Canonical Inflammasome Research Articles

Functional interplay between non-canonical inflammasomes and autophagy in inflammatory responses and diseases.

The inflammasome is a cytosolic multiprotein platform that plays a key role in the inflammatory response, an essential innate immune response that protects the body from pathogens and cellular danger signals. Autophagy is a fundamental cellular mechanism that maintains homeostasis through the elimination and recycling of dysfunctional molecules and subcellular elements. Many previous studies have demonstrated a functional interplay between canonical inflammasomes that were earlier discovered and autophagy in inflammatory responses and diseases. Given the increasing evidence that non-canonical inflammasomes are unique and key factors in inflammatory responses, the functional interplay between non-canonical inflammasomes and autophagy is noteworthy. Recent studies have demonstrated that non-canonical inflammasomes and autophagy are functionally correlated with inflammatory responses and diseases. This review comprehensively discusses recent studies that have investigated the functional interplay of non-canonical inflammasomes, such as mouse caspase-11 and human caspase-4, with autophagy and autophagy-related proteins in inflammatory responses and diseases and provides insight into the development of novel anti-inflammatory therapeutics by modulating the functional interplay between non-canonical inflammasomes and autophagy.

Antagonistic nanobodies implicate mechanism of GSDMD pore formation and potential therapeutic application

Inflammasome activation results in the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases. The N-terminal domains (GSDMDNT) oligomerize and assemble pores penetrating the target membrane. As methods to study pore formation in living cells are insufficient, the order of conformational changes, oligomerization, and membrane insertion remained unclear. We have raised nanobodies (VHHs) against human GSDMD and find that cytosolic expression of VHHGSDMD-1 and VHHGSDMD-2 prevents oligomerization of GSDMDNT and pyroptosis. The nanobody-stabilized GSDMDNT monomers partition into the plasma membrane, suggesting that membrane insertion precedes oligomerization. Inhibition of GSDMD pore formation switches cell death from pyroptosis to apoptosis, likely driven by the enhanced caspase-1 activity required to activate caspase-3. Recombinant antagonistic nanobodies added to the extracellular space prevent pyroptosis and exhibit unexpected therapeutic potential. They may thus be suitable to treat the ever-growing list of diseases caused by activation of (non-) canonical inflammasomes.

Neisseria meningitidis activates pyroptotic pathways in a mouse model of meningitis: role of a two-partner secretion system.

There is evidence that in infected cells in vitro the meningococcal HrpA/HrpB two-partner secretion system (TPS) mediates the exit of bacteria from the internalization vacuole and the docking of bacteria to the dynein motor resulting in the induction of pyroptosis. In this study we set out to study the role of the HrpA/HrpB TPS in establishing meningitis and activating pyroptotic pathways in an animal model of meningitis using a reference serogroup C meningococcal strain, 93/4286, and an isogenic hrpB knockout mutant, 93/4286ΩhrpB. Survival experiments confirmed the role of HrpA/HrpB TPS in the invasive meningococcal disease. In fact, the ability of the hrpB mutant to replicate in brain and spread systemically was impaired in mice infected with hrpB mutant. Furthermore, western blot analysis of brain samples during the infection demonstrated that: i. N. meningitidis activated canonical and non-canonical inflammasome pyroptosis pathways in the mouse brain; ii. the activation of caspase-11, caspase-1, and gasdermin-D was markedly reduced in the hrpB mutant; iii. the increase in the amount of IL-1β and IL-18, which are an important end point of pyroptosis, occurs in the brains of mice infected with the wild-type strain 93/4286 and is strongly reduced in those infected with 93/4286ΩhrpB. In particular, the activation of caspase 11, which is triggered by cytosolic lipopolysaccharide, indicates that during meningococcal infection pyroptosis is induced by intracellular infection after the exit of the bacteria from the internalizing vacuole, a process that is hindered in the hrpB mutant. Overall, these results confirm, in an animal model, that the HrpA/HrpB TPS plays a role in the induction of pyroptosis and suggest a pivotal involvement of pyroptosis in invasive meningococcal disease, paving the way for the use of pyroptosis inhibitors in the adjuvant therapy of the disease.

Immunomodulatory Effects and Regulatory Mechanisms of (R)-6-HITC, an Isothiocyanate from Wasabi (Eutrema japonicum), in an Ex Vivo Mouse Model of LPS-Induced Inflammation.

