Background: Macrophage migration inhibitory factor (MIF), a pluripotent immune regulator, is an emerging mediator in alcohol-related liver disease (ALD). MIF is associated with ALD progression through its chemokine-and cytokine-like activities.Methods: Mechanistic studies into the role of MIF in ethanol (EtOH)-induced liver injury were performed in Mif À/À mice and in C57BL/6J mice treated with a small-molecule MIF antagonist, MIF098, after Gao-Binge (acute-on-chronic) EtOH feeding, an EtOH feeding protocol associated with hepatic neutrophilia and induction of the unfolded protein response (UPR).Results: The MIF axis, for example, MIF and MIF receptors invariant polypeptide of major histocompatibility complex, class II antigen-associated (CD74), CXCR2, CXCR4, and CXCR7, was enhanced in the livers of alcoholic hepatitis (AH) patients as compared to healthy controls. Mif À/À mice were protected from hepatocellular injury after Gao-Binge feeding, independent of neutrophilia and inflammation, but were associated with the UPR. Interestingly, the UPR signature in AH patients and in mice following Gao-Binge feeding was biased toward cell death with increased expression of pro-cell death CCAAT-enhancer-binding protein homologous protein (CHOP) and decreased prosurvival GRP78. The UPR and liver injury 6 hours after binge were prevented both in Mif À/À mice and in MIF098-treated mice. However, both MIF interventions led to increased liver injury and exacerbated the hepatic UPR 9 hours after binge. Induction of upstream UPR signaling and expression of CHOP protein by thapsigargin in alpha mouse liver 12 hepatocytes were blunted by coexposure to MIF098, directly connecting MIF to UPR in hepatocytes.Conclusions: The current study revealed that, in addition to its cytokine/chemokine functions, MIF is an upstream regulator of UPR in response to EtOH feeding in mice. Importantly, both MIF and UPR can either protect or contribute to liver injury, dependent upon the stage or severity of EtOH-induced liver injury.

Background Sepsis and septic shock kill over 270,000 patients per year in the United States. Sepsis transitions from a hyper‐inflammatory to a hypo‐inflammatory phase. Alcohol dependence is a risk factor for mortality from sepsis. Ethanol (EtOH) exposure impairs pathogen clearance through mechanisms that are not fully understood. Sirtuin 2 (SIRT2) interferes with pathogen clearance in immune cells but its role in the effects of EtOH on sepsis is unknown. We studied the effect of EtOH exposure on hyper‐ and hypo‐inflammation and the role of SIRT2 in mice. Methods We exposed C57Bl/6 (WT) mice to EtOH via drinking water and used intraperitoneal cecal slurry (CS)‐induced sepsis to study: (i) 7‐day survival, (ii) leukocyte adhesion (LA) in the mesenteric microcirculation during hyper‐ and hypo‐inflammation, (iii) peritoneal cavity bacterial clearance, and (iv) SIRT2 expression in peritoneal macrophages. Using EtOH‐exposed and lipopolysaccharide (LPS)‐stimulated RAW 264.7 (RAW) cell macrophages for 4 hours or 24 hours, we studied: (i) tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), interleukin‐10 (IL‐10), and SIRT2 expression, and (ii) the effect of the SIRT2 inhibitor AK‐7 on inflammatory response at 24 hours. Lastly, we studied the effect of EtOH on sepsis in whole body Sirt2 knockout (SIRT2KO) mice during hyper‐ and hypo‐inflammation, bacterial clearance, and 7‐day survival. Results WT EtOH‐sepsis mice showed: (i) Decreased survival, (ii) Muted LA in the microcirculation, (iii) Lower plasma TNF‐α and IL‐6 expression, (iv) Decreased bacterial clearance, and (v) Increased SIRT2 expression in peritoneal macrophages versus vehicle‐sepsis. EtOH‐exposed LPS‐stimulated RAW cells showed: (i) Muted TNF‐α, IL‐6, and increased IL‐10 expression at 4 hours, (ii) endotoxin tolerance at 24 hours, and (iii) reversal of endotoxin tolerance with the SIRT2 inhibitor AK‐7. EtOH‐exposed SIRT2KO‐sepsis mice showed greater 7‐day survival, LA, and bacterial clearance than WT EtOH‐sepsis mice. Conclusion EtOH exposure decreases survival and reduces the inflammatory response to sepsis via increased SIRT2 expression. SIRT2 is a potential therapeutic target in EtOH with sepsis.

