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As cytoplasmic protein complexes that are pivotal for innate immunity, inflammasomes act primarily through the detection of pathogen- or danger-associated molecular patterns. Nucleotide oligomerisation domain-like receptor family and caspase activation recruitment domain-containing protein 4 (NLRC4) inflammasomes identify and eliminate intracellular pathogens, a process contingent on the ligand-recognition capabilities of neuronal apoptosis inhibitory proteins (NAIPs). Upon detection of specific molecules indicative of intracellular infection, NAIPs discern distinct pathogenic components and subsequently transmit signals to NLRC4, thus initiating their activation and triggering an inflammatory response. However, the mechanisms underlying NLRC4 inflammasome remain unclear. In this study, we elucidated the critical role of ATG16L2 in activating the NLRC4 inflammasome. ATG16L2-deficient macrophages exhibited reduced NLRC4 inflammasome activation, characterised by decreased oligomerisation of apoptosis-associated speck-like protein containing a CARD and attenuated cleavage of Pro-caspase-1, Pro-IL-1β and gasdermin D. Co-immunoprecipitation assays revealed an interaction between ATG16L2 and NAIPs. Furthermore, ATG16L2 enhanced the association between NAIPs and NLRC4 by binding to NAIPs. For ATG16L2-knockout mice infected with Salmonella typhimurium, pathogen clearance and survival rates markedly decreased. Collectively, our findings suggest that ATG16L2 is a significant modulator of the innate immune system, influencing the activity of the NLRC4 inflammasome and the host's defensive response to intracellular pathogens., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

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., (© 2024. Springer Nature Limited.)

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ernst et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The NLR family caspase activation and recruitment domain-containing 4 (NLRC4) inflammasome is a critical cytosolic innate immune machine formed upon the direct sensing of bacterial infection and in response to cell stress during sterile chronic inflammation. Despite its major role in instigating the subsequent host immune response, a more complete understanding of the molecular events in the formation of the NLRC4 inflammasome in humans is lacking. Here we identify Bacillus thailandensis type III secretion system needle protein (Needle) as a potent trigger of the human NLR family apoptosis inhibitory protein (NAIP)/NLRC4 inflammasome complex formation and determine its structural features by cryogenic electron microscopy. We also provide a detailed understanding of how type III secretion system pathogen components are sensed by human NAIP to form a cascade of NLRC4 protomer through a critical lasso-like motif, a 'lock-key' activation model and large structural rearrangement, ultimately forming the full human NLRC4 inflammasome. These results shed light on key regulatory mechanisms specific to the NLRC4 inflammasome assembly, and the innate immune modalities of pathogen sensing in humans., (© 2024. The Author(s).)

Inflammasomes are multimeric protein complexes that have crucial functions in innate immunity. Here, we present a protocol to reconstitute the PELO-driven assembly of NAIP5-NLRC4 inflammasome in vitro. We describe steps for expression and purification of recombinant PELO and flagellin, preparation of native cell lysate containing NAIP5-NLRC4, and in vitro assembly of NAIP5-NLRC4 inflammasome. We then detail analysis of NAIP5-NLRC4 inflammasome by blue native polyacrylamide gel electrophoresis and immunoblotting. This protocol can be adapted to monitor the oligomeric assembly of other inflammasome types. For complete details on the use and execution of this protocol, please refer to Wu et al. (2023). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Inflammasomes are large protein complexes that, once activated, initiate inflammatory responses by activating the caspase-1 protease. They play pivotal roles in host defense against pathogens. The well-established role of NAIP/NLRC4 inflammasome in bacterial infections involves NAIP proteins functioning as sensors for their ligands. However, recent reports have indicated the involvement of NLRC4 in non-bacterial infections and sterile inflammation, even though the role of NAIP proteins and the exact molecular mechanisms underlying inflammasome activation in these contexts remain to be elucidated. In this study, we investigated the activation of the NAIP/NLRC4 inflammasome in response to Trypanosoma cruzi , the protozoan parasite responsible for causing Chagas disease. This parasite has been previously demonstrated to activate NLRP3 inflammasomes. Here we found that NAIP and NLRC4 proteins are also required for IL-1β and Nitric Oxide (NO) release in response to T. cruzi infection, with their absence rendering macrophages permissive to parasite replication. Moreover, Nlrc4 -/- and Nlrp3 -/- macrophages presented similar impaired responses to T. cruzi , underscoring the non-redundant roles played by these inflammasomes during infection. Notably, it was the live trypomastigotes rather than soluble antigens or extracellular vesicles (EVs) secreted by them, that activated inflammasomes in a cathepsins-dependent manner. The inhibition of cathepsins effectively abrogated caspase-1 cleavage, IL-1β and NO release, mirroring the phenotype observed in Nlrc4 -/- / Nlrp3 -/- double knockout macrophages. Collectively, our findings shed light on the pivotal role of the NAIP/NLRC4 inflammasome in macrophage responses to T. cruzi infection, providing new insights into its broader functions that extend beyond bacterial infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Amaral, Cardoso, de Farias, de Souza, Matteucci, Torrecilhas and Bortoluci.)

Introduction: The perturbations of gut microbiota could interact with excessively activated immune responses and play key roles in the etiopathogenesis of ulcerative colitis (UC). Desulfovibrio, the most predominant sulfate reducing bacteria (SRB) resided in the human gut, was observed to overgrow in patients with UC. The interactions between specific gut microbiota and drugs and their impacts on UC treatment have not been demonstrated well., Objectives: This study aimed to elucidate whether Desulfovibrio vulgaris (D. vulgaris, DSV) and its flagellin could activate nucleotide-binding oligomerization domain-like receptors (NLR) family of apoptosis inhibitory proteins (NAIP) / NLR family caspase activation and recruitment domain-containing protein 4 (NLRC4) inflammasome and promote colitis, and further evaluate the efficacy of eugeniin targeting the interaction interface of D. vulgaris flagellin (DVF) and NAIP to attenuate UC., Methods: The abundance of DSV and the occurrence of macrophage pyroptosis in human UC tissues were investigated. Colitis in mice was established by dextran sulfate sodium (DSS) and gavaged with DSV or its purified flagellin. NAIP/NLRC4 inflammasome activation and macrophage pyroptosis were evaluated in vivo and in vitro. The effects of eugeniin on blocking the interaction of DVF and NAIP/NLRC4 and relieving colitis were also assessed., Results: The abundance of DSV increased in the feces of patients with UC and was found to be associated with disease activity. DSV and its flagellin facilitated DSS-induced colitis in mice. Mechanistically, RNA sequencing showed that gene expression associated with inflammasome complex and pyroptosis was upregulated after DVF treatment in macrophages. DVF was further demonstrated to induce significant macrophage pyroptosis in vitro, depending on NAIP/NLRC4 inflammasome activation. Furthermore, eugeniin was screened as an inhibitor of the interface between DVF and NAIP and successfully alleviated the proinflammatory effect of DVF in colitis., Conclusion: Targeting DVF-induced NAIP/NLRC4 inflammasome activation and macrophage pyroptosis ameliorates UC. This finding is of great significance for exploring the gut microbiota-host interactions in UC development and providing new insights for precise treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Intracellular immune complexes known as inflammasomes sense breaches of cytosolic sanctity. Inflammasomes promote downstream proinflammatory events, including interleukin-1 (IL-1) family cytokine release and pyroptotic cell death. The nucleotide-binding leucine-rich repeat family, apoptosis inhibitory protein/nucleotide-binding leucine-rich repeat family, caspase recruitment domain (CARD) domain-containing protein 4 (NAIP/NLRC4) inflammasome is involved in a range of pathogenic and protective inflammatory processes in mammalian hosts. In particular, the NAIP/NLRC4 inflammasome responds to flagellin and components of the virulence-associated type III secretion (T3SS) apparatus in the host cytosol, thereby allowing it to be a critical mediator of host defense during bacterial infection. Notable species- and cell type-specific differences exist in NAIP/NLRC4 inflammasome responses to bacterial pathogens. With a focus on Salmonella enterica serovar Typhimurium as a model pathogen, we review differences between murine and human NAIP/NLRC4 inflammasome responses. Differences in NAIP/NLRC4 inflammasome responses across species and cell types may have arisen in part due to evolutionary pressures., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

