LRRFIP2 negatively regulates NLRP3 inflammasome activation in macrophages by promoting Flightless-I-mediated caspase-1 inhibition.
ABSTRACT: The NLRP3 inflammasome is the most characterized inflammasome activated by cellular infection or stress, which is responsible for the maturation of proinflammatory cytokines IL-1? and IL-18. The precise molecular mechanism for negative regulation of NLRP3 inflammasome activation needs to be further defined. Here we identify leucine-rich repeat Fli-I-interacting protein 2 (LRRFIP2) as an NLRP3-associated protein and an inhibitor for NLRP3 inflammasome activation. LRRFIP2 binds to NLRP3 via its N terminus upon NLRP3 inflammasome activation, and also interacts with Flightless-I, a pseudosubstrate of caspase-1, via its Coil motif. Knockdown of Flightless-I significantly promotes NLRP3 inflammasome activation. LRRFIP2 enhances the interaction between Flightless-I and caspase-1, facilitating the inhibitory effect of Flightless-I on caspase-1 activation. Furthermore, silencing of Flightless-I abrogates the inhibitory effect of LRRFIP2 on NLRP3 inflammasome. These data demonstrate that LRRFIP2 inhibits NLRP3 inflammasome activation by recruiting the caspase-1 inhibitor Flightless-I, thus outlining a new mechanism for negative regulation of NLRP3 inflammasome.
Project description:NLRP3 and ASC are able to form a large multimeric complex called inflammasome in response to a number danger signals. The NLRP3 inflammasome is required for the activation of caspase-1 and subsequent maturation of pro-IL-1? into active IL-1?. Although the mechanisms regulating the formation and activity of NLRP3 inflammasome are yet not fully elucidated, data suggest that the assembly of NLRP3 inflammasome requires microtubules to induce the proximity of ASC and NLRP3. In this study we show that microfilaments (F-actin) inhibit NLRP3 inflammasome activity and interact with NLRP3 and ASC. We demonstrate that the inhibition depends on the actin polymerization state but not on the active polymerization process. In ATP- or nigericin-activated macrophages, our data further indicate that Flightless-I (FliI) and leucine-rich repeat FliI-interaction protein 2 (LRRFIP2) are required for the co-localization of NLRP3, ASC and F-actin. We also established that the ability of Ca(2+) to accentuate the activity of NLRP3 inflammasome is abrogated in FliI- and LRRFIP2-knockdown macrophages, suggesting that Ca(2+) signaling requires the presence of FliI and LRRFIP2. Accordingly, we observed that Ca(2+)/FliI-dependent severing of F-actin suppresses F-actin/FliI/LRRFIP2-dependent NLRP3 inflammasome inhibition leading to increase IL-1? production. Altogether, our results unveil a new function of F-actin in the regulation of NLRP3 inflammasome activity strengthening the importance of cytoskeleton in the regulation of inflammation.
Project description:B cell adaptor for phosphoinositide 3-kinase (PI3K) (BCAP) is a signaling adaptor that activates the PI3K pathway downstream of B cell receptor signaling in B cells and Toll-like receptor (TLR) signaling in macrophages. BCAP binds to the regulatory p85 subunit of class I PI3K and is a large, multidomain protein. We used proteomic analysis to identify other BCAP-interacting proteins in macrophages and found that BCAP specifically associated with the caspase-1 pseudosubstrate inhibitor Flightless-1 and its binding partner leucine-rich repeat flightless-interacting protein 2. Because these proteins inhibit the NLRP3 inflammasome, we investigated the role of BCAP in inflammasome function. Independent of its effects on TLR priming, BCAP inhibited NLRP3- and NLRC4-induced caspase-1 activation, cell death, and IL-1? release from macrophages. Accordingly, caspase-1-dependent clearance of a Yersinia pseudotuberculosis mutant was enhanced in BCAP-deficient mice. Mechanistically, BCAP delayed the recruitment and activation of pro-caspase-1 within the NLRP3/ASC preinflammasome through its association with Flightless-1. Thus, BCAP is a multifunctional signaling adaptor that inhibits key pathogen-sensing pathways in macrophages.
