G-CSF increases calprotectin expression, liver damage and neuroinflammation in a murine model of alcohol-induced ACLF

Background and aims: Granulocyte colony-stimulating factor (G-CSF) has been proposed as a therapeutic option for patients with ACLF, however clinical outcomes are controversial. We aimed at dissecting the role of G-CSF in an alcohol-induced murine model of ACLF.Methods: ACLF was triggered by a single alcohol binge (5 g/kg) in a bile duct ligation (BDL) liver fibrosis model. A subgroup of mice received two G-CSF (200 μg/kg) or vehicle injections prior to acute decompensation with alcohol. Liver, blood and brain tissues were assessed.Results: Alcohol binge administered to BDL-fibrotic mice resulted in features of ACLF indicated by a significant increase in liver damage and systemic inflammation compared to BDL alone. G-CSF treatment in ACLF mice induced an increase in liver regeneration and neutrophil infiltration in the liver compared to vehicle-treated ACLF mice. Moreover, liver-infiltrating neutrophils in G-CSF-treated mice exhibited an activated phenotype indicated by increased expression of CXC motif chemokine receptor 2, leukotriene B4 receptor 1, and calprotectin. In the liver, G-CSF triggered increased oxidative stress, type I interferon response, extracellular matrix remodeling and inflammasome activation. Circulating IL-1β was also increased after G-CSF treatment. In the cerebellum, G-CSF increased neutrophil infiltration and S100a8/9 expression, induced microglia proliferation and reactive astrocytes, which was accompanied by oxidative stress, and inflammasome activation compared to vehicle-treated ACLF mice.Conclusion: In our novel ACLF model triggered by alcohol binge that mimics ACLF pathophysiology, neutrophil infiltration and S100a8/9 expression in the liver and brain indicate increased tissue damage, accompanied by oxidative stress and inflammasome activation after G-CSF treatment

Down-regulation of miR-15a/b accelerates fibrotic remodelling in the Type 2 diabetic human and mouse heart

Correspondence: Rajesh Katare ([email protected]) Aim: Myocardial fibrosis is a well-established cause of increased myocardial stiffness and subsequent diastolic dysfunction in the diabetic heart. The molecular regulators that drive the process of fibrotic events in the diabetic heart are still unknown. We determined the role of the microRNA (miR)-15 family in fibrotic remodelling of the diabetic heart. Methods and results: Right atrial appendage (RAA) and left ventricular (LV) biopsy tissues collected from diabetic and non-diabetic (ND) patients undergoing coronary artery bypass graft surgery showed significant down-regulation of miR-15a and -15b. This was associated with marked up-regulation of pro-fibrotic transforming growth factor-β receptor-1 (TGFβR1) and connective tissue growth factor (CTGF), direct targets for miR-15a/b and pro-senescence p53 protein. Interestingly, down-regulation of miR-15a/b preceded the development of diastolic dysfunction and fibrosis in Type 2 diabetic mouse heart. Therapeutic restoration of miR-15a and -15b in HL-1 cardiomyocytes reduced the activation of pro-fibrotic TGFβR1 and CTGF, and the pro-senescence p53 protein expression, confirming a causal regulation of these fibrotic and senescence mediators by miR-15a/b. Moreover, conditioned medium (CM) collected from cardiomyocytes treated with miR-15a/b markedly diminished the differentiation of diabetic human cardiac fibroblasts. Conclusion: Our results provide first evidence that early down-regulation of miR-15a/b activates fibrotic signalling in diabetic heart, and hence could be a potential target for the treatment/prevention of diabetes-induced fibrotic remodelling of the heart

Identification of the host histone deacetylase 1 and 2 as novel anti-influenza A virus factors