NOD-like receptors (NLRs) are pattern recognition receptors for diverse innate immune responses. Self-oligomerization after engagement with a ligand is a generally accepted model for the activation of each NLR. We report here that a catalyzer was required for NLR self-oligomerization. PELO, a well-known surveillance factor in translational quality control and/or ribosome rescue, interacted with all cytosolic NLRs and activated their ATPase activity. In the case of flagellin-initiated NLRC4 inflammasome activation, flagellin-bound NAIP5 recruited the first NLRC4 and then PELO was required for correctly assembling the rest of NLRC4s into the NLRC4 complex, one by one, by activating the NLRC4 ATPase activity. Stoichiometric and functional data revealed that PELO was not a structural constituent of the NLRC4 inflammasome but a powerful catalyzer for its assembly. The catalytic role of PELO in the activation of cytosolic NLRs provides insight into NLR activation and provides a direction for future studies of NLR family members., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Gout is a disease caused by uric acid (UA) accumulation in the joints, causing inflammation. Two UA forms - monosodium urate (MSU) and soluble uric acid (sUA) have been shown to interact physically with inflammasomes, especially with the nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3), albeit the role of the immune response to UA is poorly understood, given that asymptomatic hyperuricemia does also exist. Macrophage phagocytosis of UA activate NLRP3, lead to cytokines release, and ultimately, lead to chemoattract neutrophils and lymphocytes to the gout flare joint spot. Genetic variants of inflammasome genes and of genes encoding their molecular partners may influence hyperuricemia and gout susceptibility, while also influencing other comorbidities such as metabolic syndrome and cardiovascular diseases. In this review, we summarize the inflammatory responses in acute and chronic gout, specifically focusing on innate immune cell mechanisms and genetic and epigenetic characteristics of participating molecules. Unprecedently, a novel UA binding protein - the neuronal apoptosis inhibitor protein (NAIP) - is suggested as responsible for the asymptomatic hyperuricemia paradox. Abbreviation: β2-integrins: leukocyte-specific adhesion molecules; ABCG2: ATP-binding cassete family/breast cancer-resistant protein; ACR: American college of rheumatology; AIM2: absent in melanoma 2, type of pattern recognition receptor; ALPK1: alpha-protein kinase 1; ANGPTL2: angiopoietin-like protein 2; ASC: apoptosis-associated speck-like protein; BIR: baculovirus inhibitor of apoptosis protein repeat; BIRC1: baculovirus IAP repeat-containing protein 1; BIRC2: baculoviral IAP repeat-containing protein 2; C5a: complement anaphylatoxin; cAMP: cyclic adenosine monophosphate; CARD: caspase activation and recruitment domains; CARD8: caspase recruitment domain-containing protein 8; CASP1: caspase 1; CCL3: chemokine (C-C motif) ligand 3; CD14: cluster of differentiation 14; CD44: cluster of differentiation 44; Cg05102552: DNA-methylation site, usually cytosine followed by guanine nucleotides; contains arbitrary identification code; CIDEC: cell death-inducing DNA fragmentation factor-like effector family; CKD: chronic kidney disease; CNV: copy number variation; CPT1A: carnitine palmitoyl transferase - type 1a; CXCL1: chemokine (CXC motif) ligand 1; DAMPs: damage associated molecular patterns; DC: dendritic cells; DNMT(1): maintenance DNA methyltransferase; eQTL: expression quantitative trait loci; ERK1: extracellular signal-regulated kinase 1; ERK2: extracellular signal-regulated kinase 2; EULAR: European league against rheumatism; GMCSF: granulocyte-macrophage colony-stimulating factor; GWAS: global wide association studies; H3K27me3: tri-methylation at the 27th lysine residue of the histone h3 protein; H3K4me1: mono-methylation at the 4th lysine residue of the histone h3 protein; H3K4me3: tri-methylation at the 4th lysine residue of the histone h3 protein; HOTAIR: human gene located between hoxc11 and hoxc12 on chromosome 12; IκBα: cytoplasmatic protein/Nf-κb transcription inhibitor; IAP: inhibitory apoptosis protein; IFNγ: interferon gamma; IL-1β: interleukin 1 beta; IL-12: interleukin 12; IL-17: interleukin 17; IL18: interleukin 18; IL1R1: interleukin-1 receptor; IL-1Ra: interleukin-1 receptor antagonist; IL-22: interleukin 22; IL-23: interleukin 23; IL23R: interleukin 23 receptor; IL-33: interleukin 33; IL-6: interleukin 6; IMP: inosine monophosphate; INSIG1: insulin-induced gene 1; JNK1: c-jun n-terminal kinase 1; lncRNA: long non-coding ribonucleic acid; LRR: leucine-rich repeats; miR: mature non-coding microRNAs measuring from 20 to 24 nucleotides, animal origin; miR-1: miR followed by arbitrary identification code; miR-145: miR followed by arbitrary identification code; miR-146a: miR followed by arbitrary identification code, "a" stands for mir family; "a" family presents similar mir sequence to "b" family, but different precursors; miR-20b: miR followed by arbitrary identification code; "b" stands for mir family; "b" family presents similar mir sequence to "a" family, but different precursors; miR-221: miR - followed by arbitrary identification code; miR-221-5p: miR followed by arbitrary identification code; "5p" indicates different mature miRNAs generated from the 5' arm of the pre-miRNA hairpin; miR-223: miR followed by arbitrary identification code; miR-223-3p: mir followed by arbitrary identification code; "3p" indicates different mature miRNAs generated from the 3' arm of the pre-miRNA hairpin; miR-22-3p: miR followed by arbitrary identification code, "3p" indicates different mature miRNAs generated from the 3' arm of the pre-miRNA hairpin; MLKL: mixed lineage kinase domain-like pseudo kinase; MM2P: inductor of m2-macrophage polarization; MSU: monosodium urate; mTOR: mammalian target of rapamycin; MyD88: myeloid differentiation primary response 88; n-3-PUFAs: n-3-polyunsaturated fatty-acids; NACHT: acronym for NAIP (neuronal apoptosis inhibitor protein), C2TA (MHC class 2 transcription activator), HET-E (incompatibility locus protein from podospora anserina) and TP1 (telomerase-associated protein); NAIP: neuronal apoptosis inhibitory protein (human); Naip1: neuronal apoptosis inhibitory protein type 1 (murine); Naip5: neuronal apoptosis inhibitory protein type 5 (murine); Naip6: neuronal apoptosis inhibitory protein type 6 (murine); NBD: nucleotide-binding domain; Nek7: smallest NIMA-related kinase; NET: neutrophil extracellular traps; Nf-κB: nuclear factor kappa-light-chain-enhancer of activated b cells; NFIL3: nuclear-factor, interleukin 3 regulated protein; NIIMA: network of immunity in infection, malignancy, and autoimmunity; NLR: nod-like receptor; NLRA: nod-like receptor NLRA containing acidic domain; NLRB: nod-like receptor NLRA containing BIR domain; NLRC: nod-like receptor NLRA containing CARD domain; NLRC4: nod-like receptor family CARD domain containing 4; NLRP: nod-like receptor NLRA containing PYD domain; NLRP1: nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 1; NLRP12: nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 12; NLRP3: nod-like receptor family pyrin domain containing 3; NOD2: nucleotide-binding oligomerization domain; NRBP1: nuclear receptor-binding protein; Nrf2: nuclear factor erythroid 2-related factor 2; OR: odds ratio; P2X: group of membrane ion channels activated by the binding of extracellular; P2X7: p2x purinoceptor 7 gene; p38: member of the mitogen-activated protein kinase family; PAMPs: pathogen associated molecular patters; PBMC: peripheral blood mononuclear cells; PGGT1B: geranylgeranyl transferase type-1 subunit beta; PHGDH: phosphoglycerate dehydrogenase; PI3-K: phospho-inositol; PPARγ: peroxisome proliferator-activated receptor gamma; PPARGC1B: peroxisome proliferative activated receptor, gamma, coactivator 1 beta; PR3: proteinase 3 antigen; Pro-CASP1: inactive precursor of caspase 1; Pro-IL1β: inactive precursor of interleukin 1 beta; PRR: pattern recognition receptors; PYD: pyrin domain; RAPTOR: regulatory associated protein of mTOR complex 1; RAS: renin-angiotensin system; REDD1: regulated in DNA damage and development 1; ROS: reactive oxygen species; rs000*G: single nuclear polymorphism, "*G" is related to snp where replaced nucleotide is guanine, usually preceded by an id number; SLC2A9: solute carrier family 2, member 9; SLC7A11: solute carrier family 7, member 11; SMA: smooth muscular atrophy; Smac: second mitochondrial-derived activator of caspases; SNP: single nuclear polymorphism; Sp3: specificity protein 3; ST2: serum stimulation-2; STK11: serine/threonine kinase 11; sUA: soluble uric acid; Syk: spleen tyrosine kinase; TAK1: transforming growth factor beta activated kinase; Th1: type 1 helper T cells; Th17: type 17 helper T cells; Th2: type 2 helper T cells; Th22: type 22 helper T cells; TLR: tool-like receptor; TLR2: toll-like receptor 2; TLR4: toll-like receptor 4; TNFα: tumor necrosis factor alpha; TNFR1: tumor necrosis factor receptor 1; TNFR2: tumor necrosis factor receptor 2; UA: uric acid; UBAP1: ubiquitin associated protein; ULT: urate-lowering therapy; URAT1: urate transporter 1; VDAC1: voltage-dependent anion-selective channel 1.

