LAUSR

Peripartum dairy cows experience negative energy balance, characterized by high concentrations of blood free fatty acids (FFA) and immune dysfunction. Palmitic acid (PA), the most abundant saturated fatty acid in cow blood, is not only an energy precursor, but causes cellular dysfunction when in excess. Neutrophil extracellular traps (NET) are one of the arsenals of weapons neutrophils use to fight invading pathogens. However, given the marked increase in circulating PA during the peripartum period, it remains to be determined what effect (if any) PA has on NET release. Thus, the objective of this study was to evaluate the effect of PA on NET release and the underlying mechanism in vitro. Phorbol-12-myristate-13-acetate (PMA; 100 ng/mL, 3 h) was used to induce the release of NET in vitro. We isolated neutrophils from the peripheral blood of 5 healthy postpartum dairy cows with similar parity (median = 3, range = 2-4), milk yield (median = 27.84 kg/d per cow, range = 25.79-31.43 kg/d per cow), days in milk (median = 7 d, range = 4-10 d), and serum FFA <0.25 mM, β-hydroxybutyric acid <0.6 mM, and glucose >3.5 mM. Inhibition of double-stranded DNA (dsDNA) level, a marker of NET release, in response to PA was used to determine an optimal incubation time and concentration for in vitro experiments. Cells were maintained in RPMI-1640 basic medium without phenol red, treated with 600 μM PA for different times (4, 5, 6, and 7 h) in the presence or absence of PMA. There was a decrease for dsDNA level in the supernatant due to increased duration of PA treatment, with a peak response at 6 h. Thus, 6 h was selected as the challenge time. Then, cells were treated with different concentrations of PA (100, 200, 400, and 600 μM) for 6 h in the presence or absence of PMA. There was a decrease for dsDNA level in the supernatant due to increased dose of PA, with a peak response at 400 μM. Finally, 400 μM PA for 6 h was selected as the treatment for subsequent experiments. Protein abundance of citrullinated histone in the presence or absence of PMA was markedly lower in response to incubation with PA. Morphological observations by laser confocal microscopy and scanning electron microscopy showed that the ratio of NET-releasing cells decreased in response to incubation with PA. Autophagy is a potential key intermediate process in the regulation of NET by PA. To investigate the effect of PA on autophagy, we used chloroquine to block lysosomal degradation. Exogenous PA led to accumulation of sequestosome-1 and microtubule-associated protein 1 light chain 3-II, and no further accumulation in the presence of chloroquine, all of which suggested an impairment of autophagic flux. To verify the role of autophagy in NET, we used rapamycin to promote autophagic flux; 100 nM rapamycin attenuated the suppressive effect of PA on NET release indicated by greater dsDNA levels, accumulation of citrullinated histone, and ratio of NET-releasing neutrophils. Overall, these data demonstrate PA inhibits NET release by suppressing autophagic flux, which provides information for understanding the immune dysfunction in postpartum cows.