Figure 3. Effect of recombinant and endogenous NhhA on macrophage apoptosis. A: The RAW 264.7 mouse macrophage cell line was incubated with 0–400 nM recombinant NhhA for 20 hours. B: RAW 264.7 cells were incubated with 400 nM NhhA for 0–20 hours. C: RAW 264.7 or PMA-differentiated THP-1 cells were infected with the N. meningitidis serogroup C strain FAM20 or a NhhA-deficient mutant of N. meningitidis NhhA Induces Macrophage Apoptosis FAM20 (DNhhA) at MOI = 100 for 20 hours. Control cells were uninfected. 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 experi- ments. *, p,0.05 versus untreated control(panel A+B) *, p,0.05 versus FAM20 treated cells (panel C). doi:10.1371/journal.pone.0029586.g003 was determined. A significant increase in the number of apoptotic cells could be observed in undifferentiated as well as PMA-treated THP-1 cells (Fig. 4B). Differentiation to macrophages resulted in a striking increase in the sensitivity to NhhA-induced apoptosis indicating that macrophages might be the main target for NhhA- induced apoptosis. As a complement to the microscopy-based apoptosis assays we also analysed apoptosis in undifferentiated THP-1 cells by flow cytometry (Fig. 4C). In order to further investigate the possibility of LPS contam- ination, NhhA was heated at 95u for 10 min. before treatment of PMA-differentiated THP-1 cells. Heat-treatment reduced the apoptosis-inducing activity of NhhA (400 nM) by 96.566.8%. In contrast, heat-treatment of LPS only reduced the apoptosis- inducing activity by 2.365.1%. Since LPS is highly resistant to heat inactivation [17] these data indicate that NhhA-induced apoptosis did not result from LPS contamination. Nitric oxide and cytokine signaling during NhhA-induced apoptosis Macrophages can produce the cytokine TNF-a that plays a role in proapoptotic signaling [10]. We investigated if NhhA- dependent release of TNF-a was of importance for induction of apoptosis by NhhA. However, the presence of a monoclonal antibody capable of blocking TNF-a bioactivity did not influence the proapoptotic effect of NhhA (Fig. 5). In contrast, and in agreement with previous studies [9], LPS-induced apoptosis was partially inhibited in the presence of the TNF-a antibody. We conclude that NhhA-induced apoptosis occurred independently of TNF-a signaling. We also investigated if TNF-a antibodies could block bacteria-induced apoptosis in PMA-differentiated THP-1 cells (Fig S1). We did not see any apoptosis-reducing effect of the antibodies suggesting that LOS might not play a leading role in meningococcal induction of macrophage apoptosis. Nitrosative stress has been reported to be involved in some forms of bacterially induced apoptosis [18]. We investigated if exposure of macrophages to NhhA induced nitric oxide (NO) synthesis. Nitrite, a stable NO metabolite, was measured using Griess assay. We found significantly increased levels of nitrite in NhhA-exposed cells as compared to untreated control cells (Fig. 6A). NhhA-induced nitrite production was inhibited by the Nitric Oxide Synthase inhibitor S- methylisothiourea (SMT). Production of NO was accompanied by induction of iNOS as judged by immunoblot analysis (Fig. 6B). Since NO production can contribute to proapoptotic signaling through the mitochondrial pathway we investigated if NO production was of importance for NhhA-induced apoptosis. However, we could not detect any change in the number of apoptotic cells when macrophages were pretreated with SMT before exposure to NhhA (Fig. 6D). Thus, NO production appears not to be of importance for NhhA-induced apoptosis in macrophages. Since apoptosis-related release of the cytokine high mobility group box 1 (HMGB1) by macrophages is a crucial mediator of organ damage in sepsis [19] we determined if HMGB1 release is altered during apoptosis induced by wildtype and NhhA-deficient meningococci (Fig S2). We observed statistically significant decreases in the extracellular levels of HMGB1 when cells were exposed to NhhA-deficient bacteria as compared to wildtype bacteria. PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e29586