ABCC4 impacts megakaryopoiesis and protects megakaryocytes against 6-mercaptopurine induced cytotoxicity.

The role of ABCC4, an ATP-binding cassette transporter, in the process of platelet formation, megakaryopoiesis, is unknown. Here, we show that ABCC4 is highly expressed in megakaryocytes (MKs). Mining of public genomic data (ATAC-seq and genome wide chromatin interactions, Hi-C) revealed that key megakaryopoiesis transcription factors (TFs) interacted with ABCC4 regulatory elements and likely accounted for high ABCC4 expression in MKs. Importantly these genomic interactions for ABCC4 ranked higher than for genes with known roles in megakaryopoiesis suggesting a role for ABCC4 in megakaryopoiesis. We then demonstrate that ABCC4 is required for optimal platelet formation as in vitro differentiation of fetal liver derived MKs from Abcc4-/- mice exhibited impaired proplatelet formation and polyploidization, features required for optimal megakaryopoiesis. Likewise, a human megakaryoblastic cell line, MEG-01 showed that acute ABCC4 inhibition markedly suppressed key processes in megakaryopoiesis and that these effects were related to reduced cAMP export and enhanced dissociation of a negative regulator of megakaryopoiesis, protein kinase A (PKA) from ABCC4. PKA activity concomitantly increased after ABCC4 inhibition which was coupled with significantly reduced GATA-1 expression, a TF needed for optimal megakaryopoiesis. Further, ABCC4 protected MKs from 6-mercaptopurine (6-MP) as Abcc4-/- mice show a profound reduction in MKs after 6-MP treatment. In total, our studies show that ABCC4 not only protects the MKs but is also required for maximal platelet production from MKs, suggesting modulation of ABCC4 function might be a potential therapeutic strategy to regulate platelet production.

A PPIX-binding probe facilitates discovery of PPIX-induced cell death modulation by peroxiredoxin.

While heme synthesis requires the formation of a potentially lethal intermediate, protoporphyrin IX (PPIX), surprisingly little is known about the mechanism of its toxicity, aside from its phototoxicity. The cellular protein interactions of PPIX might provide insight into modulators of PPIX-induced cell death. Here we report the development of PPB, a biotin-conjugated, PPIX-probe that captures proteins capable of interacting with PPIX. Quantitative proteomics in a diverse panel of mammalian cell lines reveal a high degree of concordance for PPB-interacting proteins identified for each cell line. Most differences are quantitative, despite marked differences in PPIX formation and sensitivity. Pathway and quantitative difference analysis indicate that iron and heme metabolism proteins are prominent among PPB-bound proteins in fibroblasts, which undergo PPIX-mediated death determined to occur through ferroptosis. PPB proteomic data (available at PRIDE ProteomeXchange # PXD042631) reveal that redox proteins from PRDX family of glutathione peroxidases interact with PPIX. Targeted gene knockdown of the mitochondrial PRDX3, but not PRDX1 or 2, enhance PPIX-induced death in fibroblasts, an effect blocked by the radical-trapping antioxidant, ferrostatin-1. Increased PPIX formation and death was also observed in a T-lymphoblastoid ferrochelatase-deficient leukemia cell line, suggesting that PPIX elevation might serve as a potential strategy for killing certain leukemias.

Extracellular Vesicles from Human Papilloma Virus-Infected Cervical Cancer Cells Enhance HIV-1 Replication in Differentiated U1 Cell Line.

In the current study, we hypothesized that extracellular vesicles (EVs) secreted from human papilloma virus (HPV)-infected cervical cancer cells exacerbate human immunodeficiency virus (HIV)-1 replication in differentiated U1 cell line through an oxidative stress pathway. To test the hypothesis, we treated an HIV-1-infected macrophage cell line (U1) with HPV-infected Caski cell culture supernatant (CCS). We observed a significant increase in HIV-1 replication, which was associated with an increase in the expression of cytochrome P450 (CYPs 1A1 and 2A6) in the CCS-treated U1 cells. Furthermore, we isolated EVs from CCS (CCS-EVs), which showed the presence of CYPs (1A1, 2A6), superoxide dismutase 1 (SOD1), and HPV oncoproteins HPV16 E6. CCS-EVs when exposed to the U1 cells also significantly increased HIV-1 replication. Treatment of antioxidant, CYP1A1 and CYP2A6 inhibitors, and chemodietary agents with antioxidant properties significantly reduced the CCS and CCS-EVs mediated HIV-1 replication in U1 cells. Altogether, we demonstrate that cervical cancer cells exacerbate HIV-1 replication in differentiated U1 cell line via transferring CYPs and HPV oncoproteins through EVs. We also show that the viral replication occurs via CYP and oxidative stress pathways, and the viral replication is also reduced by chemodietary agents. This study provides important information regarding biological interactions between HPV and HIV-1 via EVs leading to enhanced HIV-1 replication.

