Article Figures & Data
Figures
- FIG. 1.
Effect of F. nucleatum and P. gingivalis extracts on HIV-1 promoter activation in monocytes/macrophages. BF24 and THP89GFP cells (2.5 × 105/ml) were either exposed or not (mock) to bacterium extracts. Parental THP-1 (WT) cells were used as a negative control for FACS. (A) HIV-1 CAT promoter activation in BF24 cells challenged with several extract concentrations of each bacterium for 16 h was determined by ELISA. (B) Induction of HIV-1/EGFP in THP89GFP monocytes/macrophages incubated with 10 μg/ml bacterial extracts for 24 h was visualized by light microscopy and fluorescence microscopy using ×100 magnification. Percentage of EGFP-positive cells (C) and mean fluorescence intensity (MFI) of HIV-1/EGFP cells (D) described in the legend to panel B were quantified by flow cytometry as described in Materials and Methods. The data are representative of two independent experiments with triplicate determinations (n = 6). Data are expressed as means ± standard deviations. *, P < 0.01 compared to the results for the control (mock), as determined by Student's t test.
- FIG. 2.
Effect of Toll-like receptor agonists on HIV-1 CAT promoter activation in BF24 monocytes/macrophages. Cells were incubated with either 10 μg/ml bacterial extracts or different concentrations of LPS from P. gingivalis (TLR2 ligand), LPS from E. coli (TLR4 ligand), or CpG ODN2006 (TLR9 ligand). IL-8 production in culture supernatants was analyzed by ELISA, and HIV-1 promoter activation was measured by determination of CAT levels in cell lysates as described in Materials and Methods. The data are representative of three independent experiments with triplicate determinations (n = 9). Data are expressed as means ± standard deviations. *, P < 0.01 compared to the results for the control (mock), as determined by Student's t test.
- FIG. 3.
TLR9 activation by DNA from F. nucleatum is involved in HIV-1 reactivation in monocytes andmacrophages. (A) BF24 cells were incubated with either purified DNA or whole bacterial extracts from F. nucleatum or P. gingivalis for 16 h, and HIV-1 CAT promoter activation in cell lysates was determined by ELISA. ND denotes “nondetected” (values below the level of detection). (B) HIV-1 CAT promoter activity induced by 10 μg/ml DNA from F. nucleatum in BF24 cells preincubated (or not) for 1 h with the TLR-9 inhibitor chloroquine. (C) Induction of HIV-1/EGFP promoter activity in cell lysates (gray bars) and p24 levels in supernatants (black bars) by several concentrations of DNA from F. nucleatum for 24 h was determined in THP89GFP cells by fluorometry and ELISA, respectively, as described in Materials and Methods. MCF, mean cumulative fluorescence. (D) Effect of enzymatic pretreatment of DNA from F. nucleatum with DNase or RNase in HIV-1 reactivation after 24 h of incubation is shown. The data are representative of three independent experiments with triplicate determinations (n = 9). Data are expressed as means ± standard deviations. *, P < 0.01 compared to the results for the control (mock), as determined by Student's t test.
- FIG. 4.
Blocking of TLR2 inhibits bacterium-induced HIV-1 reactivation. Cells (2.5 × 105/ml) were preincubated for 1 h with different concentrations of either a monoclonal anti-TLR2 neutralizing antibody or its respective isotype control before challenge with 10 μg/ml of bacterial extracts. Effect of TLR2 neutralization in HIV-1 CAT promoter activation induced by F. nucleatum (A) and P. gingivalis (B) in BF24 cells exposed to bacteria for 16 h is shown. α, anti-. (C) Effects of TLR2 neutralization in HIV-1/EGFP promoter activation (gray bars) in cell lysates and p24 levels (black bars) in supernatants of THP89GFP cells exposed to bacteria for 48 h are shown. The data are representative of two independent experiments with triplicate determinations (n = 6). Data are expressed as means ± standard deviations. *, P < 0.01 compared to the results for the cells challenged with bacteria in the presence of anti-TLR2 versus the isotype control, as determined by Student's t test.
- FIG. 5.
TNF-α, but not IL-1β, produced by THP89GFP cells in response to F. nucleatum and P. gingivalis is involved in HIV-1/EGFP promoter reactivation. THP89GFP cells (2.5 × 105/ml) were incubated 24 h with 10 μg/ml of bacterial extracts either in the presence of a specific neutralizing antibody (gray bars) or its matched isotype control (black bars). Effects of TNF-α (A and B) and IL-1β neutralization (C and D) in HIV-1/EGFP activity induced by F. nucleatum and P. gingivalis extracts are shown. The data are representative of two independent experiments with triplicate determinations (n = 6). Data are expressed as means ± standard deviations. *, P < 0.01 compared to the results for the cells challenged with bacteria in the presence of neutralizing antibody versus the isotype control, as determined by Student's t test.
- FIG. 6.
HIV-1 reactivation induced by F. nucleatum and P. gingivalis is positively regulated by NF-κB and Sp1. BF24 or THP89GFP monocytes/macrophages were challenged with 10 μg/ml of F. nucleatum or P. gingivalis extracts either in the presence or absence of different concentrations of the specific NF-κB inhibitor BAY 11-7082 dissolved in DMSO. (A) HIV-1 CAT promoter activity in BF24 cell lysates was determined by ELISA. (B) p24 levels were determined in THP89GFP cell supernatants as described in Materials and Methods. (C) Western blot analyses of the total cell extract from THP89GFP cells transfected individually with two different Sp1siRNAs (Sp1siRNA 5 and 2) or with an NS siRNA as described in Materials and Methods. Detection of β-actin was used as a loading control. HIV-1/EGFP promoter activation (D) and viral replication (p24 levels) (E) induced by F. nucleatum (gray bars) and P. gingivalis (black bars) in THP89GFP cells differentially transfected are shown. The data are representative of two independent experiments with triplicate determinations (n = 6). Data are expressed as means ± standard deviations. *, P < 0.01 compared to the results for the cells transfected with Sp1 siRNA or NS siRNA versus nontransfected cells, as determined by Student's t test.
