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

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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.