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Two letters to the editor are presented in response the article "A Severe Case of Chronic Infantile Neurologic, Cutaneous, Articular Syndrome Treated With Biologic Agents," published in a 2006 issue of "Arthritis Rheumatology."

Familial cold autoinflammatory syndrome (FCAS, MIM 120100), commonly known as familial cold urticaria (FCU), is an autosomal-dominant systemic inflammatory disease characterized by intermittent episodes of rash, arthralgia, fever and conjunctivitis after generalized exposure to cold. FCAS was previously mapped to a 10-cM region on chromosome 1q44 (refs. 5,6). Muckle-Wells syndrome (MWS; MIM 191900), which also maps to chromosome 1q44, is an autosomal-dominant periodic fever syndrome with a similar phenotype except that symptoms are not precipitated by cold exposure and that sensorineural hearing loss is frequently also present. To identify the genes for FCAS and MWS, we screened exons in the 1q44 region for mutations by direct sequencing of genomic DNA from affected individuals and controls. This resulted in the identification of four distinct mutations in a gene that segregated with the disorder in three families with FCAS and one family with MWS. This gene, called CIAS1, is expressed in peripheral blood leukocytes and encodes a protein with a pyrin domain, a nucleotide-binding site (NBS, NACHT subfamily) domain and a leucine-rich repeat (LRR) motif region, suggesting a role in the regulation of inflammation and apoptosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Patients with chronic kidney disease (CKD) are often 25(OH)D3 and 1,25(OH)2D3 insufficient. We studied whether vitamin D repletion could correct aberrant adipose tissue and muscle metabolism in a mouse model of CKD-associated cachexia. Intraperitoneal administration of 25(OH)D3 and 1,25(OH)2D3 (75 μg/kg/day and 60 ng/kg/day respectively for 6 weeks) normalized serum concentrations of 25(OH)D3 and 1,25(OH)2D3 in CKD mice. Vitamin D repletion stimulated appetite, normalized weight gain, and improved fat and lean mass content in CKD mice. Vitamin D supplementation attenuated expression of key molecules involved in adipose tissue browning and ameliorated expression of thermogenic genes in adipose tissue and skeletal muscle in CKD mice. Furthermore, repletion of vitamin D improved skeletal muscle fiber size and in vivo muscle function, normalized muscle collagen content and attenuated muscle fat infiltration as well as pathogenetic molecular pathways related to muscle mass regulation in CKD mice. RNAseq analysis was performed on the gastrocnemius muscle. Ingenuity Pathway Analysis revealed that the top 12 differentially expressed genes in CKD were correlated with impaired muscle and neuron regeneration, enhanced muscle thermogenesis and fibrosis. Importantly, vitamin D repletion normalized the expression of those 12 genes in CKD mice. Vitamin D repletion may be an effective therapeutic strategy for adipose tissue browning and muscle wasting in CKD patients. [ABSTRACT FROM AUTHOR]

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. • LPS stimulates choline uptake and phosphorylation via CTL1 induction • Impaired choline uptake or phosphorylation reduces mitochondrial ATP synthesis • Reduced ATP synthesis leads to AMPK activation and induction of mitophagy • By causing mitophagy, ChoKα inhibition prevents NLRP3-dependent inflammation Sanchez-Lopez et al. have found that inhibition of choline uptake and phosphorylation in activated macrophages prevent NLRP3 inflammasome activation and IL-1β and IL-18 production. Inhibition of choline incorporation into phosphatidylcholine in the mitochondrial membranes and decreased ATP synthase activity lead to enhanced AMPK-dependent mitophagy that prevents acute and chronic inflammation. [ABSTRACT FROM AUTHOR]

Neutrophil extracellular traps (NETs) not only counteract bacterial and fungal pathogens but can also promote thrombosis, autoimmunity, and sterile inflammation. The presence of citrullinated histones, generated by the peptidylarginine deiminase 4 (PAD4), is synonymous with NETosis and is considered independent of apoptosis. Mitochondrial- and death receptor-mediated apoptosis promote gasdermin E (GSDME)-dependent calcium mobilization and membrane permeabilization leading to histone H3 citrullination (H3Cit), nuclear DNA extrusion, and cytoplast formation. H3Cit is concentrated at the promoter in bone marrow neutrophils and redistributes in a coordinated process from promoter to intergenic and intronic regions during apoptosis. Loss of GSDME prevents nuclear and plasma membrane disruption of apoptotic neutrophils but prolongs early apoptosisinduced cellular changes to the chromatin and cytoplasmic granules. Apoptotic signaling engages PAD4 in neutrophils, establishing a cellular state that is primed for NETosis, but that occurs only upon membrane disruption by GSDME, thereby redefining the end of life for neutrophils. [ABSTRACT FROM AUTHOR]

