Brain Microglial Cells Are Highly Susceptible to HIV-1 Infection and Spread

Abstract

Macrophages are a target of human immunodeficiency virus type 1 (HIV-1) infection and may serve as an important reservoir of the virus in the body, particularly after depletion of CD4⁺ T cells in HIV/AIDS. Recently, sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) was identified as the major restriction factor of HIV-1 infection in myeloid cells. SAMHD1 is targeted for proteolytic degradation by Vpx, a viral protein encoded by HIV-2 and many simian immunodeficiency viruses but not HIV-1. In this study, we assessed SAMHD1 restriction in in vitro differentiated macrophages and in freshly isolated macrophages from the lungs, abdomen, and brain. We found that infection and spread in in vitro cultured monocyte-derived macrophages were highly limited and that Vpx largely relieved the restriction to initial infection, as expected. We observed nearly identical infection and restriction profiles in freshly isolated peripheral blood monocytes, as well as lung (alveolar) and abdominal (peritoneal) macrophages. In contrast, under the same infection conditions, primary brain macrophages (microglia) were highly susceptible to HIV-1 infection despite levels of endogenous SAMHD1 comparable to the other macrophage populations. Addition of Vpx further increased HIV-1 infection under conditions of limiting virus input, and viral spread was robust whether or not SAMHD1 was depleted. These results suggest that HIV-1 infection of peripherally circulating macrophages is effectively restricted by SAMHD1; however, microglia are highly susceptible to infection despite SAMHD1 expression. These data may explain the long-standing observation that HIV-1 infection is often detected in macrophages in the brain, but seldom in other tissues of the body.

Keywords: HIV-1; macrophage; microglia.

FIG. 1.

SAMHD1 restricts HIV-1 infection in MDM. MDM were infected with the HIV-1 strains HIV^NLAD8 (NLAD8) (A) or HIV^{NLAD8-GFP-Nef} (NLAD8-GFP) (B) alone or in the presence of SIV-VLP (+Vpx) as indicated. Cells were stained and analyzed by flow cytometry 4 and 8 dpi. Circled populations are HIV p24-positive (HIV^NLAD8) or GPF-positive (HIV^{NLAD8-GFP-Nef}), CD4 downregulated productively infected cells. Numbers in the plots indicate percent of total cells infected. (C) MDM were cultured on glass coverslips and infected with HIV^{NLAD8-GFP-Nef} without or with SIV-VLP (+Vpx), fixed 4 or 8 dpi and stained for SAMHD1 (red) and nuclear DNA (blue). Adjacent fields were imaged, stitched together, and rendered into single whole cell volume projections. GFP (green) marks productively infected cells. Scale bar: 15 μm. (D) MDM were infected with HIV^{NLAD8-GFP-Nef} without or with SIV-VLP (+Vpx) for the indicated time and quantified by flow cytometry as above. MVC (2.5 μM) was added 5 dpi and every 3 days thereafter where indicated to block the potential spread of infection. Infection was quantified using flow cytometry as in (A). Data shown are representative of at least three independent experiments. dpi, day postinfection; HIV-1, human immunodeficiency virus type 1; MDM, monocyte-derived macrophages; MVC, Maraviroc; SAMHD1, sterile alpha motif and histidine/aspartic acid domain-containing protein 1; SIV-VLP, simian immunodeficiency virus–virus-like particles.

FIG. 2.

SAMHD1 restricts HIV-1 infection of monocytes and AM. AM and PBMC were obtained from the same healthy donor. CD14⁺ cells were selected from the PBMC, cultured in the presence of M-CSF overnight (monocytes) and infected (A, top) or cultured an additional 6 days (MDM) before infection (B, top). AM were cultured without cytokines overnight, washed vigorously to remove contaminants, and infected 1 day (A, bottom) or 1 week (B, bottom) after isolation. Cells were infected with HIV^NLAD8 in the absence or presence of SIV-VLP (+Vpx) as indicated. (A, B) Cells were stained and analyzed by flow cytometry 4 dpi. Circled populations are HIV p24 positive, CD4 downregulated, productively infected cells. Numbers in the plots indicate percent of total cells infected. (C, D) Productively infected cells from (A and B) were quantified and displayed as bar graphs in C (infected 1 day after isolation) and D (infected 1 week after isolation). Data are representative of three experiments. AM, alveolar macrophages; M-CSF, macrophage-colony stimulating factor; PBMC, peripheral blood mononuclear cells.

