Intracellular invasion of Orientia tsutsugamushi activates inflammasome in asc-dependent manner

Orientia tsutsugamushi, a causative agent of scrub typhus, is an obligate intracellular bacterium, which escapes from the endo/phagosome and replicates in the host cytoplasm. O. tsutsugamushi infection induces production of pro-inflammatory mediators including interleukin-1β (IL-1β), which is secreted mainly from macrophages upon cytosolic stimuli by activating cysteine protease caspase-1 within a complex called the inflammasome, and is a key player in initiating and maintaining the inflammatory response. However, the mechanism for IL-1β maturation upon O. tsutsugamushi infection has not been identified. In this study, we show that IL-1 receptor signaling is required for efficient host protection from O. tsutsugamushi infection. Live Orientia, but not heat- or UV-inactivated Orientia, activates the inflammasome through active bacterial uptake and endo/phagosomal maturation. Furthermore, Orientia-stimulated secretion of IL-1β and activation of caspase-1 are ASC- and caspase-1- dependent since IL-1β production was impaired in Asc- and caspase-1-deficient macrophages but not in Nlrp3-, Nlrc4- and Aim2-deficient macrophages. Therefore, live O. tsutsugamushi triggers ASC inflammasome activation leading to IL-1β production, which is a critical innate immune response for effective host defense.     

Inflammasome sensor Nlrp1b-dependent resistance to anthrax is mediated by caspase-1, IL-1 signaling and neutrophil recruitment

Bacillus anthracis infects hosts as a spore, germinates, and disseminates in its vegetative form. Production of anthrax lethal and edema toxins following bacterial outgrowth results in host death. Macrophages of inbred mouse strains are either sensitive or resistant to lethal toxin depending on whether they express the lethal toxin responsive or non-responsive alleles of the inflammasome sensor Nlrp1b (Nlrp1b(S/S) or Nlrp1b(R/R), respectively). In this study, Nlrp1b was shown to affect mouse susceptibility to infection. Inbred and congenic mice harboring macrophage-sensitizing Nlrp1b(S/S) alleles (which allow activation of caspase-1 and IL-1β release in response to anthrax lethal toxin challenge) effectively controlled bacterial growth and dissemination when compared to mice having Nlrp1b(R/R) alleles (which cannot activate caspase-1 in response to toxin). Nlrp1b(S)-mediated resistance to infection was not dependent on the route of infection and was observed when bacteria were introduced by either subcutaneous or intravenous routes. Resistance did not occur through alterations in spore germination, as vegetative bacteria were also killed in Nlrp1b(S/S) mice. Resistance to infection required the actions of both caspase-1 and IL-1β as Nlrp1b(S/S) mice deleted of caspase-1 or the IL-1 receptor, or treated with the Il-1 receptor antagonist anakinra, were sensitized to infection. Comparison of circulating neutrophil levels and IL-1β responses in Nlrp1b(S/S),Nlrp1b(R/) (R) and IL-1 receptor knockout mice implicated Nlrp1b and IL-1 signaling in control of neutrophil responses to anthrax infection. Neutrophil depletion experiments verified the importance of this cell type in resistance to B. anthracis infection. These data confirm an inverse relationship between murine macrophage sensitivity to lethal toxin and mouse susceptibility to spore infection, and establish roles for Nlrp1b(S), caspase-1, and IL-1β in countering anthrax infection.     

An inflammasome-independent role for epithelial-expressed Nlrp3 in renal ischemia-reperfusion injury

Cytoplasmic innate immune receptors are important therapeutic targets for diseases associated with overproduction of proinflammatory cytokines. One cytoplasmic receptor complex, the Nlrp3 inflammasome, responds to an extensive array of molecules associated with cellular stress. Under normal conditions, Nlrp3 is autorepressed, but in the presence of its ligands, it oligomerizes, recruits apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc), and triggers caspase 1 activation and the maturation of proinflammatory cytokines such as IL-1β and IL-18. Because ischemic tissue injury provides a potential source for Nlrp3 ligands, our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in components of the Nlrp3 inflammasome (Nlrp3(-/-) and Asc(-/-) mice). To examine the role of the inflammasome in renal ischemia-reperfusion injury (IRI) we also tested its downstream targets caspase 1, IL-1β, and IL-18. Both Nlrp3 and Asc were highly expressed in renal tubular epithelium of humans and mice, and the absence of Nlrp3, but not Asc or the downstream inflammasome targets, dramatically protected from kidney IRI. We conclude that Nlrp3 contributes to renal IRI by a direct effect on renal tubular epithelium and that this effect is independent of inflammasome-induced proinflammatory cytokine production.     

