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. 2017 Mar 9;14(1):17.
doi: 10.1186/s12977-017-0344-7.

A mature macrophage is a principal HIV-1 cellular reservoir in humanized mice after treatment with long acting antiretroviral therapy

Mariluz Araínga  1 , Benson Edagwa  1 , R Lee Mosley  1 , Larisa Y Poluektova  1 , Santhi Gorantla  1 , Howard E Gendelman  2
Affiliations

A mature macrophage is a principal HIV-1 cellular reservoir in humanized mice after treatment with long acting antiretroviral therapy

Mariluz Araínga et al. Retrovirology. .

Abstract

Background: Despite improved clinical outcomes seen following antiretroviral therapy (ART), resting CD4+ T cells continue to harbor latent human immunodeficiency virus type one (HIV-1). However, such cells are not likely the solitary viral reservoir and as such defining where and how others harbor virus is imperative for eradication measures. To such ends, we used HIV-1ADA-infected NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice reconstituted with a human immune system to explore two long-acting ART regimens investigating their abilities to affect viral cell infection and latency. At 6 weeks of infection animals were divided into four groups. One received long-acting (LA) cabotegravir (CAB) and rilpivirine (RVP) (2ART), a second received LA CAB, lamivudine, abacavir and RVP (4ART), a third were left untreated and a fourth served as an uninfected control. After 4 weeks of LA ART treatment, blood, spleen and bone marrow (BM) cells were collected then phenotypically characterized. CD4+ T cell subsets, macrophages and hematopoietic progenitor cells were analyzed for HIV-1 nucleic acids by droplet digital PCR.

Results: Plasma viral loads were reduced by two log10 or to undetectable levels in the 2 and 4ART regimens, respectively. Numbers and distributions of CD4+ memory and regulatory T cells, macrophages and hematopoietic progenitor cells were significantly altered by HIV-1 infection and by both ART regimens. ART reduced viral DNA and RNA in all cell and tissue compartments. While memory cells were the dominant T cell reservoir, integrated HIV-1 DNA was also detected in the BM and spleen macrophages in both regimen-treated mice.

Conclusion: Despite vigorous ART regimens, HIV-1 DNA and RNA were easily detected in mature macrophages supporting their potential role as an infectious viral reservoir.

Keywords: Antiretroviral therapy; HIV-1; Humanized mice; Monocyte–macrophage; T effector cells; Viral reservoirs.

