NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice

Abstract

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.

Targeting the NLRP3 inflammasome to Reduce Diet-induced Metabolic Abnormalities in Mice

“…Another unresolved question is whether the NLRP3 inflammasome activation occurs at the level of resident cells, since results so far obtained from preclinical models of obesity and insulin resistance are quite contrasting. There is good evidence that both hematopoietic and nonhematopoietic cells are involved in NLRP3 inflammasome activation in both renal and hepatic tissues (38)(39)(40)(41). The above data provide evidence in support ) and standardized to the corresponding β-actin contents.…”

Hepatocyte-specific Bid depletion reduces tumor development by suppressing inflammation-related compensatory proliferation

“…Total RNA was isolated from liver tissue and analyzed as previously described. 42 The sequences of the primers used for quantitative PCR are given in Supplementary Table 1.…”

“…Immunoblot analysis was performed as previously described. 42 Anti-Bid (Cell Signaling, Boston, MA, USA) antibody was used in combination with appropriate peroxidase-conjugated secondary antibody. Protein load was verified with an actin antibody (dilution 1 : 10 000) (Santa Cruz, Dallas, TX, USA).…”

Estrogen suppresses hepatocellular carcinoma cells through ERβ-mediated upregulation of the NLRP3 inflammasome

“…16,17 Recent study showed that pyroptotic cell death of hepatocyte mediated liver damage. 30 Another study showed that NLRP3 inflammasome activation contributed to the progression of non-alcoholic fatty liver disease. 31 Both of these reports contributed to our understanding of the pathogenesis of liver disease mediated by inflammasome activation.…”