The nucleotide-binding domain (NBD), leucine rich repeat (LRR) domain containing protein family (NLR family) apoptosis inhibitory proteins (NAIPs) are cytosolic receptors that play critical roles in the host defense against bacterial infection. NAIPs interact with conserved bacterial ligands and activate the NLR family caspase recruitment domain containing protein 4 (NLRC4) to initiate the NAIP-NLRC4 inflammasome pathway. Here we found the process of NAIP activation is completely different from NLRC4. Our cryo-EM structure of unliganded mouse NAIP5 adopts an unprecedented wide-open conformation, with the nucleating surface fully exposed and accessible to recruit inactive NLRC4. Upon ligand binding, the winged helix domain (WHD) of NAIP5 undergoes roughly 20° rotation to form a steric clash with the inactive NLRC4, which triggers the conformational change of NLRC4 from inactive to active state. We also show the rotation of WHD places the 17-18 loop at a position that directly bind the active NLRC4 and stabilize the NAIP5-NLRC4 complex. Overall, these data provide structural mechanisms of inactive NAIP5, the process of NAIP5 activation and NAIP-dependent NLRC4 activation., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The NAIP/NLRC4 inflammasome is classically associated with the detection of bacterial invasion to the cytosol. However, recent studies have demonstrated that NAIP/NLRC4 is also activated in non-bacterial infections, and in sterile inflammation. Moreover, in addition to the well-established model for the detection of bacterial proteins by NAIP proteins, the participation of other cytosolic pathways in the regulation of NAIP/NLRC4-mediated responses has been reported in distinct contexts. Using pharmacological inhibition and genetic deletion, we demonstrate here that cathepsins, well known for their involvement in NLRP3 activation, also regulate NAIP/NLRC4 responses to cytosolic flagellin in murine and human macrophages. In contrast to that observed for NLRP3 agonists, cathepsins inhibition did not reduce ASC speck formation or caspase-1 maturation in response to flagellin, ruling out their participation in the effector phase of NAIP/NLRC4 activation. Moreover, cathepsins had no impact on NF-κB-mediated priming of pro-IL-1β, thus suggesting these proteases act downstream of the NAIP/NLRC4 inflammasome activation. IL-1β levels secreted in response to flagellin were reduced in the absence of either cathepsins or Gasdermin-D (GSDMD), a molecule involved in the induction of pyroptosis and cytokines release. Notably, IL-1β secretion was abrogated in the absence of both GSDMD and cathepsins, demonstrating their non-redundant roles for the optimal IL-1β release in response to cytosolic flagellin. Given the central role of NAIP/NLRC4 inflammasomes in controlling infection and, also, induction of inflammatory pathologies, many efforts have been made to uncover novel molecules involved in their regulation. Thus, our findings bring together a relevant contribution by describing the role of cathepsins as players in the NAIP/NLRC4-mediated responses., (© 2022. The Author(s).)

Innate immunity is a primary defense system against microbial infections. Innate immune pattern recognition receptors (PRRs) play pivotal roles in detection of invading pathogens. When pathogens, such as bacteria and viruses, invade our bodies, their components are recognized by PRRs as pathogen-associated molecular patterns (PAMPs), activating the innate immune system. Cellular components such as DNA and RNA, acting as damage-associated molecular patterns (DAMPs), also activate innate immunity through PRRs under certain conditions. Activation of PRRs triggers inflammatory responses, interferon-mediated antiviral responses, and the activation of acquired immunity. Research on innate immune receptors is progressing rapidly. A variety of these receptors has been identified, and their regulatory mechanisms have been elucidated. Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) constitute a major family of intracellular PRRs and are involved in not only combating pathogen invasion but also maintaining normal homeostasis. Some NLRs are known to form multi-protein complexes called inflammasomes, a process that ultimately leads to the production of inflammatory cytokines and induces pyroptosis through the proteolytic cascade. The aberrant activation of NLRs has been found to be associated with autoimmune diseases. Therefore, NLRs are considered targets for drug discovery, such as for antiviral drugs, immunostimulants, antiallergic drugs, and autoimmune disease drugs. This review summarizes our recent understanding of the activation and regulation mechanisms of NLRs, with a particular focus on their structural biology. These include NOD2, neuronal apoptosis inhibitory protein (NAIP)/NLRC4, NLR family pyrin domain containing 1 (NLRP1), NLRP3, NLRP6, and NLRP9. NLRs are involved in a variety of diseases, and their detailed activation mechanisms based on structural biology can aid in developing therapeutic agents in the future., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ohto.)

Hosts rely on the innate immune system to clear pathogens in response to infection. Pathogen-associated molecular patterns bind to innate immune receptors and engage activation of downstream signaling to initiate a host immune response to fight infection. A key component of this innate response is programmed cell death. Recent work has highlighted significant cross-talk and functional redundancy between cell death pathways, leading to the discovery of PANoptosis, an inflammatory programmed cell death pathway dependent on PANoptosomes, which are innate immune danger-sensing complexes that activate inflammatory cell death and contain caspases with or without inflammasome components and receptor interacting protein homotypic interaction motif-containing proteins. Although PANoptosis has been characterized in response to a growing number of pathogens, inflammatory diseases, and cancer, its role and the functional consequences of PANoptotic component modulation during NLR family CARD domain-containing protein 4 (NLRC4) activation by Pseudomonas aeruginosa infection remain unknown. In this study, we show that P. aeruginosa can induce PANoptosis in mouse bone marrow-derived macrophages (BMDMs). Only the combined deletion of caspase-1, -11, -8, and RIPK3 protected mouse BMDMs from cell death. Moreover, we showed that PANoptotic components act in a compensatory manner; in the absence of NAIP5 and NLRC4 during P. aeruginosa challenge, activation of caspase-1, -3, -7, and -8 was reduced, whereas alternative cell death molecules such as RIPK1 and MLKL were activated in mouse BMDMs. Taken together, these data highlight the extensive cross-talk between cell death signaling molecules and showcase the plasticity of the system., (Copyright © 2022 The Authors.)

The gut relies on the complex interaction between epithelial, stromal and immune cells to maintain gut health in the face of food particles and pathogens. Innate sensing by the intestinal epithelium is critical for maintaining epithelial barrier function and also orchestrating mucosal immune responses. Numerous innate pattern recognition receptors (PRRs) are involved in such sensing. In recent years, several Nucleotide-binding-domain and Leucine-rich repeat-containing receptors (NLRs) have been found to partake in pathogen or damage sensing while also being implicated in gut pathologies, such as colitis and colorectal cancer (CRC). Here, we discuss the current literature focusing on NLR family apoptosis inhibitory proteins (NAIPs) and other NLRs that have non-inflammasome roles in the gut. The mechanisms behind NLR-mediated protection often converges on similar signalling pathways, such as STAT3, MAPK and NFκB. Further understanding of how these NLRs contribute to the maintenance of gut homeostasis will be important for understanding gut pathologies and developing new therapies., (© 2021 The Author(s).)

DDX17 binds short interspersed nuclear element RNAs to nucleate a dual NLRC4/NLRP3 inflammasome, highlighting the emerging role of endogenous ligands in NLRC4 activation (see the related Research Article by Wang et al .).

