Clinical effectiveness of bidirectional fecal microbiota transfer in the treatment of recurrent Clostridioides difficile infections.

BACKGROUND: Fecal microbiota transfer (FMT) has become a standard of care in the prevention of multiple recurrent Clostridioides difficile (rCDI) infection. AIM: While primary cure rates range from 70-80% following a single treatment using monodirectional approaches, cure rates of combination treatment remain largely unknown. METHODS: In a retrospective case-control study, outcomes following simultaneous bidirectional FMT (bFMT) with combined endoscopic application into the upper and lower gastrointestinal tract, compared to standard routes of application (endoscopy via upper or lower gastrointestinal tract and oral capsules; abbreviated UGIT, LGIT and CAP) on day 30 and 90 after FMT were assessed. Statistical matching partners were identified using number of recurrences (<3; ≥3), age and gender. RESULTS: Primary cure rates at D30 and D90 for bFMT were 100% (p=.001). The matched control groups showed cure rates of 81.3% for LGIT (p=.010), 62.5% for UGIT (p=.000) and 78.1% for CAP (p=.005) on D30 and 81.3% for LGIT (p=.010), 59.4% for UGIT (p=.000) and 71.9% for CAP (p=.001) on D90. CONCLUSION: In our analysis, bFMT on the same day significantly increased primary cure rate at D30 and D90. These data require prospective confirmation but suggest that route of application may play a significant role in optimizing patient outcomes. ClinicalTrials.gov no: NCT02681068.

Faecal Micro-RNAs in Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: Faecal biomarkers are used as indicators of disease activity in inflammatory bowel diseases [IBD], which include Crohn's disease [CD] and ulcerative colitis [UC]. Micro-RNAs [miRNAs] are small non-coding RNAs detectable in extracellular fluids and can be used as clinical biomarkers. The aim of this study was to determine if faecal miRNA composition is altered in IBD. METHODS: More than 800 different human faecal miRNAs were measured in stool samples from control individuals and patients with active CD by using NanoString technology. Selected miRNAs were quantified by qRT-PCR in faeces, serum and intestinal tissue of controls [n = 23] and patients with inactive or active CD [n = 22, n = 22] or UC [n = 11, n = 24] as well as patients with Clostridium difficile infection [CDI, n = 8]. RESULTS: In total, 150 miRNAs were significantly detected in faeces from controls and patients, and multivariate analyses showed that CD patients with high disease activities had a distinct miRNA profile and that miR-223 and miR-1246 were distinct from other faecal miRNAs. In a larger cohort, active UC patients displayed significantly higher levels of miR-223 and miR-1246 than controls while patients with CDI had higher levels of faecal miR-1246 but not miR-223. No differences were noted in serum samples. CONCLUSIONS: To our knowledge, this is the first comprehensive screen of faecal miRNAs performed in IBD. Further investigation will aim to confirm these findings in a larger cohort and to understand the biological function and cellular sources of faecal miRNAs.

Fecal Eosinophil Cationic Protein Is a Diagnostic and Predictive Biomarker in Young Adults with Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Fecal biomarkers are important non-invasive markers monitoring disease activity in inflammatory bowel disease (IBD). We compared the significance of fecal eosinophil cationic protein (fECP) and fecal calprotectin (fCal). METHODS: fECP and fCal were measured in patients with Crohn's disease (CD, n = 97), ulcerative colitis (UC, n = 53), Clostridioides difficile infection (CDI, n = 9), primary food allergy (PFA, n = 11), pollen-associated food allergy (n = 25) and non-inflammatory controls (n = 78). Results were correlated with clinical and endoscopic IBD activity scores. RESULTS: fECP was significantly elevated in CD, UC, CDI and PFA compared to controls. fCal was significantly increased in CD, UC and CDI. fECP had lower diagnostic accuracy than fCal (area under the curve (AUC) = 0.88) in differentiating between endoscopically active and inactive patients with IBD (AUC = 0.77, ROC analysis). In contrast to fCal, fECP correlated negatively with age and levels were also elevated in clinically and endoscopically inactive patients with IBD <45 years (endoscopically inactive IBD vs controls; AUC for fECP = 0.86; AUC for fCal = 0.62). However, in those patients with low inflammatory activity (fCal <250 mg/kg), high fECP indicated the need for treatment modification or surgery (fECP <200 µg/kg = 22%; 200-600 µg/kg = 44%; >600 µg/kg = 82%) at month 48 of follow-up. CONCLUSIONS: fECP is a diagnostic and prognostic marker in young patients with IBD in remission.

