Promyelocytic leukemia protein interacts with the apoptosis-associated speck-like protein to limit inflammasome activation.

The apoptosis-associated speck-like protein containing a caspase-activating recruitment domain (ASC) is an essential component of several inflammasomes, multiprotein complexes that regulate caspase-1 activation and inflammation. We report here an interaction between promyelocytic leukemia protein (PML) and ASC. We observed enhanced formation of ASC dimers in PML-deficient macrophages. These macrophages also display enhanced levels of ASC in the cytosol. Furthermore, IL-1ß production was markedly enhanced in these macrophages in response to both NLRP3 and AIM2 inflammasome activation and following bone marrow-derived macrophage infection with herpes simplex virus-1 (HSV-1) and Salmonella typhimurium. Collectively, our data indicate that PML limits ASC function, retaining ASC in the nucleus.

Extracellular acidosis is a novel danger signal alerting innate immunity via the NLRP3 inflammasome.

BACKGROUND: Local acidosis has been demonstrated in ischemic tissues and at inflammatory sites. RESULTS: Acidic extracellular pH triggers NLRP3 inflammasome activation and interleukin-1ß secretion in human macrophages. CONCLUSION: Acidic pH represents a novel danger signal alerting the innate immunity. SIGNIFICANCE: Local acidosis may promote inflammation at ischemic and inflammatory sites. Local extracellular acidification has been demonstrated at sites of ischemia and inflammation. IL-1ß is one of the key proinflammatory cytokines, and thus, its synthesis and secretion are tightly regulated. The NLRP3 (nucleotide-binding domain leucine-rich repeat containing family, pyrin domain containing 3) inflammasome complex, assembled in response to microbial components or endogenous danger signals, triggers caspase-1-mediated maturation and secretion of IL-1ß. In this study, we explored whether acidic environment is sensed by immune cells as an inflammasome-activating danger signal. Human macrophages were exposed to custom cell culture media at pH 7.5-6.0. Acidic medium triggered pH-dependent secretion of IL-1ß and activation of caspase-1 via a mechanism involving potassium efflux from the cells. Acidic extracellular pH caused rapid intracellular acidification, and the IL-1ß-inducing effect of acidic medium could be mimicked by acidifying the cytosol with bafilomycin A1, a proton pump inhibitor. Knocking down the mRNA expression of NLRP3 receptor abolished IL-1ß secretion at acidic pH. Remarkably, alkaline extracellular pH strongly inhibited the IL-1ß response to several known NLRP3 activators, demonstrating bipartite regulatory potential of pH on the activity of this inflammasome. The data suggest that acidic environment represents a novel endogenous danger signal alerting the innate immunity. Low pH may thus contribute to inflammation in acidosis-associated pathologies such as atherosclerosis and post-ischemic inflammatory responses.

NOD2-nitric oxide-responsive microRNA-146a activates Sonic hedgehog signaling to orchestrate inflammatory responses in murine model of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a debilitating chronic inflammatory disorder of the intestine. The interactions between enteric bacteria and genetic susceptibilities are major contributors of IBD etiology. Although genetic variants with loss or gain of NOD2 functions have been linked to IBD susceptibility, the mechanisms coordinating NOD2 downstream signaling, especially in macrophages, during IBD pathogenesis are not precisely identified. Here, studies utilizing the murine dextran sodium sulfate model of colitis revealed the crucial roles for inducible nitric-oxide synthase (iNOS) in regulating pathophysiology of IBDs. Importantly, stimulation of NOD2 failed to activate Sonic hedgehog (SHH) signaling in iNOS null macrophages, implicating NO mediated cross-talk between NOD2 and SHH signaling. NOD2 signaling up-regulated the expression of a NO-responsive microRNA, miR-146a, that targeted NUMB gene and alleviated the suppression of SHH signaling. In vivo and ex vivo studies confirmed the important roles for miR-146a in amplifying inflammatory responses. Collectively, we have identified new roles for miR-146a that established novel cross-talk between NOD2-SHH signaling during gut inflammation. Potential implications of these observations in therapeutics could increase the possibility of defining and developing better regimes to treat IBD pathophysiology.

