An altered expression of miR-376a-3p and miR-20a-5p in peripheral blood exosomes regulates the autophagy and inflammatory systemic substrates, and relates to the smoking habit and age in Crohn's disease.

miRNAs are short single-stranded noncoding RNAs that participate as epigenetic regulators in inflammatory bowel disease. Most miRNAs detectable in serum are concentrated in exosomes, with relevant cargo for immunobiological processes. We set to evaluate the exosomes miRNAs content in the serum of patients with Crohn's disease (CD) and run a prospective observational study on CD patients on biological monotherapy and healthy controls. miRNA cargo was evaluated in peripheral blood-derived exosomes. Serum autophagy and inflammatory substrates were measured. Patients were followed for 6 months. Patients (n = 28) showed an overexpression of miR-376a-3p and a downregulation of miR-20a-5p compared to controls (n = 10), without significant differences between patients according to biologics. Serum autophagy substrates ATG4C (r = .57; p = .001) and ACRV1C (r = .66; p = .001) inversely correlated with miR-376a-3p expression, whereas IGF1R correlated with miR-20a-5p expression (r = .42; p = .02). Th1-related cytokines correlated with miR-376a-3p expression, whereas the Th17-associated cytokines inversely correlated with miR-20a-5p expression. Smoking (ß = -2.301 CI 95% -3.790/-0.811, p = .004) remained as independent factor related to the overexpression of miR-376a-3p, whereas diagnosis before 16 years of age (ß = 2.044 CI 95% 0.934/3.154, p = .001) and a younger age of patients (ß = -.720 CI 95% -0.108/-0.035, p = .001) were related to decreased miR-20a-5p expression. Seven patients (25%) had a flare in the 6-month follow-up. Patients with overexpression of miR-376a-3p at the baseline showed an increased risk of flare during this period (OR 0.475 [0.237-0.950], p = .035). Finally, a comparative miRNA signature between biologic monotherapies was also explored. Targeting miR-376a-3p and miR-20a-5p epigenetic regulators may yield homeostatic effects on relevant biological processes related to disease progression in CD patients.

Aripiprazole Cytotoxicity Coincides with Activation of the Unfolded Protein Response in Human Hepatic Cells.

Schizophrenia is a mental disease that results in decreased life expectancy and well-being by promoting obesity and sedentary lifestyles. Schizophrenia is treated by antipsychotic drugs. Although the second-generation antipsychotics (SGA), Olanzapine and Aripiprazole, are more effective in treating schizophrenia, they display a higher risk of metabolic side effects, mostly by development of diabetes and insulin resistance, weight gain, and dyslipidemia. Endoplasmic reticulum (ER) stress is induced when ER homeostasis of lipid biosynthesis and protein folding is impaired. This leads to the activation of the unfolded protein response (UPR), a signaling cascade that aims to restore ER homeostasis or initiate cell death. Chronic conditions of ER stress in the liver are associated with diabetes and perturbed lipid metabolism. These metabolic dysfunctions resemble the pharmacological side effects of SGAs. We therefore investigated whether SGAs promote the UPR in human and mouse hepatocytes. We observed full-fledged activation of ER stress by Aripiprazole not by Olanzapine. This occurred at low micromolar concentrations and to variable intensities in different cell types, such as hepatocellular carcinoma, melanoma, and glioblastoma. Mechanistically, Aripiprazole caused depletion of ER calcium, leading to activation of inositol-requiring enzyme 1 (IRE1)and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), two major transducers of the UPR. Cells underwent apoptosis with Aripiprazole treatment, which coincided with UPR induction, and this effect was reduced by adding glutathione without affecting UPR itself. Deletion of IRE1 from HepG2, a human liver cancer cell line, protected cells from Aripiprazole toxicity. Our study reveals for the first time a cytotoxic effect of Aripiprazole that involves the induction of ER stress. SIGNIFICANCE STATEMENT: The antischizophrenic drug Aripiprazole exerts cytotoxic properties at high concentrations. This study shows that this cytotoxicity is associated with the induction of endoplasmic reticulum (ER) stress and IRE1 activation, mechanisms involved in diet-induced obesity. Aripiprazole induced ER stress and calcium mobilization from the ER in human and mouse hepatocytes. Our study highlights a new mechanism of Aripiprazole that is not related to its effect on dopamine signaling.

ß-Hydroxybutyrate protects from alcohol-induced liver injury via a Hcar2-cAMP dependent pathway.