The gut epithelium is a critical protective barrier. Its NAIP/NLRC4 inflammasome senses infection by Gram-negative bacteria, including Salmonella Typhimurium (S.Tm) and promotes expulsion of infected enterocytes. During the first ~12-24 h, this reduces mucosal S.Tm loads at the price of moderate enteropathy. It remained unknown how this NAIP/NLRC4-dependent tradeoff would develop during subsequent infection stages. In NAIP/NLRC4-deficient mice, S.Tm elicited severe enteropathy within 72 h, characterized by elevated mucosal TNF (>20 pg/mg) production from bone marrow-derived cells, reduced regeneration, excessive enterocyte loss, and a collapse of the epithelial barrier. TNF-depleting antibodies prevented this destructive pathology. In hosts proficient for epithelial NAIP/NLRC4, a heterogeneous enterocyte death response with both apoptotic and pyroptotic features kept S.Tm loads persistently in check, thereby preventing this dire outcome altogether. Our results demonstrate that immediate and selective removal of infected enterocytes, by locally acting epithelium-autonomous NAIP/NLRC4, is required to avoid a TNF-driven inflammatory hyper-reaction that otherwise destroys the epithelial barrier.

The gut epithelium serves to maximize the surface for nutrient and fluid uptake, but at the same time must provide a tight barrier to pathogens and remove damaged intestinal epithelial cells (IECs) without jeopardizing barrier integrity. How the epithelium coordinates these tasks remains a question of significant interest. We used imaging and an optical flow analysis pipeline to study the dynamicity of untransformed murine and human intestinal epithelia, cultured atop flexible hydrogel supports. Infection with the pathogen Salmonella Typhimurium ( S Tm) within minutes elicited focal contractions with inward movements of up to ∼1,000 IECs. Genetics approaches and chimeric epithelial monolayers revealed contractions to be triggered by the NAIP/NLRC4 inflammasome, which sensed type-III secretion system and flagellar ligands upon bacterial invasion, converting the local tissue into a contraction epicenter. Execution of the response required swift sublytic Gasdermin D pore formation, ion fluxes, and the propagation of a myosin contraction pulse across the tissue. Importantly, focal contractions preceded, and could be uncoupled from, the death and expulsion of infected IECs. In both two-dimensional monolayers and three-dimensional enteroids, multiple infection-elicited contractions coalesced to produce shrinkage of the epithelium as a whole. Monolayers deficient for Caspase-1(-11) or Gasdermin D failed to elicit focal contractions but were still capable of infected IEC death and expulsion. Strikingly, these monolayers lost their integrity to a markedly higher extent than wild-type counterparts. We propose that prompt NAIP/NLRC4/Caspase-1/Gasdermin D/myosin-dependent contractions allow the epithelium to densify its cell packing in infected regions, thereby preventing tissue disintegration due to the subsequent IEC death and expulsion process., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

NLRC4 inflammasome activation and the subsequent maturation of IL-1β and IL-18 are critical for protection against infection by bacterial pathogens. The epigenetic regulator Brd4 has emerged as a key player in inflammation by regulating the expression of inflammatory cytokines. However, whether Brd4 has any role in inflammasome activation remains undetermined. Here, we demonstrated that Brd4 is an important regulator of NLRC4 inflammasome activation in response to Salmonella typhimurium infection. Brd4-deficient bone marrow-derived macrophages (BMDMs) displayed impaired caspase-1 activation, ASC oligomerization, IL-1β maturation, gasdermin-D cleavage, and pyroptosis in response to S.typhimurium infection. RNA sequencing and RT-PCR results revealed that the transcription of Naips was decreased in Brd4-deficient BMDMs. Brd4 formed a complex with IRF8/PU.1 and bound to the IRF8 and PU.1 binding motifs on the promoters of Naips to maintain the expression of Naips. Furthermore, myeloid lineage-specific Brd4 conditional knockout mice were more susceptible to S.typhimurium infection with increased mortality, bacterial loads, and tissue damage; impaired inflammasome-dependent cytokine production; and pyroptosis. Our studies identify a novel function of Brd4 in innate immunity by controlling inflammasome-mediated cytokine release and pyroptosis to effectively battle S.typhimurium infection., (© 2021 Dong et al.)

Uric acid (UA), a product of purine nucleotide degradation able to initiate an immune response, represents a breakpoint in the evolutionary history of humans, when uricase, the enzyme required for UA cleavage, was lost. Despite being inert in human cells, UA in its soluble form (sUA) can increase the level of interleukin-1β (IL-1β) in murine macrophages. We, therefore, hypothesized that the recognition of sUA is achieved by the Naip1-Nlrp3 inflammasome platform. Through structural modelling predictions and transcriptome and functional analyses, we found that murine Naip1 expression in human macrophages induces IL-1β expression, fatty acid production and an inflammation-related response upon sUA stimulation, a process reversed by the pharmacological and genetic inhibition of Nlrp3. Moreover, molecular interaction experiments showed that Naip1 directly recognizes sUA. Accordingly, Naip may be the sUA receptor lost through the human evolutionary process, and a better understanding of its recognition may lead to novel anti-hyperuricaemia therapies.

The neuronal apoptosis inhibitory protein (NAIP) is a constituent of the NLRC4 inflammasome, which plays a key role in innate immunity, and an antiapoptotic protein. Recently, we reported the previously undescribed role of NAIP in cell division. The liver is one of the body's most actively regenerative organs. Given the novel mitotic role of NAIP, we examined its expression in hepatic mass restoration. The major liver lobe of Wistar rats was removed, and samples from both newly formed liver tissue, assessed by positive Ki67 immunostaining, and the remnant, intact liver lobes from hepatectomized rats were taken 3 and 7 days after surgery. Naip5 and Naip6 mRNA levels were significantly higher in regenerating hepatic tissue than in intact liver lobe tissue, and this increase was also observed at the protein level. Naip5 and Naip6 mRNA in situ hybridization showed that this increase occurred in the hepatic parenchyma. The histology of the regenerated liver tissue was normal, with the exception of a noticeable deficiency of hepatic lobule central veins. The results of this study suggest the involvement of NAIP in liver mass restoration following partial hepatectomy.

Infection of human macrophages with Salmonella enterica serovar Typhimurium ( S. Typhimurium) leads to inflammasome activation. Inflammasomes are multiprotein complexes facilitating caspase-1 activation and subsequent gasdermin D-mediated cell death and IL-1β and IL-18 cytokine release. The NAIP/NLRC4 inflammasome is activated by multiple bacterial protein ligands, including flagellin from the flagellum and the needle protein PrgI from the S. Typhimurium type III secretion system. In this study, we show that transfected ultrapure flagellin from S Typhimurium induced cell death and cytokine secretion in THP-1 cells and primary human monocyte-derived macrophages. In THP-1 cells, NAIP/NLRC4 and NLRP3 played redundant roles in inflammasome activation during infection with S. Typhimurium. Knockout of NAIP or NLRC4 in THP-1 cells revealed that flagellin, but not PrgI, now activated the NLRP3 inflammasome through a reactive oxygen species- and/or cathepsin-dependent mechanism that was independent of caspase-4/5 activity. In conclusion, our data suggest that NLRP3 can be activated by flagellin to act as a "safety net" to maintain inflammasome activation under conditions of suboptimal NAIP/NLRC4 activation, as observed in THP-1 cells, possibly explaining the redundant role of NLRP3 and NAIP/NLRC4 during S. Typhimurium infection., (Copyright © 2021 The Authors.)

