Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor-{kappa}B activation in muramyl dipeptide recognition

Nucleotide oligomerization domain (NOD) 2 functions as a mammalian cytosolic pathogen recognition molecule, and mutant forms have been genetically linked to Crohn's disease (CD). NOD2 associates with the caspase activation and recruitment domain of RIP-like interacting caspase-like apoptosis regulatory protein kinase (RICK)/RIP2 and activates nuclear factor (NF)-kappaB in epithelial cells and macrophages, whereas NOD2 mutant 3020insC, which is associated with CD, shows an impaired ability to activate NF-kappaB. To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants. NOD2, but not NOD2 3020insC mutant, associated with cell surface membranes of intestinal epithelial cells. Membrane targeting and subsequent NF-kappaB activation are mediated by two leucine residues and a tryptophan-containing motif in the COOH-terminal domain of NOD2. The membrane targeting of NOD2 is required for NF-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.     

Centaurin beta1 down-regulates nucleotide-binding oligomerization domains 1- and 2-dependent NF-kappaB activation

Centaurin beta1 (CENTB1), a GTPase-activating protein, is a member of the ADP-ribosylation factor family encoded by a gene located on the short arm of human chromosome 17. A yeast two-hybrid screen first suggested a direct interaction between CENTB1 and NOD2. Co-immunoprecipitation experiments confirmed direct interaction between CENTB1 and NOD2 and demonstrated similar interaction between CENTB1 and NOD1. We also demonstrate that endogenous CENTB1 interacts with endogenous NOD2 and NOD1 in SW480 and HT-29 intestinal epithelial cells. CENTB1 partially co-localized with NOD2 and NOD1 proteins in the cytoplasm of mammalian cells. CENTB1 expression in epithelial cells was highly induced by tumor necrosis factor alpha, interleukin 1beta, and the NOD1 and NOD2 ligands (gamma-d-glutamyl-meso-diaminopimelic acid and muramyl dipeptide, respectively). In addition, CENTB1 mRNA level is increased in the inflamed mucosa of patients with inflammatory bowel disease. Functionally, CENTB1 overexpression inhibited NOD1- and NOD2-dependent activation of NF-kappaB, whereas small inhibitory RNA against CENTB1 increased NF-kappaB activation following NOD1- or NOD2-mediated recognition of the bacterial components gamma-d-glutamyl-meso-diaminopimelic acid and muramyl dipeptide, respectively. In contrast, CENTB1 had no effect on NF-kappaB activation induced by Toll-like receptors. In conclusion, CENTB1 selectively down-regulates NF-kappaB activation via NODs pathways, creating a "feedback" loop and suggesting a novel role of CENTB1 in innate immune responses to bacteria and inflammatory responses.     

A major role for intestinal epithelial nucleotide oligomerization domain 1 (NOD1) in eliciting host bactericidal immune responses to Campylobacter jejuni

Campylobacter jejuni is the foremost cause of bacterial-induced diarrhoeal disease worldwide. Although it is well established that C. jejuni infection of intestinal epithelia triggers host innate immune responses, the mechanism(s) involved remain poorly defined. Innate immunity can be initiated by families of structurally related pattern-recognition receptors (PRRs) that recognize specific microbial signature motifs. Here, we demonstrated maximal induction of epithelial innate responses during infection with live C. jejuni cells. In contrast when intestinal epithelial cells (IECs) were exposed to paraformaldehyde-fixed bacteria, host responses were minimal and a marked reduction in the number of intracellular bacteria was noted in parallel. These findings suggested a role for intracellular host-C. jejuni interactions in eliciting early innate immunity. We therefore investigated the potential involvement of a family of intracellular, cytoplasmic PRRs, the nucleotide-binding oligomerization domain (NOD) proteins in C. jejuni recognition. We identified NOD1, but not NOD2, as a major PRR for C. jejuni in IEC. We also found that targeting intestinal epithelial NOD1 with small interfering RNA resulted in an increase in number of intracellular C. jejuni, thus highlighting a critical role for NOD1-mediated antimicrobial defence mechanism(s) in combating this infection at the gastrointestinal mucosal surface.     

