Giving a Nod2 the Right Target

A MODEL FOR NOD: © 2003 Nature Publishing Group Phagocytosed microorganisms release ligands recognized by NOD proteins and peptides that associate with MHC II molecules. Recognition of intracellular ligands by NOD proteins and extracellular ligands by Toll-like receptors mediates the activation of signaling pathways including those downstream of NF-κB like cytokine secretion, and co-stimulatory receptor expression (e.g. CD80 and CD86). (From N. Inohara, G. Nuñez, Nat Rev Immuno l, 3:371–82, 2003.) Nod1 and Nod2 are no strangers to Hot Papers. Nor is Gabriel Nuñez, the University of Michigan pathologist whose back-to-back discoveries concerning the cytosolic proteins were featured last year. The 2001 papers by Nuñez and colleagues demonstrated Nod1 and Nod2 (also referred to as CARD15 and CARD4, respectively) sense intracellular pathogens and activate NF-κB, a transcription factor central to immunoregulation and inflammation, and that mutations in the Nod2 gene were a risk factor for Crohn disease.1 At the time, there was some controversy as to what the Nod proteins sensed. Nuñez's assertion that the target was lipopolysaccharide (LPS), a bacterial cell-wall constituent, met with criticism from scientists who study LPS sensing. Data derived from the Science Watch/Hot Papers database and the Web of Science (Thomson Scientific, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. "Host recognition of bacterial muramyl dipeptide mediated through Nod2," Inohara N, J Biol Chem , 2003 Vol 278, 5509-12 (Cited in 115 papers) (HistCite Analysis) "Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection," Girardin SE, J Biol Chem , 2003 Vol 278, 8869-72 (Cited in 113 papers) (HistCite Analysis) In this issue's Hot Papers, Nuñez's group,2 as well as Dana Philpott and colleagues at the Pasteur Institute and INSERM,3 Paris, show that Nod2 senses a contaminant of LPS preparations, a peptidoglycan motif called muramyl dipeptide. MDP is also found in the cell walls of both Gram-positive and Gram-negative bacteria, and was recognized for years as a potent adjuvant. Both papers, published in back-to-back issues of the Journal of Biological Chemistry, used transfection studies to show that Nod2 recognizes MDP and that Crohn disease-associated mutants are defective in the MDP response. Both papers inspired a disparate group of researchers, including gastroenterologists, geneticists, and immunologists, to take a closer look at Nod2's function in innate immunity and inflammatory bowel disease. "We cloned this gene in 2001 and have given this plasmid to 100 laboratories. This is the most popular plasmid that I have," notes Nuñez. The identification of MDP as the target for Nod2 activation led to new types of research on the mechanism of Crohn disease. "The MDP discovery has altered the experiments that one does, it's altered the concept of what the pathogen might be in Crohn's disease, [and] it's altered what experiments one might do in vitro to see the factors that are responsible for Nod2 activation," comments gastroenterologist Jack Satsangi of the University of Edinburgh. The discovery has also inspired a new generation of researchers to focus more closely on the Nod proteins and related protein families. "Since those papers came out there's been much more focus on [Nod proteins] as key sensors of bacteria," says Luke O'Neill of Trinity College, Dublin. Whereas prior researchers looked at membrane-bound Toll-like receptors (TLRs) as the main drivers of the innate immune response, cytosolic Nod proteins represented a novel set of receptors. "Many people have built on that Nod2 paper to study some of their own bacterial molecules, or to study the function that Nod2 might have more broadly during host defense," says O'Neill. A FAMILY AFFAIR Philpott and coworkers used Nod1 knockout mice to study how this molecule recognizes the extracellular Gram-negative gut bacterium Helicobacter pylori in epithelial cells. They found that like Nod2, Nod1 responds to a breakdown product of bacterial peptidoglycan (specifically, a muropeptide fragment called GM-tri-DAP) by activating the NF-κB signaling pathway.4 "It's an important publication, because it integrates Nod1 into the story ... in relation to host defense against infection," says Warren Strober of the National Institute of Allergy and Infectious Diseases. Along similar lines, research by Jurg Tschopp and colleagues at the University of Lausanne, Switzerland, demonstrated another role for MDP: activation of the NALP3 inflammasome. NALPs are a family of proteins structurally similar to Nods. that NALPs contain leucine-rich repeats (LRRs), which trigger the innate immune response by detecting specific bacterial components. But unlike the Nod proteins, "the NALP proteins ... don't really activate NF-κB and inflammation transcriptionally; they induce inflammation by increasing the processing of interleukin [IL]-1-β," explains Philpott. Moreover, MDP triggers both responses. Tschopp and