Involvement of lncRNA TUG1 in HIV-1 Tat-Induced Astrocyte Senescence.

HIV-1 infection in the era of combined antiretroviral therapy has been associated with premature aging. Among the various features of HIV-1 associated neurocognitive disorders, astrocyte senescence has been surmised as a potential cause contributing to HIV-1-induced brain aging and neurocognitive impairments. Recently, lncRNAs have also been implicated to play essential roles in the onset of cellular senescence. Herein, using human primary astrocytes (HPAs), we investigated the role of lncRNA TUG1 in HIV-1 Tat-mediated onset of astrocyte senescence. We found that HPAs exposed to HIV-1 Tat resulted in significant upregulation of lncRNA TUG1 expression that was accompanied by elevated expression of p16 and p21, respectively. Additionally, HIV-1 Tat-exposed HPAs demonstrated increased expression of senescence-associated (SA) markers-SA-ß-galactosidase (SA-ß-gal) activity and SA-heterochromatin foci-cell-cycle arrest, and increased production of reactive oxygen species and proinflammatory cytokines. Intriguingly, gene silencing of lncRNA TUG1 in HPAs also reversed HIV-1 Tat-induced upregulation of p21, p16, SA-ß gal activity, cellular activation, and proinflammatory cytokines. Furthermore, increased expression of astrocytic p16 and p21, lncRNA TUG1, and proinflammatory cytokines were observed in the prefrontal cortices of HIV-1 transgenic rats, thereby suggesting the occurrence of senescence activation in vivo. Overall, our data indicate that HIV-1 Tat-induced astrocyte senescence involves the lncRNA TUG1 and could serve as a potential therapeutic target for dampening accelerated aging associated with HIV-1/HIV-1 proteins.

Alcohol and HIV-Derived Hepatocyte Apoptotic Bodies Induce Hepatic Stellate Cell Activation.

Recently, we found that both HIV and acetaldehyde, an alcohol metabolite, induce hepatocyte apoptosis, resulting in the release of large extracellular vesicles called apoptotic bodies (ABs). The engulfment of these hepatocyte ABs by hepatic stellate cells (HSC) leads to their profibrotic activation. This study aims to establish the mechanisms of HSC activation after engulfment of ABs from acetaldehyde and HIV-exposed hepatocytes (ABAGS+HIV). In vitro experiments were performed on Huh7.5-CYP (RLW) cells to generate hepatocyte ABs and LX2 cells were used as HSC. To generate ABs, RLW cells were pretreated for 24 h with acetaldehyde, then exposed overnight to HIV1ADA and to acetaldehyde for 96 h. Thereafter, ABs were isolated from cell suspension by a differential centrifugation method and incubated with LX2 cells (3:1 ratio) for profibrotic genes and protein analyses. We found that HSC internalized ABs via the tyrosine kinase receptor, Axl. While the HIV gag RNA/HIV proteins accumulated in ABs elicited no productive infection in LX2 and immune cells, they triggered ROS and IL6 generation, which, in turn, activated profibrotic genes via the JNK-ERK1/2 and JAK-STAT3 pathways. Similarly, ongoing profibrotic activation was observed in immunodeficient NSG mice fed ethanol and injected with HIV-derived RLW ABs. We conclude that HSC activation by hepatocyte ABAGS+HIV engulfment is mediated by ROS-dependent JNK-ERK1/2 and IL6 triggering of JAK-STAT3 pathways. This can partially explain the mechanisms of liver fibrosis development frequently observed among alcohol abusing PLWH.

Alcohol: basic and translational research; 15th annual Charles Lieber &1st Samuel French satellite symposium.

