Antiretroviral drug transporters and metabolic enzymes in human testicular tissue: potential contribution to HIV-1 sanctuary site.

OBJECTIVES: The testes are a potential viral sanctuary site for HIV-1 infection. Our study aims to provide insight into the expression and localization of key drug transporters and metabolic enzymes relevant to ART in this tissue compartment. METHODS: We characterized gene and protein expression of 12 representative drug transporters and two metabolic enzymes in testicular tissue samples obtained from uninfected (n = 8) and virally suppressed HIV-1-infected subjects on ART (n = 5) and quantified antiretroviral drug concentrations in plasma and testicular tissues using LC/MS/MS from HIV-1-infected subjects. RESULTS: Our data demonstrate that key ABC drug transporters (permeability glycoprotein, multidrug-resistance protein 1, 2 and 4, and breast cancer resistance protein), solute carrier transporters (organic anion transporting polypeptides 1B1 and 2B1, organic anion transporter 1, concentrative nucleoside transporter 1, equilibrative nucleoside transporter 2) and cytochrome P450 metabolic enzymes (CYP3A4 and CYP2D6) previously shown to interact with many commonly used antiretroviral drugs are expressed at the mRNA and protein level in the testes of both subject groups and localize primarily at the blood-testis barrier, with no significant differences between the two groups. Furthermore, we observed that PIs known to be substrates for ATP-binding cassette membrane transporters, displayed variable testicular tissue penetration, with darunavir concentrations falling below therapeutic values. In contrast, the NRTIs emtricitabine, lamivudine and tenofovir displayed favourable tissue penetration, reaching concentrations comparable to plasma levels. We also demonstrated that nuclear receptors, peroxisome proliferator-activated receptors α and γ exhibited higher gene expression in the testicular tissue compared with pregnane X receptor and constitutive androstane receptor, suggesting a potential regulatory pathway governing drug transporter and metabolic enzyme expression in this tissue compartment. CONCLUSIONS: Our data suggest the testes are a complex pharmacological compartment that can restrict the distribution of certain antiretroviral drugs and potentially contribute to HIV-1 persistence.

Immunosuppressive Tryptophan Catabolism and Gut Mucosal Dysfunction Following Early HIV Infection.

BACKGROUND: Tryptophan (Trp) catabolism into kynurenine (Kyn) contributes to immune dysfunction in chronic human immunodeficiency virus (HIV) infection. To better define the relationship between Trp catabolism, inflammation, gut mucosal dysfunction, and the role of early antiretroviral therapy (ART), we prospectively assessed patients early after they acquired HIV. METHODS: Forty patients in the early phase of infection were longitudinally followed for 12 months after receiving a diagnosis of HIV infection; 24 were untreated, and 16 were receiving ART. Kyn/Trp ratio, regulatory T-cells (Tregs) frequency, T-cell activation, dendritic cell counts, and plasma levels of gut mucosal dysfunction markers intestinal-type fatty acid-binding protein, soluble suppression of tumorigenicity 2, and lipopolysaccharide were assessed. RESULTS: Compared with healthy subjects, patients in the early phase of infection presented with elevated Kyn/Trp ratios, which further increased in untreated patients but normalized in ART recipients. Accordingly, in untreated subjects, the elevated Treg frequency observed at baseline continued to increase over time. The highest CD8(+) T-cell activation was observed during the early phase of infection and decreased in untreated patients, whereas activation normalized in ART recipients. The Kyn/Trp ratio was positively associated with CD8(+) T-cell activation and levels of inflammatory cytokines (interleukin 6, interferon γ-inducible protein 10, interleukin 18, and tumor necrosis factor α) and negatively associated with dendritic cell frequencies at baseline and in untreated patients. However, ART did not normalize plasma levels of gut mucosal dysfunction markers. CONCLUSIONS: Early initiation of ART normalized enhanced Trp catabolism and immune activation but did not improve plasma levels of gut mucosal dysfunction markers.

Expanded ATXN3 frameshifting events are toxic in Drosophila and mammalian neuron models.