Comparative study on pattern recognition receptors in non-teleost ray-finned fishes and their evolutionary significance in primitive vertebrates.

Pattern recognition receptors (PRRs) play important roles in innate immunity system and trigger the specific pathogen recognition by detecting the pathogen-associated molecular patterns. The main four PRRs components including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs) and C-type lectin receptors (CLRs) were surveyed in the five genomes of non-teleost ray-finned fishes (NTR) including bichir (Polypterus senegalus), American paddlefish (Polyodon spathula), alligator gar (Atractosteus spatula), spotted gar (Lepisosteus oculatus) and bowfin (Amia calva), representing all the four major basal groups of ray-finned fishes. The result indicates that all the four PRRs components have been well established in these NTR fishes. In the RLR-MAVS signal pathway, which detects intracellular RNA ligands to induce production of type I interferons (IFNs), the MAVS was lost in bichir particularly. Also, the essential genes of recognition of Lipopolysaccharide (LPS) commonly in mammals like MD2, LY96 and LBP could not be identified in NTR fishes. It is speculated that TLR4 in NTR fishes may act as a cooperator with other PRRs and has a different pathway of recognizing LPS compared with that in mammals. In addition, we provide a survey of NLR and CLR in NTR fishes. The CLRs results suggest that Group V receptors are absent in fishes and Group II and VI receptors are well established in the early vertebrate evolution. Our comprehensive research of PRRs involving NTR fishes provides a new insight into PRR evolution in primitive vertebrate.

Involvement of NLRP3 and NLRC4 Inflammasome in Uropathogenic E. coli Mediated Urinary Tract Infections.

BACKGROUND: Inflammatory response during urinary tract infection (UTI) is mediated by innate immune defense. Nod like receptors (NLRs) have been proposed to work simultaneously beside TLR pathways to mediate pro-inflammatory response and maintain tissue homeostasis. Some in vitro reports have showed the involvement of NLRP3 inflammasome during uropathogenic Escherichia coli (UPEC) mediated UTI. So we have sought to determine the status of various inflammasomes and their components in UPEC mediated UTI. METHODS: A total of 186 females experiencing the first episode of UTI were recruited for the study and forty were found to be positive for UPEC (≥105 CFU/ml) in their urine (N = 40). Further, we analyzed the expression of NLRP3, NLRC4, NAIP, AIM2, ASC, CASPASE-4, and CASPASE-1 gene at mRNA and protein level in the blood of UPEC confirmed study subjects through real time qPCR and immunoblotting. Healthy females (N = 40) visiting the OPD for health checkups, family planning advice and subjects undergoing routine medical examinations, were recruited as healthy control subjects. Pro-inflammatory cytokines (IL-6, IL-8, IFN-γ, TNF-α and MCP-1) were measured in the plasma of patients and controls through ELISA. For investigation of the involvement of NLRC4 and NLRP3 inflammasome, in vitro studies were performed using co-immunoprecipitation and confocal microscopy. RESULTS: Most of the inflammatory regulators studied (i.e., NLRP3, NAIP, NLRC4, ASC, and CASPASE-1) were found to be up-regulated at both mRNA and protein levels in the UPEC infected UTI patients. Also, pro-inflammatory cytokines (IL-6, IL-8, IFN-γ, TNF-α, and MCP-1) were found to be up-regulated in the patients group. However, no significant difference was observed in the expression of AIM2 and CASPASE-4 genes at both mRNA and protein levels. Further, in vitro studies have shown the involvement of NLRC4 inflammasome in UPEC infected THP1 derived macrophages. CONCLUSION: Involvement of NLRP3 and NLRC4 inflammasomes in UPEC infected UTI is evident from our findings. This is the first report showing levels of inflammasome and its components in UTI patients suggesting a possible role during UPEC mediated UTI. We have also reported the involvement of NLRC4 inflammasome for the first time during UTI infection.

