The Impact of HIV-and Art-Induced Mitochondrial Dysfunction in Cellular Senescence and Aging

According to the WHO, 38 million individuals were living with human immunodeficiency virus (HIV), 25.4 million of which were using antiretroviral therapy (ART) at the end of 2019. Despite ART-mediated suppression of viral replication, ART is not a cure and is associated with viral persistence, residual inflammation, and metabolic disturbances. Indeed, due to the presence of viral reservoirs, lifelong ART therapy is required to control viremia and prevent disease progression into acquired immune deficiency syndrome (AIDS). Successful ART treatment allows people living with HIV (PLHIV) to achieve a similar life expectancy to uninfected individuals. However, recent studies have illustrated the presence of increased comorbidities, such as accelerated, premature immune aging, in ART-controlled PLHIV compared to uninfected individuals. Studies suggest that both HIV-infection and ART-treatment lead to mitochondrial dysfunction, ultimately resulting in cellular exhaustion, senescence, and apoptosis. Since mitochondria are essential cellular organelles for energy homeostasis and cellular metabolism, their compromise leads to decreased oxidative phosphorylation (OXPHOS), ATP synthesis, gluconeogenesis, and beta-oxidation, abnormal cell homeostasis, increased oxidative stress, depolarization of the mitochondrial membrane potential, and upregulation of mitochondrial DNA mutations and cellular apoptosis. The progressive mitochondrial damage induced by HIV-infection and ART-treatment likely contributes to accelerated aging, senescence, and cellular dysfunction in PLHIV. This review discusses the connections between mitochondrial compromise and cellular dysfunction associated with HIV-and ART-induced toxicities, providing new insights into how HIV and current ART directly impact mitochondrial functions and contribute to cellular senescence and aging in PLHIV. Identifying this nexus and potential mechanisms may be beneficial in developing improved therapeutics for treating PLHIV

HIV-1 Latency and Viral Reservoirs: Existing Reversal Approaches and Potential Technologies, Targets, and Pathways Involved in HIV Latency Studies

Eradication of latent human immunodeficiency virus (HIV) infection is a global health challenge. Reactivation of HIV latency and killing of virus-infected cells, the so-called “kick and kill” or “shock and kill” approaches, are a popular strategy for HIV cure. While antiretroviral therapy (ART) halts HIV replication by targeting multiple steps in the HIV life cycle, including viral entry, integration, replication, and production, it cannot get rid of the occult provirus incorporated into the host-cell genome. These latent proviruses are replication-competent and can rebound in cases of ART interruption or cessation. In general, a very small population of cells harbor provirus, serve as reservoirs in ART-controlled HIV subjects, and are capable of expressing little to no HIV RNA or proteins. Beyond the canonical resting memory CD4+ T cells, HIV reservoirs also exist within tissue macrophages, myeloid cells, brain microglial cells, gut epithelial cells, and hematopoi-etic stem cells (HSCs). Despite a lack of active viral production, latently HIV-infected subjects con-tinue to exhibit aberrant cellular signaling and metabolic dysfunction, leading to minor to major cellular and systemic complications or comorbidities. These include genomic DNA damage; telo-mere attrition; mitochondrial dysfunction; premature aging; and lymphocytic, cardiac, renal, he-patic, or pulmonary dysfunctions. Therefore, the arcane machineries involved in HIV latency and its reversal warrant further studies to identify the cryptic mechanisms of HIV reservoir formation and clearance. In this review, we discuss several molecules and signaling pathways, some of which have dual roles in maintaining or reversing HIV latency and reservoirs, and describe some evolving strategies and possible approaches to eliminate viral reservoirs and, ultimately, cure/eradicate HIV infection

