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From studies of individual families to global collaborative efforts, the NLRP3 inflammasome is now recognized to be a key regulator of innate immunity. Activated by a panoply of pathogen-associated and endogenous triggers, NLRP3 serves as an intracellular sensor that drives carefully coordinated assembly of the inflammasome, and downstream inflammation mediated by IL-1 and IL-18. Initially discovered as the cause of the autoinflammatory spectrum of cryopyrin-associated periodic syndrome (CAPS), NLRP3 is now also known to play a role in more common diseases including cardiovascular disease, gout, and liver disease. We have seen cohesion in results from clinical studies in CAPS patients, ex vivo studies of human cells and murine cells, and in vivo murine models leading to our understanding of the downstream pathways, cytokine secretion, and cell death pathways that has solidified the role of autoinflammation in the pathogenesis of human disease. Recent advances in our understanding of the structure of the inflammasome have provided ways for us to visualize normal and mutant protein function and pharmacologic inhibition. The subsequent development of targeted therapies successfully used in the treatment of patients with CAPS completes the bench to bedside translational loop which has defined the study of this unique protein.

Background & aimsBreast regression protein 39 (BRP39) (Chi3L1) and its human homolog YKL-40, is an established biomarker of liver fibrosis in nonalcoholic steatohepatitis (NASH) patients, but its role in NASH pathogenesis remains unclear. We recently identified Chi3L1 as one of the top up-regulated genes in mice with inducible gain-of-function NOD-like receptor protein 3 (NLRP3) activation that mimics several liver features of NASH. This study aimed to investigate the effects of BRP39 deficiency on NLRP3-induced liver inflammation using tamoxifen-inducible Nlrp3 knockin mice sufficient (Nlrp3A350V CRT) and deficient for BRP39 (Nlrp3A350V/BRP-/- CRT).MethodsUsing Nlrp3A350V CRT mice and Nlrp3A350V BRP-/- CRT, we investigated the consequences of BRP39 deficiency influencing NLRP3-induced liver inflammation.ResultsOur results showed that BRP39 deficiency in NLRP3-induced inflammation improved body weight and liver weight. Moreover, liver inflammation, fibrosis, and hepatic stellate cell activation were reduced significantly, corresponding to significantly decreased Ly6C+ infiltrating macrophages, CD68+ osteopontin-positive hepatic lipid-associated macrophages, and activated Lymphocyte antigen 6 complex locus G6D positive (Ly6G+) and citrullinated histone H3 postivie (H3Cit+) neutrophil accumulation in the liver. Further investigation showed that circulatory neutrophils from NLRP3-induced BRP39-deficient mice have impaired chemotaxis and migration ability, and this was confirmed by RNA bulk sequencing showing reduced immune activation, migration, and signaling responses in neutrophils.ConclusionsThese data showcase the importance of BRP39 in regulating the NLRP3 inflammasome during liver inflammation and fibrotic NASH by altering cellular activation, recruitment, and infiltration during disease progression, and revealing BRP39 to be a potential therapeutic target for future treatment of inflammatory NASH and its associated diseases.

Background and aimsNucleotide-binding oligomerization domain-like receptor-family pyrin domain-containing 3 (NLRP3) inflammasome activation has been shown to result in liver fibrosis. Mechanisms and downstream signaling remain incompletely understood. Here, we studied the role of IL-18 in hepatic stellate cells (HSCs), and its impact on liver fibrosis.Approach and resultsWe observed significantly increased serum levels of IL-18 (128.4 pg/ml vs. 74.9 pg/ml) and IL-18 binding protein (BP; 46.50 ng/ml vs. 15.35 ng/ml) in patients with liver cirrhosis compared with healthy controls. Single cell RNA sequencing data showed that an immunoregulatory subset of murine HSCs highly expresses Il18 and Il18r1 . Treatment of cultured primary murine HSC with recombinant mouse IL-18 accelerated their transdifferentiation into myofibroblasts. In vivo , IL-18 receptor-deficient mice had reduced liver fibrosis in a model of fibrosis induced by HSC-specific NLRP3 overactivation. Whole liver RNA sequencing analysis from a murine model of severe NASH-induced fibrosis by feeding a choline-deficient, L-amino acid-defined, high fat diet showed that genes related to IL-18 and its downstream signaling were significantly upregulated, and Il18-/- mice receiving this diet for 10 weeks showed protection from fibrotic changes with decreased number of alpha smooth muscle actin-positive cells and collagen deposition. HSC activation triggered by NLRP3 inflammasome activation was abrogated when IL-18 signaling was blocked by its naturally occurring antagonist IL-18BP. Accordingly, we observed that the severe inflammatory phenotype associated with myeloid cell-specific NLRP3 gain-of-function was rescued by IL-18BP.ConclusionsOur study highlights the role of IL-18 in the development of liver fibrosis by its direct effect on HSC activation identifying IL-18 as a target to treat liver fibrosis.

Although Kawasaki disease (KD) and multisystem inflammatory syndrome in children (MIS-C) share some clinical manifestations, their cardiovascular outcomes are different, and this may be reflected at the level of the endothelial cell (EC). We performed RNA-seq on cultured ECs incubated with pre-treatment sera from KD (n = 5), MIS-C (n = 7), and healthy controls (n = 3). We conducted a weighted gene co-expression network analysis (WGCNA) using 935 transcripts differentially expressed between MIS-C and KD using relaxed filtering (unadjusted p < 0.05, >1.1-fold difference). We found seven gene modules in MIS-C, annotated as an increased TNFα/NFκB pathway, decreased EC homeostasis, anti-inflammation and immune response, translation, and glucocorticoid responsive genes and endothelial-mesenchymal transition (EndoMT). To further understand the difference in the EC response between MIS-C and KD, stringent filtering was applied to identify 41 differentially expressed genes (DEGs) between MIS-C and KD (adjusted p < 0.05, >2-fold-difference). Again, in MIS-C, NFκB pathway genes, including nine pro-survival genes, were upregulated. The expression levels were higher in the genes influencing autophagy (UBD, EBI3, and SQSTM1). Other DEGs also supported the finding by WGCNA. Compared to KD, ECs in MIS-C had increased pro-survival transcripts but reduced transcripts related to EndoMT and EC homeostasis. These differences in the EC response may influence the different cardiovascular outcomes in these two diseases.