Background: The chemokine system of ligands and receptors is implicated in the progression of Alcohol-associated hepatitis (AH). Finding upstream regulators could lead to novel therapies. Methods: The coordinated expression of chemokines in livers of healthy controls (HC) and patients with AH in two distinct cohorts of patients with various chronic liver diseases. Studies in cultured hepatocytes and in tissue-specific knockouts were used for mechanistic insight into a potential upstream regulator of chemokine expression in AH.Results: Selected C-X-C chemokine members of the Interleukin 8 (IL8) chemokine family and C-C chemokine CCL20 were highly associated with AH compared to HC, but not in patients with liver diseases of other etiologies (NAFLD or HCV). Our previous studies implicate Macrophage migration inhibitory factor (MIF) as a pleiotropic cytokine/chemokine with the potential to coordinately regulate chemokine expression in AH. LPS-stimulated expression of multiple chemokines in cultured hepatocytes was dependent on MIF. Gao-binge ethanol feeding to mice induced a similar coordinated chemokine expression in livers of wild-type mice; this was prevented in hepatocyte-specific Mif knockout (Mif ΔHep ) mice. Conclusion:This study demonstrates that patients with AH exhibit a specific, coordinately expressed chemokine signature and hepatocyte-derived MIF might drive this inflammatory response.

Obesity increases morbidity and resource utilization in sepsis patients. The immune response in sepsis transitions from an endotoxin-responsive hyper- to an endotoxin-tolerant hypo-inflammatory phase. The majority of sepsis mortality occurs during hypo-inflammation. We reported prolonged hypo-inflammation with increased sirtuin 2 (SIRT2) expression in obese-septic mice. The effect of direct exposure to high-fat/free fatty acid (FFA) and the role of SIRT2 in immune cells during the transition to hypo-inflammation is not well-understood. Autophagy, a degradation process of damaged protein/organelles, is dysregulated during sepsis. Here, we investigated the effect of direct FFA exposure and the role of SIRT2 expression on autophagy as macrophages transition from hyper-to hypo-inflammation. We found, FFA-exposed RAW 264.7 cells with lipopolysaccharide (LPS) stimulation undergo endotoxin-sensitive (“sensitive”) hyper- followed by endotoxin tolerant (“tolerant”) hypo-inflammatory phases; SIRT2 expression increases significantly in tolerant cells. Autophagy proteins LC3b-II, and beclin-1 increase in FFA-sensitive and decrease in tolerant cells; p62 expressions continue to accumulate in tolerant cells. We observed that SIRT2 directly deacetylates α-tubulin and impairs autophagy clearance. Importantly, we find SIRT2 inhibitor AK-7 treatment during endotoxin tolerant phase reverses autophagy dysregulation with improved autophagy clearance in FFA-tolerant cells. Thus, we report impaired autophagosome formation and autophagy clearance via increased SIRT2 expression in FFA-exposed tolerant macrophages.

Alcohol abuse, reported by 1/8th critically ill patients, is an independent risk factor for death in sepsis. Sepsis kills over 270,000 patients/year in the US. We reported that the ethanol-exposure suppresses innate-immune response, pathogen clearance, and decreases survival in sepsis-mice via sirtuin 2 (SIRT2). SIRT2 is an NAD+-dependent histone-deacetylase with anti-inflammatory properties. We hypothesized that in ethanol-exposed macrophages, SIRT2 suppresses phagocytosis and pathogen clearance by regulating glycolysis. Immune cells use glycolysis to fuel increased metabolic and energy demand of phagocytosis. Using ethanol-exposed mouse bone marrow- and human blood monocyte-derived macrophages, we found that SIRT2 mutes glycolysis via deacetylating key glycolysis regulating enzyme phosphofructokinase-platelet isoform (PFKP), at mouse lysine 394 (mK394, human: hK395). Acetylation of PFKP at mK394 (hK395) is crucial for PFKP function as a glycolysis regulating enzyme. The PFKP also facilitates phosphorylation and activation of autophagy related protein 4B (Atg4B). Atg4B activates microtubule associated protein 1 light chain-3B (LC3). LC3 is a driver of a subset of phagocytosis, the LC3-associated phagocytosis (LAP), which is crucial for segregation and enhanced clearance of pathogens, in sepsis. We found that in ethanol-exposed cells, the SIRT2-PFKP interaction leads to decreased Atg4B-phosphorylation, decreased LC3 activation, repressed phagocytosis and LAP. Genetic deficiency or pharmacological inhibition of SIRT2 reverse PFKP-deacetylation, suppressed LC3-activation and phagocytosis including LAP, in ethanol-exposed macrophages to improve bacterial clearance and survival in ethanol with sepsis mice.

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