Spinocerebellar ataxia type 3 is caused by the expansion of the coding CAG repeat in the ATXN3 gene. Interestingly, a -1 bp frameshift occurring within an (exp)CAG repeat would henceforth lead to translation from a GCA frame, generating polyalanine stretches instead of polyglutamine. Our results show that transgenic expression of (exp)CAG ATXN3 led to -1 frameshifting events, which have deleterious effects in Drosophila and mammalian neurons. Conversely, transgenic expression of polyglutamine-encoding (exp)CAA ATXN3 was not toxic. Furthermore, (exp)CAG ATXN3 mRNA does not contribute per se to the toxicity observed in our models. Our observations indicate that expanded polyglutamine tracts in Drosophila and mouse neurons are insufficient for the development of a phenotype. Hence, we propose that -1 ribosomal frameshifting contributes to the toxicity associated with (exp)CAG repeats.

Influence of Hepatitis C Virus Sustained Virological Response on Immunosuppressive Tryptophan Catabolism in ART-Treated HIV/HCV Coinfected Patients.

BACKGROUND: We previously reported an association between tryptophan (Trp) catabolism and immune dysfunction in HIV monoinfection. Coinfection with HIV is associated with more rapid evolution of hepatitis C virus (HCV)-associated liver disease despite antiretroviral therapy (ART), possibly due to immune dysregulation. We hypothesized that liver fibrosis in HIV/HCV coinfection would be associated with immune dysfunction and alterations in Trp metabolism. METHODS: Trp catabolism and inflammatory soluble markers were assessed in plasma samples from ART-treated HIV/HCV-coinfected patients (n = 90) compared with ART-treated HIV-monoinfected patients and noninfected subjects. Furthermore, 17 additional coinfected patients with sustained virological response (SVR) were assessed longitudinally 6 months after completion of interferon-α/ribavirin treatment. RESULTS: HIV/HCV patients had higher Trp catabolism compared with HIV-monoinfected and healthy individuals. Elevated kynurenine levels in HIV/HCV patients with liver fibrosis correlated with the prognostic aspartate aminotransaminase to platelet ratio (APRI scores) and insulin levels. Furthermore, HIV/HCV patients had elevated levels of disease progression markers interleukin-6 and induced protein 10 and shared similar levels of markers of microbial translocation (intestinal fatty acid-binding protein, soluble CD14 and lipopolysaccharide-binding protein) compared with HIV-monoinfected and healthy individuals. Successful HCV treatment improved APRI score and markers of disease progression and microbial translocation although elevated Trp catabolism remained unchanged 6 months after SVR. CONCLUSION: ART-treated HIV/HCV-coinfected patients had elevated immunosuppressive Trp catabolism when compared with monoinfected HIV-treated patients, which did not normalize after SVR. These findings suggest that a necroinflammatory liver syndrome persists through inflammation by Trp catabolism after 6 month of SVR.

Antiretroviral Therapy in Primary HIV-1 Infection: Influences on Immune Activation and Gut Mucosal Barrier Dysfunction.

The recent advances in the understanding of primary HIV-1 infection and the development of well-tolerated antiretroviral therapies have highlighted the potential impact of early treatment initiation on immune responses and gut mucosal barrier dysfunction. Immunological and virological assessments in the blood as well as in gut-associated lymphoid tissues during the early phase of infection indicate the crucial role of HIV-1-specific responses and non-specific immune activation in disease progression. The clinical benefit of early antiretroviral therapy has been somewhat disappointing, with the exception of a small group of patients exhibiting sustained control of HIV replication after transient antiretroviral therapy, designated as post-treatment controllers. This review focuses on the influences of antiretroviral therapy initiated early or very early in the course of infection on the clinical and immunological outcomes as well as its impact on the gut mucosal dysfunction. The evidence presented herein paves the way for the development of immunological interventions combined with early antiretroviral therapy to enhance HIV-1-specific responses and to modulate immune activation that will contribute to HIV remission and cure.

HIV and the gut microbiota, partners in crime: breaking the vicious cycle to unearth new therapeutic targets.

The gut microbiota plays a key role in health and immune system education and surveillance. The delicate balance between microbial growth and containment is controlled by the immune system. However, this balance is disrupted in cases of chronic viral infections such as HIV. This virus is capable of drastically altering the immune system and gastrointestinal environment leading to significant changes to the gut microbiota and mucosal permeability resulting in microbial translocation from the gut into the peripheral blood. The changes made locally in the gut have far-reaching consequences on the other organs of the body starting in the liver, where microbes and their products are normally filtered out, and extending to the blood and even brain. Microbial translocation and their downstream effects such as increased indolamine 2,3-dioxygenase (IDO) enzyme expression and activity create a self-sustaining feedback loop which enhances HIV disease progression and constitute a vicious cycle of inflammation and immune activation combining viral and bacterial factors. Understanding this self-perpetuating cycle could be a key element in developing new therapies aimed at the gut microbiota and its fallout after infection.

CD4:CD8 ratio as a frontier marker for clinical outcome, immune dysfunction and viral reservoir size in virologically suppressed HIV-positive patients.

INTRODUCTION: Absolute CD4 T cell count and plasma viral load have been established as predictors of HIV disease progression, and CD4 T cell count is used as an indicator for initiation of antiretroviral therapy. Following long-term therapy, patients generally present with significant CD4 T cell recovery contrasting with persistently elevated CD8 T cell counts, which leads to a partial restoration of CD4:CD8 ratio. This review focuses on the relevance of the CD4:CD8 ratio on clinical outcomes, immune dysfunction and HIV reservoir size in long-term treated patients. METHOD: We conducted a comprehensive literature review of publications in English language using major electronic databases. Our search was focused on factors contributing to CD4:CD8 T cell ratio and clinical outcome in adult HIV-positive patients in the context of treated infection. DISCUSSION: Low CD4:CD8 ratio has been linked to ageing and acts as a predictor of mortality in the general population. This ratio may represent the combined effects of inflammation and immunological changes called "inflammaging." Although the mechanisms underlying partial correction of the CD4:CD8 ratio and persistently elevated CD8 T cell count in long-term treated patients remain poorly understood, it has been recently indicated that patients with optimal CD4 T cell recovery and low CD4:CD8 ratio still harbour increased immune activation, an immune senescent phenotype and have a higher risk of non-AIDS morbidity and mortality. This review reconsiders CD4:CD8 ratio in the light of advances in the understanding of immune dysfunction and examines its pathophysiological features and implications on clinical outcome and HIV reservoir size in long-term treated HIV-positive adults. CONCLUSION: The CD4:CD8 ratio can contribute to the immunological evaluation of treated patients in a long-term follow-up and may be applied for monitoring both immune dysfunction and viral reservoir size in immune-based clinical trials.

Clinical Relevance of Kynurenine Pathway in HIV/AIDS: An Immune Checkpoint at the Crossroads of Metabolism and Inflammation.

Tryptophan degradation along the kynurenine pathway is associated with a wide variety of pathophysiological processes, of which tumor tolerance and immune dysfunction in several chronic viral infections including HIV are well known. The kynurenine pathway is at the crossroads of metabolism and immunity and plays an important role in inflammation while also playing an opposing role in the control of acute and chronic infections. In this review we have summarized findings from recent studies reporting modulation of tryptophan degrading the kynurenine pathway in the context of HIV infection. This immuno-metabolic pathway is modulated by three distinct inducible enzymes: indoleamine 2,3-dioxygenase 1 and 2 and tryptophan 2,3-dioxygenase. Increased expression of these enzymes by antigen-presenting cells leads to local or systemic tryptophan depletion, resulting in a mechanism of defense against certain microorganisms. Conversely, it can also lead to immunosuppression by antigen-specific T-cell exhaustion and recruitment of T regulatory cells. Recently, among these enzymes, indoleamine 2,3-dioxygenase 1 has been recognized to be an immune response checkpoint that plays an important role in HIV immune dysfunction, even in the context of antiretroviral therapy. In addition to the activation of the kynurenine pathway by HIV proteins Tat and Nef, the tryptophan-degrading bacteria present in the intestinal flora have been associated with dysfunction of gut mucosal CD4 Th17/Th22 cells, leading to microbial translocation and creating a systemic kynurenine pathway activation cycle. This self-sustaining feedback loop has deleterious effects on disease progression and on neurocognitive impairment in HIV-infected patients. Therapy designed to break the vicious cycle of induced tryptophan degradation is warranted to revert immune exhaustion in HIV-infected persons.

HIV-1 Recruits UPF1 but Excludes UPF2 to Promote Nucleocytoplasmic Export of the Genomic RNA.

