Joao G. Magalhaes

Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry

Autophagy is emerging as a crucial defense mechanism against bacteria, but the host intracellular sensors responsible for inducing autophagy in response to bacterial infection remain unknown. Here we demonstrated that the intracellular sensors Nod1 and Nod2 are critical for the autophagic response to invasive bacteria. By a mechanism independent of the adaptor RIP2 and transcription factor NF-κB, Nod1 and Nod2 recruited the autophagy protein ATG16L1 to the plasma membrane at the bacterial entry site. In cells homozygous for the Crohns disease–associated NOD2 frameshift mutation, mutant Nod2 failed to recruit ATG16L1 to the plasma membrane and wrapping of invading bacteria by autophagosomes was impaired. Our results link bacterial sensing by Nod proteins to the induction of autophagy and provide a functional link between Nod2 and ATG16L1, which are encoded by two of the most important genes associated with Crohns disease.

NLRX1 is a mitochondrial NOD-like receptor that amplifies NF-κB and JNK pathways by inducing reactive oxygen species production

NOD‐like receptors (NLRs) are a family of intracellular sensors of microbial‐ or danger‐associated molecular patterns. Here, we report the identification of NLRX1, which is a new member of the NLR family that localizes to the mitochondria. NLRX1 alone failed to trigger most of the common signalling pathways, including nuclear factor‐κB (NF)‐κB‐ and type I interferon‐dependent cascades, but could potently trigger the generation of reactive oxygen species (ROS). Importantly, NLRX1 synergistically potentiated ROS production induced by tumour necrosis factor α, Shigella infection and double‐stranded RNA, resulting in amplified NF‐κB‐dependent and JUN amino‐terminal kinases‐dependent signalling. Together, these results identify NLRX1 as a NLR that contributes to the link between ROS generation at the mitochondria and innate immune responses.

Identification of an innate T helper type 17 response to intestinal bacterial pathogens

Interleukin 17 (IL-17) is a central cytokine implicated in inflammation and antimicrobial defense. After infection, both innate and adaptive IL-17 responses have been reported, but the type of cells involved in innate IL-17 induction, as well as their contribution to in vivo responses, are poorly understood. Here we found that Citrobacter and Salmonella infection triggered early IL-17 production, which was crucial for host defense and was mediated by CD4+ T helper cells. Enteric innate T helper type 17 (iTH17) responses occurred principally in the cecum, were dependent on the Nod-like receptors Nod1 and Nod2, required IL-6 induction and were associated with a decrease in mucosal CD103+ dendritic cells. Moreover, imprinting by the intestinal microbiota was fully required for the generation of iTH17 responses. Together, these results identify the Nod-iTH17 axis as a central element in controlling enteric pathogens, which may implicate Nod-driven iTH17 responses in the development of inflammatory bowel diseases.

Nod-like proteins in inflammation and disease.

The field of innate immunity has undergone an enormous upheaval during the last decade. The discovery of different groups of proteins, called pattern recognition molecules (PRMs), which detect microbial components, so‐called pathogen‐associated molecular patterns (PAMPs) and trigger protective responses, had a huge impact on the understanding of innate immune responses. Among the PRMs, the intracellular Nod‐like receptors (NLRs) have recently been identified as key mediators of inflammatory and immune responses. The NLR family is divided into subfamilies on the basis of their different signal transduction domains, and recent studies have highlighted the role of certain NLRs, including Nod1, Nod2, Nalp3, Ipaf and Naip5, in the detection of intracellular microbes and possibly ‘danger signals’. In this review, we summarize the current knowledge on the function of these proteins in immunity and inflammation, with a focus on their participation in different disease pathologies. Copyright