The inhibitors of apoptosis protein (IAP)/baculoviral IAP repeat containing (BIRC) gene families are necessary for cell protection, and most of these genes act as endogenous inhibitors of apoptosis. In some cancers, the over-expression of the BIRC gene is associated with cancer progression, multidrug resistance, poor prognosis and short-term survival. In this study, we aimed to assess the effect of the BIRC family in pan-cancer. We downloaded transcriptome and clinical data from 33 types of TCGA tumor samples and adjacent tissues. Then, the expression characteristics of IAP family members BIRC2, BIRC3, BIRC5, BIRC6 and BIRC7 in pan-cancer were analyzed. R packet and Cox regression were used to analyze the clinical correlation. In addition, the transcription level of BIRC and immune subtypes, stem cells, immune tumor microenvironment (TME) and drug sensitivity were analyzed by multidimensional correlation. Our studies have shown that the expression of IAP family members BIRC2, BIRC3, BIRC5, BIRC6, and BIRC7 is different in different tumor types, and the heterogeneity is obvious in cancers. Overall, our analysis showed that BIRC2, BIRC3, BIRC6, and BIRC7 were mainly down-regulated in tumors, whereas BIRC5 was mainly up-regulated in tumors. The expression of IAP family members is related to the overall survival of patients. However, the direction of the association varies depending on specific IAP subtypes and specific types of cancer. More specifically, BIRC5 is mainly related to poor prognosis. The rest of the IAP family showed either a survival advantage or a survival disadvantage, depending on the type of cancer. In addition, BIRC2, BIRC3, BIRC5, BIRC6 and BIRC7 were significantly correlated with immune infiltration subtypes and had different degrees of correlation with the degree of interstitial cell infiltration and tumor cell dryness. Finally, our study revealed that BIRC2, BIRC5, and BIRC7 genes may be related to drug resistance of tumor cells. Our systematic analysis of (IAP) gene expression and its relationship with immune infiltration, TME, cancer stem cells, drug sensitivity and prognosis of cancer patients highlights the need to study IAP family members as separate entities in each specific cancer type. In addition, our study confirmed that IAP family genes are promising therapeutic targets for cancer and potential prognostic indicators for clinical application, although further laboratory verification is needed.

In this review we give an overview of the NAIP/NLRC4 activation mechanism as well as the described roles of this inflammasome, with a focus on in vivo infection and pathology. After ligand recognition by NAIP sensor proteins the NAIP/NLRC4 inflammasome forms through oligomerization with the NLRC4 adaptor to activate Caspase-1. The activating ligands are intracellular bacterial flagellin or type-3 secretion system components, delivered by pathogens. In vivo experiments indicate a role in macrophages during lung, spleen and liver infection and systemic sepsis like conditions, as well as in intestinal epithelial cells. Upon NAIP/NLRC4 activation in the intestine, epithelial cell extrusion is triggered in addition to the canonical inflammasome outcomes of cytokine cleavage and pyroptosis. Human patients with auto-activating mutations in NLRC4 present with an autoinflammatory syndrome including enterocolitis. Although one of the better understood inflammasomes in terms of mechanism, tissue specific functions of NAIP/NLRC4 are only beginning to be understood., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cytosolic innate immune sensing is a cornerstone of innate immunity in mammalian cells and provides a surveillance system for invading pathogens and endogenous danger signals. The NAIP-NLRC4 inflammasome responds to cytosolic flagellin, and the inner rod and needle proteins of the type 3 secretion system of bacteria. This complex induces caspase-1-dependent proteolytic cleavage of the proinflammatory cytokines IL-1β and IL-18, and the pore-forming protein gasdermin D, leading to inflammation and pyroptosis, respectively. Localized responses triggered by the NAIP-NLRC4 inflammasome are largely protective against bacterial pathogens, owing to several mechanisms, including the release of inflammatory mediators, liberation of concealed intracellular pathogens for killing by other immune mechanisms, activation of apoptotic caspases, caspase-7, and caspase-8, and expulsion of an entire infected cell from the mammalian host. In contrast, aberrant activation of the NAIP-NLRC4 inflammasome caused by de novo gain-of-function mutations in the gene encoding NLRC4 can lead to macrophage activation syndrome, neonatal enterocolitis, fetal thrombotic vasculopathy, familial cold autoinflammatory syndrome, and even death. Some of these clinical manifestations could be treated by therapeutics targeting inflammasome-associated cytokines. In addition, the NAIP-NLRC4 inflammasome has been implicated in the pathogenesis of colorectal cancer, melanoma, glioma, and breast cancer. However, no consensus has been reached on its function in the development of any cancer types. In this review, we highlight the latest advances in the activation mechanisms and structural assembly of the NAIP-NLRC4 inflammasome, and the functions of this inflammasome in different cell types. We also describe progress toward understanding the role of the NAIP-NLRC4 inflammasome in infectious diseases, autoinflammatory diseases, and cancer., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Preeclampsia (PE) is a pregnancy-related syndrome and has become the leading cause of maternal and neonatal morbidity and mortality. LncRNA has been elucidated to play critical roles in the phenotype of trophoblast cells. However, the effect of AK002210 has not been reported. We aim to investigate the effect of AK002210 on the phenotype of trophoblast cells. Quantitative reverse transcription PCR was used to assess the gene expression. CCK-8 assay was used to evaluate the cell proliferation. Transwell assay was performed to detect the migration and invasion of trophoblast cells. Luciferase assay and rescue experiment were carried out to verify the interaction between miR-590-3p and AK002210 as well as NLR family apoptosis inhibitory protein (NAIP). The results revealed that AK002210 promoted the proliferation, migration and invasion of trophoblast cell while AK002210 knockdown inhibited that. Mechanically, we found that AK002210 was targeted by miR-590-3p. Moreover, miR-590-3p also directly targets NAIP which served as a ceRNA of AK002210. Rescue experiment showed that miR-590-3p reversed the effect of AK002210 which further confirmed their interaction. Moreover, AK002210 was proved to participated in the regulation of ERK/MMP-2 signal axis. In conclusion, we found that AK002210 knockdown may play a critical role in the progression of PE via miR-590-3p/NAIP and ERK/MMP signaling. It has potential to be a novel prognostic or therapeutic marker of PE., Competing Interests: Declaration of competing interest All authors declare that no conflict of interest exists in the manuscript., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Inflammasomes can prevent systemic dissemination of enteropathogenic bacteria. As adapted pathogens including Salmonella Typhimurium (S. Tm) have evolved evasion strategies, it has remained unclear when and where inflammasomes restrict their dissemination. Bacterial population dynamics establish that the NAIP/NLRC4 inflammasome specifically restricts S. Tm migration from the gut to draining lymph nodes. This is solely attributable to NAIP/NLRC4 within intestinal epithelial cells (IECs), while S. Tm evades restriction by phagocyte NAIP/NLRC4. NLRP3 and Caspase-11 also fail to restrict S. Tm mucosa traversal, migration to lymph nodes, and systemic pathogen growth. The ability of IECs (not phagocytes) to mount a NAIP/NLRC4 defense in vivo is explained by particularly high NAIP/NLRC4 expression in IECs and the necessity for epithelium-invading S. Tm to express the NAIP1-6 ligands-flagella and type-III-secretion-system-1. Imaging reveals both ligands to be promptly downregulated following IEC-traversal. These results highlight the importance of intestinal epithelial NAIP/NLRC4 in blocking bacterial dissemination in vivo, and explain why this constitutes a uniquely evasion-proof defense against the adapted enteropathogen S. Tm.

Cataract is the leading cause of blindness in the world while the molecular mechanisms of cataracts pathogenesis are not well elucidated. Dysregulated microRNA (miRNA) expressions have been implicated in cataract. However, the precise role of miR-4328 in cataract is still unknown. We compared the expression level of total miR-4328 between clinical samples from healthy people and cataract patient and between UV-irradiated and control lens epithelial cells. We determined the effect of miR-4328 on lens epithelial cell proliferation and apoptosis by suppressing miR-4328. We further predicted NLR Family Apoptosis Inhibitory Protein (NAIP) as potential target of miR-4328 and continued to evaluate the effects of NAIP on cell proliferation and apoptosis. MiR-4328 was up-regulated in cataract sample and in UV-irradiated lens epithelial cells. Suppressing MiR-4328 promoted cell proliferation and inhibited apoptosis. MiR-4328 targeted NAIP and suppressed its expression. Knocking down NAIP abolished the effects of miR-4328 on cell proliferation and inhibited apoptosis. MiR-4328 promotes lens epithelial cell apoptosis by targeting NAIP in age-related cataract. SIGNIFICANCE OF THE STUDY: MiR-4328 targeted NAIP and suppressed its expression. Knocking down NAIP abolished the effects of miR-4328 on cell proliferation and inhibited apoptosis. MiR-4328 promotes lens epithelial cell apoptosis by targeting NAIP in age-related cataract., (© 2019 John Wiley & Sons, Ltd.)

