Protective role of cGAS in NASH is related to the maintenance of intestinal homeostasis.

BACKGROUND & AIMS: Various intracellular pathways regulate inflammation in NASH. Cyclic GMP-AMP synthase (cGAS) is a DNA sensor that activates STING and plays a role in inflammatory diseases. Here, we explored the role of cGAS in hepatic damage, steatosis, inflammation, and liver fibrosis in mouse models of NASH. METHODS: cGAS deficient (cGAS-KO) and STING deficient (STING-KO) mice received high fat-high cholesterol-high sugar diet (HF-HC-HSD) or relevant control diets. Livers were evaluated after 16 or 30 weeks. RESULTS: HF-HC-HSD diet, both at 16 and 30 weeks, resulted in increased cGAS protein expression as well as in increased ALT, IL-1ß, TNF-α and MCP-1 in wild-type (WT) mice compared to controls. Surprisingly, liver injury, triglyceride accumulation, and inflammasome activation were greater in HF-HC-HSD cGAS-KO compared to WT mice at 16 and to a lesser extent at 30 weeks. STING, a downstream target of cGAS was significantly increased in WT mice after HF-HC-HSD. In STING-KO mice after HF-HC-HSD feeding, we found increased ALT and attenuated MCP1 and IL-1ß expression compared to WT mice. Markers of liver fibrosis were increased in cGAS- and STING-KO mice compared to WT on HF-HC-HSD. We discovered that cGAS-KO mice had a significant increase in circulating endotoxin levels on HF-HC-HSD that correlated with changes in intestinal morphology which was exacerbated by HF-HC-HSD compared to WT mice. CONCLUSION: Our findings indicate that cGAS or STING deficiency exacerbate liver damage, steatosis, and inflammation in HF-HC-HSD diet-induced NASH, which might be linked to the disruption of the gut barrier.

Alcohol-induced extracellular ASC specks perpetuate liver inflammation and damage in alcohol-associated hepatitis even after alcohol cessation.

BACKGROUND AIMS: Prolonged systemic inflammation contributes to poor clinical outcomes in severe alcohol-associated hepatitis (AH) even after the cessation of alcohol use. However, mechanisms leading to this persistent inflammation remain to be understood. APPROACH RESULTS: We show that while chronic alcohol induces nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation in the liver, alcohol binge results not only in NLRP3 inflammasome activation but also in increased circulating extracellular apoptosis-associated speck-like protein containing a caspase recruitment domain (ex-ASC) specks and hepatic ASC aggregates both in patients with AH and in mouse models of AH. These ex-ASC specks persist in circulation even after the cessation of alcohol use. Administration of alcohol-induced-ex-ASC specks in vivo in alcohol-naive mice results in sustained inflammation in the liver and circulation and causes liver damage. Consistent with the key role of ex-ASC specks in mediating liver injury and inflammation, alcohol binge failed to induce liver damage or IL-1ß release in ASC-deficient mice. Our data show that alcohol induces ex-ASC specks in liver macrophages and hepatocytes, and these ex-ASC specks can trigger IL-1ß release in alcohol-naive monocytes, a process that can be prevented by the NLRP3 inhibitor, MCC950. In vivo administration of MCC950 reduced hepatic and ex-ASC specks, caspase-1 activation, IL-1ß production, and steatohepatitis in a murine model of AH. CONCLUSIONS: Our study demonstrates the central role of NLRP3 and ASC in alcohol-induced liver inflammation and unravels the critical role of ex-ASC specks in the propagation of systemic and liver inflammation in AH. Our data also identify NLRP3 as a potential therapeutic target in AH.

Neutrophil extracellular traps contribute to liver damage and increase defective low-density neutrophils in alcohol-associated hepatitis.