BACKGROUND & AIMS: Sterile inflammation resulting in alcoholic hepatitis (AH) occurs unpredictably after many years of excess alcohol intake. The factors responsible for the development of AH are not known but mitochondrial damage with loss of mitochondrial function are common features. Hcar2 is a G-protein coupled receptor which is activated by ß-hydroxybutyrate (BHB). We aimed to determine the relevance of the BHB-Hcar2 pathway in alcoholic liver disease. METHODS: We tested if loss of BHB production can result in increased liver inflammation. We further tested if BHB supplementation is protective in AH through interaction with Hcar2, and analyzed the immune and cellular basis for protection. RESULTS: Humans with AH have reduced hepatic BHB, and inhibition of BHB production in mice aggravated ethanol-induced AH, with higher plasma alanine aminotransferase levels, increased steatosis and greater neutrophil influx. Conversely supplementation of BHB had the opposite effects with reduced alanine aminotransferase levels, reduced steatosis and neutrophil influx. This therapeutic effect of BHB is dependent on the receptor Hcar2. BHB treatment increased liver Il10 transcripts, and promoted the M2 phenotype of intrahepatic macrophages. BHB also increased the transcriptional level of M2 related genes in vitro bone marrow derived macrophages. This skewing towards M2 related genes is dependent on lower mitochondrial membrane potential (Δψ) induced by BHB. CONCLUSIONS: Collectively, our data shows that BHB production during excess alcohol consumption has an anti-inflammatory and hepatoprotective role through an Hcar2 dependent pathway. This introduces the concept of metabolite-based therapy for AH. LAY SUMMARY: Alcoholic hepatitis is a life-threatening condition with no approved therapy that occurs unexpectedly in people who consume excess alcohol. The liver makes many metabolites, and we demonstrate that loss of one such metabolite ß-hydroxybutyrate occurs in patients with alcoholic hepatitis. This loss can increase alcohol-induced liver injury, and ß-hydroxybutyrate can protect from alcohol-induced liver injury via a receptor on liver macrophages. This opens the possibility of metabolite-based therapy for alcoholic hepatitis.

Treatment with non-selective beta-blockers affects the systemic inflammatory response to bacterial DNA in patients with cirrhosis.

BACKGROUND & AIMS: The use of non-selective beta-blockers has been associated with lower rates of infection and reduced infection-associated morbidity in patients with cirrhosis. However, it is unknown if these drugs modify the systemic inflammatory response to circulating bacterial DNA. METHODS: Sixty-three patients with cirrhosis were included during an episode of decompensation by ascites. Thirty of those patients were on beta-blockers. Blood samples were obtained after each patient had been in the supine position for at least 30 minutes in a quiet atmosphere. Bacterial DNA, serum cytokines, nitric oxide, and LPS were determined. Phagocytic and oxidative burst activities were determined in polymorphonuclear cells from the patients. RESULTS: The detection rate of bacterial DNA in the blood was the same (33%) for patients not treated and treated with non-selective beta-blockers. Patients naive to non-selective beta-blockers showed significantly higher serum levels of IL6, IFN-gamma and IL10 in response to the presence of bacterial DNA. Patients treated with non-selective beta-blockers showed higher basal inflammatory activity that did not change with the presence of bacterial DNA. Monocytes and granulocytes from patients treated with non-selective beta-blockers showed a significantly increased phagocytic capacity in the presence of bacterial DNA. CONCLUSIONS: In patients with cirrhosis, chronic treatment with beta-blockers is associated with a higher unstimulated production of serum cytokines and an increased phagocytic activity in the presence of bacterial DNA.

Absent in melanoma 2 triggers a heightened inflammasome response in ascitic fluid macrophages of patients with cirrhosis.

BACKGROUND & AIMS: Inflammation is a common event in the pathogenesis of liver cirrhosis. The inflammasome pathway has acquired significant relevance in the pathogenesis of inflammation, but its role in the inflammatory response in patients with decompensated cirrhosis remains unexplored. METHODS: We performed a prospective study in which 44 patients with decompensated cirrhosis and 12 healthy volunteers were included. We isolated macrophages from blood and ascitic fluid and assessed the expression and activation of the inflammasome, its response to priming by bacterial products, and its association with the degree of liver disease. RESULTS: Macrophages from sterile ascitic fluids showed constitutive activation of caspase-1 and a marked increase in the expression of IL-1ß, IL-18, and absent in melanoma 2 (AIM2) when compared to blood macrophages. Pre-stimulation of blood-derived macrophages from cirrhotic patients with bacterial DNA increased the expression of AIM2 and induced a higher AIM2-mediated inflammasome response than priming with other bacterial products such as lipopolysaccharide. By contrast, activation of the AIM2 inflammasome did not require a priming signal in ascitic fluid-derived macrophages, demonstrating the preactivated state of the inflammasome in these cells. Last, higher IL-1ß and IL-18 production by ascitic fluid macrophages correlated with a more advanced Child-Pugh score. CONCLUSIONS: The inflammasome is highly activated in the ascitic fluid of cirrhotic patients, which may explain the exacerbated inflammatory response observed in these patients under non-infected conditions. Clinically, activation of the inflammasome is associated with a higher degree of liver disease.

