Direct Bacterial Killing In Vitro by Recombinant Nod2 Is Compromised by Crohn's Disease-Associated Mutations

Background: A homeostatic relationship with the intestinal microflora is increasingly appreciated as essential for human health and wellbeing. Mutations in the leucine-rich repeat (LRR) domain of Nod2, a bacterial recognition protein, are associated with development of the inflammatory bowel disorder, Crohn’s disease. We investigated the molecular mechanisms underlying disruption of intestinal symbiosis in patients carrying Nod2 mutations. Methodology/Principal Findings: In this study, using purified recombinant LRR domains, we demonstrate that Nod2 is a direct antimicrobial agent and this activity is generally deficient in proteins carrying Crohn’s-associated mutations. Wildtype, but not Crohn’s-associated, Nod2 LRR domains directly interacted with bacteria in vitro, altered their metabolism and disrupted the integrity of the plasma membrane. Antibiotic activity was also expressed by the LRR domains of Nod1 and other pattern recognition receptors suggesting that the LRR domain is a conserved anti-microbial motif supporting innate cellular immunity. Conclusions/Significance: The lack of anti-bacterial activity demonstrated with Crohn’s-associated Nod2 mutations in vitro, supports the hypothesis that a deficiency in direct bacterial killing contributes to the association of Nod2 polymorphism

A Crucial Role of Flagellin in the Induction of Airway Mucus Production by Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. Flagellin is a P. aeruginosa virulence factor involved in host response to this pathogen. We examined the role of flagellin in P. aeruginosa-induced mucus secretion. Using a mouse model of pulmonary infection we showed that PAK, a wild type strain of P. aeruginosa, induced airway mucus secretion and mucin muc5ac expression at higher levels than its flagellin-deficient mutant (ΔFliC). PAK induced expression of MUC5AC and MUC2 in both human airway epithelial NCI-H292 cell line and in primary epithelial cells. In contrast, ΔFliC infection had lower to no effect on MUC5AC and MUC2 expressions. A purified P. aeruginosa flagellin induced MUC5AC expression in parallel to IL-8 secretion in NCI-H292 cells. Accordingly, ΔFliC mutant stimulated IL-8 secretion at significantly lower levels compared to PAK. Incubation of NCI-H292 cells with exogenous IL-8 induced MUC5AC expression and pre-incubation of these cells with an anti-IL-8 antibody abrogated flagellin-mediated MUC5AC expression. Silencing of TLR5 and Naip, siRNA inhibited both flagellin-induced MUC5AC expression and IL-8 secretion. Finally, inhibition of ERK abolished the expression of both PAK- and flagellin-induced MUC5AC. We conclude that: (i) flagellin is crucial in P. aeruginosa-induced mucus hyper-secretion through TLR5 and Naip pathways; (ii) this process is mediated by ERK and amplified by IL-8. Our findings help understand the mechanisms involved in mucus secretion during pulmonary infectious disease induced by P. aeruginosa, such as in cystic fibrosis

A Novel Protein Isoform of the Multicopy Human NAIP Gene Derives from Intragenic Alu SINE Promoters

The human neuronal apoptosis inhibitory protein (NAIP) gene is no longer principally considered a member of the Inhibitor of Apoptosis Protein (IAP) family, as its domain structure and functions in innate immunity also warrant inclusion in the Nod-Like Receptor (NLR) superfamily. NAIP is located in a region of copy number variation, with one full length and four partly deleted copies in the reference human genome. We demonstrate that several of the NAIP paralogues are expressed, and that novel transcripts arise from both internal and upstream transcription start sites. Remarkably, two internal start sites initiate within Alu short interspersed element (SINE) retrotransposons, and a third novel transcription start site exists within the final intron of the GUSBP1 gene, upstream of only two NAIP copies. One Alu functions alone as a promoter in transient assays, while the other likely combines with upstream L1 sequences to form a composite promoter. The novel transcripts encode shortened open reading frames and we show that corresponding proteins are translated in a number of cell lines and primary tissues, in some cases above the level of full length NAIP. Interestingly, some NAIP isoforms lack their caspase-sequestering motifs, suggesting that they have novel functions. Moreover, given that human and mouse NAIP have previously been shown to employ endogenous retroviral long terminal repeats as promoters, exaptation of Alu repeats as additional promoters provides a fascinating illustration of regulatory innovations adopted by a single gene

Inhibitor of apoptosis proteins, NAIP, cIAP1 and cIAP2 expression during macrophage differentiation and M1/M2 polarization

Monocytes and macrophages constitute the first line of defense of the immune system against external pathogens. Macrophages have a highly plastic phenotype depending on environmental conditions; the extremes of this phenotypic spectrum are a pro-inflammatory defensive role (M1 phenotype) and an anti-inflammatory tissue-repair one (M2 phenotype). The Inhibitor of Apoptosis (IAP) proteins have important roles in the regulation of several cellular processes, including innate and adaptive immunity. In this study we have analyzed the differential expression of the IAPs, NAIP, cIAP1 and cIAP2, during macrophage differentiation and polarization into M1 or M2. In polarized THP-1 cells and primary human macrophages, NAIP is abundantly expressed in M2 macrophages, while cIAP1 and cIAP2 show an inverse pattern of expression in polarized macrophages, with elevated expression levels of cIAP1 in M2 and cIAP2 preferentially expressed in M1. Interestingly, treatment with the IAP antagonist SMC-LCL161, induced the upregulation of NAIP in M2, the downregulation of cIAP1 in M1 and M2 and an induction of cIAP2 in M1 macrophages.This work was supported by Universidad de Granada, Plan Propio 2015;#P3B: FAM, VMC (http://investigacion.ugr.es/pages/planpropio/2015/ resoluciones/p3b_def_28072015); Universidad de Granada CEI BioTic;#CAEP2-84: VMC (http:// biotic.ugr.es/pages/resolucionprovisional enseaanzapractica22demayo/!); and Canadian nstitutes of Health Research;#231421, #318176, #361847: STB, ECL, RK (http://www.cihr-irsc.gc. ca/e/193.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A Novel Role for the NLRC4 Inflammasome in Mucosal Defenses against the Fungal Pathogen Candida albicans

Candida sp. are opportunistic fungal pathogens that colonize the skin and oral cavity and, when overgrown under permissive conditions, cause inflammation and disease. Previously, we identified a central role for the NLRP3 inflammasome in regulating IL-1β production and resistance to dissemination from oral infection with Candida albicans. Here we show that mucosal expression of NLRP3 and NLRC4 is induced by Candida infection, and up-regulation of these molecules is impaired in NLRP3 and NLRC4 deficient mice. Additionally, we reveal a role for the NLRC4 inflammasome in anti-fungal defenses. NLRC4 is important for control of mucosal Candida infection and impacts inflammatory cell recruitment to infected tissues, as well as protects against systemic dissemination of infection. Deficiency in either NLRC4 or NLRP3 results in severely attenuated pro-inflammatory and antimicrobial peptide responses in the oral cavity. Using bone marrow chimeric mouse models, we show that, in contrast to NLRP3 which limits the severity of infection when present in either the hematopoietic or stromal compartments, NLRC4 plays an important role in limiting mucosal candidiasis when functioning at the level of the mucosal stroma. Collectively, these studies reveal the tissue specific roles of the NLRP3 and NLRC4 inflammasome in innate immune responses against mucosal Candida infection