The present study aimed to investigate the effects of (R)-(-)-1-isothiocyanato-6-(methylsulfinyl)-hexane [(R)-6-HITC], the major isothiocyanate present in wasabi, in an ex vivo model of inflammation using lipopolysaccharide-stimulated murine peritoneal macrophages. (R)-6-HITC improved the immune response and mitigated oxidative stress, which involved suppression of reactive oxygen species, nitric oxide, and pro-inflammatory cytokines (IL-1β, IL-6, IL-17, IL-18, and TNF-α) production and downregulation of pro-inflammatory enzymes such as inducible nitric oxide synthase, COX-2, and mPGES-1. In addition, (R)-6-HITC was able to activate the Nrf2/HO-1 axis while simultaneously inhibiting key signaling pathways, including JAK2/STAT3, mitogen-activated protein kinases, and canonical and noncanonical inflammasome pathways, orchestrating its potent immunomodulatory effects. Collectively, these findings demonstrate the potential of (R)-6-HITC as a promising nutraceutical for the management of immuno-inflammatory diseases and justify the need for further in vivo validation studies.

Comparative Analysis of Canonical Inflammasome Activation by Flow Cytometry, Imaging Flow Cytometry and High-Content Imaging.

Inflammasome activation occurs in various diseases, including rare diseases that require multicenter studies for investigation. Flow cytometric analysis of ASC speck+ cells in patient samples can be used to detect cell type-specific inflammasome activation. However, this requires standardized sample processing and the ability to compare data from different flow cytometers. To address this issue, we analyzed stimulated and unstimulated PBMCs from healthy donors using seven different flow cytometers. Additionally, human PBMCs were analyzed by fluorescence microscopy, imaging flow cytometry and high-content imaging (HCI). Flow cytometers differed significantly in their ability to detect ASC speck+ cells. AriaIII, Astrios EQ, and Canto II performed best in separating ASC speck+ from diffuse ASC cells. Imaging flow cytometry and HCI provided additional insight into ASC speck formation based on image-based parameters. For optimal results, the ability to separate cells with diffuse ASC from ASC speck+ cells is decisive. Image-based parameters can also differentiate cells with diffuse ASC from ASC speck+ cells. For the first time, we analyzed ASC speck detection by HCI in PBMCs and demonstrated advantages of this technique, such as high-throughput, algorithm-driven image quantification and 3D-rendering. Thus, inflammasome activation by ASC speck formation can be detected by various technical methods. However, the results may vary depending on the device used.

Genetic deletion of the apoptosis associated speck like protein containing a card in LysM+ macrophages attenuates spinal cord injury by regulating M1/M2 polarization through ASC-dependent inflammasome signaling axis

Apoptosis associated speck like protein containing a card (ASC), the key adaptor protein of the assembly and activation of canonical inflammasomes, has been found to play a significant role in neuroinflammation after spinal cord injury (SCI). The previous studies indicated that widely block or knockout ASC can ameliorate SCI. However, ASC is ubiquitously expressed in infiltrated macrophages and local microglia, so further exploration is needed on which type of cell playing the key role. In this study, using the LysMcre;Ascflox/flox mice with macrophage-specifc ASC conditional knockout (CKO) and contusive SCI model, we focus on evaluating the specific role of ASC in lysozyme 2 (LysM)+ myeloid cells (mainly infiltrated macrophages) in this pathology. The results revealed that macrophage-specifc Asc CKO exhibited the follow effects: (1) A significant reduction in the numbers of infiltrated macrophages in the all phases of SCI, and activated microglia in the acute and subacute phases. (2) A significant reduction in ASC, caspase-1, interleukin (IL)-1β, and IL-18 compared to control mice. (3) In the acute and subacute phases of SCI, M1 subset differentiation was inhibited, and M2 differentiation was increased. (4) Histology and hindlimb motor recoveries were improved. In conclusion, this study elucidates that macrophage-specific ASC CKO can improve nerve function recovery after SCI by regulating M1/M2 polarization through inhibiting ASC-dependent inflammasome signaling axis. This indicates that ASC in peripheral infiltrated macrophages may play an important role in SCI pathology, at least in mice, could be a potential target for treatment.

Can We Exploit Inflammasomes for Host-Directed Therapy in the Fight against Mycobacterium tuberculosis Infection?

