Suppression of macrophage-derived nitric-oxide production by A.actinomycetemcomitans cytolethal distending toxin

Production of nitric oxide (NO) by activated macrophages is one of the most relevant mechanisms for bacterial control. Cytolethal distending toxin (CDT) is produced by the periodontopathogenic organism Aggregatibacter actinomycetemcomitans (A.a). CDT leads to cell growth arrest and apoptosis of several cells types, but its effect on macrophages is poorly understood. Objective: The purpose of this study was to evaluate the effect of A.aCDT on NO production by activated macrophages. Methods: Purified CDTA, CDTB and CDTC were obtained using a His-tag expression vector system and were reconstituted to form the CDT holotoxin. Different concentrations of CDTA/CDTB/CDTC were used (1.875/0.75/1.875; 3.75/1.25/3.75; 7.5/2.5/7.5; 12.5/4/12.5; 15/5/15; 25/8/25 and 30/10/30 respectively) (µg/ml). Separated fractions or the holotoxin were incubated in vitro with LPS-activated Raw246.7 macrophages (2x10⁵cells/well). In Con A-activated resident peritoneal macrophages from C3H/HePas (TLR4-sufficient) or from C3H/HeJ (TLR4-deficient) mice (2x10⁶cells/well), 12.5/4/12.5; 15/5/15; 25/8/25 concentrations of CDT were added. After 48h, NO concentration was determined and cell viability was assessed by MTT and crystal violet assays. Results: The production of NO was observed in both LPS-activated and non-activated RAW cells receiving the separated fractions of the CDT. However, the holotoxin CDT inhibited NO production in a dose-dependent manner in Raw246.7 cells (ANOVA-Tukey-p<0.05) without loss of cell viability, except for the highest concentration of toxin (30/10/30). Con A-activated macrophages of C3H/HeJ and C3H/HePas mice produced significant amounts of NO. Addition of CDT inhibited NO production in dose-dependent manner (ANOVA-Tukey-p<0.05) without loss of cell viability. Conclusion: The results suggest that A.aCDT inhibits NO production by macrophages and this may be an additional factor in the persistence of A.actinomycetemcomitans infection.