Human Primary Macrophages Derived In Vitro from Circulating Monocytes Comprise Adherent and Non-Adherent Subsets with Differential Expression of Siglec-1 and CD4 and Permissiveness to HIV-1 Infection

Abstract

Macrophages are a major target for human immunodeficiency virus type 1 (HIV-1) infection. However, macrophages are largely heterogeneous and may exhibit differences in permissiveness to HIV-1 infection. This study highlights the interplay of macrophage heterogeneity in HIV-1 pathogenesis. We show that monocyte-derived macrophages (MDMs) could be divided into two distinct subsets: CD14⁺Siglec-1^hiCD4⁺ (non-adherent MDM) and CD14⁺Siglec-1^LoCD4^- (adherent MDM). The CD14⁺Siglec-1^hiCD4⁺MDM subset represented the smaller proportion in the macrophage pool, and varied among different donors. Fractionation and subsequent exposure of the two MDM subsets to HIV-1 revealed opposite outcomes in terms of HIV-1 capture and infection. Although the CD14⁺Siglec-1^hiCD4⁺MDM captured significantly more HIV-1, infection was significantly higher in the CD14⁺Siglec-1^LoCD4^-MDM subset. Thus, CD14⁺Siglec-1^hiCD4⁺MDM were less permissive to infection. Depletion of CD14⁺Siglec-1^hiCD4⁺MDM or a decrease in their percentage, resulted in increased infection of MDM, suggestive of a capacity of these cells to capture and sequester HIV-1 in an environment that hinders its infectivity. Increased expression of innate restriction factors and cytokine genes were observed in the non-adherent CD14⁺Siglec-1^hiCD4⁺MDM, both before and after HIV-1 infection, compared to the adherent CD14⁺Siglec-1^LoCD4^-MDM. We speculate that the differential expression of gene expression profiles in the two macrophage subsets may provide an explanation for the differences observed in HIV-1 infectivity.

Keywords: CD4; RNA-Seq; Siglec-1; human immunodeficiency virus type 1; monocyte-derived macrophages; restriction factors.

Figure 1

Monocyte-derived macrophage (MDM) segregate into two distinct subsets: Non-adherent Siglec-1^hiCD4⁺MDM and adherent Siglec-1^LoCD4⁻MDM. Triplicate wells of primary human monocytes from nine human immunodeficiency virus (HIV)-seronegative donors (#048, #130, #170, #008, #002, #202, #132, #124, and #040) were differentiated into MDM following in vitro culture in M-CSF media for 5 days. Cultures were harvested, stained, and analyzed by flow cytometry. (A) Plots show percentage of Siglec-1^hiCD4⁺MDM and Siglec-1^LoCD4⁻MDM within the gated CD14⁺ cells. A representative plot of one of the two independent experiments is shown. (B) Plots show that gated CD14⁺ cells are not contaminated with CD3⁺ T cells. (C,D) In vitro cultures of M-CSF-derived MDM contain adherent and non-adherent MDM. (C) Adherent and non-adherent MDM were pooled, stained, and the gated CD14⁺ cells were sorted into Siglec-1^hiCD4⁺MDM and Siglec-1^LoCD4⁻MDM. (D) Non-adherent MDM were separated from adherent MDM following repeated washing with media. Adherent MDM were detached with Accutase. Both fractions were washed, stained, and the gated CD14⁺ cells were analyzed separately for the expression of Siglec-1 and CD4. Plots show that non-adherent fraction represented the Siglec-1^hiCD4⁺MDM subset, whereas the adherent fraction comprised the Siglec-1^LoCD4⁻MDM subset. (E) Histograms show the expression of CCR5 and CD163 on the gated Siglec-1^LoCD4⁻MDM (blue) and Siglec-1^hiCD4⁺MDM (red) subsets. Values in the histograms denote the mean fluorescent intensity (MFI) of the specific receptors for each of the subsets. Each experiment was done twice in triplicate and the data from one of the two experiments are shown.

Figure 2

Presence of Siglec-1^hiCD4⁺MDM dampens the degree of human immunodeficiency virus type 1 (HIV-1) infection. Monocytes from three donors were differentiated into monocyte-derived macrophage (MDM) with M-CSF media. (A) Panels show plots of unfractionated MDM and their Siglec-1^hiCD4⁺MDM-depleted counterparts. (B) Unfractionated MDM and Siglec-1^hiCD4⁺MDM-depleted cultures were infected with purified HIV-1 (BaL, JRFL, or US-1). Cells were harvested on day 3 postinfection, stained, and analyzed by flow cytometry. Panels show plots of HIV-1 infection in the unfractionated MDM cultures and in their Siglec-1^hiCD4⁺MDM-depleted counterparts. Values in the upper right quadrant(s) represent the percentage of infected MDM. Data represent one of the triplicate wells from one of two independent experiments. (C) Bar graph is derived from the experiment performed in Figure 2B and includes the data from the triplicate wells of two independent experiments and shows the% p24⁺MDM (mean ± SD) in unfractionated MDM (filled bars) or in the Siglec-1^hiCD4⁺MDM-depleted MDM (open bars) following infection with HIV-1. Significance is indicated by *p ≤ 0.05.