BACKGROUND & AIMS: In alcohol-associated hepatitis (AH), inflammation and neutrophil counts correlate with poor clinical outcomes. Here, we investigated how neutrophils contribute to liver damage in AH. METHODS: We isolated blood neutrophils from individuals with AH to examine neutrophil extracellular traps (NETs) and performed RNA sequencing to explore their unique characteristics. RESULTS: We observed a significant increase in NET production in AH. We also observed a unique low-density neutrophil (LDN) population in individuals with AH and alcohol-fed mice that was not present in healthy controls. Transcriptome analysis of peripheral LDNs and high-density neutrophils (HDNs) from individuals with AH revealed that LDNs exhibit a functionally exhausted phenotype, while HDNs are activated. Indeed, AH HDNs exhibited increased resting reactive oxygen species (ROS) production and produced more ROS upon lipopolysaccharide stimulation than control HDNs, whereas AH LDNs failed to respond to lipopolysaccharide. We show that LDNs are generated from HDNs after alcohol-induced NET release in vitro, and this LDN subset has decreased functionality, including reduced phagocytic capacity. Moreover, LDNs showed reduced homing capacity and clearance by macrophage efferocytosis; therefore, dysfunctional neutrophils could remain in the circulation and liver. Depletion of both HDNs and LDNs in vivo prevented alcohol-induced NET production and liver damage in mice. Granulocyte-colony stimulating factor treatment also ameliorated alcohol-induced liver injury in mice. CONCLUSION: Neutrophils contribute to liver damage through increased NET formation which increases defective LDNs in AH. Alcohol induces phenotypic changes in neutrophils; HDNs are activated whereas LDNs are defective. Our findings provide mechanistic insights that could guide the development of therapeutic interventions for AH. IMPACT AND IMPLICATIONS: In this study we discovered heterogeneity of neutrophils in alcohol-associated hepatitis, including high-density and low-density neutrophils that show hyper-activated or exhausted transcriptomic profiles, respectively. We found that alcohol induces neutrophil extracellular trap (NET) formation, which contributes to liver damage. NET release by high-density neutrophils resulted in low-density neutrophils that reside in the liver and escape clean-up by macrophages. Our findings help to understand the opposing neutrophil phenotypes observed in individuals with alcohol-associated hepatitis and provide mechanistic insights that could guide therapeutic strategies targeting neutrophils.

Granulocyte colony-stimulating factor attenuates liver damage by M2 macrophage polarization and hepatocyte proliferation in alcoholic hepatitis in mice.

Massive inflammation and liver failure are main contributors to the high mortality in alcohol-associated hepatitis (AH). In recent clinical trials, granulocyte colony-stimulating factor (G-CSF) therapy improved liver function and survival in patients with AH. However, the mechanisms of G-CSF-mediated beneficial effects in AH remain elusive. In this study, we evaluated effects of in vivo G-CSF administration, using a mouse model of AH. G-CSF treatment significantly reduced liver damage in alcohol-fed mice even though it increased the numbers of liver-infiltrating immune cells, including neutrophils and inflammatory monocytes. Moreover, G-CSF promoted macrophage polarization toward an M2-like phenotype and increased hepatocyte proliferation, which was indicated by an increased Ki67-positive signal colocalized with hepatocyte nuclear factor 4 alpha (HNF-4α) and cyclin D1 expression in hepatocytes. We found that G-CSF increased G-CSF receptor expression and resulted in reduced levels of phosphorylated ß-catenin in hepatocytes. In the presence of an additional pathogen-associated molecule, lipopolysaccharide (LPS), which is significantly increased in the circulation and liver of patients with AH, the G-CSF-induced hepatoprotective effects were abolished in alcohol-fed mice. We still observed increased Ki67-positive signals in alcohol-fed mice following G-CSF treatment; however, Ki67 and HNF-4α did not colocalize in LPS-challenged mice. Conclusion: G-CSF treatment increases immune cell populations, particularly neutrophil counts, and promotes M2-like macrophage differentiation in the liver. More importantly, G-CSF treatment reduces alcohol-induced liver injury and promotes hepatocyte proliferation in alcohol-fed mice. These data provide new insights into the understanding of mechanisms mediated by G-CSF and its therapeutic effects in AH.

Two Faces of Neutrophils in Liver Disease Development and Progression.

Neutrophils, the most abundant type of leukocyte in human blood, play a major role in host defense against invading pathogens and in sterile injury. Neutrophil infiltration is characteristic of inflammation because of its antimicrobial and cytotoxic activities. Neutrophils also actively participate in the resolution of inflammation and subsequent tissue repair by acting as a critical mediator between the inflammation and resolution phases of tissue damage. However, neutrophils that are consistently exposed to inflammatory conditions lose their self-resolving capabilities and maintain an inflammatory phenotype, further exacerbating tissue damage. The current review describes how neutrophils interact with tissue microenvironments and acquire disease-specific phenotypes under chronic inflammatory conditions. Here, we aim to provide a better understanding of neutrophil-mediated pathogenesis of various liver diseases.