Proteomic Profiling of Exosomes Derived from Plasma of HIV-Infected Alcohol Drinkers and Cigarette Smokers.

Abuse of alcohol and tobacco could exacerbate HIV pathogenesis by transferring materials through exosomes (small nanovesicles). Exosomes present a stable and accessible source of information concerning the health and/or disease status of patients, which can provide diagnostic and prognostic biomarkers for myriad conditions. Therefore, we aimed to study the specific exosomal proteins that are altered in both HIV-infected subjects and alcohol/tobacco users. Exosomes were isolated from plasma of the following subjects: a) HIV-negative subjects (healthy), b) HIV-positive subjects (HIV), c) HIV-negative alcohol drinkers (drinkers), d) HIV-negative tobacco smokers (smokers), e) HIV-positive drinkers (HIV + drinkers), and f) HIV-positive smokers (HIV + smokers). Quantitative proteomic profiling was then performed from these exosomes. Sixteen proteins were significantly altered in the HIV group, ten in drinkers, four in HIV + drinkers, and fifteen in smokers compared to healthy subjects. Only one protein, fibulin-1 (FBLN1), was significantly altered in HIV + smokers. Interestingly, hemopexin was not significantly altered in drinkers or HIV patients but was significantly altered in HIV + drinkers. Further, our study is the first to show properdin expression in plasma exosomes, which was decreased in HIV + smokers and HIV + drinkers compared to HIV patients. The present findings suggest that hemopexin and properdin show potential as markers for physiological effects that may arise in HIV-infected individuals who abuse alcohol and tobacco. Graphical abstract This study presents a proteomic analysis of plasma-derived exosomes from HIV-infected alcohol drinkers and smokers. Among the proteins altered due to drug-abuse, hemopexin and properdin were of highest significance. These proteins can be potential biomarkers for co-morbid conditions associated with drug abuse in HIV-patients.

The role of cytochrome P450 2E1 on ethanol-mediated oxidative stress and HIV replication in human monocyte-derived macrophages.

BACKGROUND: Alcohol consumption is considered to be a major health problem among people living with HIV/AIDS. Our previous reports have shown that ethanol reduced intracellular concentrations of antiretroviral drugs elvitegravir and darunavir in the HIV-1-infected U1 cell line. Ethanol also increased HIV-1 replication despite the presence of elvitegravir. Our previous finding has also shown that the levels of cytochrome P450 enzyme 2E1 (CYP2E1) and oxidative stress in blood monocytes were induced, while the concentration of alcohol in the plasma was reduced in HIV-1-infected alcohol users compared to uninfected alcohol users. However, the role of CYP2E1 in ethanol-enhanced oxidative stress and HIV-1 replication is still unclear. METHODS: This study examined the chronic effects (14 days) of ethanol on HIV viral load, oxidative DNA damage, expression of CYP2E1, expression of antioxidant enzymes (AOEs), expression of reactive oxygen species (ROS) in human monocyte-derived macrophages (MDM). Further, to evaluate the role of CYP2E1 in mediating ethanol-induced viral replication, CYP2E1 siRNA and CYP2E1 selective inhibitor were used in the HIV-1-infected U1 cell line following ethanol treatment. RESULTS: Chronic ethanol exposure demonstrated an increase in oxidative DNA damage and CYP2E1 expression in both non-infected and HIV-1-infected MDM. Our results showed that ethanol chronic exposure increased HIV-1 replication by ~3-fold in HIV-1-infected MDM. This ethanol-enhanced HIV-1 replication was associated with an increased oxidative DNA damage, an increased expression of CYP2E1, and a decreased expression of antioxidant enzyme PRDX6. In HIV-1-infected U1 cell line, we observed a decreased viral replication (~30%) and a decreased DNA damage (~100%) after repression of CYP2E1 by siRNA, upon ethanol exposure. We also observed a decreased viral replication (~25%) after inhibition of CYP2E1 by using selective CYP2E1 inhibitor. CONCLUSIONS: The data suggest that chronic ethanol exposure increases HIV-1 replication in MDM, at least in part, through CYP2E1-mediated oxidative stress. These results are clinically relevant to potentially find effective treatment strategies for HIV-1-infected alcohol users.

