Lipopolysaccharide induces and activates the Nalp3 inflammasome in the liver

AIM:To examine the activation of the Nalp3 inflammasome and its downstream targets following lipopolysaccharide(LPS) -induced stimulation in the liver. METHODS:Six-to-eight-week-old C57BL/6 chow fed mice were injected intraperitoneally with 0.5μg/g bodyweight LPS and sacrificed 2,4,6,18 or 24 h later. LPS-induced liver damage was confirmed by a biochemical assay to detect alanine aminotransferase(ALT) levels.To determine if LPS stimulation in the liver led to activation of the inflammasome,real-time quantitative polymerase chain reaction was used to evaluate the mRNA expression of components of the Nalp3 inflammasome.Enzyme-linked immunosorbent assays were used to determine the protein expression levels of several downstream targets of the Nalp3 inflammasome,including caspase-1 and two cytokine targets of caspase-1,interleukin(IL) -1βand IL-18. RESULTS:We found that LPS injection resulted in liver damage as indicated by elevated ALT levels.This was associated with a significant increase in both mRNA and protein levels of the proinflammatory cy-tokine tumor necrosis factor(TNF) -αin the liver,as well as increased levels of TNFs in serum.We showed that LPS stimulation led to upregulation of mRNA levels in the liver for all the receptor components of the inflammasome,including Nalp3,Nalp1,pannexin-1 and the adaptor molecule apoptosis-associated specklike,caspase recruitment domain-domain containing protein.We also found increased levels of mRNA and protein for caspase-1,a downstream target of the inflammasome.In addition,LPS challenge led to increased levels of both mRNA and protein in the liver for two cytokine targets of caspase-1,IL-1βand IL-18. Interestingly,substantial baseline expression of pre-IL1βand pre-IL-18 was found in the liver.Inflammasome and caspase-1 activation was indicated by the significant increase in the active forms of IL-1βand IL-18 after LPS stimulation. CONCLUSION:Our results show that the Nalp3 inflammasome is upregulated and activated in the liver in response to LPS stimulation.

High fat diet feeding results in gender specific steatohepatitis and inflammasome activation

AIM:To develop an animal model that encompasses the different facets of non-alcoholic steatohepatitis(NASH),which has been a challenge.METHODS:In this study,we used a high fat diet(HFD)feeding supplemented with fructose and sucrose in the water mimicking the high-fructose corn syrup that is abundant in the diet in the United States.We used C57Bl/6 wild-type mice for short and long-term feedings of 6 and 16 wk respectively,and evaluated the extent of liver damage,steatosis,and inflammasome activation.Our methods included histopathological analysis to assess liver damage and steatosis,which involved H and E and oil-red-o staining;biochemical studies to look at ALT and triglyceride levels;RNA analysis using quantitative polymerase chain reaction;and cytokine analysis,which included the enzyme-linked immunosorbent assay method to look at interleukin(IL)-1βand tumor necrosis factor-α(TNFα)levels.Furthermore,at each length of feeding we also looked at insulin resistance and glucose tolerance using insulin tolerance tests(ITT)and glucose tolerance tests.RESULTS:There was no insulin resistance,steatosis,or inflammasome activation at 6 wk.In contrast,at16 wk we found significant insulin resistance demonstrated by impaired glucose and ITT in male,but not female mice.In males,elevated alanine aminotransferase and triglyceride levels,indicated liver damage and steatosis,respectively.Increased liver TNFαand monocyte chemoattractant protein-1 mRNA and protein,correlated with steatohepatitis.The inflammasome components,adaptor molecule,Aim2,and NOD-like receptor 4,increased at the mRNA level,and functional inflammasome activation was indicated by increased caspase-1 activity and IL-1βprotein levels in male mice fed a long-term HFD.Male mice on HFD had increasedα-smooth muscle actin and pro-collagen-1 mRNA indicating evolving fibrosis.In contrast,female mice displayed only elevated triglyceride levels,steatosis,and no fibrosis.CONCLUSION:Our data indicate gender differences in NASH.Male mice fed a long-term HFD display steatohepatitis and inflammasome activation,whereas female mice have steatosis without inflammation.