The activity of inflammasomes is increased after DGalN plus LPS challenge
To characterize the involvement of inflammasomes in DGalN plus LPS induced liver damage, the hepatic expressions of inflammasomes including NLRP-1, 2, 3, 6, 10, 12, AIM2, and NLRC4 were examined by quantitative PCR. Results exhibited that DGalN/LPS treatment upregulated the mRNA levels of hepatic NLRP2, NLRP3, and AIM2 significantly (NLRP1 not detected) (Fig. 1a). The hepatic expression and activity of caspase1 were increased remarkably after DGalN/LPS injection (Fig. 1b). Moreover, the results showed a marked rise of serum IL1β and IL18 levels in DGalN/LPS treated mice (Fig. 1c). The hepatic mRNA levels of IL1β and IL18 also increased significantly upon DGalN/LPS administration (Fig. 1d). Consistently, the expressions of NLRP3, pro-IL1β, mature IL1β, and IL18 were notably upregulated after DGalN/LPS injection (Fig. 1e). These data indicate that inflammasomes, especially NLRP3 inflammasome, are activated during DGalN/LPS challenge.
Role of NLRP3 inflammasome in DGalN/LPS caused fatal hepatitis
To address the importance of NLRP3 in DGalN/LPS caused hepatitis, we established a mouse model of DGalN/LPS-driven ALF by using Nlrp3−/− mice. Our results showed that a lethal dose of DGalN/LPS treatment caused 90% mortality in WT mice and 100% mortality in Nlrp3−/− mice within 9 h (Fig. 2a). Accordingly, representative pictures showed comparable severity of liver damage between WT and Nlrp3−/− mice challenged with a sublethal dose of DGalN/LPS (Fig. 2b). Moreover, WT and Nlrp3−/− mice had similar levels of serum aminotransferases after DGalN/LPS administration (Fig. 2c). Histological analysis exhibited that the extent of liver damage in Nlrp3−/− mice was not significantly different from control animals at 6 h after DGalN/LPS injection (Fig. 2d), characterized by similar areas of necrosis (Fig. 2e). Our data indicate that NLRP3 ablation does not protect mice from DGalN plus LPS caused fatal hepatitis.
NLRP3 deficiency has no effect on hepatocyte apoptosis and pyroptosis induced by DGalN/LPS
We next investigated whether NLRP3 deficiency affects hepatocyte apoptosis and pyroptosis induced by DGalN/LPS using TUNEL assay, IHC staining, and western blot assay. DGalN/LPS treatment induced a remarkable increase of TUNEL positive cells in both WT and Nlrp3−/− mice livers; however, no significant differences were observed between these two groups (Fig. 3a-b). Consistently, IHC analysis of hepatic cleaved caspase3 showed that the number of positively stained hepatocytes did not differ between Nlrp3−/− and WT mice (Fig. 3c-d). Additionally, both WT and Nlrp3−/− mice displayed similar levels of increased hepatocyte pyroptosis after DGalN/LPS administration, as assessed by hepatic GSDMDNterm peptide production (a marker of pyroptosis) (Fig. 3e). These data suggest that NLRP3 deficiency does not appear to affect the hepatocyte apoptosis and pyroptosis induced by DGalN/LPS.
NLRP3 inactivation has no significant effect on hepatic infiltration of proinflammatory cells in DGalN/LPS treated mice
DGalN/LPS-induced acute liver injury was associated with liver infiltration with remarkable immune cells. Our results showed that deficiency of NLRP3 does not affect the numbers of CD11b+ cells (Fig. 4a-b), macrophages (F4/80+) (Fig. 4c-d), and neutrophils (Ly6G+) (Fig. 4e-f) in mice livers under physiological conditions, as assessed by IHC staining. We found that WT mice livers displayed significantly increased numbers of CD11b+ cells (Fig. 4a-b), F4/80+ cells (Fig. 4c-d), and Ly6G+ cells (Fig. 4e-f) after DGalN/LPS treatment, but the numbers of these cells were comparable with those in Nlrp3−/− mice. These data indicate that NLRP3 does not play a major role in DGalN/LPS induced intrahepatic proinflammatory cell infiltration.
Nlrp3−/− mice have similar TNFα, but reduced IL1β levels compared with WT mice after DGalN/LPS treatment
We next examined whether NLRP3 ablation affects proinflammatory cytokines production in DGalN/LPS treated mice. The serum TNFα, IL6, and MCP-1 levels were remarkably increased in WT mice upon DGalN plus LPS treatment, but the levels in Nlrp3−/− mice were similar to those in WT mice (Fig. 5a). Consistently, no significant differences were observed in hepatic TNFα, IL6, and MCP-1 levels between Nlrp3−/− and WT mice after DGalN/LPS administration (Fig. 5b). Quantitative PCR also showed similar mRNA levels of TNFα, IL6, and MCP-1 between two groups (Fig. 5c). The increased mRNA level of IL1β also has no difference between Nlrp3−/− and WT mice liver after DGalN/LPS treatment (Fig. 5d). In contrast, the hepatic mature IL1β was elevated in two genotype mice after DGalN/LPS injection, but the increase was suppressed in Nlrp3−/− mice (Fig. 5e). Consistently, DGalN/LPS treatment induced rapid increases of serum IL1β levels in WT mice, however, the rise was significantly blunted in NLRP3 deficient mice (Fig. 5f). Nevertheless, in contrast to LPS treated alone, DGalN plus LPS administration displayed a much milder reduction of IL1β in NLRP3 deficient mice [33]. The incomplete blockage of IL1β production in Nlrp3−/− mice suggested that there might be NLRP3-independent pathways to produce IL1β after DGalN/LPS treatment. As predicted, NLRP2 and NLRP12 mRNA levels were markedly upregulated in the liver of NLRP3 deficient mice after DGalN plus LPS challenge (Fig. 5g). Collectively, the results indicate a limited role of NLRP3 in regulating DGalN/LPS leaded proinflammatory cytokines production.