Impact of HIV infection on cytokine secretion and maturation of monocyte-derived macrophages and monocyte-derived DCs.

<p>(<b>A</b> and <b>B</b>) HIV-1 infection of monocyte-derived macrophages (MØ) or monocyte-derived DCs. Cells were infected at day 1 after the initiation of APC differentiation without (Mock) or with the R5 HIV-1_Bal (100 pg/ml of p24). At day 5, (<b>A</b>) the percentage of p24⁺ cells was determined by flow cytometry. Values shown are means ± SEM (n = 6). Significant differences are indicated by an asterisk (p<0.05). (<b>B</b>) HIV viral proteins were detected by western blotting using HIV-1⁺ sera. (<b>C</b>) HIV-1 decreases pro-inflammatory cytokines production. Cells at day 5 were stimulated with LPS (10 ng/ml) and IFN-γ (10³ U/ml) overnight. Cells incubated in the absence of R5 HIV-1_Bal and in the absence of stimulation represent the negative control (Med). Supernatants were collected and assessed for the presence of IL-1β, IL-6, IL-8, and TNF-α by flow cytometry using bead array. Values shown are means ± SEM (n = 3). Significant differences are indicated by an asterisk (p<0.05). (<b>D</b> and <b>E</b>) HIV-1 decreases CD86 expression at the surface of stimulated (<b>D</b>) MØ or (<b>E</b>) DCs. Cells were stained with specific CD86 mAbs, and cell surface density was assessed by flow cytometry. One representative experiment out of three is shown; the mean of fluorescence intensity is indicated. CD86 expression values shown are means ± SEM (n = 3). Significant differences are indicated by an asterisk (p<0.05).</p

HIV-1 sensitizes monocyte-derived macrophages and monocyte-derived DCs for Death receptor ligands.

<p>(<b>A</b>) Monocyte-derived macrophages (MØ) or (<b>B</b>) monocyte-derived DCs were incubated with R5 HIV-1_Bal (100 pg/ml of p24) or Mock at day 1 after the initiation of APC differentiation. At day 5, the cells were then cultured overnight in the absence or presence of recombinant TNF-α, TRAIL and FasL (100 ng/ml). A positive control of cell death was performed in the presence of Actinomycin D (10 µg/ml). Apoptosis was determined by flow cytometry using FITC-labeled Annexin V. Percentages of apoptotic cells shown are means ± SEM (n = 3). Statistical significant differences are indicated by an asterisk (p<0.05). (<b>C</b>) Dose response of FasL and Trail. Percentages of apoptotic cells shown are means ± SEM (n = 3). Statistical significant differences as compared to untreated cells are indicated by an asterisk (p<0.05). (<b>D</b>) Cells, before infection, were incubated in the absence or presence of ddI (5 µM). At day 5, the cells were then cultured overnight in the absence or presence of either TRAIL or FasL (100 ng/ml). (<b>E</b>) Cells incubated with R5 HIV-1_Bal were stimulated at day 5 with LPS (10 ng/ml) and IFN-γ (10³ U/ml) overnight in the absence or presence of death receptor antagonists: TNF-R1, TRAIL-R1/TRAIL-R2, Fas-Fc (10 µg/ml). Preventive effect was calculated as follows: ((% of MØ/DC apoptosis - % of MØ/DC apoptosis in the presence of decoy receptors)/(% of MØ/DC apoptosis)) X 100. Values are means ± SEM (n = 3). Statistical significant differences as compared to untreated cells are indicated by an asterisk (p<0.05).</p

Expression of pro- and anti-apoptotic molecules in monocytes and mDCs during primary SIV infection.

<p>(<b>A</b>) Expression of FLIP and Mcl-1 in purified CD14⁺ (MØ) from healthy RM (SIV⁻) and SIV-infected RMs (SIV⁺). After isolation, the cells were lysed and the proteins were immunoblotted with specific antibodies against the anti-apoptotic molecules FLIP and Mcl-1. Actin was used as a control for equal protein loading. Values represent the ratio of the FLIP and Mcl-1 bands and normalized with respect to the loading control. (<b>B</b>) Flow cytometric analysis of the active form of the pro-apoptotic molecules Bax and Bak in CD4⁺ T cells, and monocytes (MØ) at days 0 and 14. (<b>C</b>) Percentage of active form of Bax and Bak among monocyte and DC populations at days 0 and 14. Values are means ± sem (n = 6); Significantly different from day 0 (*, p<0.05). (<b>D</b> and <b>E</b>) PBMC from SIV-infected RMs were incubated without or with Q-VD-OPH (10 µM) and then stimulated with LPS (10 ng/ml) overnight. (<b>D</b>) Fold increase in surviving cells incubated with Q-VD-OPH is shown. (<b>E</b>) Numbers of HLA-DR⁺CD3⁻CD20⁻ expressing TNF-α in the absence or presence of Q-VD-OPH after stimulation is shown. Bars show the mean ± SEM of three independent experiments. Statistical significant differences as compared to untreated cells are indicated by an asterisk (p<0.05).</p

Frequency of SIV-DNA⁺ cells in SIV-infected macaques.

