A Weaning Reaction to Microbiota Is Required for Resistance to Immunopathologies in the Adult.

Microbes colonize all body surfaces at birth and participate in the development of the immune system. In newborn mammals, the intestinal microbiota is first shaped by the dietary and immunological components of milk and then changes upon the introduction of solid food during weaning. Here, we explored the reactivity of the mouse intestinal immune system during the first weeks after birth and into adulthood. At weaning, the intestinal microbiota induced a vigorous immune response-a "weaning reaction"-that was programmed in time. Inhibition of the weaning reaction led to pathological imprinting and increased susceptibility to colitis, allergic inflammation, and cancer later in life. Prevention of this pathological imprinting was associated with the generation of RORγt+ regulatory T cells, which required bacterial and dietary metabolites-short-chain fatty acids and retinoic acid. Thus, the weaning reaction to microbiota is required for immune ontogeny, the perturbation of which leads to increased susceptibility to immunopathologies later in life.

Perinatal foodborne titanium dioxide exposure-mediated dysbiosis predisposes mice to develop colitis through life.

BACKGROUND: Perinatal exposure to titanium dioxide (TiO2), as a foodborne particle, may influence the intestinal barrier function and the susceptibility to develop inflammatory bowel disease (IBD) later in life. Here, we investigate the impact of perinatal foodborne TiO2 exposure on the intestinal mucosal function and the susceptibility to develop IBD-associated colitis. Pregnant and lactating mother mice were exposed to TiO2 until pups weaning and the gut microbiota and intestinal barrier function of their offspring was assessed at day 30 post-birth (weaning) and at adult age (50 days). Epigenetic marks was studied by DNA methylation profile measuring the level of 5-methyl-2'-deoxycytosine (5-Me-dC) in DNA from colic epithelial cells. The susceptibility to develop IBD has been monitored using dextran-sulfate sodium (DSS)-induced colitis model. Germ-free mice were used to define whether microbial transfer influence the mucosal homeostasis and subsequent exacerbation of DSS-induced colitis. RESULTS: In pregnant and lactating mice, foodborne TiO2 was able to translocate across the host barriers including gut, placenta and mammary gland to reach embryos and pups, respectively. This passage modified the chemical element composition of foetus, and spleen and liver of mothers and their offspring. We showed that perinatal exposure to TiO2 early in life alters the gut microbiota composition, increases the intestinal epithelial permeability and enhances the colonic cytokines and myosin light chain kinase expression. Moreover, perinatal exposure to TiO2 also modifies the abilities of intestinal stem cells to survive, grow and generate a functional epithelium. Maternal TiO2 exposure increases the susceptibility of offspring mice to develop severe DSS-induced colitis later in life. Finally, transfer of TiO2-induced microbiota dysbiosis to pregnant germ-free mice affects the homeostasis of the intestinal mucosal barrier early in life and confers an increased susceptibility to develop colitis in adult offspring. CONCLUSIONS: Our findings indicate that foodborne TiO2 consumption during the perinatal period has negative long-lasting consequences on the development of the intestinal mucosal barrier toward higher colitis susceptibility. This demonstrates to which extent environmental factors influence the microbial-host interplay and impact the long-term mucosal homeostasis.

Nod2: The intestinal gate keeper.

Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular pattern recognition receptor that senses bacterial peptidoglycan (PGN)-conserved motifs in cytosol and stimulates host immune response. The association of NOD2 mutations with a number of inflammatory pathologies, including Crohn disease (CD), Graft-versus-host disease (GVHD), and Blau syndrome, highlights its pivotal role in host-pathogen interactions and inflammatory response. Stimulation of NOD2 by its ligand (muramyl dipeptide) activates pro-inflammatory pathways such as nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPKs), and Caspase-1. A loss of NOD2 function may result in a failure in the control of microbial infection, thereby initiating systemic responses and aberrant inflammation. Because the ligand of Nod2 is conserved in both gram-positive and gram-negative bacteria, NOD2 detects a wide variety of microorganisms. Furthermore, current literature evidences that NOD2 is also able to control viruses' and parasites' infections. In this review, we present and discuss recent developments about the role of NOD2 in shaping the gut commensal microbiota and pathogens, including bacteria, viruses, and parasites, and the mechanisms by which Nod2 mutations participate in disease occurrence.

Imprinting of the immune system by the microbiota early in life.

The ontogeny and maturation of the immune system is modulated by the microbiota. During fetal life, the mother's microbiota produces compounds that are transferred to the fetus and offspring, and enhance the generation of innate immune cells. After birth, the colonizing microbiota induces the development of intestinal lymphoid tissues and maturation of myeloid and lymphoid cells, and imprints the immune system with a reactivity level that persists long after weaning into adulthood. When the cross-talk between host and microbiota is perturbed early in life, a pathological imprinting may develop that is characterized by excessive immune reactivity in adulthood, which translates into increased susceptibility to inflammatory pathologies. In this review, we discuss the recent data that demonstrate the existence of a time window of opportunity early in life during which mice and human have to be exposed to microbiota in order to develop a balanced immune system. We also discuss the factors involved in imprinting, such as the microbiota, immune cells and stromal cells, as well as the nature of imprinting.

