[show abstract][hide abstract] ABSTRACT: Recognition of intracellular pathogenic bacteria by members of the nucleotide-binding domain and leucine-rich repeat containing (NLR) family triggers immune responses against bacterial infection. A major response induced by several Gram-negative bacteria is the activation of caspase-1 via the Nlrc4 inflammasome. Upon activation, caspase-1 regulates the processing of proIL-1β and proIL-18 leading to the release of mature IL-1β and IL-18, and induction of pyroptosis. The activation of the Nlrc4 inflammasome requires the presence of an intact type III or IV secretion system that mediates the translocation of small amounts of flagellin or PrgJ-like rod proteins into the host cytosol to induce Nlrc4 activation. Using the Salmonella system, it was shown that Naip2 and Naip5 link flagellin and the rod protein PrgJ, respectively, to Nlrc4. Furthermore, phosphorylation of Nlrc4 at Ser533 by Pkcδ was found to be critical for the activation of the Nlrc4 inflammasome. Here, we show that Naip2 recognizes the Shigella T3SS inner rod protein MxiI and induces Nlrc4 inflammasome activation. The expression of MxiI in primary macrophages was sufficient to induce pyroptosis and IL-1β release, which were prevented in macrophages deficient in Nlrc4. In the presence of MxiI or Shigella infection, MxiI associated with Naip2, and Naip2 interacted with Nlrc4. siRNA-mediated knockdown of Naip2, but not Naip5, inhibited Shigella-induced caspase-1 activation, IL-1β maturation and Asc pyroptosome formation. Notably, the Pkcδ kinase was dispensable for caspase-1 activation and secretion of IL-1β induced by Shigella or Salmonella infection. These results indicate that activation of caspase-1 by Shigella is triggered by the rod protein MxiI that interacts with Naip2 to induce activation of the Nlrc4 inflammasome independently of the Pkcδ kinase.
[show abstract][hide abstract] ABSTRACT: Although imbalances in gut microbiota composition, or "dysbiosis," are associated with many diseases, the effects of gut dysbiosis on host systemic physiology are less well characterized. We report that gut dysbiosis induced by antibiotic (Abx) treatment promotes allergic airway inflammation by shifting macrophage polarization in the lung toward the alternatively activated M2 phenotype. Adoptive transfer of alveolar macrophages derived from Abx-treated mice was sufficient to increase allergic airway inflammation. Abx treatment resulted in the overgrowth of a commensal fungal Candida species in the gut and increased plasma concentrations of prostaglandin E2 (PGE2), which induced M2 macrophage polarization in the lung. Suppression of PGE2 synthesis by the cyclooxygenase inhibitors aspirin and celecoxib suppressed M2 macrophage polarization and decreased allergic airway inflammatory cell infiltration in Abx-treated mice. Thus, Abx treatment can cause overgrowth of particular fungal species in the gut and promote M2 macrophage activation at distant sites to influence systemic responses including allergic inflammation.
[show abstract][hide abstract] ABSTRACT: Crohn's disease (CD) is a multifactorial pathology associated with the presence of adherent-invasive Escherichia coli (AIEC) and NLRP3 polymorphic variants. The presence of intracellular E. coli in other intestinal pathologies (OIP) and the role of NLRP3-inflammasome in the immune response activated by these bacteria have not been investigated. In this study, we sought to characterize intracellular strains isolated from patients with CD, ulcerative colitis (UC) and OIP, and analyze NLRP3-inflammasome role in the immune response and bactericidal activity induced in macrophages exposed to invasive bacteria. For this, intracellular E. coli isolation from ileal biopsies, using gentamicin-protection assay, revealed a prevalence and CFU/biopsy of E. coli higher in biopsies from CD, UC and OIP patients than in controls. To characterization of bacterial isolates, pulsed-field gel electrophoresis (PFGE) patterns, virulence genes, serogroup and phylogenetic group were analyzed. We found out that bacteria isolated from a given patient were closely related and shared virulence factors; however, strains from different patients were genetically heterogeneous. AIEC characteristics in isolated strains, such as invasive and replicative properties, were assessed in epithelial cells and macrophages, respectively. Some strains from CD and UC demonstrated AIEC properties, but not strains from OIP. Furthermore, the role of NLRP3 in pro-inflammatory cytokines production and bacterial elimination was determined in macrophages. E. coli strains induced IL-1β through NLRP3-dependent mechanism; however, their elimination by macrophages was independent of NLRP3. Invasiveness of intracellular E. coli strains into the intestinal mucosa and IL-1β production may contribute to CD and UC pathogenesis.