<p>Frequency of SIV-DNA⁺ CD4⁺ T cells, CD14⁺ cells and DCs. Prism version 3.0 (GraphPad Software) was used to calculate means ± SD at days 7, 11, 14 and 60 post-infection.</p

Increased apoptosis of monocytes and DCs during primary SIV infection.

<p>PBMC from healthy (<b>SIV⁻</b>) and SIV-infected RM at day 14 (<b>SIV⁺</b>) were incubated overnight in the absence or presence of death ligands. (<b>A</b>) The percentages of apoptotic CD4⁺ T cells were analyzed by flow cytometry using FITC-labeled Annexin V. (<b>B</b>) Gating strategy to analyze apoptotic HLA-DR⁺ and CD14⁺ cells. Cells were first analyzed on HLA-DR <i>versus</i> SSC (gate R1) and HLA-DR <i>versus</i> Lin+(CD3⁺CD14⁺CD20⁺) cells (gate R2), or CD14 <i>versus</i> Lin+(CD3⁺CD20⁺) cells (gate R3) (<b>C</b>) The percentages of apoptotic HLA-DR⁺Lin⁻ (CD3⁻CD14⁻CD20⁻) cells was determined by flow cytometry using FITC-labeled Annexin V gated on R1 and R2; (<b>D</b>) Percentage of apoptotic CD4⁺ and HLA-DR⁺Lin⁻ cells. Values shown are means ± SEM (n = 4 for SIV⁻ and SIV⁺); Significantly different compared to medium controls (*, p<0.05). (<b>E</b>) Percentage of apoptotic monocytes (MØ) and DCs at days 0, 11, 14 and 60; values are means ± SEM (n = 6); Significantly different from day 0 (*, p<0.05). (<b>F</b>) PBMC from healthy and SIV-infected AGM at day 14. The percentages of apoptotic CD4⁺ T cells and HLA-DR⁺CD3⁻CD20⁻ cells were analyzed by flow cytometry using FITC-labeled Annexin V. (<b>G</b>) Quantitation of FasL in the sera of SIV-infected RMs and AGM at different time points post-infection. Statistical significant differences as compared to day 0 are indicated by an asterisk. (<b>H</b>) Preventive effect of death receptor antagonists. PBMC from SIV-infected RMs (day 14) were incubated overnight with antagonists of death receptors: TRAIL-R1, TRAIL-R2, Fas-Fc and TNF-R1 (10 µg/ml). Apoptosis of monocytes and DCs was quantified using FITC-Annexin-V. Preventive effect was calculated as follows: ((% of MØ/DC apoptosis - % of MØ/DC apoptosis in the presence of decoy receptors)/(% of MØ/DC apoptosis)) X 100. Values are means ± SEM (n = 5); Significantly different from samples incubated with medium alone (*, p<0.05).</p

Expression of pro- and anti-apoptotic molecules in HIV-1 infected monocyte-derived macrophages and monocyte-derived DCs.

<p>(<b>A</b>) Monocytes-derived MØ or monocytes-derived DCs were incubated with R5 HIV-1_Bal (100 pg/ml of p24) or Mock at day 1 after the initiation of APC differentiation. At day 5, the cells were lysed and then the proteins were detected by immunoblotting with specific antibodies against FLIP and Mcl-1. Actin was used as a control for equal protein loading. Values represent the ratio of the protein bands normalized with respect to the loading control, analyzed with GeneTools (SynGene). Mock (non-infected cells) was considered arbitrary equivalent to 1, and bands are compared between (Mock) and HIV-infected cells (HIV). (<b>B</b>) Ratio of FLIP, Mcl-1 and Mcl-1_Exon-1 proteins. Bars show the mean ± SEM of three independent experiments. Statistical significant differences as compared to non-infected cells are indicated by an asterisk (p<0.05). (<b>C</b>) Expression of pro-apoptotic Bax and Bak molecules in the enriched mitochondrial fraction. Hsp60 was used as a control for equal protein loading. Values represent the ratio of the protein bands normalized with respect to the loading control (Hsp60), analyzed with GeneTools (SynGene). Mock (non-infected cells) was considered arbitrary equivalent to 1, and bands are compared between (Mock) and HIV-infected cells (HIV). (<b>D</b>) Ratio of Bax and Bak proteins. Bars show the mean ± SEM of three independent experiments. Statistical significant differences as compared to non-infected cells are indicated by an asterisk (p<0.05).</p

Alteration of BBB functions in the infected hCMEC/D3 cells.