Chronic alcohol-induced neuroinflammation involves CCR2/5-dependent peripheral macrophage infiltration and microglia alterations.

BACKGROUND: Chronic alcohol consumption is associated with neuroinflammation, neuronal damage, and behavioral alterations including addiction. Alcohol-induced neuroinflammation is characterized by increased expression of proinflammatory cytokines (including TNFα, IL-1ß, and CCL2) and microglial activation. We hypothesized chronic alcohol consumption results in peripheral immune cell infiltration to the CNS. Since chemotaxis through the CCL2-CCR2 signaling axis is critical for macrophage recruitment peripherally and centrally, we further hypothesized that blockade of CCL2 signaling using the dual CCR2/5 inhibitor cenicriviroc (CVC) would prevent alcohol-induced CNS infiltration of peripheral macrophages and alter the neuroinflammatory state in the brain after chronic alcohol consumption. METHODS: C57BL/6J female mice were fed an isocaloric or 5% (v/v) ethanol Lieber DeCarli diet for 6 weeks. Some mice received daily injections of CVC. Microglia and infiltrating macrophages were characterized and quantified by flow cytometry and visualized using CX3CR1eGFP/+ CCR2RFP/+ reporter mice. The effect of ethanol and CVC treatment on the expression of inflammatory genes was evaluated in various regions of the brain, using a Nanostring nCounter inflammation panel. Microglia activation was analyzed by immunofluorescence. CVC-treated and untreated mice were presented with the two-bottle choice test. RESULTS: Chronic alcohol consumption induced microglia activation and peripheral macrophage infiltration in the CNS, particularly in the hippocampus. Treatment with CVC abrogated ethanol-induced recruitment of peripheral macrophages and partially reversed microglia activation. Furthermore, the expression of proinflammatory markers was upregulated by chronic alcohol consumption in various regions of the brain, including the cortex, hippocampus, and cerebellum. Inhibition of CCR2/5 decreased alcohol-mediated expression of inflammatory markers. Finally, microglia function was impaired by chronic alcohol consumption and restored by CVC treatment. CVC treatment did not change the ethanol consumption or preference of mice in the two-bottle choice test. CONCLUSIONS: Together, our data establish that chronic alcohol consumption promotes the recruitment of peripheral macrophages into the CNS and microglia alterations through the CCR2/5 axis. Therefore, further exploration of the CCR2/5 axis as a modulator of neuroinflammation may offer a potential therapeutic approach for the treatment of alcohol-associated neuroinflammation.

Role of non-Genetic Risk Factors in Exacerbating Alcohol-related organ damage.

This review provides a summary of the symposium titled "Role of Non-Genetic Risk Factors in Exacerbating Alcohol-Related Organ Damage", which was held at the 42nd Annual Meeting of the Research Society on Alcoholism. The goals of the symposium were to provide newer insights into the role of non-genetic factors, including specific external factors, notably infectious agents or lifestyle factors, that synergistically act to exacerbate alcohol pathogenicity to generate more dramatic downstream biological defects. This summary of the symposium will benefit junior/senior basic scientists and clinicians currently investigating/treating alcohol-induced organ pathology, as well as undergraduate, graduate, and post-graduate students and fellows.

Factors Associated with Metabolic Syndrome among Middle-Aged Women in their 50s: Based on National Health Screening Data.

This study aimed to identify the risk factors associated with metabolic syndrome among middle-aged women in their 50s to provide a strategy for managing the metabolic syndrome of those whose prevalence is rapidly increasing. Secondary data from the 2012 Korean National Health Insurance Service Medical check-up cohort database were analyzed. Participants included 36,582 middle-aged women in their 50s from the cohort who received a general medical check-up. The risk factors were estimated using logistic regression analysis. Metabolic syndrome was identified in 14.6% of the surveyed persons among middle-aged women in their 50s. Working women, low household income levels, country residents, high body mass index (BMI), total cholesterol of over 240 mg/dl, non-drinker, non-exerciser, history of diabetes or hypertension, and family history of diabetes were associated with increased risk of metabolic syndrome. It is necessary to prepare a strategy to increase access to health care services so that socioeconomic vulnerability does not lead to negative health behavior such as obesity and lack of physical activity. In particular, we recommend active interventions at workplaces for the working women who have a higher risk of metabolic syndrome.

