Background/Purpose

Neutrophil extracellular traps (NETs), the extrusion of chromatin to capture microbes, has recently emerged as a possible mechanism that may increase the autoantigenic burden as well as  promote the prominent type I interferon (IFN) signature seen in most patients with systemic lupus erythematosus (SLE). Apparent spontaneous NETosis is observed ex vivo in low-density granulocytes, a cell population markedly increased in SLE. In vitro SLE RNA-containing immune complexes (RNA ICs) are reported to induce NETs from neutrophils obtained from SLE, but not healthy individuals. Since the precise requirements for RNA ICs and neutrophils in the induction of NETs have not been defined, we investigated the requirements for IC-induced NETosis in normal neutrophils, analyzed the crosstalk between FcgRs and TLRs, and characterized the released DNA material with regard to its source and oxidation.

Methods

Neutrophils, isolated from healthy individuals, were stimulated with RNA ICs, and DNA release and 8-OHdG content, analyzed by fluorescence microscopy and ELISA. The origin of the released DNA was assessed by analyzing 16S (mitochondria) and 18S (chromosomal) expression by qPCR. Phagocytosis was measured by flow cytometry.

Results

Immobilized IgG mimicking tissue-deposited ICs readily induced NETosis in neutrophils in a NADPH oxidase- and PAD4-dependent, but TLR-independent manner. In contrast, soluble endotoxin-free aggregated IgG (HAGG) did not induce NETosis whereas soluble RNA ICs were potent inducers of NETs. This process was dependent on TLR activation since addition of RNases efficiently degraded the RNA contained within the IC, and completely abrogated the ability of soluble RNA ICs to induce NETosis. Despite the fact that degradation of RNA inhibited NETosis, removal of the TLR ligand by RNase markedly increased the phagocytosis of RNA ICs by neutrophils suggesting that TLR activation suppressed phagocytosis. Consistent with this hypothesis, addition of a TLR agonist (R848) inhibited phagocytosis of ICs, but not beads, in neutrophils. Flow cytometric analysis revealed that the underlying mechanism involved rapid down-regulation of FcgRIIA on the neutrophil cell surface upon TLR activation, which reduced its phagocytic capacity and promoted progression into NETosis. The released DNA from RNA IC-activated neutrophils had high 8-OHdG content and was mainly of chromosomal origin, although some mitochondrial DNA was present as well. The chromosomal DNA was enriched for MPO, whereas the mitochondrial DNA was enriched for 8-OHdG oxidation.

Conclusion

i) Extensive aggregation of FcgR on neutrophils, as may occur in tissue deposits, appears sufficient to induce NETosis; ii) In the fluid phase, both FcgR aggregation and TLR ligation are necessary to stimulate NETosis; iii) TLR-ligand activation in neutrophils downregulates FcgRIIA expression thereby inhibiting further phagocytosis of ICs but enabling NETosis; iv) ICs induce release of oxidized DNA of chromosomal and mitochondrial origin. Deciphering the underlying signaling pathways regulating the crosstalk between FcgRs and TLRs in induction of NETosis may provide novel therapeutic targets.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rna-containing-immune-complexes-shift-human-neutrophils-from-phagocytosing-cells-to-efficient-releasers-of-oxidized-dna-in-a-process-requiring-crosstalk-between-toll-like-receptors-and-fc-ga/