H1R Signaling and Immune Deviation in EAE

描述（由申请人提供）：组胺是一种有效的炎症介质和先天和适应性免疫反应的调节剂，以及自身免疫性疾病，如实验性过敏性脑脊髓炎（EAE），多发性硬化症（MS）的主要自身免疫性模型。EAE是一种中枢神经系统的炎症性脱髓鞘疾病，具有关键的遗传成分。我们确定Bphs是EAE的易感性位点，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，CD4+ T细胞通过产生干扰素-g和白细胞介素-17等细胞因子在MS和EAE中发挥关键作用。易感等位基因（BphsS/H1RS）的T细胞特异性表达允许在H1RKO背景下发生EAE，而耐药等位基因（BphsR/H1RR）以相同方式表达则不允许。H1R等位基因在第三个细胞内环中有三个氨基酸的差异（H1RS: Pro263Val312Pro330; H1RR: Leu263Met312Ser330）。该结构域与信号转导有关，但在蛋白质折叠和运输中也可能很重要，就像其他g蛋白偶联受体（gpcr）的细胞内结构域一样。在转染研究中，H1RR表面表达明显低于H1RS， H1RR在内质网（ER）中表现出细胞内滞留。在Aim 1中，我们将分析导致H1RR等位基因er保留的机制，以及药物伴侣是否可以恢复H1RR表面表达并影响体内疾病。在体外，药物伴侣可以恢复多态gpcr的表面表达和功能，从体内实验中获得的知识可能对gpcr疾病具有广泛的意义。我们也有证据表明，一种功能性的互补机制可以克服BphsR小鼠的内质网保留过程。对近100个自交系小鼠的Hrh1序列数据和Bphs数据进行了整理，发现了表型上为BphsS，但携带H1RR等位基因的菌株。初步的定位研究将该基因座与Hrh1联系起来，因为它能纠正BphsR，我们将该基因称为bphs增强子（Bphse）。SJL/J小鼠表现出另一种hrh1相关表型，自发组胺敏感性（Shs）。我们假设Bphse和Shs存在于与Hrh1相关的功能连锁不平衡（LD）域中，并且可能包含与H1R折叠、运输或信号传导相关的基因。在Aim 2中，我们将使用正向遗传学方法来定位克隆和鉴定Bphse和Shs。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gpcr来说，持续的MAPK激活涉及通过b-阻滞蛋白进行的非g蛋白信号传导。由于我们发现等位基因H1R- ga融合蛋白的g蛋白激活没有差异，这表明H1R信号也可能涉及一种新的非g蛋白b抑制蛋白途径。因此，在Aim 3中，我们将研究H1R下游的非g蛋白信号是否需要EAE的发展。gpcr介导的非g蛋白信号传导是一种新兴的模式，新的药理学正在开发中，以选择性地靶向这些途径。

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.