N. meningitidis NhhA Induces Macrophage Apoptosis Figure 4. NhhA-induced apoptosis in different cell types. A: The pharyngeal epithelial cell line (FaDu) and the HL-60 promyelocytic leukemia cell line were exposed to 400 nM NhhA for 20 h. HL-60 cells were undifferentiated or differentiated to neutrophil-like cells by retinoic acid treatment (1 mM for 7 days, RA). B: The human monocytic cell line THP-1 was exposed to 400 nM NhhA. If indicated, THP-1 cells were differentiated to macrophages by treatment with PMA. 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. C: The human monocytic cell line THP-1 was exposed to 400 nM NhhA and apoptosis was measured by TCTF staining and flow cytometry. One representative experiment is shown. doi:10.1371/journal.pone.0029586.g004 Discussion The results presented here provide new insights on the role of NhhA during host-pathogen interaction. We found that NhhA induced caspase-dependent apoptosis in mouse and human macrophages, and to a lesser extent in monocytes. In contrast, endothelial cells and other myeloid cells appeared not to be targets of NhhA. Phosphatidylserine translocation to the outer layer of the cell membrane, a hallmark of apoptosis, was detected by two independent methods. A highly increased rate of apoptosis could be observed and after 20 hours exposure to 400 nM NhhA around 20% of the cells were found to be apoptotic. A significant increase in the number of apoptotic cells was observed already at 10 nM NhhA. In order to confirm that endogenous meningococcal NhhA regulates apoptosis we infected cells with wildtype and NhhA- deficient meningococci. We found that NhhA-deficient meningo- cocci less efficiently induced apoptosis in macrophages, confirming that endogenous NhhA controls apoptosis in macrophages. It was recently shown that NhhA can bind to vitronectin and thereby increase serum resistance by regulating complement activity [20]. Since vitronectin is abundant in serum the vitronectin-NhhA interaction could potentially regulate other actions of NhhA, such as its macrophage apoptosis-inducing activity described here. Bovine vitronectin binds to NhhA with similar affinity as human vitronectin [20]. Since our apoptosis experiments were carried out in the presence of bovine serum, we draw the conclusion that NhhA-induced macrophage apoptosis is not prevented by vitronectin. Further studies will be needed to elucidate to what extent the different functions of NhhA influence each other. Macrophages produce TNF-a, a cytokine involved in proa- poptotic signaling [10]. Meningococcal LOS induces TNF-a release which in turn contributes to induction of apoptosis in host cells [9]. A novel mechanism whereby meningococci regulate apoptosis was recently described, it was shown that strains associated with asymptomatic carriage promote shedding of TNF-a receptors whereby TNF-a is chelated and proapoptotic signaling prevented [9]. However, inhibition of TNF-a bioactivity did not affect NhhA-induced apoptosis. It appears that in contrast to LOS-induced apoptosis, NhhA-induced apoptosis does not PLoS ONE | www.plosone.org 5 January 2012 | Volume 7 | Issue 1 | e29586