Recent findings related to immune responses against leptospirosis and novel strategies to prevent infection.

What are the new approaches and emerging ideas to prevent leptospirosis, a neglected bacterial re-emerging zoonotic disease? How do Leptospira interrogans escape the host defenses? We aim here to review and discuss the most recent literature that provides some answers to these questions, in particular data related to a better understanding of adaptive and innate immunity towards leptospires, and design of vaccines. This is an opinion paper, not a comprehensive review. We will try to highlight the new strategies and technologies boosting the search for drugs and vaccines. We will also address the bottlenecks and difficulties impairing the search for efficient vaccines and the many gaps in our knowledge of immunity against leptospirosis. Finally, we aim to delineate how Leptospira spp. escape the innate immune responses of Toll-Like receptors (TLR) and Nod-Like receptors (NLR). The rational use of TLR and NLR agonists as adjuvants could be key to design future vaccines against pathogenic leptospires.

Deviant Behavior: Tick-Borne Pathogens and Inflammasome Signaling.

In the face of an assault, host cells mount an immediate response orchestrated by innate immunity. Two of the best described innate immune signaling networks are the Toll- and the Nod-like receptor pathways. Extensive work has been done characterizing both signaling cascades with several recent advances on the forefront of inflammasome biology. In this review, we will discuss how more commonly-studied pathogens differ from tick-transmitted microbes in the context of Nod-like receptor signaling and inflammasome formation. Because pathogens transmitted by ticks have unique characteristics, we offer the opinion that these microbes can be used to uncover novel principles of Nod-like receptor biology.

For Whom the Bell Tolls (and Nods): Spit-acular Saliva.

Having emerged during the early part of the Cretaceous period, ticks are an ancient group of hematophagous ectoparasites with significant veterinary and public health importance worldwide. The success of their life strategy can be attributed, in part, to saliva. As we enter into a scientific era where the collection of massive data sets and structures for biological application is possible, we suggest that understanding the molecular mechanisms that govern the life cycle of ticks is within grasp. With this in mind, we discuss what is currently known regarding the manipulation of Toll-like (TLR) and Nod-like (NLR) receptor signaling pathways by tick salivary proteins, and how these molecules impact pathogen transmission.

TLR4 and NLRP3 inflammasome activation in monocytes by N-propionyl cysteaminylphenol-maleimide-dextran (NPCMD).

BACKGROUND: N-propionyl cysteaminylphenol-maleimide-dextran (NPCMD) is a toxic tyrosinase substrate developed to treat melanoma. OBJECTIVE: We investigated the effect of NPCMD on innate immune responses in monocytes. METHODS: CD14⁺ monocytes and a monocytic cell line, THP-1, were stimulated with NPCMD in vitro. Cytokines in the culture supernatants were determined by ELISA and flow cytometry. RESULTS: NPCMD stimulated CD14⁺ monocytes and THP-1 cells to secrete TNFα, IL-6 and IL-8, but not IL-10 or IL-12. TNFα secretion from THP-1 cells stimulated with NPCMD was inhibited by addition of an anti-TLR4 mAb in culture. Moreover, NPCMD stimulated production of pro-IL-1ß in CD14⁺ monocytes and monocytic cell line THP-1 cells and activated the NLRP3-inflammasome, resulting in production of mature IL-1ß. Use of ASC and NLRP3-deficient THP-1 cell lines established involvement of the NLRP3 inflammasome in an IL-1ß secretion in treatment with NPCMD. Inhibition of IL-1ß secretion by an endocytosis inhibitor, cytochalasin B, and a lysosomal enzyme cathepsin B inhibitor, CA-074 Me, suggested the involvement of lysosomal rupture and leakage of cathepsin B into the cytosol in NLRP3 activation by NPCMD. CONCLUSION: The immunopotentiating effect of NPCMD mediated by TLR4 and NLRP3 inflammasome activation could be useful for eliciting effective adaptive immune responses against melanoma and other tumors.