The NLRP3 inflammasome is an intracellular, multiprotein complex that promotes the auto-catalytic activation of caspase-1 and the subsequent maturation and secretion of the pro-inflammatory cytokines, IL-1β and IL-18. Persistent activation of the NLRP3 inflammasome has been implicated in the pathophysiology of a number of inflammatory and autoimmune diseases, including neuroinflammation, cardiovascular disease, non-alcoholic steatohepatitis, lupus nephritis and severe asthma. Here we describe the preclinical profile of JT002, a novel small molecule inhibitor of the NLRP3 inflammasome. JT002 potently reduced NLRP3-dependent proinflammatory cytokine production across a number of cellular assays and prevented pyroptosis, an inflammatory form of cell death triggered by active caspase-1. JT002 demonstrated in vivo target engagement at therapeutically relevant concentrations when orally dosed in mice and prevented body weight loss and improved inflammatory and fibrotic endpoints in a model of Muckle–Wells syndrome (MWS). In two distinct models of neutrophilic airway inflammation, JT002 treatment significantly reduced airway hyperresponsiveness and airway neutrophilia. These results provide a rationale for the therapeutic targeting of the NLRP3 inflammasome in severe asthma and point to the use of JT002 in a variety of inflammatory disorders. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. The differential binding of NLRP3 to non-oxidized and Ox-mtDNA is characterized, which provides insight into the mechanisms of inflammasome assembly [ABSTRACT FROM AUTHOR]

Background: Individuals 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. Conclusions: Collectively, 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). [ABSTRACT FROM AUTHOR]

BACKGROUND. Multisystem 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. METHODS. The 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. RESULTS. Circulating 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. CONCLUSIONS. Activated neutrophils expressing IL-1β can be targeted by IVIG, supporting its use in both KD and MIS-C to ameliorate inflammation. [ABSTRACT FROM AUTHOR]

Cytokines such as IL-6, TNF-α and IL-1β trigger inflammatory cascades which may play a role in the pathogenesis of chronic kidney disease (CKD)-associated cachexia. CKD was induced by 5/6 nephrectomy in mice. We studied energy homeostasis in Il1β−/−/CKD, Il6−/−/CKD and Tnfα−/−/CKD mice and compared with wild type (WT)/CKD controls. Parameters of cachexia phenotype were completely normalized in Il1β−/−/CKD mice but were only partially rescued in Il6−/−/CKD and Tnfα−/−/CKD mice. We tested the effects of anakinra, an IL-1 receptor antagonist, on CKD-associated cachexia. WT/CKD mice were treated with anakinra (2.5 mg/kg/day, IP) or saline for 6 weeks and compared with WT/Sham controls. Anakinra normalized food intake and weight gain, fat and lean mass content, metabolic rate and muscle function, and also attenuated molecular perturbations of energy homeostasis in adipose tissue and muscle in WT/CKD mice. Anakinra decreased serum and muscle expression of IL-6, TNF-α and IL-1β in WT/CKD mice. Anakinra attenuated browning of white adipose tissue in WT/CKD mice. Moreover, anakinra normalized gastrocnemius weight and fiber size as well as attenuated muscle fat infiltration in WT/CKD mice. This was accompanied by correcting the increased muscle wasting signaling pathways while promoting the decreased myogenesis process in gastrocnemius of WT/CKD mice. We performed qPCR analysis for the top 20 differentially expressed muscle genes previously identified via RNAseq analysis in WT/CKD mice versus controls. Importantly, 17 differentially expressed muscle genes were attenuated in anakinra treated WT/CKD mice. In conclusion, IL-1 receptor antagonism may represent a novel targeted treatment for adipose tissue browning and muscle wasting in CKD. [ABSTRACT FROM AUTHOR]

