Fig 1: The reduction of HIV-1 titers is independent of classic PMN effector functions(A) Schematic overview of the different PMN effector functions and drugs or blocking antibodies used.(B) Quantification of NET formation by PMNs incubated with HIV-1 particles. PMNs were co-cultured with medium, 100 nM PMA, or different amounts of viral particles on fibronectin-coated cover glasses for 3 h and 24 h. Samples were stained with anti-MPO antibody and Hoechst. 5 random images (see Figure S5A for images) were taken per condition and time point, and NET-forming cells were counted manually. In PMA-treated samples, differentiation between NET-forming cells and normal PMNs was not possible because of the overall coverage of the selected areas with NETs. NET formation in these samples was set as an approximate value of 100%. For the other samples, PMNs were categorized as lobular nucleus (red), round nucleus (gray), and NET forming (blue). Depicted are mean values + SD of 4 and 3 independent experiments for 3 h and 24 h, respectively.(C) HLACs were infected with X4 HIV-1, and supernatant for determination of the viral load by SG-PERT was harvested on days 4 and 7 pi. Depicted are mean values + SD of 8 independent experiments performed in triplicates. Dotted lines indicate the number of viral particles used in (B).(D) Production of NE. HLACs were infected with X4 HIV-1 or left uninfected (mock) and cultured in the presence of 1 × 106 PMNs for 3 h and 24 h. As a positive control, 20 nM PMA was added to the mock culture. At the indicated time point, NE activity was determined (n3h = 5, n24h = 3). Shown are mean values of experiments performed in duplicates or triplicates.(E) 5 U/mL DNase I was added to X4 HIV-1-infected HLACs cultured with or without 1 × 106 PMNs. Each symbol designates the AUC of HIV-1 replication of 4–5 independent experiments performed in triplicates, hostogram bars indicate mean values + SD.(F) ROS production; experiment as in (D) but analyzed for ROS production by flow cytometry. Shown are mean values + SD of experiments performed in triplicates (n3h = 4, n24h = 2) relative to mock samples (set to 1).(G–M) Experiment as in (E) but using the following agents to affect PMN activity: (G) 1,000 U/mL catalase, (H) 100 µM 4-ABAH (n = 6), (I) 1 µM iODN 2088 (n = 4), (J) 50 µM AEBSF (n = 3), (K) 300 µM W054011 (n = 4), (L) 10 µM BAPTA-AM (n = 2), and (M) 100 µg/mL blocking antibody against CD18, CD11a, and CD11b (n = 1 or 2). Cells were treated for 30 min before addition to X4 HIV-1 infected HLACs, and experiments were performed in the presence of the inhibitors. Shown are mean values + SD of experiments performed in duplicates or triplicates.Open circles indicate mean values of data points of experiments in which the cells of the control experiment could not be analyzed because of cell death. Gray bars with errors indicate the mean + SD of all experiments. Data points are mean values + SD of triplicate infections of cells from the same donor. Statistical significance was assessed by Mann-Whitney U test (*p < 0.05, **p < 0.01). See also Figure S5.