Influences of the enteric microbiota on intestinal stem cell biology

描述（由申请方提供）：克罗恩病（CD）是一种潜在的胃肠道慢性炎症性疾病。此外，目前的药物治疗 这些疾病往往是具有潜在严重副作用的广谱免疫抑制剂。因此，有充分的理由为CD开发毒性较小的治疗选择，特别是在可能接受数十年强效免疫抑制治疗的儿科患者中。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关键作用。因此，这项研究的长期目标是开发更安全的CD疗法，增强肠上皮功能，而不是抑制免疫系统。肠干细胞（ISCs）位于肠隐窝底部，在调控肠上皮细胞的增殖和分化中起着重要作用。虽然宿主调控ISC功能的途径正在逐步阐明，外源性因素对ISC生物学的影响知之甚少。特别是，肠道微生物群对ISC生物学的精确影响尚未被表征。因此，本提案的目的是阐明肠道微生物群如何调节ISC功能。这一目标将通过测试本研究的中心假设来实现，即肠道微生物群是ISC维持和增殖的关键调节因子。该假设是根据申请人生成的支持性初步数据制定的，并将通过两个特定目标进行测试。首先，将在健康小鼠中检查肠道微生物群在调节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互补的体外和体内技术来完成，以比较常规（CV）与无菌（GF）小鼠中的ISC功能。其次，潘氏细胞（PC）作为微生物ISC相互作用的关键介质的作用将被探讨。这一目标是建立在PC是ISC生态位的关键组成部分的基础上，并且能够直接感知肠道细菌。为了实现这一目标，将使用已建立的分离PC和ISC的技术来评估GF与CV PC在体外支持ISC生长的能力。这些数据将通过Irgm 1-/-小鼠中ISC功能的体内表征来增强，所述小鼠具有明显的PC异常。所提出的方法是创新的，因为它专注于肠道微生物群的新调节作用，即其调节PC生态位信号传导到ISC隔室的能力。这与以前的工作相反，以前的工作集中在微生物群作为PC抗菌功能的调节剂的作用上。这项研究意义重大，因为它 有望导致微生物调节策略的发展，该策略将允许出于治疗目的操纵ISC功能。最终，这有可能最大限度地减少CD患者的免疫毒性治疗，同时促进粘膜愈合。重要的是，通过这项提议建立的数据和技术将为进一步的独立研究奠定基础，这些研究将确定肠道细菌调节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.