Ligation of TLR2, but not of TLR4 or other Toll-like receptors, induces neutrophil extracellular trap formation in humans

Neutrophils are usually the first cells recruited to sites of injury or infection where they mount antimicrobial responses, including the release of neutrophil extracellular traps (NETs). Toll-like receptors play a major role in the recognition of pathogen-associated molecular patterns and all but TLR3 are expressed in neutrophils. Numerous studies have reported that LPS can trigger NET formation; in nearly all cases, however, the ligand was not purified enough to target only TLR4. There also exist isolated reports on the ability of other TLRs to induce NETs. Here we comprehensively revisited the issue of TLR-elicited NET generation using ultrapure ligands. We now report that in humans, NETs are only induced following TLR2 ligation whereas engagement of other TLRs is ineffective (despite eliciting other cellular responses). A widely used (but incompletely purified) LPS preparation potently induced NET generation by binding TLR4, TLR2, and possibly other receptors, confirming previous data from other groups. By contrast, murine NETs are formed upon either TLR2 or TLR4 engagement. Mechanistically, TLR2-triggered NET formation is controlled by signaling kinases that are mobilized early (TAK1, MEK, p38 MAPK) or belatedly (Syk, PI3K, PLCγ2); acts through endogenous factors that bind the RAGE receptor; and involves PAD4 as well as endogenous reactive oxygen species, whereas elastase is dispensable. Conversely, we provide evidence that TLR4 negatively regulates NET formation in humans. Our study shows the surprisingly restricted repertoire of TLRs that can elicit NET formation in humans, and further illustrates how this emblematic neutrophil response differs between humans and rodents.