Activation of the Nlrp3 inflammasome by mitochondrial reactive oxygen species: A novel mechanism of albumin-induced tubulointerstitial inflammation

Albuminuria is not only an important marker of chronic kidney disease but also a crucial contributor to tubulointerstitial inflammation (TIF). In this study, we determined whether activation of the Nlrp3 inflammasome is involved in albuminuria induced-TIF and the underlying mechanisms of inflammasome activation by mitochondrial reactive oxygen species (mROS). We established an albumin-overload induced rat nephropathy model characterised by albuminuria, renal infiltration of inflammatory cells, tubular dilation and atrophy. The renal expression levels of the Nlrp3 inflammasome, IL-1β and IL-18 were significantly increased in this animal model. In vitro, albumin time- and dose-dependently increased the expression levels of the Nlrp3 inflammasome, IL-1β and IL18. Moreover, the silencing of the Nlrp3 gene or the use of the caspase-1 inhibitor Z-VAD-fmk significantly attenuated the albumin-induced increase in IL-1β and IL-18 expression in HK2 cells. In addition, mROS generation was elevated by albumin stimulation, whereas the ROS scavenger N-acetyl-l-cysteine (NAC) inhibited Nlrp3 expression and the release of IL-1β and IL-18. In kidney biopsy specimens obtained from patients with IgA nephropathy, Nlrp3 expression was localised to the proximal tubular epithelial cells, and this result is closely correlated with the extent of proteinuria and TIF. In summary, this study demonstrates that albuminuria may serve as an endogenous danger-associated molecular pattern (DAMP) that stimulates TIF via the mROS-mediated activation of the cytoplasmic Nlrp3 inflammasome.     

TLR2 signaling contributes to rapid inflammasome activation during F. novicida infection

BACKGROUND: Early detection of microorganisms by the innate immune system is provided by surface-expressed and endosomal pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). Detection of microbial components by TLRs initiates a signaling cascade leading to the expression of proinflammatory cytokines including IL-6 and IL-1β. Some intracellular bacteria subvert the TLR response by rapidly escaping the phagosome and entering the cytosol. However, these bacteria may be recognized by the inflammasome, a multi-protein complex comprised of a sensor protein, ASC and the cysteine protease caspase-1. Inflammasome activation leads to release of the proinflammatory cytokines IL-1β and IL-18 and death of the infected cell, an important host defense that eliminates the pathogen's replicative niche. While TLRs and inflammasomes are critical for controlling bacterial infections, it is unknown whether these distinct host pathways cooperate to activate defenses against intracellular bacteria. METHODOLOGY/SIGNIFICANT FINDINGS: Using the intracellular bacterium Francisella novicida as a model, we show that TLR2(-/-) macrophages exhibited delayed inflammasome activation compared to wild-type macrophages as measured by inflammasome assembly, caspase-1 activation, cell death and IL-18 release. TLR2 also contributed to inflammasome activation in response to infection by the cytosolic bacterium Listeria monocytogenes. Components of the TLR2 signaling pathway, MyD88 and NF-κB, were required for rapid inflammasome activation. Furthermore, TLR2(-/-) mice exhibited lower levels of cell death, caspase-1 activation, and IL-18 production than wild-type mice upon F. novicida infection. CONCLUSIONS/SIGNIFICANCE: These results show that TLR2 is required for rapid inflammasome activation in response to infection by cytosolic bacterial pathogens. In addition to further characterizing the role of TLR2 in host defense, these findings broaden our understanding of how the host integrates signals from spatiotemporally separated PRRs to coordinate an innate response against intracellular bacteria.     