Increased 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. We used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3 KICreA mice, and Gsdmd KO 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). We 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. Nlrp3 KICreA 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. These results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development. Our 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. • Human and murine hepatocytes undergo pyroptosis and release NLRP3 inflammasome proteins. • Pyroptotic cell death in hepatocytes is dependent on caspase-1 and gasdermin D activation. • Caspase-1 activity is increased in livers and serum from NASH patients. • Nlrp3 KICreA mice develop fibrosis and show increased sensitivity to liver damage. • Human hepatic stellate cells internalise extracellular NLRP3-YFP oligomeric particles. • Extracellular NLRP3 oligomeric particles perpetuate inflammation and fibrogenesis. [ABSTRACT FROM AUTHOR]

The article presents research report on inhibition of the Nod-Like Receptor Family Pyrin Domain Containing 3 (NLRP3) inflammasome prevents ovarian aging. Topics include ablation of NLRP3 improved the survival and pregnancy rates and increased anti-Müllerian hormone levels and autophagy rates in ovaries; and age-dependent loss of female fertility and position this inflammasome as a potential new therapeutic target for the treatment of infertility.

The NLRP3 inflammasome is a protein complex responsible for caspase-1-dependent maturation of the proinflammatory cytokines IL-1β and IL-18. Gain-of-function missense mutations in NLRP3 cause the disease spectrum known as the cryopyrin-associated periodic syndromes (CAPS). In this study, we generated Nlrp3-knockin mice on various KO backgrounds including Il1b/Il18-, caspase-1-, caspase-11- (Casp1/11-), and Tnf-deficient strains. The Nlrp3L351P Il1b-/- Il18-/- mutant mice survived and grew normally until adulthood and, at 6 months of age, exhibited marked splenomegaly and leukophilia. Injection of these mice with low-dose LPS resulted in elevated serum TNF levels compared with Nlrp3L351P Casp1/11-/- mice and Il1b-/- Il18-/- littermates. Treatment of Nlrp3A350V mice with the TNF inhibitor etanercept resulted in all pups surviving to adulthood, with normal body and spleen/body weight ratios. Nlrp3A350V Tnf-/- mice showed a similar phenotypic rescue, with marked reductions in serum IL-1β and IL-18, reduced myeloid inflammatory infiltrate in the skin and spleen, and substantial decreases in splenic mRNA expression of both inflammasome components (Nlrp3, Pycard, pro-Casp1) and pro-cytokines (Il1b, Il18). Likewise, we observed a reduction in the expression of both pro-Casp1 and pro-Il1b in cultured Nlrp3A350V Tnf-/- BM-derived DCs. Our data show that TNF is an important transcriptional regulator of NLRP3 inflammasome components in murine inflammasomopathies. Moreover, these results may have therapeutic implications for CAPS patients with partial responses to IL-1-targeted therapies. [ABSTRACT FROM AUTHOR]

Chronic liver disease with cirrhosis is the 12th leading cause of death in the United States, and alcoholic liver disease accounts for approximately half of all cirrhosis deaths. Chronic alcohol consumption is associated with intestinal bacterial dysbiosis, yet we understand little about the contribution of intestinal fungi, or mycobiota, to alcoholic liver disease. Here we have demonstrated that chronic alcohol administration increases mycobiota populations and translocation of fungal β-glucan into systemic circulation in mice. Treating mice with antifungal agents reduced intestinal fungal overgrowth, decreased β-glucan translocation, and ameliorated ethanol-induced liver disease. Using bone marrow chimeric mice, we found that β-glucan induces liver inflammation via the C-type lectin-like receptor CLEC7A on Kupffer cells and possibly other bone marrow-derived cells. Subsequent increases in IL-1β expression and secretion contributed to hepatocyte damage and promoted development of ethanol-induced liver disease. We observed that alcohol-dependent patients displayed reduced intestinal fungal diversity and Candida overgrowth. Compared with healthy individuals and patients with non-alcohol-related cirrhosis, alcoholic cirrhosis patients had increased systemic exposure and immune response to mycobiota. Moreover, the levels of extraintestinal exposure and immune response correlated with mortality. Thus, chronic alcohol consumption is associated with an altered mycobiota and translocation of fungal products. Manipulating the intestinal mycobiome might be an effective strategy for attenuating alcohol-related liver disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 butwhose 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. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