Influenza virus continues to pose serious medical and economic challenges to global public health as novel influenza viruses regularly emerge in human population. A universal influenza virus vaccine is not available yet and currently available influenza type- and subtype-specific vaccines need regular updating. Therefore, antiviral drugs are the first line of defence against a novel influenza virus. However, currently-approved anti-influenza drugs target viral components and influenza has mutated those components to acquire the drug resistance. Therefore, there is a need to develop alternative, effective, and long-lasting antiviral strategies to overcome the continuously emerging novel and drug-resistant influenza viruses in humans. One approach is to selectively target specific interactions of influenza virus with host pro-viral and anti-viral factors to inhibit virus replication. This strategy will less likely induce the viral resistance. This PhD project was designed to determine the role of host histone deacetylase (HDAC) 1 and 2 in the infection of influenza A virus (IAV), the most significant influenza virus. The rationale of this project was based upon the recent discovery in our lab indicating that host histone deacetylases (HDACs) are potentially a family of novel anti-IAV factors. HDACs catalyse the deacetylation of a variety of cytoplasmic and nuclear proteins, consequently regulating diverse cellular processes. Mammalian HDACs have been divided into four classes. The HDAC1 and 2 belong to class I, and are the first and second discovered HDACs, respectively. Earlier, our lab demonstrated that HDAC6, a class II HDAC, possesses an anti-IAV property and IAV downregulates HDAC6 activity to potentially undermine its antiviral function. Based on these findings, we proposed the hypothesis that class I HDACs also have similar properties. HDAC1 is a prototypic member of class I and HDAC2 has about 86% amino acid sequence similarity with HDAC1. Hence, we investigated the role of HDAC1 and 2 in IAV infection using primarily the human lung epithelial cells and IAV PR/8/34(H1N1) and WSN/1933(H1N1) strains as a model. We have found that IAV downregulates the HDAC1 expression (both at mRNA and polypeptide level) as well as its deacetylase activity, which, consistent with our hypothesis indicated an anti-IAV role of host HDAC1. Indeed, silencing of HDAC1 expression by RNA interference augmented the IAV infection by more than 6-fold, and conversely, the ectopic expression of HDAC1 from a plasmid decreased it by more than half. To efficiently replicate, IAV has evolved multiple strategies to circumvent the host innate antiviral response. The dysregulation of host HDACs could be part of that strategy as HDACs have been shown to be an important component of host innate response in a heterologous system. Consistent with this hypothesis, treatment of infected cells with trichostatin A (TSA), a widely-used HDAC inhibitor resulted in the downregulation of the phosphorylation of STAT1, a critical component of host innate antiviral response and expression of interferon-stimulated genes, IFITM3, ISG15, and viperin, which have been previously reported to have the anti-IAV function. Consequently, TSA treatment also resulted in the enhancement in IAV infection by more than 5-fold. Further, knockdown of HDAC1 expression resulted in decreased level of phosphorylated interferon regulatory factor 3, a key molecule in interferon signalling and subsequently the reduced expression of interferon α. Furthermore, the expression of viperin was also reduced or enhanced by about 58% or 55% in HDAC1-depleted or HDAC1-overexpressing cells, respectively. Similarly, HDAC2 mRNA and polypeptide expression was also downregulated in IAV infected cells, albeit by a mechanism distinct to HDAC1 downregulation. Nevertheless, the knockdown of HDAC2 expression resulted in about 4 fold increase in IAV infection. In addition, there was a modest, but consistent decrease in the level of phosphorylated STAT1 in HDAC2-depleted cells and consequently, a decrease in viperin expression. In summary, this PhD study has demonstrated an anti-IAV role of host HDAC1 and 2 and provided a significant insight into their antiviral mechanism. Evolutionary, HDAC1 and 2 are similar proteins and both were found to have the anti-IAV properties. However, HDAC1 and 2 seem to have a slightly distinct and independent interaction with IAV. Based upon the experimental evidence presented here, further mechanistic roles of these HDACs in IAV infection has been discussed and relevant future research directions have been outlined. In conclusion, both HDAC1 and HDAC2 provide a cellular refractory state to IAV infection by regulating the host innate immune response. The data presented here will contribute to further molecular understanding of the IAV-HDACs interactions