Bacterial flagellin is a well-known agonist of the innate immune system that induces proinflammatory responses through the TLR5 and Naip5/6 recognition pathways. Several clinical trials investigating flagellin fusion proteins have demonstrated promising results for inducing protective immunity toward influenza virus, which has been largely attributed to flagellin's ability to activate TLR5. Our laboratory previously demonstrated that the Salmonella enterica serovar Typhimurium flagellin protein, FliC, induces Ab responses in mice through a third pathway that is independent of TLR5, Casp1/11, and MyD88. In this study, we further define the structural features of FliC that contribute to this unknown third pathway. By destroying the Naip5/6 and TLR5 recognition sites, we demonstrate that neither were required for the TLR5-, inflammasome- and MyD88-independent Ab responses toward FliC. In contrast, deletion of FliC's D3 or D0/D1 domains eliminated primary anti-flagellin Ab responses. For optimal primary and secondary anti-flagellin Ab responses we show that TLR5, inflammasome recognition, and the D3 domain of FliC are essential for flagellin's robust immunogenicity. Our data demonstrate that the D3 domain of FliC influences immunogenicity independent of the known innate recognition sites in the D0/D1 domains to augment Ab production. Our results suggest full-length FliC is critical for optimal immunogenicity and Ab responses in flagellin-based vaccines., (Copyright © 2019 The Authors.)

Inflammasomes are multiprotein signaling complexes that are assembled by cytosolic sensors upon the detection of infectious or noxious stimuli. These complexes activate inflammatory caspases to induce host cell death and cytokine secretion and are an essential part of antimicrobial host defense. In this review, I discuss how intracellular bacteria are detected by inflammasomes, how the specific sensing mechanism of each inflammasome receptor restricts the ability of bacteria to evade immune recognition, and how host cell death is used to control bacterial replication in vivo .

The NAIP-NLRC4 family of inflammasomes are components of the innate immune system that sound a molecular alarm in the presence of intracellular pathogens. In this chapter, we provide an in-depth guide to using cryo-electron microscopy (cryo-EM) to investigate these inflammasomes, focusing especially on the techniques we used in our recent structural analysis of the NAIP5-NLRC4 inflammasome. We explain how to circumvent specific obstacles we encountered at each step, from sample preparation through data processing. The methods described here will be useful for further studies of the NAIP5-NLRC4 inflammasome and related supracomplexes involved in innate immune surveillance; they may also be useful for unrelated complexes that present similar issues, such as preferential orientations and compositional heterogeneity., (© 2019 Elsevier Inc. All rights reserved.)

Inflammasomes are supramolecular signaling platforms integral to innate immune defense against invading pathogens. The NOD-like receptor (NLR) family apoptosis inhibitory protein (NAIP)·NLR family caspase-recruiting domain (CARD) domain-containing 4 (NLRC4) inflammasome recognizes intracellular bacteria and induces the polymerization of the caspase-1 protease, which in turn executes maturation of interleukin-1β (IL-1β) and pyroptosis. Several high-resolution structures of the fully assembled NAIP·NLRC4 complex are available, but these structures do not resolve the architecture of the CARD filament in atomic detail. Here, we present the cryo-EM structure of the filament assembled by the CARD of human NLRC4 (NLRC4 CARD ) at 3.4 Å resolution. The structure revealed that the helical architecture of the NLRC4 CARD filament is essentially identical to that of the downstream filament assembled by the CARD of caspase-1 (casp1 CARD ), but deviates from the split washer-like assembly of the NAIP·NLRC4 oligomer. Our results suggest that architectural complementarity is a major driver for the recognition between upstream and downstream CARD assemblies in inflammasomes. Furthermore, a Monte Carlo simulation of the NLRC4 CARD filament assembly rationalized why an (un)decameric NLRC4 oligomer is optimal for assembling the helical base of the NLRC4 CARD filament. Together, our results explain how symmetric and asymmetric supramolecular assemblies enable high-fidelity signaling in inflammasomes., (© 2018 Matyszewski et al.)

Adrenocortical dysplasia (ACD) is a shelterin protein involved in the maintenance of telomere length and in cancer radioresistance. This study investigated the expression profile of ACD in human gliomas and its role in radioresistance of glioma cells. The expression of ACD was analyzed in 62 different grades of glioma tissues and correlated with prognosis. A radioresistant cell line was generated from U87MG cells. For mechanistic studies, ACD was inhibited by small interfering RNA-targeting ACD and the effect on cell radioresistance, telomerase activity, cyclinD1, caspase-3, hTERT, and BIRC1 was evaluated. Clonogenic assay was performed after irradiation, to investigate the effect of ACD silencing on radiation sensitivity. ACD expression appeared strongly upregulated in higher grade gliomas, and its expression was significantly correlated to grading and poor prognosis. In glioma cell lines, ACD expression pattern was similar to those observed in glioma tissues. In irradiated cells, ACD expression was increased in an ionizing radiation dose-dependent manner. A higher expression of ACD was observed in the radioresistant clones than parental cells. Silencing of ACD led to the enhanced radiation sensitivity, decreased telomerase activity and cyclin D1 expression, reduced expression of BIRC1, and finally to the upregulation of caspase-3. This study represents the first report, which demonstrated the expression pattern of ACD in gliomas and its prognostic value. Our results suggested that ACD is involved in glioblastoma radioresistance, likely through the modulation of telomerase activity, proliferation, and apoptosis. ACD might represent a potential molecular biomarker and a novel therapeutic target in glioblastoma., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Inflammasome activation is critical for host defenses against various microbial infections. Activation of the NLRC4 inflammasome requires detection of flagellin or type III secretion system (T3SS) components by NLR family apoptosis inhibitory proteins (NAIPs); yet how this pathway is regulated is unknown. Here, we found that interferon regulatory factor 8 (IRF8) is required for optimal activation of the NLRC4 inflammasome in bone-marrow-derived macrophages infected with Salmonella Typhimurium, Burkholderia thailandensis, or Pseudomonas aeruginosa but is dispensable for activation of the canonical and non-canonical NLRP3, AIM2, and Pyrin inflammasomes. IRF8 governs the transcription of Naips to allow detection of flagellin or T3SS proteins to mediate NLRC4 inflammasome activation. Furthermore, we found that IRF8 confers protection against bacterial infection in vivo, owing to its role in inflammasome-dependent cytokine production and pyroptosis. Altogether, our findings suggest that IRF8 is a critical regulator of NAIPs and NLRC4 inflammasome activation for defense against bacterial infection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Monocytes and macrophages constitute the first line of defense of the immune system against external pathogens. Macrophages have a highly plastic phenotype depending on environmental conditions; the extremes of this phenotypic spectrum are a pro-inflammatory defensive role (M1 phenotype) and an anti-inflammatory tissue-repair one (M2 phenotype). The Inhibitor of Apoptosis (IAP) proteins have important roles in the regulation of several cellular processes, including innate and adaptive immunity. In this study we have analyzed the differential expression of the IAPs, NAIP, cIAP1 and cIAP2, during macrophage differentiation and polarization into M1 or M2. In polarized THP-1 cells and primary human macrophages, NAIP is abundantly expressed in M2 macrophages, while cIAP1 and cIAP2 show an inverse pattern of expression in polarized macrophages, with elevated expression levels of cIAP1 in M2 and cIAP2 preferentially expressed in M1. Interestingly, treatment with the IAP antagonist SMC-LCL161, induced the upregulation of NAIP in M2, the downregulation of cIAP1 in M1 and M2 and an induction of cIAP2 in M1 macrophages.

The inflammasome is a complex of proteins that through the activity of caspase-1 and the downstream substrates gasdermin D, IL-1β, and IL-18 execute an inflammatory form of cell death termed pyroptosis. Activation of this complex often involves the adaptor protein ASC and upstream sensors including NLRP1, NLRP3, NLRC4, AIM2, and pyrin, which are activated by different stimuli including infectious agents and changes in cell homeostasis. Here we discuss new regulatory mechanisms that have been identified for the canonical inflammasomes, the most recently identified NLRP9b inflammasome, and the new gasdermin family of proteins that mediate pyroptosis and other forms of regulated cell death., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The delineation of the molecular pathology underlying amyotrophic lateral sclerosis (ALS) is being hampered by the lack of suitable biomarkers. We have previously reported that bromocriptine upregulates the endogenous antioxidative factor, neuronal apoptosis inhibitory protein (NAIP), sustains motor function and slows disease progression in ALS patients, implying the NAIP's implication in ALS. Here, we aimed to verify a correlation of NAIP level with disease progression in ALS patients. The amount of NAIP in mononuclear cells (MNC) from peripheral blood from ALS patients (n = 18) and the age matched healthy controls (n = 12) was validated by NAIP-Dot blotting. Notably, the MNC-NAIP level in ALS patients (0.62 ± 0.29 ng) was nearly half of that in the healthy controls (1.34 ± 0.61 ng, P = 0.0019). Furthermore, the MNC-NAIP level in ALS patients and their ALS Functional Rating Scale-Revised (ALSFRS-R) score were evaluated through 1 year. Regression analysis of the MNC-NAIP vs ALSFRS-R indicated that a higher amount of MNC-NAIP was associated with a smaller change in ALSFRS-R at 12 months (R 2  = 0.799; P = 0.016), suggesting that a progressive increment of the MNC-NAIP led to slower ALS progression. Our present report implies that NAIP will have broad implications for ALS symptoms as a risk factor and a promising prognostic biomarker.

