Fig 2: Murine ortholog of immunity-related GTPase family M (IRGM1) regulation by Sirtuin 1 (SIRT1) with or without interferon gamma (IFN-γ) or CD154 at different time points. RAW 264.7 macrophages were either infected with RH tachyzoites at a ratio of 5:1 (parasite/host cell) for 2 h or left in the culture plate without infection, followed by incubation with IFN-γ (1 μg/ml), CD154 (200 ng/ml), SRT1720 (5 μM), EX527 (10 μM), IFN-γ (1 μg/ml) and SRT1720 (5 μM), or IFN-γ (1 μg/ml) and EX527 (10 μM) for 2, 18, or 24 h before the collection of the cell lysates. We adjusted the protein concentration of each group of samples to be consistent and then carried out the experiment. Anti-IRGM1 antibody and anti-beta-Actin antibody were used at a 1:1,000 dilution. (A) The expression of IRGM1 in the Toxoplasma gondii infection group increased at 2 h but decreased at 18 and 24 h compared to that for the control group. Infected macrophages treated with IFN-γ displayed an upregulation of IRGM1 at 2, 18, and 24 h compared to that of the infection-only group. (B) Infected macrophages treated with CD154 showed no significant changes of IRGM1 compared to that for the infection-only group. (C) Infected macrophages treated with RH tachyzoites showed an upregulation of IRGM at 2 h and a downregulation at 18 or 24 h compared to that of the control group. Meanwhile, IRGM1 production was found to be decreased under SRT1720 stimulation and increased with the existence of EX527 at 2 h. However, both SRT1720 and EX527 failed to interfere with the IRGM1 level at 18 and 24 h. (D) When combining SIRT1 interference and IFN-γ stimulation, infected macrophages treated with IFN-γ and SRT720 showed a downregulation of IRGM at 2, 18, and 24 h compared to that of the IFN-γ group. Meanwhile, infected macrophages treated with IFN-γ and EX527 showed an upregulation of IRGM at 2, 18, and 24 h compared to that of the IFN-γ group. These results were obtained from three independent experiments. RH, T. gondii RH strain. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 3: Rapamycin-induced autophagy ameliorates LPS-induced AML12 hepatocyte injury model pretreated with Irgm1 shRNA.a Irgm1 knockdown by shRNA decreased the hepatocyte expression of Irgm1 and increased the concentration of ALT and AST in the supernatant, which was significantly decreased by pretreatment with rapamycin (all P < 0.05). b Increased expression of LC3II and the degradation of P62 in the LPS-induced AML12 hepatocyte injury model were inhibited by Irgm1 shRNA but were significantly reversed by pretreatment with rapamycin (all P < 0.05). c Expression of NLRP3, ASC, and caspase-1 was significantly increased in the LPS-induced AML12 hepatocyte injury model after Irgm1 knockdown but were significantly decreased by pretreatment with rapamycin (all P < 0.05). Compared to controls and the LPS-induced AML12 hepatocyte injury model, the levels of intrahepatic NLRP3, ASC, caspase-1, IL-1β, and TNF-α mRNA were upregulated after Irgm1 knockdown but were significantly recovered by pretreatment with rapamycin (all P < 0.05). All cell experiments were averaged from triplicate wells and repeated five times.

Fig 4: Intrahepatic Irgm1 expression, serum ALT/AST levels, and hepatic histological activity index (HAI) score in ALF mice.a Intrahepatic Irgm1 expression significantly decreased in ALF mice compared to controls (both P < 0.05). b Immunohistochemical staining (200×) showed that the Irgm1 protein was mainly expressed in hepatocytes and that its expression significantly decreased in ALF mice. c Representative appearance and H&E staining (200×) showing severe intrahepatic inflammation and necrosis, accompanied by increased hepatic HAI scores and serum ALT and AST levels (all P < 0.05).

Fig 5: The intrahepatic expression of IRGM, autophagy-related proteins (LC3II, P62), NLRP3, ASC, and caspase-1 in HBV-ACLF patients and HCs.a IRGM expression was decreased in HBV-ACLF patients compared to HCs (P < 0.05). b Expression of LC3II and degradation of P62 were decreased in HBV-ACLF patients compared to HCs (both P < 0.05). c Intrahepatic expression of NLRP3 inflammasome, ASC, and caspase-1, as well as serum levels of IL-1β and TNF-α were increased in HBV-ACLF patients compared to HCs (all P < 0.05). It should be noted that the Western Blot banding randomly selected from three cases of HBV-ACLF patients and HCs in each group were presented.