Use of proton pump inhibitors decrease cellular oxidative burst in patients with decompensated cirrhosis.

BACKGROUND AND AIMS: Proton pump inhibitors (PPIs) are commonly used antisecretory drugs and have been linked to an increased risk of bacterial infections in cirrhosis. We investigated whether the treatment with PPIs in cirrhosis affects the oxidative burst activity of granulocytes and monocytes and its possible interference with serum norfloxacin (Nflx) levels in these patients. METHODS: 70 patients with cirrhosis and ascitic fluid and 24 healthy controls were included in the study and distributed into groups according to the regular use of PPIs and/or norfloxacin. The blood granulocyte and monocyte's phagocytic activity and oxidative burst were evaluated by flow cytometry. Blood levels of norfloxacin were measured by HPLC and bacterial translocation was evaluated by detection of bacterial DNA in blood. RESULTS: Use of PPIs was associated with a decreased granulocyte and monocyte oxidative burst, but not of phagocytic activity, as compared with patients not receiving PPIs. PPIs use did not affect serum norfloxacin levels in patients. A not significant trend to an increased bacterial DNA translocation was observed in patients receiving PPIs, including patients simultaneously receiving norfloxacin. CONCLUSIONS: PPIs significantly decrease cellular oxidative burst in cirrhosis. This fact may provide a pathogenic explanation to the reported high rates of bacterial infections in this setting, and strongly suggests that PPIs should only be used in patients with cirrhosis when clinically indicated.

Specific targeting of the NRF2/ß-TrCP axis promotes beneficial effects in NASH.

Non-alcoholic steatohepatitis (NASH) is a common chronic liver disease that compromises liver function, for which there is not a specifically approved medicine. Recent research has identified transcription factor NRF2 as a potential therapeutic target. However, current NRF2 activators, designed to inhibit its repressor KEAP1, exhibit unwanted side effects. Alternatively, we previously introduced PHAR, a protein-protein interaction inhibitor of NRF2/ß-TrCP, which induces a mild NRF2 activation and selectively activates NRF2 in the liver, close to normal physiological levels. Herein, we assessed the effect of PHAR in protection against NASH and its progression to fibrosis. We conducted experiments to demonstrate that PHAR effectively activated NRF2 in hepatocytes, Kupffer cells, and stellate cells. Then, we used the STAM mouse model of NASH, based on partial damage of endocrine pancreas and insulin secretion impairment, followed by a high fat diet. Non-invasive analysis using MRI revealed that PHAR protects against liver fat accumulation. Moreover, PHAR attenuated key markers of NASH progression, including liver steatosis, hepatocellular ballooning, inflammation, and fibrosis. Notably, transcriptomic data indicate that PHAR led to upregulation of 3 anti-fibrotic genes (Plg, Serpina1a, and Bmp7) and downregulation of 6 pro-fibrotic (including Acta2 and Col3a1), 11 extracellular matrix remodeling, and 8 inflammatory genes. Overall, our study suggests that the mild activation of NRF2 via the protein-protein interaction inhibitor PHAR holds promise as a strategy for addressing NASH and its progression to liver fibrosis.

Saturated fatty acid-enriched small extracellular vesicles mediate a crosstalk inducing liver inflammation and hepatocyte insulin resistance.