The NLRP3 inflammasome is a key mediator of the innate immune response to sterile tissue injury and is involved in many chronic and acute diseases. Physically and chemically diverse agents activate the NLRP3 inflammasome. Here, we show that NLRP3 binds non-oxidized and Ox-mtDNA differentially, with a half maximum inhibitory concentration (IC50) for non-oxidized and Ox-mtDNA of 4 nM and 247.2 nM, respectively. The NLRP3 Neonatal-Onset Multisystem Inflammatory Disease (NOMIDFCAS) gain of function mutant could bind non-oxidized mtDNA but had higher affinity for Ox-mtDNA compared to WT with an IC50 of 8.1 nM. NLRP3 lacking the pyrin domain can bind both oxidized and non-oxidized mtDNA. Isolated pyrin domain prefers Ox-mtDNA. The NLRP3 pyrin domain shares a protein fold with DNA glycosylases and generate a model for DNA binding based on the structure and sequence alignment to Clostridium acetobutylicum and human OGG1, an inhibitor of Ox-mtDNA generation, 8-oxoguanine DNA glycosylases. We provide a new model for how NLRP3 interacts with Ox-mtDNA supported by DNA binding in the presence of a monoclonal antibody against the pyrin domain. These results give new insights into the mechanism of inflammasome assembly, and into the function of reactive oxygen species in establishing a robust immune response.

BackgroundIndividuals with certain chronic inflammatory lung diseases have a higher risk of developing lung cancer (LC). However, the underlying mechanisms remain largely unknown. Here, we hypothesized that chronic exposure to house dust mites (HDM), a common indoor aeroallergen associated with the development of asthma, accelerates LC development through the induction of chronic lung inflammation (CLI).  METHODS: The effects of HDM and heat-inactivated HDM (HI-HDM) extracts were evaluated in two preclinical mouse models of LC (a chemically-induced model using the carcinogen urethane and a genetically-driven model with oncogenic KrasG12D activation in lung epithelial cells) and on murine macrophages in vitro. Pharmacological blockade or genetic deletion of the Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, caspase-1, interleukin-1β (IL-1β), and C-C motif chemokine ligand 2 (CCL2) or treatment with an inhaled corticosteroid (ICS) was used to uncover the pro-tumorigenic effect of HDM.  RESULTS: Chronic intranasal (i.n) instillation of HDM accelerated LC development in the two mouse models. Mechanistically, HDM caused a particular subtype of CLI, in which the NLRP3/IL-1β signaling pathway is chronically activated in macrophages, and made the lung microenvironment conducive to tumor development. The tumor-promoting effect of HDM was significantly decreased by heat treatment of the HDM extract and was inhibited by NLRP3, IL-1β, and CCL2 neutralization, or ICS treatment.ConclusionsCollectively, these data indicate that long-term exposure to HDM can accelerate lung tumorigenesis in susceptible hosts (e.g., mice and potentially humans exposed to lung carcinogens or genetically predisposed to develop LC).

Mitochondrial DNA (mtDNA) escaping stressed mitochondria provokes inflammation via cGAS-STING pathway activation and, when oxidized (Ox-mtDNA), it binds cytosolic NLRP3, thereby triggering inflammasome activation. However, it is unknown how and in which form Ox-mtDNA exits stressed mitochondria in non-apoptotic macrophages. We found that diverse NLRP3 inflammasome activators rapidly stimulated uniporter-mediated calcium uptake to open mitochondrial permeability transition pores (mPTP) and trigger VDAC oligomerization. This occurred independently of mtDNA or reactive oxygen species, which induce Ox-mtDNA generation. Within mitochondria, Ox-mtDNA was either repaired by DNA glycosylase OGG1 or cleaved by the endonuclease FEN1 to 500-650 bp fragments that exited mitochondria via mPTP- and VDAC-dependent channels to initiate cytosolic NLRP3 inflammasome activation. Ox-mtDNA fragments also activated cGAS-STING signaling and gave rise to pro-inflammatory extracellular DNA. Understanding this process will advance the development of potential treatments for chronic inflammatory diseases, exemplified by FEN1 inhibitors that suppressed interleukin-1β (IL-1β) production and mtDNA release in mice.

Background Damage to the coronary arteries during the acute phase of Kawasaki disease (KD) is linked to inflammatory cell infiltration, myointimal proliferation, and endothelial cell (EC) dysfunction. To understand the response of ECs to KD treatment, we studied the genome-wide transcriptional changes in cultured ECs incubated with KD sera before and after treatment with or without atorvastatin. Methods and Results RNA sequencing of human umbilical vein ECs incubated with pooled sera from patients with acute KD before or after treatment with intravenous immunoglobulin and infliximab revealed differentially expressed genes in interleukin-1, tumor necrosis factor-α, and inflammatory cell recruitment pathways. Subacute sera pooled from patients treated with intravenous immunoglobulin, infliximab, and atorvastatin uniquely induced expression of NOS3, Kruppel like factor (KLF2, and KLF4 (promotes EC homeostasis and angiogenesis) and ZFP36 ring finger protein (ZFP36) and suppressor of cytokine signaling 3 (SOCS3) (suppresses inflammation), and suppressed expression of TGFB2 and DKK1 (induces endothelial-mesenchymal transition) and sphingosine kinase 1 (SPHK1) and C-X-C motif chemokine ligand 8 (CXCL8) (induces inflammation). Conclusions These results suggest that atorvastatin treatment of patients with acute KD may improve EC health, reduce mediators of inflammation produced by ECs, and block KD-induced myofibroblast proliferation.

