H1R Signaling and Immune Deviation in EAE

描述(申请人提供)：组胺是一种有效的炎症介质，并调节先天和适应性免疫反应，以及自身免疫性疾病，如实验性变态反应性脑脊髓炎(EAE)，多发性硬化症(MS)的主要自身免疫模型。EAE是一种中枢神经系统炎症性脱髓鞘疾病，具有关键的遗传成分。我们确定BPHS是EAE的易感基因，定位候选基因克隆证实BPHS是组胺H1受体(HRH1/H1R)。与许多自身免疫性疾病一样，CD4T细胞通过产生干扰素-g和白介素17等细胞因子在MS和EAE中发挥关键作用。易感(BphsS/H1RS)等位基因的T细胞特异性表达允许在H1RKO背景下发生EAE，而抗病(BphsR/H1RR)等位基因的表达不允许以相同的方式表达。H1R等位基因在第三个细胞内环上有三个氨基酸的差异(H1RS：Pro263Val312Pro330；H1RR：Leu263Met312Ser330)。这个结构域与信号转导有关，但也可能在蛋白质的折叠和运输中发挥重要作用，就像其他G蛋白偶联受体(GPCRs)的胞内结构域一样。在转染研究中，H1RR的表面表达显著低于H1RS，H1RR显示内质网(ER)的细胞内滞留。在目标1中，我们将剖析(S)导致H1RR等位基因ER滞留的机制，以及药物伴侣是否能够恢复H1RR表面的表达并在体内影响疾病。药理伴侣在体外恢复了多态GPCRs的表面表达和功能，从拟议的体内实验中获得的知识可能对GPCR病具有广泛的意义。我们也有证据表明，一种功能互补的机制可以克服BphsR小鼠的内质网滞留过程。收集了100个近交系小鼠的HRH1序列数据和BPHS数据，发现这些品系表型为BphsS，但携带H1RR等位基因。初步的定位研究将该基因与HRH1联系起来，因为它纠正了BphsR，所以我们将这个基因称为BPHS-增强子(Bphse)。SJL/J小鼠表现出另一种与HRH1相关的表型，即自发性组胺敏感性(SHS)。我们假设Bphse和SHS位于与HRH1连锁的功能连锁不平衡(LD)结构域中，可能包含与H1R折叠、运输或信号转导相关的基因。在目标2中，我们将使用正向遗传学方法对Bphse和SHS进行定位克隆和鉴定。H1R信号通过p38丝裂原活化蛋白激酶(MAPK)在EAE易感性中起重要作用。对于许多GPCRs来说，持续的MAPK激活涉及到非G蛋白通过b-拦阻蛋白的信号传递。由于我们没有发现等位基因H1R-Ga融合蛋白在G蛋白激活方面的差异，这表明H1R信号可能还涉及一条新的非G蛋白b-arrestin途径。因此，在目标3中，我们将研究H1R下游的非G蛋白信号是否是EAE发展所必需的。通过GPCRs的非G蛋白信号转导是一个新兴的范例，正在开发新的药理学来选择性地针对这些途径。 公共卫生相关性：G蛋白偶联受体(GPCRs)，如组胺H1受体(H1R)，其功能可以通过它们的亚细胞定位和它们引发的信号通路来调节。GPCRs的正确折叠和细胞表面表达是配体结合和信号传递所必需的。导致不正确折叠和/或细胞内转运的突变构成了导致疾病的最大类别的GPCR突变。我们的发现表明，控制自身免疫性疾病易感性的H1R等位基因表现出细胞表面的差异表达和细胞内转运的改变，而抗性等位基因保留在内质网内，这是影响GPCR转运和细胞表面表达的多态可以调节免疫功能的第一个证据。了解导致H1R等位基因转运和细胞表面表达差异的机制，以及如何利用这种自然发生的小鼠模型在体内操纵它们，无疑将有助于开发新的疾病治疗策略，在这些疾病中，获得或丧失功能突变导致GPCR错误折叠和/或不正当的细胞内转运。

DESCRIPTION (provided by applicant): Histamine is a potent mediator of inflammation and regulator of innate and adaptive immune responses, and autoimmune diseases such as experimental allergic encephalomyelitis (EAE), the principal autoimmune model of multiple sclerosis (MS). EAE is an inflammatory demyelinating disease of the central nervous system with a critical genetic component. We identified Bphs as a susceptibility locus for EAE and positional candidate gene cloning demonstrated Bphs to be the histamine H1 receptor (Hrh1/H1R). As in many autoimmune diseases, CD4+ T cells play a key role in MS and EAE through production of cytokines such as interferon-g and interleukin-17. T cell-specific expression of the susceptible (BphsS/H1RS) allele allowed for EAE development on a H1RKO background, while expression of the resistant (BphsR/H1RR) allele in the same fashion did not. H1R alleles differ by three amino acids in the third intracellular loop (H1RS: Pro263Val312Pro330; H1RR: Leu263Met312Ser330). This domain is associated with signal transduction, but may also be important in protein folding and trafficking, like the intracellular domains of other G-protein coupled receptors (GPCRs). In transfection studies, H1RR surface expression was substantially lower than H1RS, with H1RR exhibiting intracellular retention in the endoplasmic reticulum (ER). In Aim 1 we will dissect the mechanism(s) responsible for the ER-retention of the