A feedback loop sustaining neutrophil extracellular trap formation involves S100 proteins, histones, TLR2 and RAGE, and is restrained by albumin.

Neutrophil extracellular trap (NET) generation must be tightly controlled as this essential antimicrobial response can also cause tissue damage and contribute to various pathologies. We previously described that neutrophils undergoing NET formation release endogenous mediators that feedback on the cells via the RAGE receptor to drive the response. We now identify S100 proteins and histones as prominent endogenous NET inducers. Mass spectrometry analyses of culture supernatant from NETing neutrophils revealed the presence of seven species of S100 proteins and ten histone variants, all of which could bind to RAGE. In addition, endogenous NET inducers were found to act via TLR2 since antagonism of the receptor for up to 3 h post-stimulation hindered NET formation elicited by various classes of physiological stimuli. By comparison, antagonism of TLR4 did not inhibit NET generation. Conversely, we show that serum or equivalent albumin concentrations restrain NET formation by sequestering endogenous NET inducers. Serum depleted of albumin indeed loses its ability to prevent NET formation; likewise, the belated addition of HSA to culture medium blocks NET generation. Prior exposure to serum or albumin renders endogenous NET inducers unable to trigger the response; mass spectrometry analyses confirmed that albumin captures S100 proteins. Collectively, our data unveil a feedback loop of NET formation involving endogenous S100 proteins and histones, acting through RAGE and/or TLR2, that can be physiologically counteracted by albumin.