Background & aimsNonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. The NLRP3 inflammasome, a platform for caspase-1 activation and release of interleukin 1β, is increasingly recognized in the induction of inflammation and liver fibrosis during NAFLD. However, the cell-specific contribution of NLRP3 inflammasome activation in NAFLD remains unknown.MethodsTo investigate the role of NLRP3 inflammasome activation in hepatocytes, hepatic stellate cells (HSCs) and myeloid cells, a conditional Nlrp3 knock-out mouse was generated and bred to cell-specific Cre mice. Both acute and chronic liver injury models were used: lipopolysaccharide/adenosine-triphosphate to induce in vivo NLRP3 activation, choline-deficient, L-amino acid-defined high-fat diet, and Western-type diet to induce fibrotic nonalcoholic steatohepatitis (NASH). In vitro co-culture studies were performed to dissect the crosstalk between myeloid cells and HSCs.ResultsMyeloid-specific deletion of Nlrp3 blunted the systemic and hepatic increase in interleukin 1β induced by lipopolysaccharide/adenosine-triphosphate injection. In the choline-deficient, L-amino acid-defined high-fat diet model of fibrotic NASH, myeloid-specific Nlrp3 knock-out but not hepatocyte- or HSC-specific knock-out mice showed significant reduction in inflammation independent of steatosis development. Moreover, myeloid-specific Nlrp3 knock-out mice showed ameliorated liver fibrosis and decreased HSC activation. These results were validated in the Western-type diet model. In vitro co-cultured studies with human cell lines demonstrated that HSC can be activated by inflammasome stimulation in monocytes, and this effect was significantly reduced if NLRP3 was downregulated in monocytes.ConclusionsThe study provides new insights in the cell-specific role of NLRP3 in liver inflammation and fibrosis. NLRP3 inflammasome activation in myeloid cells was identified as crucial for the progression of NAFLD to fibrotic NASH. These results may have implications for the development of cell-specific strategies for modulation of NLRP3 activation for treatment of fibrotic NASH.

BACKGROUNDMultisystem inflammatory syndrome in children (MIS-C) is a rare but potentially severe illness that follows exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Kawasaki disease (KD) shares several clinical features with MIS-C, which prompted the use of intravenous immunoglobulin (IVIG), a mainstay therapy for KD. Both diseases share a robust activation of the innate immune system, including the IL-1 signaling pathway, and IL-1 blockade has been used for the treatment of both MIS-C and KD. The mechanism of action of IVIG in these 2 diseases and the cellular source of IL-1β have not been defined.METHODSThe effects of IVIG on peripheral blood leukocyte populations from patients with MIS-C and KD were examined using flow cytometry and mass cytometry (CyTOF) and live-cell imaging.RESULTSCirculating neutrophils were highly activated in patients with KD and MIS-C and were a major source of IL-1β. Following IVIG treatment, activated IL-1β+ neutrophils were reduced in the circulation. In vitro, IVIG was a potent activator of neutrophil cell death via PI3K and NADPH oxidase, but independently of caspase activation.CONCLUSIONSActivated neutrophils expressing IL-1β can be targeted by IVIG, supporting its use in both KD and MIS-C to ameliorate inflammation.FUNDINGPatient Centered Outcomes Research Institute; NIH; American Asthma Foundation; American Heart Association; Novo Nordisk Foundation; NIGMS; American Academy of Allergy, Asthma and Immunology Foundation.

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Background & aimsIncreased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is unknown.MethodsWe used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3KICreA mice, and GsdmdKO mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia).ResultsWe demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3KICreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1β secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B.ConclusionsThese results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development.Lay summaryOur findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.

Autoinflammatory diseases are monogenic and polygenic disorders due to dysregulation of the innate immune system. The inherited conditions have been clustered with primary immunodeficiencies in the latest practice parameters; however, these diseases have unique clinical presentations, genetics, and available therapies. Given the presentation of fevers, rashes, and mucosal symptoms observed in many of these syndromes, patients are likely to present to an allergist/immunologist. Although there has been attention in the literature to diagnosis and treatment of rare, genetically defined autoinflammatory disorders, physicians are challenged by increasing numbers of patients with intermittent or periodic fevers who face unnecessary morbidities due to a lack of a diagnosis. The broad differential of diseases presenting with fever includes autoinflammatory syndromes, infections associated with immunodeficiency and/or allergies complicated by infection, and less commonly, autoimmune disorders or malignancy. To address this challenge, we review the history of the medical approach to fever, current diagnostic paradigms, and controversies in management. We describe the spectrum of disorders referred to a recurrent fever disorders clinic established in an Allergy/Immunology division at a tertiary pediatric care center. Finally, we provide practical recommendations including historical features and initial laboratory investigations that can help clinicians appropriately manage these patients.

B cell development is a highly regulated process involving multiple differentiation steps, yet many details regarding this pathway remain unknown. Sequencing of patients with B cell-restricted immunodeficiency reveals autosomal dominant mutations in TOP2B. TOP2B encodes a type II topoisomerase, an essential gene required to alleviate topological stress during DNA replication and gene transcription, with no previously known role in B cell development. We use Saccharomyces cerevisiae, and knockin and knockout murine models, to demonstrate that patient mutations in TOP2B have a dominant negative effect on enzyme function, resulting in defective proliferation, survival of B-2 cells, causing a block in B cell development, and impair humoral function in response to immunization.

