Method to Measure Ubiquitination of NLRs.

Posttranslational modifications are crucial in determining the functions of proteins in the cell. Modification of the NLRP3 inflammasome by the ubiquitin system has recently emerged as a new level of regulation of the inflammasome complex. Here we describe a method to detect poly-ubiquitination of NRLP3 using two different approaches: (i) detection with a ubiquitin antibody or (ii) using TUBEs (Tandem Ubiquitin Binding entities). This approach can be used to detect ubiquitination of other NLRs or other proteins.

NLRP3 at the crossroads between immune/inflammatory responses and enteric neuroplastic remodelling in a mouse model of diet-induced obesity.

BACKGROUND AND PURPOSE: Enteric neurogenic/inflammation contributes to bowel dysmotility in obesity. We examined the role of NLRP3 in colonic neuromuscular dysfunctions in mice with high-fat diet (HFD)-induced obesity. EXPERIMENTAL APPROACH: Wild-type C57BL/6J and NLRP3-KO (Nlrp3-/- ) mice were fed with HFD or standard diet for 8 weeks. The activation of inflammasome pathways in colonic tissues from obese mice was assessed. The role of NLRP3 in in vivo colonic transit and in vitro tachykininergic contractions and substance P distribution was evaluated. The effect of substance P on NLRP3 signalling was tested in cultured cells. KEY RESULTS: HFD mice displayed increased body and epididymal fat weight, cholesterol levels, plasma resistin levels and plasma and colonic IL-1ß levels, colonic inflammasome adaptor protein apoptosis-associated speck-like protein containing caspase-recruitment domain (ASC) and caspase-1 mRNA expression and ASC immunopositivity in macrophages. Colonic tachykininergic contractions were enhanced in HFD mice. HFD NLRP3-/- mice developed lower increase in body and epididymal fat weight, cholesterol levels, systemic and bowel inflammation. In HFD Nlrp3-/- mice, the functional alterations of tachykinergic pathways and faecal output were normalized. In THP-1 cells, substance P promoted IL-1ß release. This effect was inhibited upon incubation with caspase-1 inhibitor or NK1 antagonist and not observed in ASC-/- cells. CONCLUSION AND IMPLICATIONS: In obesity, NLRP3 regulates an interplay between the shaping of enteric immune/inflammatory responses and the activation of substance P/NK1 pathways underlying the onset of colonic dysmotility. Identifying NLRP3 as a therapeutic target for the treatment of bowel symptoms related to obesity.

Prodromal Intestinal Events in Alzheimer's Disease (AD): Colonic Dysmotility and Inflammation Are Associated with Enteric AD-Related Protein Deposition.

Increasing evidence suggests that intestinal dysfunctions may represent early events in Alzheimer's disease and contribute to brain pathology. This study examined the relationship between onset of cognitive impairment and colonic dysfunctions in a spontaneous AD model before the full development of brain pathology. SAMP8 mice underwent Morris water maze and assessment of faecal output at four, six and eight months of age. In vitro colonic motility was examined. Faecal and colonic Aß, tau proteins, α-synuclein and IL-1ß were assessed by ELISA. Colonic citrate synthase activity was assessed by spectrophotometry. Colonic NLRP3, caspase-1 and ASC expression were evaluated by Western blotting. Colonic eosinophil density and claudin-1 expression were evaluated by immunohistochemistry. The effect of Aß on NLRP3 signalling and mitochondrial function was tested in cultured cells. Cognitive impairment and decreased faecal output occurred in SAMP8 mice from six months. When compared with SAMR1, SAMP8 animals displayed: (1) impaired in vitro colonic contractions; (2) increased enteric AD-related proteins, IL-1ß, active-caspase-1 expression and eosinophil density; and (3) decreased citrate synthase activity and claudin-1 expression. In THP-1 cells, Aß promoted IL-1ß release, which was abrogated upon incubation with caspase-1 inhibitor or in ASC-/- cells. Aß decreased mitochondrial function in THP-1 cells. In SAMP8, enteric AD-related proteins deposition, inflammation and impaired colonic excitatory neurotransmission, occurring before the full brain pathology development, could contribute to bowel dysmotility and represent prodromal events in AD.

The three cytokines IL-1ß, IL-18, and IL-1α share related but distinct secretory routes.

Interleukin (IL)-1 family cytokines potently regulate inflammation, with the majority of the IL-1 family proteins being secreted from immune cells via unconventional pathways. In many cases, secretion of IL-1 cytokines appears to be closely coupled to cell death, yet the secretory mechanisms involved remain poorly understood. Here, we studied the secretion of the three best-characterized members of the IL-1 superfamily, IL-1α, IL-1ß, and IL-18, in a range of conditions and cell types, including murine bone marrow-derived and peritoneal macrophages, human monocyte-derived macrophages, HeLa cells, and mouse embryonic fibroblasts. We discovered that IL-1ß and IL-18 share a common secretory pathway that depends upon membrane permeability and can operate in the absence of complete cell lysis and cell death. We also found that the pathway regulating the trafficking of IL-1α is distinct from the pathway regulating IL-1ß and IL-18. Although the release of IL-1α could also be dissociated from cell death, it was independent of the effects of the membrane-stabilizing agent punicalagin, which inhibited both IL-1ß and IL-18 release. These results reveal that in addition to their role as danger signals released from dead cells, IL-1 family cytokines can be secreted in the absence of cell death. We propose that models used in the study of IL-1 release should be considered context-dependently.

