Anaplasma phagocytophilum inhibits human neutrophil apoptosis via upregulation of bfl-1, maintenance of mitochondrial membrane potential and prevention of caspase 3 activation.
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 approximately 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.
Ge
Yan
Y
Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA.
Yoshiie
Kiyotaka
K
Kuribayashi
Futoshi
F
Lin
Mingqun
M
Rikihisa
Yasuko
Y
D041081:Anaplasma phagocytophilum / Q000472:pathogenicity / Q000502:physiology*; D017209:Apoptosis; D001562:Benzimidazoles / Q000378:metabolism; D002232:Carbocyanines / Q000378:metabolism; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008564:Membrane Potentials; D015778:Minor Histocompatibility Antigens; D008928:Mitochondria / Q000378:metabolism* / Q000502:physiology; D009504:Neutrophils / Q000378:metabolism / Q000382:microbiology*; D009930:Organic Chemicals; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D015854:Up-Regulation
C096816:BCL2-related protein A1; D001562:Benzimidazoles; D002232:Carbocyanines; D061945:Caspase Inhibitors; D005456:Fluorescent Dyes; D015778:Minor Histocompatibility Antigens; D009930:Organic Chemicals; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; C107472:red dye CMXRos; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases