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Withaferin A alleviates fulminant hepatitis by targeting macrophage and NLRP3.

ABSTRACT: Fulminant hepatitis (FH) is an incurable clinical syndrome where novel therapeutics are warranted. Withaferin A (WA), isolated from herb Withania Somnifera, is a hepatoprotective agent. Whether and how WA improves D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced FH is unknown. This study was to evaluate the hepatoprotective role and mechanism of WA in GalN/LPS-induced FH. To determine the preventive and therapeutic effects of WA, wild-type mice were dosed with WA 0.5?h before or 2?h after GalN treatment, followed by LPS 30?min later, and then killed 6?h after LPS treatment. To explore the mechanism of the protective effect, the macrophage scavenger clodronate, autophagy inhibitor 3-methyladenine, or gene knockout mouse lines NLR family pyrin domain containing 3 (Nlrp3)-null, nuclear factor-erythroid 2-related factor 2 (Nrf2)-null, liver-specific AMP-activated protein kinase (Ampk)a1 knockout (Ampka1^?Hep) and liver-specific inhibitor of KB kinase ? (Ikkb) knockout (Ikkb^?Hep) mice were subjected to GalN/LPS-induced FH. In wild-type mice, WA potently prevented GalN/LPS-induced FH and inhibited hepatic NLRP3 inflammasome activation, and upregulated NRF2 and autophagy signaling. Studies with Nrf2-null, Ampka1^?Hep, and Ikkb^?Hep mice demonstrated that the hepatoprotective effect was independent of NRF2, hepatic AMPK?1, and I??B. Similarly, 3-methyladenine cotreatment failed to abolish the hepatoprotective effect of WA. The hepatoprotective effect of WA against GalN/LPS-induced FH was abolished after macrophage depletion, and partially reduced in Nlrp3-null mice. Consistently, WA alleviated LPS-induced inflammation partially dependent on the presence of NLRP3 in primary macrophage in vitro. Notably, WA potently and therapeutically attenuated GalN/LPS-induced hepatotoxicity. In conclusion, WA improves GalN/LPS-induced hepatotoxicity by targeting macrophage partially dependent on NLRP3 antagonism, while largely independent of NRF2 signaling, autophagy induction, and hepatic AMPK?1 and I??B. These results support the concept of treating FH by pharmacologically targeting macrophage and suggest that WA has the potential to be repurposed for clinically treating FH as an immunoregulator.

SUBMITTER: Xia Y

PROVIDER: S-EPMC7878893 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Withaferin A alleviates fulminant hepatitis by targeting macrophage and NLRP3.

Xia Yangliu Y Wang Ping P Yan Nana N Gonzalez Frank J FJ Yan Tingting T

Cell death & disease 20210211 2

Fulminant hepatitis (FH) is an incurable clinical syndrome where novel therapeutics are warranted. Withaferin A (WA), isolated from herb Withania Somnifera, is a hepatoprotective agent. Whether and how WA improves D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced FH is unknown. This study was to evaluate the hepatoprotective role and mechanism of WA in GalN/LPS-induced FH. To determine the preventive and therapeutic effects of WA, wild-type mice were dosed with WA 0.5 h before or 2 h after ...[more]

