N. meningitidis NhhA Induces Macrophage Apoptosis Figure 2. NhhA induces caspase-dependent apoptosis in macrophages. A: The RAW 264.7 mouse macrophage cell line was incubated with or without 400 nM recombinant NhhA and apoptotic cells were visualized by staining with an AnnexinV antibody and PI. The relative number of AnnexinV-positive and PI-negative cells were determined by fluorescence microscopy. B: Cells were incubated with NhhA as above or with 0.5 mg/ml E. coli LPS for 20 hours. If indicated, cells were pretreated with 1 mM Polymyxin B (PMB) or with the caspase inhibitor ZVAD-FMK (1 mM) for 1 h. Apoptotic cell were visualized using the APOPercentage kit. The relative numbers of apoptotic cells were determined using light microscopy by counting at least 100 cells per sample. Values indicate mean6SD of three independent experiments. *, p,0.05 versus untreated control. doi:10.1371/journal.pone.0029586.g002 (Fig. 4A). In agreement with previous publications, retinoic acid- differentiated HL-60 cells were found to have a high level of background apoptosis [16]. NhhA did not alter the rate of apoptosis in any of these cells indicating that NhhA might specifically act on macrophages. In order to investigate if human macrophages might be a target of NhhA we differentiated the monocytic cell line THP-1 to macrophage-like cells with phorbol 12-myristate 13-acetate (PMA). Undifferentiated and PMA-differentiated THP-1 cells were treated with NhhA and the relative number of apoptotic cells PLoS ONE | www.plosone.org 3 January 2012 | Volume 7 | Issue 1 | e29586