Real-Time Imaging of Interactions of Neutrophils with Cryptococcus neoformans Demonstrates a Crucial Role of Complement C5a-C5aR Signaling.
Neutrophils have been shown to efficiently kill Cryptococcus neoformans, a causative agent of meningoencephalitis. Here, using live-cell imaging, we characterize the dynamic interactions of neutrophils with C. neoformans and the underlying mechanisms in real time. Neutrophils were directly seen to chase C. neoformans cells and then rapidly internalize them. Complement C5a-C5aR signaling guided neutrophils to migrate to the yeast cells, resulting in optimal phagocytosis and subsequent killing of the organisms. The addition of recombinant complement C5a enhanced neutrophil movement but did not induce chemotaxis, suggesting that the C5a gradient is crucial. Incubation with C. neoformans resulted in enhanced activation of Erk and p38 mitogen-activated protein (MAP) kinases (MAPKs) in neutrophils. Inhibition of the p38 MAPK pathway, but not the Erk pathway, significantly impaired neutrophil migration and its subsequent killing of C. neoformans. Deficiency of CD11b or blocking of CD11b did not affect the migration of neutrophils toward C. neoformans but almost completely abolished phagocytosis and killing of the organisms by neutrophils. C5a-C5aR signaling induced enhanced surface expression of CD11b. Interestingly, the original surface expression of CD11b was essential and sufficient for neutrophils to attach to C. neoformans but was unable to mediate phagocytosis. In contrast, the enhanced surface expression of CD11b induced by C5a-C5aR signaling was essential for neutrophil phagocytosis and subsequent killing of yeast cells. Collectively, this is the first report of the dynamic interactions of neutrophils with C. neoformans, demonstrating a crucial role of C5a-C5aR signaling in neutrophil killing of C. neoformans in real time.
Division of Immunology, Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, USA.
Donglei
D
Sun
Division of Immunology, Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, USA Department of Immunology, Nanjing Medical University, Nanjing, China.
Mingshun
M
Zhang
Division of Immunology, Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, USA.
Gongguan
G
Liu
Division of Immunology, Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, USA.
Hui
H
Wu
Division of Immunology, Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, USA.
Xiaoping
X
Zhu
Department of Immunology, Nanjing Medical University, Nanjing, China.
Hong
H
Zhou
Division of Immunology, Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, USA mshi@umd.edu.
Meiqing
M
Shi
D000818:Animals; D039481:CD11b Antigen / Q000096:biosynthesis; D002465:Cell Movement / Q000276:immunology; D002478:Cells, Cultured; D003176:Complement C3 / Q000235:genetics / Q000276:immunology; D015936:Complement C5a / Q000235:genetics / Q000276:immunology*; D003453:Cryptococcosis / Q000276:immunology / Q000382:microbiology; D003455:Cryptococcus neoformans / Q000276:immunology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008590:Meningoencephalitis / Q000276:immunology / Q000382:microbiology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009504:Neutrophils / Q000276:immunology*; D010587:Phagocytosis / Q000276:immunology*; D044087:Receptor, Anaphylatoxin C5a / Q000235:genetics / Q000276:immunology*; D011951:Receptors, Complement / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction / Q000276:immunology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
C488033:C5AR1 protein, human; D039481:CD11b Antigen; D003176:Complement C3; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; C085567:complement C3a receptor; D015936:Complement C5a; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases