PKC/ROS-Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania Zinc-Metalloprotease during Infection

Parasites of the Leishmania genus infect and survive within macrophages by inhibiting several microbicidal molecules, such as nitric oxide and pro-inflammatory cytokines. In this context, various species of Leishmania have been reported to inhibit or reduce the production of IL-1β both in vitro and in vivo. However, the mechanism whereby Leishmania parasites are able to affect IL-1β production and secretion by macrophages is still not fully understood. Dependent on the stimulus at hand, the maturation of IL-1β is facilitated by different inflammasome complexes. The NLRP3 inflammasome has been shown to be of pivotal importance in the detection of danger molecules such as inorganic crystals like asbestos, silica and malarial hemozoin, (HZ) as well as infectious agents. In the present work, we investigated whether Leishmania parasites modulate NLRP3 inflammasome activation. Using PMA-differentiated THP-1 cells, we demonstrate that Leishmania infection effectively inhibits macrophage IL-1β production upon stimulation. In this context, the expression and activity of the metalloprotease GP63 - a critical virulence factor expressed by all infectious Leishmania species - is a prerequisite for a Leishmania-mediated reduction of IL-1β secretion. Accordingly, L. mexicana, purified GP63 and GP63-containing exosomes, caused the inhibition of macrophage IL-1β production. Leishmania-dependent suppression of IL-1β secretion is accompanied by an inhibition of reactive oxygen species (ROS) production that has previously been shown to be associated with NLRP3 inflammasome activation. The observed loss of ROS production was due to an impaired PKC-mediated protein phosphorylation. Furthermore, ROS-independent inflammasome activation was inhibited, possibly due to an observed GP63-dependent cleavage of inflammasome and inflammasome-related proteins. Collectively for the first time, we herein provide evidence that the protozoan parasite Leishmania, through its surface metalloprotease GP63, can significantly inhibit NLRP3 inflammasome function and IL-1β production.     

Mycoplasma hyorhinis Activates the NLRP3 Inflammasome and Promotes Migration and Invasion of Gastric Cancer Cells

Background

Mycoplasma hyorhinis (M.hyorhinis, M.hy) is associated with development of gastric and prostate cancers. The NLRP3 inflammasome, a protein complex controlling maturation of important pro-inflammatory cytokines interleukin (IL)-1β and IL-18, is also involved in tumorigenesis and metastasis of various cancers.

Methodology/Principal Findings

To clarify whether M.hy promoted tumor development via inflammasome activation, we analyzed monocytes for IL-1β and IL-18 production upon M.hy challenge. When exposed to M.hy, human monocytes exhibited rapid and robust IL-1β and IL-18 secretion. We further identified that lipid-associated membrane protein (LAMP) from M.hy was responsible for IL-1β induction. Applying competitive inhibitors, gene specific shRNA and gene targeted mice, we verified that M.hy induced IL-1β secretion was NLRP3-dependent in vitro and in vivo. Cathepsin B activity, K⁺ efflux, Ca²⁺ influx and ROS production were all required for the NLRP3 inflammasome activation by M.hy. Importantly, it is IL-1β but not IL-18 produced from macrophages challenged with M.hy promoted gastric cancer cell migration and invasion.

Conclusions

Our data suggest that activation of the NLRP3 inflammasome by M.hy may be associated with its promotion of gastric cancer metastasis, and anti-M.hy therapy or limiting NLRP3 signaling could be effective approach for control of gastric cancer progress.     

Caspase-11 Activation in Response to Bacterial Secretion Systems that Access the Host Cytosol

Inflammasome activation is important for antimicrobial defense because it induces cell death and regulates the secretion of IL-1 family cytokines, which play a critical role in inflammatory responses. The inflammasome activates caspase-1 to process and secrete IL-1β. However, the mechanisms governing IL-1α release are less clear. Recently, a non-canonical inflammasome was described that activates caspase-11 and mediates pyroptosis and release of IL-1α and IL-1β. Caspase-11 activation in response to Gram-negative bacteria requires Toll-like receptor 4 (TLR4) and TIR-domain-containing adaptor-inducing interferon-β (TRIF)-dependent interferon production. Whether additional bacterial signals trigger caspase-11 activation is unknown. Many bacterial pathogens use specialized secretion systems to translocate effector proteins into the cytosol of host cells. These secretion systems can also deliver flagellin into the cytosol, which triggers caspase-1 activation and pyroptosis. However, even in the absence of flagellin, these secretion systems induce inflammasome activation and the release of IL-1α and IL-1β, but the inflammasome pathways that mediate this response are unclear. We observe rapid IL-1α and IL-1β release and cell death in response to the type IV or type III secretion systems of Legionella pneumophila and Yersinia pseudotuberculosis. Unlike IL-1β, IL-1α secretion does not require caspase-1. Instead, caspase-11 activation is required for both IL-1α secretion and cell death in response to the activity of these secretion systems. Interestingly, whereas caspase-11 promotes IL-1β release in response to the type IV secretion system through the NLRP3/ASC inflammasome, caspase-11-dependent release of IL-1α is independent of both the NAIP5/NLRC4 and NLRP3/ASC inflammasomes as well as TRIF and type I interferon signaling. Furthermore, we find both overlapping and non-redundant roles for IL-1α and IL-1β in mediating neutrophil recruitment and bacterial clearance in response to pulmonary infection by L. pneumophila. Our findings demonstrate that virulent, but not avirulent, bacteria trigger a rapid caspase-11-dependent innate immune response important for host defense.     

Cholesterol Crystals Activate the NLRP3 Inflammasome in Human Macrophages: A Novel Link between Cholesterol Metabolism and Inflammation

Background

Chronic inflammation of the arterial wall is a key element in the pathogenesis of atherosclerosis, yet the factors that trigger and sustain the inflammation remain elusive. Inflammasomes are cytoplasmic caspase-1-activating protein complexes that promote maturation and secretion of the proinflammatory cytokines interleukin(IL)-1β and IL-18. The most intensively studied inflammasome, NLRP3 inflammasome, is activated by diverse substances, including crystalline and particulate materials. As cholesterol crystals are abundant in atherosclerotic lesions, and IL-1β has been linked to atherogenesis, we explored the possibility that cholesterol crystals promote inflammation by activating the inflammasome pathway.

Principal Findings

Here we show that human macrophages avidly phagocytose cholesterol crystals and store the ingested cholesterol as cholesteryl esters. Importantly, cholesterol crystals induced dose-dependent secretion of mature IL-1β from human monocytes and macrophages. The cholesterol crystal-induced secretion of IL-1β was caspase-1-dependent, suggesting the involvement of an inflammasome-mediated pathway. Silencing of the NLRP3 receptor, the crucial component in NLRP3 inflammasome, completely abolished crystal-induced IL-1β secretion, thus identifying NLRP3 inflammasome as the cholesterol crystal-responsive element in macrophages. The crystals were shown to induce leakage of the lysosomal protease cathepsin B into the cytoplasm and inhibition of this enzyme reduced cholesterol crystal-induced IL-1β secretion, suggesting that NLRP3 inflammasome activation occurred via lysosomal destabilization.

Conclusions

The cholesterol crystal-induced inflammasome activation in macrophages may represent an important link between cholesterol metabolism and inflammation in atherosclerotic lesions.     

Virulent Mycobacterium bovis Beijing Strain Activates the NLRP7 Inflammasome in THP-1 Macrophages

Mycobacterium bovis is the causative agent of tuberculosis in a wide range of mammals, including humans. Macrophages are the first line of host defense. They secrete proinflammatory cytokines, such as interleukin-1 beta (IL-1β), in response to mycobacterial infection, but the underlying mechanisms by which human macrophages are activated and release IL-1β following M. bovis infection are poorly understood. Here we show that the ‘nucleotide binding and oligomerization of domain-like receptor (NLR) family pyrin domain containing 7 protein’ (NLRP7) inflammasome is involved in IL-1β secretion and caspase-1 activation induced by M. bovis infection in THP-1 macrophages. NLRP7 inflammasome activation promotes the induction of pyroptosis as well as the expression of tumor necrosis factor alpha (TNF-α), Chemokine (C-C motif) ligand 3 (CCL3) and IL-1β mRNAs. Thus, the NLRP7 inflammasome contributes to IL-1β secretion and induction of pyroptosis in response to M. bovis infection in THP-1 macrophages.     

Zinc depletion regulates the processing and secretion of IL-1β

Sterile inflammation contributes to many common and serious human diseases. The pro-inflammatory cytokine interleukin-1β (IL-1β) drives sterile inflammatory responses and is thus a very attractive therapeutic target. Activation of IL-1β in sterile diseases commonly requires an intracellular multi-protein complex called the NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome. A number of disease-associated danger molecules are known to activate the NLRP3 inflammasome. We show here that depletion of zinc from macrophages, a paradigm for zinc deficiency, also activates the NLRP3 inflammasome and induces IL-1β secretion. Our data suggest that zinc depletion damages the integrity of lysosomes and that this event is important for NLRP3 activation. These data provide new mechanistic insight to how zinc deficiency contributes to inflammation and further unravel the mechanisms of NLRP3 inflammasome activation.     

Staphylococcus aureus α-Hemolysin Activates the NLRP3-Inflammasome in Human and Mouse Monocytic Cells

Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal α-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus α-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant α-hemolysin, we now demonstrate that α-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant α-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to α-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by α-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs) to induce secretion of pro-inflammatory cytokines IL-1β and IL-18. Additionally, α-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1). These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation.     

NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy in a Type 2 Diabetes Rat Model

Background

Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is associated with metabolic disorder and cell death, which are important triggers in diabetic cardiomyopathy (DCM). We aimed to explore whether NLRP3 inflammasome activation contributes to DCM and the mechanism involved.

Methods

Type 2 diabetic rat model was induced by high fat diet and low dose streptozotocin. The characteristics of type 2 DCM were evaluated by metabolic tests, echocardiography and histopathology. Gene silencing therapy was used to investigate the role of NLRP3 in the pathogenesis of DCM. High glucose treated H9c2 cardiomyocytes were used to determine the mechanism by which NLRP3 modulated the DCM. The cell death in vitro was detected by TUNEL and EthD-III staining. TXNIP-siRNA and pharmacological inhibitors of ROS and NF-kB were used to explore the mechanism of NLRP3 inflammasome activation.

Results

Diabetic rats showed severe metabolic disorder, cardiac inflammation, cell death, disorganized ultrastructure, fibrosis and excessive activation of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), pro-caspase-1, activated caspase-1 and mature interleukin-1β (IL-1β). Evidence for pyroptosis was found in vivo, and the caspase-1 dependent pyroptosis was found in vitro. Silencing of NLRP3 in vivo did not attenuate systemic metabolic disturbances. However, NLRP3 gene silencing therapy ameliorated cardiac inflammation, pyroptosis, fibrosis and cardiac function. Silencing of NLRP3 in H9c2 cardiomyocytes suppressed pyroptosis under high glucose. ROS inhibition markedly decreased nuclear factor-kB (NF-kB) phosphorylation, thioredoxin interacting/inhibiting protein (TXNIP), NLRP3 inflammasome, and mature IL-1β in high glucose treated H9c2 cells. Inhibition of NF-kB reduced the activation of NLRP3 inflammasome. TXNIP-siRNA decreased the activation of caspase-1 and IL-1β.

Conclusion

NLRP3 inflammasome contributed to the development of DCM. NF-κB and TXNIP mediated the ROS-induced caspase-1 and IL-1β activation, which are the effectors of NLRP3 inflammasome. NLRP3 gene silencing may exert a protective effect on DCM.     

CD36 promotes NLRP3 inflammasome activation via the mtROS pathway in renal tubular epithelial cells of diabetic kidneys

Tubulointerstitial inflammation plays a key role in the pathogenesis of diabetic nephropathy (DN). Interleukin-1β (IL-1β) is the key proinflammatory cytokine associated with tubulointerstitial inflammation. The NLRP3 inflammasome regulates IL-1β activation and secretion. Reactive oxygen species (ROS) represents the main mediator of NLRP3 inflammasome activation. We previously reported that CD36, a class B scavenger receptor, mediates ROS production in DN. Here, we determined whether CD36 is involved in NLRP3 inflammasome activation and explored the underlying mechanisms. We observed that high glucose induced-NLRP3 inflammasome activation mediate IL-1β secretion, caspase-1 activation, and apoptosis in HK-2 cells. In addition, the levels of CD36, NLRP3, and IL-1β expression (protein and mRNA) were all significantly increased under high glucose conditions. CD36 knockdown resulted in decreased NLRP3 activation and IL-1β secretion. CD36 knockdown or the addition of MitoTempo significantly inhibited ROS production in HK-2 cells. CD36 overexpression enhanced NLRP3 activation, which was reduced by MitoTempo. High glucose levels induced a change in the metabolism of HK-2 cells from fatty acid oxidation (FAO) to glycolysis, which promoted mitochondrial ROS (mtROS) production after 72 h. CD36 knockdown increased the level of AMP-activated protein kinase (AMPK) activity and mitochondrial FAO, which was accompanied by the inhibition of NLRP3 and IL-1β. The in vivo experimental results indicate that an inhibition of CD36 could protect diabetic db/db mice from tubulointerstitial inflammation and tubular epithelial cell apoptosis. CD36 mediates mtROS production and NLRP3 inflammasome activation in db/db mice. CD36 inhibition upregulated the level of FAO-related enzymes and AMPK activity in db/db mice. These results suggest that NLRP3 inflammasome activation is mediated by CD36 in renal tubular epithelial cells in DN, which suppresses mitochondrial FAO and stimulates mtROS production.Subject terms: Biochemistry, Cell biology     

Participation of c-FLIP in NLRP3 and AIM2 inflammasome activation

Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of caspase-8 and is required for macrophage survival. Recent studies have revealed a selective role of caspase-8 in noncanonical IL-1β production that is independent of caspase-1 or inflammasome. Here we demonstrated that c-FLIP_L is an unexpected contributor to canonical inflammasome activation for the generation of caspase-1 and active IL-1β. Hemizygotic deletion of c-FLIP impaired ATP- and monosodium uric acid (MSU)-induced IL-1β production in macrophages primed through Toll-like receptors (TLRs). Decreased IL-1β expression was attributed to a reduced activation of caspase-1 in c-FLIP hemizygotic cells. In contrast, the production of TNF-α was not affected by downregulation in c-FLIP. c-FLIP_L interacted with NLRP3 or procaspase-1. c-FLIP is required for the full NLRP3 inflammasome assembly and NLRP3 mitochondrial localization, and c-FLIP is associated with NLRP3 inflammasome. c-FLIP downregulation also reduced AIM2 inflammasome activation. In contrast, c-FLIP inhibited SMAC mimetic-, FasL-, or Dectin-1-induced IL-1β generation that is caspase-8-mediated. Our results demonstrate a prominent role of c-FLIP_L in the optimal activation of the NLRP3 and AIM2 inflammasomes, and suggest that c-FLIP could be a valid target for treatment of inflammatory diseases caused by over-activation of inflammasomes.     

The colonic pathogen Entamoeba histolytica activates caspase-4/1 that cleaves the pore-forming protein gasdermin D to regulate IL-1β secretion

A hallmark of Entamoeba histolytica (Eh) invasion in the gut is acute inflammation dominated by the secretion of pro-inflammatory cytokines TNF-α and IL-1β. This is initiated when Eh in contact with macrophages in the lamina propria activates caspase-1 by recruiting the NLRP3 inflammasome complex in a Gal-lectin and EhCP-A5-dependent manner resulting in the maturation and secretion of IL-1β and IL-18. Here, we interrogated the requirements and mechanisms for Eh-induced caspase-4/1 activation in the cleavage of gasdermin D (GSDMD) to regulate bioactive IL-1β release in the absence of cell death in human macrophages. Unlike caspase-1, caspase-4 activation occurred as early as 10 min that was dependent on Eh Gal-lectin and EhCP-A5 binding to macrophages. By utilizing CRISPR-Cas9 gene edited CASP4/1, NLRP3 KO and ASC-def cells, caspase-4 activation was found to be independent of the canonical NLRP3 inflammasomes. In CRISPR-Cas9 gene edited CASP1 macrophages, caspase-4 activation was significantly up regulated that enhanced the enzymatic cleavage of GSDMD at the same cleavage site as caspase-1 to induce GSDMD pore formation and sustained bioactive IL-1β secretion. Eh-induced IL-1β secretion was independent of pyroptosis as revealed by pharmacological blockade of GSDMD pore formation and in CRISPR-Cas9 gene edited GSDMD KO macrophages. This was in marked contrast to the potent positive control, lipopolysaccharide + Nigericin that induced high expression of predominantly caspase-1 that efficiently cleaved GSDMD with high IL-1β secretion/release associated with massive cell pyroptosis. These results reveal that Eh triggered “hyperactivated macrophages” allowed caspase-4 dependent cleavage of GSDMD and IL-1β secretion to occur in the absence of pyroptosis that may play an important role in disease pathogenesis.     

