FcgRIIA shedding requires reactive oxygen species. (A) Neutrophils were activated with R848, and FcgRIIA and CD66b levels were analyzed by flow cytometry. The experiment was repeated eight times; combined results are shown and compared using paired Student’s t test (P < 0.0001). (B) Heat-map illustrating phosphoproteins modified upon TLR7/8 activation by R848 and RNP-ICs. Results are expressed as fold change as compared with nonstimulated neutrophils with green representing decreased phosphorylation and red indicating increased phosphorylation. (C) Phosphorylated ncf1 (p47 phox) at S345 upon R848 activation as determined by phosphoproteomics. The experiment was repeated three times; combined results are shown and compared using paired Student’s t test (P = 0.044). (D) Neutrophils were incubated with R848 in the absence or presence of the PI3K inhibitor Ly294002 and analyzed for pS345 or total levels of p47 phox using Western Blot. The experiment was repeated four times; combined results are shown and compared using paired t test (No stim, P = 0.03; R848+LY294002, P = 0.0011). (E) Neutrophils were treated with inhibitors of NADPH oxidase before addition of R848 and analyzed for cell surface expression of FcgRIIA by flow cytometry. The experiment was repeated six times; combined results are shown and compared using paired t test (DPI, P = 0.0042; Apocynin, P = 0.0044). (F) Neutrophils from healthy individuals (HV, n = 18) and CGD patients (n = 4) were stimulated with R848 and analyzed for FcgRIIA levels by flow cytometry. The data are analyzed using paired Student’s t test (HC, P < 0.0001) and unpaired Student’s t test (HC vs. CGD, P = 0.0097). (G) Neutrophils from healthy individuals (HV, n = 13) and CGD patients (n = 3) were activated by R848 and analyzed for CD66b expression by flow cytometry using paired Student’s t test (HC, P < 0.0001; CGD, P = 0.039). (H) Neutrophils were activated with LPS or PAM3CSK4 in presence of DPI and analyzed for FcgRIIA levels by flow cytometry. The experiment was repeated four times; combined results are shown and compared using paired Student’s t test (LPS, P = 0.0062; PAM, P = 0.0003). (I) Neutrophils were activated with R848, RNP-ICs, or PMA and analyzed for cellular localization for the ROS generation by flow cytometry and fluorimetry. The experiment was repeated five (extracellular) and eight (intracellular) times; combined results are shown and compared using paired Student’s t test (Extracellular: PMA, P = 0.007; Intracellular: PMA, P < 0.0001; RNP-IC, P = 0.0007; R848, P < 0.0001). (J) Phosphorylation of Akt and S6 was determined by flow cytometry upon TLR7/8 activation. The experiment was repeated four times; combined results are shown and compared using paired t test (pS6, P = 0.042; pAkt, P = 0.037). (K) Neutrophils, pretreated with inhibitors of PI3K (LY294002, 10 µM) or NADPH oxidase (DPI, 25 µM), were activated with R848 and analyzed for ROS generation by flow cytometry using DHR123. The experiment was repeated three times; combined results are shown and compared using paired Student’s t test (R848, P = 0.0049; R848+LY294002, P = 0.004; R848+DPI, P = 0.0031). (L) Neutrophils were pretreated with the PI3K inhibitor LY294002 (10 µM) and analyzed for R848-mediated shedding of FcgRIIA by flow cytometry. The experiment was repeated eight times; combined results are shown and compared using paired Student’s t test (P < 0.0001). (M–O) Neutrophils, with or without pretreatment with LY294002, were activated with heat-aggregated IgG (HAGG) and analyzed for (M) CD66b, (N) FcgRIIA shedding, and (O) pS6 expression by flow cytometry. In M and N, the experiments were repeated eight times. In O, the experiment was repeated five times; combined results are shown and compared using paired Student’s t test (M: R848, P < 0.0001; HAGG, P = 0.017; R848 vs. HAGG, P = 0.0001; N: R848, P < 0.0001; HAGG, P = 0.0002; R848 vs. HAGG, P = 0.0029; HAGG vs. HAGG+LY294002, P = 0.018; O: HAGG, P = 0.0024; R848, P = 0.0314; RNP-IC, P = 0.011). *, P < 0.05; **, P < 0.01; ***, P < 0.001.