IL-6-dependent mucosal protection prevents establishment of a microbial niche for attaching/effacing lesion-forming enteric bacterial pathogens

Enteric infections with attaching/effacing lesion-inducing bacterial pathogens are a worldwide health problem. A murine infection model with one such pathogen, Citrobacter rodentium, was used to elucidate the importance of the pleiotropic immune regulator, IL-6, in the pathogenesis of infection. IL-6 was strongly induced in colonic epithelial cells and macrophages upon C. rodentium infection and was required for effective host defense, because mice lacking IL-6 failed to control bacterial numbers 2-3 wk after infection and exhibited increased mortality. IL-6 was not needed for mounting effective T and B cell responses to the pathogens, nor was it important for induction of IFN-gamma or TNF-alpha, cytokines involved in host defense against the bacteria, or the antibacterial effector, NO. Instead, IL-6 played a key role in mucosal protection, since its absence was associated with marked infection-induced apoptosis in the colonic epithelium and subsequent ulcerations. Cell culture studies confirmed that IL-6 protected colon epithelial cells directly against inducible apoptosis, which was accompanied by increased expression of an array of genes encoding antiapoptotic proteins, including Bcl-x(L), Mcl-1, cIAP-2, and Bcl-3. Ulcerations appeared to be pathogenetically important, because bacteria localized preferentially to those regions, and chemically induced colonic ulcerations promoted bacterial colonization. Furthermore, blood components likely present in ulcer exudates, particularly alanine, asparagine, and glycine, promoted bacterial growth. Thus, IL-6 is an important regulator of host defense against C. rodentium by protecting the mucosa against ulcerations which can act as a microbial niche for the bacteria.     

Modulation of inflammasome pathways by bacterial and viral pathogens

Inflammasomes are emerging as key regulators of the host response against microbial pathogens. These cytosolic multiprotein complexes recruit and activate the cysteine protease caspase-1 when microbes invade sterile tissues or elicit cellular damage. Inflammasome-activated caspase-1 induces inflammation by cleaving the proinflammatory cytokines IL-1β and IL-18 into their biologically active forms and by releasing the alarmin HMGB1 into the extracellular milieu. Additionally, inflammasomes counter bacterial replication and clear infected immune cells through an inflammatory cell death program termed pyroptosis. As a countermeasure, bacterial and viral pathogens evolved virulence factors to antagonize inflammasome pathways. In this review, we discuss recent progress on how inflammasomes contribute to host defense against bacterial and viral pathogens, and we review how viruses and bacteria modulate inflammasome function to their benefit.     

Early administration of probiotic Lactobacillus acidophilus and/or prebiotic inulin attenuates pathogen-mediated intestinal inflammation and Smad 7 cell signaling

Immaturity of gut-associated immunity may contribute to pediatric mortality associated with enteric infections. A murine model to parallel infantile enteric disease was used to determine the effects of probiotic, Lactobacillus acidophilus (La), prebiotic, inulin, or both (synbiotic, syn) on pathogen-induced inflammatory responses, NF-κB, and Smad 7 signaling. Newborn mice were inoculated bi-weekly for 4?weeks with La, inulin, or syn and challenged with Citrobacter rodentium (Cr) at 5?weeks. Mouse intestinal epithelial cells (CMT93) were exposed to Cr to determine temporal alterations in NF-Kappa B and Smad 7 levels. Mice with pretreatment of La, inulin, and syn show reduced intestinal inflammation following Cr infection compared with controls, which is associated with significantly reduced bacterial colonization in La, inulin, and syn animals. Our results further show that host defense against Cr infection correlated with enhanced colonic IL-10 and transforming growth factor-β expression and inhibition of NF-κB in syn-treated mice, whereas mice pretreated with syn, La, or inulin had attenuation of Cr-induced Smad 7 expression. There was a temporal Smad 7 and NF-κB intracellular accumulation post-Cr infection and post-tumor necrosis factor stimulation in CMT93 cells. These results, therefore, suggest that probiotic, La, prebiotic inulin, or synbiotic may promote host-protective immunity and attenuate Cr-induced intestinal inflammation through mechanisms affecting NF-κB and Smad 7 signaling.     

