Exploring the role and mechanisms of action of UC pouchitis-associated pathobionts (PAP) to gain insights into the etiopathogenesis of Inflammatory Bowel Diseases

项目摘要/摘要 炎症性肠病(IBD)在工业化国家和新兴发展中国家日益受到关注，因为 环境、生活方式和饮食都发生了变化。IBD涉及宿主与宿主之间复杂的相互作用 环境、饮食、遗传和肠道微生物区系，每一个都是必要的，但不足以导致疾病。然而，它 目前尚不清楚是什么引发了这些疾病，这些因素在 IBD的发病机制，以及这些疾病慢性的原因。目前的治疗方法仍然不精确，而且 针对高危患者的预防疾病战略是不存在的。一些证据表明，这种变化 在肠道内，微生物区系可以影响风险并引发IBD。我们的实验室使用了一种人体模型，包括溃烂 结肠炎(UC)患者接受结肠切除，然后建立回肠袋-肛门吻合术(IPAA)。在……里面 理论上，IPAA应该是治愈的，但近一半的患者会发展为回肠炎。 眼袋称为膀胱炎，而接受同样手术的非IBD患者很少发生膀胱炎。 这些患者被纵向跟踪和采样，元基因组数据显示了病原体的大量繁殖。 如在膀胱炎发生前和发展过程中的脆弱类杆菌。关于抗生素诱导的 缓解后，脆弱杆菌的相对丰度下降，这意味着它可能是一种潜在的触发和维持 膀胱炎。所提出的研究策略检验了一种假说，即膀胱炎相关病原体(PAP) 就像脆弱类杆菌引起的IPAA相关性膀胱炎一样。值得注意的是，所有从 UC膀胱炎患者是非产肠毒素菌株，甚至在IPAA出现之前就已经存在 已执行。因此，我们相信通过这些研究获得的洞察力将直接与 人类炎症性肠病的发病机制。作为人类膀胱炎模型的对应物，我们采用了无菌(GF) 以及植入PAP B的SPF(无特定病原体)野生型(WT)和IL10基因敲除(IL10-/-)小鼠。 后者中的脆性纤毛与结肠炎的发病率和严重性增加有关。我会决定B。 脆性肠杆菌通过对结肠炎的成员和功能造成干扰而直接和/或间接促进结肠炎 土生土长的结肠微生物群。SPF，而不是GF，IL10-/-小鼠在遗传上易患IBD。 人PAP B.Fragilis菌株很容易移植到SPF IL10-/-小鼠的微生物区系中，但不能移植到WT小鼠的微生物区系中，并且 促进结肠炎的发展。使用Bulk-RNA测序分析和流式细胞术，我将评估 脆弱芽孢杆菌的存在如何改变宿主的免疫反应和GF胃肠道中的基因表达 和SPF级IL-10-/-小鼠。粪便样本、区域肠腔和粘膜内容物将接受16秒 RRNA、元基因组和代谢组学分析以评估脆弱杆菌是否会导致促进慢性生物失调的疾病 IL10-/-小鼠模型中的结肠炎。这项研究提供了对发展之前的事件的关键洞察力 易患IBD的宿主的慢性炎症。此外，这些研究将揭示潜在的发展潜力。 生物标记物可以预测风险，帮助定义预防策略，并指导治疗管理。

PROJECT SUMMARY/ABSTRACT Inflammatory Bowel Diseases (IBD) are a growing concern in industrialized and newly developing countries as the environment, lifestyles, and diets change. IBD involves a complex interplay between the host's environment, diet, genetics, and gut microbiota, each necessary but insufficient to cause disease. However, it is unclear what initiates these diseases, what the relative contributions of these factors play in the pathogenesis of IBD, and what makes these diseases chronic. Current therapies are still imprecise, and preventative disease strategies for high-risk patients are non-existent. Some evidence suggests that changes in the gut microbiota can influence the risk and trigger IBD. Our lab uses a human model involving ulcerative colitis(UC) patients who undergo colectomy followed by creating an ileal pouch-anal anastomosis (IPAA). In theory, IPAA should be curative, but nearly half of these patients develop an inflammatory condition of the ileal pouch called pouchitis, whereas non-IBD patients undergoing the same procedure rarely develop pouchitis. These patients were followed and sampled longitudinally, and metagenomic data shows blooms of pathobionts such as Bacteroides fragilis before and during the development of pouchitis. Upon antibiotic-induced remission, the relative abundance of B. fragilis decreased, implicating it as a potential trigger and sustainer of pouchitis. The proposed research strategy tests the hypothesis that pouchitis-associated pathobionts (PAP) like Bacteroides fragilis cause in IPAA-associated pouchitis. Notably all the B. fragilis strains cultivated from UC pouchitis patients are non-enterotoxigenic strains that were present even before the IPAA was performed. Thus, we believe that insight gained through these studies will directly relate to the etiopathogenesis of human IBD. As a counterpart to the human pouchitis model, we employ germ-free (GF) and SPF (specific pathogen-free) wild-type (WT) and IL10 knockout (IL10-/-) mice where engraftment of PAP B. fragilis in the latter is associated with increased incidence and severity of colitis. I will determine whether B. fragilis promotes colitis directly and/or indirectly by causing perturbations in the membership and function of the indigenous colonic microbiota. SPF, but not GF, IL10-/- mice are genetically predisposed to developing IBD. Human PAP B. fragilis strains readily engraft into SPF IL10-/- mouse microbiota, but not that of WT mice, and promote the development of colitis. Using Bulk-RNA Sequencing analysis and flow cytometry, I will evaluate how the presence of B. fragilis alters the host's immune responses and gene expression in the GI tract of GF and SPF IL-10-/- mice. Fecal samples, regional intestinal luminal, and mucosal content will be subjected to 16S rRNA, metagenomic, and metabolomic analyses to assess if B. fragilis leads to dysbiosis–promoting chronic colitis in the IL10-/- mouse model. This study provides critical insight into the events prior to the development of chronic inflammation in an IBD-susceptible host. In addition, these studies will shed light on potential biomarkers that can predict risk, help define preventative strategies, and guide therapeutic management.