Inflammasomes are cytosolic multiprotein complexes that initiate host defense against bacterial pathogens by activating caspase-1-dependent cytokine secretion and cell death. In mice, specific nucleotide-binding domain, leucine-rich repeat-containing family, apoptosis inhibitory proteins (NAIPs) activate the nucleotide-binding domain, leucine-rich repeat-containing family, CARD domain-containing protein 4 (NLRC4) inflammasome upon sensing components of the type III secretion system (T3SS) and flagellar apparatus. NAIP1 recognizes the T3SS needle protein, NAIP2 recognizes the T3SS inner rod protein, and NAIP5 and NAIP6 recognize flagellin. In contrast, humans encode a single functional NAIP, raising the question of whether human NAIP senses one or multiple bacterial ligands. Previous studies found that human NAIP detects both flagellin and the T3SS needle protein and suggested that the ability to detect both ligands was achieved by multiple isoforms encoded by the single human NAIP gene. Here, we show that human NAIP also senses the Salmonella Typhimurium T3SS inner rod protein PrgJ and that T3SS inner rod proteins from multiple bacterial species are also detected. Furthermore, we show that a single human NAIP isoform is capable of sensing the T3SS inner rod, needle, and flagellin. Our findings indicate that, in contrast to murine NAIPs, promiscuous recognition of multiple bacterial ligands is conferred by a single human NAIP., Competing Interests: The authors declare no conflict of interest.

Aim: To clarify the long-term natural history of SMA in Japanese patients by investigating the peak motor milestones of cases 7months through 57years of age, in efforts to contribute to evaluating outcomes of new therapeutic interventions., Methods: We sub-classified 112 SMA type I-III cases into type Ia, type Ib, type IIa, type IIb, type IIIa and type IIIb, according to peak motor milestone achieved, and analyzed the SMN1, SMN2 and NAIP genes in relation to clinical subtypes., Results: In type I cases, there was a significant difference (p<0.0001), depending on whether or not head control was obtained, in the time of ventilation support being required. In type II cases as well, the time at which the ability to maintain the sitting position independently was lost also differed significantly (p<0.01) between those acquiring the ability to sit unaided within eight months after birth and those acquiring this ability after eight months of age. In type III cases, being able versus unable to climb stairs was associated with a significant difference (p=0.02) in the median time until loss of walking independently. Positive correlations were also seen between copy numbers and the clinical severity of SMA., Conclusion: Our long-term results show peak motor milestone evaluations distinguishing between subtypes to be useful not only as outcome measures for assessing treatment efficacy in clinical trials but also for predicting the clinical courses of Japanese SMA patients., (Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

B-cell CLL/lymphoma 6 (BCL6) exerts oncogenic effects in several human hematopoietic malignancies including chronic myeloid leukemia (CML), where BCL6 expression was shown to be essential for CML stem cell survival and self-renewal during imatinib mesylate (IM) treatment. As several lines of evidence suggest that interferon γ (IFNγ) production in CML patients might have a central role in the response to tyrosine kinase inhibitor (TKI) therapy, we analyzed if IFNγ modulates BCL6 expression in CML cells. Although separate IFNγ or IM treatment only slightly upregulated BCL6 expression, combined treatment induced remarkable BCL6 upregulation in CML lines and primary human CD34+ CML stem cells. We proved that during combined treatment, inhibition of constitutive signal transducer and activator of transcription (STAT) 5 activation by IM allowed the specific enhancement of the STAT1 dependent, direct upregulation of BCL6 by IFNγ in CML cells. By using colony-forming assay, we found that IFNγ enhanced the ex vivo colony or cluster-forming capacity of human CML stem cells in the absence or presence of IM, respectively. Furthermore, inhibition of the transcriptional repressor function of BCL6 in the presence of IM and IFNγ almost completely blocked the cluster formation of human CML stem cells. On the other hand, by using small interfering RNA knockdown of BCL6, we demonstrated that in an IM-treated CML line the antiapoptotic effect of IFNγ was independent of BCL6 upregulation. We found that IFNγ also upregulated several antiapoptotic members of the BCL2 and BIRC gene families in CML cells, including the long isoform of MCL1, which proved to be essential for the antiapoptotic effect of IFNγ in an IM-treated CML line. Our results suggest that combination of TKIs with BCL6 and MCL1 inhibitors may potentially lead to the complete eradication of CML stem cells.

While NLRC4-dependent sensing of intracellular Gram-negative pathogens such as Salmonella enterica serovar typhimurium is a beneficial host response, NLRC4-dependent sensing of the Pseudomonas aeruginosa type 3 secretion system (T3SS) has been shown to be involved in pathogenicity. In mice, different pathogen-associated microbial patterns are sensed by the combination of the NLRC4-inflammasome with different neuronal apoptosis inhibitory proteins (NAIPs). NAIP2 is involved in sensing PscI, an inner-rod protein of the P. aeruginosa T3SS. Surprisingly, only a single human NAIP (hNAIP) has been found. Moreover, there is no description of hNAIP-NLRC4 inflammasome recognition of T3SS inner-rod proteins in humans. Here, we show that the P. aeruginosa T3SS inner-rod protein PscI and needle protein PscF are both sensed by the hNAIP-NLRC4 inflammasome in human macrophages and PBMCs from healthy donors, allowing caspase-1 and IL-1β maturation and resulting in a robust inflammatory response. TLR4 and TLR2 are involved in redundantly sensing these two T3SS components., (© The Japanese Society for Immunology. 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

To remove dying or unwanted cells from an epithelium while preserving the barrier function of the layer, epithelia use a unique process called cell extrusion. To extrude, the cell fated to die emits the lipid Sphingosine 1 Phosphate (S1P), which binds the G-protein-coupled receptor Sphingosine 1 Phosphate receptor 2 (S1P 2 ) in the neighboring cells that activates Rho-mediated contraction of an actomyosin ring circumferentially and basally. This contraction acts to squeeze the cell out apically while drawing together neighboring cells and preventing any gaps to the epithelial barrier. Epithelia can extrude out cells targeted to die by apoptotic stimuli to repair the barrier in the face of death or extrude live cells to promote cell death when epithelial cells become too crowded. Indeed, because epithelial cells naturally turn over by cell death and division at some of the highest rates in the body, epithelia depend on crowding-induced live cell extrusion to preserve constant cell numbers. If extrusion is defective, epithelial cells rapidly lose contact inhibition and form masses. Additionally, because epithelia act as the first line of defense in innate immunity, preservation of this barrier is critical for preventing pathogens from invading the body. Given its role in controlling constant cell numbers and maintaining barrier function, a number of different pathologies can result when extrusion is disrupted. Here, we review mechanisms and signaling pathways that control epithelial extrusion and discuss how defects in these mechanisms can lead to multiple diseases. We also discuss tactics pathogens have devised to hijack the extrusion process to infect and colonize epithelia., (Published by Elsevier Ltd.)

