NODS-Like receptors in infection and immunity

Rationale. The gut flora (also referred as the microbiota) is composed of a huge number of diverse microbial communities. One can consider the microbiota as our “extended self”, in as much as this multicellular organ consumes, stores and redistributes energy harvested from nutrients. With this reductionist organ centred-view in mind, it is interesting to contemplate the dynamics of symbiosis (defined as an association between organisms of two different species in which each is benefited). Symbiosis is well exemplified by the co-evolutionary, beneficial interactions between the microbiota and the intestinal barrier, which has widely differing living requirements. One can also hypothesize that not all commensal lineages (of which Bacteroidetes is the most prominent), have the same ability to act as a failed pathogen for the benefit of the host infrastructure. The physiological impact of the microbiota is indeed intrinsically determined by the microbiome – a term introduced by the Nobel laureate Joshua Lederberg to describe the collective genomic content of the gut microbiota. It is worth mentioning Bacteroides thetaiotaomicron was found to behave as a more versatile commensal, in that genetically predisposed hosts may perceived it to be an opportunistic microorganism. Those that do are referred to herein as pathobionts. By contrast, secretion of polysaccharide A by Bacteroides fragilis confers its ability to function as a true symbiotic microorganism by exploiting innate immunity. In the absence of any commensal lineages, the colonic mucosa displays defects in goblet cell differentiation and lymphoid tissue architecture (e.g., isolated lymphoid follicles). Throughout the host’s entire life, the coexistence of certain resident commensals with a single layer of intestinal epithelial cells (IECs) has indeed a beneficial impact on post-natal maturation of the gut innate and adaptive immune barrier, as illustrated by the key observation on acid-producing lactic bacilli by the 1908 Nobel laureate Elie Metchnikoff. However, despite an extensive research in this field, it is still not yet known (i) how successive waves of colonization can comprehensively shape the mucosal healing response to environmental stress and (ii) which selective genetic and diet forces ensure stability and resilience of symbionts from the first days of life onwards.

Main achievements. Our knowledge of bacterial symbiosis now suggests that a recently described cytosolic surveillance mechanism (the NOD-like receptors) has a key role. The Nod-like receptors family consists of about 23 members in mammals, all of which share similarities with the superfamily of plant disease-resistance proteins. Although some of the NLRs are not fully characterized, these molecules are thought to play an essential role in detecting a diversified set of threats that primarily originate from microorganisms and cell remnants. At the Fondation Jean Dausset, Dr. Chamaillard contributed to the identification of NOD2 as the first Crohn’s disease predisposing gene through a positional cloning strategy (Nature. 2001 May 31;411(6837):599-603), providing a proof-of-concept on gene discovery in human complex illnesses. More recently, we conducted a meta-analysis of six ulcerative colitis genome-wide association study datasets and increased the number of confirmed inflammatory bowel disease risk loci to 99 (Nat Genet. 2011 Mar;43(3):246-52). Thanks to a functional genomic approach, Dr. Chamaillard next revealed how allelic heterogeneity in Nod2 may predispose to either Crohn’s disease or Blau syndrome – another granulomatous disorder that affect solely aseptic sites (Nat Genet. 2001 Sep;29(1):19-20 and Proc Natl Acad Sci U S A. 2003 Mar 18;100(6):3455-60). Mechanistically, Dr. Chamaillard also contributed to the identification of MurNAc-L-Ala-D-isoGln (MDP) as the essential microbial structure sensed by NOD2 in vitro and in vivo, which led to the publication in two high-ranking publications (> 100 citations per year since 2003) (J Biol Chem. 2003 Mar 14;278(11):8869-72 and Science. 2005 Feb 4;307(5710):731-4). That paradigm is of importance in that it unveiled a link between the pathogenesis of Crohn’s disease and impaired bacterial sensing in the gut. Dr. Chamaillard next identified NOD1 as a sensor of a specific bacterial subset, which is characterized by a diaminopimelic acid-containing peptidoglycan (Nat Immunol. 2003 Jul;4(7):702-7). By using a systematic mutational analysis, Dr. Chamaillard also contributed to the identification of a general mechanism by which NOD1 and NOD2 may confer differential host discrimination of bacterial peptidoglycan through their leucine-rich repeats (EMBO J. 2004 Apr 7;23(7):1587-97). The N-terminal and C-terminal leucine-rich repeats were differentially required in the engagement of NOD1/2-mediated signalling by specific muropeptides. Notably, the residues thought to be involved in the bstrand/bturn of the C-terminal leucine-rich repeats are essential for the muropeptide-induced NF-kB activation. We also identified specific residues and essential regulatory domains of NOD1 and NOD2 involved in NF-kB activation.

