Medico - Scientific Background 

The gut microbiota is composed of a huge number of diverse bacterial communities (Bacteroidetes and Firmicutes are the dominant phyla) and of abundant fungi. In humans, the coexistence of resident commensals and host cells play a beneficial role in regulating both energy harvesting from nutrients and host defense. Notably, the intestinal microbiota is able to regulate virulence of enteric bacterial pathogens, including Citrobacter rodentium. However, not all commensals are able to maintain quiescent and protective immunity, arguing for the need to decipher the nature and mechanisms of these processes.

The nucleotide-binding oligomerization domain containing protein 2 (Nod2) is thought to play a decisive role in maintaining microbial tolerance and host defense at the intestinal barrier through two adaptor proteins, namely Ripk2 and Card9, but its role in inciting innate and adaptive immunity is complex. Noteworthy, Nod2 and Card9 variants have been associated with Crohn’s disease (CD). More recently, we unveiled a key role of Nod2-driven dysbiosis in predisposing to intestinal inflammation (Couturier-Maillard, et al JCI 2013). More importantly, genetic ablation of either Nod2 (preliminary data) or Card9 (Sokol et al. Gastroenterology 2013) results in abnormal host defense against C. rodentium.

Based on such current state of the art, the C. rodentium-driven colitis model is of high interest as appropriate response to such enteric bacterial pathogen requires many major biological processes involved in the maintenance of intestinal homeostasis and that are altered in CD.

However, the cellular and molecular mechanism whereby Nod2 and/or Card9 may control pathogen virulence by shaping a non-permissive microbiota has not been investigated yet.

Through a multidisciplinary approach, the main objective of the current proposal is therefore to decipher the microbiota-dependent and -independent mechanisms by which the pathogenesis of enteric pathogen bacteria is regulated.

To achieve our objective, we envisage an integrated “microbiome/immunology” approach by taking advantage of gnotobiotic animals and by using cre-lox and high-throughput sequencing technologies, as well as developing multidimensional statistical models to decipher the dynamic balance established between the commensal microbiota and the host immune system. Notably, we will use transgenic models of dysbiotic microbiota (ie. Nod2 and Card9 deficient mice) and C. rodentium, as an experimental model microorganism. Whereas most of the studies on this topic so far analyzed either the bacterial gut microbiota or the host response, our ambitious strategy is mainly based on the concomitant analysis of gut microbiota (bacterial but also fungal part) and host compartments.

Collectively, our project is highly innovative as it will explore the gut-microbiota crosstalk within the gut in a new dimension by integrating the spatio-temporal host response through combination of up to date genetically engineered mice, gnotobiotic mice, transcriptomics, sequencing technologies and systems biology. Deciphering the complex interactions between the gut microbiota and its host will improve our understanding of human diseases pathogenesis and our discoveries of new therapeutic targets. The intestinal microbiota has been indeed pointed out as a major player in an increasing number of diseases including inflammatory bowel diseases, rheumatoid arthritis, multiple sclerosis, type1 diabetes, obesity or non-alcoholic fatty liver disease.

Work Packages

To achieve our objective, we envisage an integrated "microbiome/immunology" approach by taking advantage of gnotobiotic animals and by using cre-lox and high-throughput sequencing technologies, as well as developing multidimensional statistical models to decipher the dynamic balance established between the commensal microbiota and the host immune system. The four tasks proposed for this project can be done independently but gathering the results will allow us getting a global insight in the physiological role of Card9 and Ripk2 on the Nod2-mediated control of C. rodentium infection. These tasks are respectively:

Task 1: Identify molecular and cellular mechanisms shaping onset of a disease predisposing microbiota.

The respective contributions of Nod2, Ripk2 and Card9 as well as the cellular interaction involved in the genesis of gut microbiota alterations will be determined using not only already generated but also novel genetically engineered mice that are not yet published. This will allow us to identify the common disease-predisposing core within dysbiotic microbiota in the absence of Nod2, Ripk2 or Card9.

Task 2: Identify how disease-predisposing dysbiosis regulates C. rodentium susceptibility.

The intrinsic contribution of Nod2-, Ripk2- and Card9-driven bacterial and fungal dysbiosis on the virulence of C. rodentium will be assessed.

Task 3: Identify gut microbiota-independent role of Nod2, Ripk2 and Card9 on control of Citrobacter rodentium colitis.

The role of Nod2, Ripk2 and Card9 in host defense against C. rodentium will be investigated by using newly-generated germ-free animals.

CombInnate Publications