Regulation of DMBT1 via NOD2 and TLR4 in intestinal epithelial cells modulates bacterial recognition and invasion

Mucosal epithelial cell layers are constantly exposed to a complex resident microflora. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by pathogen binding. This report describes the regulation and function of DMBT1 in intestinal epithelial cells, which form the primary immunological barrier for invading pathogens. We report that intestinal epithelial cells up-regulate DMBT1 upon proinflammatory stimuli (e.g., TNF-alpha, LPS). We demonstrate that DMBT1 is a target gene for the intracellular pathogen receptor NOD2 via NF-kappaB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn's disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF-kappaB activation and cytokine secretion in vitro. Thus, DMBT1 may play an important role in the first line of mucosal defense conferring immune exclusion of bacterial cell wall components. Dysregulated intestinal DMBT1 expression due to mutations in the NOD2/CARD15 gene may be part of the complex pathophysiology of barrier dysfunction in Crohn's disease.     

NOD2/CARD15 mediates induction of the antimicrobial peptide human beta-defensin-2

Production of inducible antimicrobial peptides offers a first and rapid defense response of epithelial cells against invading microbes. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide induced in various epithelia upon extracellular as well as intracellular bacterial challenge. Nucleotide-binding oligomerization domain protein 2 (NOD2/CARD15) is a cytosolic protein involved in intracellular recognition of microbes by sensing peptidoglycan fragments (e.g. muramyl dipeptide). We used luciferase as a reporter gene for a 2.3-kb hBD-2 promoter to test the hypothesis that NOD2 mediates the induction of hBD-2. Activation of NOD2 in NOD2-overexpressing human embryonic kidney 293 cells through its ligand muramyl dipeptide (MDP) induced hBD-2 expression. In contrast, overexpression of NOD2 containing the 3020insC frame-shift mutation, the most frequent NOD2 variant associated with Crohn disease, resulted in defective induction of hBD-2 through MDP. Luciferase gene reporter analyses and site-directed mutagenesis experiments demonstrated that functional binding sites for NF-kappaB and AP-1 in the hBD-2 promoter are required for NOD2-mediated induction of hBD-2 through MDP. Moreover, the NF-kappaB inhibitor Helenalin as well as a super-repressor form of the NF-kappaB inhibitor IkappaB strongly inhibited NOD2-mediated hBD-2 promoter activation. Expression of NOD2 was detected in primary keratinocytes, and stimulation of these cells with MDP induced hBD-2 peptide release. In contrast, small interference RNA-mediated down-regulation of NOD2 expression in primary keratinocytes resulted in a defective induction of hBD-2 upon MDP treatment. Together, these data suggest that NOD2 serves as an intracellular pattern recognition receptor to enhance host defense by inducing the production of antimicrobial peptides such as hBD-2.     

The c-Jun N-terminal Kinase (JNK)-binding Protein (JNKBP1) Acts as a Negative Regulator of NOD2 Protein Signaling by Inhibiting Its Oligomerization Process

NOD2 is one of the best characterized members of the cytosolic NOD-like receptor family. NOD2 is able to sense muramyl dipeptide, a specific bacterial cell wall component, and to subsequently induce various signaling pathways leading to NF-κB activation and autophagy, both events contributing to an efficient innate and adaptive immune response. Interestingly, loss-of-function NOD2 variants were associated with a higher susceptibility for Crohn disease, which highlights the physiological importance of proper regulation of NOD2 activity. We performed a biochemical screen to search for new NOD2 regulators. We identified a new NOD2 partner, c-Jun N-terminal kinase-binding protein 1 (JNKBP1), a scaffold protein characterized by an N-terminal WD-40 domain. JNKBP1, through its WD-40 domain, binds to NOD2 following muramyl dipeptide activation. This interaction attenuates NOD2-mediated NF-κB activation and IL-8 secretion as well as NOD2 antibacterial activity. JNKBP1 exerts its repressor effect by disturbing NOD2 oligomerization and RIP2 tyrosine phosphorylation, both steps required for downstream NOD2 signaling. We furthermore showed that JNKBP1 and NOD2 are co-expressed in the human intestinal epithelium and in immune cells recruited in the lamina propria, which suggests that JNKBP1 contributes to maintain NOD2-mediated intestinal immune homeostasis.     

