title: NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways.
abstract: Neutrophils deposit antimicrobial proteins, such as myeloperoxidase and proteases on chromatin, which they release as neutrophil extracellular traps (NETs). Neutrophils also carry key components of the complement alternative pathway (AP) such as properdin or complement factor P (CFP), complement factor B (CFB), and C3. However, the contribution of these complement components and complement activation during NET formation in the presence and absence of bacteria is poorly understood. We studied complement activation on NETs and a Gram-negative opportunistic bacterial pathogen Pseudomonas aeruginosa (PA01, PAKwt, and PAKgfp). Here, we show that anaphylatoxin C5a, formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA), which activates NADPH oxidase, induce the release of CFP, CFB, and C3 from neutrophils. In response to PMA or P. aeruginosa, neutrophils secrete CFP, deposit it on NETs and bacteria, and induce the formation of terminal complement complexes (C5b-9). A blocking anti-CFP antibody inhibited AP-mediated but not non-AP-mediated complement activation on NETs and P. aeruginosa. Therefore, NET-mediated complement activation occurs via both AP- and non AP-based mechanisms, and AP-mediated complement activation during NETosis is dependent on CFP. These findings suggest that neutrophils could use their "AP tool kit" to readily activate complement on NETs and Gram-negative bacteria, such as P. aeruginosa, whereas additional components present in the serum help to fix non-AP-mediated complement both on NETs and bacteria. This unique mechanism may play important roles in host defense and help to explain specific roles of complement activation in NET-related diseases.
authors.affiliation: Cell Biology Program, The Hospital for Sick Children Research Institute, Toronto, ON, Canada; Program in Physiology and Experimental Medicine, The Hospital for Sick Children Research Institute, Toronto, ON, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
authors.forename: Joshua
authors.initials: J
authors.lastname: Yuen
authors.affiliation: Cell Biology Program, The Hospital for Sick Children Research Institute, Toronto, ON, Canada; Division of Haematology/Oncology, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: Fred G
authors.initials: FG
authors.lastname: Pluthero
authors.affiliation: Program in Physiology and Experimental Medicine, The Hospital for Sick Children Research Institute, Toronto, ON, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
authors.forename: David N
authors.initials: DN
authors.lastname: Douda
authors.affiliation: Cell Biology Program, The Hospital for Sick Children Research Institute , Toronto, ON , Canada.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Riedl
authors.affiliation: Cell Biology Program, The Hospital for Sick Children Research Institute , Toronto, ON , Canada.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Cherry
authors.affiliation: Division of Medical Sciences, Northern Ontario School of Medicine, Lakehead University , Thunder Bay, ON , Canada.
authors.forename: Marina
authors.initials: M
authors.lastname: Ulanova
authors.affiliation: Cell Biology Program, The Hospital for Sick Children Research Institute, Toronto, ON, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; Division of Haematology/Oncology, The Hospital for Sick Children, Toronto, ON, Canada; Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada; Department of Biochemistry, University of Toronto, Toronto, ON, Canada; Department of Paediatrics, University of Toronto, Toronto, ON, Canada.
authors.forename: Walter H A
authors.initials: WH
authors.lastname: Kahr
authors.affiliation: Program in Physiology and Experimental Medicine, The Hospital for Sick Children Research Institute, Toronto, ON, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada.
authors.forename: Nades
authors.initials: N
authors.lastname: Palaniyar
authors.affiliation: Cell Biology Program, The Hospital for Sick Children Research Institute, Toronto, ON, Canada; Program in Physiology and Experimental Medicine, The Hospital for Sick Children Research Institute, Toronto, ON, Canada; Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada; Department of Paediatrics, University of Toronto, Toronto, ON, Canada; Division of Nephrology, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: Christoph
authors.initials: C
authors.lastname: Licht
keywords: NETosis; Pseudomonas aeruginosa; alternative pathway; complement system; neutrophil extracellular traps; properdin