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tb), is a major global health issue, with around 10 million new cases annually. Advances in TB immunology have improved our understanding of host signaling pathways, leading to innovative therapeutic strategies. Inflammasomes, protein complexes organized by cytosolic pattern recognition receptors (PRRs), play a crucial role in the immune response to M. tb by activating caspase 1, which matures proinflammatory cytokines IL1β and IL18. While inflammation is necessary to fight infection, excessive or dysregulated inflammation can cause tissue damage, highlighting the need for precise inflammasome regulation. Drug-resistant TB strains have spurred research into adjunctive host-directed therapies (HDTs) that target inflammasome pathways to control inflammation. Canonical and non-canonical inflammasome pathways can trigger excessive inflammation, leading to immune system exhaustion and M. tb spread. Novel HDT interventions can leverage precision medicine by tailoring treatments to individual inflammasome responses. Studies show that medicinal plant derivatives like silybin, andrographolide, and micheliolide and small molecules such as OLT1177, INF39, CY-09, JJ002, Ac-YVAD-cmk, TAK-242, and MCC950 can modulate inflammasome activation. Molecular tools like gene silencing and knockouts may also be used for severe TB cases. This review explores these strategies as potential adjunctive HDTs in fighting TB.

Distinct sequential death complexes regulate pyroptosis and IL-1β release in response to Yersinia blockade of immune signaling.

Pathogen infection of host cells triggers an inflammatory cell death termed pyroptosis via activation of inflammatory caspases. However, blockade of immune signaling kinases by the Yersinia virulence factor YopJ triggers cell death involving both apoptotic caspase-8 and pyroptotic caspase-1. While caspase-1 is normally activated within inflammasomes, Yersinia-induced caspase-1 activation is independent of known inflammasome components. We report that caspase-8 is an essential initiator, while caspase-1 is an essential amplifier of its own activation through two feed-forward loops involving caspase-1 auto-processing and caspase-1-dependent activation of gasdermin D and NLPR3. Notably, while Yersinia-induced caspase-1 activation and cell death are inflammasome-independent, IL-1β release requires NLPR3 inflammasome activation. Mechanistically, caspase-8 is rapidly activated within multiple foci throughout the cell, followed by assembly of a canonical inflammasome speck, indicating that caspase-8 and canonical inflammasome complex assemblies are kinetically and spatially distinct. Our findings reveal that functionally interconnected but distinct death complexes mediate pyroptosis and IL-1β release in response to pathogen blockade of immune signaling.

A Synthetic Small Molecule, LGM2605: A Promising Modulator of Increased Pro-Inflammatory Cytokine and Osteoclast Differentiation by Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin.

Our research explores the interplay between Aggregatibacter actinomycetemcomitans (Aa) cytolethal distending toxin (Cdt) and the host's inflammatory response in molar/incisor pattern periodontitis (MIPP). Cdt disrupts phosphatidylinositol-3,4,5-triphosphate (PIP3) signaling, influencing cytokine expression through canonical and non-canonical inflammasome activation as well as nuclear factor-κB (NF-κB) activation, leading to inflammation in MIPP. THP-1 differentiated macrophages (TDMs) exposed to Cdt exhibited an upregulation of pro-inflammatory genes and subsequent cytokine release. We analyzed the ability of a small molecule therapeutic, LGM2605, known for its anti-inflammatory properties, to reduce pro-inflammatory gene expression and cytokine release in Cdt-exposed and Aa-inoculated TDMs. LGM2605's mechanism of action involves inhibiting NF-κB while activating the Nrf2-transcription factor and antioxidants. Herein, we show that this small molecule therapeutic mitigates Cdt-induced pro-inflammatory cytokine expression and secretion. Our study also further defines Cdt's impact on osteoclast differentiation and maturation in MIPP. Cdt promotes increased TRAP+ cells, indicating heightened osteoclast differentiation, specific to Cdt's phosphatase activity. Cathepsin K levels rise during this process, reflecting changes in TRAP distribution between control and Cdt-treated cells. Exploring LGM2605's effect on Cdt-induced osteoclast differentiation and maturation, we found TRAP+ cells significantly reduced with LGM2605 treatment compared to Cdt alone. Upon LGM2605 treatment, immunocytochemistry revealed a decreased TRAP intensity and number of multinucleated cells. Moreover, immunoblotting showed reduced TRAP and cathepsin K levels, suggesting LGM2605's potential to curb osteoclast differentiation and maturation by modulating inflammatory cytokines, possibly involving Nrf2 activation. In summary, our research reveals the intricate connections between Cdt, pro-inflammatory cytokines, and osteoclast differentiation, offering novel therapeutic possibilities for managing these conditions.