Unspliced, genomic HIV-1 RNA (vRNA) is a component of several ribonucleoprotein complexes (RNP) during the viral replication cycle. In earlier work, we demonstrated that the host upframeshift protein 1 (UPF1), a key factor in nonsense-mediated mRNA decay (NMD), colocalized and associated to the viral structural protein Gag during viral egress. In this work, we demonstrate a new function for UPF1 in the regulation of vRNA nuclear export. OPEN ACCESS Biomolecules 2015, 5 2809 We establish that the nucleocytoplasmic shuttling of UPF1 is required for this function and demonstrate that UPF1 exists in two essential viral RNPs during the late phase of HIV-1 replication: the first, in a nuclear export RNP that contains Rev, CRM1, DDX3 and the nucleoporin p62, and the second, which excludes these nuclear export markers but contains Gag in the cytoplasm. Interestingly, we observed that both UPF2 and the long isoform of UPF3a, UPF3aL, but not the shorter isoforms UPF3aS and UPF3b, are excluded from the UPF1-Rev-CRM1-DDX3 complex as they are negative regulators of vRNA nuclear export. In silico protein-protein docking analyses suggest that Rev binds UPF1 in a region that overlaps the UPF2 binding site, thus explaining the exclusion of this negative regulatory factor by HIV-1 that is necessary for vRNA trafficking. This work uncovers a novel and unique regulatory circuit involving several UPF proteins that ultimately regulate vRNA nuclear export and trafficking.

Immune Suppression by Myeloid Cells in HIV Infection: New Targets for Immunotherapy.

Over thirty years of extensive research has not yet solved the complexity of HIV pathogenesis leading to a continued need for a successful cure. Recent immunotherapy-based approaches are aimed at controlling the infection by reverting immune dysfunction. Comparatively less appreciated than the role of T cells in the context of HIV infection, the myeloid cells including macrophages monocytes, dendritic cells (DCs) and neutrophils contribute significantly to immune dysfunction. Host restriction factors are cellular proteins expressed in these cells which are circumvented by HIV. Guided by the recent literature, the role of myeloid cells in HIV infection will be discussed highlighting potential targets for immunotherapy. HIV infection, which is mainly characterized by CD4 T cell dysfunction, also manifests in a vicious cycle of events comprising of inflammation and immune activation. Targeting the interaction of programmed death-1 (PD-1), an important regulator of T cell function; with PD-L1 expressed mainly on myeloid cells could bring promising results. Macrophage functional polarization from pro-inflammatory M1 to anti-inflammatory M2 and vice versa has significant implications in viral pathogenesis. Neutrophils, recently discovered low density granular cells, myeloid derived suppressor cells (MDSCs) and yolk sac macrophages provide new avenues of research on HIV pathogenesis and persistence. Recent evidence has also shown significant implications of neutrophil extracellular traps (NETs), antimicrobial peptides and opsonizing antibodies. Further studies aimed to understand and modify myeloid cell restriction mechanisms have the potential to contribute in the future development of more effective anti-HIV interventions that may pave the way to viral eradication.

eEF2 and Ras-GAP SH3 domain-binding protein (G3BP1) modulate stress granule assembly during HIV-1 infection.

Stress granules (SG) are translationally silent sites of RNA triage induced by environmental stresses including viral infection. Here we show that HIV-1 Gag blocks SG assembly irrespective of eIF2α phosphorylation and even when SG assembly is forced by overexpression of Ras-GAP SH3 domain-binding protein (G3BP1) or TIAR. The overexposed loops in the amino-terminal capsid domain of Gag and host eukaryotic elongation factor 2 (eEF2) are found to be critical for the SG blockade via interaction. Moreover, cyclophilin A (CypA) stabilizes the Gag-eEF2 association. eEF2 depletion not only lifts the SG blockade but also results in impaired virus production and infectivity. Gag also disassembles preformed SGs by recruiting G3BP1, thereby displacing eEF2, revealing another unsuspected virus-host interaction involved in the HIV-1-imposed SG blockade. Understanding how HIV-1 counters anti-viral stress responses will lay the groundwork for new therapeutic strategies to bolster host cell immune defences against HIV-1 and other pathogens.

Detection of viral RNA by fluorescence in situ hybridization (FISH).

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles. FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev), FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus. Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.