Summary 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.

Inflammasome activation has been recently recognized to play 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 constitutively activated NLRP3. To study the presence and significance of NLRP3 initiated pyroptotic cell death, we separated hepatocytes from non-parenchymal cells and developed a novel flow cytometry-based (FACS) strategy to detect and quantify pyroptosis in vivo based on detection of active caspase1 and propidium iodide (PI) positive cells. Liver inflammation was quantified histologically, by FACS and via gene expression analysis. Liver fibrosis was assessed by Sirius-Red-staining and qPCR 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 fivefold increase in active caspase1-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. Conclusions Our data demonstrates 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.

SUMMARY Cryopyrin (NALP3) mediates formation of the inflammasome, a protein complex responsible for cleavage of pro-IL-1β to its active form. Mutations in the cryopyrin gene, NLRP3, cause the autoinflammatory disease spectrum: cryopyrin-associated periodic syndromes (CAPS). The central role of IL-1β in CAPS is supported by the remarkable response to IL-1 targeted therapy. We developed two novel Nlrp3 mutant knock-in mouse strains to model CAPS to examine the role of other inflammatory mediators and adaptive immune responses in an innate immune driven disease. These mice had systemic inflammation and poor growth, similar to some human CAPS patients, and demonstrated early mortality, primarily mediated by myeloid cells. Mating these mutant mice to various knock-out backgrounds confirmed the mouse disease phenotype required an intact inflammasome, was only partially dependent on IL-1β, and was independent of T cells. This data suggests CAPS are true inflammasomopathies and provide insight for more common inflammatory disorders.

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.

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ORMDL3 (orosomucoid like 3) has been strongly linked with asthma in genetic association studies. As allergen challenge induces lung ORMDL3 expression in WT 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 spontaneous increased AHR to methacholine compared to WT 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. In addition, 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 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).

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.

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 airwayhyperresponsiveness (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, hGSDMB Zp3-Cre mice showed increases in the same remodeling and AHR mediators (TGF-β1, 5-LO) observed in vitro in GSDMBoverexpressing 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.GSDMB | asthma | airway-hyperresponsiveness | remodeling | inflammation