2, 6-dimethoxy-1, 4-benzoquinone alleviates septic shock in mice by inhibiting NLRP3 inflammasome activation

To investigate the mechanism of 2, 6-dimethoxy-1, 4-benzoquinone (DMQ), an active ingredients in fermented wheat germ extract, for inhibiting NLRP3 inflammasome activation and alleviating septic shock in mice. Cultured murine bone marrow-derived macrophages (BMDM) stimulated with lipopolysaccharide (LPS) were treated with DMQ, followed by treatment with Nigericin, ATP, and MSU for activating the canonical NLRP3 inflammasome; the noncanonical NLRP3 inflammasome was activated by intracellular transfection of LPS, and AIM2 inflammasome was activated using Poly A: T.In human monocytic THP-1 cells, the effect of Nigericin on inflammasome activation products was examined using Western blotting and ELISA.Co-immunoprecipitation was performed to explore the mechanism of DMQ-induced blocking of NLRP3 inflammasome activation.In a male C57BL/6J mouse model of LPS-induced septic shock treated with 20 and 40 mg/kg DMQ, the levels of IL-1β and TNF-α in the serum and peritoneal lavage fluid were determined using ELISA, and the survival time of the mice within 36 h was observed. Treatment with DMQ effectively inhibited LPS-induced activation of canonical NLRP3 inflammasome in mouse BMDM and human THP-1 cells and also inhibited non-canonical NLRP3 inflammasome activation in mouse BMDM, but produced no significant effect on AIM2 inflammasome activation.DMQ significantly blocked the binding between ASC and NLRP3.In the mouse models of septic shock, DMQ treatment significantly reduced the levels of IL-1β in the serum and peritoneal fluid and obviously prolonged survival time of the mice. DMQ can effectively block ASC-NLRP3 interaction to inhibit NLRP3 inflammasome activation and alleviate LPSinduced septic shock in mice.

Daidzein and Equol: Ex Vivo and In Silico Approaches Targeting COX-2, iNOS, and the Canonical Inflammasome Signaling Pathway.

The inflammasome is a cytosolic multiprotein complex associated with multiple autoimmune diseases. Phytochemical compounds in soy (Glycine max) foods, such as isoflavones, have been reported for their anti-inflammatory properties. the anti-inflammatory activity of DZ (daidzein) and EQ (equol) were investigated in an ex vivo model of LPS-stimulated murine peritoneal macrophages and by molecular docking correlation. Cells were pre-treated with DZ (25, 50, and 100 µM) or EQ (5, 10, and 25 µM), followed by LPS stimulation. The levels of PGE2, NO, TNF-α, IL-6, and IL-1β were analyzed by ELISA, whereas the expressions of COX-2, iNOS, NLRP3, ASC, caspase 1, and IL-18 were measured by Western blotting. Also, the potential for transcriptional modulation by targeting NF-κB, COX-2, iNOS, NLRP3, ASC, and caspase 1 was investigated by molecular docking. The anti-inflammatory responses observed may be due to the modulation of NF-κB due to the binding of DZ or EQ, which is translated into decreased TNF-α, COX-2, iNOS, NLRP3, and ASC levels. This study establishes that DZ and EQ inhibit LPS-induced inflammatory responses in peritoneal murine macrophages via down-regulation of NO and PGE2 generation, as well as the inhibition of the canonical inflammasome pathway, regulating NLRP3, and consequently decreasing IL-1β and IL-18 activation.