Macrophage-Specific NF-κB Activation Dynamics Can Segregate Inflammatory Bowel Disease Patients.

The heterogeneous nature of inflammatory bowel disease (IBD) presents challenges, particularly when choosing therapy. Activation of the NF-κB transcription factor is a highly regulated, dynamic event in IBD pathogenesis. Using a lentivirus approach, NF-κB-regulated luciferase was expressed in patient macrophages, isolated from frozen peripheral blood mononuclear cell samples. Following activation, samples could be segregated into three clusters based on the NF-κB-regulated luciferase response. The ulcerative colitis (UC) samples appeared only in the hypo-responsive Cluster 1, and in Cluster 2. Conversely, Crohn's disease (CD) patients appeared in all Clusters with their percentage being higher in the hyper-responsive Cluster 3. A positive correlation was seen between NF-κB-induced luciferase activity and the concentrations of cytokines released into medium from stimulated macrophages, but not with serum or biopsy cytokine levels. Confocal imaging of lentivirally-expressed p65 activation revealed that a higher proportion of macrophages from CD patients responded to endotoxin lipid A compared to controls. In contrast, cells from UC patients exhibited a shorter duration of NF-κB p65 subunit nuclear localization compared to healthy controls, and CD donors. Analysis of macrophage cytokine responses and patient metadata revealed a strong correlation between CD patients who smoked and hyper-activation of p65. These in vitro dynamic assays of NF-κB activation in blood-derived macrophages have the potential to segregate IBD patients into groups with different phenotypes and may therefore help determine response to therapy.

Subcellular antigen localization in commensal E. coli is critical for T cell activation and induction of specific tolerance.

Oral tolerance to soluble antigens is critically important for the maintenance of immunological homeostasis in the gut. The mechanisms of tolerance induction to antigens of the gut microbiota are still less well understood. Here, we investigate whether the subcellular localization of antigens within non-pathogenic E. coli has a role for its ability to induce antigen-specific tolerance. E. coli that express an ovalbumin (OVA) peptide in the cytoplasm, at the outer membrane or as secreted protein were generated. Intestinal colonization of mice with non-pathogenic E. coli expressing OVA at the membrane induced the expansion of antigen-specific Foxp3+ Tregs and mediated systemic immune tolerance. In contrast, cytoplasmic OVA was ignored by antigen-specific CD4+ T cells and failed to induce tolerance. In vitro experiments revealed that surface-displayed OVA of viable E. coli was about two times of magnitude more efficient to activate antigen-specific CD4+ T cells than soluble antigens, surface-displayed antigens of heat-killed E. coli or cytoplasmic antigen of viable or heat-killed E. coli. This effect was independent of the antigen uptake efficiency in dendritic cells. In summary, our results show that subcellular antigen localization in viable E. coli strongly influences antigen-specific CD4+ cell expansion and tolerance induction upon intestinal colonization.

Desmoglein 2, but not desmocollin 2, protects intestinal epithelia from injury.

Desmosomes are the least understood intercellular junctions in the intestinal epithelia and provide cell-cell adhesion via the cadherins desmoglein (Dsg)2 and desmocollin (Dsc)2. We studied these cadherins in Crohn's disease (CD) patients and in newly generated conditional villin-Cre DSG2 and DSC2 knockout mice (DSG2ΔIEC; DSC2ΔIEC). CD patients exhibited altered desmosomes and reduced Dsg2/Dsc2 levels. The intestines of both transgenic animal lines were histopathologically inconspicuous. However, DSG2ΔIEC, but not DSC2ΔIEC mice displayed an increased intestinal permeability, a wider desmosomal space as well as alterations in desmosomal and tight junction components. After dextran sodium sulfate (DSS) treatment and Citrobacter rodentium exposure, DSG2ΔIEC mice developed a more-pronounced colitis, an enhanced intestinal epithelial barrier disruption, leading to a stronger inflammation and activation of epithelial pSTAT3 signaling. No susceptibility to DSS-induced intestinal injury was noted in DSC2ΔIEC animals. Dsg2 interacted with the cytoprotective chaperone Hsp70. Accordingly, DSG2ΔIEC mice had lower Hsp70 levels in the plasma membrane compartment, whereas DSC2ΔIEC mice displayed a compensatory recruitment of galectin 3, a junction-tightening protein. Our results demonstrate that Dsg2, but not Dsc2 is required for the integrity of the intestinal epithelial barrier in vivo.

