© 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 Immunol, 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...


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


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."



© 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."

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