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Inflammasomes are cytosolic protein complexes, which orchestrate the maturation of active IL-1 beta by proteolytic cleavage via caspase-1. Although many principles of inflammasome activation have been described, mechanisms that limit inflammasome-dependent immune responses remain poorly defined. Here, we show that the thiol-specific peroxidase peroxiredoxin-4 (Prdx4) directly regulates IL-1 beta generation by interfering with caspase-1 activity. We demonstrate that caspase-1 and Prdx4 form a redox-sensitive regulatory complex via caspase-1 cysteine 397 that leads to caspase-1 sequestration and inactivation. Mice lacking Prdx4 show an increased susceptibility to LPS-induced septic shock. This effect was phenocopied in mice carrying a conditional deletion of Prdx4 in the myeloid lineage (Prdx4-Delta LysMCre). Strikingly, we demonstrate that Prdx4 co-localizes with inflammasome components in extracellular vesicles (EVs) from inflammasome-activated macrophages. Purified EVs are able to transmit a robust IL-1 beta-dependent inflammatory response in vitro and also in recipient mice in vivo. Loss of Prdx4 boosts the pro-inflammatory potential of EVs. These findings identify Prdx4 as a critical regulator of inflammasome activity and provide new insights into remote cell-to-cell communication function of inflammasomes via macrophage-derived EVs.

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-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.

Objective The genetic basis of inflammatory bowel disease (IBD) is incompletely understood. The aim of this study was to identify rare genetic variants involved in the pathogenesis of IBD. Design Exome sequencing and immunological profiling were performed in a patient with early onset Crohn9s disease (CD). The coding region of the gene encoding X-linked inhibitor of apoptosis protein ( XIAP ) was sequenced in samples of 275 paediatric IBD and 1047 adult-onset CD patients. XIAP genotyping was performed in samples of 2680 IBD patients and 2864 healthy controls. Functional effects of the variants identified were investigated in primary cells and cultured cell lines. Results Our results demonstrate the frequent occurrence of private variants in XIAP in about four percent of male patients with paediatric-onset CD. While XIAP mutations are known to be associated with the primary immunodeficiency (PID) X-linked lymphoproliferative disease type 2 (XLP2), CD patients described here exhibited intestinal inflammation in the absence of XLP2 and harboured a spectrum of mutations partially distinct from that observed in XLP2. The majority of XIAP variants identified was associated with a selective defect in NOD1/2 signalling, impaired NOD1/2-mediated activation of NF-κB, and altered NF-κB-dependent cytokine production. Conclusions This study reveals the unanticipated, frequent occurrence of XIAP variants in male paediatric-onset CD. The link between XIAP and NOD1/2, and the association of XIAP variants with XLP2, support the concept of PID in a subset of IBD patients. Moreover, these studies provide a rationale for the implementation of XIAP sequencing in clinical diagnostics in male patients with severe CD.

The recognition of autophagy related 16-like 1 (ATG16L1) as a genetic risk factor has exposed the critical role of autophagy in Crohn's disease. Homozygosity for the highly prevalent ATG16L1 risk allele, or murine hypomorphic (HM) activity, causes Paneth cell dysfunction. As Atg16l1(HM) mice do not develop spontaneous intestinal inflammation, the mechanism(s) by which ATG16L1 contributes to disease remains obscure. Deletion of the unfolded protein response (UPR) transcription factor X-box binding protein-1 (Xbp1) in intestinal epithelial cells, the human orthologue of which harbours rare inflammatory bowel disease risk variants, results in endoplasmic reticulum (ER) stress, Paneth cell impairment and spontaneous enteritis. Unresolved ER stress is a common feature of inflammatory bowel disease epithelium, and several genetic risk factors of Crohn's disease affect Paneth cells. Here we show that impairment in either UPR (Xbp1(ΔIEC)) or autophagy function (Atg16l1(ΔIEC) or Atg7(ΔIEC)) in intestinal epithelial cells results in each other's compensatory engagement, and severe spontaneous Crohn's-disease-like transmural ileitis if both mechanisms are compromised. Xbp1(ΔIEC) mice show autophagosome formation in hypomorphic Paneth cells, which is linked to ER stress via protein kinase RNA-like endoplasmic reticulum kinase (PERK), elongation initiation factor 2α (eIF2α) and activating transcription factor 4 (ATF4). Ileitis is dependent on commensal microbiota and derives from increased intestinal epithelial cell death, inositol requiring enzyme 1α (IRE1α)-regulated NF-κB activation and tumour-necrosis factor signalling, which are synergistically increased when autophagy is deficient. ATG16L1 restrains IRE1α activity, and augmentation of autophagy in intestinal epithelial cells ameliorates ER stress-induced intestinal inflammation and eases NF-κB overactivation and intestinal epithelial cell death. ER stress, autophagy induction and spontaneous ileitis emerge from Paneth-cell-specific deletion of Xbp1. Genetically and environmentally controlled UPR function within Paneth cells may therefore set the threshold for the development of intestinal inflammation upon hypomorphic ATG16L1 function and implicate ileal Crohn's disease as a specific disorder of Paneth cells.