Perturbation of azurophilic granule integrity drives NLRP3-independent IL-1b processing and release in neutrophils

Abstract IL-1β is an inflammatory cytokine secreted by myeloid cells in response to infection or sterile tissue damage. Non-canonical secretion of IL-1β from monocytes downstream of activated NLRP3 inflammasomes is the best-characterized model; this is mediated by caspase-1 cleavage of GSDMD and N-GSDMD plasma membrane (PM) pore formation to permit release of IL-1β and induction of pyroptosis. NLRP3 is a cytosolic sensor of cellular homeostasis including [K+]. In monocytes, K+ efflux mediated NLRP3 activation is triggered by agents that disrupt lysosomal integrity. Neutrophils assemble competent NLRP3 inflammasomes and release IL-1β via GSDMD-dependent mechanisms, but they resist N-GSDMD PM pore formation and pyroptosis. We tested whether lysosome-disrupting stimuli in neutrophils would phenocopy the monocyte mechanism of NLRP3-dependent IL-1β release. Surprisingly, our data indicates that lysosome disruption induces release of mature IL-1β and lytic cell death in an NLRP3-independent manner. Kinetic analysis demonstrates an alternate mechanism of release such that early phase IL-1β release is NLRP3-dependent, but prolonged stimulation leads to NLRP3-independent release. Collectively, our data supports a signaling mechanism by which azurophilic granule disruption and cytosolic accumulation of neutrophil serine proteases disrupt canonical NLRP3 inflammasome assembly and directly cleave proIL-1β as part of a novel neutrophil-specific signaling mechanism of IL-1β processing and export.

Abstract 1115: Novel Mechanism For Extrahepatic Inflammation In Progressive Familial Intrahepatic Cholestasis 1

Background: Human mutations in ATP8b1, a member of the P4-type ATPase family, cause Progressive Familial Intrahepatic Cholestasis I (PFIC1). PFIC1 patients often show extrahepatic inflammatory manifestations such as atherosclerosis, pancreatitis, and hearing loss. Even after liver transplantation, which rescues PFIC1 hepatic symptoms, the extrahepatic inflammatory symptoms persist. The mechanism of how ATP8b1 regulates inflammation is not clear. Pyroptosis executor Gasdermin D (GsdmD) plays a major role in promoting inflammation. GsdmD can be cleaved via a caspase-1/Nlrp3 dependent canonical inflammasome pathway, or a caspase-11 (in mice) or caspase-4/5 (in humans) dependent non-canonical inflammasome pathway. Goal: To decipher the mechanism for ATP8b1-mediated regulation of inflammasome activity in immune cells. Methods & Results: We employed Crispr-Cas9 generated homozygous ATP8b1 knockout human monocytes/macrophages to determine the status of inflammasome activity. Human monocytes/macrophages lacking ATP8b1 released significantly more IL-1β upon exposure to LPS (1μg/ml) + Nigericin (5 mM) vs. control cells (mean±SD, N=3-6, p<0.0001 by t-test.) Interestingly, ATP8b1 -/- cells showed GsdmD cleavage with LPS priming alone without a secondary signal, and markedly increased IL-1β release vs. WT cells (N=3-6, mean±SD, p<0.001). Mechanistically, LPS gained cytoplasmic access in ATP8b1 -/- immune cells, leading to cleavage of GsdmD in an NLRP3-independent, and caspase 4/5 dependent non-canonical inflammasome. The ATP8b1 -/- immune cells showed impaired lysosomal acidification, though the lysosomal number and lysosomal membrane integrity were not compromised. ATP8b1 -/- immune cells showed defects in inflammasome-resolving pathways such as phagocytosis and efferocytosis. Atomic force microscopy of ATP8b1 -/- cells showed a reduced order state of the plasma membrane with Young’s modulus of elasticity for THP-1 ATP8b1 -/- (Ey= 296±10.42 Pa) vs. WT macrophages (Ey =680±25.27 Pa, N =30, mean±SD, p<0.0001). Conclusions: ATP8b1 negatively regulates GsdmD cleavage via a non-canonical inflammasome pathway. GsdmD may serve as a therapeutic target for resolving inflammation in PFIC1 patients.

