Anaplasma phagocytophilum inhibits human neutrophil apoptosis via upregulation of bfl‐1, maintenance of mitochondrial membrane potential and prevention of caspase 3 activation

Summary The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis. Intracellular signalling pathways through which the obligatory intracellular bacterium Anaplasma phagocytophilum inhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated. bfl‐1 mRNA levels in uninfected neutrophils after 12 h in culture were reduced to ≈ 5–25% of 0 h levels, but remained high in infected neutrophils. The eukaryotic RNA synthesis inhibitor, actinomycin D, prevented the maintenance of bfl‐1 mRNA levels by A. phagocytophilum. Differences in mcl‐1, bax, bcl‐w, bad or bak mRNA levels in infected versus uninfected neutrophils were not remarkable. By using mitochondrial fluorescent dyes, Mitotracker Red and JC‐1, it was found that most uninfected neutrophils lost mitochondrial membrane potential after 10–12 h incubation, whereas A. phagocytophilum‐infected neutrophils maintained high membrane potential. Caspase 3 activity and the degree of apoptosis were lower in dose‐dependent manner in A. phagocytophilum‐infected neutrophils at 16 h post infection, as compared to uninfected neutrophils. Anti‐active caspase 3 antibody labelling showed less positively stained population in infected neutrophils compared to those in uninfected neutrophils after 12 h incubation. These results suggest that A. phagocytophilum inhibits human neutrophil apoptosis via transcriptional upregulation of bfl‐1 and inhibition of mitochondria‐mediated activation of caspase 3.