Successful Fecal Microbiota Transplantation in a Patient with Severe Complicated Clostridium difficile Infection after Liver Transplantation.

Clostridium difficile infection (CDI) represents one of the most common healthcare-associated infections. Due to increasing numbers of recurrences and therapy failures, CDI has become a major disease burden. Studies have shown that fecal microbiota transplantation (FMT) can both be a safe and highly efficacious therapy for patients with therapy-refractory CDI. However, patients undergoing solid organ transplantation are at high risk for CDI due to long-term immunosuppression, previous antibiotic therapy, and proton pump inhibitor use. Additionally, these patients may be especially prone to adverse events related to FMT. Here, we report a successful FMT in a patient with severe therapy-refractory CDI after liver transplantation.

Glycan-Functionalized Microgels for Scavenging and Specific Binding of Lectins.

Lectins are proteins with a well-defined carbohydrate recognition domain. Many microbial proteins such as bacterial toxins possess lectin or lectin-like binding domains to interact with cell membranes that are decorated with glycan recognition motifs. We report a straightforward way to prepare monodisperse and biocompatible polyethylene glycol microgels, which carry glycan motifs for specific binding to lectins. The sugar-functionalized colloids exhibit a wide mesh size and a highly accessible volume. The microgels are prepared via drop-based microfluidics combined with radical polymerization. GSII and ECL are used as model lectins that bind specifically to the corresponding carbohydrates, namely, GlcNAc and LacNAc. LacNAc microgels bind ECL with a high capacity and high affinity (Kd ≈ 0.5 to 1 µM), suggesting multivalent binding of the lectin to the LacNAc-decorated flexible microgel network. Glycan-functionalized microgels present a useful tool for lectin scavenging in biomedical applications.

Anti-food and anti-microbial IgG subclass antibodies in inflammatory bowel disease.

OBJECTIVES: Inflammatory bowel disease (IBD), particularly Crohn's disease (CD), is associated with increased microbial-specific IgG and IgA antibodies, whereas alterations of anti-food antibodies are still disputed. The knowledge about IgG subclass antibodies in IBD is limited. In this study we analysed IgG subclass antibodies specific for nutritional and commensal antigens in IBD patients and controls. METHODS: Serum IgG1, IgG2, IgG3 and IgG4 specific for wheat and milk extracts, purified ovalbumin, Escherichia coli and Bacteroides fragilis lysates and mannan from Saccharomyces cerevisiae were analysed by ELISA in patients with CD (n = 56), ulcerative colitis (UC; n = 29), acute gastroenteritis/colitis (n = 12) as well as non-inflammatory controls (n = 62). RESULTS: Anti-Saccharomyces cerevisiae antibodies (ASCA) of all IgG subclasses and anti-B. fragilis IgG1 levels were increased in CD patients compared to UC patients and controls. The discriminant validity of ASCA IgG2 and IgG4 was comparable with that of ASCA pan-IgG and IgA, whereas it was inferior for ASCA IgG1/IgG3 and anti-B. fragilis IgG1. Complicated CD defined by the presence of perianal, stricturing or penetrating disease phenotypes was associated with increased ASCA IgG1/IgG3/IgG4, anti-B. fragilis IgG1 and anti-E. coli IgG1 levels. Anti-food IgG subclass levels were not different between IBD patients and controls and did not correlate with food intolerance. In contrast to anti-microbial Abs, food-specific IgG responses were predominately of the IgG4 isotype and all food-specific IgG subclass levels correlated negatively with age. CONCLUSION: Our study supports the notion that the adaptive immune recognition of food and commensal antigens are differentially regulated.

Biliary Mucosal Barrier and Microbiome.