Nod2-Dependent Th2 Polarization of Antigen-Specific Immunity

While a number of microbial-associated molecular patterns have been known for decades to act as adjuvants, the mechanisms and the signaling pathways underlying their action have remained elusive. Here, we examined the unfolding of the adaptive immune response induced by Nod2 in vivo upon activation by its specific ligand, muramyl dipeptide, a component of peptidoglycan. Our findings demonstrate that this bacterial sensor triggers a potent Ag-specific immune response with a Th2-type polarization profile, characterized by the induction of IL-4 and IL-5 by T cells and IgG1 Ab responses. Nod2 was also found to be critical for the induction of both Th1- and Th2-type responses following costimulation with TLR agonists. Importantly, the synergistic responses to Nod2 and TLR agonists seen in vivo were recapitulated by dendritic cells in vitro, suggesting that these cells likely play a central role in the integration of Nod2- and TLR-dependent signals for driving the adaptive immune response. Taken together, our results identify Nod2 as a critical mediator of microbial-induced potentiation and polarization of Ag-dependent immunity. Moreover, these findings affect our understanding of Crohn’s diseases pathogenesis, where lack of Nod2-dependent Th2 signaling in a subset of these patients might explain heightened Th1-mediated inflammation at the level of the intestinal mucosa.

Murine Nod1 but not its human orthologue mediates innate immune detection of tracheal cytotoxin

Tracheal cytotoxin (TCT) was originally described as the minimal effector that was able to reproduce the cytotoxic response of Bordetella pertussis on ciliated epithelial cells. This molecule triggers pleiotropic effects such as immune stimulation or slow‐wave sleep modulation. Further characterization identified TCT as a specific diaminopimelic acid (DAP)‐containing muropeptide, GlcNAc‐(anhydro)MurNAc‐L‐Ala‐D‐Glu‐mesoDAP‐D‐Ala. Here, we show that the biological activity of TCT depends on Nod1, an intracellular sensor of bacterial peptidoglycan. However, Nod1‐dependent detection of TCT was found to be host specific, as human Nod1 (hNod1) poorly detected TCT, whereas mouse Nod1 (mNod1) did so efficiently. More generally, hNod1 required a tripeptide (L‐Ala‐D‐Glu‐mesoDAP) for efficient sensing of peptidoglycan, whereas mNod1 detected a tetrapeptide structure (L‐Ala‐D‐Glu‐mesoDAP‐D‐Ala). In murine macrophages, TCT stimulated cytokine secretion and NO production through Nod1. Finally, in vivo, injection of the tetrapeptide structure in mice triggered a transient yet strong release of cytokines into the bloodstream and the maturation of macrophages, in a Nod1‐dependent manner. This study thereby identifies Nod1 as the long sought after sensor of TCT in mammals.

NLRC5 Limits the Activation of Inflammatory Pathways

Nod-like receptors (NLRs) are intracellular sentinel proteins that are implicated in the detection of microbes and danger signals, thereby controlling several key innate immune pathways. The human genome encodes 22 NLR proteins, the function of many of which remains unknown. In this study, we present the identification and characterization of NLRC5, a NLR protein whose expression is found predominantly in cells of the myeloid and lymphoid lineages. NLRC5 expression was strongly induced by IFN-γ and more modestly by LPS and polyinosinic:polycytidylic acid. Overexpression of NLRC5 in HEK293T cells resulted in a global dampening of NF-κB–, AP-1–, and type I IFN-dependent signaling, most likely through transcriptional repression. Accordingly, NLRC5 was found to shuttle between the cytosol and the nucleus in a CrmA-dependent manner. Knocking down NLRC5 expression in RAW264.7 murine macrophages resulted in a potent upregulation of the proinflammatory responses to IFN-γ and LPS, including increased secretion of TNF, IL-6, and IL-1β, as well as cell surface expression of CD40. Strikingly, NLRC5 expression was also found to be critical for LPS-induced IL-10 production in RAW264.7 macrophages. Collectively, our results identify NLRC5 as a negative modulator of inflammatory pathways.

