Yasunori Ogura

A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease

Crohns disease is a chronic inflammatory disorder of the gastrointestinal tract, which is thought to result from the effect of environmental factors in a genetically predisposed host. A gene location in the pericentromeric region of chromosome 16, IBD1, that contributes to susceptibility to Crohns disease has been established through multiple linkage studies, but the specific gene(s) has not been identified. NOD2, a gene that encodes a protein with homology to plant disease resistance gene products is located in the peak region of linkage on chromosome 16 (ref. 7). Here we show, by using the transmission disequilibium test and case-control analysis, that a frameshift mutation caused by a cytosine insertion, 3020insC, which is expected to encode a truncated NOD2 protein, is associated with Crohns disease. Wild-type NOD2 activates nuclear factor NF-κB, making it responsive to bacterial lipopolysaccharides; however, this induction was deficient in mutant NOD2. These results implicate NOD2 in susceptibility to Crohns disease, and suggest a link between an innate immune response to bacterial components and development of disease.

Nod2, a Nod1/Apaf-1 Family Member That Is Restricted to Monocytes and Activates NF-κB

Apaf-1 and Nod1 are members of a protein family, each of which contains a caspase recruitment domain (CARD) linked to a nucleotide-binding domain, which regulate apoptosis and/or NF-κB activation. Nod2, a third member of the family, was identified. Nod2 is composed of two N-terminal CARDs, a nucleotide-binding domain, and multiple C-terminal leucine-rich repeats. Although Nod1 and Apaf-1 were broadly expressed in tissues, the expression of Nod2 was highly restricted to monocytes. Nod2 induced nuclear factor κB (NF-κB) activation, which required IKKγ and was inhibited by dominant negative mutants of IκBα, IKKα, IKKβ, and IKKγ. Nod2 interacted with the serine-threonine kinase RICK via a homophilic CARD-CARD interaction. Furthermore, NF-κB activity induced by Nod2 correlated with its ability to interact with RICK and was specifically inhibited by a truncated mutant form of RICK containing its CARD. The identification of Nod2 defines a subfamily of Apaf-1-like proteins that function through RICK to activate a NF-κB signaling pathway.

An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid

Nucleotide-binding oligomerization domain protein 1 (NOD1) belongs to a family that includes multiple members with NOD and leucine-rich repeats in vertebrates and plants. NOD1 has been suggested to have a role in innate immune responses, but the mechanism involved remains unknown. Here we report that NOD1 mediates the recognition of peptidoglycan derived primarily from Gram-negative bacteria. Biochemical and functional analyses using highly purified and synthetic compounds indicate that the core structure recognized by NOD1 is a dipeptide, γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP). Murine macrophages deficient in NOD1 did not secrete cytokines in response to synthetic iE-DAP and did not prime the lipopolysaccharide response. Thus, NOD1 mediates selective recognition of bacteria through detection of iE-DAP-containing peptidoglycan.*Note: In the version of this article initially published online, one authors first name and last name were reversed. The correct author name should be Su Qiu. This mistake has been corrected for the HTML and print versions of the article.

Inflammasome recognition of influenza virus is essential for adaptive immune responses

Influenza virus infection is recognized by the innate immune system through Toll like receptor (TLR) 7 and retinoic acid inducible gene I. These two recognition pathways lead to the activation of type I interferons and resistance to infection. In addition, TLR signals are required for the CD4 T cell and IgG2a, but not cytotoxic T lymphocyte, responses to influenza virus infection. In contrast, the role of NOD-like receptors (NLRs) in viral recognition and induction of adaptive immunity to influenza virus is unknown. We demonstrate that respiratory infection with influenza virus results in the activation of NLR inflammasomes in the lung. Although NLRP3 was required for inflammasome activation in certain cell types, CD4 and CD8 T cell responses, as well as mucosal IgA secretion and systemic IgG responses, required ASC and caspase-1 but not NLRP3. Consequently, ASC, caspase-1, and IL-1R, but not NLRP3, were required for protective immunity against flu challenge. Furthermore, we show that caspase-1 inflammasome activation in the hematopoietic, but not stromal, compartment was required to induce protective antiviral immunity. These results demonstrate that in addition to the TLR pathways, ASC inflammasomes play a central role in adaptive immunity to influenza virus.

