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RNAi screening identifies mediators of NOD2 signaling: implications for spatial specificity of MDP recognition.

ABSTRACT: The intracellular nucleotide-binding oligomerization domain-2 (NOD2) receptor detects bacteria-derived muramyl dipeptide (MDP) and activates the transcription factor NF-?B. Here we describe the regulatome of NOD2 signaling using a systematic RNAi screen. Using three consecutive screens, we identified a set of 20 positive NF-?B regulators including the known pathway members RIPK2, RELA, and BIRC4 (XIAP) as well as FRMPD2 (FERM and PDZ domain-containing 2). FRMPD2 interacts with NOD2 via leucine-rich repeats and forms a complex with the membrane-associated protein ERBB2IP. We demonstrate that FRMPD2 spatially assembles the NOD2-signaling complex, hereby restricting NOD2-mediated immune responses to the basolateral compartment of polarized intestinal epithelial cells. We show that genetic truncation of the NOD2 leucine-rich repeat domain, which is associated with Crohn disease, impairs the interaction with FRMPD2, and that intestinal inflammation leads to down-regulation of FRMPD2. These results suggest a structural mechanism for how polarity of epithelial cells acts on intestinal NOD-like receptor signaling to mediate spatial specificity of bacterial recognition and control of immune responses.

SUBMITTER: Lipinski S

PROVIDER: S-EPMC3535590 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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RNAi screening identifies mediators of NOD2 signaling: implications for spatial specificity of MDP recognition.

Lipinski Simone S Grabe Nils N Jacobs Gunnar G Billmann-Born Susanne S Till Andreas A Häsler Robert R Aden Konrad K Paulsen Maren M Arlt Alexander A Kraemer Lars L Hagemann Nina N Erdmann Kai Sven KS Schreiber Stefan S Rosenstiel Philip P

Proceedings of the National Academy of Sciences of the United States of America 20121203 52

The intracellular nucleotide-binding oligomerization domain-2 (NOD2) receptor detects bacteria-derived muramyl dipeptide (MDP) and activates the transcription factor NF-κB. Here we describe the regulatome of NOD2 signaling using a systematic RNAi screen. Using three consecutive screens, we identified a set of 20 positive NF-κB regulators including the known pathway members RIPK2, RELA, and BIRC4 (XIAP) as well as FRMPD2 (FERM and PDZ domain-containing 2). FRMPD2 interacts with NOD2 via leucine-r ...[more]

PMID: 23213202

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Project description:Background & aimsNucleotide oligomerization domain 2 (Nod2) polymorphisms increase the risk of developing Crohn's disease, which is characterized by chronic intestinal inflammation. Bacterial peptidoglycan products chronically stimulate Nod2 in the intestine. Recent studies found that chronic Nod2 stimulation in human macrophages down-regulates proinflammatory cytokines on Nod2 or Toll-like receptor (TLR) restimulation. Therefore, an emerging hypothesis is that Nod2-mediated cytokine down-regulation is required for intestinal homeostasis, but the mechanisms mediating this down-regulation are incompletely understood.MethodsUtilizing primary human macrophages, we examined secretory mediators as a mechanism of Nod2-mediated tolerance by inhibiting their function and assessing tolerance reversal through cytokine secretion. Signaling pathways contributing to secretory mediator induction and Nod2-mediated tolerance were identified through pathway inhibition.ResultsWe found that chronic Nod2 stimulation cross-tolerizes not only to TLRs but also to the interleukin (IL)-1 receptor. Moreover, chronic IL-1β stimulation down-regulates Nod2 responses. Accordingly, IL-1β blockade partially reverses Nod2-mediated tolerance. We found that an additional essential mechanism for Nod2-mediated tolerance is the early secretion of the anti-inflammatory mediators IL-10, transforming growth factor β, and IL-1Ra. Importantly, the mammalian target of rapamycin (mTOR) pathway, involved in cell growth, differentiation, and activation, significantly contributes to Nod2-induced anti-inflammatory as opposed to proinflammatory cytokines and to Nod2-mediated tolerance.ConclusionsInflammatory responses through the IL-1R are down-regulated upon chronic Nod2 stimulation, secretory mediators are a critical mechanism for Nod2-mediated cytokine down-regulation, and the mTOR pathway is crucial for Nod2-mediated tolerance. These results further contribute to our understanding of the mechanisms through which Nod2, a protein critical to intestinal homeostasis, down-regulates cytokine responses.

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RNAi screening identifies mediators of NOD2 signaling: implications for spatial specificity of MDP recognition.

Publications

RNAi screening identifies mediators of NOD2 signaling: implications for spatial specificity of MDP recognition.

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