PMID: 33574236

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Project description:BACKGROUND & AIMS:The macrophage scavenger receptor 1 (Msr1, also called SRA) is a pattern recognition receptor primarily expressed on myeloid cells, which plays an important role in the maintenance of immune homeostasis. Since MSR1 expression was upregulated in the livers of patients with fulminant hepatitis (FH), we investigated the functional mechanism of Msr1 in FH pathogenesis. METHODS:Msr1-deficient (Msr1-/-) mice and their wild-type (WT) littermates were infected with mouse hepatitis virus strain-A59 (MHV-A59) to induce FH, and the levels of tissue damage, serum alanine aminotransferase, inflammatory cytokines and complement component 5a (C5a) were measured and compared. Liver injury was studied after MHV infection with or without neutrophil depletion. RESULTS:Our results showed that Msr1-/- mice were resistant to MHV-induced hepatitis. Treatment with the C5a receptor antagonist (C5aRa) diminished the differences in inflammatory responses and liver injury between MHV-infected wild-type and Msr1-/- mice, suggesting that C5a-induced pro-inflammatory response plays a critical role in the Msr1-mediated regulation of FH pathogenesis. We demonstrated that Msr1 efficiently enhanced transforming growth factor-activated kinase-1 phosphorylation in neutrophils upon MHV-A59 stimulation, thereby promoting the activation of the extracellular signal-regulated kinase pathway and subsequent NETosis formation. Moreover, we provided evidence that blockage of Msr1 attenuated the liver damage caused by MHV-A59 infection. CONCLUSIONS:Msr1 promotes the pathogenesis of virus-induced FH by enhancing induction of neutrophil NETosis and subsequent complement activation. Targeting Msr1 may be employed as a new immunotherapeutic strategy for FH. LAY SUMMARY:Virus-induced fulminant hepatitis (FH) is a disease with a high mortality worldwide. Enhanced levels of macrophage scavenger receptor 1 (Msr1) in the liver of patients with FH and of murine experimental FH indicated Msr1 plays a role in the pathogenesis of FH. Herein, we demonstrate that mice deficient in Msr1 are resistant to FH induced by MHV-A59, and the Msr1 inhibitor fucoidan suppresses the progression of FH in mice. Our study suggests that use of drugs inhibiting MSR1 function could be beneficial to patients with FH.

Project description:Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1.

Project description:BACKGROUND & AIMS:The innate immune system responds not only to bacterial signals, but also to non-infectious danger-associated molecular patterns that activate the NLRP3 inflammasome complex after tissue injury. Immune functions vary over the course of the day, but it is not clear whether these changes affect the activity of the NLRP3 inflammasome. We investigated whether the core clock component nuclear receptor subfamily 1 group D member 1 (NR1D1, also called Rev-erb?) regulates expression, activity of the NLRP3 inflammasome, and its signaling pathway. METHODS:We collected naïve peritoneal macrophages and plasma, at multiple times of day, from Nr1d1-/- mice and their Nr1d1+/+ littermates (controls) and analyzed expression NLRP3, interleukin 1? (IL1B, in plasma), and IL18 (in plasma). We also collected bone marrow-derived primary macrophages from these mice. Levels of NR1D1 were knocked down with small hairpin RNAs in human primary macrophages. Bone marrow-derived primary macrophages from mice and human primary macrophages were incubated with lipopolysaccharide (LPS) to induce expression of NLRP3, IL1B, and IL18; cells were incubated with LPS and adenosine triphosphate to activate the NLRP3 complex. We analyzed caspase 1 activity and cytokine secretion. NR1D1 was activated in primary mouse and human macrophages by incubation with SR9009; some of the cells were also incubated with an NLRP3 inhibitor or inhibitors of caspase 1. Nr1d1-/- mice and control mice were given intraperitoneal injections of LPS to induce peritoneal inflammation; plasma samples were isolated and levels of cytokines were measured. Nr1d1-/- mice, control mice, and control mice given injections of SR9009 were given LPS and D-galactosamine to induce fulminant hepatitis and MCC950 to specifically inhibit NLRP3; plasma was collected to measure cytokines and a marker of liver failure (alanine aminotransferase); liver tissues were collected and analyzed by quantitative polymerase chain reaction, immunohistochemistry, and flow cytometry. RESULTS:In peritoneal macrophages, expression of NLRP3 and activation of its complex varied with time of day (circadian rhythm)-this regulation required NR1D1. Primary macrophages from Nr1d1-/- mice and human macrophages with knockdown of NR1D1 had altered expression patterns of NLRP3, compared to macrophages that expressed NR1D1, and altered patterns of IL1B and 1L18 production. Mice with disruption of Nr1d1 developed more-severe acute peritoneal inflammation and fulminant hepatitis than control mice. Incubation of macrophage with the NR1D1 activator SR9009 reduced expression of NLRP3 and secretion of cytokines. Mice given SR9009 developed less-severe liver failure and had longer survival times than mice given saline (control). CONCLUSIONS:In studies of Nr1d1-/- mice and human macrophages with pharmacologic activation of NR1D1, we found NR1D1 to regulate the timing of NLRP3 expression and production of inflammatory cytokines by macrophages. Activation of NR1D1 reduced the severity of peritoneal inflammation and fulminant hepatitis in mice.

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Withaferin A alleviates fulminant hepatitis by targeting macrophage and NLRP3.

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Withaferin A alleviates fulminant hepatitis by targeting macrophage and NLRP3.

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