The present review is based on the research presented at the symposium dedicated to the legacy of the two scientists that made important discoveries in the field of alcohol-induced liver damage: Professors C.S. Lieber and S.W. French. The invited speakers described pharmacological, toxicological and patho-physiological effects of alcohol misuse. Moreover, genetic biomarkers determining adverse drug reactions due to interactions between therapeutics used for chronic or infectious diseases and alcohol exposure were discussed. The researchers presented their work in areas of alcohol-induced impairment in lipid protein trafficking and endocytosis, as well as the role of lipids in the development of fatty liver. The researchers showed that alcohol leads to covalent modifications that promote hepatic dysfunction and injury. We concluded that using new advanced techniques and research ideas leads to important discoveries in science.

Ethanol attenuates presentation of cytotoxic T-lymphocyte epitopes on hepatocytes of HBV-infected humanized mice.

BACKGROUND AND AIMS: Approximately 3.5% of the global population is chronically infected with Hepatitis B Virus (HBV), which puts them at high risk of end-stage liver disease, with the risk of persistent infection potentiated by alcohol consumption. However, the mechanisms underlying the effects of alcohol on HBV persistence remain unclear. Here, we aimed to establish in vivo/ex vivo evidence that alcohol suppresses HBV peptides-major histocompatibility complex (MHC) class I antigen display on primary human hepatocytes (PHH), which diminishes the recognition and clearance of HBV-infected hepatocytes by cytotoxic T-lymphocytes (CTLs). METHODS: We used fumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chain knock-out (FRG-KO) humanized mice transplanted with human leukocyte antigen-A2 (HLA-A2)-positive hepatocytes. The mice were HBV-infected and fed control and alcohol diets. Isolated hepatocytes were exposed ex vivo to HBV 18-27-HLA-A2-restricted CTLs to quantify cytotoxicity. For mechanistic studies, we measured proteasome activities, unfolded protein response (UPR), and endoplasmic reticulum (ER) stress in hepatocytes from HBV-infected humanized mouse livers. RESULTS AND CONCLUSIONS: We found that alcohol feeding attenuated HBV core 18-27-HLA-A2 complex presentation on infected hepatocytes due to the suppression of proteasome function and ER stress induction, which diminished both the processing of HBV peptides and trafficking of HBV-MHC class I complexes to the hepatocyte surface. This alcohol-mediated decrease in MHC class I-restricted antigen presentation of the CTL epitope on target hepatocytes reduced the CTL-specific elimination of infected cells, potentially leading to HBV-infection persistence, which promotes end-stage liver disease outcomes.

Alcohol-Induced Lysosomal Damage and Suppression of Lysosome Biogenesis Contribute to Hepatotoxicity in HIV-Exposed Liver Cells.

Although the causes of hepatotoxicity among alcohol-abusing HIV patients are multifactorial, alcohol remains the least explored "second hit" for HIV-related hepatotoxicity. Here, we investigated whether metabolically derived acetaldehyde impairs lysosomes to enhance HIV-induced hepatotoxicity. We exposed Cytochrome P450 2E1 (CYP2E1)-expressing Huh 7.5 (also known as RLW) cells to an acetaldehyde-generating system (AGS) for 24 h. We then infected (or not) the cells with HIV-1ADA then exposed them again to AGS for another 48 h. Lysosome damage was assessed by galectin 3/LAMP1 co-localization and cathepsin leakage. Expression of lysosome biogenesis-transcription factor, TFEB, was measured by its protein levels and by in situ immunofluorescence. Exposure of cells to both AGS + HIV caused the greatest amount of lysosome leakage and its impaired lysosomal biogenesis, leading to intrinsic apoptosis. Furthermore, the movement of TFEB from cytosol to the nucleus via microtubules was impaired by AGS exposure. The latter impairment appeared to occur by acetylation of α-tubulin. Moreover, ZKSCAN3, a repressor of lysosome gene activation by TFEB, was amplified by AGS. Both these changes contributed to AGS-elicited disruption of lysosome biogenesis. Our findings indicate that metabolically generated acetaldehyde damages lysosomes and likely prevents their repair and restoration, thereby exacerbating HIV-induced hepatotoxicity.