Figure 3

Human immunodeficiency virus type 1 (HIV-1) infection is lower in Siglec-1^hiCD4⁺MDM despite higher virus capture. Equal numbers (3 × 10⁵ cells) of Siglec-1^hiCD4⁺MDM and Siglec-1^LoCD4⁻MDM from three HIV-seronegative donors (#130, #170, #132) were incubated with HIV-1 (BaL, JRFL, or US-1) for 3 h at 37°C/5% CO₂. (A) Cell lysates were subjected to qRT-PCR to detect gag RNA transcripts, indicative of virus capture. Uninfected cell lysates were negative for gag RNA transcripts (not shown). Bar graphs show the delta Ct (normalized) gag RNA in Siglec-1^hiCD4⁺MDM (red bars) and Siglec-1^LoCD4⁻MDM (blue bars) for each donor and with each virus. Data are representative of three independent experiments and show the mean values (***p ≤ 0.001). (B,C) Siglec-1^hiCD4⁺MDM and Siglec-1^LoCD4⁻MDM were infected with HIV-1. Cultures were harvested on day 4 postinfection, washed, stained, and analyzed for the presence of intracellular p24 by flow cytometry. (B) Panels show plots of HIV-1 infection in gated Siglec-1^hiCD4⁺MDM and Siglec-1^LoCD4⁻MDM from a representative donor. (C) Bar graph shows % p24⁺MDM of triplicate wells of three independent experiments (mean ± SD; *p ≤ 0.05) in Siglec-1^LoCD4⁻MDM (blue bars) and in the Siglec-1^hiCD4⁺MDM (red bars) following infection with US-1, BaL, or JRFL. (D) Supernatants from cultures of HIV-1-infected Siglec-1^hiCD4⁺MDM and Siglec-1^LoCD4⁻MDM were harvested on day 4 postinfection, and analyzed by ELISA for the presence of extracellular p24. Bar graphs show the p24 concentration of triplicate wells of two independent experiments (mean ± SD; *p ≤ 0.05) in the supernatants of HIV-1-infected Siglec-1^LoCD4⁻MDM (blue bars) and Siglec-1^hiCD4⁺MDM (red bars) for each donor and with each virus. (E,F) Siglec-1^LoCD4⁻MDM (#130) were left uninfected, or (F) were infected with HIV-1. Plots show the presence of Siglec-1^hiCD4⁺MDM both in (E) the uninfected Siglec-1^LoCD4⁻MDM cultures and in (F) their HIV-1-infected Siglec-1^LoCD4⁻MDM cultures. (G,H) The HIV-1-infected monocyte-derived macrophage (MDM) described in (F) were analyzed for localization of HIV-1 on the basis of their Siglec-1^LoCD4⁻MDM versus Siglec-1^hiCD4⁺MDM phenotype. Panels show that the presence of HIV-1 infection only in (G) gated Siglec-1^LoCD4⁻MDM and not in the (H) gated Siglec-1^hiCD4⁺MDM. Values in the upper right quadrant(s) represent the percentage of infected MDM. A representative plot of three independent experiments is shown.

Figure 4

Siglec-1^hiCD4⁺MDM transfer human immunodeficiency virus type 1 (HIV-1) to Siglec-1^LoCD4⁻MDM. (A) M-CSF-derived monocyte-derived macrophage (MDM) from three donors were incubated with media or with HIV-1 (BaL) for 1 h at 37°C/5% CO₂. Cells were washed and fractionated into Siglec-1^hiCD4⁺MDM and Siglec-1^LoCD4⁻MDM. The infected Siglec-1^LoCD4⁻MDM were subsequently labeled with PKH-26. Both subsets were cultured at 37°C/5% CO₂. (A) Plots show the percentage of infected cells at 3 days postculture in the infected Siglec-1^hiCD4⁺MDM and (B) the infected Siglec-1^LoCD4⁻MDM. (C) HIV-1 exposed Siglec-1^hiCD4⁺MDM were cocultured with PKH-67-labeled uninfected Siglec-1^LoCD4⁻MDM. Plots show evidence of HIV-1 infection in both the Siglec-1^hiCD4⁺MDM and the PKH-67-labeled Siglec-1^LoCD4⁻MDM. (D) In parallel, PKH-26-labeled HIV-1-exposed Siglec-1^LoCD4⁻MDM were cocultured with uninfected Siglec-1^hiCD4⁺MDM. Plots show evidence of HIV-1 infection mostly in the PKH-26-labeled Siglec-1^LoCD4⁻MDM.

Figure 5

Heat map of expression of human immunodeficiency virus type 1 (HIV-1) restriction factors and cytokine genes in uninfected and HIV-1-infected Siglec-1^hiCD4⁺MDM and Siglec-1^LoCD4⁻MDM. M-CSF-derived monocyte-derived macrophage (MDM) from two donors were fractionated into Siglec-1^hiCD4⁺MDM and Siglec-1^LoCD4⁻MDM. The MDM subsets were incubated with media or with HIV-1 (BaL) for 3 h at 37°C/5% CO₂. The expression levels of cellular restriction factors, insulin growth factor-1 (IGF-1), IL-13, and IL-12-related genes were analyzed by RNA-Seq. Relative expression levels of HIV-1 restriction genes are shown for uninfected and infected Siglec-1^hiCD4⁺MDM and Siglec-1^LoCD4⁻MDM samples with a self-clustering heatmap generated by the R/CRAN package heatmap3. Samples and genes are clustered by their respective expression profiles and every row is rescaled to have a mean of zero and standard deviation of 0 in order to illustrate relative expression changes across samples on a scale similar to log 2 fold-change. Blue indicates low expression, and red indicates high expression.