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.
Inflammasome 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. Conclusions: Our 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. (Hepatology 2014;59:898–910)
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The NLRP3 inflammasome, a caspase‐1 activation platform, plays a key role in the modulation of liver inflammation and fibrosis. Here, we tested the hypothesis that interleukin 17 (IL‐17) and tumor ...necrosis factor (TNF) are key cytokines involved in amplifying and perpetuating the liver damage and fibrosis resulting from NLRP3 activation. To address this hypothesis, gain‐of‐function Nlrp3A350V knock‐in mice were bred onto il17a and Tnf knockout backgrounds allowing for constitutive Nlrp3 activation in myeloid derived cells in mice deficient in IL‐17 or TNF. Livers of Nlrp3A350V knock‐in mice exhibited severe liver inflammatory changes characterized by infiltration with neutrophils, increased expression of chemokine (C‐X‐C motif) ligand (CXCL) 1 and CXCL2 chemokines, activated inflammatory macrophages, and elevated levels of IL‐17 and TNF. Mutants with ablation of il17a signal showed fewer neutrophils when compared to intact Nlrp3A350V mutants, but still significant inflammatory changes when compared to the nonmutant il17a knockout littermates. The severe inflammatory changes associated with mutant Nlrp3 were almost completely rescued by Tnf knockout in association with a marked decrease in circulating IL‐1β levels. Intact Nlrp3A350Vmutants showed changes in liver fibrosis, as evidenced by morphometric quantitation of Sirius Red staining and increased mRNA levels of profibrotic genes, including connective tissue growth factor and tissue inhibitor of matrix metalloproteinase 1. Il17a lacking mutants exhibited amelioration of the aforementioned fibrosis, whereas Tnf‐deficient mutants showed no signs of fibrosis when compared to littermate controls. Conclusion: Our study uncovers key roles for TNF and, to a lesser extent, IL‐17 as mediators of liver inflammation and fibrosis induced by constitutive NLRP3 inflammasome activation in myeloid‐derived cells. These findings may lead to therapeutic strategies aimed at halting the progression of liver injury and fibrogenesis in various liver pathogeneses driven by NLRP3 activation. (Hepatology 2018;67:736‐749).
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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.
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).
Activating‐mutations in NOD‐like receptor (NLR) family, pyrin domain‐containing 3 (NLRP3) cause neonatal‐onset multisystem inflammatory disease. However, the ontogeny of skeletal anomalies in this ...disorder is poorly understood. Mice globally expressing the D301N mutation in Nlrp3 (D303N in human) model the human phenotype, including systemic inflammation and skeletal deformities. To gain insights into the skeletal manifestations, we generated mice in which the expression of D301N Nlrp3 (Nlrp3D301N) is restricted to myeloid cells. These mice exhibit systemic inflammation and severe osteopenia (~60% lower bone mass) similar to mice globally expressing the knock‐in mutation, consistent with the paradigm of innate immune‐driven cryopyrinopathies. Because systemic inflammation may indirectly affect bone homeostasis, we engineered mice in which Nlrp3D301N is expressed specifically in osteoclasts, the cells that resorb bone. These mice also develop ~50% lower bone mass due to increased osteolysis, but there is no systemic inflammation and no change in osteoclast number. Mechanistically, aside from its role in IL‐1β maturation, Nlrp3D301N expression enhances osteoclast bone resorbing ability through reorganization of actin cytoskeleton while promoting the degradation of poly(ADP‐ribose) polymerase 1, an inhibitor of osteoclastogenesis. Thus, NLRP3 inflammasome activation is not restricted to the production of proinflammatory mediators but also leads to cytokine‐autonomous responses.—Qu, C., Bonar, S. L., Hickman‐Brecks, C. L., Abu‐Amer, S., McGeough, M. D., Peña, C. A., Broderick, L., Yang, C., Grimston, S., K., Kading, J., Abu‐Amer, Y., Novack, D. V., Hoffman, H. M., Civitelli, R., Mbalaviele, G. NLRP3 mediates osteolysis through inflammation‐dependent and ‐independent mechanisms. FASEB J. 29, 1269‐1279 (2015). www.fasebj.org
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The spectrum of the clinical presentation and severity of malaria infections is broad, ranging from uncomplicated febrile illness to severe forms of disease such as cerebral malaria (CM), acute lung ...injury (ALI), acute respiratory distress syndrome (ARDS), pregnancy-associated malaria (PAM) or severe anemia (SA). Rodent models that mimic human CM, PAM and SA syndromes have been established. Here, we show that DBA/2 mice infected with P. berghei ANKA constitute a new model for malaria-associated ALI. Up to 60% of the mice showed dyspnea, airway obstruction and hypoxemia and died between days 7 and 12 post-infection. The most common pathological findings were pleural effusion, pulmonary hemorrhage and edema, consistent with increased lung vessel permeability, while the blood-brain barrier was intact. Malaria-associated ALI correlated with high levels of circulating VEGF, produced de novo in the spleen, and its blockage led to protection of mice from this syndrome. In addition, either splenectomization or administration of the anti-inflammatory molecule carbon monoxide led to a significant reduction in the levels of sera VEGF and to protection from ALI. The similarities between the physiopathological lesions described here and the ones occurring in humans, as well as the demonstration that VEGF is a critical host factor in the onset of malaria-associated ALI in mice, not only offers important mechanistic insights into the processes underlying the pathology related with malaria but may also pave the way for interventional studies.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The 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.
We 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).
The 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.
Recent studies in pigs have detected copy number variants (CNVs) using the Comparative Genomic Hybridization technique in arrays designed to cover specific porcine chromosomes. The goal of this study ...was to identify CNV regions (CNVRs) in swine species based on whole genome SNP genotyping chips.
We used predictions from three different programs (cnvPartition, PennCNV and GADA) to analyze data from the Porcine SNP60 BeadChip. A total of 49 CNVRs were identified in 55 animals from an Iberian x Landrace cross (IBMAP) according to three criteria: detected in at least two animals, contained three or more consecutive SNPs and recalled by at least two programs. Mendelian inheritance of CNVRs was confirmed in animals belonging to several generations of the IBMAP cross. Subsequently, a segregation analysis of these CNVRs was performed in 372 additional animals from the IBMAP cross and its distribution was studied in 133 unrelated pig samples from different geographical origins. Five out of seven analyzed CNVRs were validated by real time quantitative PCR, some of which coincide with well known examples of CNVs conserved across mammalian species.
Our results illustrate the usefulness of Porcine SNP60 BeadChip to detect CNVRs and show that structural variants can not be neglected when studying the genetic variability in this species.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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