FRMBP2 directs NOD2 to the membrane.

T he immune system acts as a shield against harmful microbes and requires exquisite control of the activation and resolution of multiple signaling pathways. Tight regulation of these processes is required not only to produce an effective response, but also to maintain immune homeostasis and prevent autoinflammatory disease. In the intestine, this process is further complicated by the residence of a beneficial community of microbes. One major question is how the immune system determines “friend from foe,” restricting antimicrobial responses to the intestinal microbiota but retaining protective antimicrobial responses against pathogens. Answering this question is critical to uncovering mechanisms that go awry in autoinflammatory disease. In PNAS, Lipinsky et al. (1) provide mechanistic insights into how polarized intestinal epithelial cells restrict antibacterial responses to the basolateral compartment through directing the subcellular localization the bacterial sensor NOD2 (nucleotide-binding oligomerization domain-2). NOD2 is a member of the phylogenetically conserved NOD-like receptor family, which recognizes microbe-associated molecular patterns (i.e., bacterial peptidoglycan, flagellin, and so forth) and danger-associated molecular patterns (i.e., reactive oxygen species, ATP, and others.) in the cell cytosol (2). The NOD2 protein is primarily expressed in granulocytes, antigen-presenting cells, Paneth cells, and intestinal epithelial cells. NOD2 is activated by the muramyl dipeptide (MDP) component of the peptidoglycan cell wall present in most bacteria (3, 4). Like other NODlike receptors, NOD2 forms a dynamic macromolecular platform with multiple proteins upon activation, which dictates cellular responses to bacteria. These responses include the production of cytokines, chemokines, and antimicrobial peptides, activation of autophagy, and stimulation of antigen presentation. The critical role of NOD2 in immune homeostasis is demonstrated by the association of both gain-of-function and loss-of-function NOD2 genetic variants with chronic inflammatory diseases, such as Crohn disease and Blau syndrome. Because of its pivotal role in maintaining gut homeostasis, it is critical to understand the mechanisms involved in appropriate activation of NOD2-induced signaling. To this end, several global screening methodologies (i.e., yeast twohybrid, biochemical purification, and RNAi) have been applied to identify an expanding pool of potential NOD2 modulators. Although these studies have identified some key players involved in NOD2 signaling, they generally fail to provide a good picture of how these regulators affect NOD2 function in a physiologic context. Commonly, the analyses are performed in easy to manipulate but not functionally relevant cell types using overexpressed proteins. The intracellular location and integration of regulators with other known NOD2 pathway components is also not usually examined. Although these more informative studies are technically challenging because of the low expression of NOD2 and the poor quality of NOD2-specific reagents (i.e., antibodies), these types of analyses are essential to provide significant, physiologic insights and are what make the study by Lipinski et al. (1) outstanding. The authors initially perform multiple, systematic layers of screening and confirmation to identify novel components of NOD2 signaling, followed by functional studies in relevant cell types using a combination of cell biology, molecular, and biochemical techniques. The authors then relate these findings to alterations in NOD2 signaling observed in the inflammatory bowel disease, Crohn disease. In the report by Lipiniski et al. (1), the authors searched for proteins that modify NOD2 signaling using an RNAi libraryscreening approach. The authors performed an initial RNAi screen targeting a limited set of “druggable” genes in HEK293 cells expressing exogenous NOD2, and analyzed the effect of the RNAi knockdown on the activation of an NFκB reporter gene in response to the NOD2 ligand, MDP (screen 1). The initial pool of candidate genes was reconfirmed using the same system to rule out any nonreproducible results (screen 2). This process was followed by a third screen using a different RNAi library containing siRNAs designed by a different algorithm targeting the 69 reproducible candidates, resulting in 20 candidate-positive regulators of NFκB signaling (screen 3). Additionally, all of the data from these screens were subjected to network analysis to determine the relevance of the candidate genes to known NOD2 pathway components, which resulted in a stepwise increase in hits closer to the NOD2 Fig. 1. FRMPD2 localizes NOD2 to the basolateral membrane in polarized intestinal epithelial cells and dictates spatial specificity to NOD2 signaling. (A) Under normal conditions in the gut, NOD2 localizes to the basolateral membrane through interactions with FRMPD2 and is nonresponsive to commensal bacteria present on apical surfaces through an interaction with ERBB2IP. (B) When injury or pathogenic infection occurs, cells are exposed to bacteria (MDP) basolaterally. Membrane-associated NOD2 senses MDP and is released from ERBB2IP repression, which results in recruitment of RIP2 to the membrane, initiation of signaling through NFκB, and stimulation of a proinflammatory and antibacterial response. (C) In a disease context [i.e., FRMPD2 expression is decreased by inflammatory signals (Left) or a Crohn disease-associated NOD2 variant (L1007fs) is expressed], NOD2 is mislocalized and is unable to respond effectively and clear the bacterial infection.