Influences of the enteric microbiota on intestinal stem cell biology

描述(由申请人提供)：克罗恩病(CD)是一种潜在的胃肠道慢性炎症性疾病。此外，目前治疗这一问题的药物 疾病往往是广谱的免疫抑制剂，有可能产生严重的副作用。因此，有一个强有力的理由来开发毒性较低的CD治疗方案，特别是对于可能接受数十年有效免疫抑制治疗的儿科患者。虽然CD的确切病因尚不清楚，但上皮功能改变在其发病机制中起着关键作用。因此，这项研究的长期目标是开发更安全的CD治疗方法，增强肠道上皮功能，而不是抑制免疫系统。位于肠隐窝底部的肠干细胞对肠上皮细胞的增殖和分化起着重要的调控作用。虽然调控ISC功能的宿主途径正在逐步阐明，但外源因素对ISC生物学的影响却知之甚少。特别是，共生肠道微生物区系对ISC生物学的确切影响还没有被表征。因此，本提案的目的是阐明肠道微生物区系如何调节ISC功能。这一目标将通过检验本研究的中心假设来实现，即肠道微生物区系是ISC维持和增殖的关键调节因子。这一假设是基于申请者提供的支持初步数据而提出的，并将通过两个具体目标进行检验。首先，将在健康的小鼠身上检查肠道微生物区系在调节IS生物学中的作用。这将使用已建立的流式细胞术方法以及补充的体外和体内技术来实现，以比较常规(CV)和无菌(GF)小鼠的ISC功能。第二，潘氏细胞(PC)作为微生物-ISC相互作用的关键媒介的作用将被探索。这一目标建立在PC是ISC利基的关键组件的理论基础上，并且能够直接感知共生肠道细菌。为了实现这一目标，将使用成熟的分离PC和ISCs的技术来评估GF和CV PC支持ISC体外生长的能力。这些数据将通过对Irgm1-/-小鼠的ISC功能的活体表征而得到加强，这些小鼠有明显的PC异常。拟议的方法是创新的，因为它集中在肠道微生物区系的一个新的调节角色，即它调节PC生态位信号到ISC隔室的能力。这与以前集中在微生物区系作为PC抗菌功能调节器的作用的工作形成了对比。这项研究具有重要意义，因为它 预计将导致微生物调节策略的开发，从而允许出于治疗目的操纵ISC功能。最终，这有可能将CD患者的免疫毒性治疗降至最低，同时促进粘膜愈合。重要的是，通过这一提议建立的数据和技术将为进一步的独立研究奠定基础，这些研究确定肠道细菌调节ISC生物学的机制。

DESCRIPTION (provided by applicant): Crohn's disease (CD) is a potentially debilitating, chronic inflammatory disorder of the gastrointestinal tract. Moreover, current medications for this disorder tend to be broad-spectrum immunosuppressive agents with the potential for profound side effects. Thus there is a strong rationale to develop less toxic therapeutic options for CD, particularly in pediatric patients who may receive decades of potent immunosuppressive therapy. Although the precise etiology of CD remains unclear, altered epithelial function plays a key role in its pathogenesis. Therefore, the long-term goal of this research is to develop safer therapies for CD that augment intestinal epithelial function, as opposed to suppressing the immune system. Intestinal stem cells (ISCs) located at the base of intestinal crypts play a central role governing proliferation and differentiation of the gut epithelium. Although host pathways that regulate ISC function are progressively being elucidated, the effects of exogenous factors on ISC biology are poorly understood. In particular, the precise influences of the commensal enteric microbiota on ISC biology have not been characterized. Hence, the objective of the current proposal is to elucidate how the gut microbiota modulates ISC function. This objective will be achieved by testing the central hypothesis of this study, namely that the intestinal microbiota is a key regulator of ISC maintenance and proliferation. This hypothesis has been formulated based upon supporting preliminary data generated by the applicant, and will be tested via two specific aims. First, the role of the intestinal microbiota in regulating IS biology will be examined in healthy mice. This will be accomplished using established flow cytometry approaches, as well as complementary in vitro and in vivo techniques to compare ISC function in conventional (CV) versus germ-free (GF) mice. Second, the role of Paneth cells (PCs) as critical mediators of microbial-ISC interactions will be explored. This aim is built upon the rationale that PCs are a key component of the ISC niche, and are able to directly sense commensal gut bacteria. To accomplish this aim, established techniques for isolating PCs and ISCs will be used to assess the ability of GF versus CV PCs to support ISC growth in vitro. These data will be enhanced by in vivo characterization of ISC function in Irgm1-/- mice, which have clear PC abnormalities. The proposed approach is innovative because it focuses on a novel regulatory role for the gut microbiota, namely its ability to modulate PC niche signaling to the ISC compartment. This is in contrast to previous work that has concentrated on the role of the microbiota as a regulator of PC antimicrobial function. This research is significant because it is expected to lead to the development of microbial modulation strategies that will allow for the manipulation of ISC function for therapeutic purposes. Ultimately, this has the potential to minimize immunotoxic therapies for patients with CD, while concomitantly promoting mucosal healing. Importantly, the data and techniques established through this proposal will form the foundation for further independent studies that define the mechanisms by which intestinal bacteria regulate ISC biology.