Background & Aims: Lipotoxicity triggers non-alcoholic fatty liver disease (NAFLD) progression owing to the accumulation of toxic lipids in hepatocytes including saturated fatty acids (SFAs), which activate pro-inflammatory pathways. We investigated the impact of hepatocyte- or circulating-derived small extracellular vesicles (sEV) secreted under NAFLD conditions on liver inflammation and hepatocyte insulin signalling. Methods: sEV released by primary mouse hepatocytes, characterised and analysed by lipidomics, were added to mouse macrophages/Kupffer cells (KC) to monitor internalisation and inflammatory responses. Insulin signalling was analysed in hepatocytes exposed to conditioned media from sEV-loaded macrophages/KC. Mice were i.v. injected sEV to study liver inflammation and insulin signalling. Circulating sEV from mice and humans with NAFLD were used to evaluate macrophage-hepatocyte crosstalk. Results: Numbers of sEV released by hepatocytes increased under NAFLD conditions. Lipotoxic sEV were internalised by macrophages through the endosomal pathway and induced pro-inflammatory responses that were ameliorated by pharmacological inhibition or deletion of Toll-like receptor-4 (TLR4). Hepatocyte insulin signalling was impaired upon treatment with conditioned media from macrophages/KC loaded with lipotoxic sEV. Both hepatocyte-released lipotoxic sEV and the recipient macrophages/KC were enriched in palmitic (C16:0) and stearic (C18:0) SFAs, well-known TLR4 activators. Upon injection, lipotoxic sEV rapidly reached KC, triggering a pro-inflammatory response in the liver monitored by Jun N-terminal kinase (JNK) phosphorylation, NF-κB nuclear translocation, pro-inflammatory cytokine expression, and infiltration of immune cells into the liver parenchyma. sEV-mediated liver inflammation was attenuated by pharmacological inhibition or deletion of TLR4 in myeloid cells. Macrophage inflammation and subsequent hepatocyte insulin resistance were also induced by circulating sEV from mice and humans with NAFLD. Conclusions: We identified hepatocyte-derived sEV as SFA transporters targeting macrophages/KC and activating a TLR4-mediated pro-inflammatory response enough to induce hepatocyte insulin resistance. Impact and Implications: Small extracellular vesicles (sEV) released by the hepatocytes under non-alcoholic fatty liver disease (NAFLD) conditions cause liver inflammation and insulin resistance in hepatocytes via paracrine hepatocyte-macrophage-hepatocyte crosstalk. We identified sEV as transporters of saturated fatty acids (SFAs) and potent lipotoxic inducers of liver inflammation. TLR4 deficiency or its pharmacological inhibition ameliorated liver inflammation induced by hepatocyte-derived lipotoxic sEV. Evidence of this macrophage-hepatocyte interactome was also found in patients with NAFLD, pointing to the relevance of sEV in SFA-mediated lipotoxicity in NAFLD.

Hypothalamic JNK1-hepatic fatty acid synthase axis mediates a metabolic rewiring that prevents hepatic steatosis in male mice treated with olanzapine via intraperitoneal: Additional effects of PTP1B inhibition.

Olanzapine (OLA), a widely used second-generation antipsychotic (SGA), causes weight gain and metabolic alterations when administered orally to patients. Recently, we demonstrated that, contrarily to the oral treatment which induces weight gain, OLA administered via intraperitoneal (i.p.) in male mice resulted in body weight loss. This protection was due to an increase in energy expenditure (EE) through a mechanism involving the modulation of hypothalamic AMPK activation by higher OLA levels reaching this brain region compared to those of the oral treatment. Since clinical studies have shown hepatic steatosis upon chronic treatment with OLA, herein we further investigated the role of the hypothalamus-liver interactome upon OLA administration in wild-type (WT) and protein tyrosine phosphatase 1B knockout (PTP1B-KO) mice, a preclinical model protected against metabolic syndrome. WT and PTP1B-KO male mice were fed an OLA-supplemented diet or treated via i.p. Mechanistically, we found that OLA i.p. treatment induces mild oxidative stress and inflammation in the hypothalamus in a JNK1-independent and dependent manner, respectively, without features of cell dead. Hypothalamic JNK activation up-regulated lipogenic gene expression in the liver though the vagus nerve. This effect concurred with an unexpected metabolic rewiring in the liver in which ATP depletion resulted in increased AMPK/ACC phosphorylation. This starvation-like signature prevented steatosis. By contrast, intrahepatic lipid accumulation was observed in WT mice treated orally with OLA; this effect being absent in PTP1B-KO mice. We also demonstrated an additional benefit of PTP1B inhibition against hypothalamic JNK activation, oxidative stress and inflammation induced by chronic OLA i.p. treatment, thereby preventing hepatic lipogenesis. The protection conferred by PTP1B deficiency against hepatic steatosis in the oral OLA treatment or against oxidative stress and neuroinflammation in the i.p. treatment strongly suggests that targeting PTP1B might be also a therapeutic strategy to prevent metabolic comorbidities in patients under OLA treatment in a personalized manner.

Metformin reduces macrophage HIF1α-dependent proinflammatory signaling to restore brown adipocyte function in vitro.

Therapeutic potential of metformin in obese/diabetic patients has been associated to its ability to combat insulin resistance. However, it remains largely unknown the signaling pathways involved and whether some cell types are particularly relevant for its beneficial effects. M1-activation of macrophages by bacterial lipopolysaccharide (LPS) promotes a paracrine activation of hypoxia-inducible factor-1α (HIF1α) in brown adipocytes which reduces insulin signaling and glucose uptake, as well as ß-adrenergic sensitivity. Addition of metformin to M1-polarized macrophages blunted these signs of brown adipocyte dysfunction. At the molecular level, metformin inhibits an inflammatory program executed by HIF1α in macrophages by inducing its degradation through the inhibition of mitochondrial complex I activity, thereby reducing oxygen consumption in a reactive oxygen species (ROS)-independent manner. In obese mice, metformin reduced inflammatory features in brown adipose tissue (BAT) such as macrophage infiltration, proinflammatory signaling and gene expression, and restored the response to cold exposure. In conclusion, the impact of metformin on macrophages by suppressing a HIF1α-dependent proinflammatory program is likely responsible for a secondary beneficial effect on insulin-mediated glucose uptake and ß-adrenergic responses in brown adipocytes.