Biogenesis, physiological functions and potential applications of extracellular vesicles in substance use disorders.

Substance use disorder (SUD) is a growing health problem that affects several millions of people worldwide, resulting in negative socioeconomic impacts and increased health care costs. Emerging evidence suggests that extracellular vesicles (EVs) play a crucial role in SUD pathogenesis. EVs, including exosomes and microvesicles, are membrane-encapsulated particles that are released into the extracellular space by most types of cells. EVs are important players in mediating cell-to-cell communication through transfer of cargo such as proteins, lipids and nucleic acids. The EV cargo can alter the status of recipient cells, thereby contributing to both physiological and pathological processes; some of these play critical roles in SUD. Although the functions of EVs under several pathological conditions have been extensively reviewed, EV functions and potential applications in SUD remain less studied. In this review, we provide an overview of the current knowledge of the role of EVs in SUD, including alcohol, cocaine, heroin, marijuana, nicotine and opiate abuse. The review will focus on the biogenesis and cargo composition of EVs as well as the potential use of EVs as biomarkers of SUD or therapeutic targets in SUD.

Pancreatogenic Diabetes: Triggering Effects of Alcohol and HIV.

Multiorgan failure may not be completely resolved among people living with HIV despite HAART use. Although the chances of organ dysfunction may be relatively low, alcohol may potentiate HIV-induced toxic effects in the organs of alcohol-abusing, HIV-infected individuals. The pancreas is one of the most implicated organs, which is manifested as diabetes mellitus or pancreatic cancer. Both alcohol and HIV may trigger pancreatitis, but the combined effects have not been explored. The aim of this review is to explore the literature for understanding the mechanisms of HIV and alcohol-induced pancreatotoxicity. We found that while premature alcohol-inducing zymogen activation is a known trigger of alcoholic pancreatitis, HIV entry through C-C chemokine receptor type 5(CCR5)into pancreatic acinar cells may also contribute to pancreatitis in people living with HIV (PLWH). HIV proteins induce oxidative and ER stresses, causing necrosis. Furthermore, infiltrative immune cells induce necrosis on HIV-containing acinar cells. When necrotic products interact with pancreatic stellate cells, they become activated, leading to the release of both inflammatory and profibrotic cytokines and resulting in pancreatitis. Effective therapeutic strategies should block CCR5 and ameliorate alcohol's effects on acinar cells.

Alcohol-and-HIV-Induced Lysosomal Dysfunction Regulates Extracellular Vesicles Secretion in Vitro and in Liver-Humanized Mice.

BACKGROUND: Alcohol abuse is common in people living with HIV-1 and dramaticallyenhances the severity of HIV-induced liver damage by inducing oxidative stress and lysosomaldysfunction in the liver cells. We hypothesize that the increased release of extracellular vesicles(EVs) in hepatocytes and liver humanized mouse model is linked to lysosome dysfunction. METHODS: The study was performed on primary human hepatocytes and human hepatoma RLWXP-GFP (Huh7.5 cells stably transfected with CYP2E1 and XPack-GFP) cells and validated on ethanol-fed liverhumanizedfumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chainknockout (FRG-KO) mice. Cells and mice were infected with HIV-1ADA virus. RESULTS: We observedan increase in the secretion of EVs associated with a decrease in lysosomal activity and expressionof lysosomal-associated membrane protein 1. Next-generation RNA sequencing of primary humanhepatocytes revealed 63 differentially expressed genes, with 13 downregulated and 50 upregulatedgenes in the alcohol-HIV-treated group. Upstream regulator analysis of differentially expressedgenes through Ingenuity Pathway Analysis identified transcriptional regulators affecting downstreamgenes associated with increased oxidative stress, lysosomal associated disease, and function andEVs biogenesis. Our in vitro findings were corroborated by in vivo studies on human hepatocytetransplantedhumanized mice, indicating that intensive EVs' generation by human hepatocytes andtheir secretion to serum was associated with increased oxidative stress and reduction in lysosomalactivities triggered by HIV infection and ethanol diet. CONCLUSION: HIV-and-ethanol-metabolisminducedEVs release is tightly controlled by lysosome status in hepatocytes and participates in thedevelopment of double-insult-induced liver injury.