Inhibition of the Inflammasome Signaling Cascade Reduces Alcohol Consumption in Female But Not Male Mice.

BACKGROUND: Alcohol use disorder is a significant societal and medical burden that is associated with both organ pathology and addiction. Excessive alcohol use results in neuroinflammation characterized by activation of the inflammasome, a multiprotein complex, and IL-1ß increase in the brain. Recent studies suggest that inflammation could contribute to alcohol addiction. Here, we targeted components of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome cascade, which senses and responds to immunologic stimuli, to determine whether NLRP3 inhibition modulates alcohol consumption. METHODS: C57BL/6J male and female mice were provided a 2-bottle choice of alcohol at increasing concentrations (3, 6, 9, and 12%, 4 days each) or water, and some were treated with daily injections of an NLRP3 inhibitor (MCC950), a caspase-1 inhibitor (VX765), IL-1 receptor antagonist (IL-1ra; anakinra), or vehicle injection. RESULTS: Treatment with VX765, MCC950, and IL-1ra significantly reduced alcohol consumption and preference in female mice (p < 0.05). Treatment with MCC950 and IL-1ra reduced alcohol consumption, while IL-1ra reduced alcohol preference in male mice (p < 0.05). VX765 did not affect alcohol consumption or preference in male mice. CONCLUSIONS: These findings highlight gender differences in alcohol preference and demonstrate that inhibition of different steps in inflammasome signaling can reduce alcohol consumption in females. Inhibition of NLRP3 inflammasome activation and the inflammasome-IL-1ß cascade opens novel insights into the development of new therapies to address alcohol use disorder in an era of targeted and precision medicine.

Pharmacological Inhibition of CCR2/5 Signaling Prevents and Reverses Alcohol-Induced Liver Damage, Steatosis, and Inflammation in Mice.

Kupffer cell and macrophage (MØ) activation contributes to steatosis, inflammation, and fibrosis in alcoholic liver disease (ALD). We found increased frequency of MØ, T cells, and expression of C-C chemokine receptor type 2 (Ccr2) and C-C chemokine receptor type 5 (Ccr5) in the livers of patients with ALD, and increased circulating chemokines, C-C chemokine ligand types 2 (CCL2), and C-C chemokine ligand types 5 (CCL5) in patients with alcoholic hepatitis. We hypothesized that inhibition of CCL2 signaling with the dual CCR2/5 inhibitor, cenicriviroc (CVC), would attenuate ALD. In a mouse model of ALD, liver injury (alanine aminotransferase [ALT]) and steatosis were prevented by CVC whether administered as "prevention" throughout the alcohol feeding or as "treatment" started after the development of ALD. Alcohol-induced increases in early liver fibrosis markers (sirius red, hydroxyproline, and collagen-1) were normalized by both modes of CVC administration. We found that prevention and treatment with CVC reversed alcohol-related increases in liver mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and CCL2. CVC administration regimens prevented the increase in infiltrating MØ (F4/80lo CD11bhi ) and reduced proinflammatory Ly6Chi MØ in livers of alcohol-fed mice. CVC increased liver T-cell numbers and attenuated Il-2 expression without an effect on CD69+ or CD25+ T-cell expression. In vitro, CVC inhibited CCL2-induced increases in hepatocyte fatty acid synthase (Fasn) and adipose differentiation-related protein (Adrp), whereas it augmented acyl-coenzyme A oxidase 1 (Acox-1), proliferator-activated receptor gamma co-activator alpha (Pgc1α) and uncoupling protein 2 expression, suggesting mechanisms for attenuated hepatocyte steatosis. We found that CCL2 and CCL5 sensitized hepatocytes to lipopolysaccharide-induced liver injury (TNF-α, ALT, and lactate dehydrogenase release). Alcohol feeding induced apoptosis (poly ADP-ribose polymerase [PARP] and caspase-3 [CASP-3] cleavage) and pyroptosis (gasdermin D [GSDMD] cleavage) in livers, and CVC prevented both of these forms of cell death. Conclusion: Together, our data demonstrate preclinical evidence for CCR2/CCR5 inhibition with CVC as a potent intervention to ameliorate alcohol-induced steatohepatitis and liver damage.

