Regulation of Nfil3 in innate lymphocyte-mediated host immunity

摘要 先天淋巴细胞是宿主抵抗病原体免疫的关键早期哨兵。研究最多的人 先天淋巴细胞是自然杀伤(NK)细胞，专门针对病毒感染的宿主细胞。人类 由于对疱疹病毒感染的易感性，NK细胞缺乏会导致严重的健康并发症。此外 对于NK细胞，一类天然淋巴样细胞(ILC)最近被证明在 防止肠道、肺和皮肤等屏障表面的病原体感染。我们的长期计划 目的是了解这些先天发育的分子机制。 淋巴细胞，这将增加我们对这些细胞在感染期间的功能的了解。为此， 我们最近发现了转录因子Nfil3(也称为E4BP4)在发育中的一个新角色 NK细胞和ILC家族的所有成员。使用老鼠病原体准确地模拟人类感染 疾病，以及新产生的去除Nfil3基因或 进化保守的调节域，这一R01拨款建议调查Nfil3是如何控制 自然杀伤细胞和白细胞介素性细胞的发育及其抗病原体功能。在目标1中，我们将调查Nfil3是如何 染色质免疫沉淀(CHIP)对先天淋巴细胞祖细胞转录调控的研究 转录因子STAT5、PU.1、TCF-1和Ets-1，然后进行qPCR或深度测序(Seq)。在目标2中，我们 将使用两个新的小鼠品系，其中包含CRISPR/Cas9靶向删除保守的非编码 序列(称为Nfil3、CNS1和CNS2)，以确定内含子调节区对先天性的影响 淋巴细胞的发育和抗病毒、蠕虫和细菌感染的效应器功能。在《目标3》中，我们将 使用芯片序列、rna-seq和质谱仪来确定nfil3控制和结合的基因。 影响Nfil3介导的对这些靶基因的调控的伙伴。总之，这项提案中的研究 不仅增加了我们对NK细胞和ILC的一般分子机制的了解 在病原体入侵期间发展和促进宿主防御，但也建立新的临床范例 关于如何利用先天淋巴细胞室来治疗感染性疾病 疾病。

ABSTRACT Innate lymphocytes represent early sentinels critical in host immunity against pathogens. The best studied innate lymphocyte is the natural killer (NK) cell, which specifically targets virally-infected host cells. Humans deficient in NK cells have severe health complications due to susceptibility to herpesvirus infections. In addition to NK cells, a family of innate lymphoid cells (ILC) has recently been demonstrated to play an important role in protection against pathogen infection at barrier surfaces such as the intestine, lung, and skin. Our long term goals are to understand the the molecular mechanisms underlying the development of these innate lymphocytes, which will increase our understanding of the function of these cells during infection. To this end, we have recently identified a novel role for the transcription factor Nfil3 (also known as E4BP4) in development of NK cells and all members of the ILC family. Using mouse pathogens that accurately model human infectious diseases, along with newly-generated transgenic mouse models that ablate either the Nfil3 gene or evolutionarily-conserved regulatory domains, this R01 grant proposes to investigate how Nfil3 is controling the development and anti-pathogen functions of NK cells and ILCs. In Aim 1, we will investigate how Nfil3 is transcriptionally regulated in innate lymphocyte progenitors by chromatin immunoprecipitation (ChIP) for the transcription factors STAT5, PU.1, TCF-1, and Ets-1 followed by qPCR or deep sequencing (seq). In Aim 2, we will use two novel mouse strains containing a CRISPR/Cas9-targeted deletion of conserved noncoding sequences (termed Nfil3 CNS1 and CNS2) to determine the influence of intronic regulatory regions on innate lymphocyte development and effector function against virus, helminth, and bacterial infection. In Aim 3, we will use ChIP-seq, RNA-seq, and mass spectrometry to determine the genes that Nfil3 is controlling, and binding partners that influence Nfil3-mediated regulation of these target genes. Together, the studies in this proposal will not only increase our understanding of the general molecular mechanisms whereby NK cells and ILCs develop and contribute to host defense during pathogen invasion, but also establish novel clinical paradigms for how the innate lymphocyte compartment may be harnessed for therapeutic strategies against infectious disease.