Mechanistic Characterization of the IBD Risk Gene, PTPN2, as a Novel Susceptibility Marker for Increased SARS-CoV-2 Infection

摘要/摘要 2019 年冠状病毒病 (COVID-19) 全球大流行已在 220 年影响了超过 7000 万人 国家（www.who.int）。 COVID-19 的一个主要临床混杂因素是缺乏对宿主因素的了解 促进某些患者对 SARS-CoV-2 感染的易感性和更严重的胃肠道症状。 此外，还缺乏减轻这些风险的干预措施。我们的初步研究表明 对多种肠道疾病易感性的遗传标记，rs1893217 功能丧失的 PTPN2 变体， 炎症性肠病肠道活检中 SARS-CoV-2 受体 ACE2 水平升高 （炎症性肠病）患者。我们假设携带 PTPN2 功能丧失变异的个体可能更容易 易受 SARS-CoV-2 感染。我们已经在功能上验证了 IBD 的初步发现 PTPN2 基因活性丧失导致 ACE2 表达和 SARS-Cov-2 增加的患者 使用体外和/或体内模型，刺突蛋白进入细胞。我们的总体目标是 从机制上确定 PTPN2 活性丧失如何通过上调促进 SARS-CoV-2 细胞进入 ACE2 表达的影响，以及临床批准的 JAK 抑制剂托法替尼是否可以减轻这种敏感性。 目标 1 将解决 PTPN2 如何限制 ACE2 和其他宿主病毒进入辅助因子的表达，以发挥功能 限制病毒进入 PTPN2 降低或表达 IBD 风险的人肠上皮细胞 (IEC) 系 PTPN2 变体 rs1893217，以及来自 Ptpn2 缺陷小鼠的肠样蛋白。目标 2 将确定 PTPN2 如何 IEC 的缺乏会改变小鼠适应的 SARS-CoV-2 以及肠道细菌感染的严重程度 与 COVID-19 患者腹泻症状相关的结果。目标 3 将机械地确定 JAK 是否 抑制剂托法替布可以在体外、体内使 ACE2 水平正常化，以减少病毒进入 IEC、人类和小鼠体内 肠类、Ptpn2 缺陷小鼠和来自 PTPN2 基因型 IBD 患者的细胞。我们还将确定托法替尼是否 可以改变 IBD 患者的 ACE2 水平。我们已经建立了新的小鼠品系和体外模型系统 这项研究。我们将在一系列创新和成熟的方法中使用这些模型系统，使我们能够 从机制上定义 PTPN2 对 SARS-CoV-2 和上皮细胞之间基本相互作用的调节 病毒进入并随后导致 COVID-19 所需的细胞。这些实验代表了 令人兴奋的新方向，可以协同调查团队的专业知识。这些研究的结果是 有望在识别以下方面取得重大进展：1）遗传风险变异如何与多种因素高度相关 NIDDK 疾病会增加对 SARS-CoV-2 感染的易感性； 2) SARS-CoV-2 如何破坏上皮细胞 促进腹泻的完整性和体内平衡； 3) 如何将已批准的 IBD 治疗药物重新用于 减轻这些风险。因此，这项研究的临床意义包括确定一种新机制 可以通过现有的 JAK 抑制剂进行预防性或治疗性给药以及基因筛查 进行 SARS-CoV-2 疫苗试验的个体。

SUMMARY/ABSTRACT The global coronavirus disease 2019 (COVID-19) pandemic has affected over 70 million individuals in 220 countries (www.who.int). A major clinical confounder of COVID-19 is the lack of knowledge of host factors that promote susceptibility to SARS-CoV-2 infection and more severe gastrointestinal symptoms in some patients. Moreover, there is also a lack of interventions to mitigate these risks. Our preliminary studies identified that a genetic marker of susceptibility to several intestinal diseases, the rs1893217 loss-of-function PTPN2 variant, increased levels of the SARS-CoV-2 receptor, ACE2, in intestinal biopsies from inflammatory bowel disease (IBD) patients. We hypothesize that individuals harboring PTPN2 loss-of-function variants may be more susceptible to SARS-CoV-2 infection. We have already functionally validated our initial findings in IBD patients that loss of activity of the PTPN2 gene results in increased ACE2 expression and SARS-Cov-2 spike protein cellular entry, using in vitro and/or in vivo models. Our overall objective will be to mechanistically determine how PTPN2 loss-of-activity promotes SARS-CoV-2 cellular entry through upregulation of ACE2 expression, and if this susceptibility can be mitigated by the clinically approved JAK inhibitor, tofacitinib. Aim 1 will address how PTPN2 restricts expression of ACE2, and other host virus entry co-factors, to functionally restrict virus entry in human intestinal epithelial cell (IEC) lines with reduced PTPN2 or expressing the IBD risk PTPN2 variant rs1893217, as well as enteroids from Ptpn2-deficient mice. Aim 2 will o determine how PTPN2 deficiency in IECs alters the severity of infection with a mouse-adapted SARS-CoV-2, as well as intestinal outcomes relevant to diarrheal symptoms in COVID-19 patients. Aim 3 will mechanistically determine if the JAK inhibitor, tofacitinib can normalize ACE2 levels in vitro, in vivo, to reduce virus entry in IEC, human and mouse enteroids, Ptpn2-deficient mice, and cells from PTPN2-genotyped IBD patients. We will also identify if tofacitinib can modify ACE2 levels in IBD patients. We have established novel mouse lines and in vitro model systems for this study. We will use these model systems in a series of innovative and established approaches, to allow us to mechanistically define PTPN2 regulation of the fundamental interactions between SARS-CoV-2 and epithelial cells that are required for virus entry and subsequently cause COVID-19. These experiments represent an exciting new direction that synergizes the expertise of the investigative team. The results from these studies are poised to generate significant advances in identifying 1) how a genetic risk variant of high relevance to several NIDDK diseases can increase susceptibility to SARS-CoV-2 infection; 2) how SARS-CoV-2 can disrupt epithelial integrity and homeostasis to promote diarrhea; 3) how an approved IBD therapeutic can be repurposed to mitigate these risks. Therefore, the clinical implications of this study include identifying a novel mechanism that can be targeted by existing JAK inhibitors for prophylactic or therapeutic administration, and genetic screening of individuals for SARS-CoV-2 vaccine trials.