Insights Into Extracellular Vesicles as Biomarker of NAFLD Pathogenesis.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease around the world estimated to affect up to one-third of the adult population and is expected to continue rising in the coming years. Nonalcoholic fatty liver disease is considered as the hepatic manifestation of the metabolic syndrome because it is strongly associated with obesity, insulin resistance, type 2 diabetes mellitus, and cardiovascular complications. Despite its high prevalence, factors leading to NAFLD progression from simple steatosis to nonalcoholic steatohepatitis, cirrhosis, and, ultimately hepatocellular carcinoma remain poorly understood. To date, no treatment has proven efficacy, and also no reliable method is currently available for diagnosis or staging of NAFLD beyond the highly invasive liver biopsy. Recently, extracellular vesicles (EVs) have emerged as potential candidate biomarkers for the diagnosis of NAFLD. Extracellular vesicles are circulating, cell-derived vesicles containing proteins and nucleic acids, among other components, that interact with and trigger a plethora of responses in neighbor or distant target cells. Several mechanisms implicated in NAFLD progression, such as inflammation, fibrosis, and angiogenesis, all related to metabolic syndrome-associated lipotoxicity, trigger EV production and release by liver cells. As hepatocytes represent ~80% of the liver volume, in this review we will focus on hepatocyte-derived EVs as drivers of the interactome between different liver cell types in NAFLD pathogenesis, as well as in their role as noninvasive biomarkers for NAFLD diagnosis and progression. Based on that, we will highlight the research that is currently available on EVs in this topic, the current limitations, and future directions for implementation in a clinical setting as biomarkers or targets of liver disease.

Impact of global PTP1B deficiency on the gut barrier permeability during NASH in mice.

OBJECTIVE: Non-alcoholic steatohepatitis (NASH) is characterized by a robust pro-inflammatory component at both hepatic and systemic levels together with a disease-specific gut microbiome signature. Protein tyrosine phosphatase 1 B (PTP1B) plays distinct roles in non-immune and immune cells, in the latter inhibiting pro-inflammatory signaling cascades. In this study, we have explored the role of PTP1B in the composition of gut microbiota and gut barrier dynamics in methionine and choline-deficient (MCD) diet-induced NASH in mice. METHODS: Gut features and barrier permeability were characterized in wild-type (PTP1B WT) and PTP1B-deficient knockout (PTP1B KO) mice fed a chow or methionine/choline-deficient (MCD) diet for 4 weeks. The impact of inflammation was studied in intestinal epithelial and enteroendocrine cells. The secretion of GLP-1 was evaluated in primary colonic cultures and plasma of mice. RESULTS: We found that a shift in the gut microbiota shape, disruption of gut barrier function, higher levels of serum bile acids, and decreased circulating glucagon-like peptide (GLP)-1 are features during NASH. Surprisingly, despite the pro-inflammatory phenotype of global PTP1B-deficient mice, they were partly protected against the alterations in gut microbiota composition during NASH and presented better gut barrier integrity and less permeability under this pathological condition. These effects concurred with higher colonic mucosal inflammation, decreased serum bile acids, and protection against the decrease in circulating GLP-1 levels during NASH compared with their WT counterparts together with increased expression of GLP-2-sensitive genes in the gut. At the molecular level, stimulation of enteroendocrine STC-1 cells with a pro-inflammatory conditioned medium (CM) from lipopolysaccharide (LPS)-stimulated macrophages triggered pro-inflammatory signaling cascades that were further exacerbated by a PTP1B inhibitor. Likewise, the pro-inflammatory CM induced GLP-1 secretion in primary colonic cultures, an effect augmented by PTP1B inhibition. CONCLUSION: Altogether our results have unraveled a potential role of PTP1B in the gut-liver axis during NASH, likely mediated by increased sensitivity to GLPs, with potential therapeutic value.

Impact of Bacterial Translocation on Sarcopenia in Patients with Decompensated Cirrhosis.

