Figure 5. NhhA-induced apoptosis does not require TNF-a signaling. The RAW 264.7 mouse macrophage cell line was incubated with 400 nM recombinant NhhA or 0.5 mg/ml E. coli LPS for 20 hours. In addition, cells were incubated with an inhibitory TNF-a antibody or treated with an irrelevant isotype-matched antibody. Apoptotic cells were visualized using the APOPercentage kit and the relative numbers of apoptotic cells were determined by light microscopy. Values indicate mean6SD of three independent experiments. *, p,0.05. doi:10.1371/journal.pone.0029586.g005 N. meningitidis NhhA Induces Macrophage Apoptosis require autocrine or paracrine TNF-a signaling. Instead, NhhA- induced apoptosis might involve other caspase-dependent path- ways such as the intrinsic/mitochondrial pathway or the FasL or TRAIL pathways [21]. Exposure of macrophages to NhhA also triggered induction of iNOS and production of NO. Pretreatment of cells with a NO synthase inhibitor blocked NO production but not NhhA-induced apopotosis. Therefore, NO production appears not to be required for NhhA-dependent apoptosis. NO production is of major importance for host-defense mechanisms against N. meningitidis [22] and other bacteria. Thus, NhhA-induced NO production might instead contribute to the bactericidal activity of macrophages and other cells. Systemic meningococcal infections are associated with a strong proinflammatory response that by itself can contribute to tissue injury and mortality. Several publications have indicated a relationship between organ failure in sepsis and apoptosis [23–24]. In severe sepsis, high levels of the cytokine high mobility group box 1 (HMGB1) are accumulated leading to lethal organ failure due to epithelial cell dysfunction [25,26,27]. Macrophages appear to play a key role in these events by their ability to respond to the presence of apoptotic cells by releasing HMGB1 [19]. It was shown that HMGB1 is a critical cytokine mediator of organ damage in severe sepsis since monoclonal antibodies against HMGB1 protected Figure 6. NhhA-induced apoptosis does not require NO. The RAW 264.7 mouse macrophage cell line was incubated with or without 400 nM recombinant NhhA for 20 hours. If indicated cells were pretreated for 1 h with the NO synthase inhibitor S-methylisothiourea (50 mM, SMT). A: Nitrite levels in cell supernatants were determined using Griess assay. B: The expression of iNOS was detected by immunoblot analysis. C+D: Apoptotic cells were visualized using the APOPercentage kit and the relative numbers of apoptotic cells were determined by light microscopy. Values indicate mean6SD of three independent experiments. *, p,0.05 versus untreated control. doi:10.1371/journal.pone.0029586.g006 PLoS ONE | www.plosone.org 6 January 2012 | Volume 7 | Issue 1 | e29586