International journal of medical microbiology: IJMM 01/2014; · 4.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: The microbiota is an important factor in the development of the immune response. The interaction between the gastrointestinal tract and resident microbiota is well-balanced in healthy individuals, but its breakdown can lead to intestinal and extra-intestinal disease. We review current knowledge about the mechanisms that regulate the interaction between the immune system and the microbiota, focusing on the role of resident intestinal bacteria in the development of immune responses. We also discuss mechanisms that prevent immune responses against resident bacteria, and how the indigenous bacteria stimulate the immune system to protect against commensal pathobionts and exogenous pathogens. Unraveling the complex interactions between resident intestinal bacteria and the immune system could improve our understanding of disease pathogenesis and lead to new therapeutic approaches.
[show abstract][hide abstract] ABSTRACT: The NLRP3 inflammasome is a critical component of the innate immune system. NLRP3 activation is induced by diverse stimuli associated with bacterial infection or tissue damage, but its inappropriate activation is involved in the pathogenesis of inherited and acquired inflammatory diseases. However, the mechanism by which NLRP3 is activated remains poorly understood. In this study, we explored the role of kinases in NLRP3 inflammasome activation by screening a kinase inhibitor library and identified 3,4-Methylenedioxy-β-nitrostyrene (MNS) as an inhibitor for NLRP3 inflammasome activation. Notably, MNS did not affect the activation of the NLRC4 or AIM2 inflammasome. Mechanistically, MNS specifically prevented NLRP3-mediated ASC speckle formation and oligomerization without blocking potassium efflux induced by NLRP3 agonists. Surprisingly, Syk kinase, the reported target of MNS, did not mediate the inhibitory activity of MNS on NLRP3 inflammasome activation. We also found that the nitrovinyl group of MNS is essential for the inhibitory activity of MNS. Immunoprecipitation, mass spectrometry and mutation studies suggest that both the NOD domain and the LRR domain of NLRP3 were the intracellular targets of MNS. Administration of MNS also inhibited NLRP3 ATPase activity in vitro, suggesting that MNS blocks the NLRP3 inflammasome by directly targeting NLRP3 or NLRP3-associated complexes. These studies identified a novel chemical probe for studying the molecular mechanism of NLRP3 inflammasome activation which may advance the development of novel strategies to treat diseases associated with abnormal activation of NLRP3 inflammasome.