Therapeutic inhibition of miR-155 attenuates liver fibrosis via STAT3 signaling

Most chronic liver diseases progress to liver fibrosis, which, when left untreated, can lead to cirrhosis and hepatocellular carcinoma. MicroRNA (miRNA)-targeted therapeutics have become attractive approaches to treat diseases. In this study, we investigated the therapeutic effect of miR-155 inhibition in the bile duct ligation (BDL) mouse model of liver fibrosis and evaluated the role of miR-155 in chronic liver fibrosis using miR-155-deficient (miR-155 knockout [KO]) mice. We found increased hepatic miR-155 expression in patients with cirrhosis and in the BDL- and CCl4-induced mouse models of liver fibrosis. Liver fibrosis was significantly reduced in miR-155 KO mice after CCl4 administration or BDL. To assess the therapeutic potential of miR-155 inhibition, we administered an rAAV8-anti-miR-155 tough decoy in vivo that significantly reduced liver damage and fibrosis in BDL. BDL-induced protein levels of transforming growth factor β (TGF-β), p-SMAD2/3, and p-STAT3 were attenuated in anti-miR-155-treated compared with control mice. Hepatic stellate cells from miR-155 KO mice showed attenuation in activation and mesenchymal marker expression. In vitro, miR-155 gain- and loss-of-function studies revealed that miR-155 regulates activation of stellate cells partly via STAT3 signaling. Our study suggests that miR-155 is the key regulator of liver fibrosis and might be a potential therapeutic target to attenuate fibrosis progression

Combined Insults of a MASH Diet and Alcohol Binges Activate Intercellular Communication and Neutrophil Recruitment via the NLRP3-IL-1β Axis in the Liver

Binge drinking in obese patients positively correlates with accelerated liver damage and liver-related death. However, the underlying mechanism and the effect of alcohol use on the progression of metabolic-dysfunction-associated steatotic liver disease (MASLD) remain unexplored. Here, we show that short-term feeding of a metabolic-dysfunction-associated steatohepatitis (MASH) diet plus daily acute alcohol binges for three days induce liver injury and activation of the NLRP3 inflammasome. We identify that a MASH diet plus acute alcohol binges promote liver inflammation via increased infiltration of monocyte-derived macrophages, neutrophil recruitment, and NET release in the liver. Our results suggest that both monocyte-derived macrophages and neutrophils are activated via NLRP3, while the administration of MCC950, an NLRP3 inhibitor, dampens these effects.In this study, we reveal important intercellular communication between hepatocytes and neutrophils. We discover that the MASH diet plus alcohol induces IL-1β via NLRP3 activation and that IL-1β acts on hepatocytes and promotes the production of CXCL1 and LCN2. In turn, the increase in these neutrophils recruits chemokines and causes further infiltration and activation of neutrophils in the liver. In vivo administration of the NLRP3 inhibitor, MCC950, improves the early phase of MetALD by preventing liver damage, steatosis, inflammation, and immune cells recruitment

Transcriptional regulation of Acsl1 by CHREBP and NF-kappa B in macrophages during hyperglycemia and inflammation.

Acyl-CoA synthetase 1 (ACSL1) is an enzyme that converts fatty acids to acyl-CoA-derivatives for lipid catabolism and lipid synthesis in general and can provide substrates for the production of mediators of inflammation in monocytes and macrophages. Acsl1 expression is increased by hyperglycemia and inflammatory stimuli in monocytes and macrophages, and promotes the pro-atherosclerotic effects of diabetes in mice. Yet, surprisingly little is known about the mechanisms underlying Acsl1 transcriptional regulation. Here we demonstrate that the glucose-sensing transcription factor, Carbohydrate Response Element Binding Protein (CHREBP), is a regulator of the expression of Acsl1 mRNA by high glucose in mouse bone marrow-derived macrophages (BMDMs). In addition, we show that inflammatory stimulation of BMDMs with lipopolysaccharide (LPS) increases Acsl1 mRNA via the transcription factor, NF-kappa B. LPS treatment also increases ACSL1 protein abundance and localization to membranes where it can exert its activity. Using an Acsl1 reporter gene containing the promoter and an upstream regulatory region, which has multiple conserved CHREBP and NF-kappa B (p65/RELA) binding sites, we found increased Acsl1 promoter activity upon CHREBP and p65/RELA expression. We also show that CHREBP and p65/RELA occupy the Acsl1 promoter in BMDMs. In primary human monocytes cultured in high glucose versus normal glucose, ACSL1 mRNA expression was elevated by high glucose and further enhanced by LPS treatment. Our findings demonstrate that CHREBP and NF-kappa B control Acsl1 expression under hyperglycemic and inflammatory conditions