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Final Report Summary - 6-SENSE (The role of NLRP6 sensing in regulation of intestinal host-microbiome interactions)

Interactions between the gut microbiome and the human host are increasingly recognized to be fundamentally important in governing our health as well as a variety of common. ‘multi-factorial diseases ranging from infection, inflammation, auto-immune disorders, cancer and even neurodegeneration. Elucidation of the molecular basis of these interactions is critically important in identifying new therapeutic targets in these disorders. In this Marie Curie project, we aimed to decipher the molecular mechanism by which the host gut epithelial cell layer senses components of the gut microbiome in deciding between tolerance induction in times of peace to initiation of a potent inflammatory response when pathogens are introduced into the system. We previously identified a multi-protein complex in intestinal epithelial cells, termed the NLRP6 inflammasome, as a critical innate immune regulator of the intestinal microbiome, a diverse ecosystem, whose poorly understood interactions with the host influence a multitude of physiological and pathological processes. We found that NLRP6 inflammasome disruption results in dramatic alterations in microbiome composition & function, in turn leading to auto-inflammation, and a propensity to the development of the metabolic syndrome. However, the basic mechanisms governing NLRP6 activation & regulation of host-microbiome interactions remain unknown. Using the NLRP6 system as a proof-of-principle model, and innovative robotic high-throughput modalities, gnotobiotics, metagenomics and multiple genetically altered mouse models, our project was aimed at deciphering the codes comprising the host-microbiome communication network and factors initiating loss of homeostasis (dysbiosis). Specifically, our goals were to (A) elucidate the host-derived microbiome recognition signaling pathway at its entirety, from its upstream activators to the downstream effector molecules controlling microbial ecology; (B) decipher the cell-specific mechanisms of NLRP6 regulation of the intestinal crypt niche; and (C) uncover mechanisms by which the innate immune arm regulates the composition and function of the gut commensal eco-system in health and disease. In our mid-term report, we described our findings (Wladorska et al, Cell 2014), that NLRP6 is critically important for anti-pathogen immune responses. These responses are via inflammasome-independent mechanisms such as regulation of goblet cell function, as well as through inflammasome-dependent mechanisms such as IL-18 mediated regulation of intestinal anti-microbial peptides (AMP). We further described the importance of NLRP6-dependent regulatory pathways in mucus secretion from goblet cells, and eventual autophagy, which are critical processes to clearing enteric pathogens. We highlighted the role of the NLRP6 inflammasome as the major regulator of steady-state IL-18 levels, which in turn control host secretion of anti microbial peptides. Finally, we reported that NLRP6 activity is dependent on 'healthy' gut microbiome colonization. In this final report, we highlight our subsequent work (Levy et al, Cell 2015) demonstrating the role of the microbiota in signaling for NLRP6 inflammasome assembly and downstream activity, including the regulation of IL-18 secretion from the intestinal epithelium. We also identified specific AMP released in response to IL-18 secretion, which have a direct effect on the composition of the microbiota. In the other direction, we describe how specific microbiota-derived metabolites affect NLRP6 inflammasome activity. In sum, our work has comprehensively illustrated a mechanistic pathway for the determination of gut microbiota composition, involving host and microbial components. Our demonstration of the critical role for the NLRP6 inflammasome in this process may provide insights toward understanding dysbiotic-associated pathologies, as well as direction toward potential therapeutic interventions. On a broader range, Marie Curie support of this ambitious project enabled me to establish my independent lab, progress in the lab’s first major project until its fruition as two high profile publications, thereby greatly assisting me in exceling my early stage career and disseminating my lab’s discoveries. As such, it was a fundamentally important support in my integration process into the European immunological community as an indepenedent young investigator.
Deciphering the molecular basis of host-microbiome interaction is critically important in understanding how the intestinal niche is formed and regulated, and how the host and its microbiome integrate their activity to ensure a proper and mutually beneficial niche structure. In this project we focused on the NLRP6 inflammasome, which we previously found to be highly active in the intestinal niche under homeostatic conditions, orchestrating normal niche construction and the composition and function of the symbiotic immune response. Our goal was to uncover the mechanisms by which the host and its microbiome regulate NLRP6 inflammasome activity with its downstream effector functions, and how these functions contribute to regulation of the intestinal niche and the commensal microbiome.
We followed the following specific aims:
Aim 1: Identifying microbial NLRP6 inflammasome activators during homeostatic and infectious conditions. This aim was focused on NLRP6’s mechanisms of regulation of the anti-microbial immune responses. It involved in-vivo study to determine (A) the components of the NLRP6 inflammasome pathway participate in host sensing of C. rodentium infection, (B) the microbial effector molecules eliciting the NLRP6 inflammasome protective response, and (C) the molecular mechanism underlying pathogen recognition by the NLRP6 inflammasome, using high throughput in-vitro readout systems designed to detect inflammasome activation. For this reporting period, our objective was the identification of microbiota-derived NLRP6 inflammasome regulators that are involved in the maintenance of healthy or dysbiotic microbione composition.