Background: Eosinophilic 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. Methods: Genotype 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. Results: The 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<0.05) and tryptase (CC subjects = 145 per mm2: TT = 307 per mm2) (p<0.05) positive cells and higher epithelial remodeling scores (2.4 vs 3.7, p<0.001) than CC subjects. The differences in TGFβ1 and tryptase positive cells as well as fibrosis were significantly increased when there was concurrent food sensitization. Food sensitization alone did not associate with any parameters of inflammation or remodeling. Conclusions: Our data support a gene-environment interaction between food and genotype at C-509 that modulates disease severity in EoE. Since EoE subjects often continue to consume foods to which they are sensitized, these findings may have clinical relevance for disease management. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Ca2+ uptake via MCU triggers IMM mPTP opening to induce OMM VDAC oligomerization • Ox-mtDNA is repaired by OGG1 or cleaved by FEN1 to fragments that exit mitochondria • Cytosolic Ox-mtDNA activates NLRP3 inflammasome and cGAS-STING and escapes cells • mtOGG1 and FEN1 inhibitors suppress acute peritonitis and reduce circulating mtDNA Ox-mtDNA enables NLRP3 inflammasome activation, but how Ox-mtDNA reaches the cytoplasm is unclear. In this issue of Immunity , Xian et al. show that Ox-mtDNA cleaved by FEN1 escapes mitochondria via mPTP and VDAC channels to activate NLRP3 inflammasome and STING. [ABSTRACT FROM AUTHOR]

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 mat uration. We hypothesized that fetal lung macrophages mature into distinct subpopulations during mouse development, and that ac tivation could influence macrophage maturation. Expression of the fetal macrophage markers CD68, CD86, CD206, Yml, 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, CDllblowF4/80high 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. [ABSTRACT FROM AUTHOR]

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 (hORMDL3zp3-Cre) mice that overexpress human ORMDL3 universally to investigate the role of ORMDL3 in regulating airway inflammation and remodeling. These hORMDL3zp3-Cre mice have significantly increased levels of airway remodeling, including increased airway smooth muscle, subepithelial fibrosis, and mucus. hORMDL3zp3-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 hORMDL3zp3-Cre mice. Additionally, increased levels of expression of genes associated with airway remodeling (TGF-b1, ADAM8) were detected in airway epithelium of these mice. Increased levels of airway remodeling preceded increased levels of airway inflammation in hORMDL3zp3-Cre mice. hORMDL3zp3-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). [ABSTRACT FROM AUTHOR]

The inflammasome is a cytoplasmic multiprotein complex that promotes proinflammatory cytokine maturation in response to host- and pathogen-derived signals. Missense mutations in cryopyrin (NLRP3) result in a hyperactive inflammasome that drives overproduction of the proinflammatory cytokines IL-1β and IL-18, leading to the cryopyrin-associated periodic syndromes (CAPS) disease spectrum. Mouse lines harboring CAPS-associated mutations in Nlrp3 have elevated levels of IL-1β and IL-18 and closely mimic human disease. To examine the role of inflammasome-driven IL-18 in murine CAPS, we bred Nlrp3 mutations onto an Il18r-null background. Deletion of Il18r resulted in partial phenotypic rescue that abolished skin and visceral disease in young mice and normalized serum cytokines to a greater extent than breeding to Il1r-null mice. Significant systemic inflammation developed in aging Nlrp3 mutant Il18r-null mice, indicating that IL-1 and IL-18 drive pathology at different stages of the disease process. Ongoing inflammation in double-cytokine knockout CAPS mice implicated a role for caspase-1-mediated pyroptosis and confirmed that CAPS is inflammasome dependent. Our results have important implications for patients with CAPS and residual disease, emphasizing the need to explore other NLRP3-mediated pathways and the potential for inflammasome-targeted therapy. [ABSTRACT FROM AUTHOR]