Obeticholic acid attenuates human immunodeficiency virus/alcohol metabolism-induced pro-fibrotic activation in liver cells.

BACKGROUND: The morbidity and mortality of human immunodeficiency virus (HIV)-infection is often associated with liver disease, which progresses slowly into severe liver dysfunction. There are multiple insults which exacerbate HIV-related liver injury, including HIV-associated dysregulation of lipid metabolism and fat turnover, co-infections with hepatotropic viruses and alcohol abuse. As we reported before, exposure of hepatocytes to HIV and alcohol metabolites causes high oxidative stress, impairs proteasomal and lysosomal functions leading to accumulation of HIV in these cells, which end-ups with apoptotic cell death and finally promotes development of liver fibrosis. AIM: To study whether obeticholic acid (OCA) prevents HIV/ethanol metabolism-induced hepatotoxicity and subsequent activation of hepatic stellate cells (HSC) by HIV+ apoptotic hepatocyte engulfment. METHODS: Huh7.5-CYP (RLW) cells were exposed to HIV and acetaldehyde-generating system (AGS) in the presence or absence of OCA. In the cells, we measured the expression of HIV-related markers: HIVgagRNA-by real-time polymerase chain reaction (PCR), p24- by western blot, HIV DNA-by semi-nested PCR, integrated HIV DNA-by ddPCR. Lysosomal and proteasomal activities were measured using fluorometrically-labeled substrates. For hepatocyte apoptosis, cleaved caspase 3 and cleaved PARP were visualized by western blot and cytokeratin 18- by M30 ELISA-in supernatants. Apoptotic bodies were generated from untreated and HIV-treated RLW cells exposed to UV light. Pro-fibrotic activation of HSC was characterized by Col1A1 and transforming growth factor-ß mRNAs, while inflammasome activation- by NLRP3, caspase 1, interleukin (IL)-6, IL-1ß mRNA levels. RESULTS: In RLW cells, OCA treatment attenuated HIV-AGS-induced accumulation of HIVgagRNA, HIV DNA and p24. OCA suppressed reactive oxygen species production and restored chymotrypsin-like proteasome activity as well as cathepsin B lysosome activity. OCA also decreased HIV-AGS-triggered apoptosis in RLW cells. Exposure of HIV-containing apoptotic hepatocytes to HSC prevented activation of inflammasome and induced pro-fibrotic activation in these cells. CONCLUSION: We conclude that by suppressing oxidative stress and restoring proteasomal and lysosomal functions impaired by HIV and ethanol metabolism, OCA decreases accumulation of HIV in hepatocytes, leading to down-regulation of apoptosis in these cells. In addition, OCA reverses pro-fibrotic and inflammasome-related activation of HSC triggered by engulfment of HIV-containing apoptotic hepatocytes, potentially contributing to suppression of liver fibrosis development.

Neuronal-derived extracellular vesicles are enriched in the brain and serum of HIV-1 transgenic rats.

Despite the efficacy of combination antiretroviral therapy (ART) in controlling human immunodeficiency virus (HIV-1) replication, cytotoxic viral proteins such as HIV-1 transactivator of transcription (Tat) persist in tissues such as the brain. Although HIV-1 does not infect neuronal cells, it is susceptible to viral Tat protein-mediated toxicity, leading to neuroinflammation that underlies HIV-associated neurocognitive disorders (HAND). Given the role of extracellular vesicles (EVs) in both cellular homoeostasis and under pathological conditions, we sought to investigate the alterations in the quantity of neuronal-derived EVs in the brain - as defined by the presence of cell adhesion molecule L1 (L1CAM) and to evaluate the presence of L1CAM+ EVs in the peripheral circulation of HIV-1 transgenic (HIV-1 Tg) rats. The primary goal of this study was to investigate the effect of long-term exposure of HIV-1 viral proteins on the release of neuronal EVs in the brain and their transfer in the systemic compartment. Brain and serum EVs were isolated from both wild type and HIV-1 Tg rats using differential ultracentrifugation with further purification using the Optiprep gradient method. The subpopulation of neuronal EVs was further enriched using immunoprecipitation. The current findings demonstrated increased presence of L1CAM+ neuronal-derived EVs both in the brain and serum of HIV-1 Tg rats.

Intranasal Delivery of lincRNA-Cox2 siRNA Loaded Extracellular Vesicles Decreases Lipopolysaccharide-Induced Microglial Proliferation in Mice.

Long non-coding RNAs (lncRNAs), including long intergenic non-coding RNAs (lincRNAs), play an important regulatory role in controlling various biological processes. Both in vitro and in vivo studies have demonstrated that lincRNA-Cox2 plays a global regulatory role in regulating the expression of immune genes. Extracellular vesicles (EVs) are cell-derived nanosized membrane vesicles that have gained increasing attention in recent years due to their ability to efficiently deliver therapeutics to specific target organs or cell types. In this study, we found that lincRNA-Cox2 controls the expression of a set of cell cycle genes in lipopolysaccharide (LPS)-stimulated microglial cells. Our in vitro study suggested that knocking down lincRNA-Cox2 reversed LPS-induced microglial proliferation. In addition, our in vivo study demonstrated that intranasally delivered lincRNA-Cox2-siRNA loaded EVs could reach the brain resulting in a significant decrease in the expression of lincRNA-Cox2 in the microglia. Importantly, lincRNA-Cox2-siRNA loaded EVs also decreased LPS-induced microglial proliferation in mice. These findings indicate that intranasal delivery of EV-loaded small RNA could be developed as therapeutics for treatment of a multitude of CNS disorders.

Strategies for the use of Extracellular Vesicles for the Delivery of Therapeutics.

Extracellular vesicles (EVs) are nanosized, membrane-bound vesicles released from eukaryotic and prokaryotic cells that can transport cargo containing DNA, RNA, lipids and proteins, between cells as a means of intercellular communication. Although EVs were initially considered to be cellular debris deprived of any essential biological functions, emerging literature highlights the critical roles of EVs in the context of intercellular signaling, maintenance of tissue homeostasis, modulation of immune responses, inflammation, cancer progression, angiogenesis, and coagulation under both physiological and pathological states. Based on the ability of EVs to shuttle proteins, lipids, carbohydrates, mRNAs, long non-coding RNAs (lncRNAs), microRNAs, chromosomal DNA, and mitochondrial DNA into target cells, the presence and content of EVs in biofluids have been exploited for biomarker research in the context of diagnosis, prognosis and treatment strategies. Additionally, owing to the characteristics of EVs such as stability in circulation, biocompatibility as well as low immunogenicity and toxicity, these vesicles have become attractive systems for the delivery of therapeutics. More recently, EVs are increasingly being exploited as conduits for delivery of therapeutics for anticancer strategies, immunomodulation, targeted drug delivery, tissue regeneration, and vaccination. In this review, we highlight and discuss the multiple strategies that are employed for the use of EVs as delivery vehicles for therapeutic agents, including the potential advantages and challenges involved. Graphical abstract.

Alcohol Metabolism Potentiates HIV-Induced Hepatotoxicity: Contribution to End-Stage Liver Disease.