Intestinal epithelial cells (IECs) form a critical barrier against pathogen invasion. By generation of mice in which inflammasome expression is restricted to IECs, we describe a coordinated epithelium-intrinsic inflammasome response in vivo. This response was sufficient to protect against Salmonella tissue invasion and involved a previously reported IEC expulsion that was coordinated with lipid mediator and cytokine production and lytic IEC death. Excessive inflammasome activation in IECs was sufficient to result in diarrhea and pathology. Experiments with IEC organoids demonstrated that IEC expulsion did not require other cell types. IEC expulsion was accompanied by a major actin rearrangement in neighboring cells that maintained epithelium integrity but did not absolutely require Caspase-1 or Gasdermin D. Analysis of Casp1 -/- Casp8 -/- mice revealed a functional Caspase-8 inflammasome in vivo. Thus, a coordinated IEC-intrinsic, Caspase-1 and -8 inflammasome response plays a key role in intestinal immune defense and pathology., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Benign prostatic hyperplasia (BPH) treatment includes the apoptosis machinery modulation through the direct inhibition of caspase cascade. We previously demonstrated that Serenoa repens (Ser) with lycopene (Ly) and selenium (Se) reawakened apoptosis by reducing survivin and neuronal apoptosis inhibitory protein (NAIP) levels in rats. The aim of this study was to evaluate the effectiveness of Ser-Se-Ly association on survivin and NAIP expression in BPH patients. Ninety patients with lower urinary tract symptoms (LUTS) due to clinical BPH were included in this randomized, double-blind, placebo-controlled trial. Participants were randomly assigned to receive placebo (Group BPH + placebo, n = 45) or Ser-Se-Ly association (Group BPH + Ser-Se-Ly; n = 45) for 3 months. At time 0, all patients underwent prostatic biopsies. After 3 months of treatment, they underwent prostatic re-biopsy and specimens were collected for molecular, morphological, and immunohistochemical analysis. After 3 months, survivin and NAIP were significantly decreased, while caspase-3 was significantly increased in BPH patients treated with Ser-Se-Ly when compared with the other group. In BPH patients treated with Ser-Se-Ly for 3 months, the glandular epithelium was formed by a single layer of cuboidal cells. PSA showed high immunoexpression in all BPH patients and a focal positivity in Ser-Se-Ly treated patients after 3 months. Evident prostate specific membrane antigen (PSMA) immunoexpression was shown in all BPH patients, while no positivity was present after Ser-Se-Ly administration. Ser-Se-Ly proved to be effective in promoting apoptosis in BPH patients.

The neuronal apoptosis inhibitory protein (NAIP) is a constituent of the inflammasome and a key component of the innate immune system. Here we use immunofluorescence to position NAIP within the cytokinetic apparatus, contiguous to chromosomal passenger complex (CPC), Centralspindlin, PRC1 and KIF4A. During metaphase, NAIP accumulates in the mitotic spindle poles and is shown in spindle microtubules; in anaphase NAIP is detected in the middle of the central spindle. At the end of cytokinesis, NAIP is localized in the outlying region of the stem body, the center of the intercellular bridge formed between daughter cells prior to cellular abscission. We also describe the sustained presence of NAIP mRNA and protein throughout the cell cycle with a significant increase observed in the G2/M phase. Consistent with a role for NAIP in cytokinesis, NAIP overexpression in HeLa cells promotes the acquisition of a multinuclear phenotype. Conversely, NAIP siRNA gene silencing results in an apoptotic lethal phenotype. Our confocal and super resolution stimulated-emission-depletion (STED) examination of mammalian cell cytokinesis demonstrate a potential new role for NAIP in addition to anti-apoptotic and innate immunology functions.

Background: Innate immune genes tend to be highly conserved in metazoans, even in early divergent lineages such as Cnidaria (jellyfish, corals, hydroids and sea anemones) and Porifera (sponges). However, constant and diverse selection pressures on the immune system have driven the expansion and diversification of different immune gene families in a lineage-specific manner. To investigate how the innate immune system has evolved in a subset of sea anemone species (Order: Actiniaria), we performed a comprehensive and comparative study using 10 newly sequenced transcriptomes, as well as three publically available transcriptomes, to identify the origins, expansions and contractions of candidate and novel immune gene families., Results: We characterised five conserved genes and gene families, as well as multiple novel innate immune genes, including the newly recognised putative pattern recognition receptor CniFL. Single copies of TLR, MyD88 and NF-κB were found in most species, and several copies of IL-1R-like, NLR and CniFL were found in almost all species. Multiple novel immune genes were identified with domain architectures including the Toll/interleukin-1 receptor (TIR) homology domain, which is well documented as functioning in protein-protein interactions and signal transduction in immune pathways. We hypothesise that these genes may interact as novel proteins in immune pathways of cnidarian species. Novelty in the actiniarian immunome is not restricted to only TIR-domain-containing proteins, as we identify a subset of NLRs which have undergone neofunctionalisation and contain 3-5 N-terminal transmembrane domains, which have so far only been identified in two anthozoan species., Conclusions: This research has significance in understanding the evolution and origin of the core eumetazoan gene set, including how novel innate immune genes evolve. For example, the evolution of transmembrane domain containing NLRs indicates that these NLRs may be membrane-bound, while all other metazoan and plant NLRs are exclusively cytosolic receptors. This is one example of how species without an adaptive immune system may evolve innovative solutions to detect pathogens or interact with native microbiota. Overall, these results provide an insight into the evolution of the innate immune system, and show that early divergent lineages, such as actiniarians, have a diverse repertoire of conserved and novel innate immune genes.

NLRs (nucleotide-binding domain, leucine-rich repeat containing receptors) are pattern recognition receptors associated with immunity and inflammation in response to endogenous and exogenous pathogen and damage associated molecular patterns (PAMPs and DAMPs respectively). Dysregulated NLR function is associated with several diseases including cancers, metabolic diseases, autoimmune disorders and autoinflammatory syndromes. In the last decade, distinct cell and organ specific roles for NLRs have been identified however; their roles in cancer initiation, development and progression remain controversial. This review summarizes the emerging role of NLRs in cancer and their possible future as targets for cancer therapeutics.

Flagellin has the capacity to activate both Toll-like receptor 5 (TLR5) and Nod-like receptor C4 (NLRC4)/neuronal apoptosis inhibitory protein 5 (NAIP5) inflammasome signaling. We fused E7m (the inactivated E7 of human papillomavirus) to either end of the flagellin protein, and the resulting recombinant flagellin-E7m proteins (rFliCE7m and rE7mFliC) were used as immunogens. Both fusion proteins activated receptor signaling to different degrees. rE7mFliC-induced TLR5 activity was 10-fold higher than that of rFliCE7m, whereas rFliCE7m activated the NLRC4/NAIP5 pathway more strongly. Therefore, these recombinant proteins provided a tool to investigate which signaling pathway is critical for the induction of antigen-specific T cell responses and anti-tumor immunity. We demonstrated that rFliCE7m induced higher levels of E7-specific IFN-gamma-secreting cells and cytotoxic T lymphocytes (CTLs) than rE7mFliC, and a single injection with rFliCE7m but not rE7mFliC inhibited E7-expressing tumor growth in vivo. Furthermore, we confirmed that CD8(+) T cells played a major role in the anti-tumor immunity induced by rFliCE7m. These findings suggested that the NLRC4/NAIP5 intracellular signaling pathway was critical for the induction of anti-tumor immunity. These observations provide important information for the rational design of flagellin-based immunotherapy.

Previous studies have indicated that interleukin (IL)‑1β has an important role in the development of allergic diseases. Therefore, the present study aimed to investigate the upstream pathway underlying IL‑1β production in an experimental model of allergy. BALB/c mice (female, 6‑8 weeks old) were sensitized to recombinant (r)Che a 2 by intraperitoneal injection of rChe a 2 adsorbed onto an alum gel suspension on days 0, 7, 14 and 21. In the control group, mice received an injection of 20 mM phosphate‑buffered saline absorbed onto alum via the same route. The allergic status of the mice was confirmed serologically by measuring allergen‑specific immunoglobulin (Ig)E levels. The protein expression levels of IL‑1β and the mRNA expression levels of inflammasome compartments were measured by enzyme‑linked immunosorbent assay and semi‑quantitative reverse transcription polymerase chain reaction, respectively. In addition, caspase‑1 activity was determined by fluorometric assay. Sensitized mice exhibited significantly increased levels of specific IgE (P<0.05). IL‑1β production and caspase‑1 activity were significantly higher in the sensitized mice compared with the control group. In addition, no significant differences were observed between the control and sensitized mice in the expression of genes associated with the inflammasome, including NLR family, pyrin domain containing 3; apoptosis‑associated speck‑like protein; and NLR family, apoptosis inhibitory protein 5. However, IL‑1β converting enzyme protease‑activating factor (IPAF) expression was significantly increased in sensitized mice compared with in the control group (P<0.05). These data indicate that caspase‑1 activation and IL‑1β expression are associated with the IPAF inflammasome. Therefore, based on this association, the IPAF inflammasome may be considered for IL‑1β production in the experimental model of allergy.