Journal of Biological Chemistry 11/2013; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Atopic dermatitis is a chronic inflammatory skin disease that affects 15-30% of children and approximately 5% of adults in industrialized countries. Although the pathogenesis of atopic dermatitis is not fully understood, the disease is mediated by an abnormal immunoglobulin-E immune response in the setting of skin barrier dysfunction. Mast cells contribute to immunoglobulin-E-mediated allergic disorders including atopic dermatitis. Upon activation, mast cells release their membrane-bound cytosolic granules leading to the release of several molecules that are important in the pathogenesis of atopic dermatitis and host defence. More than 90% of patients with atopic dermatitis are colonized with Staphylococcus aureus in the lesional skin whereas most healthy individuals do not harbour the pathogen. Several staphylococcal exotoxins can act as superantigens and/or antigens in models of atopic dermatitis. However, the role of these staphylococcal exotoxins in disease pathogenesis remains unclear. Here we report that culture supernatants of S. aureus contain potent mast-cell degranulation activity. Biochemical analysis identified δ-toxin as the mast cell degranulation-inducing factor produced by S. aureus. Mast cell degranulation induced by δ-toxin depended on phosphoinositide 3-kinase and calcium (Ca(2+)) influx; however, unlike that mediated by immunoglobulin-E crosslinking, it did not require the spleen tyrosine kinase. In addition, immunoglobulin-E enhanced δ-toxin-induced mast cell degranulation in the absence of antigen. Furthermore, S. aureus isolates recovered from patients with atopic dermatitis produced large amounts of δ-toxin. Skin colonization with S. aureus, but not a mutant deficient in δ-toxin, promoted immunoglobulin-E and interleukin-4 production, as well as inflammatory skin disease. Furthermore, enhancement of immunoglobulin-E production and dermatitis by δ-toxin was abrogated in Kit(W-sh/W-sh) mast-cell-deficient mice and restored by mast cell reconstitution. These studies identify δ-toxin as a potent inducer of mast cell degranulation and suggest a mechanistic link between S. aureus colonization and allergic skin disease.
[show abstract][hide abstract] ABSTRACT: Inflammation is a critical player in the development of both colitis-associated and sporadic colon cancers. Several studies suggest that the microbiota contribute to inflammation and tumorigenesis; however, studies to understand the role of the microbiota in colon tumor development in germfree (GF) mice are limited. We therefore studied the effects of the microbiota on the development of inflammation and tumors in germfree and conventionally-raised specific pathogen-free (SPF) mice treated with azoxymethane (AOM) and dextran sulfate sodium (DSS). We discovered that GF mice developed significantly more and larger tumors compared to that in SPF mice after AOM and DSS treatment despite the lack of early acute inflammation in response to chemically-induced injury by DSS. Although the extent of intestinal epithelial damage and apoptosis was not significantly different in GF and SPF mice, there was a delay in intestinal epithelial repair to DSS-induced injury in GF mice resulting in a late onset of proinflammatory and protumorigenic responses and increased epithelial proliferation and microadenoma formation. Recolonization of GF mice with commensal bacteria or administration of LPS reduced tumorigenesis. Thus, although commensal bacteria are capable of driving chronic inflammation and tumorigenesis, the gut microbiota also have important roles in limiting chemically-induced injury and proliferative responses that lead to tumor development.
[show abstract][hide abstract] ABSTRACT: Decreased levels or function of nucleotide-binding oligomerization domain 2 (NOD2) are associated with Crohn's disease. NOD2 regulates intestinal inflammation, and is also expressed by human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), to regulate their differentiation. We investigated whether NOD2 is required for the anti-inflammatory activities of MSCs in mice with colitis.
Colitis was induced in mice by administration of dextran sulfate sodium or trinitrobenzene sulfonic acid. Mice were then given intraperitoneal injections of NOD2-activated hUCB-MSCs; colon tissues and mesenteric lymph nodes were collected for histologic analyses. A bromodeoxyuridine assay was used to determine the ability of hUCB-MSCs to inhibit proliferation of human mononuclear cells in culture.
Administration of hUCB-MSCs reduced the severity of colitis in mice. The anti-inflammatory effects of hUCB-MSCs were greatly increased by activation of NOD2 by its ligand, muramyl dipeptide (MDP). Administration of NOD2-activated hUCB-MSCs increased anti-inflammatory responses in colons of mice, such as production of interleukin (IL)10 and infiltration by T regulatory (Treg) cells, and reduced production of inflammatory cytokines. Proliferation of mononuclear cells was significantly inhibited by co-culture with hUCB-MSCs that had been stimulated with MDP. MDP induced prolonged production of prostaglandin (PG)E2 in hUCB-MSCs via the NOD2-RIP2 pathway, which suppressed proliferation of mononuclear cells derived from hUCB. PGE2 produced by hUCB-MSCs in response to MDP increased production of IL10 and Treg cells. In mice, production of PGE2 by MSCs and subsequent production of IL10 were required to reduce the severity of colitis.