BACKGROUND: The biliary system is in continuous contact with the complex microbiota of the intestine. Microbial products have recently been proposed as potential triggers for biliary diseases. METHODS: The aim of this review is to provide a summary of the current knowledge regarding the role of the biliary and intestinal microbiome in biliary inflammatory diseases. RESULTS: Previously, it was suggested that the healthy biliary system is a sterile organ, while acute cholangitis and cholecystitis may occur from ascending infections. Although non-inflammatory biliary colonization by certain bacteria such as Salmonella spp. has been already recognized since several decades, human and animal studies indicated only very recently that the gallbladder harbors a complex microbiota also under non-pathologic conditions. Novel findings suggested that - similar to the situation in the intestine - the biliary mucosa features a chemical, mechanical, and immunological barrier, ensuring immunological tolerance against commensals. However, microbial triggers might influence acute and chronic inflammatory disease of the biliary system and the whole liver. CONCLUSION: Although yet undefined, dysbiosis of the biliary or intestinal microbiota rather than a single microorganism may influence disease progression.

CX3CR1 is a gatekeeper for intestinal barrier integrity in mice: Limiting steatohepatitis by maintaining intestinal homeostasis.

UNLABELLED: Nonalcoholic fatty liver disease is seen as the hepatic manifestation of the metabolic syndrome and represents the most common liver disease in Western societies. The G protein-coupled chemokine receptor CX3CR1 plays a central role in several metabolic syndrome-related disease manifestations and is involved in maintaining intestinal homeostasis. Because diet-induced intestinal dysbiosis is a driver for nonalcoholic fatty liver disease, we hypothesized that CX3CR1 may influence the development of steatohepatitis. In two independent models of diet-induced steatohepatitis (high-fat diet and methionine/choline-deficient diet), CX3CR1 protected mice from excessive hepatic steatosis and inflammation, as well as systemic glucose intolerance. Lack of Cx3cr1 expression was associated with significantly altered intestinal microbiota composition, which was linked to an impaired intestinal barrier. Concomitantly, endotoxin levels in portal serum and inflammatory macrophages in liver were increased in Cx3cr1-/- mice, indicating an increased inflammatory response. Depletion of intestinal microbiota by administration of broad-spectrum antibiotics suppressed the number of infiltrating macrophages and promoted macrophage polarization in liver. Consequently, antibiotic-treated mice demonstrated a marked improvement of steatohepatitis. CONCLUSION: Microbiota-mediated activation of the innate immune responses through CX3CR1 is crucial for controlling steatohepatitis progression, which recognizes CX3CR1 as an essential gatekeeper in this scenario.

PRR-signaling pathways: Learning from microbial tactics.

Recognition of bacterial pathogens by the mammalian host relies on the induction of early innate immune responses initiated by the activation of pattern-recognition receptors (PRRs) upon sensing of their cognate microbe-associated-patterns (MAMPs). Successful pathogens have evolved to intercept PRR activation and signaling at multiple steps. The molecular dissection of the underlying mechanisms revealed many of the basic mechanisms used by the immune system. Here we provide an overview of the different strategies used by bacterial pathogens and commensals to subvert and reprogram PPR-mediated innate immune responses. A particular attention is given to recent discoveries highlighting novel molecular details of the host inflammatory response in mammalian cells and current advances in our understanding of the interaction of commensals with PRR-mediated responses.

Isolation and characterization of human intestinal mast cells.

Mast cells are granulated immune cells typically located at barrier sites of the body, such as the skin and the mucosa of the respiratory, urogenital, and gastrointestinal tract. They are well known for their capacity to participate in the orchestration of inflammatory and immune responses by releasing a broad array of mediators as a consequence of IgE-dependent and IgE-independent activation. Mast cells derive from myeloid progenitors, but in contrast to other myeloid cells, they leave the bone marrow in an immature state; therefore, mast cells are not visible in the blood under normal conditions. For full maturation, the tissue environment is necessary. Thus, mature mast cells can be only isolated from tissue such as skin or mucosal sites, which makes mast cell isolation complicated. This chapter describes methods to isolate, purify, and culture mast cells from the human intestinal mucosa. Human mucosal mast cells can be used to characterize their mediators and to study the mechanisms of human mast cell activation, signal transduction, and exocytosis in response to specific stimuli.

Distinct patterns of IgG and IgA against food and microbial antigens in serum and feces of patients with inflammatory bowel diseases.