Project description:Shikonin is a highly lipophilic naphtoquinone found in the roots of Lithospermum erythrorhizon used for its pleiotropic effects in traditional Chinese medicine. Based on its reported antipyretic and anti-inflammatory properties, we investigated whether shikonin suppresses the activation of NLRP3 inflammasome. Inflammasomes are cytosolic protein complexes that serve as scaffolds for recruitment and activation of caspase-1, which, in turn, results in cleavage and secretion of proinflammatory cytokines IL-1? and IL-18. NLRP3 inflammasome activation involves two steps: priming, i.e. the activation of NF-?B pathway, and inflammasome assembly. While shikonin has previously been reported to suppress the priming step, we demonstrated that shikonin also inhibits the second step of inflammasome activation induced by soluble and particulate NLRP3 instigators in primed immortalized murine bone marrow-derived macrophages. Shikonin decreased NLRP3 inflammasome activation in response to nigericin more potently than acetylshikonin. Our results showed that shikonin also inhibits AIM2 inflammasome activation by double stranded DNA. Shikonin inhibited ASC speck formation and caspase-1 activation in murine macrophages and suppressed the activity of isolated caspase-1, demonstrating that it directly targets caspase-1. Complexing shikonin with ?-lactoglobulin reduced its toxicity while preserving the inhibitory effect on NLRP3 inflammasome activation, suggesting that shikonin with improved bioavailability might be interesting for therapeutic applications in inflammasome-mediated conditions.
Project description:Caspase-1 and caspase-11 are proinflammatory caspases that regulate cytokine production and leukocyte migration during pathogen infection. In an attempt to identify new intracellular regulators of caspase-11, we found that Flightless-I, a member of the gelsolin superfamily of actin-remodeling proteins, interacts and regulates both caspase-11 and caspase-1. Flightless-I targets caspase-11 to the Triton X-100-insoluble cytoskeleton fraction and the cell leading edge. In addition, Flightless-I inhibits caspase-1 activation and caspase-1-mediated interleukine-1beta (IL-1beta) maturation. The physiological relevance of these findings is supported by the opposing effects of Flightless-I overexpression and knockdown on caspase-1 activity and IL-1beta maturation. Our results suggest that Flightless-I may be a bona fide caspase-1 inhibitor that acts through a mechanism similar to that of cytokine response modifier A, a potent caspase-1 inhibitor from the cowpox virus. Our study provides a new mechanism controlling the localization and activation of proinflammatory caspases.
Project description:The NOD-leucine rich repeat and pyrin containing protein 3 (NLRP3) inflammasome is a multi-protein complex, aimed at producing IL-1β in response to danger signals which must be tightly regulated. Here we investigated the importance of the stress sensor, Heat Shock Protein 70 (HSP70) on NLRP3 inflammasome activation. HSP70 deficiency leads to the worsening of NLRP3-dependent peritonitis in mice. HSP70 deficiency also enhances caspase-1 activation and IL-1β production in murine Bone Marrow-Derived Macrophages (BMDMs) under NLRP3 activator treatment in vitro. This observation is associated with an increased number and size of Apoptosis associated Speck-like protein containing a CARD domain (ASC)/NLRP3 specks. Conversely, the overexpression of HSP70 in BMDMs decreases caspase-1 activation and IL-1β production under NLRP3 activator treatment. HSP70 interacts with NLRP3 and this interaction is lost upon NLRP3 inflammasome activation. Heat shock inhibits NLRP3 inflammasome activation in vitro and inhibits peritonitis in mice. Therefore this study provides evidence on the inhibitory role of HSP70 on NLRP3 inflammasome and open the possibility of treating inflammatory diseases via HSP70 induction and/or by hyperthermia.
Project description:Non-alcoholic steatohepatitis (NASH), a type of non-alcoholic fatty liver disease, is characterized as steatosis and inflammation in the liver. NLRP3 inflammasome activation is associated with NASH pathology. We hypothesized that suppressing the NLRP3 inflammasome could be effective in preventing NASH. We searched substances that could inhibit the activation of the NLRP3 inflammasome and identified sweroside as an NLRP3 inhibitor. We investigated whether sweroside can be applied to prevent the pathological symptoms associated with NASH in a methionine-choline-deficient (MCD) diet-induced NASH mouse model. The activation of the NLRP3 inflammasome was determined by detecting the production of caspase-1 and IL-1? from pro-caspase-1 and pro-IL-1? in primary mouse macrophages and mouse liver. In a NASH model, mice were fed an MCD diet for two weeks with daily intraperitoneal injections of sweroside. Sweroside effectively inhibited NLRP3 inflammasome activation in primary macrophages as shown by a decrease in IL-1? and caspase-1 production. In a MCD diet-induced NASH mouse model, intraperitoneal injection of sweroside significantly reduced serum aspartate transaminase and alanine transaminase levels, hepatic immune cell infiltration, hepatic triglyceride accumulation, and liver fibrosis. The improvement of NASH symptoms by sweroside was accompanied with its inhibitory effects on the hepatic NLRP3 inflammasome as hepatic IL-1? and caspase-1 were decreased. Furthermore, sweroside blocked de novo synthesis of mitochondrial DNA in the liver, contributing to suppression of the NLRP3 inflammasome. These results suggest that targeting the NLRP3 inflammasome with sweroside could be beneficially employed to improve NASH symptoms.