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Figures

Fig. 1
Fig. 1
PVL quantification in the plasma of HIV-1 infected and ART treated humanized mice. Humanized NSG mice were infected with HIV-1ADA and after 6 weeks of infection, VL was determined. Then, infected animals were distributed into three groups: untreated control, treated with 2ART or 4ART regimens. Plasma VL was measured after 4 weeks of treatment (10 weeks of HIV-1 infection). The figures represent viral copies/mL detected in plasma, using the COBAS Amplilink detection system. Two-way ANOVA for means (bar) of log10 copies/mL for 4–9 mice/group showed an effect of treatment by time (P = 0.00003). P values from pairwise comparison with Tukey’s HSD post hoc test are shown above each pre- and post-treatment pair groups
Fig. 2
Fig. 2
Lymphocytes phenotypes are illustrated during HIV-1 infection and ART treatment. Humanized NSG mice were infected with HIV-1ADA, and after 10 weeks of infection, cellular phenotyping were detected by flow cytometry. a First, percentages of CD4+ and CD8+ T cells were determined from total human CD45+ CD3+ gate from blood, spleen and BM of uninfected, infected and infected and treated animal with two or four ART regimens. Results shown are at 10 weeks post HIV-1 infection. b Then spleen and BM CD4+ cells were investigated to determine the phenotype for TSCM, TCM, TEM and TREG populations in all the groups as explained in a. Cell suspensions were labelled with anti-human monoclonal antibodies (mAb) targeting the following cell-surface markers: CD45, CD3, CD19, CD4, CD8, CD25, CD127, CD45RA, CD45RO, CD95, CCR7 (all from BD Biosciences). Data shows the percentage of the specific human CD4+ cells population. c Schematic description of the frequencies of TSCM, TCM and TEM and TREG during HIV-1 infection and ART treatment in humanized mice. All acquisitions were performed on a LSRII flow cytometer (Beckman Coulter) and data were analysed by FlowJo software. a, b Comparisons of means (±SEM) for 4–9 mice per treatment group were determined by one-way ANOVA and pairwise significance by Fisher’s LSD post hoc test. P ≤ 0.05 compared with auninfected, binfected, or cinfected and treated with 2ART
Fig. 3
Fig. 3
Monocyte–macrophage frequencies during HIV-1 and ART treatments. Humanized NSG mice were uninfected, infected, or infected and treated with 2ART or 4ART regimens. Cells from blood, spleen and BM were identified as CD3−/CD20−/CD8−/HLA-DR+/CD14+CD16+ by flow cytometry, as described in methods. Data were analyzed with FlowJo software. Mean ± SEM for 4–9 mice per treatment group were compared by one-way ANOVA, and pairwise comparisons were determined by Fisher’s LSD post hoc test. P ≤ 0.05 compared with auninfected, binfected, or cinfected and treated with 2ART
Fig. 4
Fig. 4
Identification of progenitor CD34+ cells. Multi-color flow cytometric analysis was performed on spleen and BM cells from humanized NSG mice that were uninfected, infected with HIV-1ADA or infected and treated with 2ART or 4ART regimens. Cells from each tissue were incubated with labeled antibodies for the identification of CD34 and Lineage-1 cellular markers. Lin-CD34+ progenitor cells were determined by the expression of CD34 marker, excluding all Lineage-1 cells. Data were analyzed with FlowJo software. Mean ± SEM of 4–9 mice per treatment group were compared by one way ANOVA, and pairwise comparisons were determined by Fisher’s LSD post hoc test. P ≤ 0.05 compared with auninfected, binfected, or cinfected and treated with 2ART
Fig. 5
Fig. 5
Defining the levels and species of HIV-1 infection in immune CD4+ subsets from spleen and BM, without and with 2ART or 4ART. Tissues were collected and cells in suspension were incubated with anti-human CD45 magnetic beads for isolating human CD45+ cells previous to FACS. Specific antibodies were applied and TSCM, TCM, TEM, and TREG CD4+ cells were sorted. Then, sorted cells from 10 weeks HIV-1 infected humanized mice (4 weeks after ART) were used for detecting both viral RNA (msRNA and usRNA) and total viral DNA and inDNA as explained in methods, using the ddPCR system. The numbers indicate nucleic acid viral copies, RNA or DNA, per mL per each specific sorted subsets and frequencies for each group
Fig. 6
Fig. 6
HIV-1 infection in monocyte–macrophages from spleen and BM and intervention of ART in the frequencies on infected cells. Sorted monocyte–macrophages CD14+CD16+ cells were processed for RNA and DNA isolation and examined by ddPCR system as described in methods. Coloured dots are representations for the frequency of viral RNA or DNA of different treatment groups from spleen and BM cells. Dots in blue indicate the HIV-1 infected control group, dots in red are the HIV-1 infected and 2ART drug-treated group and dots in green represent HIV-1 infected and 4ART drug-treated group
Fig. 7
Fig. 7
Frequency of infected progenitor CD34+ cells during HIV-1 with or without ART in humanized mice. At 10 weeks post HIV-1 infection, spleen and BM cells were sorted for Lin-CD34+ and were collected for RNA and DNA isolation for the detection of HIV-1 using the ddPCR system. Coloured dots are representations for the frequency of viral RNA or DNA of different treatment groups from spleen and BM cells. Dots in blue indicate HIV-1 infected control group, dots in red are for HIV-1 and 2ART and dots in green represent HIV-1 and 4ART regimens
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