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

摘要/摘要 2019年全球冠状病毒病(新冠肺炎)大流行已影响220名患者中的7000多万人 国家/地区(www.hor.int)。新冠肺炎的一个主要临床混淆因素是缺乏对宿主因素的了解 增加一些患者对SARS-CoV-2感染和更严重的胃肠道症状的易感性。 此外，也缺乏缓解这些风险的干预措施。我们的初步研究发现， 几种肠道疾病易感性的遗传标记，rs1893217功能丧失的PTPN2变体， 炎症性肠病患者肠道活检组织中SARS-CoV-2受体ACE2水平升高 (IBD)患者。我们假设携带PTPN2功能丧失变异体的个体可能比 容易感染SARS-CoV-2。我们已经在功能上验证了我们在IBD上的初步发现 患者PTPN2基因活性丧失导致ACE2表达增加和SARS-CoV-2 使用体外和/或体内模型，刺激性蛋白进入细胞。我们的总体目标将是 从机制上确定PTPN2失活如何通过上调促进SARS-CoV-2细胞进入 如果这种敏感性可以被临床批准的JAK抑制剂tofacitinib缓解。 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变异体rs1893217，以及ptpn2基因缺陷小鼠的肠样组织。目标2将决定PTPn2如何 IECS缺陷改变了小鼠适应的SARS-CoV-2感染的严重程度，以及肠道 新冠肺炎患者的结果与腹泻症状相关。AIM 3将机械地确定JAK是否 抑制剂托法替尼在体外和体内均可使ACE2水平正常化，以减少病毒在IEC、人和小鼠中的侵入 肠样细胞、PTPN2缺陷小鼠和来自PTPN2基因分型的IBD患者的细胞。我们还将确定tofacitinib是否 可以改变IBD患者的ACE2水平。我们已经建立了新的小鼠系和体外模型系统 这项研究。我们将在一系列创新和既定的方法中使用这些模型系统，以使我们能够 对SARS-CoV-2与上皮细胞之间基本相互作用的PTPN2调节的机械定义 病毒进入并随后导致新冠肺炎所需的细胞。这些实验代表了一种 令人振奋的新方向，协同调查团队的专业知识。这些研究的结果是 有望在识别1)与几个基因高度相关的遗传风险变量方面取得重大进展 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 完整性和动态平衡以促进腹泻；3)已批准的IBD治疗方法如何改变用途 缓解这些风险。因此，这项研究的临床意义包括确定一种新的机制， 可以被现有的JAK抑制剂靶向用于预防性或治疗性给药和基因筛查 参加SARS-CoV-2疫苗试验的个人。

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.