Intracellular pathogen sensor NOD2 programs macrophages to trigger Notch1 activation

Intracellular pathogen sensor, NOD2, has been implicated in regulation of wide range of anti-inflammatory responses critical during development of a diverse array of inflammatory diseases; however, underlying molecular details are still imprecisely understood. In this study, we demonstrate that NOD2 programs macrophages to trigger Notch1 signaling. Signaling perturbations or genetic approaches suggest signaling integration through cross-talk between Notch1-PI3K during the NOD2-triggered expression of a multitude of immunological parameters including COX-2/PGE(2) and IL-10. NOD2 stimulation enhanced active recruitment of CSL/RBP-Jk on the COX-2 promoter in vivo. Intriguingly, nitric oxide assumes critical importance in NOD2-mediated activation of Notch1 signaling as iNOS(-/-) macrophages exhibited compromised ability to execute NOD2-triggered Notch1 signaling responses. Correlative evidence demonstrates that this mechanism operates in vivo in brain and splenocytes derived from wild type, but not from iNOS(-/-) mice. Importantly, NOD2-driven activation of the Notch1-PI3K signaling axis contributes to its capacity to impart survival of macrophages against TNF-α or IFN-γ-mediated apoptosis and resolution of inflammation. Current investigation identifies Notch1-PI3K as signaling cohorts involved in the NOD2-triggered expression of a battery of genes associated with anti-inflammatory functions. These findings serve as a paradigm to understand the pathogenesis of NOD2-associated inflammatory diseases and clearly pave a way toward development of novel therapeutics.     

Internalization-dependent recognition of Mycobacterium avium ssp. paratuberculosis by intestinal epithelial cells

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease, a highly prevalent chronic intestinal infection in domestic and wildlife ruminants. The microbial pathogenesis of MAP infection has attracted additional attention due to an association with the human enteric inflammatory Crohn's disease. MAP is acquired by the faecal–oral route prompting us to study the interaction with differentiated intestinal epithelial cells. MAP was rapidly internalized and accumulated in a late endosomal compartment. In contrast to other opportunistic mycobacteria or M. bovis, MAP induced significant epithelial activation as indicated by a NF-κB-independent but Erk-dependent chemokine secretion. Surprisingly, MAP-induced chemokine production was completely internalization-dependent as inhibition of Rac-dependent bacterial uptake abolished epithelial activation. In accordance, innate immune recognition of MAP by differentiated intestinal epithelial cells occurred through the intracellularly localized pattern recognition receptors toll-like receptor 9 and NOD1 with signal transduction via the adaptor molecules MyD88 and RIP2. The internalization-dependent innate immune activation of intestinal epithelial cells is in contrast to the stimulation of professional phagocytes by extracellular bacterial constituents and might significantly contribute to the histopathological changes observed during enteric MAP infection.     

TRIM27 negatively regulates NOD2 by ubiquitination and proteasomal degradation

NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohn's disease. We found that TRIM27 expression is increased in Crohn's disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus.We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases.     

The Cofilin Phosphatase Slingshot Homolog 1 (SSH1) Links NOD1 Signaling to Actin Remodeling

NOD1 is an intracellular pathogen recognition receptor that contributes to anti-bacterial innate immune responses, adaptive immunity and tissue homeostasis. NOD1-induced signaling relies on actin remodeling, however, the details of the connection of NOD1 and the actin cytoskeleton remained elusive. Here, we identified in a druggable-genome wide siRNA screen the cofilin phosphatase SSH1 as a specific and essential component of the NOD1 pathway. We show that depletion of SSH1 impaired pathogen induced NOD1 signaling evident from diminished NF-κB activation and cytokine release. Chemical inhibition of actin polymerization using cytochalasin D rescued the loss of SSH1. We further demonstrate that NOD1 directly interacted with SSH1 at F-actin rich sites. Finally, we show that enhanced cofilin activity is intimately linked to NOD1 signaling. Our data thus provide evidence that NOD1 requires the SSH1/cofilin network for signaling and to detect bacterial induced changes in actin dynamics leading to NF-κB activation and innate immune responses.     