H1RR allele and whether pharmacological chaperones can restore H1RR surface expression and affect disease in vivo. Pharmacological chaperones restore surface expression and function of polymorphic GPCRs in vitro, and knowledge gained from the proposed in vivo experiments may have broad implications for GPCR-diseases. We also have evidence of a functional, complementary mechanism that can overcome the ER retention process in BphsR mice. Compilation of Hrh1 sequence data and Bphs data from ~100 inbred mouse strains, revealed strains that were phenotypically BphsS, but bear the H1RR allele. Preliminary mapping studies linked the locus to Hrh1 and because it corrects BphsR, we refer to this gene as Bphs-enhancer (Bphse). SJL/J mice exhibit another Hrh1-linked phenotype, spontaneous histamine sensitivity (Shs). We hypothesize that Bphse and Shs reside within a functional linkage disequilibrium (LD)-domain linked to Hrh1 and may contain genes related to H1R folding, trafficking, or signaling. In Aim 2 we will use a forward genetics approach to positionally clone and identify Bphse and Shs. H1R signaling through p38 mitogen activated protein kinase (MAPK) is important in EAE susceptibility. For many GPCRs sustained MAPK activation involves non-G-protein signaling through b-arrestins. Since we found no difference in G-protein activation of allelic H1R-Ga fusion proteins, this suggested that H1R signaling may also involve a novel non-G-protein b-arrestin pathway. Thus in Aim 3 we will examine whether non-G-protein signaling downstream of the H1R is required for EAE development. Non-G-protein signaling by GPCRs is an emerging paradigm and new pharmacologics are being developed to selectively target these pathways. PUBLIC HEALTH RELEVANCE: The function of G-protein coupled receptors (GPCRs) such as the histamine H1 receptor (H1R) can be regulated by their subcellular localization and the signaling pathways they elicit. Proper folding and cell surface expression of GPCRs is required for ligand binding and signaling. Mutations that lead to improper folding and/or intracellular trafficking comprise the largest class of GPCR mutations that result in disease. Our finding that H1R alleles controlling susceptibility to autoimmune disease exhibit differential cell surface expression and altered intracellular trafficking, with the resistant allele being retained within the endoplasmic reticulum, was the first demonstration that polymorphisms influencing GPCR trafficking and cell surface expression can regulate immune functions. Understanding the mechanisms leading to differences in the trafficking and cell surface expression of the H1R alleles, and how they can be manipulated in vivo using this naturally occurring mouse model, will undoubtedly aid in the development of new therapeutic strategies for diseases in which gain- or loss-of-function mutants leading to GPCR misfolding and/or improper intracellular trafficking are implicated.