NOD1 Activators Link Innate Immunity to Insulin Resistance

OBJECTIVE Insulin resistance associates with chronic inflammation, and participatory elements of the immune system are emerging. We hypothesized that bacterial elements acting on distinct intracellular pattern recognition receptors of the innate immune system, such as bacterial peptidoglycan (PGN) acting on nucleotide oligomerization domain (NOD) proteins, contribute to insulin resistance. RESEARCH DESIGN AND METHODS Metabolic and inflammatory properties were assessed in wild-type (WT) and NOD1/2−/− double knockout mice fed a high-fat diet (HFD) for 16 weeks. Insulin resistance was measured by hyperinsulinemic euglycemic clamps in mice injected with mimetics of meso-diaminopimelic acid–containing PGN or the minimal bioactive PGN motif, which activate NOD1 and NOD2, respectively. Systemic and tissue-specific inflammation was assessed using enzyme-linked immunosorbent assays in NOD ligand–injected mice. Cytokine secretion, glucose uptake, and insulin signaling were assessed in adipocytes and primary hepatocytes exposed to NOD ligands in vitro. RESULTS NOD1/2−/− mice were protected from HFD-induced inflammation, lipid accumulation, and peripheral insulin intolerance. Conversely, direct activation of NOD1 protein caused insulin resistance. NOD1 ligands induced peripheral and hepatic insulin resistance within 6 h in WT, but not NOD1−/−, mice. NOD2 ligands only modestly reduced peripheral glucose disposal. NOD1 ligand elicited minor changes in circulating proinflammatory mediators, yet caused adipose tissue inflammation and insulin resistance of muscle AS160 and liver FOXO1. Ex vivo, NOD1 ligand caused proinflammatory cytokine secretion and impaired insulin-stimulated glucose uptake directly in adipocytes. NOD1 ligand also caused inflammation and insulin resistance directly in primary hepatocytes from WT, but not NOD1−/−, mice. CONCLUSIONS We identify NOD proteins as innate immune components that are involved in diet-induced inflammation and insulin intolerance. Acute activation of NOD proteins by mimetics of bacterial PGNs causes whole-body insulin resistance, bolstering the concept that innate immune responses to distinctive bacterial cues directly lead to insulin resistance. Hence, NOD1 is a plausible, new link between innate immunity and metabolism.

Shigella Induces Mitochondrial Dysfunction and Cell Death in Nonmyleoid Cells

Shigella rapidly kills myeloid cells via a caspase-1 inflammasome-dependent cell death mechanism. However, despite a critical role for nonmyeloid cells in the physiopathology of Shigella infection, the mechanism by which Shigella kills nonmyeloid cells remains uncharacterized. Here we demonstrate that, in nonmyeloid cells, Shigella infection induces loss of mitochondrial inner membrane potential, mitochondrial damage, and necrotic cell death through a pathway dependent on Bnip3 and cyclophilin D, two molecules implicated in the host oxidative stress responses. This mitochondrial cell death mechanism was potently counterbalanced by a Nod1-dependent Rip2/IKKbeta/NF-kappaB signaling pathway activated by the pathogen in the first hours of infection. Our results suggest that in nonmyeloid cells, oxidative stress pathways and signaling triggered by an intracellular bacterial pathogen are tightly linked and demonstrate the existence of specific Shigella-induced prodeath and prosurvival pathways converging at the mitochondria to control a necrotic cell death program.

Nod1 and Nod2 Regulation of Inflammation in the Salmonella Colitis Model

ABSTRACT The pattern recognition molecules Nod1 and Nod2 play important roles in intestinal homeostasis; however, how these proteins impact on the development of inflammation during bacterial colitis has not been examined. In the streptomycin-treated mouse model of Salmonella colitis, we found that mice deficient for both Nod1 and Nod2 had attenuated inflammatory pathology, reduced levels of inflammatory cytokines, and increased colonization of the mucosal tissue. Nod1 and Nod2 from both hematopoietic and nonhematopoietic sources contributed to the pathology, and all phenotypes were recapitulated in mice deficient for the signaling adaptor protein Rip2. However, the influence of Rip2 was strictly dependent on infection conditions that favored expression of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS), as Rip2 was dispensable for inflammation when mice were infected with bacteria grown under conditions that promoted expression of the SPI-1 TTSS. Thus, Nod1 and Nod2 can modulate inflammation and mediate efficient clearance of bacteria from the mucosal tissue during Salmonella colitis, but their role is dependent on the expression of the SPI-2 TTSS.