Leucine-rich repeat (LRR) domains are evolutionarily conserved in proteins that function in development and immunity. Here we report strict exonic modularity of LRR domains of several human gene families, which is a precondition for alternative splicing (AS). We provide evidence for AS of LRR domain within several Nod-like receptors, most prominently the inflammasome sensor NLRP3. Human NLRP3, but not mouse NLRP3, is expressed as two major isoforms, the full-length variant and a variant lacking exon 5. Moreover, NLRP3 AS is stochastically regulated, with NLRP3 ∆ exon 5 lacking the interaction surface for NEK7 and hence loss of activity. Our data thus reveals unexpected regulatory roles of AS through differential utilization of LRRs modules in vertebrate innate immunity.

Jacobsen syndrome (OMIM #147791) is a rare contiguous gene disorder caused by deletions in distal 11q. The clinical phenotype is variable and can include dysmorphic features, varying degrees of intellectual disability, behavioral problems including autism and attention deficit hyperactivity disorder, congenital heart defects, structural kidney defects, genitourinary problems, immunodeficiency, and a bleeding disorder due to impaired platelet production and function. Previous studies combining both human and animal systems have implicated several disease-causing genes in distal 11q that contribute to the Jacobsen syndrome phenotype. One gene, ETS1, has been implicated in causing congenital heart defects, structural kidney defects, and immunodeficiency. We performed a comprehensive phenotypic analysis on a patient with congenital heart disease previously found to have a de novo frameshift mutation in ETS1, resulting in the loss of the DNA-binding domain of the protein. Our results suggest that loss of Ets1 causes a "partial Jacobsen syndrome phenotype" including congenital heart disease, facial dysmorphism, intellectual disability, and attention deficit hyperactivity disorder.

Choline is a vitamin-like nutrient that is taken up via specific transporters and metabolized by choline kinase, which converts it to phosphocholine needed for de novo synthesis of phosphatidylcholine (PC), the main phospholipid of cellular membranes. We found that Toll-like receptor (TLR) activation enhances choline uptake by macrophages and microglia through induction of the choline transporter CTL1. Inhibition of CTL1 expression or choline phosphorylation attenuated NLRP3 inflammasome activation and IL-1β and IL-18 production in stimulated macrophages. Mechanistically, reduced choline uptake altered mitochondrial lipid profile, attenuated mitochondrial ATP synthesis, and activated the energy sensor AMP-activated protein kinase (AMPK). By potentiating mitochondrial recruitment of DRP1, AMPK stimulates mitophagy, which contributes to termination of NLRP3 inflammasome activation. Correspondingly, choline kinase inhibitors ameliorated acute and chronic models of IL-1β-dependent inflammation.

Distinct macrophage subsets populate the developing embryo and fetus in distinct waves. However little is known about the functional differences between in utero macrophage populations or how they might contribute to fetal and neonatal immunity. Here we tested the innate immune response of mouse macrophages derived from the embryonic yolk sac and from fetal liver. When isolated from liver or lung, CD11bHI fetal liver derived macrophages responded to the TLR4 agonist LPS by expressing and releasing inflammatory cytokines. However F4/80HI macrophages from the yolk sac did not respond to LPS treatment. While differences in TLR4 expression did not appear to explain these data, F4/80HI macrophages had much lower NLRP3 inflammasome expression compared to CD11bHI macrophages. Gene expression profiling also demonstrated LPS-induced expression of inflammatory genes in CD11bHI macrophages, but not in F4/80HI cells. Genes expressed in LPS-treated CD11bHI macrophages were more likely to contain predicted NF-κB binding sites in their promoter regions. Our data show that CD11bHI macrophages derived from fetal liver are the major pro-inflammatory cells in the developing fetus. These findings could have important implications in better understanding the fetal inflammatory response and the unique features of neonatal immunity.

Inflammation accompanies heart failure and is a mediator of cardiac fibrosis. CaMKIIδ plays an essential role in adverse remodeling and decompensation to heart failure. We postulated that inflammation is the mechanism by which CaMKIIδ contributes to adverse remodeling in response to nonischemic interventions. We demonstrate that deletion of CaMKIIδ in the cardiomyocyte (CKO) significantly attenuates activation of NF-κB, expression of inflammatory chemokines and cytokines, and macrophage accumulation induced by angiotensin II (Ang II) infusion. The inflammasome was activated by Ang II, and this response was also diminished in CKO mice. These events occurred prior to any evidence of Ang II-induced cell death. In addition, CaMKII-dependent inflammatory gene expression and inflammasome priming were observed as early as the third hour of infusion, a time point at which macrophage recruitment was not evident. Inhibition of either the inflammasome or monocyte chemoattractant protein 1 (MCP1) signaling attenuated macrophage accumulation, and these interventions, like cardiomyocyte CaMKIIδ deletion, diminished the fibrotic response to Ang II. Thus, activation of CaMKIIδ in the cardiomyocyte represents what we believe to be a novel mechanism for initiating inflammasome activation and an inflammatory gene program that leads to macrophage recruitment and ultimately to development of fibrosis.

Group A Streptococcus (GAS) is among the top ten causes of infection-related mortality in humans. M protein is the most abundant GAS surface protein, and M1 serotype GAS strains are associated with invasive infections, including necrotizing fasciitis and toxic shock syndrome. Here, we report that released, soluble M1 protein triggers programmed cell death in macrophages (Mϕ). M1 served as a second signal for caspase-1-dependent NLRP3 inflammasome activation, inducing maturation and release of proinflammatory cytokine interleukin-1β (IL-1β) and macrophage pyroptosis. The structurally dynamic B-repeat domain of M1 was critical for inflammasome activation, which involved K+ efflux and M1 protein internalization by clathrin-mediated endocytosis. Mouse intraperitoneal challenge showed that soluble M1 was sufficient and specific for IL-1β activation, which may represent an early warning to activate host immunity against the pathogen. Conversely, in systemic infection, hyperinflammation associated with M1-mediated pyroptosis and IL-1β release could aggravate tissue injury.