MyD88 signalling plays a critical role in host defence by controlling pathogen burden and promoting epithelial cell homeostasis during Citrobacter rodentium-induced colitis

Myeloid differentiation factor (MyD)88, an adaptor protein shared by the Toll-interleukin 1 receptor superfamily, plays a critical role in host defence during many systemic bacterial infections by inducing protective inflammatory responses that limit bacterial growth. However, the role of innate responses during gastrointestinal (GI) infections is less clear, in part because the GI tract is tolerant to commensal antigens. The current study investigated the role of MyD88 following infection by the murine bacterial pathogen, Citrobacter rodentium . MyD88-deficient mice suffered a lethal colitis coincident with colonic mucosal ulcerations and bleeding. Their susceptibility was associated with an overwhelming bacterial burden and selectively impaired immune responses in colonic tissues, which included delayed inflammatory cell recruitment, reduced iNOS and abrogated production of TNF-α and IL-6 from MyD88-deficient macrophages and colons cultured ex vivo . Immunostaining for Ki67 and BrDU revealed that MyD88 signalling mediated epithelial hyper-proliferation in response to C. rodentium infection. Thus, MyD88-deficient mice could not promote epithelial cell turnover and repair, leading to deep bacterial invasion of colonic crypts, intestinal barrier dysfunction and, ultimately, widespread mucosal ulcerations. In conclusion, MyD88 signalling within the GI tract plays a critical role in mediating host defence against an enteric bacterial pathogen, by controlling bacterial numbers and promoting intestinal epithelial homeostasis.     

Porphyromonas gingivalis Mediates Inflammasome Repression in Polymicrobial Cultures through a Novel Mechanism Involving Reduced Endocytosis

The interleukin (IL)-1β-processing inflammasome has recently been identified as a target for pathogenic evasion of the inflammatory response by a number of bacteria and viruses. We postulated that the periodontal pathogen, Porphyromonas gingivalis may suppress the inflammasome as a mechanism for its low immunogenicity and pathogenic synergy with other, more highly immunogenic periodontal bacteria. Our results show that P. gingivalis lacks signaling capability for the activation of the inflammasome in mouse macrophages. Furthermore, P. gingivalis can suppress inflammasome activation by another periodontal bacterium, Fusobacterium nucleatum. This repression affects IL-1β processing, as well as other inflammasome-mediated processes, including IL-18 processing and cell death, in both human and mouse macrophages. F. nucleatum activates IL-1β processing through the Nlrp3 inflammasome; however, P. gingivalis repression is not mediated through reduced levels of inflammasome components. P. gingivalis can repress Nlrp3 inflammasome activation by Escherichia coli, and by danger-associated molecular patterns and pattern-associated molecular patterns that mediate activation through endocytosis. However, P. gingivalis does not suppress Nlrp3 inflammasome activation by ATP or nigericin. This suggests that P. gingivalis may preferentially suppress endocytic pathways toward inflammasome activation. To directly test whether P. gingivalis infection affects endocytosis, we assessed the uptake of fluorescent particles in the presence or absence of P. gingivalis. Our results show that P. gingivalis limits both the number of cells taking up beads and the number of beads taken up for bead-positive cells. These results provide a novel mechanism of pathogen-mediated inflammasome inhibition through the suppression of endocytosis.     

Toll or Interleukin-1 Receptor (TIR) Domain-containing Adaptor Inducing Interferon-β (TRIF)-mediated Caspase-11 Protease Production Integrates Toll-like Receptor 4 (TLR4) Protein- and Nlrp3 Inflammasome-mediated Host Defense against Enteropathogens

Enteric pathogens represent a major cause of morbidity and mortality worldwide. Toll-like receptor (TLR) and inflammasome signaling are critical for host responses against these pathogens, but how these pathways are integrated remains unclear. Here, we show that TLR4 and the TLR adaptor TRIF are required for inflammasome activation in macrophages infected with the enteric pathogens Escherichia coli and Citrobacter rodentium. In contrast, TLR4 and TRIF were dispensable for Salmonella typhimurium-induced caspase-1 activation. TRIF regulated expression of caspase-11, a caspase-1-related protease that is critical for E. coli- and C. rodentium-induced inflammasome activation, but dispensable for inflammasome activation by S. typhimurium. Thus, TLR4- and TRIF-induced caspase-11 synthesis is critical for noncanonical Nlrp3 inflammasome activation in macrophages infected with enteric pathogens.     