coworkers showed that addition of synthetic MDP to THP-1 cells increased secretion of pro-IL-1β as a result of NALP3 activation.6 SOLVING A PARADOX The implication of Nod2's role in Crohn disease led to an apparent paradox: Cells that harbor mutations in the MDP recognition site of Nod2 do not activate NF-κB, a major component of the inflammatory signaling pathway. Nevertheless, patients with Crohn disease have more inflammation as a result of increased NF-κB activation. An early explanation suggested that because defective Nod2 leads to lower NF-κB activation, organisms that infect the gut proliferate and cause inflammation through a pathway not involving Nod2. But recent work by Strober and colleagues led to a different explanation. Using experiments in Nod2 knockout mice, Strober showed that Nod2 modulates activation of NF-κB in response to ligands to toll-like receptor 2 (TLR-2).5 In this pathway, peptidoglycan stimulates NF-κB in macrophages via TLR-2 and is then taken up into endosomes and broken down to release MDP. MDP then stimulates Nod2, which modulates the TLR-2 response by decreasing NF-κB activation. "Ordinarily, you react to peptidoglycan present in the gut, and that reaction occurring through TLR-2 is modified and actually downregulated through Nod2," says Strober. In the absence of downregulation, responses to peptidoglycan increase, leading to increased production of IL-12 and IL-18 and the development of disease in patients. Philpott explains that Strober's work demonstrates a relationship between toll-like receptors, which are membrane-based and detect extracellular microbial products, and cytoplasmic Nod proteins. "What his paper showed is that there's a tight interaction between these two systems, where Nod2 can influence the signaling downstream of toll-like receptor 2 ... showing that there's a cross-talk between these two sensing systems," she says. Although some contradictory data exist in the literature, Strober's knockout experiments offer the best explanation for how Nod2 mutants increase inflammatory activity in the gut, says Satsangi. "To my mind that's the best hypothesis that we have at the present time." WHAT'S NEXT INFLAMMATION WITHOUT NOD: © 2004 Nature Publishing Group (A) Some data support a model whereby NOD2 senses MDP, a in the cytosol limiting TLR2 signaling activated at the cell surface. This affects NF-κB activation leading to modulation in IL-12 production. (B) Mutant NOD2 in Crohn disease, however, is unable to sense MDP, removing this constraint on the TLR2 pathway and enhancing IL-12 production. (From L.A.J. O'Neil, Nat Immunol, 5:776–8, 2004.) One central question about the mechanism of Nod2 recognition of MDP remained unanswered after Nuñez and Philpott's initial discovery. "One difficulty with respect to MDP was how it would get into the cell to be involved in activating Nod2, which is purely an intracellular protein," says Satsangi. This problem recently was addressed by a group of researchers at the University of Chicago, who found that the intestinal transporter hPepT1 transports MDP, allowing its uptake by intestinal epithelial cells.7 Other questions remain. Researchers are still investigating the identity of other proteins that interact with the Nod proteins to initiate inflammation. "We're still uncertain as to whether another mechanism is being missed completely," says Satsangi, who adds that his laboratory has identified several promising candidates that interact directly with Nod2. At least one competing theory has been suggested regarding the role of Nod2 deficiency in the development of Crohn disease. Some have noted Nod2 expression in paneth cells, which are located at the base of intestinal crypts and produce small antibacterial molecules called a-defensins.8This indicates that in the absence of Nod2, signaling results in fewer a-defensins being produced, and more infection occurs at the base of the crypts, leading to disease. "It's not a bad idea, but it's unproven. And it's not as compelling as the effect of Nod2 deficiency on antigen-presenting cells," says Strober. Recent knockout mouse work from Nuñez's lab and Micheal Karin's group at the University of California, San Diego, supports a defensin-deficiency hypothesis.910 Others point out that Nod2 is not the only part of the Crohn disease story, as only 15% of patients with the disease harbor mutations in the NOD2 gene. "We know that there must be other genes involved, because simply having mutations in NOD2 in humans or in animals isn't sufficient to give you inflammatory bowel disease," says Bruce Beutler of the Scripps Institute, La Jolla, Calif. "It's a polygenic disease," he adds, "and so we'd very much like to know what else is going on to create the conditions whereby these mutations can cause disease."

Giving a Nod2 the Right Target

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Aileen Constans

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February 2005

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