Malaria-Induced NLRP12/NLRP3-Dependent Caspase-1 Activation Mediates Inflammation and Hypersensitivity to Bacterial Superinfection

Cyclic paroxysm and high fever are hallmarks of malaria and are associated with high levels of pyrogenic cytokines, including IL-1β. In this report, we describe a signature for the expression of inflammasome-related genes and caspase-1 activation in malaria. Indeed, when we infected mice, Plasmodium infection was sufficient to promote MyD88-mediated caspase-1 activation, dependent on IFN-γ-priming and the expression of inflammasome components ASC, P2X7R, NLRP3 and/or NLRP12. Pro-IL-1β expression required a second stimulation with LPS and was also dependent on IFN-γ-priming and functional TNFR1. As a consequence of Plasmodium-induced caspase-1 activation, mice produced extremely high levels of IL-1β upon a second microbial stimulus, and became hypersensitive to septic shock. Therapeutic intervention with IL-1 receptor antagonist prevented bacterial-induced lethality in rodents. Similar to mice, we observed a significantly increased frequency of circulating CD14⁺CD16⁻Caspase-1⁺ and CD14^dimCD16⁺Caspase-1⁺ monocytes in peripheral blood mononuclear cells from febrile malaria patients. These cells readily produced large amounts of IL-1β after stimulation with LPS. Furthermore, we observed the presence of inflammasome complexes in monocytes from malaria patients containing either NLRP3 or NLRP12 pyroptosomes. We conclude that NLRP12/NLRP3-dependent activation of caspase-1 is likely to be a key event in mediating systemic production of IL-1β and hypersensitivity to secondary bacterial infection during malaria.     

Malarial Hemozoin Activates the NLRP3 Inflammasome through Lyn and Syk Kinases

The intraerythrocytic parasite Plasmodium—the causative agent of malaria—produces an inorganic crystal called hemozoin (Hz) during the heme detoxification process, which is released into the circulation during erythrocyte lysis. Hz is rapidly ingested by phagocytes and induces the production of several pro-inflammatory mediators such as interleukin-1β (IL-1β). However, the mechanism regulating Hz recognition and IL-1β maturation has not been identified. Here, we show that Hz induces IL-1β production. Using knockout mice, we showed that Hz-induced IL-1β and inflammation are dependent on NOD-like receptor containing pyrin domain 3 (NLRP3), ASC and caspase-1, but not NLRC4 (NLR containing CARD domain). Furthermore, the absence of NLRP3 or IL-1β augmented survival to malaria caused by P. chabaudi adami DS. Although much has been discovered regarding the NLRP3 inflammasome induction, the mechanism whereby this intracellular multimolecular complex is activated remains unclear. We further demonstrate, using pharmacological and genetic intervention, that the tyrosine kinases Syk and Lyn play a critical role in activation of this inflammasome. These findings not only identify one way by which the immune system is alerted to malarial infection but also are one of the first to suggest a role for tyrosine kinase signaling pathways in regulation of the NLRP3 inflammasome.     

Distinct Licensing of IL-18 and IL-1β Secretion in Response to NLRP3 Inflammasome Activation

Inflammasome activation permits processing of interleukins (IL)-1β and 18 and elicits cell death (pyroptosis). Whether these responses are independently licensed or are “hard-wired” consequences of caspase-1 (casp1) activity has not been clear. Here, we show that that each of these responses is independently regulated following activation of NLRP3 inflammasomes by a “non-canonical” stimulus, the secreted Listeria monocytogenes (Lm) p60 protein. Primed murine dendritic cells (DCs) responded to p60 stimulation with reactive oxygen species (ROS) production and secretion of IL-1β and IL-18 but not pyroptosis. Inhibitors of ROS production inhibited secretion of IL-1β, but did not impair IL-18 secretion. Furthermore, DCs from caspase-11 (casp11)-deficient 129S6 mice failed to secrete IL-1β in response to p60 but were fully responsive for IL-18 secretion. These findings reveal that there are distinct licensing requirements for processing of IL-18 versus IL-1β by NLRP3 inflammasomes.     