The Sex Specific Effect of Alcohol Consumption on Circulating Levels of CTRP3

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The goal of this project was to establish the effect of alcohol consumption on the circulating levels of the adipose tissue derived protein C1q TNF Related Protein 3 (CTRP3). Adipose tissue secretes several adipokines, such as adiponectin and leptin, which exert a multitude of biological effects important for human health. However, adipose tissue is extremely sensitive to alcohol consumption, leading not only to disrupted fat storage, but also to disruptions in adipokine production. Changes to adipokine secretion could have widespread biological effects and potentially contribute to alcohol-induced ailments, such as alcoholic fatty liver disease (ALD). CTRP3 has been previously demonstrated to attenuate fatty liver disease, and suppression of CTRP3 with alcohol consumption could contribute to development of and progression to alcoholic fatty liver disease. To examine the effect of ethanol consumption on circulating adipokine levels, male and female mice were fed an ethanol containing diet (Lieber- DeCarli 5% (v/v) ethanol diet) for 10-days followed by a single gavage of 5 g/kg ethanol (the NIAAA model), or for 6-weeks with no binge added (chronic model). In female mice, adiponectin levels increased ~2-fold in both models of ethanol feeding, but in male mice increased adiponectin levels were only observed after chronic ethanol feeding. On the other hand, in female mice, circulating CTRP3 levels decreased by ~75% and ~50% in the NIAAA and chronic model, respectively, with no changes observed in the male mice in either feeding model. Leptin levels were unchanged with ethanol feeding regardless of model or sex of mice. Lastly, chronic ethanol feeding led to a significant increase in mortality (~50%) in female mice, with no difference in relative ethanol consumption. These findings indicate that ethanol consumption can dysregulate adipokine secretion, but that the effects vary by sex of animal, method of ethanol consumption, and adipokine examined. These findings also indicate that female mice are more sensitive to the chronic effects of ethanol than male mice. Notably, this is the first study to document the effects of ethanol consumption on the circulating levels of CTRP3. Understanding the impact of excessive alcohol consumption on adipokine production and secretion could identify novel mechanisms of alcohol-induced human disease. However, the mechanism responsible for the increased sensitivity remains elusive

LMP1 Signaling Pathway Activates IRF4 in Latent EBV Infection and a Positive Circuit Between PI3K and Src Is Required

Interferon (IFN) regulatory factors (IRFs) have crucial roles in immune regulation and oncogenesis. We have recently shown that IRF4 is activated through c-Src-mediated tyrosine phosphorylation in virus-transformed cells. However, the intracellular signaling pathway triggering Src activation of IRF4 remains unknown. In this study, we provide evidence that Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) promotes IRF4 phosphorylation and markedly stimulates IRF4 transcriptional activity, and that Src mediates LMP1 activation of IRF4. As to more precise mechanism, we show that LMP1 physically interacts with c-Src, and the phosphatidylinositol 3 kinase (PI3K) subunit P85 mediates their interaction. Depletion of P85 by P85-specific short hairpin RNAs disrupts their interaction and diminishes IRF4 phosphorylation in EBV-transformed cells. Furthermore, we show that Src is upstream of PI3K for activation of both IRF4 and Akt. In turn, inhibition of PI3K kinase activity by the PI3K-speicfic inhibitor LY294002 impairs Src activity. Our results show that LMP1 signaling is responsible for IRF4 activation, and further characterize the IRF4 regulatory network that is a promising therapeutic target for specific hematological malignancies

Protein Phosphatase 1 Abrogates IRF7-Mediated Type I IFN Response In Antiviral Immunity

Interferon (IFN) regulatory factor 7 (IRF7) plays a key role in the production of IFN‐α in response to viral infection, and phosphorylation at IRF7 C‐terminal serine sites is prelude to its function. However, phosphatases that negatively regulate IRF7 phosphorylation and activity have not been reported. In this study, we have identified a conserved protein phosphatase 1 (PP1)‐binding motif in human and mouse IRF7 proteins, and shown that PP1 physically interacts with IRF7. Exogenous expression of PP1 subunits (PP1α, β, or γ) ablates IKKε‐stimulated IRF7 phosphorylation and dramatically attenuates IRF7 transcriptional activity. Inhibition of PP1 activity significantly increases IRF7 phosphorylation and IRF7‐mediated IFN‐α production in response to Newcastle disease virus (NDV) infection or Toll‐like receptor 7 (TLR7) challenge, leading to impaired viral replication. In addition, IFN treatment, TLR challenges and viral infection induce PP1 expression. Our findings disclose for the first time a pivotal role for PP1 in impeding IRF7‐mediated IFN‐α production in host immune responses

Induction of p38- and gc1qr-Dependent IL-8 Expression in Pulmonary Fibroblasts by Soluble Hepatitis c Core Protein