BACKGROUND: Inflammatory bowel disease (IBD) is associated with a defective intestinal barrier and enhanced adaptive immune responses against commensal microbiota. Immune responses against food antigens in IBD patients remain poorly defined. METHODS: IgG and IgA specific for food and microfloral antigens (wheat and milk extracts; purified ovalbumin; Escherichia coli and Bacteroides fragilis lysates; mannan from Saccharomyces cerevisiae) were analyzed by ELISA in the serum and feces of patients with Crohn's disease (CD; n = 52 for serum and n = 20 for feces), ulcerative colitis (UC; n = 29; n = 17), acute gastroenteritis/colitis (AGE; n = 12; n = 9) as well as non-inflammatory controls (n = 61; n = 39). RESULTS: Serum anti-Saccharomyces cerevisiae antibodies (ASCA) and anti-B. fragilis IgG and IgA levels were increased in CD patients whereas antibody (Ab) levels against E. coli and food antigens were not significantly different within the patient groups and controls. Subgroup analysis revealed that CD patients with severe diseases defined by stricturing and penetrating lesions have slightly higher anti-food and anti-microbial IgA levels whereas CD and UC patients with arthropathy have decreased anti-food IgG levels. Treatment with anti-TNF-α Abs in CD patients was associated with significantly decreased ASCA IgG and IgA and anti-E. coli IgG. In the feces specific IgG levels against all antigens were higher in CD and AGE patients while specific IgA levels were higher in non-IBD patients. Anti-food IgG and IgA levels did not correlate with food intolerance. SUMMARY: In contrast to anti-microbial Abs, we found only minor changes in serum anti-food Ab levels in specific subgroups of IBD patients. Fecal Ab levels towards microbial and food antigens show distinct patterns in controls, CD and UC patients.

Interferon-γ regulates growth and controls Fcγ receptor expression and activation in human intestinal mast cells.

BACKGROUND: Development and function of tissue resident mast cells (MCs) is tightly controlled by various cytokines, most of which belong to the typical T helper (Th) 2-type cytokines such as IL-3 and IL-4. The effects of the Th1-type cytokine IFN-γ on human MCs is less clear. RESULTS: Here, we analyzed the effects of IFN-γ on tissue-derived, mature human MCs. We found that INF-γ decreases proliferation, without affecting apoptosis in human intestinal MCs cultured in the presence of optimal concentrations of stem cell factor (SCF) or SCF and IL-4. However, in the absence of growth factors or at suboptimal concentrations of SCF, INF-γ promotes survival through inhibition of MC apoptosis. Interestingly, we found that INF-γ has no effect on FcϵRI expression and FcϵRI-mediated release of histamine and leukotriene (LT)C4, while it has profound effects on FcγR expression and activation. We show that intestinal MCs express FcγRI, FcγRIIa, and FcγRIIc, whereas FcγRIIb expression was found in only 40% of the isolates and FcγRIII was never detectable. INF-γ strongly increases FcγRI and decreases FcγRIIa expression. INF-γ-naïve MCs produce LTC4 but fail to degranulate upon crosslinking of surface-bound monomeric IgG. In contrast, INF-γ-treated MCs rapidly release granule-stored histamine in addition to de novo-synthesized LTC4. CONCLUSION: In summary, we identify INF-γ as an important regulator of tissue-resident human MCs. IFN-γ displays a dual function by blocking extensive MC proliferation, while decreasing apoptosis in starving MCs and enhancing FcγRI expression and activation. These results emphasize the involvement of mucosal MCs in Th1-mediated disorders.

Shigella impairs T lymphocyte dynamics in vivo.

