Physiological concentrations of short-chain fatty acids induce the formation of neutrophil extracellular traps in vitro

Neutrophils represent the first line of host cellular defense against various pathogens. The most recently described microbicidal mechanism of these cells is the release of neutrophil extracellular traps (NET). Currently, a wide range of chemical and biological stimuli are known to induce this response; however, the effect of short-chain fatty acids (SCFAs) on the induction of NET is still unknown. SCFAs are produced mainly by bacterial fermentation of dietary fiber and are found in host tissues and blood. This study aimed to determine whether physiological levels of SCFAs can induce the formation of NET. Previously reported concentrations of SCFAs (as found in the colonic lumen and peripheral blood in postprandial and basal states) were used to stimulate the neutrophils. In order to determine the signaling pathway utilized by SCFAs, we tested the inhibition of the Free Fatty Acid 2 Receptor (FFA2R) expressed in neutrophils using CATPB, the inhibitor of FFA2R, genistein, an inhibitor of the downstream Gα/q11 proteins and DPI, an inhibitor of the NADPH oxidase complex. The SCFAs at colonic intestinal lumen concentrations were able to induce the formation of NET, and when tested at concentrations found in the peripheral blood, only acetic acid at 100 μM (fasting equivalent) and 700 μM (postprandial equivalent) was found to induce the formation of NET. The administration of the competitive inhibitor against the receptor or blockade of relevant G protein signaling and the inhibition of NADPH oxidase complex decreased NET release. SCFAs stimulate NET formation in vitro and this effect is mediated, in part, by the FFA2R.