Nod2 Protects the Gut From Experimental Colitis Spreading to Small Intestine.

BACKGROUND AND AIMS: Nucleotide oligomerization domain 2 [NOD2] mutations are key risk factors for Crohn's disease [CD]. NOD2 contributes to intestinal homeostasis by regulating innate and adaptive immunity together with intestinal epithelial function. However, the exact roles of NOD2 in CD and other NOD2-associated disorders remain poorly known. METHODS: We initially observed that NOD2 expression was increased in epithelial cells away from inflamed areas in CD patients. To explore this finding, Nod2 mRNA expression, inflammation, and cytokines expression were examined in the small bowel of wild-type [WT], Nod2 knockout and Nod2 mutant mice after rectal instillation of 2,4,6-trinitrobenzene sulphonic acid [TNBS]. RESULTS: In WT mice, Nod2 upregulation upstream to rectal injury was associated with pro-inflammatory cytokine expression but no overt histological inflammatory lesions. Conversely, in Nod2-deficient mice the inflammation spread from colitis to ileum and duodenum. CONCLUSIONS: Nod2 protects the gut from colitis spreading to small intestine.

Excess calorie intake early in life increases susceptibility to colitis in adulthood.

Epidemiological data reveal an association between obesity and inflammatory bowel disease (IBD). Furthermore, animal models demonstrate that maternal high-fat diet (HFD) and maternal obesity increase susceptibility to IBD in offspring. Here we report that excess calorie intake by neonatal mice, as a consequence of maternal HFD, forced feeding of neonates or low litter competition, leads to an increase during weaning in intestinal permeability, expression of pro-inflammatory cytokines and hydrogen sulfide production by the microbiota. These intestinal changes engage in mutual positive feedback that imprints increased susceptibility to colitis in adults. The pathological imprinting is prevented by the neutralization of IFN-γ and TNF-α or the production of hydrogen sulfide, or by normalization of intestinal permeability during weaning. We propose that excess calorie intake by neonates leads to multiple causally linked perturbations in the intestine that imprint the individual with long-term susceptibility to IBD.

NOD2 Expression in Intestinal Epithelial Cells Protects Toward the Development of Inflammation and Associated Carcinogenesis.

Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular pattern recognition receptor that senses bacterial peptidoglycan-conserved motifs in cytosol and stimulates host immune response including epithelial and immune cells. The association of NOD2 mutations with a number of inflammatory pathologies including Crohn's disease (CD), graft-versus-host diseases, or Blau syndrome, highlights its pivotal role in inflammatory response and the associated-carcinogenesis development. Since its identification in 2001 and its association with CD, the role of NOD2 in epithelial cells and immune cells has been investigated extensively but the precise mechanism by which NOD2 mutations lead to CD and the associated carcinogenesis development is largely unknown. In this review, we present and discuss recent developments about the role of NOD2 inside epithelial cells on the control of the inflammatory process and its linked carcinogenesis development.

GAPs in early life facilitate immune tolerance.

Goblet cells deliver microbial antigens to generate regulatory T cells before and during weaning to induce long-term tolerance to symbionts.

Complementary Roles of Nod2 in Hematopoietic and Nonhematopoietic Cells in Preventing Gut Barrier Dysfunction Dependent on MLCK Activity.

BACKGROUND: Crohn's disease (CD) pathogenesis is multifactorial involving genetic and environmental factors. Loss of function mutations in the nucleotide oligomerization domain 2 (NOD2) gene are the main genetic risk factor for CD. Like patients with CD, Nod2 mice are characterized by an enhanced Th1 immune response and a defective mucosal barrier function evidenced by increased intestinal permeability. We previously showed that the latter is related to hematopoietic Nod2 deficiency. Our aim was to explore the mechanisms by which Nod2 expressed in the hematopoietic and in the nonhematopoietic compartments interplay to control epithelial paracellular permeability. METHODS: Depletion of CD4 T cells in Nod2 mice and treatments with inhibitors were conducted in chimeric mice transplanted with bone marrow cells from Nod2-deficient donors into Nod2-sufficient recipients or vice versa. Caco-2 cells overexpressing a NOD2 gene which did or did not include a CD-associated polymorphism were treated with inhibitors or siRNAs and cocultured with hematopoietic cells from Peyer's patches. RESULTS: In vivo and in vitro Nod2 in hematopoietic cells regulates epithelial paracellular permeability through cytokine production influencing myosin light chain kinase (MLCK) activity. Indeed, tumor necrosis factor-α and interferon-γ secretion by CD4 T cells upregulated expression and activity of epithelial MLCK leading to increased epithelial tight junction opening. When stimulated by muramyl dipeptide, Nod2 in the nonhematopoietic compartment normalized the permeability and T-cell cytokine secretion and regulated MLCK activity. This MLCK regulation is mediated by TAK1 and RICK-dependent mechanisms. CONCLUSIONS: Our study demonstrates how hematopoietic and nonhematopoietic Nod2 regulate intestinal barrier function, improving our knowledge on the mechanisms involved in CD pathogenesis.