Advanced liver disease is associated with a persistent inflammatory state, derived from abnormal bacterial translocation from the gut, which may contribute to the development of sarcopenia in cirrhosis. We aim to document the association of chronic inflammation and bacterial translocation with the presence of sarcopenia in cirrhosis. We prospectively followed cirrhotic patients aged 18-70 years with medically refractory ascites at a single tertiary care center in Toronto, Canada. The baseline data included patient demographic variables, the presence of bacterial DNA in serum/ascitic fluid, systemic inflammatory response syndrome (SIRS) status, and nutritional assessment. Thirty-one patients were enrolled, 18 (58.1%) were sarcopenic, 9 (29%) had bacterial DNA in serum and ascites fluid. The mean MELD score was 11.5 ± 4.0 (6-23). Sarcopenic and non-sarcopenic patients did not differ significantly in their baseline MELD scores, caloric intake, resting energy expenditure, the incidence of bacterial translocation, or SIRS. While sarcopenia was not linked to increased hospital admissions or death, it was strongly associated with increased episodes of acute kidney injury (3 vs. 0, p = 0.05). This pilot study did not demonstrate an association between sarcopenia and SIRS or bacterial translocation. These results should be confirmed in future larger studies, encompassing a greater number of chronic inflammation events and quantifying levels of bacterial DNA.

Ammonia-Induced Brain Edema Requires Macrophage and T Cell Expression of Toll-Like Receptor 9.

BACKGROUND & AIM: Ammonia is central in the pathogenesis of brain edema in acute liver failure (ALF) with infection and systemic inflammation expediting development of intracranial hypertension (ICH). Patients with acetaminophen-induced ALF have increased neutrophil TLR9 expression which can be induced by ammonia. We determined whether ammonia-induced brain edema and immune dysfunction are mediated by TLR9 and if this could be prevented in a TLR9-deficient mouse model. METHODS: Ammonium acetate (NH4-Ac; 4mmol/kg) was injected intraperitoneally in wild type (WT), Tlr9-/- and Lysm-Cre Tlr9fl/fl mice (TLR9 absent in neutrophils and macrophages including Kupffer cells) and compared to controls. Six hours after NH4-Ac injection, intracellular cytokine production was determined in splenic macrophages, CD4+ and CD8+ T cells. Brain water (BW) and total plasma DNA (tDNA) were also measured. The impact of the TLR9 antagonist ODN2088 (50µg/mouse) was evaluated. RESULTS: Following NH4-Ac injection, BW, macrophage and T cell cytokine production increased (P < .0001) in WT but not Tlr9-/- mice (P < .001). ODN2088 inhibited macrophage and T cell cytokine production (P < .05) and prevented an increase in BW (P < .0001). Following NH4-Ac injection, macrophage cytokine production and BW were ameliorated in Lysm-Cre Tlr9fl/fl mice compared to WT mice (P < .05) but there was no difference compared to Tlr9-/- mice. Following NH4-Ac injection, plasma tDNA levels increased in WT and Tlr9-/- mice (P < .05) suggesting that TLR9 may be activated by DNA released from ammonia-stimulated cells. CONCLUSION: Ammonia-induced brain edema requires macrophage and T cell expression of TLR9. Amelioration of brain edema and lymphocyte cytokine production by ODN2088 supports exploration of TLR9 antagonism in early ALF to prevent progression to ICH.

A leukocyte activation test identifies food items which induce release of DNA by innate immune peripheral blood leucocytes.

BACKGROUND: Leukocyte activation (LA) testing identifies food items that induce a patient specific cellular response in the immune system, and has recently been shown in a randomized double blinded prospective study to reduce symptoms in patients with irritable bowel syndrome (IBS). We hypothesized that test reactivity to particular food items, and the systemic immune response initiated by these food items, is due to the release of cellular DNA from blood immune cells. METHODS: We tested this by quantifying total DNA concentration in the cellular supernatant of immune cells exposed to positive and negative foods from 20 healthy volunteers. To establish if the DNA release by positive samples is a specific phenomenon, we quantified myeloperoxidase (MPO) in cellular supernatants. We further assessed if a particular immune cell population (neutrophils, eosinophils, and basophils) was activated by the positive food items by flow cytometry analysis. To identify the signaling pathways that are required for DNA release we tested if specific inhibitors of key signaling pathways could block DNA release. RESULTS: Foods with a positive LA test result gave a higher supernatant DNA content when compared to foods with a negative result. This was specific as MPO levels were not increased by foods with a positive LA test. Protein kinase C (PKC) inhibitors resulted in inhibition of positive food stimulated DNA release. Positive foods resulted in CD63 levels greater than negative foods in eosinophils in 76.5% of tests. CONCLUSION: LA test identifies food items that result in release of DNA and activation of peripheral blood innate immune cells in a PKC dependent manner, suggesting that this LA test identifies food items that result in release of inflammatory markers and activation of innate immune cells. This may be the basis for the improvement in symptoms in IBS patients who followed an LA test guided diet.

SIRT1 Controls Acetaminophen Hepatotoxicity by Modulating Inflammation and Oxidative Stress.