Cytokine profiling of exosomes derived from the plasma of HIV-infected alcohol drinkers and cigarette smokers.

Cytokines and chemokines circulate in plasma and may be transferred to distant sites, via exosomes. HIV infection is associated with dysregulation of cytokines and chemokines, which subsequently contribute to the pathogenesis of HIV. Alcohol and tobacco exposure, which are prevalent in HIV-infected individuals, may induce changes in the expression of cytokines and chemokines. Therefore, our aim in this study was to quantify plasma exosomal cytokines and chemokines that we expect to exacerbate toxicity or disease progression in HIV-positive drug abusers. We measured the levels of cytokines and chemokines in the plasma and plasma exosomes of 39 patients comprising six groups: HIV-negative and HIV-positive non drug abusers, HIV-negative and HIV-positive alcohol users, and HIV-negative and HIV positive tobacco smokers. We measured six cytokines (TNF-α, IL-1ß, IL-8, IL-6, IL-1ra, IL-10) and two chemokines (MCP-1 and RANTES). All were present in exosomes of healthy subjects, but their levels varied between different study groups. HIV-positive alcohol drinkers had higher levels of plasma IL-8 compared to those of HIV-positive non-drinkers. The IL-1ra level was significantly higher in exosomes of non-HIV-infected alcohol drinkers compared to those of HIV-positive alcohol drinkers. Interestingly, the IL-10 level was higher in exosomes compared with their respective plasma levels in all study groups except HIV-positive non-alcohol drinkers. IL-10 was completely packaged in exosomes of HIV-positive smokers. HIV-positive smokers had significantly higher levels of plasma IL-8 compared with HIV-positive non-smokers and significantly higher exosomal IL-6 levels compared with HIV-negative subjects. HIV-positive smokers had significantly increased plasma levels of IL-1ra compared to HIV-positive non-smokers. The MCP-1 levels in the plasma of HIV-positive smokers was significantly higher than in either HIV-positive non-drug abusers or HIV-negative smokers. Overall, the findings suggest that plasma cytokines and chemokines are packaged in exosomes at varying degrees in different study groups. Exosomal cytokines and chemokines are likely to have a significant biological role at distant sites including cells in the brain.

Potential neuroprotective role of astroglial exosomes against smoking-induced oxidative stress and HIV-1 replication in the central nervous system.

INTRODUCTION: HIV-1-infected smokers are at risk of oxidative damage to neuronal cells in the central nervous system by both HIV-1 and cigarette smoke. Since neurons have a weak antioxidant defense system, they mostly depend on glial cells, particularly astrocytes, for protection against oxidative damage and neurotoxicity. Astrocytes augment the neuronal antioxidant system by supplying cysteine-containing products for glutathione synthesis, antioxidant enzymes such as SOD and catalase, glucose for antioxidant regeneration via the pentose-phosphate pathway, and by recycling of ascorbic acid. Areas covered: The transport of antioxidants and energy substrates from astrocytes to neurons could possibly occur via extracellular nanovesicles called exosomes. This review highlights the neuroprotective potential of exosomes derived from astrocytes against smoking-induced oxidative stress, HIV-1 replication, and subsequent neurotoxicity observed in HIV-1-positive smokers. Expert opinion: During stress conditions, the antioxidants released from astrocytes either via extracellular fluid or exosomes to neurons may not be sufficient to provide neuroprotection. Therefore, we put forward a novel strategy to combat oxidative stress in the central nervous system, using synthetically developed exosomes loaded with antioxidants such as glutathione and the anti-aging protein Klotho.

Benzo(a)pyrene in Cigarette Smoke Enhances HIV-1 Replication through NF-κB Activation via CYP-Mediated Oxidative Stress Pathway.

Smoking aggravates HIV-1 pathogenesis and leads to decreased responses to antiretroviral therapy. In this study, we aim to find a molecular mechanism that would explain smoking-induced HIV-1 replication. Benzo(a)pyrene (BaP), a major carcinogen in cigarette, requires metabolic activation through cytochrome P450s (CYPs) to exert its toxic effects. We hypothesized that CYP-mediated BaP metabolism generates reactive oxygen species (ROS), and the resultant oxidative stress aggravates HIV-1 replication. As expected, we observed ~3 to 4-fold increase in HIV-1 replication in U1 cells and human primary macrophages after chronic BaP exposure. We also observed ~30-fold increase in the expression of CYP1A1 at mRNA level, ~2.5-fold increase in its enzymatic activity as well as elevated ROS and cytotoxicity in U1 cells. The knock-down of the CYP1A1 gene using siRNA and treatment with selective CYP inhibitors and antioxidants significantly reduced HIV-1 replication. Further, we observed a nuclear translocation of NF-κB subunits (p50 and p65) after chronic BaP exposure, which was reduced by treatment with siRNA and antioxidants/CYP inhibitors. Suppression of NF-κB pathway using specific NF-κB inhibitors also significantly reduced HIV-1 replication. Altogether, our results suggest that BaP enhances HIV-1 replication in macrophages by a CYP-mediated oxidative stress pathway followed by the NF-κB pathway.