In this study, we used cre-lox techniques to generate mice selectively deficient in ORMDL3 in airway epithelium (Ormdl3Δ2-3/Δ2-3/CC10) to simulate an inhaled therapy that effectively inhibited ORMDL3 expression in the airway. In contrast to the anticipated reduction in airway hyperresponsiveness (AHR), OVA allergen-challenged Ormdl3Δ2-3/Δ2-3/CC10 mice had a significant increase in AHR compared with wild-type mice. Levels of airway inflammation, mucus, fibrosis, and airway smooth muscle were no different in Ormdl3Δ2-3/Δ2-3/CC10 and wild-type mice. However, levels of sphingosine-1-phosphate (S1P) were significantly increased in Ormdl3Δ2-3/Δ2-3/CC10 mice as well as in airway epithelial cells in which ORMDL3 was inhibited with small interfering RNA. Incubation of S1P with airway smooth muscle cells significantly increased contractility. Overall, Ormdl3Δ2-3/Δ2-3/CC10 mice exhibit increased allergen-induced AHR independent of inflammation and associated with increased S1P generation. These studies raise concerns for inhaled therapies that selectively and effectively inhibit ORMDL3 in airway epithelium in asthma.

Gasdermin B (GSDMB) on chromosome 17q21 demonstrates a strong genetic linkage to asthma, but its function in asthma is unknown. Here we identified that GSDMB is highly expressed in lung bronchial epithelium in human asthma. Overexpression of GSDMB in primary human bronchial epithelium increased expression of genes important to both airway remodeling [TGF-β1, 5-lipoxygenase (5-LO)] and airway-hyperresponsiveness (AHR) (5-LO). Interestingly, hGSDMBZp3-Cre mice expressing increased levels of the human GSDMB transgene showed a significant spontaneous increase in AHR and a significant spontaneous increase in airway remodeling, with increased smooth muscle mass and increased fibrosis in the absence of airway inflammation. In addition, hGSDMBZp3-Cre mice showed increases in the same remodeling and AHR mediators (TGF-β1, 5-LO) observed in vitro in GSDMB-overexpressing epithelial cells. GSDMB induces TGF-β1 expression via induction of 5-LO, because knockdown of 5-LO in epithelial cells overexpressing GSDMB inhibited TGF-β1 expression. These studies demonstrate that GSDMB, a gene highly linked to asthma but whose function in asthma is previously unknown, regulates AHR and airway remodeling without airway inflammation through a previously unrecognized pathway in which GSDMB induces 5-LO to induce TGF-β1 in bronchial epithelium.

Interleukin-1β (IL-1β) is a key proinflammatory cytokine that drives antimicrobial immune responses. IL-1β is aberrantly activated in autoimmune diseases, and IL-1β inhibitors are used as therapeutic agents to treat patients with certain autoimmune disorders. Review of postmarketing surveillance of patients receiving IL-1β inhibitors found a disproportionate reporting of invasive infections by group A Streptococcus (GAS). IL-1β inhibition increased mouse susceptibility to GAS infection, but IL-1β was produced independent of canonical inflammasomes. Newly synthesized IL-1β has an amino-terminal prodomain that blocks signaling activity, which is usually proteolytically removed by caspase-1, a protease activated within the inflammasome structure. In place of host caspases, the secreted GAS cysteine protease SpeB generated mature IL-1β. During invasive infection, GAS isolates may acquire pathoadaptive mutations eliminating SpeB expression to evade detection by IL-1β. Pharmacological IL-1β inhibition alleviates this selective pressure, allowing invasive infection by nonpathoadapted GAS. Thus, IL-1β is a sensor that directly detects pathogen-associated proteolysis through an independent pathway operating in parallel with host inflammasomes. Because IL-1β function is maintained across species, yet cleavage by caspases does not appear to be, detection of microbial proteases may represent an ancestral system of innate immune regulation.

ObjectiveKawasaki disease (KD) is the most common cause of acquired cardiac disease in US children. In addition to coronary artery abnormalities and aneurysms, it can be associated with systemic arterial aneurysms. We evaluated the development of systemic arterial dilatation and aneurysms, including abdominal aortic aneurysm (AAA) in the Lactobacillus casei cell-wall extract (LCWE)-induced KD vasculitis mouse model.Methods and resultsWe discovered that in addition to aortitis, coronary arteritis and myocarditis, the LCWE-induced KD mouse model is also associated with abdominal aorta dilatation and AAA, as well as renal and iliac artery aneurysms. AAA induced in KD mice was exclusively infrarenal, both fusiform and saccular, with intimal proliferation, myofibroblastic proliferation, break in the elastin layer, vascular smooth muscle cell loss, and inflammatory cell accumulation in the media and adventitia. Il1r(-/-), Il1a(-/-), and Il1b(-/-) mice were protected from KD associated AAA. Infiltrating CD11c(+) macrophages produced active caspase-1, and caspase-1 or NLRP3 deficiency inhibited AAA formation. Treatment with interleukin (IL)-1R antagonist (Anakinra), anti-IL-1α, or anti-IL-1β mAb blocked LCWE-induced AAA formation.ConclusionsSimilar to clinical KD, the LCWE-induced KD vasculitis mouse model can also be accompanied by AAA formation. Both IL-1α and IL-1β play a key role, and use of an IL-1R blocking agent that inhibits both pathways may be a promising therapeutic target not only for KD coronary arteritis, but also for the other systemic arterial aneurysms including AAA that maybe seen in severe cases of KD. The LCWE-induced vasculitis model may also represent an alternative model for AAA disease.

