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.

Piscirickettsia salmonis-Triggered Extracellular Traps Formation as an Innate Immune Response of Atlantic Salmon-Derived Polymorphonuclear Neutrophils

“…We observed an increase of cell free DNA (cf-DNA) after 10 and 50 µL of P. salmonis stimulation ( Figure 2 A). In addition, we used OA as positive control, which was previously described as a potent NETs inducer in bovine PMN [ 12 ]. To corroborate P. salmonis -induced ETs in Atlantic salmon-isolated PMN, we used a fluorescent dye for extracellular DNA staining (PicoGreen ® ), to visualize some thin extracellular web-like structures after either P. salmonis - or OA stimulation (white arrows in Figure 2 B).…”

“…P. salmonis -triggered ETs were quantified by measuring the amount of free extracellular DNA, which was stained by the cell-impermeant fluorescent dye PicoGreen ® (Invitrogen, Carlsbad, CA, USA), according to Muñoz-Caro et al [ 40 ] and Alarcón et al [ 12 ]. A total of 1 × 10 6 PMN/tube were incubated at 37 °C and then stimulated with different volumes of P. salmonis (10 and 50 µL), or 300 µM of oleic acid (OA) or vehicle (HBSS/calcium), for 60 min.…”

“…As stated before, major structural components of PMN-derived ETs are DNA, histones, and proteins from granules [neutrophil elastase (NE), myeloperoxidase (MPO), cathepsin G, lactoferrin, pentraxin, and gelatinase)] [ 5 , 11 ]. ETosis is induced by several chemical compounds such as the protein kinase C activator, phorbol myristate acetate (PMA), oleic acid (OA), hydrogen peroxide (H 2 O 2 ) from the respiratory burst, lipopolysaccharide (LPS), or bacteria [ 6 , 12 , 13 , 14 ]. This cellular process is an essential part of the mammalian inflammatory response but is also related to pathogenicity in some diseases, such as thrombosis, mastitis, arthritis, preeclampsia, appendicitis, cancer metastasis, and reproductive disorders [ 15 , 16 , 17 , 18 , 19 ].…”

Long Chain Fatty Acids as Modulators of Immune Cells Function: Contribution of FFA1 and FFA4 Receptors

“…Even though TAK-875 shows clinical efficacy in patients with type 2 diabetes, side effects, such as nasopharyngitis, upper respiratory tract inflammation, and liver toxicity, have been described (Kaku et al, 2015;Suckow and Briscoe, 2017). AMG837 (Amgen) is a β-substituted phenylpropanoic acid Hatanaka et al, 2006;Pereira et al, 2008;Carrillo et al, 2011;Hidalgo et al, 2011;Reyes-Quiroz et al, 2014;Manosalva et al, 2015;Mena et al, 2016;Medeiros-de-Moraes et al, 2018;Alarcon et al, 2020 ↑ M2 markers, NF-κB, apoE; ↓ iNOS, COX-2, TNF-α, IL1β, IL6, and apoptosis in macrophages. Huang et al, 2004;de Lima et al, 2006;Camell and Smith, 2013;Kim et al, 2017;Karasawa et al, 2018;Muller et al, 2019 Linoleic acid ↓ proliferation in T-cell; ↑ apoptosis in T-cell.…”

The Erythrocyte Membrane Lipidome of Healthy Dogs: Creating a Benchmark of Fatty Acid Distribution and Interval Values

“…The panel of 10 fatty acids represents a set of information, chosen for their biochemical, nutritional, and metabolic significance, and their reference ranges for healthy dogs can represent a benchmark to evaluate pathological conditions. It is worth to remark that the effects of fatty acids in cellular metabolism departing from the membrane release and remodeling processes are more and more studied also for immune functions ( 59 ); therefore, the knowledge of the membrane asset can be a useful indication for the examination of animal response at molecular level. Indeed, the protocol herein described, including calibration and quantitation procedures, can be applied to large animal populations, with molecular information that are ready to be combined with other medical data sets for mining techniques, which are nowadays under study for the exploitation of disease risks and prevention plans ( 60 ).…”