Project Summary Th17 cells are a subset of IL-17-producing T helper CD4+ T cells and play a pivotal role in the pathogenicity of a variety of autoimmune diseases, which are affecting over 23 million Americans with no cure. Knowledge about regulatory mechanisms of Th17 cells might offer new opportunities for treating these diseases. Based on the requirement of IL-23 for the development, Th17 cells are currently divided into IL-23-independent homeostatic and IL-23-dependent inflammatory Th17 cells, of which the latter execute both physiological (clearing extracellular pathogen infections) and pathogenic (eliciting autoimmunity) functions. Substantial efforts have been made to define gene regulatory networks that discriminate homeostatic and inflammatory Th17 cells. However, how to dissect the subsets of inflammatory Th17 cells is largely unknown. This knowledge is critically needed because non-selective suppression of inflammatory Th17 cells, as by the newly approved IL- 23 monoclonal antibody-based therapy for autoimmune diseases, inevitably increases susceptibility to fungal and bacterial infections. Herein, autoimmune and anti-infection Th17 subsets display differential responses to the small molecule Clofazimine: the autoimmune effect of Th17 subsets was repressed while the anti-infection function of Th17 subsets was not disturbed. Our overall objectives in this application are to elucidate the gene expression and metabolic programs discriminating autoimmune and anti-infection Th17 subsets. The central hypothesis is that autoimmune and anti-infection Th17 subsets are distinguishable and SHMT1 is a checkpoint of intracellular serine levels in autoimmune Th17 cells. The rationale for this project is that determination of the differences between autoimmune and anti-infection Th17 subsets is likely to offer a strong scientific framework whereby new strategies to autoimmune diseases therapy with substantially increased selectivity and reduced adverse effects can be developed. The central hypothesis will be tested by pursuing two specific aims: Aim 1) To identify gene expression signatures of anti-infection and autoimmune Th17 subsets in vivo; and Aim 2) To determine the role of SHMT1 in regulating intracellular serine in autoimmune Th17 cells. Under Aim 1, single- cell RNA-seq analysis will be utilized with verification using Cas9-based knockout assay. For Aim 2, the pathogenicity of autoimmune Th17 cells and their intracellular serine levels will be evaluated in the presence of SHMT1 modulation: pharmacologic inhibition, genetic knockout, and point mutation with inactive enzymatic activities. The research proposed in this application is innovative, because it focuses on the regulatory network discriminating anti-infection and autoimmune Th17 subsets, a heretofore-unexamined mechanism. The proposed research is significant because it is expected to provide novel opportunities to develop autoimmune Th17-selective therapeutics for autoimmune diseases. This would be extraordinarily important for patients that have been infected or exposed to certain pathogens, such as tuberculosis, since existing Th17 inhibitors cannot be used due to the risks of decreasing the patients’ control of the infection.