Opposing regulation of endolysosomal pathways by long-acting nanoformulated antiretroviral therapy and HIV-1 in human macrophages

Abstract

Background: Long-acting nanoformulated antiretroviral therapy (nanoART) is designed to improve patient regimen adherence, reduce systemic drug toxicities, and facilitate clearance of human immunodeficiency virus type one (HIV-1) infection. While nanoART establishes drug depots within recycling and late monocyte-macrophage endosomes, whether or not this provides a strategic advantage towards viral elimination has not been elucidated.

Results: We applied quantitative SWATH-MS proteomics and cell profiling to nanoparticle atazanavir (nanoATV)-treated and HIV-1 infected human monocyte-derived macrophages (MDM). Native ATV and uninfected cells served as controls. Both HIV-1 and nanoATV engaged endolysosomal trafficking for assembly and depot formation, respectively. Notably, the pathways were deregulated in opposing manners by the virus and the nanoATV, likely by viral clearance. Paired-sample z-scores, of the proteomic data sets, showed up- and down- regulation of Rab-linked endolysosomal proteins. NanoART and native ATV treated uninfected cells showed limited effects. The data was confirmed by Western blot. DAVID and KEGG bioinformatics analyses of proteomic data showed relationships between secretory, mobility and phagocytic cell functions and virus and particle trafficking.

Conclusions: We posit that modulation of endolysosomal pathways by antiretroviral nanoparticles provides a strategic path to combat HIV infection.

Figure 1

Schematic representation of the MDM phagosome network identified in HIV-1-infected (A) and HIV-1-infected and nanoATV treated cells (B). Proteins identified were compared against control uninfected MDM cultures (p < 0.05). The acquired profiles were analyzed through the bioinformatics program using a comprehensive set of functional annotation tools to uncover biological data sets behind the uncovered list of genes. Data for Annotation, Visualization and Integrated Discovery (DAVID) facilitated the linked sets of enriched functional-related protein groups. This tool was employed to identify enriched biological processes among the expressed proteins. Gene Ontology terms were used to identify related pathways with the assistance of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The KEGG database facilitated the elucidation of the functions for the MDM as derived from the proteomic datasets. Statistical significance was determined using a p-value < 0.05. Proteins in red and blue, display up- and down- regulation, respectively. Proteins in green belong to the phagosome network and not deregulated by ATV treatment. The differences in protein up- and down-regulation between HIV infection alone and HIV infection with nanoATV treatment are circled in red.

Figure 2

Changes in MDM phagosome network for uninfected cells treated with native ATV (A) or nanoATV (B). Proteins were compared to uninfected and untreated MDM controls (p < 0.05) then bioinformatics analysis performed following parallel procedures described in Figure 1. Proteins in red and blue, display up- and down- regulation, respectively. Proteins in green belong to the phagosome network but were not significantly altered by viral infection or treatment.

Figure 3

Endolysosomal proteins in HIV-1, HIV + nanoATV, nanoATV and native ATV treated MDM.

Figure 4

NanoATV treatment affects HIV-1 reverse transcriptase (RT) activity. HIV-1 RT activity was determined in treated (native ATV or nanoATV) MDM followed by HIV-1 infection at days 0, 5 or 10. HIV-1 infected cells without any treatment served as a positive control for RT activity. All samples were collected after 7 days of viral infection. Results shown are the mean of 5 replicates.

Figure 5

NanoATV and HIV-1 endosomal protein regulation. Western blot of Rab5, −7, −11, LAMP1 and β-actin was performed in cell lysates from MDM treated with native ATV or nanoATV and infected with HIV-1 at day 0, 5 or 10 post-drug treatment then incubated for 7 days. Uninfected cells and infected cells without drug treatment served as negative and positive controls for differential expression of cellular proteins during HIV-1 infection. Blots shown are from one donor and experiment, and equivalent to two independent experiments performed.

Figure 6

Subcellular localization of nanoATV, HIV-1 and endolysosomal proteins. Cellular localization of Rab7 or LAMP1 endosomal compartments (red), HIV-1p24 (yellow) and nanoATV (green) are shown by confocal microscopy. Cell nuclei were stained with DAPI (blue). Merged images showed the co-localization of all proteins. Fluorescence images were acquired with a LSM 510 confocal microscopy, 400x magnification.

Figure 7

NanoATV regulation of cytokine profiles in HIV-1 infected MDM. MDM were treated with 100 μM native ATV or nanoATV and infected with HIV-1 at day 0, 5 or 10 post-drug treatment. Untreated, uninfected cells were used as controls. After 24 hours of viral infection, cell culture media were collected and analyzed using a cytokine bead array. (A) Density plots show expression of IL-12, TNF, IL-10, IL-6, IL-1β and IL-8 for control, treated, HIV-1 infected and treated infected MDM. Cytokine levels were detected by FACSArray cytometer and data was plotted using FlowJo (version 10.7) software. (B) Levels of IL-12 and TNF after treatment and infection are shown. Data are analyzed using FCAP software and values of cytokine expression were expressed as pg/mL.