Aim 2: Deciphering the mechanisms of NLRP6 activity in orchestrating the intestinal crypt niche. Our recent studies have pointed at the intestinal epithelium as the major site of NLRP6 activity. However, the intestinal epithelium is a diverse tissue consisting of various cell types with highly specialized functions, including surface enterocytes, goblet cells, paneth cells, endocrine cells and stem cells. Our study of the cell-type specific mechanism of epithelial inflammasome activity thus necessitated a detailed functional analysis of the candidate cell types involved. For this reporting period, our objective was to define more precisely the role of the intestinal epithelium in mediating NLRP6 inflammasome activity, particularly with regard to the secretion of IL-18.

Aim 3: Deciphering the mechanisms of NLRP6 activity in modulation of the composition and activity of the microbiome ecosystem. Prior to the inception of the funded project, we highlighted a role for the NLRP6 inflammasome in shaping the composition of the intestinal microbiota eco-system (Elinav et al., Cell 2011). Extending this finding, we aimed to study the mechanisms by which NLRP6 signaling affects microbiota function using advanced computational next-generation metagenomic sequencing techniques. In addition, using germ-free NLRP6 deficient mice and gnotobiotic bacterial transfer experiments, we studied the kinetics of NLRP6 regulation of the microbiota, and how specific commensal populations regulate pathogenic infection and inflammation in susceptible mice. For this reporting period, our objective was to identify and characterize anti microbial peptides whose expression is driven by NLRP6 inflammasome activity, and which act to modulate the composition of commensal microbiome.

Project infrastructure: Throughout the period of funding, we completed the recruitment of students and post docs for this and our other projects. All purchasing of lab equipment was completed and all of our experimental systems have been set up. These include a state-of the art germ free facility encompassing over 500 mouse cages of diverse strains, the first ‘personalized germ free’ facility enabling cage autonomous germ-free conditions, which increased our capacity to house 300 more cages of GF animals (see Hecht et al, Lab Animals, 2014), an anaerobic chamber enabling experimentation with various gut-mimetic conditions, a fully automated robotic system enabling high throughput screening of molecular libraries, and next generation sequencing pipeline enabling microbiome processing, genomic library preparation, sequencing, and analysis.

In this project, we provided the first detailed molecular explication of the host genetic contribution toward dysbiosis, and identified mechanisms leading to dysbiotic microbiome takeover wild type microbiome configuration. Our results clarify some of the central mechanisms governing host and microbiome co-regulation of their mutual niche. These mechanisms explain in large part, how dysbiotic microbiomes transmit associated disease susceptibility across individuals, through direct control of their own composition, as well as through their effects on host inflammasome components.

Deciphering the mechanisms of NLRP6 signaling and their downstream effects on the mucosal immune response (1) has substantial implications on our understanding of novel mechanisms regulating mucosal tolerance and inflammation; (2) may uncover the mechanisms by which the innate immune arm regulates the composition and function of the gut commensal eco-system, and (3) potentially enable the identification of new personalized therapeutic targets for a multitude of inflammasome and microbiota-regulated diseases including infection, auto-inflammation, carcinogenesis, and the various manifestations of the metabolic syndrome.

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