Development of a novel in vitro primary human monocyte-derived macrophage model to study reactivation of HIV-1 transcription

Latent HIV reservoirs persist in people living with HIV despite effective antiretroviral therapy and contribute to rebound viremia upon treatment interruption. Macrophages are an important reservoir cell-type, but analysis of agents that modulate latency in macrophages is limited by lack of appropriate in vitro models. We therefore generated an experimental system to investigate this by purifying non-productively-infected human monocyte-derived macrophages (MDM) following in vitro infection with an M-tropic EGFP reporter HIV clone, and quantified activation of HIV transcription using live-cell fluorescence microscopy. The proportion of HIV-infected MDM was quantified by qPCR detection of HIV DNA, and GFP expression was validated as a marker of productive HIV infection by co-labelling of HIV Gag protein. HIV transcription spontaneously reactivated in latently-infected MDM at a rate of 0.22% ± 0.04 cells per day (mean ± SEM, n=10 independent donors), producing infectious virions able to infect heterologous T cells in coculture experiments, and both T cells and TZM-bl cells in a cell-free infection system using MDM culture supernatants. Polarization to an M1 phenotype with IFNγ + TNF resulted in a 2.3 fold decrease in initial HIV infection of MDM (p<0.001, n=8) and 1.4 fold decrease in spontaneous reactivation (p=0.025, n=6) whereas M2 polarization using IL-4 prior to infection led to a 1.6 fold decrease in HIV infectivity (p=0.028, n=8), but a 2.0 fold increase in the rate of HIV reactivation in latently-infected MDM (p=0.023, n=6). The latency reversing agents bryostatin and vorinostat, but not panobinostat, significantly induced HIV reactivation in latently infected MDM (p=0.031 and p=0.038, respectively, n=6). Importance: Agents which modulate latent HIV reservoirs in infected cells are of considerable interest to HIV cure strategies. The present study characterizes a robust, reproducible model enabling quantification of HIV reactivation in primary HIV-infected human MDM which is relatively insensitive to the monocyte donor source and hence suitable for evaluating latency modifiers in MDM. The rate of initial viral infection was greater than the rate of HIV reactivation, suggesting different mechanisms regulate these processes. HIV reactivation was sensitive to macrophage polarization, suggesting cellular and tissue environments influence HIV reactivation in different macrophage populations. Importantly, latently infected MDM showed different susceptibility to certain latency reversing agents known to be effective in T cells, indicating dedicated strategies may be required to target latently-infected macrophage populations in vivo .

HIV-1 Productively Infects and Integrates in Bronchial Epithelial Cells

BackgroundThe role of lung epithelial cells in HIV-1-related lung comorbidities remains unclear, and the major hurdle in curing HIV is the persistence of latent HIV reservoirs in people living with HIV (PLWH). The advent of combined antiretroviral therapy has considerably increased the life span; however, the incidence of chronic lung diseases is significantly higher among PLWH. Lung epithelial cells orchestrate the respiratory immune responses and whether these cells are productively infected by HIV-1 is debatable.MethodsNormal human bronchial epithelial cells (NHBEs) grown on air–liquid interface were infected with X4-tropic HIV-1LAV and examined for latency using latency-reversing agents (LRAs). The role of CD4 and CXCR4 HIV coreceptors in NHBEs were tested, and DNA sequencing analysis was used to analyze the genomic integration of HIV proviral genes, Alu-HIVgag-pol, HIV-nef, and HIV-LTR. Lung epithelial sections from HIV-infected humans and SHIV-infected macaques were analyzed by FISH for HIV-gag-pol RNA and epithelial cell-specific immunostaining.Results and DiscussionNHBEs express CD4 and CXCR4 at higher levels than A549 cells. NHBEs are infected with HIV-1 basolaterally, but not apically, by X4-tropic HIV-1LAV in a CXCR4/CD4-dependent manner leading to HIV-p24 antigen production; however, NHBEs are induced to express CCR5 by IL-13 treatment. In the presence of cART, HIV-1 induces latency and integration of HIV provirus in the cellular DNA, which is rescued by the LRAs (endotoxin/vorinostat). Furthermore, lung epithelial cells from HIV-infected humans and SHIV-infected macaques contain HIV-specific RNA transcripts. Thus, lung epithelial cells are targeted by HIV-1 and could serve as potential HIV reservoirs that may contribute to the respiratory comorbidities in PLWH.