Neutrophils are usually the first cells recruited to sites of injury or infection, where they exert several effector functions to protect the host, including the release of NETs. These extracellular webs of decondensed chromatin adorned with antimicrobial molecules can trap and inactivate pathogens. The presence of injury or infection can be detected via Toll-like receptors (TLRs). Neutrophils express TLR1, TLR2, TLR4, TLR5, and TLR6 on their surface, as well as TLR7, TLR8, and TLR9 on their endosomes. Here we dissect which TLRs can promote the formation of NETs using ultrapure ligands. We found that in humans NETs are only induced following TLR2 ligation, whereas TLR4, TLR5, TLR7, TLR8, and TLR9 engagement fail to do so (but do stimulate other cellular responses as expected). A widely used (but only partially purified) lipopolysaccharide preparation induced NET formation by binding TLR2 (and possibly other surface receptors), confirming data from several other groups who used the same LPS preparation and concluded that TLR4 ligation promotes NET generation by human neutrophils. By contrast, murine NETs are formed upon both TLR2 and TLR4 engagement using ultrapure ligands. We next investigated the molecules downstream of TLR2 involved in NET production in humans. Similar to other classes of physiological NET inducers, TLR2 ligand-triggered NET formation is controlled by signaling kinases (TAK1, MEK, p38 MAPK) mobilized early during this response, while Syk, PI3K, and PLCγ2 acted belatedly (+2h after stimulation). Likewise, endogenous factors are released during TLR2-induced NET formation and feed back to bind the RAGE receptor, thereby sustaining the NET response. Finally, PAD4 proves to be essential to NET production following TLR2 ligation, while elastase appears to be dispensable. Unlike other physiological NET inducers, TLR2-induced NET formation partially relies on reactive oxygen species production. Our study shows the surprisingly restricted repertoire of TLRs that can elicit NET formation in humans and illustrates how this emblematic neutrophil response differs between humans and rodents.

Heart failure with reduced (HFrEF) or preserved ejection fraction (HFpEF) is characterized by low-grade chronic inflammation. Circulating neutrophils regroup two subtypes termed high- and low-density neutrophils (HDNs and LDNs). LDNs represent less than 2% of total neutrophil under physiological conditions, but their count increase in multiple pathologies, releasing more inflammatory cytokines and neutrophil extracellular traps (NETs). We wanted to assess the differential count and role of HDNs, LDNs and NETs-related activities in HF patients. HDNs and LDNs were isolated from human blood by density gradient and purified by FACS and their counts obtained by flow cytometry. NETs formation (NETosis) was quantified by confocal microscopy. Circulating inflammatory and NETosis biomarkers were measured by ELISA. Neutrophil adhesion onto human extracellular matrix (hECM) was assessed by optical microscopy. A total of 140 individuals were enrolled, including 33 healthy volunteers (HV), 41 HFrEF (19 stable patients and 22 presenting acute decompensated HF; ADHF) and 66 HFpEF patients (36 stable patients and 30 presenting HF decompensation). HDNs and LDNs counts were significantly increased up to 39% and 2740% respectively in HF patients compared to HV. In HF patients, the correlations between LDNs counts and circulating inflammatory (CRP, IL-6 and -8), Troponin T, NT-proBNP and NETosis components were all significant. In vitro, LDNs expressed more H3Cit and NETs and were more pro-adhesive, with ADHFpEF patients presenting the highest pro-inflammatory profile. In conclusion, HFpEF patients present higher levels of circulating LDNs and NETs related activities, which are the highest in the context of acute HF decompensation.