The NLR gene family: a standard nomenclature.

Iimmune regulatory proteins such as CIITA, NAIP, IPAF, NOD1, NOD2, NALP1, cryopyrin/NALP3 are members of a family characterized by the presence of a nucleotide-binding domain (NBD) and leucine-rich repeats (LRR). Members of this gene family encode a protein structure similar to the NB-LRR subgroup of disease-resistance genes in plants and are involved in the sensing of pathogenic products and the regulation of cell signaling and apoptosis. Several members of this family have been associated with immunologic disorders. NOD2 for instance is associated with both Crohns disease and Blau syndrome. A variety of different names are currently used to describe this gene family, its subfamilies and individual genes, including CATERPILLER (CLR), NOD-LRR, NACHT-LRR, CARD, NALP, NOD, PAN and PYPAF, and this lack of consistency has led to a pressing need to unify the nomenclature. Consequently, we collectively propose the family designation NLR (nucleotide-binding domain and leucine-rich repeat containing) and provide unique and standardized gene designations for all family members.

Crohn’s disease and the NOD2 gene: a role for paneth cells

BACKGROUND & AIMS The NOD2 gene, which is strongly associated with susceptibility to Crohns disease (CD) of the terminal ileum, interacts with bacterial lipopolysaccharide (LPS), inducing cellular activation. However, the mechanisms by which NOD2 mutations cause terminal ileitis are unknown, and NOD2 is expressed most highly by peripheral blood monocytes, which are distributed ubiquitously and readily respond to LPS via cell-surface receptors. Paneth cells on the other hand, are most numerous in the terminal ileum, are critically important in enteric antibacterial defense, and respond to LPS through as yet undefined pathways. We therefore determined if these specialized intestinal epithelial cells also expressed the NOD2 gene. METHODS In situ hybridization, immunohistochemistry, and laser-capture microdissection were used to determine RNA and protein expression in tissue sections, and real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to quantitate gene expression in intestinal epithelial cells and peripheral blood mononuclear cells. RESULTS NOD2 was detected readily in monocytes, but not in mature macrophages in the lamina propria or within granulomas, and levels declined as monocytes differentiated into macrophages in vitro, so that Caco-2 cells expressed more NOD2 mRNA than macrophages. NOD2 mRNA was enriched in crypts compared with villi, and in situ, Paneth cells were the most prominent cells expressing NOD2 in normal and CD-affected intestinal tissue, where they also strongly expressed tumor necrosis factor alpha (TNFalpha) RNA. CONCLUSIONS The NOD2 gene product is most abundant in Paneth cells in the terminal ileum, which could therefore play a critical and hitherto unrecognized role in the pathogenesis of NOD2-associated CD.

Induction of Nod2 in Myelomonocytic and Intestinal Epithelial Cells via Nuclear Factor-κB Activation

Nod2, a member of the Apaf1/Nod protein family, confers responsiveness to bacterial products and activates NF-κB, a transcription factor that plays a central role in innate immunity. Recently, genetic variation in Nod2 has been associated with susceptibility to Crohns disease. Here, we report that expression of Nod2 is induced upon differentiation of CD34+hematopoietic progenitor cells into granulocyte or monocyte/macrophages. In peripheral blood cells, the highest levels of Nod2 were observed in CD14+ (monocytes), CD15+(granulocytes), and CD40+/CD86+ (dendritic cells) cell populations. Notably, stimulation of myeloblastic and epithelial cells with bacterial lipopolysaccharide or TNFα resulted in up-regulation of Nod2. A search for consensus sites within the Nod2 promoter revealed a NF-κB binding element that was required for transcriptional activity in response to TNFα. Moreover, ectopic expression of p65 induced transactivation, whereas that of dominant-negative IκBα blocked the transcriptional activity of the Nod2 promoter. Upon stimulation with TNFα or lipopolysaccharide, both p50 and p65 subunits of NF-κB were bound to the Nod2 promoter. Thus, Nod2 expression is enhanced by proinflammatory cytokines and bacterial components via NF-κB, a mechanism that may contribute to the amplification of the innate immune response and susceptibility to inflammatory disease.

