CD4 regulatory T cells augment HIV-1 expression of polarized M1 and M2 monocyte derived macrophages

Abstract

Previous in vitro studies have shown that the HIV-1 virus can alter the cytokine/chemokine profile of polarized macrophages which may lead to their increased susceptibility to viral infection. Here, we found that M2 monocyte derived macrophages (MDM) were significantly more permissive to productive infection by R5-tropic HIV-1 strains, including transmitted founder (T/F) viruses, than M1 MDM. Previous in vitro studies by our lab showed that regulatory T cells (Tregs) suppress HIV-1 infection in non-Treg CD4 T cells. Here, we investigated potential inhibitory effects of Tregs on HIV-1 infection of polarized MDM. We found that Tregs significantly increased HIV-1 infection in M1 and M2 MDM via a mechanism that was cell contact dependent. These findings suggest a potential role for Tregs in HIV-1 infection of tissue resident macrophages of M1 and M2 phenotype, which may contribute to the establishment and pathogenesis of HIV-1 disease.

Keywords: HIV-1; Macrophages; Tregs.

Fig. 1

Phenotypic Analysis of Polarized MDM by Expression of M1 and M2 MDM Associated Cell Surface Markers. The phenotype of M1 and M2 MDM were assessed 24 hrs after polarization by multi-color flow cytometry as described in the Materials and Methods. (A) A dot plot analysis of one representative experiment of 3 is shown. M1 MDM cell surface markers (CD80, CD86, and HLA-DR) and M2 MDM cell surface markers (CD36, CD209, and CD163) are indicated along the x-axis. CD14 expression is depicted along the y-axis. The percentages and mean fluorescent intensities (MFI) of cells for each quadrant are noted in the corners of the respective quadrants. (B) The MFI of M1 MDM associated cell surface markers CD80, CD86, and HLA-DR are shown for 9 or 10 independent experiments. (C) The MFI of M2 associated cell surface markers CD36, CD209, and CD163 are shown for 6 or 7 independent experiments. M1 and M2 primed MDM were used as controls. Statistical significance of differences between means of M1 and M2 MDM cell surface marker mean fluorescent intensity was determined using non-parametric Wilcoxin test and is indicated by *p < 0.05 and **p < 0.01. NS represents not statistically significant.

Fig. 2

Characterization of M1 and M2 Polarized MDM by Cytokine and Chemokine Gene Expression. The polarization state of the M1 and M2 MDM was further characterized by measuring the mRNA levels of (A) M1 associated pro-inflammatory cytokines/chemokine IL-1β, IL-6, and CCL2 (MCP-1), and (B) M2 associated anti-inflammatory cytokines/chemokine IL-10, IL-1Ra, and CCL18 using real-time RT-PCR. Messenger RNA (mRNA) levels were reported relative to GAPDH. The results are representative of 3 independent experiments. M1 and M2 primed MDM were used as controls. Statistical significance of differences between means of mRNA levels of M1 and M2 associated cytokines/chemokines was determined using nonparametric Wilcoxin test and is indicated by *p < 0.05. NS represents not statistically significant.

Fig. 3

M2 MDM Expressed Higher Levels of CCR5 HIV-1_BaL Infection Compared to M1 MDM. MDM cells were M1 and M2 primed (controls, cultured in the presence of GM-CSF or M-CSF) or were polarized with M1 (IFN-γ + LPS) or M2 (IL-4) -inducing cytokines for 24 hrs. After 24 hrs, MDM were infected at a MOI of 2 with (A) NLENG1i-BaL.ecto (GFP reporter virus) or (B) NL-LucR.T2A-BaL.ecto (Renilla Luciferase reporter virus). On day 7 post-infection, HIV-1 infection was monitored by visualizing GFP expression using fluorescent microscopy or by measuring luciferase activity, respectively. GFP fluorescence (left panel) and bright field images (right panel) of cells taken at 10× magnification are shown for one representative experiment. The relative light units (RLU) from measuring luciferase activity in M1 and M2 primed MDM and polarized MDM from 9 and 15 independent experiments, respectively, are plotted in (B). The distinct dots represent individual donors. Statistical significance of difference of means of M1 and M2 MDM relative luminescence was determined using non-parametric Wilcoxin test and is indicated by ****p < 0.0001.

Fig. 4

M2 MDM Exhibited Higher Susceptibility to HIV-1 T/F Virus Infection Compared to M1 MDM. (A) M1 and M2 primed MDM (controls, cultured in the presence of GM-CSF and M-CSF, respectively) and (B) polarized MDM were infected with R5-tropic Env-IMC-LucR viruses encoding Env of HIV-1_BaL or of T/F strains (CH077.t, THRO.c, and REJO.c) at a MOI of 2. On days 3, 7, 10, 13, and 16 post-infection, the cells were analyzed for luciferase activity. The results are representative of 5 independent experiments. Uninfected cells are represented by the symbol (HIV-1−) and infected cells by the symbol (HIV-1+). The bar graph shows relative light units (RLU) from measuring luciferase activity on day 16 post-infection in (C) M2 primed MDM (control, cultured in the presence of M-CSF) and (D) polarized M2 MDM from 5 independent donors. Statistical significance of differences between means of the M1 and M2 primed MDM and polarized M2 MDM was determined using non-parametric Wilcoxin test and is indicated by *p < 0.05. NS represents not statistically significant.

Fig. 5

Co-Culture with Autologous Tregs and Tconv Cells Significantly Increased HIV-1 Expression of Polarized MDM. (A) HIV-1 (NL-LucR.T2A-BaL.ecto) infected polarized M1 and M2 MDM were cultured alone or in a co-culture with autologous CD4 Tregs or conventional effector CD4 T cells at a 1:1 ratio for 7 days. On day 7 post-infection, the CD4 T cells were removed from the co-cultures, and the polarized M1 and M2 MDM were analyzed for luciferase activity. The means from n = 8 independent experiments are shown as bar graphs, with standard variation plotted, and individual donors are represented by unique symbols. (B and C) Fold changes in the levels of HIV-1 infection of M1 or M2 MDM co-cultured with autologous CD4 Tregs and conventional effector CD4 T cells as compared to polarized M1 or M2 MDM cultured alone. (D) CD4 Tregs and conventional effector CD4 T cells removed from three out of eight independent donor co-cultures, were lysed and analyzed for luciferase activity. Statistical significance of differences between means was determined using non-parametric Wilcoxin test and is indicated by *p < 0.05 and **p < 0.01. NS represents not statistically significant. Co-cultures are represented by the abbreviation CC.

Fig. 6

Treg-Mediated Modulation of HIV-1 Expression in M1 and M2 MDM is Cell Contact Dependent. Polarized M1 and M2 MDM were infected with NL-LucR.T2A-BaL.ecto virus. The cells were then cultured alone or co-cultured with autologous Tregs (A) or Tconv cells (B) at a 1:1 ratio in the same well, or co-cultured with Tregs and Tconv cells added to a transwell insert without cell contact between MDM and CD4 T cells. On day 7 post-infection, the polarized M1 and M2 MDM were analyzed for luciferase activity. The means and individual values from 5 independent experiments are plotted. Statistical significance of differences between means was determined using non-parametric Wilcoxin test and is indicated by *p < 0.05 and **p < 0.01. NS represents not statistically significant.