In an era of improved survival due to modern antiretroviral therapy, liver disease has become a major cause of morbidity and mortality, resulting in death in 15-17% of human immunodeficiency virus (HIV)-infected patients. Alcohol enhances HIV-mediated liver damage and promotes the progression to advanced fibrosis and cirrhosis. However, the mechanisms behind these events are uncertain. Here, we hypothesize that ethanol metabolism potentiates accumulation of HIV in hepatocytes, causing oxidative stress and intensive apoptotic cell death. Engulfment of HIV-containing apoptotic hepatocytes by non-parenchymal cells (NPCs) triggers their activation and liver injury progression. This study was performed on primary human hepatocytes and Huh7.5-CYP cells infected with HIV-1ADA, and major findings were confirmed by pilot data obtained on ethanol-fed HIV-injected chimeric mice with humanized livers. We demonstrated that ethanol exposure potentiates HIV accumulation in hepatocytes by suppressing HIV degradation by lysosomes and proteasomes. This leads to increased oxidative stress and hepatocyte apoptosis. Exposure of HIV-infected apoptotic hepatocytes to NPCs activates the inflammasome in macrophages and pro-fibrotic genes in hepatic stellate cells. We conclude that while HIV and ethanol metabolism-triggered apoptosis clears up HIV-infected hepatocytes, continued generation of HIV-expressing apoptotic bodies may be detrimental for progression of liver inflammation and fibrosis due to constant activation of NPCs.

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis.

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease's pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

Human-like NSG mouse glycoproteins sialylation pattern changes the phenotype of human lymphocytes and sensitivity to HIV-1 infection.

BACKGROUND: The use of immunodeficient mice transplanted with human hematopoietic stem cells is an accepted approach to study human-specific infectious diseases such as HIV-1 and to investigate multiple aspects of human immune system development. However, mouse and human are different in sialylation patterns of proteins due to evolutionary mutations of the CMP-N-acetylneuraminic acid hydroxylase (CMAH) gene that prevent formation of N-glycolylneuraminic acid from N-acetylneuraminic acid. How changes in the mouse glycoproteins' chemistry affect phenotype and function of transplanted human hematopoietic stem cells and mature human immune cells in the course of HIV-1 infection are not known. RESULTS: We mutated mouse CMAH in the NOD/scid-IL2Rγc-/- (NSG) mouse strain, which is widely used for the transplantation of human cells, using the CRISPR/Cas9 system. The new strain provides a better environment for human immune cells. Transplantation of human hematopoietic stem cells leads to broad B cells repertoire, higher sensitivity to HIV-1 infection, and enhanced proliferation of transplanted peripheral blood lymphocytes. The mice showed no effect on the clearance of human immunoglobulins and enhanced transduction efficiency of recombinant adeno-associated viral vector rAAV2/DJ8. CONCLUSION: NSG-cmah-/- mice expand the mouse models suitable for human cells transplantation, and this new model has advantages in generating a human B cell repertoire. This strain is suitable to study different aspects of the human immune system development, provide advantages in patient-derived tissue and cell transplantation, and could allow studies of viral vectors and infectious agents that are sensitive to human-like sialylation of mouse glycoproteins.

Matrix stiffness regulate apoptotic cell death in HIV-HCV co-infected hepatocytes: Importance for liver fibrosis progression.

HIV-HCV co-infection causes rapid progression of liver fibrosis. These outcomes to liver cirrhosis can be improved, but not stopped by specific antiviral therapies. Due to high significance of HIV-HCV interactions for morbidity and mortality in co-infected patients, our attention was attracted to the multi-component pathogenesis of fibrosis progression as the transition to end-stage liver disease development. In this study, we hypothesize that increased matrix stiffness enhances apoptosis in HCV-HIV-co-infected hepatocytes and that capturing of apoptotic bodies (AB) derived from these infected hepatocytes by hepatic stellate cells (HSC) drives the fibrosis progression. As the source of viruses, JFH1 (HCV genotype 2a) and HIV-1ADA (either purified or containing in infected macrophage supernatants) were chosen. Using Huh7.5-CYP (RLW) cells and primary human hepatocytes mono-infected with HCV and HIV or co-infected, we have shown that both HCV and HIV RNA levels were increased in co-infected cells, which was accompanied by hepatocyte apoptosis. This apoptosis was attenuated by azidothymidine treatment. The levels of both infections and apoptosis were more prominent in primary hepatocytes cultured on substrates mimicking fibrotic stiffness (24 kPa-stiff) compared to substrates mimicking healthy liver (2.4 kPa-soft). The engulfment of AB from pathogen-exposed hepatocytes activated pro-fibrotic mRNAs in HSC. Overall, the increased matrix stiffness is not only a consequence of liver inflammation/fibrosis, but the condition that further accelerates liver fibrosis development. This is attributed to the switching of HSC to pro-fibrotic phenotype by capturing of excessive amounts of apoptotic HCV- and HIV-infected hepatocytes.

