Elucidating mechanisms of fibrosis associated with the Crohns disease-associated pathogenic variant in the metal transporter ZIP8

项目摘要 炎症性肠病(IBD)，包括克罗恩病(CD)和溃疡性结肠炎，每200人中就有1人受到影响 美国人。抗肿瘤坏死因子-α疗法仍然是患者治疗的基石 IBD，但大约40%的患者将经历原发或继发性反应丧失，并且 在目前的临床实践中，有肿瘤坏死因子经验的患者不太可能对随后的所有治疗产生反应。 单细胞RNA测序发现了与抗肿瘤坏死因子不耐药相关的关键细胞特征 强烈暗示炎症巨噬细胞和活化的成纤维细胞之间的信号转导。那里仍然有一个 我们对导致这种异常巨噬细胞的患者特有因素的理解存在重大差距- 成纤维细胞激活，它如何与抗肿瘤坏死因子无反应联系，以及调节它改变为 病程和治疗反应。我们的工作集中在一种致病因子的功能含义上。 金属转运蛋白ZIP8 A391T的变异，它失调了锰的动态平衡，并与 复杂性(狭窄和穿透性)克罗恩病。在小鼠模型(ZIP8)中研究ZIP8 391-Thr 我们对结肠炎的易感性增加了，但更有趣的是，我们发现 观察到纤维化增强。这些小鼠还表现出显著的诱导IL-11，一种促炎症和促- 纤维化细胞因子是炎性巨噬细胞和激活的巨噬细胞之间异常信号传递的标志 克罗恩病患者的成纤维细胞--尤其是抗肿瘤坏死因子难治性疾病。因此，我们 假设研究异常的巨噬细胞-成纤维细胞信号是疾病发病机制的关键特征。 ZIP8 393T-Ki小鼠。建立潜在的疾病机制很重要，因为(1)高达25% 部分人群携带ZIP8391-Thr基因；(2)ZIP8393T-KI基因可服务于 作为一种新的机制研究的翻译模型，特别是与抗肿瘤坏死因子无反应相关的机制。我们会 研究这一假说有两个目的：在目标1中，我们将确定ZIP8 393T-Ki是否干扰巨噬细胞的固有功能 免疫反应；在目标2中，我们将比较ZIP8 393T-KI和WT ZIP8诱导的成纤维细胞激活 巨噬细胞。长期目标是确定ZIP8 391-Thr基因型是否具有临床意义 克罗恩病的防治及ZIP8391-Thr相关病理研究 作为阐明锰的动态平衡在人类疾病中的作用的关键机会。此R03应用程序构建自 在ZIP8 393T-KI中建立结肠炎改变的锰稳态和纤维炎症表型的K08 老鼠。这一具有很强翻译相关性的应用程序将提供对ZIP8的关键机械洞察力 基因对巨噬细胞-成纤维细胞信号转导的影响及与锰稳态的相互作用优先考虑 治疗目标，包括抑制STAT3或IL-11，以及作为R01应用一部分的患者研究。

PROJECT ABSTRACT Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis, affect 1 in 200 Americans. Anti-tumor necrosis factor (TNF)-alpha therapies remain the cornerstone of therapy for patients with IBD, but approximately 40% of patients will experience primary or secondary loss of response, and “anti- TNF-experienced” patients are less likely to respond to all subsequent therapies in current clinical practice. Single-cell RNA sequencing has uncovered key cellular signatures associated with anti-TNF refractoriness that strongly implicate signaling between inflammatory macrophages and activated fibroblasts. There remains a major gap in our understanding of the patient-specific factors that predispose to this aberrant macrophage- fibroblast activation, how it links to anti-TNF unresponsiveness, and the potential to modulate it to change to disease course and treatment response. Our work has focused on the functional implications of a pathogenic variant in a metal transporter, ZIP8 A391T, that dysregulates manganese homeostasis and is associated with complicated (stricturing and penetrating) Crohn’s disease. In studying ZIP8 391-Thr in a mouse model (Zip8 393T-knock-in (KI)), we have shown increased susceptibility to colitis, but even more interestingly, we have observed enhanced fibrosis. These mice also exhibit marked induction of Il-11, a pro-inflammatory and pro- fibrotic cytokine that is a hallmark of aberrant signaling between inflammatory macrophages and activated fibroblasts in patients with Crohn’s disease – particularly anti-TNF refractory disease. We therefore hypothesize that study of aberrant macrophage-fibroblast signaling is a key feature of disease pathogenesis in the Zip8 393T-KI mice. Establishing the underlying disease mechanisms are important because (1) up to 25% of patients with Crohn’s disease carry ZIP8 391-Thr in some populations and (2) the Zip8 393T-KI could serve as a novel translational model for mechanistic studies, particularly related to anti-TNF non-response. We will study this hypothesis in two aims: In Aim 1, we will determine if Zip8 393T-KI perturbs macrophage innate immune responses; in Aim 2, we will compare fibroblast activation induced by Zip8 393T-KI vs. WT Zip8 macrophages. The long-term goal is to establish if ZIP8 391-Thr genotype has clinical implications for the prevention of and treatment of patients with Crohn’s disease and use study of ZIP8 391-Thr-related pathology as key opportunity to elucidate the role of Mn homeostasis in human disease. This R03 application builds from a K08 that established the altered Mn homeostasis and fibroinflammatory phenotype in colitis in Zip8 393T-KI mice. This application with strong translational relevance will provide critical mechanistic insight of the ZIP8 genotypic effect on macrophage-fibroblast signaling and the interaction with Mn homeostasis to prioritize therapeutic targets, including STAT3 or IL-11 inhibition, and patient studies as part of an R01 application.