Regulation of Nfil3 in innate lymphocyte-mediated host immunity

摘要 先天性淋巴细胞代表了宿主对病原体免疫的早期哨兵。研究得最好的 先天性淋巴细胞是自然杀伤（NK）细胞，其特异性靶向病毒感染的宿主细胞。人类 缺乏NK细胞的人由于对疱疹病毒感染的易感性而具有严重的健康并发症。此外 对于NK细胞，最近已经证明先天淋巴样细胞（ILC）家族在以下方面发挥重要作用： 保护屏障表面如肠、肺和皮肤免受病原体感染。我们的长期 目标是了解这些先天性的发展背后的分子机制， 淋巴细胞，这将增加我们对这些细胞在感染过程中的功能的理解。为此， 我们最近发现了转录因子Nfil3（也称为E4BP4）在发育中的新作用， NK细胞和ILC家族的所有成员。利用小鼠病原体精确模拟人类传染病 疾病，沿着新产生的转基因小鼠模型，其消除Nfil3基因或 进化上保守的调控结构域，这个R01赠款提出调查Nfil3是如何控制 NK细胞和ILC的发育和抗病原体功能。在目标1中，我们将研究Nfil3是如何 通过染色质免疫沉淀（ChIP）在先天性淋巴细胞祖细胞中转录调节， 1、TCF-1和Ets-1，随后进行qPCR或深度测序（seq）。在目标2中， 将使用两种新的小鼠品系，其中含有CRISPR/Cas9靶向的保守非编码缺失， 序列（称为Nfil3 CNS1和CNS2），以确定内含子调控区对先天性 淋巴细胞发育和抗病毒、蠕虫和细菌感染的效应子功能。在目标3中，我们 使用ChIP-seq，RNA-seq和质谱法来确定Nfil3控制和结合的基因 影响Nfil3介导的这些靶基因调控的伴侣。总之，本提案中的研究 这不仅将增加我们对NK细胞和ILC的一般分子机制的理解， 在病原体入侵期间发展并有助于宿主防御，而且还建立了新的临床范例 如何利用先天淋巴细胞区室进行抗感染的治疗策略， 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.