Inflammasomes activate caspase-1 for processing and secretion of the cytokines interleukin-1β (IL-1β) and IL-18. Cryopyrin/NALP3/NLRP3 is an essential component of inflammasomes triggered by microbial ligands, danger-associated molecular patterns (DAMPs), and crystals. Inappropriate Cryopyrin activity has been incriminated in the pathogenesis of gouty arthritis, Alzheimer's, and silicosis. Therefore, inhibitors of the Nalp3 inflammasome offer considerable therapeutic promise. In this study, we show that the type 2 diabetes drug glyburide prevented activation of the Cryopyrin inflammasome. Glyburide's cyclohexylurea group, which binds to adenosine triphosphatase (ATP)-sensitive K[sup +] (K[sub ATP]) channels for insulin secretion, is dispensable for inflammasome inhibition. Macrophages lacking K[sub ATP] subunits or ATP-binding cassette transporters also activate the Cryopyrin inflammasome normally. Glyburide analogues inhibit ATP- but not hypothermia-induced IL-1β secretion from human monocytes expressing familial cold-associated autoinflammatory syndrome-associated Cryopyrin mutations, thus suggesting that inhibition occurs upstream of Cryopyrin. Concurrent with the role of Cryopyrin in endotoxemia, glyburide significantly delays lipopolysaccharide-induced lethality in mice. Therefore, glyburide is the first identified compound to prevent Cryopyrin activation and microbial ligand-, DAMP-, and crystal-induced IL-1β secretion. [ABSTRACT FROM AUTHOR]

Inflammation under sterile conditions is a key event in autoimmunity and following trauma. Hyaluronan, a glycosaminoglycan released from the extracellular matrix after injury, acts as an endogenous signal of trauma and can trigger chemokine release in injured tissue. Here, we investigated whether NLRP3/cryopyrin, a component of the inflammasome, participates in the inflammatory response to injury or the cytokine response to hyaluronan. Mice with a targeted deletion in cryopyrin showed a normal increase in Cxcl2 in response to sterile injuries but had decreased inflammation and release of interleukin-1β (IL-1β3). Similarly, the addition of hyaluronan to macrophages derived from cryopyrin-deficient mice increased release of Cxcl2 but did not increase IL-1β release. To define the mechanism of hyaluronan-mediated activation of cryopyrin, elements of the hyaluronan recognition process were studied in detail. IL-1β release was inhibited in peritoneal macrophages derived from CD44-deficient mice, in an MH-S macrophage cell line treated with antibodies to CD44, or by inhibitors of lysosome function. The requirement for CD44 binding and hyaluronan internalization could be bypassed by intracellular administration of hyaluronan oligosaccharides (10 -18-mer) in lipopolysaccharide-primed macrophages. Therefore, the action of CD44 and subsequent hyaluronan catabolism trigger the intracellular cryopyrin → IL-1β pathway. These findings support the hypothesis that hyaluronan works through IL-1β and the cryopyrin system to signal sterile inflammation. [ABSTRACT FROM AUTHOR]

Macrophages contribute to the induction and resolution of inflammation and play a central role in chronic low-grade inflammation in cardiovascular diseases caused by atherosclerosis. Human milk oligosaccharides (HMOs) are complex unconjugated glycans unique to human milk that benefit infant health and act as innate immune modulators. Here, we identify the HMO 3'sialyllactose (3'SL) as a natural inhibitor of Toll-Like Receptor (TLR) 4-induced low-grade inflammation in macrophages and endothelium. Transcriptome analysis in macrophages revealed that 3'SL attenuates mRNA levels of a selected set of inflammatory genes and promotes the activity of Liver X Receptor (LXR) and Sterol Regulatory Element-binding Protein-1 (SREBP). These acute anti-inflammatory effects of 3'SL were associated with reduced histone H3K27 acetylation at a subset of lipopolysaccharide (LPS)-inducible enhancers distinguished by preferential enrichment for CCCTC-binding factor (CTCF), Interferon Regulatory Factor 2 (IRF2), B-cell lymphoma 6 (BCL6), and other transcription factor recognition motifs. In a murine atherosclerosis model, both subcutaneous and oral administration of 3'SL significantly reduced atherosclerosis development and the associated inflammation. This study provides evidence that 3'SL attenuates inflammation by a transcriptional mechanism to reduce atherosclerosis development in the context of cardiovascular disease.