Bile acids attenuate hepatic inflammation during ischemia/reperfusion injury

Background & AimsPersistent cholestasis has been associated with poor prognosis after orthotopic liver transplantation (OLT). In this study, we aimed to investigate how the accumulation of tauro-beta-muricholic acid (TβMCA), resulting from the reprogramming of bile acid (BA) metabolism during liver ischemia/reperfusion (IR) stress, attenuates liver inflammation. MethodsIngenuity Pathway Analysis (IPA) was performed using transcriptome data from a murine hepatic IR model. Three different models of hepatic IR (liver warm IR, bile duct separation-IR, common bile duct ligation-IR) were employed. We generated AAV-transfected mice and CD11b-DTR mice to assess the role of BAs in regulating the myeloid S1PR2-GSDMD axis. Hepatic BA levels were analyzed using targeted metabolomics. Finally, the correlation between the reprogramming of BA metabolism and hepatic S1PR2 levels was validated through RNA-seq of human OLT biopsies. ResultsWe found that BA metabolism underwent reprogramming in murine hepatocytes under IR stress, leading to increased synthesis of TβMCA, catalyzed by the enzyme CYP2C70. The levels of hepatic TβMCA were negatively correlated with the severity of hepatic inflammation, as indicated by the serum IL-1β levels. Inhibition of hepatic CYP2C70 resulted in reduced TβMCA production, which subsequently increased serum IL-1β levels and exacerbated IR injury. Moreover, our findings suggested that TβMCA could inhibit canonical inflammasome activation in macrophages and attenuate inflammatory responses in a myeloid-specific S1PR2-GSDMD-dependent manner. Additionally, Gly-βMCA, a derivative of TβMCA, could effectively attenuate inflammatory injury in vivo and inhibit human macrophage pyroptosis in vitro. ConclusionsIR stress orchestrates hepatic BA metabolism to generate TβMCA, which attenuates hepatic inflammatory injury by inhibiting the myeloid S1PR2-GSDMD axis. Bile acids have immunomodulatory functions in liver reperfusion injury that may guide therapeutic strategies. Impact and implicationsOur research reveals that liver IR stress triggers reprogramming of BA metabolism. This functions as an adaptive mechanism to mitigate inflammatory injury by regulating the S1PR2-GSDMD axis, thereby controlling the release of IL-1β from macrophages. Our results highlight the crucial role of bile acids in regulating hepatocyte-immune cell crosstalk, which demonstrates an immunomodulatory function in liver reperfusion injury that may guide therapeutic strategies targeting bile acids and their receptors.

Proteomic analysis of P. gingivalis-Lipopolysaccharide induced neuroinflammation in SH-SY5Y and HMC3 cells.

Chronic periodontitis and its keystone pathogen, Porphyromonas gingivalis, have increasingly been linked with Alzheimer's disease (AD). However, P.gingivalis-lipopolysaccharide (LPS) mediated release of neuroinflammatory proteins contributes to AD remains underexplored. In this study, we utilized data-independent acquisition mass spectrometry to characterize P.gingivalis-LPS induced profile of differentially expressed proteins associated with the neuroinflammatory response in human neuroblastoma (SH-SY5Y) and human microglial (HMC3) cells. We reported a set of 124 proteins in SH-SY5Y cells and 96 proteins in HMC3 cells whose levels were significantly upregulated or downregulated by exposure to P. gingivalis-LPS. Our findings demonstrate that P. gingivalis-LPS contributed to the elevated expressions of dementia biomarkers and pro-inflammatory cytokines that include APP, Aβ1-42, Aβ1-40, T-Tau, p-Tau, VEGF, TGF-β, IL-1β, IL-6 and TNF-α through 2 distinct pathways of extracellular sensing by cell surface receptors and intracellular cytosolic receptors. Interestingly, intracellular signaling proteins activated with P. gingivalis-LPS transfection using Lipofectamine™ 2000 had significantly higher fold change protein expression compared to the extracellular signaling with P. gingivalis-LPS treatment. Additionally, we also explored P. gingivalis-LPS mediated activation of caspase-4 dependent non canonical inflammasome pathway in both SH-SY5Y and HMC3 cells. In summary, P. gingivalis-LPS induced neuroinflammatory protein expression in SH-SY5Y and HMC3 cells, provided insights into the specific inflammatory pathways underlying the potential link between P. gingivalis-LPS infection and the pathogenesis of Alzheimer's disease and related dementias.

Salmonella Enteritidis antitoxin DinJ inhibits NLRP3-dependent canonical inflammasome activation in macrophages.

