against organ damage but did not prevent the accumulation of apoptotic cells in the spleen [19]. NhhA-mediated macrophage apoptosis might contribute to HMGB1 release and tissue damage during meningococcal sepsis. In summary, we demonstrate that NhhA promotes caspase- dependent macrophage apoptosis. NhhA-induced macrophage apoptosis might contribute to disease severity in meningococcal infections by reducing phagocytotic and immunoregulatory activities of macrophages. Materials and Methods Bacterial strains and growth conditions FAM20, a N. meningitidis serogroup C strain, and its NhhA- deficient mutant [6], were grown at 37uC and 5% CO2 on GC medium base (Acumedia). Cloning and purification of recombinant NhhA A sequence encoding amino acids 51 to 510 of NhhA was amplified by PCR using genomic DNA from FAM20, a N. meningitidis serogroup C strain, using the following primers; 59- GCGAATGGATCCGATACCGATGAAGATG-39 and 59- CCTTTAAGCTTTGCGACGTTTGTAACATC-39. After puri- fication and digestion the PCR product was cloned into the vector pET-21a (Novagen) using restriction enzymes BamHI and HindIII. Recombinant NhhA was expressed in E. coli in fusion with a C- terminal hexahistidine tag. NhhA was purified by affinity chromatography using Talon Resin (Clontech). Recombinant NhhA was then further purified by FPLC gel filtration. Purification was monitored by SDS-PAGE with Coomassie brilliant blue staining. The meningococcal protein NMC0101 was expressed and purified in the same way to serve as a control. Cell culture and treatment Cell lines were obtained from American Type Culture Collection. The murine macrophage cell line RAW 264.7 and the human-derived pharyngeal epithelial cell line FaDu were cultured in DMEM plus 10% heat-inactivated fetal calf serum at 37uC and 5% CO2. The human monocytic cell line THP-1 and the human HL-60 promyelocytic leukemia cells were cultured in RPMI-1640 plus 10% heat-inactivated fetal calf serum at 37uC and 5% CO2. Retinoic acid treatment of HL-60 cells was with 1 mM all trans retinoic acid for seven days. THP-1 cells were differentiated to macrophages using 100 nM PMA (Sigma) for 3 days. Cells were maintained in serum-containing growth medium during the treatments described below. After seeding cells (50,000/ml) into 24- or 96-well cell culture plates or 8-well m- slides (Ibidi), cells were allowed to attach for 6 h. If indicated, cells were pretreated with 1 mM Polymyxin B (InvivoGen), 1 mM caspase inhibitor ZVAD-FMK (BD Biosciences), 50 mM S- methylisothiourea (Sigma-Aldrich), 2.5 mg/ml anti TNF-a mono- clonal antibody, clone MP6-XT22 (BD Biosciences) or 2.5 mg/ml of an unrelated isotype matched monoclonal antibody. After 1 h pretreatment cells were exposed to 0–400 nM NhhA for 5–20 h or 500 ng/ml E. coli LPS (Sigma-Aldrich) for 20 h. Alternatively, cells were exposed to wildtype FAM20 meningococci or a NhhA- deficient FAM20 mutant strain for 20 h at MOI = 100. Binding assay by Immunofluorescence microscopy 13 mm glass cover slips were coated with poly-L-lysine (5 mg/ ml in PBS). RAW 264.7 cells were grown onto these over night, reaching approximately 40% confluence. The cells were then treated with 400 nM NhhA or an unrelated His-tagged protein for 4 hours. Unbound protein was removed by washing in PBS. N. meningitidis NhhA Induces Macrophage Apoptosis Cells were then fixed in 2% paraformaldehyde for 15 min in RT, washed in PBS and permeabilized with 0.3% TritonX-100 in PBS for 10 min in RT. After washing in PBS, the cover slips were incubated in blocking buffer (3% BSA, 0.2% Tween, 16PBS) for 30 min in RT. Cells were then incubated with primary antibody monoclonal mouse a-His-tag IgG2a (GE healthcare), diluted 1:200, for 1 hour in RT. Incubation with Alexa Fluor 488-labeled secondary antibody (Invitrogen) diluted 1:200 was performed for 1 hour in RT. The cover slips were fastened onto object glasses using ProLongH Gold mounting medium with DAPI (49,69-diamidino-2-phenylidole) (Invitrogen). Images were taken with an Axiovision Cell Observer HS fluorescent microscope (Carl Zeiss). Binding assay by Immunoblot analysis RAW 264.7 cells were treated with 400 nM NhhA for 4 hours. The cells were washed in PBS, dissolved in SDS sample buffer, boiled at 95uC for 5 minutes and proteins were separated by SDS-PAGE (12%). Separated proteins were transferred to polyvinylidene floride membranes using a semidry transfer system (Bio-Rad). Membranes were blocked in 5% skim milk solution at 4uC o/n and incubated with primary antibody monoclonal mouse a-His-tag (GE healthcare), diluted 1:1000 in PBS, at RT for 1 hour. Secondary antibody was an IRDyeH 680 Goat anti- Mouse (1:15,000, LI-COR Biosciences). For detection the OdysseyH Infrared Imaging System (LI-COR Biosciences) was used. Measurement of apoptosis The relative number of cells undergoing apoptosis was determined using the APOPercentage kit (Biocolor) with the dye 3, 4, 5, 6-tetrachloro 29, 49, 59, 79 -tetraiodofluorescein (TCTF) following manufacturer instructions. Flow cytometry of TCTF- stained cells was done according to Meyer et al [28] using a LSRFortessa flow cytometer (BD biosciences). Alternatively cells were stained with propidium iodide (5 mg/ml) and a FITC- conjugated Annexin V antibody (BD Biosciences) according to manufacturer instructions. Cells were examined with an Axiovi- sion Cell Observer HS fluorescent microscope (Carl Zeiss) and the relative number of cells that were Annexin V-positive and/or propidium iodide positive were determined. Measurement of NO production, iNOS expression and HMGB1 release Production of NO was determined by measurement of nitrite in the cell supernatant using a Griess assay (Promega). iNOS expression was detected as follows. After washing twice with ice- cold PBS, cells were treated with lysis buffer containing 50 mM Tris-HCl (pH 7.6), 0.3 M Sucrose, 2 mM EDTA, 1 mM PMSF, 2 mM DTT, 20% Glycerol, 0.5% IGEPAL. The concentration was measured and equal amounts (30 mg) of protein was separated by SDS-PAGE and transferred to PVDF membrane (Santa Cruz Biotechnology). The primary antibody was a rabbit anti-mouse iNOS (1:500, MyBioSource) and as second antibody IRDyeH 680 Donkey anti-Rabbit (1:15,000, LI-COR Biosciences) was used and detected with the OdysseyH Infrared Imaging System (LI-COR Biosciences). The levels of HMGB1 in cell supernatants was determined using an indirect ELISA assay with a mouse anti- HMGB1 antibody (Santa Cruz, SC-74085). Statistical analysis Student’s t-test was used for statistical analysis. Differences between means were considered significant if P#0.05. PLoS ONE | www.plosone.org 7 January 2012 | Volume 7 | Issue 1 | e29586