Nutritional strategies and gut microbiota composition as risk factors for necrotizing enterocolitis in very-preterm infants.

Background: The pathophysiology of necrotizing enterocolitis (NEC) remains poorly understood.Objective: We assessed the relation between feeding strategies, intestinal microbiota composition, and the development of NEC.Design: We performed a prospective nationwide population-based study, EPIPAGE 2 (Etude Epidémiologique sur les Petits Ages Gestationnels), including preterm infants born at <32 wk of gestation in France in 2011. From individual characteristics observed during the first week of life, we calculated a propensity score for the risk of NEC (Bell's stage 2 or 3) after day 7 of life. We analyzed the relation between neonatal intensive care unit (NICU) strategies concerning the rate of progression of enteral feeding, the direct-breastfeeding policy, and the onset of NEC using general linear mixed models to account for clustering by the NICU. An ancillary propensity-matched case-control study, EPIFLORE (Etude Epidémiologique de la flore), in 20 of the 64 NICUs, analyzed the intestinal microbiota by culture and 16S ribosomal RNA gene sequencing.Results: Among the 3161 enrolled preterm infants, 106 (3.4%; 95% CI: 2.8%, 4.0%) developed NEC. Individual characteristics were significantly associated with NEC. Slower and intermediate rates of progression of enteral feeding strategies were associated with a higher risk of NEC, with an adjusted OR of 2.3 (95% CI: 1.2, 4.5; P = 0.01) and 2.0 (95% CI: 1.1, 3.5; P = 0.02), respectively. Less favorable and intermediate direct-breastfeeding policies were associated with higher NEC risk as well, with an adjusted OR of 2.5 (95% CI: 1.1, 5.8; P = 0.03) and 2.3 (95% CI: 1.1, 4.8; P = 0.02), respectively. Microbiota analysis performed in 16 cases and 78 controls showed an association between Clostridium neonatale and Staphylococcus aureus with NEC (P = 0.001 and P = 0.002).Conclusions: A slow rate of progression of enteral feeding and a less favorable direct-breastfeeding policy are associated with an increased risk of developing NEC. For a given level of risk assessed by propensity score, colonization by C. neonatale and/or S. aureus is significantly associated with NEC. This trial (EPIFLORE study) was registered at clinicaltrials.gov as NCT01127698.

Nod2 Deficiency Leads to a Specific and Transmissible Mucosa-associated Microbial Dysbiosis Which Is Independent of the Mucosal Barrier Defect.

BACKGROUND AND AIMS: Crohn's disease [CD] is a complex disorder characterised by an inappropriate immune response, impaired barrier function and microbial dysbiosis. Mutations in nucleotide oligomeriation domain 2 [NOD2] are CD risk factors. Increase of intestinal permeability, CD4+ T cell infiltration, and bacterial dysbiosis are also seen in Nod2-knockout [Nod2 KO] mice. However, the specificity and relationship between these Nod2-associated abnormalities remain largely unexplored. METHODS: Wild-type [WT], Nod1-knockout [Nod1 KO] and Nod2 KO mice were analysed in parallel. Microbial composition was defined by 454-pyrosequencing of bacterial 16S rRNA genes. Mucin and antimicrobial peptide expression was assessed by RT-PCR. Cell populations from Peyer's patches were determined by flow cytometry. Ussing chambers were used to measure intestinal permeability and bacterial translocation. Finally, to explore the impact of colonisation with mother's microbiota at birth, analyses were also performed in Nod2 KO and WT mice born from WT surrogate mothers after embryo transfer. RESULTS: Nod2 KO mice exhibited colonic bacterial dysbiosis different from WT and Nod1 KO mice. Altered expression of antimicrobial peptides and mucins in ileum and colon was associated with the microbial composition. Bacterial composition of Nod2 KO and WT mice obtained by embryo transfer was similar to that observed in Nod2 KO mice, arguing for a dominant effect of Nod2 KO-associated dysbiosis. In contrast, increased levels of CD4+ T cells and gut barrier defects across Peyer's patches were specific to Nod2 deficiency and independent of Microbial dysbiosis. CONCLUSIONS: Nod2 deficiency is associated with a specific dominant dysbiosis which does not drive mucosal tissue and immune alterations.