Astrocytes are HIV reservoirs in the brain: A cell type with poor HIV infectivity and replication but efficient cell-to-cell viral transfer

Abstract

The major barrier to eradicating Human immunodeficiency virus-1 (HIV) infection is the generation of tissue-associated quiescent long-lasting viral reservoirs refractory to therapy. Upon interruption of anti-retroviral therapy (ART), HIV replication can be reactivated. Within the brain, microglia/macrophages and a small population of astrocytes are infected with HIV. However, the role of astrocytes as a potential viral reservoir is becoming more recognized because of the improved detection and quantification of HIV viral reservoirs. In this report, we examined the infectivity of human primary astrocytes in vivo and in vitro, and their capacity to maintain HIV infection, become latently infected, be reactivated, and transfer new HIV virions into neighboring cells. Analysis of human brain tissue sections obtained from HIV-infected individuals under effective and prolonged ART indicates that a small population of astrocytes has integrated HIV-DNA. In vitro experiments using HIV-infected human primary astrocyte cultures confirmed a low percentage of astrocytes had integrated HIV-DNA, with poor to undetectable replication. Even in the absence of ART, long-term culture results in latency that could be transiently reactivated with histone deacetylase inhibitor, tumor necrosis factor-alpha (TNF-α), or methamphetamine. Reactivation resulted in poor viral production but efficient cell-to-cell viral transfer into cells that support high viral replication. Together, our data provide a new understanding of astrocytes' role as viral reservoirs within the central nervous system (CNS).

Keywords: NeuroHIV; anti-retroviral; astrocytes; cure; latency; reactivation; reservoirs.

Figure 1:

In situ detection and quantification of HIV-infected astrocytes in human brains obtained from HIV infected individuals under long term ART. (A) In situ staining of uninfected human brain tissues for DAPI (nucleus, blue), HIV-DNA (probe for Nef protein, green), Alu repeats (host DNA, red), and GFAP (to identify astrocytes, white staining, Cy5). DAPI staining colocalized with Alu-repeats, and no HIV-DNA staining was observed. GFAP staining was mostly cytoplasmatic and lineup blood vessels as expected (control). (B) A cluster of HIV-infected astrocytes can be observed with DAPI and Alu-repeat staining that colocalized with HIV-Nef staining (HIV). A higher GFAP expression was identified in all the cases in association with HIV-DNA signal in the nucleus. However, most of the brain was negative for HIV-DNA. (C) Quantify the numbers of GFAP positive cells with HIV-DNA signal colocalized with DAPI and Alu-repeats in brains from individuals in ART for 10-18 years versus the total numbers of cells in large pieces of the frontal cortex and subcortical areas of the brain, 5.45 cm². The total number of cells with HIV-DNA in the nucleus (HIVPre_T) was quantified in samples pre-ART or early ART era. A total of 5 different cases were analyzed (see table 1). HIV-astrocytes (HIV-Astro) corresponds to an important reservoir in the current ART era (n=8, different individuals with 10-12 different large sections per tissue analyzed). No HIV-DNA detection was found in uninfected tissues (n=7, different individuals with 10-12 different large sections per tissue analyzed). In contrast, tissues from the pre-and -early stages of the ART era indicates that the percentage of HIV-astrocytes (HIV-Astro) corresponded to a higher number for both cell types. Also, the % of cells with integrated HIV-DNA was high, reaching up to ~40% of the cells due to the large damage and cell fusion observed. Thus, long term ART reduces the size of the CNS viral reservoirs. (*p≤0.0012 as compared to uninfected conditions. #p≤0.00013 as compared to HIVAstro conditions).

Figure 2:

HIV-entry into human primary astrocytes is CD4 independent and chemokine receptor and endocytosis dependent. (A) Quantification of entry and infectivity of primary cultures in the presence and absence of soluble CD4 (sCD4, 50 ng/mL), blocking compounds to CCR5 and CXCR4 (αCCR5, TAK779, and αCXCR4, AMD3100, 100 ng/mL), and blocker for endocytosis (bafilomycin-A1, bafi, 50 nmol/L; chloroquine, CQ, 100 μg/mL; NH₄CL, 50 mm/L, and chlorpromazine, Chlorpro, 50 μg/mL). Controls with the vehicle, DMSO, or media alone did not affect the % of astrocytes with viral entry. These data are included in Fig. 2A in the HIV_ADA data. Infectivity was evaluated by in situ staining for HIV-DNA after 7 days post-infection. (B) Long term time course of HIV-replication upon HIV_ADA infection. Media was collected at different time points, and HIV-p24 ELISA was performed (n=12 different astrocyte cultures from different individuals with three replicates. *p≤0.00015 compared to HIV_ADA conditions).

Figure 3:

Long term HIV infection of astrocytes is reactivable. (A) Long term infection time course, up to 120-150 days in the presence of HIV_ADA, HIV_BaL, HIV_JRCSF, HIV_JRFL, and HIV_98UG021. The time course in Fig. 2B was not viral strain-specific and laboratory-adapted, and primary isolates behave similarly after HIV-infection of primary cultures of astrocytes. (B) Viral reactivation (React, see arrows) after 120 days in culture with SAHA (1 μM), TNF-α (10 ng/mL), or Methamphetamine (1 μM). Upon addition of the reactivating agent, HIV-p24 in the supernatant was quantified by ELISA up to 296 days post-infection (*p≤0.0024 as compared to uninfected conditions, in Graph B, all curves were significant as compared to HIV infection alone except 216, 240, and 296 days post-infection with a p≤0.004). (C to J) correspond to a representative example of staining for Uninfected astrocytes (C-F) and HIV infected astrocytes (G-J). Denote the colocalization of the HIV-Nef DNA signal with DAPI and Alu-repeats. Bar: 15 μm. (K) Quantification of the total number of astrocytes with HIV integrated DNA (HIV-DNA) expressing viral mRNA (HIV mRNA) and HIV-p24 (HIV-p24) during long-term culture. (L) Quantification of the numbers of HIV-infected astrocytes after reactivation with SAHA/TNF-α/methamphetamine. All numbers were pooled together due to the low variability. At each time point, three coverslips were fixed and stained for HIV-DNA, mRNA, and p24. Denote the increase in viral mRNA and protein expression n=5-7 independent experiments from different donors with three coverslips per time point.

Figure 4:

Cell-to-cell contact provides an effective mechanism of astrocyte-glial/myeloid/lymphoid viral infection spread. Upon infection and reactivation of the virus after 168 days in culture as described in Fig. 3B, Media from reactivated cultures with methamphetamine or uninfected cells (Un) was collected and added to uninfected cultures of human primary astrocytes (Astro), CD4 T lymphocytes (T cells), Macrophages (Macro), or CEM-SS cells (CEM) for 2 h, then cultures were washed and viral replication in the supernatant was quantified after 7, 14 and 21 days. However, if the cells could establish cell-to-cell contact through a filter to prevent the mixing of the uninfected and HIV-infected populations, it resulted in greater infection of all the cell types. (*p≤0.007, n=4, as compared to uninfected conditions. #p≤0.0031 as compared to soluble conditions).