HIV-related proteins prolong macrophage survival through induction of Triggering receptor expressed on myeloid cells-1

Abstract

Triggering receptor expressed on myeloid cells-1(TREM-1) is a member of the superimmunoglobulin receptor family. We have previously shown that TREM-1 prolongs survival of macrophages treated with lipoolysaccharide through Egr2-Bcl2 signaling. Recent studies suggest a role for TREM-1 in viral immunity. Human immunodeficiency virus-1 (HIV) targets the monocyte/macrophage lineage at varying stages of infection. Emerging data suggest that macrophages are key reservoirs for latent HIV even in individuals on antiretroviral therapy. Here, we investigated the potential role of TREM-1 in HIV latency in macrophages. Our data show that human macrophages infected with HIV show an increased expression of TREM-1. In parallel, direct exposure to the HIV-related proteins Tat or gp120 induces TREM-1 expression in macrophages and confers anti-apoptotic attributes.NF-κB p65 silencing identified that these proteins induce TREM-1 in p65-dependent manner. TREM-1 silencing in macrophages exposed to HIV-related proteins led to increased caspase 3 activation and reduced Bcl-2 expression, rendering them susceptible to apotosis. These novel data reveal that TREM-1 may play a critical role in establishing HIV reservoir in macrophages by inhibiting apoptosis. Therefore, targeting TREM-1 could be a novel therapeutic approach to enhance clearance of the HIV reservoir, at least within the macrophage pools.

Figure 1. TREM-1 expression in HIV-1- infected human monocyte-derived-macrophages (MDMs).

Human MDMs were infected with HIV-1 for 8 days. Total RNA was extracted for mRNA analysis. There was a 7.6-fold increase of TREM-1 expression in MDMs infected with HIV-1 compared to uninfected cells (*p < 0.05, n = 6–7).

Figure 2. TREM-1 expression in macrophages treated with recombinant HIV Tat and gp120.

RAW264.7 cells were treated with recombinant Tat (100 ng/ml) and gp120 (100 ng/ml) for 4 h and 24 h, respectively. (a) qPCR demonstrating an increased expression of TREM-1 at 24 hours in cells treated with Tat and gp120; (b) representative images of immunofluorescence staining demonstrating TREM-1 expression in RAW264.7 cells treated with recombinant Tat and gp120 (100 ng/ml) for 24 h. Fixed cells were stained with anti-TREM-1/rabbit anti- goat FITC Abs (green) and nucleus was counterstained with DAPI (blue). Original magnification, X40, scale bars, 50 μM. (c) representative histogram images showed increased expression of TREM-1 protein at 24 hours; (d) Mean fluorescence intensity (MFI) in HIV Tat and gp120-treated macrophages. Data are shown as means ± SE, *P < 0.05 as significant difference. (e) Peritoneal macrophages, (f) BMDM and (g) alveolar macrophages from wild type C57BL/6, were treated with HIV Tat and gp120 (100 g/ml) for 24 h, respectively. TREM-1 expression was analyzed by Q-PCR. Statistical significance was assessed by two-tailed paired Student’s test. Data are shown as means ± SE, *P < 0.05 as significant difference.

Figure 3. TREM-1 expression in macrophages induced by HIV Tat and gp120 is NF-κB p65 dependent.

RAW264.7 cells were transfected with si-NF-κB p65 or negative control siRNA (total 100 pmol of siRNA for each transfection). 24 hours later, transfected cells were treated with recombinant Tat and gp120 for 24 hours following which cells were harvested and total RNA was extracted. (a) Real-time PCR and (b) western blot assay showed NF-κB p65 expression was significantly reduced in cells with si-NF-κB p65, compared to cells that were treated with control siRNA. Statistical comparisons were performed, numbers are mean ± SE, *P < 0.05 as significant difference; (c) Detection of TREM-1 mRNA expression in NF-κB p65 silenced- RAW264.7 cells treated with HIV Tat and gp120. (d) Representative images of RAW264.7 cells stained for NF-κB p65 and TREM-1. Cells were transfected with si-NF-κB p65 or control siRNA. Expression of TREM-1 protein was significantly attenuated in cells treated with si-p65 compared to control siRNA. Original magnification 40X, scale bars: 50 μm.

Figure 4. Macrophage apoptosis in TREM-1 knockdown cells treated with recombinant HIV Tat and gp120.

RAW264.7 cells were transfected with si-TREM-1 or control siRNA for 24 hours following which they were treated with recombinant HIV Tat and gp120 (100 ng/ml).(a) TREM-1 expression was significantly reduced in cells with si-TREM-1, compared to cells that were treated with control siRNA. (b) cell apoptosis determined by TUNEL staining (green) and PI (red) scale bars, 50 μM, (c) Percentage of TUNEL-positive cells per DAPI-positive cells were measured from 200 cells for each preparation from three independent experiments, *p < 0.05 considered significant. Statistical comparisons were performed, numbers are mean ± SE, *P < 0.05 as significant difference.

Figure 5. Capsase-3 activity and Bcl2 expression in TREM-1- silenced macrophages treated with recombinant Tat and gp120 protein.

RAW264.7 cells were transfected with siRNA against TREM-1 or control siRNA for 24 hours and then treated with recombinant HIV Tat and gp120 (100 ng/ml) respectively. (a) Caspase3 activity was measured in RAW264.7 macrophages. (b) Caspase3 activity was also measured in BMDM from wild type C57BL/6 and TREM-1/3 deficient mice, *p < 0.05. Caspase3, cleaved caspase3, PARP and cleaved PARP expression, shown in (c), and Bcl-2 expression, shown in (d), were analyzed by western blot, and actin was used as internal control.