Reduced gut microbiome protects from alcohol-induced neuroinflammation and alters intestinal and brain inflammasome expression.

BACKGROUND: The end-organ effects of alcohol span throughout the entire body, from the gastrointestinal tract to the central nervous system (CNS). In the intestine, alcohol use changes the microbiome composition and increases gut permeability allowing translocation of microbial components into the circulation. Gut-derived pathogen-associated signals initiate inflammatory responses in the liver and possibly elsewhere in the body. Because previous studies showed that the gut microbiome contributes to alcohol-induced liver disease, we hypothesized that antibiotic administration to reduce the gut microbiome would attenuate alcohol-induced inflammation in the brain and small intestine (SI). METHODS: Six- to 8-week-old C57BL/6J female mice were fed alcohol in a liquid diet or a calorie-matched control diet for 10 days with an acute alcohol binge or sugar on the final day (acute-on-chronic alcohol administration). Some mice were treated with oral antibiotics daily to diminish the gut microbiome. We compared serum levels of TNFα, IL-6, and IL-1ß by ELISA; expression of cytokines Tnfα, Mcp1, Hmgb1, Il-17, Il-23, Il-6, and Cox2; and inflammasome components Il-1ß, Il-18, Casp1, Asc, and Nlrp3 in the CNS and SI by qRT-PCR. Microglial morphology was analyzed using immunohistochemical IBA1 staining in the cortex and hippocampus. RESULTS: Antibiotics dramatically reduced the gut microbiome load in both alcohol- and pair-fed mice. Alcohol-induced neuroinflammation and increase in SI cytokine expression were attenuated in mice with antibiotic treatment. Acute-on-chronic alcohol did not induce serum TNFα, IL-6, and IL-1ß. Alcohol feeding significantly increased the expression of proinflammatory cytokines such as Tnfα, Mcp1, Hmgb1, Il-17, and Il-23 in the brain and intestine. Reduction in the gut bacterial load, as a result of antibiotic treatment, attenuated the expression of all of these alcohol-induced proinflammatory cytokines in both the brain and SI. Alcohol feeding resulted in microglia activation and morphologic changes in the cortex and hippocampus characterized by a reactive phenotype. These alcohol-induced changes were abrogated following an antibiotic-induced reduction in the gut microbiome. Unexpectedly, antibiotic treatment increased the mRNA expression of some inflammasome components in both the brain and intestine. CONCLUSIONS: Our data show for the first time that the acute-on-chronic alcohol administration in mice induces both neuroinflammation and intestinal inflammation and that reduction in the intestinal bacterial load can attenuate alcohol-associated CNS and gut inflammation. Gut microbiome-derived signals contribute to neuroinflammation in acute-on-chronic alcohol exposure.

Abnormal neutrophil traps and impaired efferocytosis contribute to liver injury and sepsis severity after binge alcohol use.