The natural killer T-cell ligand alpha-galactosylceramide prevents autoimmune diabetes in non-obese diabetic mice

Diabetes in non-obese diabetic (NOD) mice is mediated by pathogenic T-helper type 1 (Th1) cells that arise because of a deficiency in regulatory or suppressor T cells. V alpha 14-J alpha 15 natural killer T (NKT) cells recognize lipid antigens presented by the major histocompatibility complex class I-like protein CD1d (refs. 3,4). We have previously shown that in vivo activation of V alpha 14 NKT cells by alpha-galactosylceramide (alpha-GalCer) and CD1d potentiates Th2-mediated adaptive immune responses. Here we show that alpha-GalCer prevents development of diabetes in wild-type but not CD1d-deficient NOD mice. Disease prevention correlated with the ability of alpha-GalCer to suppress interferon-gamma but not interleukin-4 production by NKT cells, to increase serum immunoglobulin E levels, and to promote the generation of islet autoantigen-specific Th2 cells. Because alpha-GalCer recognition by NKT cells is conserved among mice and humans, these findings indicate that alpha-GalCer might be useful for therapeutic intervention in human diseases characterized by Th1-mediated pathology such as Type 1 diabetes.     

High Glucose Induces Down-Regulated GRIM-19 Expression to Activate STAT3 Signaling and Promote Cell Proliferation in Cell Culture

Recent studies indicated that Gene Associated with Retinoid-IFN-Induced Mortality 19 (GRIM-19), a newly discovered mitochondria-related protein, can regulate mitochondrial function and modulate cell viability possibly via interacting with STAT3 signal. In the present study we sought to test: 1) whether GRIM-19 is involved in high glucose (HG) induced altered cell metabolism in both cancer and cardiac cells, 2) whether GRIM-19/STAT3 signaling pathway plays a role in HG induced biological effects, especially whether AMPK activity could be involved. Our data showed that HG enhanced cell proliferation of both HeLa and H9C2 cells, which was closely associated with down-regulated GRIM-19 expression and increased phosphorylated STAT3 level. We showed that GRIM-19 knock-down alone in normal glucose cultured cells can also result in an increase in phosphorylated STAT3 level and enhanced proliferation capability, whereas GRIM-19 over-expression can abolished HG induced STAT3 activation and enhanced cell proliferation. Importantly, both down-regulated or over-expression of GRIM-19 increased lactate production in both HeLa and H9C2 cells. The activated STAT3 was responsible for increased cell proliferation as either AG-490, an inhibitor of JAK2, or siRNA targeting STAT3 can attenuate cell proliferation increased by HG. In addition, HG increased lactate acid levels in HeLa cells, which was also observed when GRIM-19 was genetically manipulated. However, HG did not affect the lactate levels in H9C2 cells. Of note, over-expression of GRIM-19 and silencing of STAT3 both increased lactate production in H9C2 cells. As expected, HG resulted in significant decreases in phosphorylated AMPKα levels in H9C2 cells, but not in HeLa cells. Interestingy, activation of AMPKα by metformin was associated with a reversal of the suppressed GRIM-19 expression in H9C2 cells, the fold of changes in GRIM-19 expression by metformin were much less in HeLa cells. Metformin did not affect the phosphorylated STAT3 lelvels, however, decreased its levels in H9C2, especially in the setting of HG culture. Not like HG alone which resulted in no changes in lactate acid in H9C2 cells, metformin can increase lactate acid levels in H9C2 cells. Increased lactate induced by metformin was also observed in HeLa cells.     

