Store-operated Ca2+ entry-sensitive glycolysis regulates neutrophil adhesion and phagocytosis in dairy cows with subclinical hypocalcemia

Hypocalcemia in dairy cows is associated with a decrease of neutrophil adhesion and phagocytosis, an effect driven partly by changes in the expression of store-operated Ca²⁺ entry (SOCE)-related molecules. It is well-established in non-ruminants that neutrophils obtain the energy required for immune function through glycolysis. Whether glycolysis plays a role in the acquisition of energy by neutrophils during hypocalcemia in dairy cows is unknown. To address this relationship, we performed a cohort study and then a clinical trial. Neutrophils were isolated at 2 d postcalving from lactating Holstein dairy cows (average 2.83 ± 0.42 lactations, n = 6) diagnosed as clinically healthy (CON) or with plasma concentrations of Ca²⁺ < 2.0 mmol/L as a criterion for diagnosing subclinical hypocalcemia (HYP, average 2.83 ± 0.42 lactations, n = 6). In the first experiment, neutrophils were isolated from blood of CON and HYP cows and used to analyze aspects of adhesion and phagocytosis function through QRT-PCR along with confocal laser scanning microscopy, mRNA expression of the glycolysis-related gene Hexokinase 2 (HKII) and components of the SOCE moiety ORAI calcium release-activated calcium modulator 1 [ORAI1, ORAI2, ORAI3, stromal interaction molecule 1 (STIM1) and STIM2]. Results showed that adhesion and phagocytosis function were reduced in HYP cows. The mRNA expression of adhesion-related syndecan-4 (SDC4), Integrin β9 (ITGB9) and Integrin β3 (ITGB3) and phagocytosis-related molecules Complement Component 1 R subcomponent (C1R), CD36, tubulinß1 (TUBB1) were significantly decreased in the HYP group. In the second experiment, to address how glycolysis affects neutrophil adhesion and phagocytosis, neutrophils isolated from CON and HYP cows were treated with 2 μM of the HKII inhibitor Benserazide-d3 or 1 μM of the fructose-bisphosphatase 1 (FBP1) inhibitor MB05032 for 1 h. Results revealed that the HKII inhibitor Benserazide-d3 reduced phagocytosis and the mRNA abundance of ITGB9, and CD36 in the HYP group. The FBP1 inhibitor MB05032 increased adhesion and phagocytosis and increased mRNA abundance of HKII, ITGB9, and CD36 in the HYP group. Lastly, to investigate the mechanism whereby SOCE-sensitive glycolysis affects neutrophil adhesion and phagocytosis, isolated neutrophils were treated with 1 μM of the SOCE activator thapsigagin or 50 μM of the inhibitor 2-APB for 1 h. Results showed that thapsigargin increased mRNA abundance of HKII, ITGB9, and CD36, and increased adhesion and phagocytosis in the HYP group. In contrast, 2-APB decreased mRNA abundance of HKII and both adhesion and phagocytosis of neutrophils in the CON group. Overall, the data indicated that SOCE-sensitive intracellular Ca²⁺ levels affect glycolysis and help regulate adhesion and phagocytosis of neutrophils during hypocalcemia in dairy cows.

Glycolysis; adhesion; neutrophils; phagocytosis; subclinical hypocalcemia.