BACKGROUND & AIMS: Neutrophil extracellular traps (NETs) are an important strategy utilized by neutrophils to immobilize and kill invading microorganisms. Herein, we studied NET formation and the process of neutrophil cell death (NETosis), as well as the clearance of NETs by macrophages (MΦ) (efferocytosis) in acute sepsis following binge drinking. METHODS: Healthy volunteers consumed 2 ml of vodka/kg body weight, before blood endotoxin and 16 s rDNA were measured. Peripheral neutrophils were isolated and exposed to alcohol followed by phorbol 12-myristate 13-acetate (PMA) stimulation. Mice were treated with three alcohol binges and intraperitoneal lipopolysaccharide (LPS) to assess the dynamics of NET formation and efferocytosis. In vivo, anti-Ly6G antibody (IA8) was used for neutrophil depletion. RESULTS: Inducers of NETs (endotoxin and bacterial DNA) significantly increased in the circulation after binge alcohol drinking in humans. Ex vivo, alcohol alone increased NET formation, but upon PMA stimulation alcohol attenuated NET formation. Binge alcohol in mice resulted in a biphasic response to LPS. Initially, binge alcohol reduced LPS-induced NET formation and resulted in a diffuse distribution of neutrophils in the liver compared to alcohol-naïve mice. Moreover, indicators of NET formation including citrullinated histone H3, neutrophil elastase, and neutrophil myeloperoxidase were decreased at an early time point after LPS challenge in mice receiving binge alcohol, suggesting decreased NET formation. However, in the efferocytosis phase (15 h after LPS) citrullinated histone-H3 was increased in the liver in alcohol binge mice, suggesting decreased clearance of NETs. In vitro alcohol treatment reduced efferocytosis and phagocytosis of NETotic neutrophils and promoted expression of CD206 on MΦ. Finally, depletion of neutrophils prior to binge alcohol ameliorated LPS-induced systemic inflammation and liver injury in mice. CONCLUSIONS: Dysfunctional NETosis and efferocytosis following binge drinking exacerbate liver injury associated with sepsis. LAY SUMMARY: Disease severity in alcoholic liver disease (ALD) is associated with a significant presence of neutrophils (a type of immune cell) in the liver. It remains unknown how alcohol affects the capacity of neutrophils to control infection, a major hallmark of ALD. We found that binge alcohol drinking impaired important strategies used by neutrophils to contain and resolve infection, resulting in increased liver injury during ALD.

Correction: Alcohol-related changes in the intestinal microbiome influence neutrophil infiltration, inflammation and steatosis in early alcoholic hepatitis in mice.

[This corrects the article DOI: 10.1371/journal.pone.0174544.].

Alcohol-induced miR-155 and HDAC11 inhibit negative regulators of the TLR4 pathway and lead to increased LPS responsiveness of Kupffer cells in alcoholic liver disease.

Inflammation promotes the progression of alcoholic liver disease. Alcohol sensitizes KCs to gut-derived endotoxin (LPS); however, signaling pathways that perpetuate inflammation in alcoholic liver disease are only partially understood. We found that chronic alcohol feeding in mice induced miR-155, an inflammatory miRNA in isolated KCs. We hypothesized that miR-155 might increase the responsiveness of KCs to LPS via targeting the negative regulators of LPS signaling. Our results revealed that KCs that were isolated from alcohol-fed mice showed a decrease in IRAK-M, SHIP1, and PU.1, and an increase in TNF-α levels. This was specific to KCs, as no significant differences were observed in these genes in hepatocytes. We found a causal effect of miR-155 deficiency on LPS responsiveness, as KCs that were isolated from miR-155 KO mice showed a greater induction of IRAK-M, SHIP1, and suppressor of cytokine signaling 1 after LPS treatment. C/EBPß, a validated miR-155 target, stimulates IL-10 transcription. We found a higher induction of C/EBPß and IL-10 in KCs that were isolated from miR-155 KO mice after LPS treatment. Gain- and loss-of-function studies affirmed that alcohol-induced miR-155 directly regulates IRAK-M, SHIP1, suppressor of cytokine signaling 1, and C/EBPß, as miR-155 inhibition increased and miR-155 overexpression decreased these genes in LPS or alcohol-pretreated wild-type KCs. HDAC11, a regulator of IL-10, was significantly increased and IL-10 was decreased in KCs that were isolated from alcohol-fed mice. Functionally, knockdown of HDAC11 with small interfering RNA resulted in an IL-10 increase in LPS or alcohol-pretreated Mϕ. We found that acetaldehyde and NF-κB pathways regulate HDAC11 levels. Collectively, our results indicate that the alcohol-induced responsiveness of KCs to LPS, in part, is governed by miR-155 and HDAC11.

Application of radiographic images in diagnosis and treatment of deep neck infections with necrotizing fasciitis: a case report.

The advent and wide use of antibiotics have decreased the incidence of deep neck infection. When a deep neck infection does occur, however, it can be the cause of significant morbidity and death, resulting in airway obstruction, mediastinitis, pericarditis, epidural abscesses, and major vessel erosion. In our clinic, a patient with diffuse chronic osteomyelitis of mandible and fascial space abscess and necrotic fasciitis due to odontogenic infection at the time of first visit came. We successfully treated the patient by early diagnosis using contrast-enhanced CT and follow up dressing through the appropriate use of radiographic images.