Herpes Simplex Virus 2 (HSV-2) Prevents Dendritic Cell Maturation, Induces Apoptosis, and Triggers Release of Proinflammatory Cytokines: Potential Links to HSV-HIV Synergy
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Fig 1Immature moDCs support low level of HSV-2 replication. (A) Phenotypic characterization of moDCs after differentiation from CD14+ monocytes, cultured in GM-CSF and IL-4 for 7-8 days. The data are means + the standard errors of the mean (SEM) obtained from a minimum of three independent donors. Immature moDCs were exposed to HSV-2(333)ZAG (MOI = 0.01 to 10 PFU/cell, as indicated) for 1 h at 37°C. (B and C) At the specified times p.i., infection was assessed by quantifying GFP expression (10,000 live event acquisition) by flow cytometry (B), and cell viability was assessed in 10,000 total events by staining with a Live/Dead marker (C). Infected (GFP+) DCs within the live population of panel C are shown in panel D. The data are representative dot plots (B) and are means + SEM (C and D) of a minimum of five independent experiments. (E) Immature moDCs were exposed to live or UV-inactivated HSV-2(333)ZAG (MOI 5 PFU/cell) for 1 h at 37°C. At the indicated times p.i., infection was assessed by quantifying GFP expression (10,000 live event acquisition) by flow cytometry. The data are means + the SEM of seven independent experiments. (F) Immature moDCs and CaSki cells were infected with HSV-2(G) (MOI = 1 PFU/cell). At the indicated times p.i., the culture supernatants were collected, and viral yields were quantified by performing plaque assays on Vero cells. The results are presented as PFU/ml and are means + the standard deviations (SD) obtained from five independent experiments conducted in duplicate. (G) Immature or LPS-matured DCs were evaluated for CD83 expression (left) and then infected with HSV-2(333)ZAG at the indicated MOIs and at 4 h p.i. were analyzed for GFP expression as a marker of infection by flow cytometry (10,000 live event acquisition). The results are presented as means + the SEM of three independent experiments.
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Fig 2LPS, but not HSV-2, triggers DC maturation. (A and B) Immature moDCs were exposed to LPS (1 μg/ml) or HSV-2(G) (live or UV inactivated) (MOI = 5 PFU/cell) for 1 h at 37°C. The cells were analyzed by flow cytometry for surface marker CD83 expression at 4 h posttreatment. The data are representative dot plots (A) or the mean percent % CD83+ cells + the SEM from six independent experiments (B). (C and D) Further analysis of CD83 expression within infected (GFP+; darkest two segments) or bystander (GFP−; lightest two segments) populations following exposure of DCs to LPS or HSV-2(333)ZAG (MOI = 0.01 to 10 PFU/cell as indicated) at 4 and 8 h postexposure (C and D, respectively). The results are presented as the percentage of 10,000 live events acquired and are means obtained from four independent experiments. Asterisks indicate significant differences relative to mock-treated DCs (P = 0.05).
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Fig 3HSV-2-infected moDCs are impaired in antigen presentation. (A) Immature moDCs or CaSki cells were infected with HSV-2(G) (MOI = 1 PFU/cell) in the absence or presence of acyclovir (ACV) at 100 μg/ml. At the indicated times p.i., culture supernatants were collected, and viral yields were quantified by performing plaque assays on Vero cells. The results are presented as PFU/ml and are means + the SD obtained from five independent experiments conducted in duplicate. (B) 104 human moDCs were exposed to influenza virus BB.PR8 and HSV-2(G) simultaneously or to influenza virus BB.PR8 at 4 h post-HSV-2(G) challenge and then cultured with autologous CD14− T cells at a 1:10 DC/T cell ratio in the presence of ACV (200 μg/ml). IFN-γ release was measured at 40 h postcoculture. T cells stimulated with a CD3 MAb served as a positive control for the ELISpot assay (far left). The results are presented as means + the SEM spot-forming units (SPU) obtained in five independent experiments, each performed in triplicate. The asterisks indicate significant differences relative to influenza virus-challenged DCs (P < 0.05).
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Fig 4HSV-2 induces apoptosis in infected and bystander moDCs. (A) Immature moDCs were mock infected, exposed to staurosporine (ST; 1 μM) or to live and UV-inactivated HSV-2(333)ZAG (MOI = 5 PFU/cell), and analyzed by flow cytometry for apoptosis at 4 h and 8 h p.i. The results are presented as the percentages of cells that are annexin V+ 7AAD− (early apoptosis) and annexin V+ 7AAD+ (late apoptosis) and are means + the SEM of a minimum of three independent experiments. The asterisks indicate significant differences relative to mock-treated DCs (P < 0.05). (B) Dot blots of subsequent analysis of infected (GFP+) and bystander (GFP−) DC populations 4 and 8 h p.i. The results are representative data obtained from five independent experiments.