Nuclear factor κB (NF-κB), a key activator of inflammation, primes the NLRP3-inflammasome for activation by inducing pro-IL-1β and NLRP3 expression. NF-κB, however, also prevents excessive inflammation and restrains NLRP3-inflammasome activation through a poorly defined mechanism. We now show that NF-κB exerts its anti-inflammatory activity by inducing delayed accumulation of the autophagy receptor p62/SQSTM1. External NLRP3-activating stimuli trigger a form of mitochondrial (mt) damage that is caspase-1- and NLRP3-independent and causes release of direct NLRP3-inflammasome activators, including mtDNA and mtROS. Damaged mitochondria undergo Parkin-dependent ubiquitin conjugation and are specifically recognized by p62, which induces their mitophagic clearance. Macrophage-specific p62 ablation causes pronounced accumulation of damaged mitochondria and excessive IL-1β-dependent inflammation, enhancing macrophage death. Therefore, the "NF-κB-p62-mitophagy" pathway is a macrophage-intrinsic regulatory loop through which NF-κB restrains its own inflammation-promoting activity and orchestrates a self-limiting host response that maintains homeostasis and favors tissue repair.

ObjectiveKawasaki disease (KD) is the most common cause of acute vasculitis and acquired cardiac disease among US children. We have previously shown that both TLR2/MyD88 and interleukin (IL)-1β signaling are required for the Lactobacillus casei cell wall extract-induced KD vasculitis mouse model. The objectives of this study were to investigate the cellular origins of IL-1 production, the role of CD11c(+) dendritic cells and macrophages, and the relative contribution of hematopoietic and stromal cells for IL-1 responsive cells, as well the MyD88 signaling, in Lactobacillus casei cell wall extract-induced KD mouse model of vasculitis.Approach and resultsUsing mouse knockout models and antibody depletion, we found that both IL-1α and IL-1β were required for Lactobacillus casei cell wall extract-induced KD. Both dendritic cells and macrophages were necessary, and we found that MyD88 signaling was required in both hematopoietic and stromal cells. However, IL-1 response and signaling were critically required in nonendothelial stromal cells, but not in hematopoietic cells.ConclusionsOur results suggest that IL-1α and IL-1β, as well as CD11c(+) dendritic cells and macrophages, are essential for the development of KD vasculitis and coronary arteritis in this mouse model. Bone marrow chimera experiments suggest that MyD88 signaling is important in both hematopoietic and stromal cells, whereas IL-1 signaling and response are required only in stromal cells, but not in endothelial cells. Determining the role of IL-1α and IL-1β and of specific cell types in the KD vasculitis mouse model may have important implications for the design of more targeted therapies and understanding of the molecular mechanisms of KD immunopathologies.

In response to infections and tissue damage, ASC-containing inflammasome protein complexes are assembled that promote caspase-1 activation, IL-1β and IL-18 processing and release, pyroptosis, and the release of ASC particles. However, excessive or persistent activation of the inflammasome causes inflammatory diseases. Therefore, a well-balanced inflammasome response is crucial for the maintenance of homeostasis. We show that the PYD-only protein POP1 inhibited ASC-dependent inflammasome assembly by preventing inflammasome nucleation, and consequently interfered with caspase-1 activation, IL-1β and IL-18 release, pyroptosis, and the release of ASC particles. There is no mouse ortholog for POP1, but transgenic expression of human POP1 in monocytes, macrophages, and dendritic cells protected mice from systemic inflammation triggered by molecular PAMPs, inflammasome component NLRP3 mutation, and ASC danger particles. POP1 expression was regulated by TLR and IL-1R signaling, and we propose that POP1 provides a regulatory feedback loop that shuts down excessive inflammatory responses and thereby prevents systemic inflammation.

This study was designed to understand the contribution of the inflammasome and IL-1β activation in otitis media (OM). We examined the middle ear (ME) response to non-typeable Haemophilus influenzae (NTHi) in wild type (WT) mice using gene microarrays and a murine model of acute OM. Expression of members of the NOD domain-like receptor family of inflammasome genes was significantly up-regulated early in NTHi infection of the ME, potentially activating specific downstream regulatory cascades that contribute to the proliferative inflammatory response observed during OM. Expression of the pro-forms of the inflammasome targets IL-1β and IL-18 were also up-regulated. To evaluate the role of inflammasome-mediated cytokine maturation, NTHi-induced OM was examined in Asc(-/-)-deficient mice and compared with that seen in WT mice. Mice lacking the Asc gene showed near absence of IL-1β maturation in the ME and a reduction in leukocyte recruitment and infiltration to the cavity, and their macrophages exhibited reduced phagocytosis of NTHi. These inflammatory defects were linked to an increase in the degree and duration of mucosal epithelial hyperplasia in the ME of Asc(-/-) mice, as well as a delay in bacterial clearance from their MEs. These data demonstrate an important role for the inflammasome and cytokine processing in the course and resolution of OM.

AimsThe NLRP3 inflammasome is activated in the ischaemic heart promoting caspase-1 activation, inflammation, and cell death. Ischaemic injury establishes both a priming signal (transcription of inflammasome components) and a trigger (NLRP3 activation). Whether NLRP3 activation, without priming, induces cardiac dysfunction and/or failure is unknown. The aim of this study was to assess the independent and complementary roles of the priming and the triggering signals in the heart, in the absence of ischaemia or myocardial injury.Methods and resultsWe used mice with mutant NLRP3 (constitutively active), NLRP3-A350V, under the control of tamoxifen-driven expression of the Cre recombinase (Nlrp3-A350V/CreT mice). The mice were treated for 10 days with tamoxifen before measuring the activity of caspase-1, the effector enzyme in the inflammasome. Tamoxifen treatment induced the inflammasome in the spleen but not in the heart, despite expression of the mutant NLRP3-A350V. The components of the inflammasome were significantly less expressed in the heart compared with the spleen. Subclinical low-dose lipopolysaccharide (LPS; 2 mg/kg) in Nlrp3-A350V/CreT mice induced the expression of the components of the inflammasome (priming), measured using real-time PCR and western blot, leading to the formation of an active inflammasome (caspase-1 activation) in the heart and LV systolic dysfunction while low-dose LPS was insufficient to induce LV systolic dysfunction in wild-type mice (all P < 0.01 for mutant vs. wild-type mice).ConclusionThe signalling pathway governing the inflammasome formation in the heart requires a priming signal in order for an active NLRP3 to induce caspase-1 activation and LV dysfunction.