The inflammasome is a pivotal component of the innate immune system, acting as a multiprotein complex that plays an essential role in detecting and responding to microbial infections. Salmonella Enteritidis have evolved multiple mechanisms to regulate inflammasome activation and evade host immune system clearance. Through screening S. Enteritidis C50336ΔfliC transposon mutant library, we found that the insertion mutant of dinJ increased inflammasome activation. In this study, we demonstrated the genetic connection between the antitoxin DinJ and the toxin YafQ in S. Enteritidis, confirming their co-transcription. The deletion mutant ΔfliCΔdinJ increased cell death and IL-1β secretion in J774A.1 cells. Western blotting analysis further showed elevated cleaved Caspase-1 product (p10 subunits) and IL-1β secretion in cells infected with ΔfliCΔdinJ compared to cells infected with ΔfliC. DinJ was found to inhibit canonical inflammasome activation using primary bone marrow-derived macrophages (BMDMs) from Casp-/- C57BL/6 mice. Furthermore, DinJ specifically inhibited NLRP3 inflammasome activation, as demonstrated in BMDMs from Nlrp3-/- and Nlrc4-/- mice. Fluorescence resonance energy transfer (FRET) experiments confirmed the translocation of DinJ into host cells during infection. Finally, we revealed that DinJ could inhibit the secretion of IL-1β and IL-18 in vivo, contributing to S. Enteritidis evading host immune clearance. In summary, our findings provide insights into the role of DinJ in modulating the inflammasome response during S. Enteritidis infection, highlighting its impact on inhibiting inflammasome activation and immune evasion.

Mechanistic insights from inflammasome structures.

Inflammasomes are supramolecular complexes that form in the cytosol in response to pathogen-associated and damage-associated stimuli, as well as other danger signals that perturb cellular homoeostasis, resulting in host defence responses in the form of cytokine release and programmed cell death (pyroptosis). Inflammasome activity is closely associated with numerous human disorders, including rare genetic syndromes of autoinflammation, cardiovascular diseases, neurodegeneration and cancer. In recent years, a range of inflammasome components and their functions have been discovered, contributing to our knowledge of the overall machinery. Here, we review the latest advances in inflammasome biology from the perspective of structural and mechanistic studies. We focus on the most well-studied components of the canonical inflammasome - NAIP-NLRC4, NLRP3, NLRP1, CARD8 and caspase-1 - as well as caspase-4, caspase-5 and caspase-11 of the noncanonical inflammasome, and the inflammasome effectors GSDMD and NINJ1. These structural studies reveal important insights into how inflammasomes are assembled and regulated, and how they elicit the release of IL-1 family cytokines and induce membrane rupture in pyroptosis.

Roles of the Caspase-11 Non-Canonical Inflammasome in Rheumatic Diseases.

Inflammasomes are intracellular multiprotein complexes that activate inflammatory signaling pathways. Inflammasomes comprise two major classes: canonical inflammasomes, which were discovered first and are activated in response to a variety of pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), and non-canonical inflammasomes, which were discovered recently and are only activated in response to intracellular lipopolysaccharide (LPS). Although a larger number of studies have successfully demonstrated that canonical inflammasomes, particularly the NLRP3 inflammasome, play roles in various rheumatic diseases, including rheumatoid arthritis (RA), infectious arthritis (IR), gouty arthritis (GA), osteoarthritis (OA), systemic lupus erythematosus (SLE), psoriatic arthritis (PA), ankylosing spondylitis (AS), and Sjögren's syndrome (SjS), the regulatory roles of non-canonical inflammasomes, such as mouse caspase-11 and human caspase-4 non-canonical inflammasomes, in these diseases are still largely unknown. Interestingly, an increasing number of studies have reported possible roles for non-canonical inflammasomes in the pathogenesis of various mouse models of rheumatic disease. This review comprehensively summarizes and discusses recent emerging studies demonstrating the regulatory roles of non-canonical inflammasomes, particularly focusing on the caspase-11 non-canonical inflammasome, in the pathogenesis and progression of various types of rheumatic diseases and provides new insights into strategies for developing potential therapeutics to prevent and treat rheumatic diseases as well as associated diseases by targeting non-canonical inflammasomes.

