Histamine promotes adhesion of neutrophils by inhibition of autophagy in dairy cows with subacute ruminal acidosis.

Subacute ruminal acidosis (SARA), a common digestive disease in dairy cows, is accompanied by systemic inflammation and high concentrations of histamine in blood. Histamine-induced neutrophil adhesion may play an important role in the systemic inflammation experienced by cows during SARA. Autophagy, an intracellular degradation system, regulates recycling of membrane-associated integrin and may be involved in histamine-induced adhesion of bovine neutrophils. In the present study, 20 multiparous mid-lactation cows (average body weight 486 ± 24 kg) fitted with ruminal fistula were assigned to a control group (n = 10) or a SARA group (n = 10). We induced SARA by feeding different combinations of wheat-barley pellets and chopped alfalfa hay for 8 wk; SARA was defined as a ruminal pH <5.6 for at least 3 h/d. Blood samples were collected in wk 8. Compared with controls, SARA cows had greater serum concentrations of tumor necrosis factor-α, IL-6, IL-1ß, lipopolysaccharide (LPS)-binding protein, haptoglobin, and serum amyloid A. Serum concentrations of these proinflammatory factors had strong positive correlations with the concentration of serum histamine and LPS. In ex vivo adhesion experiments, the number of adherent neutrophils was greater in the SARA group. Additionally, membrane protein abundance of adhesion molecules such as integrin α-L precursor (CD11a) and integrin α-M precursor (CD11b) was greater in neutrophils of the SARA group, confirming enhanced adhesion ability. Neutrophils of SARA cows had greater number of autophagosomes, greater protein abundance of autophagy substrate sequestosome-1 (p62), and higher ratio of microtubule associated proteins 1A/1B light chain 3 (LC3)-II to LC3-I, indicating congestion during the late phase of autophagy flux. For in vitro experiments, neutrophils isolated from control cows were incubated with 0.4 endotoxin units (EU)/mL LPS or 7 µM histamine for 0, 1, 2, and 4 h, respectively. We detected linear and quadratic effects for the number of adherent neutrophils after histamine treatment with a peak response at 2 h, whereas no significant effect was detected after LPS treatment. Membrane protein abundance of CD11a and CD11b was greater after histamine treatment, suggesting that it may have an inhibitory effect on the degradation of adhesion molecules. The greater abundance of p62, higher ratio of LC3-II to LC3-I, and increased co-localization between CD11b and LC3 after histamine treatment indicated that recycling of adhesion molecules and autophagy flux were blocked. These effects were not aggravated further in the presence of chloroquine, a specific inhibitor of the late phase of autophagy flux. Overall, our results revealed that histamine increases adhesion of neutrophils by inhibiting autophagy in dairy cows with SARA.

Palmitic acid hinders extracellular traps of neutrophil from postpartum dairy cow in vitro.

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, ß-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.