Figure 1
HIV infection of human macrophages results in increased numbers of TNTs. Cultures of human macrophages were exposed to HIVADA for 24 h and then washed extensively to eliminate unbound virus. Media was collected every day to assay for secreted HIV-p24 by ELISA. In parallel, at different time points, cells were fixed for confocal microscopy or SEM. (A) Time course of formation of TNT or numbers of cells expressing TNTs using confocal microscopy. Uninfected cells contained low numbers of TNT (black line). However, HIV infection induced the formation of TNT per cell as well as the numbers of cells with TNT (red line). *p ≤ 0.0005 as compared to uninfected conditions, n = 23. (B) Time course of formation of TNT or numbers of cells expressing TNTs using SEM. Quantification of cells with TNT indicates that SEM is able to detect 1–20% higher numbers of cells with TNT than confocal microscopy. *p ≤ 0.0008 as compared to uninfected conditions. #p ≤ 0.003 as compared to the values in (A) in the presence of HIV, n = 5. (C) Representative image of macrophages infected with HIV after 3 days’ post infection and stained with DAPI, Actin, and HIV-p24. HIV-p24 (green staining) is spread by TNTs (white arrows). No background or nonspecific staining was detected using isotype-matched irrelevant antibodies (data not shown). (D) Representative image of 3 days of culture of uninfected macrophages analyzed by SEM. (E) Representative image of HIV infected cultures after 3 days post infection. (F) High magnification of the end of the TNT process in HIV infected conditions. n = 5.
