Host-microbe interactions in the intestinal ecosystem regulated through Nod proteins

在肠道生态系统中，通过宿主免疫系统和被称为微生物群的常驻细菌群落之间的严格调控的串扰来维持稳态。从宿主的角度来看，体内平衡依赖于免疫系统同时耐受微生物群并对病原攻击保持警惕的能力。特别是，先天免疫蛋白Nod1和Nod2已被证明在调节肠道炎症中发挥关键作用，通过感知上皮细胞内的细菌结构并发出适当的炎症反应信号。另一方面，微生物群可能“启动”这些传感器，但其他免疫调节活动在很大程度上是未知的。由于慢性炎症性疾病与Nod蛋白的遗传缺陷有关，并且也以细菌群落的变化为特征，因此在健康和疾病的背景下，对宿主-微生物相互作用的深入了解对科学界和公众都具有极大的兴趣。我提出这项研究的目标是更好地了解肠道生态系统是如何由肠道的微生物群落和免疫功能来定义的。野生型（WT）和nod缺陷小鼠将被用作模型系统，从微生物群的角度确定维持体内平衡的重要参数。我将首先验证与Nod信号缺乏相关的肠道状况（即减少促炎细胞因子，抗菌肽，粘液分泌等）对微生物群群落结构的影响的假设。这种方法将包括在恒温器中培养稳定的微生物群，然后接种缺乏微生物群（无菌）的小鼠肠道。细菌群落将使用基于dna的技术进行长期监测，并与免疫输出测量相关联。这种标准化所有小鼠微生物群的方法应该揭示Nod1和Nod2对塑造肠道细菌群落组成的影响的新细节。接下来，我将使用厌氧培养方法来深入了解与nod缺乏小鼠相关的微生物群的各个组成部分。各种旨在近似肠道环境条件的生长培养基将用于分离单个物种或细菌群。DNA分析将用于鉴定分离的细菌，包括分类和社区概况，代谢分析将用于深入了解其代谢功能。细菌培养以及通过使用这些互补方法获得的相关知识将为设计简化的小鼠细菌群落提供机会，其中微生物群的所有成员都是已知的。基于肠道内稳态依赖于宿主免疫系统和微生物群提供的促炎性和抗炎性信号的平衡这一前提，第三个项目将研究微生物组成的差异如何影响Nod功能背景下的内稳态。我推测，操纵群落组成有利于拟杆菌样细菌可能促进促炎免疫反应，而偏向梭状芽胞杆菌样细菌的群落可能促进抗炎反应。我进一步预计，拟杆菌倾斜的群落将增加致病性感染后肠道炎症的严重程度，特别是当加上Nod缺乏时，这些系统将缺乏恢复到感染前状态的能力。这项跨学科的研究成果将有助于粘膜免疫学和微生物生态学领域的发展和整合。

In the intestinal ecosystem, homeostasis is maintained through a tightly regulated cross-talk between the host immune system and resident bacterial communities known as the microbiota. From the host’s perspective, homeostasis relies on the immune system’s ability to simultaneously tolerate the microbiota and remain vigilant against pathogenic attack. In particular, the innate immune proteins, Nod1 and Nod2, have been shown to play key roles in regulating intestinal inflammation by sensing bacterial structures within epithelial cells and signaling for appropriate inflammatory responses. On the other hand, the microbiota may “prime” these sensors, but other immune-modulating activities are largely unknown. As chronic inflammatory diseases are linked to genetic deficiencies in Nod proteins and also characterized by shifts in bacterial communities, insights into host-microbe interactions are of great interest to the scientific community and the public in the context of both health and disease. My goal for this proposed research is to better understand how the intestinal ecosystem is defined by both the microbial community and the immune functions of the gut. Wild type (WT) and Nod-deficient mice will be used as model systems to identify parameters important for maintaining homeostasis from the perspective of the microbiota. I will first test the hypothesis that intestinal conditions associated with a lack of Nod signaling (i.e. reduced pro-inflammatory cytokines, antimicrobial peptides, mucus secretion, etc.) impact on community structure of the microbiota. This approach will involve culturing a stable microbiota in a chemostat and then inoculating the intestines of mice lacking a microbiota (germ-free). Bacterial communities will be monitored over time using DNA-based techniques and correlated with immune output measurements. This approach of standardizing the microbiota in all mice should reveal new details of the influence of Nod1 and Nod2 on shaping the composition of intestinal bacterial communities. I will next use anaerobic culturing methods to gain insights into the individual components of the microbiota associated with Nod-deficient mice. A variety of growth media intended to approximate conditions in the intestinal environment will be used to isolate individual species or groups of bacteria. DNA analysis will be used to identify isolated bacteria, both taxonomically and within community profiles, and metabolic profiling will be used to gain insights into their metabolic functions. Bacterial cultures along with the associated knowledge acquired through use of these complementary methods will provide opportunities for designing simplified bacterial communities in mice, in which all the members of the microbiota are known. Based on the premise that intestinal homeostasis depends on a balance of pro- and anti-inflammatory signals provided by both the host immune system and the microbiota, the third project will investigate how differences in microbial composition impact on homeostasis in the context of Nod functioning. I hypothesize that manipulation of community composition in favour of Bacteroides-like bacteria may promote pro-inflammatory immune responses while communities skewed towards Clostridium-like bacteria may promote anti-inflammatory responses. I further expect that Bacteroides-skewed communities will increase the severity of intestinal inflammation following pathogenic infection, especially when coupled with Nod deficiency, and that these systems will lack the ability to return to their pre-infection states. The findings from this interdisciplinary research will contribute to the advancement and integration of the fields of mucosal immunology and microbial ecology.