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.

Factor H Binds to Extracellular DNA Traps Released from Human Blood Monocytes in Response to Candida albicans

“…This is in agreement with recent reports of NETs (48, 49) and suggested that complement is regulated on the NETs to allow opsonization and phagocytosis to terminate the immune response. Clearance of extracellular traps is achieved with the help of DNase I, which cleaves the extracellular DNA and facilitates uptake and clearance of DNA by macrophages (28).…”

Mature neutrophils suppress T cell immunity in ovarian cancer microenvironment

“…However, tumor cells can secrete complement proteins that stimulate tumor growth and epithelial mesenchymal transition (47,48). Complement activation can also promote platelet-granulocyte aggregation, thrombosis, and NETosis (49,50). CR3 signaling was associated with suppression of NK cell function and increased growth of syngeneic melanoma (51).…”

Production of complement components by cells of the immune system

“…Neutrophils not only store C3 but also FP, the only complement regulator that enhances complement activation, which can be secreted upon stimulation [30,35,37]. Besides the release of C3 and FP, FB was also demonstrated to be secreted upon activation of neutrophils [38]. Locally released FP, FB and C3 could provide a local platform for further complement activation via the AP.…”