NOD2 is an intracellular receptor for the bacterial cell wall component muramyl dipeptide (MDP), and variants of NOD2 are associated with chronic inflammatory diseases of barrier organs (e. g., Crohn's disease, asthma, and atopic eczema). It is known that activation of NOD2 induces a variety of inflammatory and antibacterial factors. The exact transcriptomal signatures that define the cellular programs downstream of NOD2 activation and the influence of the Crohn-associated variant L1007fsinsC are yet to be defined. To describe the MDP-induced activation program, we analyzed the transcriptomal reactions of isogenic HEK293 cells expressing NOD2(wt) or NOD2(L1007fsinsC) to stimulation with MDP. Importantly, a clear loss of function could be observed in the cells carrying the Crohn-associated variant L1007fsinsC, whereas the NOD2(wt) cells showed differential regulation of growth factors, chemokines, and several antagonists of NF-kappa B (e. g., TNFAIP3 A20 and IER3). This genotype-dependent regulation pattern was confirmed in primary human myelomonocytic cells. The influence of TNFAIP3 and IER3 in the context of NOD2 signaling was characterized, and we could validate the predicted role as inhibitors of NOD2-induced NF-kappa B activation. We show that IER3 impairs the protective effect of NOD2(wt) against bacterial cytoinvasion. These results further our understanding of NOD2 as a first-line defense molecule and emphasize the importance of simultaneous upregulation of counter-regulatory anti-inflammatory factors as an integral part of the NOD2-induced cellular program. Lack of these regulatory events due to the L1007fsinsC variant may pivotally contribute to the induction and perpetuation of chronic inflammation. The Journal of Immunology, 2011, 186: 4027-4038.

Several coding variants of NOD2 and ATG16L1 are associated with increased risk of Crohn disease (CD). NOD2, a cytosolic receptor of the innate immune system activates pro-inflammatory signalling cascades upon recognition of bacterial muramyl dipeptide, but seems also to be involved in antiviral and antiparasitic defence programs. The CD associated variant L1007fsinsC leads to impaired pro-inflammatory signalling and diminished bacterial clearance. ATG16L1 is a protein essential for autophagosome formation at the phagophore assembly site. The CD associated T300A variant is located in the c-terminal WD40 domain, whose function is still unknown. Basal autophagy is not affected by the T300A variant, but antibacterial autophagy (xenophagy) is impaired, a finding that relates ATG16L1 as well as NOD2 to pathogen defence. Notably, combination of disease-associated alleles of ATG16L1 and NOD2/CARD15 leads to synergistically increased susceptibility for CD, indicating a possible crosstalk between NOD2- and ATG16L1-mediated processes in the pathogenesis of CD. This review surveys current research results and discusses the functional models of potential interplay between NLR-pathways and xenophagy. Interaction between pathways is discussed in the context of reactive oxygen species (ROS), membrane co-localisation, antigen processing and implications of disturbed Paneth cell vesicle export. These effects on pathogen response might imbalance the intestinal barrier epithelia towards chronic inflammation and promote development of Crohn disease. Further elucidation of NOD2/ATG16L1 interplay in xenophagy is relevant for understanding the aetiology of chronic intestinal inflammation and host-microbe interaction in general and could lead to principal new insights to xenophagy induction. (C) 2010 Elsevier GmbH. All rights reserved.

A plethora of functional and genetic studies have suggested a key role for the IL-23 pathway in chronic intestinal inflammation. Currently, pathogenic actions of IL-23 have been ascribed to specific effects on immune cells. Herein, we unveil a protective role of IL-23R signaling. Mice deficient in IL-23R expression in intestinal epithelial cells (Il23R(ΔIEC)) have reduced Reg3b expression, show a disturbed colonic microflora with an expansion of flagellated bacteria, and succumb to DSS colitis. Surprisingly, Il23R(ΔIEC) mice show impaired mucosal IL-22 induction in response to IL-23. αThy-1 treatment significantly deteriorates colitis in Il23R(ΔIEC) animals, which can be rescued by IL-22 application. Importantly, exogenous Reg3b administration rescues DSS-treated Il23R(ΔIEC) mice by recruiting neutrophils as IL-22-producing cells, thereby restoring mucosal IL-22 levels. The study identifies a critical barrier-protective immune pathway that originates from, and is orchestrated by, IL-23R signaling in intestinal epithelial cells.