Pathophysiology of NSAID-Associated Intestinal Lesions in the Rat: Luminal Bacteria and Mucosal Inflammation as Targets for Prevention.

Non-steroidal anti-inflammatory drugs (NSAIDs) can damage the small intestine, mainly through an involvement of enteric bacteria. This study examined the pathophysiology of NSAID-associated intestinal lesions in a rat model of diclofenac-enteropathy and evaluated the effect of rifaximin on small bowel damage. Enteropathy was induced in 40-week old male rats by intragastric diclofenac (4 mg/kg BID, 14 days). Rifaximin (delayed release formulation) was administered (50 mg/kg BID) 1 h before the NSAID. At the end of treatments, parameters dealing with ileal damage, inflammation, barrier integrity, microbiota composition, and TLR-NF-κB-inflammasome pathway were evaluated. In addition, the modulating effect of rifaximin on NLRP3 inflammasome was tested in an in vitro cell system. Diclofenac induced intestinal damage and inflammation, triggering an increase in tissue concentrations of tumor necrosis factor and interleukin-1ß, higher expression of TLR-2 and TLR-4, MyD88, NF-κB and activation of caspase-1. In addition, the NSAID decreased ileal occludin expression and provoked a shift of bacterial phyla toward an increase in Proteobacteria and Bacteroidetes abundance. All these changes were counterbalanced by rifaximin co-administration. This drug was also capable of increasing the proportion of Lactobacilli, a genus depleted by the NSAID. In LPS-primed THP-1 cells stimulated by nigericin (a model to study the NLRP3 inflammasome), rifaximin reduced IL-1ß production in a concentration-dependent fashion, this effect being associated with inhibition of the up-stream caspase-1 activation. In conclusion, diclofenac induced ileal mucosal lesions, driving inflammatory pathways and microbiota changes. In conclusion, rifaximin prevents diclofenac-induced enteropathy through both anti-bacterial and anti-inflammatory activities.

USP7 and USP47 deubiquitinases regulate NLRP3 inflammasome activation.

The assembly and activation of the inflammasomes are tightly regulated by post-translational modifications, including ubiquitin. Deubiquitinases (DUBs) counteract the addition of ubiquitin and are essential regulators of immune signalling pathways, including those acting on the inflammasome. How DUBs control the assembly and activation of inflammasomes is unclear. Here, we show that the DUBs USP7 and USP47 regulate inflammasome activation in macrophages. Chemical inhibition of USP7 and USP47 blocks inflammasome formation, independently of transcription, by preventing ASC oligomerisation and speck formation. We also provide evidence that the ubiquitination status of NLRP3 itself is altered by inhibition of USP7 and USP47. Interestingly, we found that the activity of USP7 and USP47 increased in response to inflammasome activators. Using CRISPR/Cas9 in the macrophage cell line THP-1, we show that inflammasome activation is reduced when both USP7 and USP47 are knocked down. Altogether, these data reveal a new post-transcriptional role for USP47 and USP7 in inflammation by regulating inflammasome activation and the release of the pro-inflammatory cytokines IL-1ß and IL-18, and implicate dual USP7 and USP47 inhibitors as potential therapeutic agents for inflammatory disease.

The inflammasomes, immune guardians at defence barriers.

As a result of its strategic location, the epithelium is constantly exposed to a wide variety of pathogen and danger signals. Traditionally, the epithelium has been perceived as a defensive but passive barrier; however, it has now become evident that the epithelium senses and actively responds to these signals in order to maintain barrier homeostasis and contributes to the inflammatory response. One way it does this is by producing pro-inflammatory cytokines including interleukin-1ß (IL-1ß) and IL-18. These two cytokines are synthesized as inactive precursors, the maturation of which is mediated by pro-inflammatory caspases after the activation and assembly of macromolecular complexes called inflammasomes. Epithelial cells express a large panel of inflammasome components, and although the molecular mechanisms underlying the activation of these complexes in haematopoietic cells are well understood, how epithelial cells react to danger signals to activate the inflammasome remains unclear. We review and discuss how different inflammasomes contribute to barrier homeostasis and inflammation at several barrier sites, their mechanisms and how their aberrant regulation contributes to disease at the different epithelia.

Method to Measure Ubiquitination of NLRs.

Posttranslational modifications are crucial in determining the functions of proteins in the cell. Modification of the NLRP3 inflammasome by the ubiquitin system has recently emerged as a new level of regulation of the inflammasome complex. Here, we describe a method to detect polyubiquitination of NRLP3 using two different approaches: (1) detection with an ubiquin antibody or (2) using TUBE (Tandem Ubiquitin Binding entities). This approach can be used to detect ubiquitination of other NLR or other components of the inflammasome.