BackgroundEosinophilic esophagitis (EoE) is a chronic antigen mediated disease associated with substantial esophageal remodeling and fibrosis. The functional TGFβ1 promoter SNP C-509 associates with renal fibrosis and asthma. The effect of TGFβ1 genotype and EoE severity or potential gene-environment interactions have not been previously reported in EoE.MethodsGenotype at TGFβ1 C-509T and remodeling was analyzed in 144 subjects with EoE. The severity of remodeling and inflammation was analyzed in the context of IgE sensitization to food antigens and C-509T genotype.ResultsThe TGFβ1 promoter C-509 genotypes CC, CT, and TT were 35%, 52%, and 13%, respectively. Sixty-six percent of subjects were sensitized to foods by positive skin prick test (SPT) or serum specific IgE. TT genotype subjects had significantly more TGFβ1 (CC subjects = 1300 per mm2; TT = 2250 per mm2) (p

Inflammasomes are intracellular sensors that couple detection of pathogens and cellular stress to activation of Caspase-1, and consequent IL-1β and IL-18 maturation and pyroptotic cell death. Here, we show that the absent in melanoma 2 (AIM2) and nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasomes trigger Caspase-1-dependent mitochondrial damage. Caspase-1 activates multiple pathways to precipitate mitochondrial disassembly, resulting in mitochondrial reactive oxygen species (ROS) production, dissipation of mitochondrial membrane potential, mitochondrial permeabilization, and fragmentation of the mitochondrial network. Moreover, Caspase-1 inhibits mitophagy to amplify mitochondrial damage, mediated in part by cleavage of the key mitophagy regulator Parkin. In the absence of Parkin activity, increased mitochondrial damage augments pyroptosis, as indicated by enhanced plasma membrane permeabilization and release of danger-associated molecular patterns (DAMPs). Therefore, like other initiator caspases, Caspase-1 activation by inflammasomes results in mitochondrial damage.

NLR inflammasomes, caspase 1 activation platforms critical for processing key pro-inflammatory cytokines, have been implicated in the development of nonalcoholic fatty liver disease (NAFLD). As the direct role of the NLRP3 inflammasome remains unclear, we tested effects of persistent NLRP3 activation as a contributor to NAFLD development and, in particular, as a modulator of progression from benign hepatic steatosis to steatohepatitis during diet-induced NAFLD. Gain of function tamoxifen-inducible Nlrp3 knock-in mice allowing for in vivo temporal control of NLRP3 activation and loss of function Nlrp3 knockout mice were placed on short-term choline-deficient amino acid-defined (CDAA) diet, to induce isolated hepatic steatosis or long-term CDAA exposure, to induce severe steatohepatitis and fibrosis, respectively. Expression of NLRP3 associated proteins was assessed in liver biopsies of a well-characterized group of patients with the full spectrum of NAFLD. Nlrp3(-/-) mice were protected from long-term feeding CDAA-induced hepatomegaly, liver injury, and infiltration of activated macrophages. More importantly, Nlrp3(-/-) mice showed marked protection from CDAA-induced liver fibrosis. After 4 weeks on CDAA diet, wild-type (WT) animals showed isolated hepatic steatosis while Nlrp3 knock-in mice showed severe liver inflammation, with increased infiltration of activated macrophages and early signs of liver fibrosis. In the liver samples of patients with NAFLD, inflammasome components were significantly increased in those patients with nonalcoholic steatohepatitis (NASH) when compared to those with non-NASH NAFLD with mRNA levels of pro-IL1 beta correlated to levels of COL1A1. Our study uncovers a crucial role for the NLRP3 inflammasome in the development of NAFLD. These findings may lead to novel therapeutic strategies aimed at halting the progression of hepatic steatosis to the more severe forms of this disease. Key message: Mice with NLRP3 inflammasome loss of function are protected from diet-induced steatohepatitis. NLRP3 inflammasome gain of function leads to early and severe onset of diet-induced steatohepatitis in mice. Patients with severe NAFLD exhibit increased levels of NLRP3 inflammasome components and levels of pro-IL1β mRNA correlate with the expression of COL1A1.

In preterm infants, exposure to inflammation increases the risk of bronchopulmonary dysplasia, a chronic, developmental lung disease. Although macrophages are the key cells that initiate lung inflammation, less is known about lung macrophage phenotype and maturation. We hypothesized that fetal lung macrophages mature into distinct subpopulations during mouse development, and that activation could influence macrophage maturation. Expression of the fetal macrophage markers CD68, CD86, CD206, Ym1, fibrinogen-like protein 2, and indolamine-2, 3-dioxygenase was developmentally regulated, with each marker having different temporal patterns. Flow cytometry analysis showed macrophages within the fetal lung were less diverse than the distinctly separate subpopulations in newborn and adult lungs. Similar to adult alveolar macrophages, fetal lung macrophages responded to the TLR4 agonist LPS and the alternative activation cytokines IL-4 and IL-13. Using a macrophage-specific constitutively active IκB Kinase transgenic model (IKFM), we demonstrated that macrophage activation increased proinflammatory gene expression and reduced the response of fetal lung macrophages to IL-4 and IL-13. Activation also increased fetal lung macrophage proliferation. Fetal IKFM lungs contained increased percentages of more mature, CD11b(low)F4/80(high) cells that also expressed higher levels of the alternative activation markers CD204 and CD206. Development of fetal lung macrophages into mature alveolar macrophages may therefore include features of both proinflammatory and alternative activation paradigms.

Orosomucoid-like (ORMDL)3 has been strongly linked with asthma in genetic association studies. Because allergen challenge induces lung ORMDL3 expression in wild-type mice, we have generated human ORMDL3 zona pellucida 3 Cre (hORMDL3(zp3-Cre)) mice that overexpress human ORMDL3 universally to investigate the role of ORMDL3 in regulating airway inflammation and remodeling. These hORMDL3(zp3-Cre) mice have significantly increased levels of airway remodeling, including increased airway smooth muscle, subepithelial fibrosis, and mucus. hORMDL3(zp3-Cre) mice had spontaneously increased airway responsiveness to methacholine compared to wild-type mice. This increased airway remodeling was associated with selective activation of the unfolded protein response pathway transcription factor ATF6 (but not Ire1 or PERK). The ATF6 target gene SERCA2b, implicated in airway remodeling in asthma, was strongly induced in the lungs of hORMDL3(zp3-Cre) mice. Additionally, increased levels of expression of genes associated with airway remodeling (TGF-β1, ADAM8) were detected in airway epithelium of these mice. Increased levels of airway remodeling preceded increased levels of airway inflammation in hORMDL3(zp3-Cre) mice. hORMDL3(zp3-Cre) mice had increased levels of IgE, with no change in levels of IgG, IgM, and IgA. These studies provide evidence that ORMDL3 plays an important role in vivo in airway remodeling potentially through ATF6 target genes such as SERCA2b and/or through ATF6-independent genes (TGF-β1, ADAM8).

UnlabelledInflammasome activation plays a central role in the development of drug-induced and obesity-associated liver disease. However, the sources and mechanisms of inflammasome-mediated liver damage remain poorly understood. Our aim was to investigate the effect of NLRP3 inflammasome activation on the liver using novel mouse models. We generated global and myeloid cell-specific conditional mutant Nlrp3 knock-in mice expressing the D301N Nlrp3 mutation (ortholog of D303N in human NLRP3), resulting in a hyperactive NLRP3. To study the presence and significance of NLRP3-initiated pyroptotic cell death, we separated hepatocytes from nonparenchymal cells and developed a novel flow-cytometry-based (fluorescence-activated cell sorting; FACS) strategy to detect and quantify pyroptosis in vivo based on detection of active caspase 1 (Casp1)- and propidium iodide (PI)-positive cells. Liver inflammation was quantified histologically by FACS and gene expression analysis. Liver fibrosis was assessed by Sirius Red staining and quantitative polymerase chain reaction for markers of hepatic stellate cell (HSC) activation. NLRP3 activation resulted in shortened survival, poor growth, and severe liver inflammation; characterized by neutrophilic infiltration and HSC activation with collagen deposition in the liver. These changes were partially attenuated by treatment with anakinra, an interleukin-1 receptor antagonist. Notably, hepatocytes from global Nlrp3-mutant mice showed marked hepatocyte pyroptotic cell death, with more than a 5-fold increase in active Casp1/PI double-positive cells. Myeloid cell-restricted mutant NLRP3 activation resulted in a less-severe liver phenotype in the absence of detectable pyroptotic hepatocyte cell death.ConclusionsOur data demonstrate that global and, to a lesser extent, myeloid-specific NLRP3 inflammasome activation results in severe liver inflammation and fibrosis while identifying hepatocyte pyroptotic cell death as a novel mechanism of NLRP3-mediated liver damage.

Cell death is crucial for maintaining tissue balance and responding to diseases. However, under pathological conditions, the surge in dying cells results in an overwhelming presence of cell debris and the release of danger signals. In the liver, this gives rise to hepatic inflammation and hepatocellular cell death, which are key factors in various liver diseases caused by viruses, toxins, metabolic issues, or autoimmune factors. Both clinical and in vivo studies strongly affirm that hepatocyte death serves as a catalyst in the progression of liver disease. This advancement is characterized by successive stages of inflammation, fibrosis, and cirrhosis, culminating in a higher risk of tumor development. In this review, we explore pivotal forms of cell death, including apoptosis, pyroptosis, and necroptosis, examining their roles in both acute and chronic liver conditions, including liver cancer. Furthermore, we discuss the significance of cell death in liver surgery and ischemia-reperfusion injury. Our objective is to illuminate the molecular mechanisms governing cell death in liver diseases, as this understanding is crucial for identifying therapeutic opportunities aimed at modulating cell death pathways. [ABSTRACT FROM AUTHOR]

Abstract Background The Cryopyrin-Associated Periodic Syndromes (CAPS) are a group of rare hereditary autoinflammatory diseases and encompass Familial Cold Autoinflammatory Syndrome (FCAS), Muckle-Wells Syndrome (MWS), and Neonatal Onset Multisystem Inflammatory Disease (NOMID). Canakinumab is a monoclonal antibody directed against IL-1 beta and approved for CAPS patients but requires post-approval monitoring due to low and short exposures during the licensing process. Creative approaches to observational methodology are needed, harnessing novel registry strategies to ensure Health Care Provider reporting and patient monitoring. Methods A web-based registry was set up to collect information on long-term safety and effectiveness of canakinumab for CAPS. Results Starting in November 2009, this registry enrolled 241 patients in 43 centers and 13 countries by December 31, 2012. One-third of the enrolled population was aged