FIG. 3.

SAMHD1 restricts HIV-1 infection of PM. Macrophages (PM) were isolated from peritoneal exudates by Ficoll centrifugation and CD14⁺ magnetic bead selection. PM were infected 1 day after isolation with HIV^{NLAD8-GFP-Nef} (NLAD8) without or with SIV-VLP (+Vpx) as indicated. (A) PM were cultured on glass coverslips, fixed, and stained for SAMHD1 (red) and nuclear DNA (blue) 4 and 8 dpi. Adjacent fields were imaged, stitched together, and rendered into single whole cell volume projections. GFP (green) marks productively infected cells. Scale bar: 15 μm. (B) Cells were cultured overnight, infected as above, and analyzed by flow cytometry 4 and 8 dpi. The mean percent infected cells from three independent experiments are shown; error bars represent standard error of the mean (n = 3). PM, peritoneal macrophages.

FIG. 4.

Microglia are highly permissive to HIV-1 infection even in the presence of SAMHD1. Human microglia were cultured overnight on chamber slides and infected with HIV^{NLAD8-GFP-Nef} (NLAD8) without or with SIV-VLP (+Vpx) as indicated. Cells were fixed and stained for SAMHD1 (red) and nuclear DNA (blue) and imaged. Adjacent fields were stitched together and rendered into single whole cell volume projections. GFP (green) marks productively infected cells. Representative images of multiple fields are shown. (A, B) Standard titer (150 ng/ml) of virus was used, and cells were analyzed 3 and 6 dpi as indicated. (C, D) Low titer (15 ng/ml = 10% of standard titer) of virus was used, and cells were analyzed 4 and 8 dpi as indicated. (B, D) GFP-positive cells in multiple fields were quantified, including those shown in (A, C), and plotted as average GFP-positive nuclei per field. Bars indicate the standard error of the mean (n = 3 fields, ∼1,500 cells). Data are representative of three independent experiments. Scale bars: 15 μm (A), 50 μm (C).

FIG. 5.

Coreceptor expression, viral release, SAMHD1 expression, and phosphorylation are similar in MDM and microglia. (A) MDM and microglia were infected with NLAD8-GFP-Nef. MDM were infected with standard titer, as in Figure 1, and microglia were infected with low titer as in Figure 4. Cells were washed 16 h p.i, supernatants were collected 72 h later, and p24 was quantified by ELISA. Infection was quantified by flow cytometry (MDM) or fluorescence microscopy (microglia). The graph shows the percent of infected cells (bars) and the corresponding p24 concentration in the supernatant (♦). (B) MDM (dark blue) and freshly isolated microglia (dark red) were stained and analyzed by flow cytometry for cell surface expression of CD4 and CCR5. Unstained MDM (light blue) and microglia (light red) controls are also shown. (C) Activated CD4⁺ T cells, naive CD4⁺ T cells, MDM, and microglia were cultured in 10 μM EdU overnight. Cells were fixed; EdU was labeled (red) and stained for nuclear DNA (blue). Adjacent fields were stitched together and rendered into single whole cell volume projections. Representative images of multiple fields are shown. (D) Whole cell extracts were prepared from MDM treated with SIV-VLP delivering Vpx (SIV-VLP^Vpx+) or SIV-VLP lacking Vpx (SIV-VLP^Vpx−) in addition to untreated MDM, microglia, naive CD4⁺ T cells, activated CD4⁺ T cells, and THP-1 cells. Extracts were separated on SDS-PAGE and analyzed by western blot using the indicated antibodies.