Project description: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-1? 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-1?. Hemizygotic deletion of c-FLIP impaired ATP- and monosodium uric acid (MSU)-induced IL-1? production in macrophages primed through Toll-like receptors (TLRs). Decreased IL-1? expression was attributed to a reduced activation of caspase-1 in c-FLIP hemizygotic cells. In contrast, the production of TNF-? 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-1? 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.
Project description:The Wnt signaling pathway acts ubiquitously in metazoans to control various aspects of embryonic development. Wnt ligands bind their receptors Frizzled and low-density lipoprotein receptor-related protein 5/6 and function through Disheveled (Dvl), Axin, adenomatous polyposis coli, glycogen synthase kinase 3beta, and casein kinase (CK) 1 to stabilize beta-catenin and induce lymphocyte enhancer-binding factor (LEF)/T cell factor (TCF)-dependent transcriptional activities. To identify previously unrecognized Wnt signaling modulators, a genome-wide functional screen was performed using large-scale arrayed cDNA collections. From this screen, both known components and previously uncharacterized regulators of this pathway were identified, including beta-catenin, Dvl1, Dvl3, Fbxw-1, Cul1, CK1epsilon, CK1delta, and gamma-catenin. In particular, a previously unrecognized activator, LRRFIP2 (leucine-rich repeat in Flightless interaction protein 2), was found that interacts with Dvl to increase the cellular levels of beta-catenin and activate beta-catenin/LEF/TCF-dependent transcriptional activity. The function of LRRFIP2 is blocked when a dominant negative Dvl (Xdd1) is coexpressed. Expression of LRRFIP2 in Xenopus embryos induced double axis formation and Wnt target gene expression; a dominant negative form of LRRFIP2 suppresses ectopic Wnt signaling in Xenopus embryos and partially inhibits endogenous dorsal axis formation. These data suggest that LRRFIP2 plays an important role in transducing Wnt signals.
Project description:ATP-dependent chromatin remodeling complexes, such as SWI/SNF, are required for transcriptional activation of specific genes and are believed to be recruited to gene promoters by direct interaction with DNA binding transcription factors. However, we report here that recruitment of SWI/SNF to target genes of estrogen receptor alpha (ERalpha) requires the previously described nuclear receptor coactivator protein Flightless-I (Fli-I). Fli-I can bind directly to both ER and BAF53, an actin-related component of the SWI/SNF complex, suggesting that Fli-I may recruit SWI/SNF to ER target genes via interaction with BAF53. Point mutations in Fli-I that disrupt binding to ER or BAF53 compromised the ability of Fli-I to enhance ER-mediated activation of a transiently transfected reporter gene. Depletion of endogenous Fli-I or BAF53 inhibited estrogen-responsive expression of endogenous target genes of ER, indicating a critical role for Fli-I and BAF53. Moreover, depletion of endogenous Fli-I or BAF53 specifically eliminated part of the complex cyclical pattern of recruitment of SWI/SNF to estrogen-responsive promoters in a way that indicates multiple roles and multiple mechanisms of recruitment for SWI/SNF in estrogen-dependent target gene expression. These results begin to establish the functional relationships and interdependencies that coordinate the actions of the many coactivators participating in the transcriptional activation process.
Project description:Astrocytes are involved in the neuroinflammation of neurodegenerative diseases, such as Parkinson's disease (PD). Among the numerous inflammatory cytokines, interleukin-1β (IL-1β) produced by astrocytic Nod-like receptor protein (NLRP) inflammasome is crucial in the pathogenesis of PD. β-arrestin2-mediated dopamine D2 receptor (Drd2) signal transduction has been regarded as a potential anti-inflammatory target. Our previous study revealed that astrocytic Drd2 suppresses neuroinflammation in the central nervous system. However, the role of Drd2 in astrocytic NLRP3 inflammasome activation and subsequent IL-1β production remains unclear. In the present study, we used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mouse model to investigate whether Drd2 could suppress astrocytic NLRP3 inflammasome activation. We showed that Drd2 agonist inhibited NLRP3 inflammasome activation, evidenced by decreased caspase-1 expression and reduced IL-1β release in the midbrain of wild type mice. The anti-inflammasome effect of Drd2 was abolished in β-arrestin2 knockout and β-arrestin2 small interfering RNA-injected mice, suggesting a critical role of β-arrestin2 in Drd2-regulated NLRP3 inflammasome activation. We also found that Drd2 agonists suppressed the upregulation of caspase-1 and IL-1β expression in primary cultured mouse astrocytes in response to the activation of NLRP3 inflammasome induced by lipopolysaccharide plus adenosine triphosphate. Furthermore, we demonstrated that β-arrestin2 mediated the inhibitory effect of Drd2 on NLRP3 inflammasome activation via interacting with NLRP3 and interfering the inflammasome assembly. Collectively, our study illustrates that astrocytic Drd2 inhibits NLRP3 inflammasome activation through a β-arrestin2-dependent mechanism, and provides a new strategy for treatment of PD.