A functional role for Nlrp6 in intestinal inflammation and tumorigenesis

The nucleotide-binding oligomerization domain-like receptor (NLR) family member, Nlrp6, has been implicated in inflammasome signaling to activate caspase-1, which is essential for the production of mature IL-1β and IL-18. However, a function for Nlrp6 in vivo has never been demonstrated. Due to the relative high expression of Nlrp6 in intestinal tissue, we hypothesized that Nlrp6 has a role in intestinal homeostasis. Indeed, Nlrp6-deficient mice are more susceptible to chemically induced colitis as well as colitis-induced tumorigenesis than wild-type (WT) mice. Nlrp6-deficient mice exhibited significantly more inflammation within the colon than WT mice after dextran sulfate sodium treatment. Their inability to resolve inflammation and repair damaged epithelium as efficiently as WT mice resulted in prolonged increases in epithelial proliferative activity that likely underlie the increased propensity for tumors in these mice during chronic inflammation. We further show that the activity of Nlrp6 in hematopoietic cells is critical for protection against inflammation-related colon tumorigenesis. This study highlights the importance of NLR function in maintaining intestinal homeostasis to prevent the development of aberrant inflammation and tumor development within the colon.     

From virus to inflammation: mechanisms of RIG-I-induced IL-1β production

Early detection of viruses by the innate immune system is critical for host defense. Antiviral immunity is initiated by germline encoded pattern recognition receptors (PRRs) that recognize viral pathogen-associated molecular patterns (PAMPs) such as nucleic acids. Intracellular PRRs then drive the production of interferons and cytokines to orchestrate immune responses. One key host factor that is critical for antiviral immunity and for systemic inflammatory reactions including fever is interleukin-1beta (IL-1β). Here we discuss current insights into the molecular mechanisms how the cytosolic RNA helicase RIG-I triggers NF-κB signaling and inflammasome activation specifically for RNA virus-induced IL-1β production.     

MicroRNA-30e regulates neuroinflammation in MPTP model of Parkinson’s disease by targeting Nlrp3

Accumulating evidences suggest that neuroinflammation is a pathological hallmark of Parkinson’s disease (PD), a neurodegenerative disorder characterized by loss of dopaminergic neurons in substantia nigra pars compacta (SNpc). MicroRNAs have been recently recognized as crucial regulators of inflammatory responses. Here, we found significant downregulation of microRNA-30e (miR-30e) in SNpc of MPTP-induced PD mice. Next, we employed miR-30e agomir to upregulate miR-30e expression in MPTP-treated mice. Our results showed that delivery of miR-30e agomir remarkably improved motor behavioral deficits and neuronal activity, and inhibited the loss of dopamine neurons. Moreover, the increased α-synuclein protein expression in SNpc of MPTP-PD mice was alleviated by the upregulation of miR-30e. Further, miR-30e agomir administration also attenuated the marked increase of inflammatory cytokines, such as TNF-α, COX-2, iNOS, and restored the decreased secretion of BDNF in SNpc. In addition, we demonstrated for the first time that miR-30e directly targeted to Nlrp3, thus suppressing Nlrp3 mRNA and protein expression. Finally, miR-30e upregulation significantly inhibited the activation of Nlrp3 inflammasome as evident from the decreased Nlrp3, Caspase-1 and ASC expressions and IL-18 and IL-1β secretions. Taken together, our study demonstrates that miR-30e ameliorates neuroinflammation in the MPTP model of PD by decreasing Nlrp3 inflammasome activity. These findings suggesting that miR30e may be a key inflammation-mediated molecule that could be a potential target for PD therapeutics.     