Deficiency of circadian clock gene Bmal1 exacerbates noncanonical inflammasome-mediated pyroptosis and lethality via Rev-erbα-C/EBPβ-SAA1 axis

Circadian arrhythmia has been linked to increased susceptibility to multiple inflammatory diseases, such as sepsis. However, it remains unclear how disruption of the circadian clock modulates molecular aspects of innate immune responses, including inflammasome signaling. Here, we examined the potential role of the circadian clock in inflammasome-mediated responses through myeloid-specific deletion of BMAL1, a master circadian clock regulator. Intriguingly, Bmal1 deficiency significantly enhanced pyroptosis of macrophages and lethality of mice under noncanonical inflammasome-activating conditions but did not alter canonical inflammasome responses. Transcriptome analysis of enriched peritoneal myeloid cells revealed that Bmal1 deficiency led to a marked reduction in Rev-erbα expression at steady state and a significant increase in serum amyloid A1 (SAA1) expression upon poly(I:C) stimulation. Notably, we found that the circadian regulator Rev-erbα is critical for poly(I:C)- or interferon (IFN)-β-induced SAA1 production, resulting in the circadian oscillation pattern of SAA1 expression in myeloid cells. Furthermore, exogenously applied SAA1 markedly increased noncanonical inflammasome-mediated pyroptosis of macrophages and lethality of mice. Intriguingly, our results revealed that type 1 IFN receptor signaling is needed for poly(I:C)- or IFN-β-induced SAA1 production. Downstream of the type 1 IFN receptor, Rev-erbα inhibited the IFN-β-induced association of C/EBPβ with the promoter region of Saa1, leading to the reduced transcription of Saa1 in macrophages. Bmal1-deficient macrophages exhibited enhanced binding of C/EBPβ to Saa1. Consistently, the blockade of Rev-erbα by SR8278 significantly increased poly(I:C)-stimulated SAA1 transcription and noncanonical inflammasome-mediated lethality in mice. Collectively, our data demonstrate a potent suppressive effect of the circadian clock BMAL1 on the noncanonical inflammasome response via the Rev-erbα-C/EBPβ-SAA1 axis.

P159 Inhibition of Caspase-1 signalling subtly affect the gut microbiota and reduces Adherent-invasive Escherichia coli (AIEC)-induced inflammasome activation

Abstract Background Inflammatory bowel disease (IBD), encompassing Crohn’s Disease and ulcerative colitis, is a chronic gastrointestinal disorder with increasing incidence worldwide. Microbial factors such as Adherent-invasive Escherichia coli (AIEC) have been suggested to play a significant role in the pathogenesis of IBD. The inflammasome, a multi-protein complex, senses environmental signals, including bacteria, resulting in the activation of caspase-1 leading to the subsequent maturation and secretion of IL-1β. In this study we examined the impact of AIEC on inflammasome activation and on the inhibition of Caspase-1 signaling in AIEC-colonised mice and on intestinal epithelial cells (IECs). Methods Human IECs (CaCO2Bee1 cells) were cultured for 3hr with AIEC (HM605 and its invasiveness mutant), washed with antibiotics and further cultured in full cell media for 13hrs, followed by analysis on cytokine and inflammasome gene expression and activation by qRT-PCR, ELISA, imaging and flow cytometry. To inhibit caspase-1 activation, cells were pre-treated for 1 hr with the caspase-1 inhibitor zVAD-fmk. To establish AIEC colonisation, C57BL/6 and IL-10-/- mice were treated with streptomycin for 24hrs followed by oral gavage of AIEC-HM605 (10-8 cfu/mL) and faecal and tissue samples were collected at 6 days post AIEC-infection. Caspase-1 inhibitor was orally gavaged every in mice. Faecal samples were prepared for 16S rRNA gene sequencing and colon tissue was analysed for cytokines and inflammasome markers by qRT-PCR and ELISA. Results IECs cultured with AIEC and AIEC-colonised C57BL/6 and IL-10-/- mice resulted in an increased expression and activation of both canonical (Caspase-1) and non-canonical inflammasome (caspase 4). The AIEC-induced inflammasome response was dependent on the invasiveness of AIEC. Treatment with a caspase-1 inhibitor reduced the AIEC-induced inflammasome markers in IECs and in C57BL/6 mice. No major changes in alpha or beta-diversity was detected in AIEC-colonised mice or upon Caspase-1 inhibition. AIEC-colonisation resulted in an increased abundance of Bacteroides and Alistipides and reduced abundance of Lachnospiraceae, alterations reversed by Caspase-1 inhibition. Conclusion We show that the invasion of AIEC in IECs and colonisation of murine intestine results in activation of caspase-1 inflammasome. AIEC colonisation provoke subtle microbiota alterations which are reversed by caspase-1 inhibition. In summary, our data supports a role of AIEC in IBD pathogenesis through the activation of caspase-1 inflammasome.