Background NETosis is an antimicrobial cell death program of neutrophils characterized by the release of Neutrophil Extracellular Traps (NETs). NETs consist of processed chromatin associated with cytoplasmic proteins and granules. NETs behave as alarmins and can directly activate Dendritic Cells (DCs) or modulate their LPS-mediated activation. Interestingly recent data demonstrate a role for NETosis in Systemic Lupus Erythematosus (SLE) and other autoimmune diseases. The underlying mechanisms are not completely clear, but defective NET-DNA degradation seems to be important. Caspase-activated DNAse (CAD) is an intracellular DNAse activated by caspases during apoptotic cell death whose absence leads to the lupus-like disease in genetically predisposed mice.
Objectives Our aim was to determine the role of CAD in NETs release. Specifically whether its absence may influence LPS-mediated DCs activation via NETs modulation. We also investigated weather NETosis in the absence of CAD was sex-biased. The latter in view of the relevance of sex in lupus
Methods Neutrophils from the bone marrow of C57bl/6 (b6) and b6.CAD (CAD)–/– mice were treated with 50 nM phorbol myristate acetate (PMA) to induce NETosis. NET-DNA was detected in supernatants by spectrophotometry after 4 and 16 hours. Pretreatment with heparin (50 ug/ml) or physiological concentrations of DNAse (20 ng/ml) was used to respectively neutralize NET-histones and degrade NET-DNA. NETting neutrophils were also visualized and counted by Immunofluorescence. Bone marrow-derived Dendritic Cells (BMDCs) were co-cultured for 20 hours with neutrophils previously treated for 4 hours as mentioned before, in presence or absence of 25 ng/ml LPS. Neutrophils and BMDCs were also treated separately as controls. DCs activation was studied by quantifying TNF-alpha from supernatants by ELISA and by staining cells were stained for flow cytometry to detect CD11b+CD11c+CD86+ DCs.
Results We found that in CAD–/–, NET-DNA was lower than b6 at 4 and 16 hours, and DNAse was not as effective as in b6 in degrading NET-DNA. NETting neutrophils didn't produce any detectable TNF-alpha. LPS-mediated DC activation was enhanced in the presence of NETting neutrophils from b6 males and CAD–/– males and females. Interestingly we found that for b6 females, heparin pretreatment increased the response. In contrast for CAD–/– females, the response was reduced by DNAse. Finally in males, NETs in the absence of CAD led to lower LPS-mediated DC activation. The latter showed decreased activation only with heparin pretreatment.
Conclusions We show that CAD activity is important for the proper release of NETs, its absence confers resistance to DNAse and its role is sex-biased. Indeed we show that NETs protect only b6 females from enhanced LPS-induced DC activation, and activation is significantly higher in absence of CAD and resistant to NET inhibition. Finally in males, histones seem to have a significant role in DC activation in b6, while in females the role of DNA seems to be more relevant in both strains. Further studies are needed to characterize the molecular features of these differences.
Pinegin B et al. Autoimmun Rev. 2015 Jul;14(7):633–40
Barrientos L et al. J Immunol. 2014 Dec 1;193(11):5689–98
Jog NR et al. Arthritis Rheum. 2012 Apr;64(4):1247–56
Disclosure of Interest None declared