Activation of NOD2 is required for the ability of hUCB-MSCs to reduce the severity of colitis in mice. NOD2 signaling increases the ability of these cells to suppress mononuclear cell proliferation by inducing production of PGE2.
[show abstract][hide abstract] ABSTRACT: Helicobacter pylori colonization of the stomach affects about half of the world population and is associated with the development of gastritis, ulcers and cancer. Polymorphisms in the IL1B gene are linked to an increased risk of H. pylori-associated cancer, but the bacterial and host factors that regulate IL-1β production in response to H. pylori infection remain unknown. Using murine bone marrow-derived DCs, we show that the bacterial virulence factors cagPAI and CagL, but not VacA or CagA, regulate the induction of pro-IL-1β and the production of mature IL-1β in response to H. pylori infection. We further show that the host receptors, TLR2 and NOD2, but not NOD1, are required for induction of pro-IL-1β and NLRP3 in H. pylori-infected DCs. In contrast, NLRP3 and the adaptor ASC were essential for the activation of caspase-1, processing of pro-IL-1β into IL-1β and IL-1β secretion. Finally, we show that mice deficient in caspase-1, IL-1β and IL-1 receptor, but not NLRP3 are impaired in the clearance of CagA-positive H. pylori from the stomach when compared with wild-type mice. These studies identify bacterial cagPAI and the cooperative interaction among host innate receptors TLR2, NOD2 and NLRP3 as important regulators of IL-1β production in H. pylori-infected DCs. This article is protected by copyright. All rights reserved.
European Journal of Immunology 07/2013; · 4.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: The NLRP3 inflammasome is an important component of the innate immune system. However, its mechanism of activation remains largely unknown. We show that NLRP3 activators including bacterial pore-forming toxins, nigericin, ATP, and particulate matter caused mitochondrial perturbation or the opening of a large membrane pore, but this was not required for NLRP3 activation. Furthermore, reactive oxygen species generation or a change in cell volume was not necessary for NLRP3 activation. Instead, the only common activity induced by all NLRP3 agonists was the permeation of the cell membrane to K(+) and Na(+). Notably, reduction of the intracellular K(+) concentration was sufficient to activate NLRP3, whereas an increase in intracellular Na(+) modulated but was not strictly required for inflammasome activation. These results provide a unifying model for the activation of the NLRP3 inflammasome in which a drop in cytosolic K(+) is the common step that is necessary and sufficient for caspase-1 activation.
[show abstract][hide abstract] ABSTRACT: A dense resident microbial community in the gut, referred as the commensal microbiota, coevolved with the host and is essential for many host physiological processes that include enhancement of the intestinal epithelial barrier, development of the immune system and acquisition of nutrients. A major function of the microbiota is protection against colonization by pathogens and overgrowth of indigenous pathobionts that can result from the disruption of the healthy microbial community. The mechanisms that regulate the ability of the microbiota to restrain pathogen growth are complex and include competitive metabolic interactions, localization to intestinal niches and induction of host immune responses. Pathogens, in turn, have evolved strategies to escape from commensal-mediated resistance to colonization. Thus, the interplay between commensals and pathogens or indigenous pathobionts is critical for controlling infection and disease. Understanding pathogen-commensal interactions may lead to new therapeutic approaches to treating infectious diseases.
[show abstract][hide abstract] ABSTRACT: Periodontitis is a common disease that is characterized by resorption of the alveolar bone and mediated by commensal bacteria that trigger host immune responses and bone destruction through unidentified mechanisms. We report that Nod1, an innate intracellular host receptor for bacterial peptidoglycan-related molecules, is critical for commensal-induced periodontitis in a mouse model. Mice lacking Nod1 exhibit reduced bone resorption as well as impaired recruitment of neutrophils to gingival tissues and osteoclasts to the alveolar bone, which mediate tissue and bone destruction. Further analysis showed that accumulation of a Nod1-stimulating commensal bacterium, NI1060, at gingival sites was sufficient to induce neutrophil recruitment and bone resorption. Genomic sequencing revealed that NI1060 is a mouse-specific bacterium that is related to bacteria associated with the development of aggressive periodontitis in humans. These findings provide insight into commensal-host interactions contributing to periodontitis and identify a potential target for preventing this common oral disease.