Enhanced activity of NLRP3 inflammasome in peripheral blood cells of patients with active rheumatoid arthritis

IntroductionInterleukin-1β (IL-1β) is a major inflammatory cytokine, produced predominantly by innate immune cells through NLRP3-inflammasome activation. Both intrinsic and extrinsic danger signals may activate NLRP3. Genetic variations in NLRP3-inflammasome components have been reported to influence rheumatoid arthritis (RA) susceptibility and severity. We sought to assess the activity of NLRP3-inflammasome in patients with active RA compared to healthy individuals.MethodIntracellular protein expression of NLRP3, ASC, pro- and active caspase-1, pro- and active IL-1β was assessed by immunoblotting both at baseline and upon inflammasome activation. NLRP3 function (IL-1β secretion) was assessed upon priming of TLR2 (Pam(3)CysSK(4), TLR3 (poly(I:C)) or TLR4 (LPS) and ATP sequential treatment. We used caspase inhibitors (casp-1, 3/7 and 8) to assess their contribution to IL-1β maturation. All experiments were performed in whole blood cells.ResultsActive RA patients (n = 11) expressed higher basal intracellular levels of NLRP3 (p < 0.008), ASC (p < 0.003), active caspase-1 (p < 0.02) and pro-IL-1β (p < 0.001). Upon priming with TLR4 (LPS) and ATP, RA-derived cell extracts (n = 7) displayed increased expression of NLRP3 (p < 0.01) and active caspase-1 (p < 0.001). Secreted IL-1β in culture supernatants from whole blood cells activated with TLR4 (LPS) or TLR3 agonist (poly(I:C)) plus ATP was higher in RA patients (n = 20) versus controls (n = 18) (p < 0.02 for both). Caspase-1 inhibition significantly reduced IL-1β secretion induced by all stimuli, whereas caspase-8 inhibition affected only TLR4 and TLR3 cell priming.ConclusionPatients with active RA have increased expression of NLRP3 and NLRP3-mediated IL-1β secretion in whole blood cells upon stimulation via TLR3 and TLR4 but not TLR2. In these patients, IL-1β secretion seems to be predominately driven by caspase-1 and caspase-8. Targeting NLRP3 or downstream caspases may be of benefit in suppressing IL-1β production in RA.     

NLRP3 Controls Trypanosoma cruzi Infection through a Caspase-1-Dependent IL-1R-Independent NO Production

Trypanosoma cruzi (T. cruzi) is an intracellular protozoan parasite and the etiological agent of Chagas disease, a chronic infectious illness that affects millions of people worldwide. Although the role of TLR and Nod1 in the control of T. cruzi infection is well-established, the involvement of inflammasomes remains to be elucidated. Herein, we demonstrate for the first time that T. cruzi infection induces IL-1β production in an NLRP3- and caspase-1-dependent manner. Cathepsin B appears to be required for NLRP3 activation in response to infection with T. cruzi, as pharmacological inhibition of cathepsin B abrogates IL-1β secretion. NLRP3^−/− and caspase1^−/− mice exhibited high numbers of T. cruzi parasites, with a magnitude of peak parasitemia comparable to MyD88^−/− and iNOS^−/− mice (which are susceptible models for T. cruzi infection), indicating the involvement of NLRP3 inflammasome in the control of the acute phase of T. cruzi infection. Although the inflammatory cytokines IL-6 and IFN-γ were found in spleen cells from NLRP3^−/− and caspase1^−/− mice infected with T. cruzi, these mice exhibited severe defects in nitric oxide (NO) production and an impairment in macrophage-mediated parasite killing. Interestingly, neutralization of IL-1β and IL-18, and IL-1R genetic deficiency demonstrate that these cytokines have a minor effect on NO secretion and the capacity of macrophages to control T. cruzi infection. In contrast, inhibition of caspase-1 with z-YVAD-fmk abrogated NO production by WT and MyD88^−/− macrophages and rendered them as susceptible to T. cruzi infection as NLRP3^−/− and caspase-1^−/− macrophages. Taken together, our results demonstrate a role for the NLRP3 inflammasome in the control of T. cruzi infection and identify NLRP3-mediated, caspase-1-dependent and IL-1R-independent NO production as a novel effector mechanism for these innate receptors.