<p>(A) Altered permeability of a monolayer of infected hCMEC/D3 cells. The endothelial cells were cocultured with irradiated MT-2 or C81-66 lymphocytes for 15 days. Then hCMEC/D3 cells were seeded on Transwell filters and permeability to FITC-dextran 70 kDa was assessed after differentiation of the monolayer. (B) Effect of endothelial cells infection on CEM lymphocyte migration. Infected endothelial cells were seeded onto filters. The migration was estimated at 24 hours of culture by fluorescence assay after labeling of lymphocytes (from the CEM, Jurkat, MT-2 of C81-66 cell-lines) with a fluorescent marker. The migration rate is expressed as ratio (%) of fluorescence intensity in the lower compartment versus total fluorescence. (C) Analysis of the expression level of ZO-1, Occludin, and viral p24 of a monolayer of hCMEC/D3 cells cocultured with irradiated MT-2 or C81-66 for 15 days. β-tubulin was used for normalization. (D) Inhibiting MLC phosphorylation has no effect on the permeability for FITC dextran 70 kDa across a monolayer of infected endothelial cells. hCMEC/D3 cells were seeded on filters. After differentiation, cultures were either left untreated of treated for 48 hours with ML-1, a specific inhibitor for MLCK activity. Permeability was then estimated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000205#s4" target="_blank">Materials and Methods</a>.</p

Productive infection of hCMEC/D3 cells by HTLV-1.

<p>(A) Kinetics of p19 viral protein secretion in the supernatant of hCMEC/D3 and irradiated HTLV-1 MT-2 lymphocyte cocultures. Cells were cultivated or not in presence of 25 µM AZT. Results are mean and standard deviation from triplicate experiments. (B) Kinetics of detection of infected hCMEC/D3 cells by irradiated MT-2 cells. Infection was assessed by FACS analysis of p24 viral protein at days 12, 14, 16 and 22 post-coculture. (C) Production of infectious viral particles by HTLV-1-infected endothelial cells using a reporter cell-line (293T-LTR-GFP). The supernatant of hCMEC/D3 infected cells was collected and ultracentrifuged and the resuspended pellet was applied on the reporter cell-line 293T-LTR-GFP. The expression of GFP was assessed 6 days later after cell fixation.</p

Syncytia formation between hCMEC/D3 cells and HTLV-1 infected MT-2 lymphocytes at 24 h post-contact.

<p>(A) Role of the viral proteins in the formation of the syncytia. Evaluation of the number and size (nb of nuclei/syncytium) of the syncytia obtained by coculture between hCMEC/D3 and MT-2 cells in the presence of serum from a HAM/TSP patient (1/7) (noted HAM/TSP) or from an uninfected individual (control, noted HTLV negative control or in the absence of any serum (w/o serum). (B) Role of the viral receptors in the formation of the syncytia. Evaluation of the number and size (nb of nuclei/syncytium) of the syncytia obtained by coculture between hCMEC/D3 and MT-2 cells in the presence of dextran sulfate in blue (that prevents the HTLV-1 Env/HSPG interaction), or in the presence of VEGF165 in green (that prevents the HTLV-1 Env/NRP-1 interaction), or in the presence of a polyclonal antibody against Glut-1 in red (that prevents the HTLV-1 Env/GLUT1 interaction). Results are representative of 3 independent experiments. (C) Detection of viral p24 protein (green) by immunofluorescence in a syncytium. Nuclei were stained with DAPI (blue). (D) Demonstration of endothelial origin of the syncytia by prior labeling of hCMEC/D3 cells with a red vital fluorescent marker (Cell-tracker, red). Nuclei were stained with DAPI (blue). Magnification (B–C): 350×.</p

Expression of HTLV-1 receptors, Glut-1 and NP-1, in the thoracic spinal cord from an uninfected individual.

<p>(A,B) Glut-1 immunostaining by immunoperoxydase technique (iPO) (DAB substrate). (C) Dual staining for Glut-1 (iPO, DAB substrate, brown color) and factor VIII (Alcaline phosphatase (AP), FastBlue substrate, blue color). (D,E) NP-1 staining by iPO technique (DAB substrate). Thoracic spinal cord specimens were cut in a cryostat, fixed in methanol and processed for iPO as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000205#s4" target="_blank">Materials and Methods</a>. (F) Dual staining for NP-1 (iPO, DAB substrate, brown color) and factor VIII (Alcaline phosphatase, FastBlue substrate, blue color). Frozen tissue samples from the thoracic spinal cord were cut on a cryostat at 10 µm, fixed in methanol and processed for immunohistochemistry as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000205#s4" target="_blank">Materials and Methods</a>. Magnification: (A,D) 5×, (B–E) 50×, (C–F) 100×.</p