The Gram-negative enteroinvasive bacterium Shigella flexneri is responsible for the endemic form of bacillary dysentery, an acute rectocolitis in humans. S. flexneri uses a type III secretion system to inject effector proteins into host cells, thus diverting cellular functions to its own benefit. Protective immunity to reinfection requires several rounds of infection to be elicited and is short-lasting, suggesting that S. flexneri interferes with the priming of specific immunity. Considering the key role played by T-lymphocyte trafficking in priming of adaptive immunity, we investigated the impact of S. flexneri on T-cell dynamics in vivo. By using two-photon microscopy to visualize bacterium-T-cell cross-talks in the lymph nodes, where the adaptive immunity is initiated, we provide evidence that S. flexneri, via its type III secretion system, impairs the migration pattern of CD4(+) T cells independently of cognate recognition of bacterial antigens. We show that bacterial invasion of CD4(+) T lymphocytes occurs in vivo, and results in cell migration arrest. In the absence of invasion, CD4(+) T-cell migration parameters are also dramatically altered. Signals resulting from S. flexneri interactions with subcapsular sinus macrophages and dendritic cells, and recruitment of polymorphonuclear cells are likely to contribute to this phenomenon. These findings indicate that S. flexneri targets T lymphocytes in vivo and highlight the role of type III effector secretion in modulating host adaptive immune responses.

Hepatocyte caspase-8 is an essential modulator of steatohepatitis in rodents.

UNLABELLED: In human and murine models of nonalcoholic steatohepatitis (NASH), increased hepatocyte apoptosis is a critical mechanism contributing to inflammation and fibrogenesis. Caspase 8 (Casp8) is essential for death-receptor-mediated apoptosis activity and therefore its modulation might be critical for the pathogenesis of NASH. The aim was to dissect the role of hepatocyte Casp8 in a murine model of steatohepatitis. We generated hepatocyte-specific Casp8 knockout (Casp8(Δhep) ) mice. Animals were fed with a methionine-choline-deficient (MCD) diet. Liver injury was assessed by histopathological analysis, apoptotic death, serum alanine aminotransferase (ALT), fluorescent-activated cell sorter (FACS), analysis of liver infiltration and inflammation, reactive oxygen species (ROS), and liver fibrosis. MCD feeding triggered steatosis, hepatic lipid storage, and accumulation of free fatty acid (FFA) in wildtype (WT) livers, which were significantly reduced in Casp8(Δhep) animals. Additionally, lack of Casp8 expression in hepatocytes reduced the MCD-dependent increase in apoptosis and decreased expression of proinflammatory cytokines as well as hepatic infiltration. As a consequence, ROS production was lower, leading to a reduction in the progression of liver fibrosis in Casp8(Δhep) livers. CONCLUSION: Selective ablation of Casp8 in hepatocytes ameliorates development of NASH by modulating liver injury. Casp8-directed therapy might be a plausible treatment for patients with steatohepatitis. (HEPATOLOGY 2013;57:2189-2201).

ARC is a novel therapeutic approach against acetaminophen-induced hepatocellular necrosis.

BACKGROUND & AIMS: Acetaminophen (AAP) overdose is the most frequent cause of drug-induced liver failure. c-Jun N-terminal kinase (JNK) is thought to play a central role in AAP-induced hepatocellular necrosis. The apoptosis repressor with caspase recruitment domain (ARC) is a death repressor that inhibits death receptor and mitochondrial apoptotic signaling. Here, we investigated ARC's therapeutic effect and molecular mechanisms on AAP-induced hepatocellular necrosis. METHODS: We tested the in vivo and in vitro effects of ARC fused with the transduction domain of HIV-1 (TAT-ARC) on murine AAP hepatotoxicity. RESULTS: Treatment with TAT-ARC protein completely abrogated otherwise lethal liver failure induced by AAP overdose in C57BL/6 mice. AAP triggered caspase-independent necrosis, as evidenced by liver histology, elevated serum transaminases, and secreted HMGB1 that was inhibited by ARC. ARC-mediated hepatoprotection was not caused by an alteration of AAP metabolism, but resulted in reduced oxidative stress. AAP overdose led to induction of RIP-dependent signaling with subsequent JNK activation. Ectopic ARC inhibited JNK activation by specific interactions between ARC and JNK1 and JNK2. Importantly, survival of mice was even preserved when ARC therapy was initiated in a delayed manner after AAP administration. CONCLUSIONS: This work identifies for the first time ARC-JNK-binding with subsequent inhibition of JNK signaling as a specific mechanism of ARC to interfere with AAP-dependent necrosis. Our data suggests that AAP-mediated induction of RIP signaling serves as a critical switch for hepatocellular necrosis. The efficacy of TAT-ARC protein transduction in murine AAP hepatotoxicity suggests its therapeutic potential for reversing AAP intoxication also in humans.

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.