Demethylase JMJD6 as a New Regulator of Interferon Signaling: Effects of HCV and Ethanol Metabolism.

BACKGROUND & AIMS: Alcohol-induced progression of hepatitis C virus (HCV) infection is related to dysfunction of innate immunity in hepatocytes. Endogenously produced interferon (IFN)α induces activation of interferon-stimulated genes (ISGs) via triggering of the Janus kinase-signal transducer and activator of transcription 1 (STAT1) pathway. This activation requires protein methyltransferase 1-regulated arginine methylation of STAT1. Here, we aimed to study whether STAT1 methylation also depended on the levels of demethylase jumonji domain-containing 6 protein (JMJD6) and whether ethanol and HCV affect JMJD6 expression in hepatocytes. METHODS: Huh7.5-CYP (RLW) cells and hepatocytes were exposed to acetaldehyde-generating system (AGS) and 50 mmol/L ethanol, respectively. JMJD6 messenger RNA and protein expression were measured by real-time polymerase chain reaction and Western blot. IFNα-activated cells either overexpressing JMJD6 or with knocked-down JMJD6 expression were tested for STAT1 methylation, ISG activation, and HCV RNA. In vivo studies have been performed on C57Bl/6 mice (expressing HCV structural proteins or not) or chimeric mice with humanized livers fed control or ethanol diets. RESULTS: AGS exposure to cells up-regulated JMJD6 expression in RLW cells. These results were corroborated by ethanol treatment of primary hepatocytes. The promethylating agent betaine reversed the effects of AGS/ethanol. Similar results were obtained in vivo, when mice were fed control/ethanol with and without betaine supplementation. Overexpression of JMJD6 suppressed STAT1 methylation, IFNα-induced ISG activation, and increased HCV-RNA levels. In contrast, JMJD6 silencing enhanced STAT1 methylation, ISG stimulation by IFNα, and attenuated HCV-RNA expression in Huh7.5 cells. CONCLUSIONS: We conclude that arginine methylation of STAT1 is suppressed by JMJD6. Both HCV and acetaldehyde increase JMJD6 levels, thereby impairing STAT1 methylation and innate immunity protection in hepatocytes exposed to the virus and/or alcohol.

Human hepatocyte depletion in the presence of HIV-1 infection in dual reconstituted humanized mice.

Human immunodeficiency virus type 1 (HIV-1) infection impairs liver function, and liver diseases have become a leading cause of morbidity in infected patients. The immunopathology of liver damage caused by HIV-1 remains unclear. We used chimeric mice dually reconstituted with a human immune system and hepatocytes to address the relevance of the model to pathobiology questions related to human hepatocyte survival in the presence of systemic infection. TK-NOG males were transplanted with mismatched human hematopoietic stem/progenitor cells and hepatocytes, human albumin concentration and the presence of human immune cells in blood were monitored for hepatocytes and immune reconstitution, and mice were infected with HIV-1. HIV-1-infected animals showed a decline in human albumin concentration with a significant reduction in percentage of human hepatocytes compared to uninfected mice. The decrease in human albumin levels correlated with a decline in CD4+ cells in the liver and with an increase in HIV-1 viral load. HIV-1 infection elicited proinflammatory response in the immunological milieu of the liver in HIV-infected mice compared to uninfected animals, as determined by upregulation of IL23, CXCL10 and multiple toll-like receptor expression. The inflammatory reaction associated with HIV-1 infection in vivo could contribute to the depletion and dysfunction of hepatocytes. The dual reconstituted TK-NOG mouse model is a feasible platform to investigate hepatocyte-related HIV-1 immunopathogenesis.This article has an associated First Person interview with the first author of the paper.