AIMS: Sirtuin 1 (SIRT1) is a key player in liver physiology and a therapeutic target against hepatic inflammation. We evaluated the role of SIRT1 in the proinflammatory context and oxidative stress during acetaminophen (APAP)-mediated hepatotoxicity. RESULTS: SIRT1 protein levels decreased in human and mouse livers following APAP overdose. SIRT1-Tg mice maintained higher levels of SIRT1 on APAP injection than wild-type mice and were protected against hepatotoxicity by modulation of antioxidant systems and restrained inflammatory responses, with decreased oxidative stress, proinflammatory cytokine messenger RNA levels, nuclear factor kappa B (NFκB) signaling, and cell death. Mouse hepatocytes stimulated with conditioned medium of APAP-treated macrophages (APAP-CM) showed decreased SIRT1 levels; an effect mimicked by interleukin (IL)1ß, an activator of NFκB. This negative modulation was abolished by neutralizing IL1ß in APAP-CM or silencing p65-NFκB in hepatocytes. APAP-CM of macrophages from SIRT1-Tg mice failed to downregulate SIRT1 protein levels in hepatocytes. In vivo administration of the NFκB inhibitor BAY 11-7082 preserved SIRT1 levels and protected from APAP-mediated hepatotoxicity. INNOVATION: Our work evidenced the unique role of SIRT1 in APAP hepatoprotection by targeting oxidative stress and inflammation. CONCLUSION: SIRT1 protein levels are downregulated by IL1ß/NFκB signaling in APAP hepatotoxicity, resulting in inflammation and oxidative stress. Thus, maintenance of SIRT1 during APAP overdose by inhibiting NFκB might be clinically relevant. Rebound Track: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16:293-296, 2012) with the following serving as open reviewers: Rafael de Cabo, Joaquim Ros, Kalervo Hiltunen, and Neil Kaplowitz. Antioxid. Redox Signal. 28, 1187-1208.

Digoxin Suppresses Pyruvate Kinase M2-Promoted HIF-1α Transactivation in Steatohepatitis.

Sterile inflammation after tissue damage is a ubiquitous response, yet it has the highest amplitude in the liver. This has major clinical consequences, for alcoholic and non-alcoholic steatohepatitis (ASH and NASH) account for the majority of liver disease in industrialized countries and both lack therapy. Requirements for sustained sterile inflammation include increased oxidative stress and activation of the HIF-1α signaling pathway. We demonstrate the ability of digoxin, a cardiac glycoside, to protect from liver inflammation and damage in ASH and NASH. Digoxin was effective in maintaining cellular redox homeostasis and suppressing HIF-1α pathway activation. A proteomic screen revealed that digoxin binds pyruvate kinase M2 (PKM2), and independently of PKM2 kinase activity results in chromatin remodeling and downregulation of HIF-1α transactivation. These data identify PKM2 as a mediator and therapeutic target for regulating liver sterile inflammation, and demonstrate a novel role for digoxin that can effectively protect the liver from ASH and NASH.

Nuevos conocimientos sobre el papel clave de las vesículas extracelulares en la enfermedad de hígado graso no alcohólica / New insights into the key role of extracellular vesicles in non-alcoholic fatty liver disease

La enfermedad del hígado graso no alcohólico (EHGNA) es la enfermedad hepática crónica más común del mundo. La EHGNA se considera la manifestación hepática del síndrome metabólico al estar directamente asociada con la obesidad, la resistencia a la insulina, la diabetes mellitus tipo 2 y las complicaciones cardiovasculares. Pese a su prevalencia, los factores que desencadenan la progresión de la EHGNA a la esteatohepatitis no alcohólica, la cirrosis y el carcinoma hepatocelular son poco conocidos. Actualmente, no existe tratamiento eficaz ni hay disponible un método fiable para su diagnóstico y estatificación más allá de la biopsia hepática altamente invasiva.Recientemente, las vesículas extracelulares (VEs) han emergido como posibles biomarcadores para el diagnóstico de la EHGNA. Las VEs son vesículas derivadas de las células que contienen proteínas y ácidos nucleicos, entre otros componentes, que interactúan y desencadenan una gran variedad de respuestas en células diana próximas o distantes. Varios mecanismos implicados en la progresión de la EHGNA, como la inflamación, la fibrosis y la angiogénesis, relacionados con la lipotoxicidad, desencadenan la secreción de VEs por las células hepáticas. En esta revisión nos centraremos en las VEs secretadas por los hepatocitos (Hep-VEs) como mensajeros del interactoma entre las diferentes células hepáticas en la patogénesis de la EHGNA, así como en su papel como biomarcadores no invasivos para su diagnóstico y estratificación. Además, destacaremos las investigaciones disponibles hasta la fecha, las limitaciones actuales y las futuras directrices para su implementación en un entorno clínico como biomarcadores o dianas terapéuticas de la enfermedad hepática. (AU)

