Background/Purpose:  Neutrophils participate in host defense 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 kill 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 signaling in neutrophil phagocytosis of RNA-containing 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.

Methods:  Neutrophils, isolated from healthy individuals, were incubated with RNA-ICs and analyzed for phagocytosis and NETosis by flow cytometry and fluorimetry, respectively, in the presence of FcgR blocking antibodies or TLR8 inhibitors (oligonucleotides, RNase). FcgRIIA cleavage on neutrophils was assessed on ex vivo isolated neutrophils from healthy controls (n=7) and SLE patients (n=19) as well upon serum incubation, using two antibody clones recognizing full length or cleaved 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. While degradation of RNA inhibited NETosis as expected, surprisingly, 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 of latex beads, by 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. Interestingly, the opposite was also true, namely that phagocytic stimuli (beads) could suppress IC-mediated NETosis (p<0.01) while promoting phagocytosis (p<0.05), suggesting plasticity of the neutrophils in adapting into specific effector cell functions. Importantly, ex vivo isolated neutrophils from SLE patients demonstrated increased shedding of FcgRIIA (p<0.0001), which correlated with neutrophil activation (r=-0.73, p=0.003) and the presence of anti-Sm/RNP antibodies (p<0.001), consistent with the in vitro data.

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

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/immune-complex-mediated-tlr8-activation-shifts-neutrophils-from-phagocytosis-to-netosis-through-furin-dependent-shedding-of-fcgriia/