[show abstract][hide abstract] ABSTRACT: The mammalian intestine is colonized by trillions of microorganisms, most of which are bacteria that have co-evolved with the host in a symbiotic relationship. The collection of microbial populations that reside on and in the host is commonly referred to as the microbiota. A principal function of the microbiota is to protect the intestine against colonization by exogenous pathogens and potentially harmful indigenous microorganisms via several mechanisms, which include direct competition for limited nutrients and the modulation of host immune responses. Conversely, pathogens have developed strategies to promote their replication in the presence of competing microbiota. Breakdown of the normal microbial community increases the risk of pathogen infection, the overgrowth of harmful pathobionts and inflammatory disease. Understanding the interaction of the microbiota with pathogens and the host might provide new insights into the pathogenesis of disease, as well as novel avenues for preventing and treating intestinal and systemic disorders.
[show abstract][hide abstract] ABSTRACT: Dendritic cells (DC) are permissive to murine norovirus (MNV) infection in vitro and in vivo. However, their roles during infection in vivo are not well defined. To determine the role of DC during infection, conventional DC were depleted from CD11c-DTR mice and infected with a persistent MNV strain. Viral titers in the intestine and secondary lymphoid organs were determined at early time points during infection, and anti-MNV antibody responses were analyzed later during infection. Depletion of conventional DC resulted in increased viral loads in intestinal tissues, impaired generation of antibody responses, and a failure of MNV to efficiently infect lymphoid tissues. These data suggest that DC play multiple roles in MNV pathogenesis, in both innate immunity and the efficient generation of adaptive immune responses against MNV, as well as by promoting the dissemination of MNV to secondary lymphoid tissues. This is the first study to probe the roles of DC in controlling and/or facilitating a norovirus infection in vivo and provides the basis for further studies aimed at defining mechanisms by which DC control MNV replication and promote viral dissemination.
Journal of General Virology 05/2013; · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Ashkenazi Jewish population has a several-fold higher prevalence of Crohn's disease (CD) compared with non-Jewish European ancestry populations and has a unique genetic history. Haplotype association is critical to CD etiology in this population, most notably at NOD2, in which three causal, uncommon and conditionally independent NOD2 variants reside on a shared background haplotype. We present an analysis of extended haplotypes that showed significantly greater association to CD in the Ashkenazi Jewish population compared with a non-Jewish population (145 haplotypes and no haplotypes with P-value <10(-3), respectively). Two haplotype regions, one each on chromosomes 16 and 21, conferred increased disease risk within established CD loci. We performed exome sequencing of 55 Ashkenazi Jewish individuals and follow-up genotyping focused on variants in these two regions. We observed Ashkenazi Jewish-specific nominal association at R755C in TRPM2 on chromosome 21. Within the chromosome 16 region, R642S of HEATR3 and rs9922362 of BRD7 showed genome-wide significance. Expression studies of HEATR3 demonstrated a positive role in NOD2-mediated NF-κB signaling. The BRD7 signal showed conditional dependence with only the downstream rare CD-causal variants in NOD2, but not with the background haplotype; this elaborates NOD2 as a key illustration of synthetic association.Genes and Immunity advance online publication, 25 April 2013; doi:10.1038/gene.2013.19.