A major challenge in understanding the physiological function of Nod-like receptors relates now to discovering how the latter coordinate the biologic dynamics of the microbiota. Since the creation of Dr Chamaillard’s independent research group in 2010, he has adopted a wide-ranging, integrated approach to studying the biology of NLR by dissecting bacterial symbiosis in a standardized animal facility via gnotobiotic, biochemical and deep-sequencing technologies. To the best of our knowledge, our studies are the first to have described an unexpected role of the major Crohn’s disease predisposing NOD2 protein in the protection against intestinal inflammation and tumorigenesis afforded by gut bacterial communities in mice. Reciprocal faecal microbiota transplantation led to long-term changes in intestinal microbial communities and decreased the vulnerability of Nod2-deficient mice to colonic injury. Conversely, disease risk was enhanced in wild-type hosts that were recolonized with dysbiotic faecal microbiota from Nod2-deficient mice (J Clin Invest. 2013 Feb 1;123(2):700-11). Last but not least, we and others also identified NLRP6 as a key molecular mechanism linking mucosal healing and maintenance of gut microbial communities (Proc Natl Acad Sci U S A. 2011 Jun 7;108(23):9601-6). Notably, we also unveiled a novel non-hematopoietic function of IL-33 in epithelial barrier function and wound healing (Gut. 2012 Dec 8. [Epub ahead of print]). Furthermore, we demonstrated efficient repair of colonic mechanical injuries requires villin F-actin severing, emphasizing the importance of villin function in homeostasis. Thus, villin severs F-actin to ensure microvillus depolarization and enterocyte remodelling upon injury. This work highlights the importance of specialized apical poles disassembly for the repolarization of epithelial cells initiating migration (Proc Natl Acad Sci U S A. 2013 Apr 9;110(15):E1380-9). From a therapeutic perspective, we unveiled a novel therapeutic strategy based on PPARgamma agonists (Proc Natl Acad Sci U S A. 2010 May 11;107(19):8772-7).

Unmet needs. The composition of gut microbial communities is controlled by co-evolutionarily conserved surveillance systems, including the Nod-like receptors. Our recent findings suggest that emerging, disease-predisposing dysbiosis can now be intentionally manipulated by targeting the major Crohn’s disease-predisposing NOD2 gene and the related molecule NLRP6. However, the lack of a multi-level approach with high spatial and temporal resolution biases our view of the bioactivity of the microbiota and the dynamics of the host’s effector/regulatory immune network that maintain microbial symbiosis (Gastroenterology. 2013 Nov;145(5):1150-1).

Research focus. Understanding the contribution that specific commensals make to impairing or failing to promote mucosal healing and how the host coordinates bacterial symbiosis is urgently needed, in order to envision the development of more efficient therapeutic interventions.

Innovative aspects and added values of our integrated approach. To address this fundamental, challenging issue, we shall seek to identify: (i) biochemical, anatomical and immunological features of commensals which overcome or preserve (either individually or as a whole organ) epithelial barrier function, and (ii) specific cellular and molecular features through which NOD2 and NLRP6 shape a protective assembly of commensal lineages against intestinal inflammation and tumorigenesis. These insights will be gained by combining germ-free and Cre-LoxP technologies with innovative in vivo imaging, deep sequencing and mass spectrometry-based proteomics.

Expected outcomes. The outputs of this integrated approach may challenge dogmas on ancestral microbial-eukaryotic symbiosis, and have a broad impact on biomedical sciences worldwide.

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