NLRP10 enhances Shigella-induced pro-inflammatory responses

Members of the NLR family evolved as intracellular sensors for bacterial and viral infection. However, our knowledge on the implication of most of the human NLR proteins in innate immune responses still remains fragmentary. Here we characterized the role of human NLRP10 in bacterial infection. Our data revealed that NLRP10 is a cytoplasmic localized protein that positively contributes to innate immune responses induced by the invasive bacterial pathogen Shigella flexneri. SiRNA-mediated knock-down studies showed that NLRP10 contributes to pro-inflammatory cytokine release triggered by Shigella in epithelial cells and primary dermal fibroblasts, by influencing p38 and NF-κB activation. This effect is dependent on the ATPase activity of NLRP10 and its PYD domain. Mechanistically, NLRP10 interacts with NOD1, a NLR that is pivotally involved in sensing of invasive microbes, and both proteins are recruited to the bacterial entry point at the plasma membrane. Moreover, NLRP10 physically interacts with downstream components of the NOD1 signalling pathway, such as RIP2, TAK1 and NEMO. Taken together, our data revealed a novel role of NLRP10 in innate immune responses towards bacterial infection and suggest that NLRP10 functions as a scaffold for the formation of the NOD1-Nodosome.     

GRIM-19: A master regulator of cytokine induced tumor suppression,metastasis and energy metabolism

Cytokines induce cell proliferation or growth suppression depending on the context. It is increasingly becoming clear that success of standard radiotherapy and/or chemotherapeutics to eradicate solid tumors is dependent on IFN signaling. In this review we discuss the molecular mechanisms of tumor growth suppression by a gene product isolated in our laboratory using a genome-wide expression knock-down strategy. Gene associated with retinoid-IFN-induced mortality −19 (GRIM-19) functions as non-canonical tumor suppressor by antagonizing oncoproteins. As a component of mitochondrial respiratory chain, GRIM-19 influences the degree of “Warburg effect” in cancer cells as many advanced and/or aggressive tumors show severely down-regulated GRIM-19 levels. In addition, GRIM-19 appears to regulate innate and acquired immune responses in mouse models. Thus, GRIM-19 is positioned at nodes that favor cell protection and/or prevent aberrant cell growth.     

NOD1 is required for Helicobacter pylori induction of IL‐33 responses in gastric epithelial cells

Helicobacter pylori (H. pylori) causes chronic inflammation which is a key precursor to gastric carcinogenesis. It has been suggested that H. pylori may limit this immunopathology by inducing the production of interleukin 33 (IL‐33) in gastric epithelial cells, thus promoting T helper 2 immune responses. The molecular mechanism underlying IL‐33 production in response to H. pylori infection, however, remains unknown. In this study, we demonstrate that H. pylori activates signalling via the pathogen recognition molecule Nucleotide‐Binding Oligomerisation Domain‐Containing Protein 1 (NOD1) and its adaptor protein receptor‐interacting serine–threonine Kinase 2, to promote production of both full‐length and processed IL‐33 in gastric epithelial cells. Furthermore, IL‐33 responses were dependent on the actions of the H. pylori Type IV secretion system, required for activation of the NOD1 pathway, as well as on the Type IV secretion system effector protein, CagA. Importantly, Nod1^+/+ mice with chronic H. pylori infection exhibited significantly increased gastric IL‐33 and splenic IL‐13 responses, but decreased IFN‐γ responses, when compared with Nod1^?/? animals. Collectively, our data identify NOD1 as an important regulator of mucosal IL‐33 responses in H. pylori infection. We suggest that NOD1 may play a role in protection against excessive inflammation.     

Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2

The nucleotide-binding domain and leucine-rich repeat containing protein NOD2 serves as a cytoplasmic pattern recognition molecule sensing bacterial muramyl dipeptide (MDP), whereas TLR2 mediates cell surface recognition of bacterial lipopeptides. In this study, we show that NOD2 stimulation activated Rac1 in human THP-1 cells and primary human monocytes. Rac1 inhibition or knock-down, or actin cytoskeleton disruption increased MDP-stimulated IL-8 secretion and NF-kappaB activation, whereas TLR2-dependent cell activation was suppressed by Rac1 inhibition. p21-activated kinase [Pak]-interacting exchange factor (beta-PIX) plays a role in this negative regulation, because knock-down of beta-PIX also led to increased NOD2-mediated but not TLR2-mediated IL-8 secretion, and coimmunoprecipitation experiments demonstrated that NOD2 interacted with beta-PIX as well as Rac1 upon MDP stimulation. Moreover, knock-down of beta-PIX or Rac1 abrogated membrane recruitment of NOD2, and interaction of NOD2 with its negative regulator Erbin. Overall, our data indicate that beta-PIX and Rac1 mediate trafficking and negative regulation of NOD2-dependent signaling which is different from Rac1's positive regulatory role in TLR2 signaling.     

Salmonella Typhimurium Type III Secretion Effectors Stimulate Innate Immune Responses in Cultured Epithelial Cells

Recognition of conserved bacterial products by innate immune receptors leads to inflammatory responses that control pathogen spread but that can also result in pathology. Intestinal epithelial cells are exposed to bacterial products and therefore must prevent signaling through innate immune receptors to avoid pathology. However, enteric pathogens are able to stimulate intestinal inflammation. We show here that the enteric pathogen Salmonella Typhimurium can stimulate innate immune responses in cultured epithelial cells by mechanisms that do not involve receptors of the innate immune system. Instead, S. Typhimurium stimulates these responses by delivering through its type III secretion system the bacterial effector proteins SopE, SopE2, and SopB, which in a redundant fashion stimulate Rho-family GTPases leading to the activation of mitogen-activated protein (MAP) kinase and NF-κB signaling. These observations have implications for the understanding of the mechanisms by which Salmonella Typhimurium induces intestinal inflammation as well as other intestinal inflammatory pathologies.     

Microbial induction of CARD15 expression in intestinal epithelial cells via toll-like receptor 5 triggers an antibacterial response loop

With the discovery of CARD15 as susceptibility gene for Crohn's disease (CD) a first link to a potential defect in the innate immune system was made. In this work we aimed to analyze enterocyte NOD2/CARD15 expression and regulation in response to bacterial motifs and the consequences of the most common CD-specific CARD15 mutation on antibacterial responses of normal intestinal epithelial cells (IEC). Under normal conditions, IEC lines and ileal enterocytes did not express NOD2/CARD15 mRNA or protein, contrary to IEC derived from inflammatory CD sections. In vitro analyses revealed that the simple contact with non-pathogenic commensal E. Coli K12 was sufficient to induced NOD2/CARD15 mRNA and protein in human IEC (HIEC). We identified bacterial flagellin interacting with TLR5 as major motif in this regulation of NOD2/CARD15. E. Coli mutants not expressing flagellin (DeltaFliC) failed to induce CARD15. Similarly, in HIEC transfected with a plasmid encoding dominant negative TLR5, no CARD15 induction was observed after K12 contact. Isolated TLR2 or TLR4 stimulation had no or only a marginal effect on NOD2/CARD15 expression. NOD2/CARD15 negative HIEC were unresponsive to muramyl dipeptide (MDP), but once NOD2/CARD15 was induced, HIEC and Caco2 cells responded to intra or extracellular MDP presentation with the activation of the NFkB pathway. IEC transfected with the Crohn-specific CARD15 mutant (F3020insC, FS) failed to activate NFkB after MDP-challenge, in contrast to CARD15WT IEC. In response to MDP, IEC induced a massive antibacterial peptide (ABP) response, seen in the apical release of CCL20. This was completely abolished in IEC carrying CARD15FS. These data suggest a critical role of NOD2/CARD15 in the bacterial clearance of the intestinal epithelium while CD-specific mutated NOD2/CARD15 causes an impaired epithelial barrier.