Curcuma longa extract reduces inflammatory and oxidative stress biomarkers in osteoarthritis of knee: a four-month, double-blind, randomized, placebo-controlled trial.

BACKGROUND AND PURPOSE: Curcuma longa L. (CL), an Indian herb, has been used to treat many disorders because of its wide spectrum of pharmacological activities. It has been shown to exhibit anti-oxidant and anti-inflammatory properties, and is being used as herbal remedy since ancient times. Osteoarthritis of knee (KOA) is a chronic painful disorder in which prolong use of non-steroidal anti-inflammatory drugs (NSAIDs) or steroids may result into many serious side effects; hence, there is a need to develop herbal drugs, having good analgesia without side effects. Therefore, we planned to evaluate the efficacy of CL in KOA. METHODS: The study was designed as a randomized, double-blind, placebo-controlled trial in patients of KOA. After obtaining ethical clearance and written informed consent, a total of 160 patients of KOA were randomly enrolled into two groups to receive either CL extract or placebo along with the standard drug regimen. The patients were assessed on day 0, day 60, and day 120. On the days of their visit, the clinical prognosis was assessed by visual analog scale (VAS) and Western Ontario and McMaster Universities (WOMAC) Osteoarthritis index. On these days, the radiographs were also taken for Kellgren and Lawrence grading and blood samples were collected for assessing the changes in levels of IL-1ß and biomarkers of oxidative stress, such as reactive oxygen species and malondialdehyde (MDA). RESULTS: Over all significant improvement was observed in the patients of CL extract group as compared to placebo group. Clinically, the VAS and WOMAC scores became better, and simultaneously, the levels of biomarkers, viz., IL-1ß, ROS, and MDA, were also significantly (p < 0.05) improved. CONCLUSION: It may be concluded that on chronic administration, CL suppresses inflammation and brings clinical improvement in patients of KOA, which may be observed by decreased level of IL-1ß and VAS/WOMAC scores, respectively. At the same time, CL decreases the oxidative stress also.

Bryostatin-1 causes radiosensitization of BMG-1 malignant glioma cells through differential activation of protein kinase-Cδ not evident in the non-malignant AA8 fibroblasts.

Bryostatin-1 (bryo-1), a non-phorbol ester, is known to sensitize mammalian cells against certain chemotherapeutic drugs. We assessed its ability to modify radiation response of mammalian cells using Chinese hamster fibroblasts AA8 cells and human malignant glioma BMG-1 cells. In the malignant glioma BMG-1 cell line, bryo-1 pre-treatment significantly enhanced radiation-induced growth inhibition and cytogenetic damage, and further reduced the clonogenic cell survival as compared to cells irradiated at the clinically relevant dose of 2 Gy. PKCδ expression increased significantly when bryo-1 pre-treated BMG-1 glioma cells were irradiated at 2 Gy and induced prolonged ERK-1/2 activation associated with p21 overexpression. Silencing PKCδ resulted in inhibition of bryo-1-induced radiosensitization. In contrast, bryo-1 failed to alter radiosensitivity (cell survival; growth inhibition; cytogenetic damage) or activate ERK1/2 pathway in the AA8 fibroblasts despite PKCδ phosphorylation at its regulatory (Y155) domain, indicating alternate mechanisms in these non-malignant cells as compared to the glioma cells. This study suggests that bryo-1 may effectively enhance the radiosensitivity of malignant cells and warrants further in-depth investigations to evaluate its radiosensitizing potential in various cell types.