Oleic and Linoleic Acids Induce the Release of Neutrophil Extracellular Traps via Pannexin 1-Dependent ATP Release and P2X1 Receptor Activation

Abstract

Non-esterified fatty acids (NEFAs) such as oleic acid (OA) and linoleic acid (LA) are associated with a higher incidence of infectious diseases such as metritis and mastitis during the bovine peripartum. Fatty acids can induce an increase in the release of ATP, and changes in the expression levels of purinergic receptors in bovine polymorphonuclears (PMN) during peripartum have also been reported. PMN respond to inflammatory processes with production of ROS, release of proteolytic and bactericidal proteins, and formation of neutrophil extracellular traps (NETs). NETs formation is known to require ATP production through glycolysis. Studies have shown that the above-mentioned metabolic changes alter innate immune responses, particularly in PMN. We hypothesized that NEFAs induce the formation of NETs through ATP release by Pannexin 1 and activation of purinergic receptors. In this study, we found that OA and LA induce NET formation and extracellular ATP release. Carbenoxolone, a pannexin-1 (PANX1) inhibitor, reduced OA- and LA-induced ATP release. We also found that P2X1, P2X4, P2X5, P2X7, and PANX1 were expressed at the mRNA level in bovine PMN. Additionally, NEFA-induced NET formation was completely abolished with exposure to NF449, a P2X1 antagonist, and partially inhibited by treatment with etomoxir, an inhibitor of fatty acid oxidation (FAO). Our results suggest that OA and LA induce NET formation and ATP release via PANX1 and activation of P2X1. These new data contribute to explaining the effects of NEFA high concentrations during the transition period of dairy cattle and further understanding of pro-inflammatory effects and outcome of postpartum diseases.

Keywords: ATP; PMN; neutrophil extracellular trap; non-esterified fatty acids; purinergic receptor.

Figure 1

Oleic acid (OA) and linoleic acid (LA) induce neutrophil extracellular trap (NET) formation independently of FFAR1 and NADPH oxidase activation. Bar graph (means ± S.E.M.) showing the relative fluorescence (RFU) of cell-free (cf)-DNA obtained from PMN treated with different concentrations of OA (A) or LA (B). Immunofluorescence of PMN treated with a 300 μM concentration of OA (C) or LA (D) using an anti-histone H4 citrulline 3 antibody as a NET marker and Sytox orange as a DNA marker. Images are representative of four independent experiments; scale bar = 20 μM. Bar graph of RFU (means ± S.E.M.) of cf-DNA obtained from PMN treated with GW1100 (FFAR1 antagonist) (E,F) or diphenyleneiodonium (DPI) (G,H) for 15 min and then stimulated for 30 min with OA or LA. n = 4; ***p < 0.001, ****p < 0.0001 compared with vehicle controls.

Figure 2

Oleic acid (OA) and linoleic acid (LA) induce ATP release. Levels of released ATP in supernatants of PMN treated with a 200 μM concentration of OA (A) or LA (B) for 0, 15, 30, and 60 s. The data were normalized using the mean of 60 s of measurement of control group. n = 4; *p < 0.05, **p < 0.01, ***p < 0.001 compared with vehicle controls.

Figure 3

Non-esterified fatty acid (NEFA)-induced ATP release is mediated via PANX1. Relative expression of PANX1 to RPS9 (housekeeping) in bovine PMN assessed by RT-qPCR (A), n = 4. Gel of mRNA amplicons of PANX1, GJA1, and RPS9 (B). N.D.: not detected. bp: base pair. RT+, RT–: reverse transcriptase +, reverse transcriptase -. Bar graph showing ATP release from PMN treated with 10 μM carbenoxolone (CBX) for 15 min and then stimulated with oleic acid (OA) (C) or linoleic acid (LA) (D) for 30 min. n = 4; *p < 0.05, ***p < 0.001, ****p < 0.0001 compared with vehicle controls.

Figure 4

Oleic acid (OA) and linoleic acid (LA) induce neutrophil extracellular trap (NET) formation via PANX1-mediated ATP release. Bar graph (means ± S.E.M.) of cell-free (cf)-DNA of PMN treated with 10 μM carbenoxolone (CBX) for 15 min and stimulated or not with OA (A) or LA (B). n = 4; **p < 0.01, ***p < 0.001 compared with OA or LA treatment alone. Representative image from four independent experiments of PMN treated with CBX and stimulated with OA or LA (C).

Figure 5

mRNA expression of the P2X receptor in bovine PMN. Relative expression of P2X1–7 to RPS9 (housekeeping) in bovine PMN (A). Representative image of PCR amplicons of P2X receptors (B). N.D., not detected.; bp, base pair; ST, standard of bp; RT+, RT–, reverse transcriptase +, reverse transcriptase –. N = 4.

Figure 6

P2X1 is involved in oleic acid (OA)-induced neutrophil extracellular trap (NET) formation. Bar graph (means ± S.E.M.) of cf-DNA of PMN treated with 1 μM NF449 for 15 min and stimulated with OA (A) or linoleic acid (LA) (B). ****p < 0.0001 compared with OA treatment alone. Representative images from four independent experiments of PMN treated with NF449 and stimulated with OA (C) or LA (C). n = 4.

Figure 7

Etomoxir partially blocks non-esterified fatty acid (NEFA)-induced neutrophil extracellular trap (NET) formation. Bar graph (means ± S.E.M.) of cell-free (cf)-DNA of PMN treated with 10 μM etomoxir for 60 min and stimulated with oleic acid (OA) (A) or linoleic acid (LA) (B). Representative image from four independent experiments of PMN treated with etomoxir and stimulated with OA or LA (C). n = 4; **p < 0.01 compared with OA or LA treatment alone.