Phagocytosis via complement receptor 3 enables microbes to evade killing by neutrophils

• Published in: Journal of leukocyte biology 2023-07, Vol.114 (1), p.1-20
• Main Authors: Smirnov, Asya, Daily, Kylene P, Gray, Mary C, Ragland, Stephanie A, Werner, Lacie M, Brittany Johnson, Morgan, Eby, Joshua C, Hewlett, Erik L, Taylor, Ronald P, Criss, Alison K
• Format: Article
• Language: English
• Subjects: Animals; Complement C3b - metabolism; Humans; Macrophage-1 Antigen - metabolism; Mice; Neutrophils; Phagocytosis; Reactive Oxygen Species - metabolism; Receptors, Complement - metabolism
• ISSN: 1938-3673; 0741-5400; 1938-3673
• DOI: 10.1093/jleuko/qiad028

CR3 (CD11b/CD18; αmβ2 integrin) is a conserved phagocytic receptor. The active conformation of CR3 binds the iC3b fragment of complement C3 as well as many host and microbial ligands, leading to actin-dependent phagocytosis. There are conflicting reports about how CR3 engagement affects the fate of phagocytosed substrates. Using imaging flow cytometry, we confirmed that binding and internalization of iC3b-opsonized polystyrene beads by primary human neutrophils was CR3-dependent. iC3b-opsonized beads did not stimulate neutrophil reactive oxygen species, and most beads were found in primary granule-negative phagosomes. Similarly, Neisseria gonorrhoeae that does not express phase-variable Opa proteins suppresses neutrophil reactive oxygen species and delays phagolysosome formation. Here, binding and internalization of Opa-deleted (Δopa) N. gonorrhoeae by adherent human neutrophils was inhibited using blocking antibodies against CR3 and by adding neutrophil inhibitory factor, which targets the CD11b I-domain. No detectable C3 was deposited on N. gonorrhoeae in the presence of neutrophils alone. Conversely, overexpressing CD11b in HL-60 promyelocytes enhanced Δopa N. gonorrhoeae phagocytosis, which required the CD11b I-domain. Phagocytosis of N. gonorrhoeae was also inhibited in mouse neutrophils that were CD11b-deficient or treated with anti-CD11b. Phorbol ester treatment upregulated surface CR3 on neutrophils in suspension, enabling CR3-dependent phagocytosis of Δopa N. gonorrhoeae. Neutrophils exposed to Δopa N. gonorrhoeae had limited phosphorylation of Erk1/2, p38, and JNK. Neutrophil phagocytosis of unopsonized Mycobacterium smegmatis, which also resides in immature phagosomes, was CR3-dependent and did not elicit reactive oxygen species. We suggest that CR3-mediated phagocytosis is a silent mode of entry into neutrophils, which is appropriated by diverse pathogens to subvert phagocytic killing.