Retinoic Acid Improves the Recovery of Replication-Competent Virus from Latent SIV Infected Cells

Abstract

The accurate estimation and eradication of Human Immunodeficiency Virus (HIV) viral reservoirs is limited by the incomplete reactivation of cells harboring the latent replication-competent virus. We investigated whether the in vitro and in vivo addition of retinoic acid (RA) enhances virus replication and improves the detection of latent virus. Peripheral blood mononuclear cells (PBMCs) from naive and anti-retroviral therapy (ART)-treated SIV-infected rhesus macaques (RMs) were cultured in vitro with anti-CD3/CD28 + IL-2 in the presence/absence of RA. Viral RNA and p27 levels were quantified using RT-qPCR and ELISA, respectively. Viral reservoirs were estimated using the Tat/Rev-Induced Limited Dilution Assay (TILDA) and Quantitative Viral Outgrowth Assay (QVOA). In vitro and in vivo measures revealed that there was also an increase in viral replication in RA-treated versus without RA conditions. In parallel, the addition of RA to either CD3/CD28 or phorbol myristate acetate (PMA)/ionomycin during QVOA and TILDA, respectively, was shown to augment reactivation of the replication-competent viral reservoir in anti-retroviral therapy (ART)-suppressed RMs as shown by a greater than 2.3-fold increase for QVOA and 1 to 2-fold increments for multi-spliced RNA per million CD4+ T cells. The use of RA can be a useful approach to enhance the efficiency of current protocols used for in vitro and potentially in vivo estimates of CD4+ T cell latent reservoirs. In addition, flow cytometry analysis revealed that RA improved estimates of various viral reservoir assays by eliciting broad CD4 T-cell activation as demonstrated by elevated CD25 and CD38 but reduced CD69 and PD-1 expressing cells.

Actions of Retinoic Acid in the Pathophysiology of HIV Infection

“…To this end, reactivation of latent HIV/SIV by AMPK-activating agents including RA has been demonstrated [92]. This hypothesis is supported by recent studies that confirmed the ability of RA to enhance the re-activation of replication-competent viral reservoirs in peripheral rhesus macaque CD4+ T cells following activation with anti-CD3/CD28 beads to activate the T-cell receptor or PMA/ionomycin [93] (Figure 7). Likewise, Zhang et al showed that supplementation of RA during assays designed to estimate viral reservoirs showed an increase in viral replication in RA-treated versus without RA conditions [95].…”

“…13, x FOR PEER REVIEW 7 of 15studies that confirmed the ability of RA to enhance the re-activation of replication-competent viral reservoirs in peripheral rhesus macaque CD4+ T cells following activation with anti-CD3/CD28 beads to activate the T-cell receptor or PMA/ionomycin[93] (Figure7).…”

Chronic Morphine Administration Differentially Modulates Viral Reservoirs in a Simian Immunodeficiency Virus SIVmac251-Infected Rhesus Macaque Model

“…The Tat/rev induced limiting dilution assay (TILDA) was performed as described earlier ( 85 , 86 ). In brief, enriched CD4 + T cells from frozen PBMCs or LN total cells were suspended in complete RPMI at a 2-million cells/ml concentration and then rested at 37°C and 5% CO 2 for a minimum of 2 h. After resting, cells were divided into two groups: one was stimulated with 100 ng/ml phorbol myristate acetate (PMA) and 1 μg/ml ionomycin, and the other group was left untreated as the control.…”

Dual role for microbial short-chain fatty acids in modifying SIV disease trajectory following anti-α4β7 antibody administration

“…This finding is consistent with natural, non-progressing SIV hosts, which can initiate gut healing despite chronic viremia [ 21 ]. In addition to its role in gut homeostasis, atRA, as well as synthetic analogues, have been proposed as latency-reversing agents promoting apoptosis in infected CD4+ T cells, thereby reducing the size of the viral reservoir [ 22 , 54–56 ]. However, this process may necessitate the presence of CD8+ T cells, which were experimentally depleted before infection in the present study [ 57 ].…”