Nlrp3: An immune sensor of cellular stress and infection

Innate immune cells rely on pathogen recognition receptors such as the nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family to mount an appropriate immune response against microbial threats. The NLR protein Nlrp3 senses microbial ligands, endogenous danger signals and crystalline substances in the cytosol to trigger the assembly of a large caspase-1-activating protein complex termed the Nlrp3 inflammasome. Autoproteolytic maturation of caspase-1 zymogens in the Nlrp3 inflammasome leads to maturation and extracellular release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18. Gain-of-function mutations in the NOD domain of Nlrp3 are associated with auto-inflammatory disorders characterized by skin rashes and prolonged episodes of fever. In addition, decreased Nlrp3 expression was recently linked with susceptibility to Crohn's disease in humans. In this review, we discuss recent developments on the role of the Nlrp3 inflammasome in innate immunity, its activation mechanisms and the auto-inflammatory disorders associated with deregulation of Nlrp3 inflammasome activity.     

The NLRP3 inflammasome detects encephalomyocarditis virus and vesicular stomatitis virus infection

Inflammasomes are cytosolic protein complexes that regulate caspase-1 activation and the secretion of interleukin-1β (IL-1β) and IL-18. Several different inflammasome complexes have been identified, but the NLRP3 inflammasome is particularly notable because of its central role in diseases of inflammation. Recent work has demonstrated an essential role for the NLRP3 inflammasome in host defense against influenza virus. We show here that two other RNA viruses, encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV), activate the NLRP3 inflammasome in dendritic cells and macrophages through a mechanism requiring viral replication. Inflammasome activation in response to both viruses does not require MDA5 or RIG-I signaling. Despite the ability of the NLRP3 inflammasome to detect EMCV and VSV, wild-type and caspase-1-deficient mice were equally susceptible to infection with both viruses. These findings indicate that the NLRP3 inflammasome may be a common pathway for RNA virus detection, but its precise role in the host response may be variable.     

A Balanced IL-1β Activity Is Required for Host Response to Citrobacter rodentium Infection

Microbial sensing plays essential roles in the innate immune response to pathogens. In particular, NLRP3 forms a multiprotein inflammasome complex responsible for the maturation of interleukin (IL)-1β. Our aim was to delineate the role of the NLRP3 inflammasome in macrophages, and the contribution of IL-1β to the host defense against Citrobacter rodentium acute infection in mice. Nlrp3^−/− and background C57BL/6 (WT) mice were infected by orogastric gavage, received IL-1β (0.5 µg/mouse; ip) on 0, 2, and 4 days post-infection (DPI), and assessed on 6 and 10 DPI. Infected Nlrp3^−/− mice developed severe colitis; IL-1β treatments reduced colonization, abrogated dissemination of bacteria to mesenteric lymph nodes, and protected epithelial integrity of infected Nlrp3^−/− mice. In contrast, IL-1β treatments of WT mice had an opposite effect with increased penetration of bacteria and barrier disruption. Microscopy showed reduced damage in Nlrp3^−/− mice, and increased severity of disease in WT mice with IL-1β treatments, in particular on 10 DPI. Secretion of some pro-inflammatory plasma cytokines was dissipated in Nlrp3^−/− compared to WT mice. IL-1β treatments elevated macrophage infiltration into infected crypts in Nlrp3^−/− mice, suggesting that IL-1β may improve macrophage function, as exogenous administration of IL-1β increased phagocytosis of C. rodentium by peritoneal Nlrp3^−/− macrophages in vitro. As well, the exogenous administration of IL-1β to WT peritoneal macrophages damaged the epithelial barrier of C. rodentium-infected polarized CMT-93 cells. Treatment of Nlrp3^−/− mice with IL-1β seems to confer protection against C. rodentium infection by reducing colonization, protecting epithelial integrity, and improving macrophage activity, while extraneous IL-1β appeared to be detrimental to WT mice. Together, these findings highlight the importance of balanced cytokine responses as IL-1β improved bacterial clearance in Nlrp3^−/− mice but increased tissue damage when given to WT mice.