[show abstract][hide abstract] ABSTRACT: Mammals are colonized by large numbers of microorganisms, including trillions of bacteria, most of which live in the intestinal tract. These indigenous microorganisms that inhabit the body of humans and animals are referred collectively to as the microbiota. Accumulating evidence indicates that the microbiota regulates the development and/or function of different types of immune cells in the intestine. For example, the microbiota drives homeostatic, pathogenic, and regulatory T cell immune responses that contribute to tissue homeostasis, but also can promote disease. The gut microbes also facilitate IgA responses, which in turn regulate the composition and function of the gut microbiota. Thus, the reciprocal regulation of the gut microbiota and the host immune system may influence the balance between homeostasis and disease in the intestine.
The Journal of Immunology 02/2013; 190(4):1389-95. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Inflammasomes are multi-protein platforms that drive the activation of caspase-1 leading to the processing and secretion of biologically active IL-1β and IL-18. Different inflammasomes including NLRP3, NLRC4 and AIM2 are activated and assembled in response to distinct microbial or endogenous stimuli. However, the mechanisms by which upstream stimuli trigger inflammasome activation remain poorly understood. PKR, a protein kinase activated by viral infection, has been recently shown to be required for the activation of the inflammasomes. Using macrophages from two different mouse strains deficient in PKR, we found that PKR is important for the induction of the inducible nitric oxide synthase (iNOS). However, PKR was dispensable for caspase-1 activation, processing of pro-IL-1β/IL-18 and secretion of IL-1β induced by stimuli that trigger the activation of NLRP3, NLRC4 and AIM2. These results indicate that PKR is not required for inflammasome activation in macrophages.
European Journal of Immunology 02/2013; · 4.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: Instability in the composition of gut bacterial communities (dysbiosis) has been linked to common human intestinal disorders, such as Crohn's disease and colorectal cancer. Here, we show that dysbiosis caused by Nod2 deficiency gives rise to a reversible, communicable risk of colitis and colitis-associated carcinogenesis in mice. Loss of either Nod2 or RIP2 resulted in a proinflammatory microenvironment that enhanced epithelial dysplasia following chemically induced injury. The condition could be improved by treatment with antibiotics or an anti-interleukin-6 receptor-neutralizing antibody. Genotype-dependent disease risk was communicable via maternally transmitted microbiota in both Nod2-deficient and WT hosts. Furthermore, reciprocal microbiota transplantation reduced disease risk in Nod2-deficient mice and led to long-term changes in intestinal microbial communities. Conversely, disease risk was enhanced in WT hosts that were recolonized with dysbiotic fecal microbiota from Nod2-deficient mice. Thus, we demonstrated that licensing of dysbiotic microbiota is a critical component of disease risk. Our results demonstrate that NOD2 has an unexpected role in shaping a protective assembly of gut bacterial communities and suggest that manipulation of dysbiosis is a potential therapeutic approach in the treatment of human intestinal disorders.
The Journal of clinical investigation 01/2013; · 15.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: The cytoplasmic receptor NOD2 (nucleotide-binding oligomerization domain 2) senses peptidoglycan fragments and triggers host defense pathways, including activation of nuclear factor κB (NF-κB) signaling, which lead to inflammatory immune responses. Dysregulation of NOD2 signaling is associated with inflammatory diseases, such as Crohn's disease and Blau syndrome. We used a genome-wide small interfering RNA screen to identify regulators of the NOD2 signaling pathway. Several genes associated with Crohn's disease risk were identified in the screen. A comparison of candidates from this screen with other "omics" data sets revealed interconnected networks of genes implicated in NF-κB signaling, thus supporting a role for NOD2 and NF-κB pathways in the pathogenesis of Crohn's disease. Many of these regulators were validated in secondary assays, such as measurement of interleukin-8 secretion, which is partially dependent on NF-κB. Knockdown of putative regulators in human embryonic kidney 293 cells followed by stimulation with tumor necrosis factor-α revealed that most of the genes identified were general regulators of NF-κB signaling. Overall, the genes identified here provide a resource to facilitate the elucidation of the molecular mechanisms that regulate NOD2- and NF-κB-mediated inflammation.