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. NAFLD, the hepatic manifestation of the metabolic syndrome, closely associates with insulin resistance, type 2 diabetes mellitus, obesity and cardiovascular disease. Until now, the specific factors involved in the progression of NAFLD from fatty liver to non-alcoholic steatohepatitis, cirrhosis and, ultimately hepatocellular carcinoma have not been totally elucidated.Also, patients have to face the lack of efficient or personalized treatments, as well as the absence of reliable diagnosis or staging methods beyond the highly invasive liver biopsy. In the last years, extracellular vesicles (VEs) are considered as potential biomarkers for the diagnosis many diseases including NAFLD. VEs are released by different cells types into the circulation and contain nucleic acids and proteins, among other components of their, that interact with surrounding or distant target cells, thereby triggering a plethora of responses. During NAFLD progression, several processes such as inflammation, fibrosis and angiogenesis, all related to MS-associated lipotoxicity, lead to VEs release by liver cells. In this review we will focus in the role of hepatocyte-derived VEs (Hep-VEs) and their interactions with non-parenchymal liver cells populations during NAFLD pathogenesis, as well as in their role as non-invasive biomarkers for disease diagnosis and progression. We will highlight the recent work currently available on VEs in the context of NAFLD, the current limitations and future directions for the implementation of VEs as biomarkers or targets of liver disease in the clinical setting. (AU)

Nuevos conocimientos sobre el papel clave de las vesículas extracelulares en la enfermedad de hígado graso no alcohólica / New insights into the key role of extracellular vesicles in non-alcoholic fatty liver disease

La enfermedad del hígado graso no alcohólico (EHGNA) es la enfermedad hepática crónica más común del mundo. La EHGNA se considera la manifestación hepática del síndrome metabólico al estar directamente asociada con la obesidad, la resistencia a la insulina, la diabetes mellitus tipo 2 y las complicaciones cardiovasculares. Pese a su prevalencia, los factores que desencadenan la progresión de la EHGNA a la esteatohepatitis no alcohólica, la cirrosis y el carcinoma hepatocelular son poco conocidos. Actualmente, no existe tratamiento eficaz ni hay disponible un método fiable para su diagnóstico y estatificación más allá de la biopsia hepática altamente invasiva.Recientemente, las vesículas extracelulares (VEs) han emergido como posibles biomarcadores para el diagnóstico de la EHGNA. Las VEs son vesículas derivadas de las células que contienen proteínas y ácidos nucleicos, entre otros componentes, que interactúan y desencadenan una gran variedad de respuestas en células diana próximas o distantes. Varios mecanismos implicados en la progresión de la EHGNA, como la inflamación, la fibrosis y la angiogénesis, relacionados con la lipotoxicidad, desencadenan la secreción de VEs por las células hepáticas. En esta revisión nos centraremos en las VEs secretadas por los hepatocitos (Hep-VEs) como mensajeros del interactoma entre las diferentes células hepáticas en la patogénesis de la EHGNA, así como en su papel como biomarcadores no invasivos para su diagnóstico y estratificación. Además, destacaremos las investigaciones disponibles hasta la fecha, las limitaciones actuales y las futuras directrices para su implementación en un entorno clínico como biomarcadores o dianas terapéuticas de la enfermedad hepática. (AU)

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. NAFLD, the hepatic manifestation of the metabolic syndrome, closely associates with insulin resistance, type 2 diabetes mellitus, obesity and cardiovascular disease. Until now, the specific factors involved in the progression of NAFLD from fatty liver to non-alcoholic steatohepatitis, cirrhosis and, ultimately hepatocellular carcinoma have not been totally elucidated.Also, patients have to face the lack of efficient or personalized treatments, as well as the absence of reliable diagnosis or staging methods beyond the highly invasive liver biopsy. In the last years, extracellular vesicles (VEs) are considered as potential biomarkers for the diagnosis many diseases including NAFLD. VEs are released by different cells types into the circulation and contain nucleic acids and proteins, among other components of their, that interact with surrounding or distant target cells, thereby triggering a plethora of responses. During NAFLD progression, several processes such as inflammation, fibrosis and angiogenesis, all related to MS-associated lipotoxicity, lead to VEs release by liver cells. In this review we will focus in the role of hepatocyte-derived VEs (Hep-VEs) and their interactions with non-parenchymal liver cells populations during NAFLD pathogenesis, as well as in their role as non-invasive biomarkers for disease diagnosis and progression. We will highlight the recent work currently available on VEs in the context of NAFLD, the current limitations and future directions for the implementation of VEs as biomarkers or targets of liver disease in the clinical setting. (AU)