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Fig 5HSV-2-induced apoptosis is associated with reduced expression of cellular-FLICE inhibitory protein. Immature moDCs were mock infected or exposed to staurosporine (1 μM) or live or UV-inactivated HSV-2(G) (MOI = 10 PFU/cell). At the indicated times postexposure, the cells were lysed and analyzed by Western blotting for the expression of caspase-3 (A) or caspase-8 (B) and β-actin as a loading control. A blot representative of results obtained from two independent experiments is shown. (C) Alternatively, cells were infected with HSV-2(G) (10 PFU/cell), medium (mock), or LPS (10 μg/ml) and, at the indicated times postexposure, the cells were lysed and blotted for expression of c-FLIP and β-actin. The blots were scanned, and the percentages of caspase-3 (A), caspase-8 (B), and c-FLIP (C), relative to β-actin, are indicated below each lane.
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Fig 6HSV-2 triggers persistent Akt activation, which does not require viral gene expression or viral replication. (A) Immature moDCs were mock infected (M) or exposed to LPS (10 μg/ml) or HSV-2(G) (MOI = 10 PFU/cell), and the cells were harvested at the indicated times posttreatment and analyzed by Western blotting for expression of phosphorylated Akt (p-Akt), total Akt, viral glycoprotein B (gB), and β-actin expression. (B) Immature moDCs were mock infected (M) or exposed to LPS (10 μg/ml) or HSV-2(G) (live and UV inactivated) (MOI = 10 PFU/cell) in the absence or presence of ACV (100 μg/ml). At the indicated times posttreatment, the cells were harvested, lysed, and analyzed by Western blotting for the expression of p-Akt, total Akt, and β-actin expression. (C) Immature moDCs were exposed to HSV-2(333)ZAG and at 4 h p.i. were sorted for GFP expression, and 106 GFP+ and GFP− cells were lysed and analyzed by immunoblotting using antibodies to p-Akt, total Akt, and β-actin. Staurosporine (1 μM)-treated cells were included as an apoptosis control. The blots were scanned, and the protein levels were quantified by using ImageJ. They are representative of three (A and C) or two (B) independent experiments, and the percentages of p-Akt relative to total Akt after scanning are indicated below each lane.
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Fig 7HSV-2-induced apoptosis and maturation block is independent of Akt phosphorylation. (A) DCs were treated with wortmannin (25 μM), rapamycin (0.1 μM), or no drug and were mock infected (M), LPS (10 μg/ml) treated, or infected with HSV-2(G) (MOI = 10 PFU/cell). Cells were harvested 4 h p.i. and analyzed by Western blotting for the expression of p-Akt, total Akt, and β-actin expression. The blot is a representative of two independent experiments. The blots were scanned, the protein levels were quantified, and the percentages of p-Akt relative to total Akt are indicated below each lane. (B and C) In parallel, DCs were challenged with HSV-2(G) (MOI = 5 PFU/cell) in the presence of wortmannin and at 4 h p.i. were analyzed by FACS for apoptosis (B) and maturation (C) (as quantified by CD83 expression). The results are presented as the percent positive for annexin V+ 7AAD− (early apoptosis) and annexin V+ 7AAD+ (late apoptosis) (B) and the fold increase in CD83 expression relative to mock-treated cells (C). The data are means + the SEM of three independent experiments. (D) DCs were treated with wortmannin or no drug and were mock infected or exposed to live or UV-inactivated HSV-2(G) (MOI = 10 PFU/cell) and, at the indicated times postexposure, the cells were lysed and analyzed by Western blotting for the expression of c-FLIP and β-actin. A blot representative of results obtained from two independent experiments is shown, and the percentage of c-FLIP relative to β-actin is indicated below each lane.
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Fig 8Live HSV-2 triggers the release of TNF-α and IL-6, whereas UV-inactivated virus triggers an IFN response. (A) Immature moDCs were mock infected, treated with LPS (1 μg/ml) or staurosporine (1 μM), or infected with live or UV-inactivated HSV-2(G) (MOI = 5 PFU/cell), and culture supernatants were harvested at 24 h p.i. and analyzed by Luminex for cytokine levels. The results are means + the SEM obtained from duplicate wells from a minimum of nine independent experiments. (B and C) Immature moDCs were exposed to the same stimuli and, 24 h postexposure, culture supernatants were assessed for the release of IFN-β by ELISA (B) or the release of IFN-γ by Luminex (C). (D) The cells were also lysed at the indicated times for the measurement of IFN-β gene expression by qRT-PCR. The data are presented as pg/ml released (A, B, and C) and gene expression (relative to the housekeeping α-tubulin gene) (D). The data are means + the SEM from three independent experiments. The asterisks indicate significant differences relative to mock-treated DCs (P < 0.05).
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Fig 9HSV-2-exposed DC culture supernatants induce HIV-1 replication in U1 cells. Human immature moDCs were mock exposed, exposed to LPS (1 μg/ml), and HSV-2G (MOI = 5 PFU/cell) (A) or UV-inactivated HSV-2G (MOI = 5 PFU/cell) (B) for 1 h at 37°C. U1 cells were cultured for 4 days in (UV-inactivated) DC supernatants harvested at 24 h post-HSV-2 exposure. TNF-α (100 U/ml) was included as a positive control for induction of HIV-1 replication. HIV release into the U1 cell culture supernatants was monitored by p24 ELISA on days 1 and 4. The results are means + the SEM obtained from six (A) and four (B) independent experiments where each condition was tested in triplicate. The asterisks indicate a significant increase in p24 relative to mock-treated cells (P < 0.05).
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