Functional Proteomic Analysis for Regulatory T Cell Surveillance of the HIV-1-Infected Macrophage

Regulatory T cells (Treg) induce robust neuroprotection in murine models of neuroAIDS, in part, through eliciting anti-inflammatory responses for HIV-1-infected brain mononuclear phagocytes (MP; macrophage and microglia). Herein, using both murine and human primary cell cultures in proteomic and cell biologic tests, we report that Treg promotes such neuroprotection by an even broader range of mechanisms than previously seen including inhibition of virus release, killing infected MP, and inducing phenotypic cell switches. Changes in individual Treg-induced macrophage proteins were quantified by iTRAQ labeling followed by mass spectrometry identifications. Reduction in virus release paralleled the upregulation of interferon-stimulated gene 15, an ubiquitin-like protein involved in interferon-mediated antiviral immunity. Treg killed virus-infected macrophages through caspase-3 and granzyme and perforin pathways. Independently, Treg transformed virus-infected macrophages from an M1 to an M2 phenotype by down- and up- regulation of inducible nitric oxide synthase and arginase 1, respectively. Taken together, Treg affects a range of virus-infected MP functions. The observations made serve to challenge the dogma of solitary Treg immune suppressor functions and provides novel insights into how Treg affects adaptive immunosurveillance for control of end organ diseases, notably neurocognitive disorders associated with advanced viral infection

Cytometric Bead Array analysis.

<p>CBA assays on IL-10 (<b>A</b>), IL-6 (<b>B</b>), TNF-α (<b>C</b>), IL-12 (<b>D</b>), IFN-γ (<b>E</b>) and MCP-1 (<b>F</b>) were shown in their respective panels. For these assays, Day 6 BMDCs were treated with LPS (1 µg/mL) and/or FTY720 (500 nM) for 24 h. The culture supernatants of each group were subjected to CBA analysis by flow cytometry. Data are shown as mean ± SEM. *<i>P</i><0.05, n = 3. The cytokine concentrations were calculated from the standard curve (<i>R</i>²>0.95) via data analysis by a four parameter linear fitting program provided by the manufacturer.</p

FTY720 alters the surface phenotype of mouse bone marrow-derived dendritic cells upon LPS activation.

<p>Expression of CD11c (<b>A</b>), MHC II molecule I-A^d (<b>B</b>), CD80 (<b>C</b>), CD86 (<b>D</b>) and CD40 (<b>E</b>) was shown in their respective panels. For these assays, mouse bone marrow cells were differentiated for 6 d to prepare BMDCs that were exposed to LPS (1 µg/mL) and/or FTY720 (500 nM) for additional 24 h. Representative results out of three independent experiments were shown. The mean fluorescence intensities (MFI) of each marker were analyzed for statistical difference. *<i>P</i><0.05, n = 3.</p

Topographical and morphological changes of BMDCs upon LPS-activation are reversible by FTY720.

<p>(<b>A</b>) Morphologies of LPS- and/or FTY720- treated BMDCs. Day 6 BMDCs were exposed to LPS (1 µg/ml) and/or FTY720 (500 nM) for additional 48 h, followed by microscopic observations. Scale bar  = 20 µm. (<b>B</b>) Statistical results on the shape indices of each group. *<i>P</i><0.05, n = 20 (20 cells randomly selected from high power fields from 3 separate experiments). Atomic force microscope observations on the untreated (<b>C</b>), FTY720 alone (<b>D</b>), LPS alone (<b>E</b>) and LPS+FTY720- (<b>F</b>) treated groups were shown as representative images. Enlarged areas were indicated in squares in dark and green, respectively. The statistical results of the cell volume (<b>G</b>), RMS roughness (<b>H</b>) and peak to valley distance (<b>I</b>) are analyzed and shown in histograms. Data are shown as mean ± SEM. *<i>P</i><0.05, n = 10 (10 cells randomly selected from 3 separate experiments).</p

CCR7 transcription, NO production as well as phagocytosis and endocytosis of BMDCs.

<p>(<b>A</b>) CCR7 mRNA production analysis. Day 6 BMDCs were treated with LPS (1 µg/mL) and/or FTY720 (500 nM) for 24 h. Total mRNA were extracted, reverse transcripted and quantified by the real-time PCR. *<i>P</i><0.05, n = 3. (<b>B</b>) Nitric oxide production assays. Supernatants of cells with similar treatments were obtained and analyzed by diazotization reaction and colorimetry. *<i>P</i><0.05, n = 3. (<b>C</b>) Phagocytosis assay of BMDCs. Day 6 BMDCs were treated with LPS (1 µg/ml) alone or FTY720 (500 nM) alone for 24 h and subjected to fluorescence-conjugated beads uptake assays. Representative of results on the percentage of phagocytes were shown in each flow chart. Statistical analysis on the percentage of phagocytotic cells were presented as mean ± SEM. *<i>P</i><0.05, n = 3. (<b>D</b>) Endocytosis assay of BMDCs. Similar experimental design to (<b>C</b>) were employed. Cells were treated with FITC-conjugated dextran and analyzed by FACS. Representative results on the percentage of endocytotic cells were shown. Statistical data were presented as mean ± SEM. *<i>P</i><0.05, n = 3.</p

Cell viability determination.

<p>(<b>A</b>) MTT assays on the bone marrow cells. Mouse bone marrow cells were isolated and exposed to FTY720 with different concentrations for 48 h. Cytotoxicity was indicated by the ratio of OD_FTY720 to OD_control. Data are shown as mean ± SEM. *<i>P</i><0.05, n = 3. (<b>B</b>) MTT assays on BMDCs. Day 6 BMDCs were treated with FTY720 at concentrations of 0, 0.1, 0.5 and 1.0 µM for 48 h. Data are shown as mean ± SEM. *<i>P</i><0.05, n = 3. (<b>C</b>) Apoptosis assays on BMDCs. The cell death rates of BMDCs was determined by Annexin V-FITC and PI staining flow cytometry. Cells were treated with FTY720 for 48 h at concentrations ranged from 0.5 to 10 µM.</p

Scanning electron microscopic observation.

<p>Day 6 BMDCs were treated with LPS (1 µg/mL) and/or FTY720 (500 nM) for additional 48 h before SEM observation. Scale bar = 5 µm.</p