Immune recognition of Pseudomonas aeruginosa mediated by the IPAF/NLRC4 inflammasome

Pseudomonas aeruginosa is a Gram-negative bacterium that causes opportunistic infections in immunocompromised individuals. P. aeruginosa employs a type III secretion system to inject effector molecules into the cytoplasm of the host cell. This interaction with the host cell leads to inflammatory responses that eventually result in cell death. We show that infection of macrophages with P. aeruginosa results in activation of caspase-1 in an IPAF-dependent, but flagellin-independent, manner. Macrophages deficient in IPAF or caspase-1 were markedly resistant to P. aeruginosa–induced cell death and release of the proinflammatory cytokine interleukin (IL)-1β. A subset of P. aeruginosa isolates express the effector molecule exoenzyme U (ExoU), which we demonstrate is capable of inhibiting caspase-1–driven proinflammatory cytokine production. This study shows a key role for IPAF and capase-1 in innate immune responses to the pathogen P. aeruginosa, and also demonstrates that virulent ExoU-expressing strains of P. aeruginosa can circumvent this innate immune response.

CorrespondenceThe NLR Gene Family: A Standard Nomenclature

Iimmune regulatory proteins such as CIITA, NAIP, IPAF, NOD1, NOD2, NALP1, cryopyrin/NALP3 are members of a family characterized by the presence of a nucleotide-binding domain (NBD) and leucine-rich repeats (LRR). Members of this gene family encode a protein structure similar to the NB-LRR subgroup of disease-resistance genes in plants and are involved in the sensing of pathogenic products and the regulation of cell signaling and apoptosis. Several members of this family have been associated with immunologic disorders. NOD2 for instance is associated with both Crohns disease and Blau syndrome. A variety of different names are currently used to describe this gene family, its subfamilies and individual genes, including CATERPILLER (CLR), NOD-LRR, NACHT-LRR, CARD, NALP, NOD, PAN and PYPAF, and this lack of consistency has led to a pressing need to unify the nomenclature. Consequently, we collectively propose the family designation NLR (nucleotide-binding domain and leucine-rich repeat containing) and provide unique and standardized gene designations for all family members.

Expression of NOD2 in Paneth cells: a possible link to Crohn’s ileitis

Background and aims: Genetic variation in NOD2 has been associated with susceptibility to Crohn’s disease (CD) and specifically with ileal involvement. The reason for the unique association of NOD2 mutations with ileal disease is unclear. To identify a possible link, we tested expression of NOD2 in intestinal tissue of CD patients and controls. Patients and methods: Fifty five specimens of ileum or colon from 21 CD patients, seven ulcerative colitis (UC) patients, and five controls with pathology other than CD or UC were stained for NOD2 using an immunoperoxidase method. Results: Using a monoclonal antibody against NOD2 developed in our laboratory, we detected uniform expression of NOD2 in terminal ileum Paneth cells from controls and patients as well as in metaplastic Paneth cells in the colon. Mechanical purification showed enriched expression of NOD2 mRNA in ileal crypts. In Paneth cells, NOD2 was located in the cytosol in close proximity to the granules that contain antimicrobial peptides. We detected minimal NOD2 in the villous epithelium of the ileum or in the colonic epithelium from both CD patients and controls. Conclusions: These results suggest a role for NOD2 in the regulation of Paneth cell mediated responses against intestinal bacteria and a plausible mechanism to explain the selective association of NOD2 mutations with ileal disease. The impaired capacity of CD associated mutations to sense luminal bacteria may result in increased susceptibility to certain gut microbes.