Recent advances in cancer outcomes in HIV-positive smokers.

HIV-infected smokers are at relatively higher risk of cancer than HIV-infected non-smokers. HIV weakens the immune system and renders infected individuals more vulnerable to the carcinogenic effects of smoking. HIV-infected smokers suffer more aggressive forms of cancers than do non-smokers because of the cumulative effects of the virus and cigarette smoke carcinogens. The major types of cancer observed in HIV-infected smokers are lung, head and neck, esophageal, anal, and cervical cancers. In this review, we will discuss the recent advances in cancer outcomes, primarily in terms of cancer incidence, prevalence, and progression in HIV patients who are smokers.

Monocyte-derived exosomes upon exposure to cigarette smoke condensate alter their characteristics and show protective effect against cytotoxicity and HIV-1 replication.

Smoking is known to exacerbate HIV-1 pathogenesis, especially in monocytes, through the oxidative stress pathway. Exosomes are known to alter HIV-1 pathogenesis through inter-cellular communication. However, the role of exosomes in smoking-mediated HIV-1 pathogenesis is unknown. In this study, we investigated the effect of cigarette smoke condensate (CSC) on the characteristics of monocyte-derived exosomes and their influence on HIV-1 replication. Initially, we demonstrated that CSC reduced total protein and antioxidant capacity in exosomes derived from HIV-1-infected and uninfected macrophages. The exosomes from CSC-treated uninfected cells showed a protective effect against cytotoxicity and viral replication in HIV-1-infected macrophages. However, exosomes derived from HIV-1-infected cells lost their protective capacity. The results suggest that the exosomal defense is likely to be more effective during the early phase of HIV-1 infection and diminishes at the latter phase. Furthermore, we showed CSC-mediated upregulation of catalase in exosomes from uninfected cells, with a decrease in the levels of catalase and PRDX6 in exosomes derived from HIV-1-infected cells. These results suggest a potential role of antioxidant enzymes, which are differentially packaged into CSC-exposed HIV-1-infected and uninfected cell-derived exosomes, on HIV-1 replication of recipient cells. Overall, our study suggests a novel role of exosomes in tobacco-mediated HIV-1 pathogenesis.

Effect of Polyaryl Hydrocarbons on Cytotoxicity in Monocytic Cells: Potential Role of Cytochromes P450 and Oxidative Stress Pathways.

BACKGROUND: Benzo(a)pyrene (BaP), naphthalene (NPh), phenanthrene (Phe), benzo(a)antharacene (BeA), and benzo(b)fluoranthene (BeF) are known carcinogenic polyaryl hydrocarbons (PAHs) present in cigarette smoke. This study was designed to examine the relative effect of these constituents on the cytotoxicity of monocytic cells and the possible mechanism of PAH-mediated cytotoxicity. METHODS: We examined the acute (6-24 hours) and chronic (7 days) effects of these PAHs on the expression of cytochromes P450 (CYPs), oxidative stress, and cytotoxicity. The treated cells were examined for mRNA and protein levels of CYPs (1A1 and 3A4) and antioxidants enzymes (AOEs) superoxide dismutase-1 (SOD1) and catalase. Further, we assessed the levels of reactive oxygen species (ROS), caspase-3 cleavage activity, and cell viability. We performed these experiments in U937 and/or primary monocytic cells. RESULTS: Of the five PAHs tested, after chronic treatment only BaP (100 nM) showed a significant increase in the expression of CYP1A1, AOEs (SOD1 and catalase), ROS generation, caspase-3 cleavage activity, and cytotoxicity. However, acute treatment with BaP showed only an increase in the mRNA expression of CYP1A1. CONCLUSIONS: These results suggest that of the five PAHs tested, BaP is the major contributor to the toxic effect of PAHs in monocytic cells, which is likely to occur through CYP and oxidative stress pathways.