Background Neutrophils participate in host defence through mechanisms including phagocytosis and formation of neutrophil extracellular traps (NETs), a neutrophil cell death process in which DNA is extruded together with cytoplasmic and granular content to trap and eliminate pathogens. Immune complex (IC)-mediated NET formation has emerged as a mechanism that may increase the autoantigenic burden as well as promote type I interferon production in patients with the autoimmune disease systemic lupus erythematosus (SLE). Although TLR agonists, such as nucleic acids, have been shown to enhance phagocytosis by macrophages and dendritic cells, the role of TLR signalling in neutrophil phagocytosis of RNA-containing SLE ICs has not been extensively studied. The aim of the current study was to explore the cross-talk between TLRs and FcgRs in the regulation of IC-mediated phagocytosis and NETosis.

Materials and methods Neutrophils, isolated from healthy individuals were incubated with RNA-containing ICs and analysed for phagocytosis and NETosis by flow cytometry and fluorimetry, respectively, in the presence of blocking antibodies or TLR8 inhibitors (oligodinucleotides, RNase). Neutrophils from healthy controls (n = 7) and SLE patients (n = 19) were analysed for FcgRIIA expression by flow cytometry, using two antibody clones, recognising full-length or shed FcgRIIA, and the results related to clinical data.

Results Both FcgRIIA- and TLR8-engagement were required for induction of NETosis by RNA-ICs, as demonstrated by FcgR blocking antibodies as well as RNase treatment. Although degradation of RNA inhibited NETosis, removal of the TLR ligand by RNase markedly increased the phagocytosis of RNA-ICs by neutrophils (p < 0.0001), suggesting that TLR activation suppressed phagocytosis. Consistent with this hypothesis, addition of TLR8 agonist (R848) inhibited phagocytosis of ICs (p < 0.0001), but not beads, in neutrophils. Mechanistically, TLR8 activation mediated furin-dependent proteolytic cleavage of the most N-terminal part of FcgRIIA reducing the phagocytic capacity while promoting progression into NETosis. Importantly, ex vivo isolated neutrophils from SLE patients demonstrated increased shedding of neutrophil FcgRIIA (p < 0.0001), which was correlated with neutrophil activation (r = −0.73, p = 0.003) and the presence of anti-Sm/RNP antibodies (p < 0.001).

Conclusions Neutrophils are not terminally differentiated cells but could shift into phagocytic or NETosing cells, partly regulated by a cross-talk between TLR8 and FcgRIIA. SLE patients have ongoing shedding of neutrophil FcgRIIA related to neutrophil activation and anti-RNA antibodies, demonstrating the in vivo relevance of our observation. Therapeutic approaches aimed at degrading the TLR8 ligand would be predicted to increase uptake of circulating ICs, while disarming their inflammatory potential and ability to induce NETs.