Background: Recent studies suggest that HCV infection is associated with progressive declines in pulmonary function in patients with underlying pulmonary diseases such as asthma and chronic obstructive pulmonary disease. Few molecular studies have addressed the inflammatory aspects of HCV-associated pulmonary disease. Because IL-8 plays a fundamental role in reactive airway diseases, we examined IL-8 signaling in normal human lung fibroblasts (NHLF) in response to the HCV nucleocapsid core protein, a viral antigen shown to modulate intracellular signaling pathways involved in cell proliferation, apoptosis and inflammation. Methods: NHLF were treated with HCV core protein and assayed for IL-8 expression, phosphorylation of the p38 MAPK pathway, and for the effect of p38 inhibition. Results: Our studies demonstrate that soluble HCV core protein induces significant increases in both IL-8 mRNA and protein expression in a dose- and time-dependent manner. Treatment with HCV core led to phosphorylation of p38 MAPK, and expression of IL-8 was dependent upon p38 activation. Using TNFα as a co-stimulant, we observed additive increases in IL-8 expression. HCV core-mediated expression of IL-8 was inhibited by blocking gC1qR, a known receptor for soluble HCV core linked to MAPK signaling. Conclusions: These studies suggest that HCV core protein can lead to enhanced p38- and gC1qR-dependent IL-8 expression. Such a proinflammatory role may contribute to the progressive deterioration in pulmonary function recently recognized in individuals chronically infected with HCV

Enhanced Virus-Specific CD8\u3csup\u3e+\u3c/sup\u3e T Cell Responses by Listeria Monocytogenes-Infected Dendritic Cells in the Context of Tim-3 Blockade

In this study, we engineered Listeria monocytogens (Lm) by deleting the LmΔactA/ΔinlB virulence determinants and inserting HCV-NS5B consensus antigens to develop a therapeutic vaccine against hepatitis C virus (HCV) infection. We tested this recombinant Lm-HCV vaccine in triggering of innate and adaptive immune responses in vitro using immune cells from HCV-infected and uninfected individuals. This live-attenuated Lm-HCV vaccine could naturally infect human dendritic cells (DC), thereby driving DC maturation and antigen presentation, producing Th1 cytokines, and triggering CTL responses in uninfected individuals. However, vaccine responses were diminished when using DC and T cells derived from chronically HCV-infected individuals, who express higher levels of inhibitory molecule Tim-3 on immune cells. Notably, blocking Tim-3 signaling significantly improved the innate and adaptive immune responses in chronically HCV-infected patients, indicating that novel strategies to enhance the potential of antigen presentation and cellular responses are essential for developing an effective therapeutic vaccine against HCV infection

Expansion of Myeloid-Derived Suppressor Cells Promotes Differentiation of Regulatory T Cells in HIV-1+ Individuals

Objective: Regulatory T cells (Tregs) contribute to HIV-1 disease progression by impairing antiviral immunity; however, the precise mechanisms responsible for the development of Tregs in the setting of HIV-1 infection are incompletely understood. Design: In this study, we provide evidence that HIV-induced expansion of monocytic myeloid-derived suppressor cells (M-MDSCs) promote the differentiation of Foxp3+ Tregs. Methods: We measured MDSC induction and cytokine expression by flow cytometry and analyzed their functions by coculturing experiments. Results: We observed a dramatic increase in M-MDSC frequencies in the peripheral blood of HIV-1 seropositive (HIV-1+) individuals, even in those on antiretroviral therapy with undetectable viremia, when compared with healthy participants. We also observed increases in M-MDSCs after incubating healthy peripheral mononuclear cells (PBMCs) with HIV-1 proteins (gp120 or Tat) or Toll-like receptor 4 ligand lipopolysaccharides in vitro, an effect that could be abrogated in the presence of the phosphorylated signal transducer and activator of transcription 3 inhibitor, STA-21. Functional analyses indicated that M-MDSCs from HIV-1+ individuals express higher levels of IL-10, tumor growth factor-β, IL-4 receptor α, p47phex, programmed death-ligand 1, and phosphorylated signal transducer and activator of transcription 3 – all of which are known mediators of myelopoiesis and immunosuppression. Importantly, incubation of healthy CD4+ T cells with MDSCs derived from HIV-1+ individuals significantly increased differentiation of Foxp3+ Tregs. In addition, depletion of MDSCs from PBMCs of HIV-1+ individuals led to a significant reduction of Foxp3+ Tregs and increase of IFNγ production by CD4+ T effector cells. Conclusions: These results suggest that HIV-induced MDSCs promote Treg cell development and inhibit T cell function – a hallmark of many chronic infectious diseases

Tim-3 Negatively Regulates IL-12 Expression by Monocytes in HCV Infection

T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is a newly identified negative immunomodulator that is up-regulated on dysfunctional T cells during viral infections. The expression and function of Tim-3 on human innate immune responses during HCV infection, however, remains poorly characterized. In this study, we report that Tim-3 is constitutively expressed on human resting CD14+ monocyte/macrophages (M/MØ) and functions as a cap to block IL-12, a key pro-inflammatory cytokine linking innate and adaptive immune responses. Tim-3 expression is significantly reduced and IL-12 expression increased upon stimulation with Toll-like receptor 4 (TLR4) ligand - lipopolysaccharide (LPS) and TLR7/8 ligand - R848. Notably, Tim-3 is over-expressed on un-stimulated as well as TLR-stimulated M/MØ, which is inversely associated with the diminished IL-12 expression in chronically HCV-infected individuals when compared to healthy subjects. Up-regulation of Tim-3 and inhibition of IL-12 are also observed in M/MØ incubated with HCV-expressing hepatocytes, as well as in primary M/MØ or monocytic THP-1 cells incubated with HCV core protein, an effect that mimics the function of complement C1q and is reversible by blocking the HCV core/gC1qR interaction. Importantly, blockade of Tim-3 signaling significantly rescues HCV-mediated inhibition of IL-12, which is primarily expressed by Tim-3 negative M/MØ. Tim-3 blockade reduces HCV core-mediated expression of the negative immunoregulators PD-1 and SOCS-1 and increases STAT-1 phosphorylation. Conversely, blocking PD-1 or silencing SOCS-1 gene expression also decreases Tim-3 expression and enhances IL-12 secretion and STAT-1 phosphorylation. These findings suggest that Tim-3 plays a crucial role in negative regulation of innate immune responses, through crosstalk with PD-1 and SOCS-1 and limiting STAT-1 phosphorylation, and may be a novel target for immunotherapy to HCV infection

p62-mediated Selective Autophagy Endows Virus-Transformed Cells With Insusceptibility to DNA Damage Under Oxidative Stress

DNA damage response (DDR) and selective autophagy both can be activated by reactive oxygen/nitrogen species (ROS/RNS), and both are of paramount importance in cancer development. The selective autophagy receptor and ubiquitin (Ub) sensor p62 plays a key role in their crosstalk. ROS production has been well documented in latent infection of oncogenic viruses including Epstein-Barr Virus (EBV). However, p62-mediated selective autophagy and its interplay with DDR have not been investigated in these settings. In this study, we provide evidence that considerable levels of p62-mediated selective autophagy are spontaneously induced, and correlate with ROS-Keap1-NRF2 pathway activity, in virus-transformed cells. Inhibition of autophagy results in p62 accumulation in the nucleus, and promotes ROS-induced DNA damage and cell death, as well as downregulates the DNA repair proteins CHK1 and RAD51. In contrast, MG132-mediated proteasome inhibition, which induces rigorous autophagy, promotes p62 degradation but accumulation of the DNA repair proteins CHK1 and RAD51. However, pretreatment with an autophagy inhibitor offsets the effects of MG132 on CHK1 and RAD51 levels. These findings imply that p62 accumulation in the nucleus in response to autophagy inhibition promotes proteasome-mediated CHK1 and RAD51 protein instability. This claim is further supported by the findings that transient expression of a p62 mutant, which is constitutively localized in the nucleus, in B cell lines with low endogenous p62 levels recaptures the effects of autophagy inhibition on CHK1 and RAD51 protein stability. These results indicate that proteasomal degradation of RAD51 and CHK1 is dependent on p62 accumulation in the nucleus. However, small hairpin RNA (shRNA)-mediated p62 depletion in EBV-transformed lymphoblastic cell lines (LCLs) had no apparent effects on the protein levels of CHK1 and RAD51, likely due to the constitutive localization of p62 in the cytoplasm and incomplete knockdown is insufficient to manifest its nuclear effects on these proteins. Rather, shRNA-mediated p62 depletion in EBV-transformed LCLs results in significant increases of endogenous RNF168-γH2AX damage foci and chromatin ubiquitination, indicative of activation of RNF168-mediated DNA repair mechanisms. Our results have unveiled a pivotal role for p62-mediated selective autophagy that governs DDR in the setting of oncogenic virus latent infection, and provide a novel insight into virus-mediated oncogenesis