Inflammation has an essential role in healing. However, over-active inflammation disrupts normal cellular functions and can be life-threatening when not resolved. The NLRP3 inflammasome, a component of the innate immune system, is an intracellular multiprotein complex that senses stress-associated signals, and, for this reason is a promising therapeutic target for treating unresolved, pathogenic inflammation. Alternative splicing of NLRP3 RNA has been suggested as a regulatory mechanism for inflammasome activation, as some spliced isoforms encode NLRP3 proteins with compromised function. Here, we take advantage of this natural regulatory mechanism and devise a way to control pathogenic inflammation using splice-switching antisense oligonucleotides (ASOs). To identify and induce NLRP3 spliced isoforms lacking inflammatory activity, we tested a series of ASOs, each targeting a different exon, to determine the most effective strategy for down-regulating NLRP3. We identify several ASOs that modulate NLRP3 splicing, reduce NLRP3 protein, and decrease inflammasome signaling in vitro . The most effective ASO suppresses systemic inflammation in vivo in mouse models of acute inflammation and cryopyrin-associated periodic syndrome (CAPS). Our results demonstrate a systematic approach to protein engineering using splice-switching ASOs to generate isoforms with altered activity, and identify an ASO that can treat pathological inflammation in mice by reducing functional NLRP3.

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.

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., (© 2023 The Authors. Immunological Reviews published by John Wiley & Sons Ltd.)

Background & Aims: Breast 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 (Nlrp3 A350V CRT) and deficient for BRP39 (Nlrp3 A350V /BRP -/- CRT)., Methods: Using Nlrp3 A350V CRT mice and Nlrp3 A350V BRP -/- CRT, we investigated the consequences of BRP39 deficiency influencing NLRP3-induced liver inflammation., Results: Our 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., Conclusions: These 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., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Neutrophil extracellular traps (NETs) not only counteract bacterial and fungal pathogens but can also promote thrombosis, autoimmunity, and sterile inflammation. The presence of citrullinated histones, generated by the peptidylarginine deiminase 4 (PAD4), is synonymous with NETosis and is considered independent of apoptosis. Mitochondrial- and death receptor-mediated apoptosis promote gasdermin E (GSDME)-dependent calcium mobilization and membrane permeabilization leading to histone H3 citrullination (H3Cit), nuclear DNA extrusion, and cytoplast formation. H3Cit is concentrated at the promoter in bone marrow neutrophils and redistributes in a coordinated process from promoter to intergenic and intronic regions during apoptosis. Loss of GSDME prevents nuclear and plasma membrane disruption of apoptotic neutrophils but prolongs early apoptosis-induced cellular changes to the chromatin and cytoplasmic granules. Apoptotic signaling engages PAD4 in neutrophils, establishing a cellular state that is primed for NETosis, but that occurs only upon membrane disruption by GSDME, thereby redefining the end of life for neutrophils.

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a multiprotein complex and component of the innate immune system that is activated by exogenous and endogenous danger signals to promote activation of caspase-1 and the maturation and release of the proinflammatory cytokines interleukin (IL)-1 β and IL-18. Inappropriate activation of NLRP3 has been implicated in the pathophysiology of multiple inflammatory and autoimmune diseases, including cardiovascular disease, neurodegenerative diseases, and nonalcoholic steatohepatitis (NASH), thus increasing the clinical interest of this target. We describe in this study the preclinical pharmacologic, pharmacokinetic, and pharmacodynamic properties of a novel and highly specific NLRP3 inhibitor, JT001 (6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-sulfonylurea). In cell-based assays, JT001 potently and selectively inhibited NLRP3 inflammasome assembly, resulting in the inhibition of cytokine release and the prevention of pyroptosis, a form of inflammatory cell death triggered by active caspase-1. Oral administration of JT001 to mice inhibited IL-1 β production in peritoneal lavage fluid at plasma concentrations that correlated with mouse in vitro whole blood potency. Orally administered JT001 was effective in reducing hepatic inflammation in three different murine models, including the Nlrp3 A350V /+ CreT model of Muckle-Wells syndrome (MWS), a diet-induced obesity NASH model, and a choline-deficient diet-induced NASH model. Significant reductions in hepatic fibrosis and cell damage were also observed in the MWS and choline-deficient models. Our findings demonstrate that blockade of NLRP3 attenuates hepatic inflammation and fibrosis and support the use of JT001 to investigate the role of NLRP3 in other inflammatory disease models. SIGNIFICANCE STATEMENT: Persistent inflammasome activation is the consequence of inherited mutations of NLRP3 and results in the development of cryopyrin-associated periodic syndromes associated with severe systemic inflammation. NLRP3 is also upregulated in nonalcoholic steatohepatitis, a metabolic chronic liver disease currently missing a cure. Selective and potent inhibitors of NLRP3 hold great promise and have the potential to overcome an urgent unmet need., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)