Fatty acids promote migration of CD4⁺ T cells through calcium release-activated calcium modulator ORAI1 sensitive glycolysis in dairy cows

Nutritional and metabolic state in dairy cows are important determinants of the immune response. During the periparturient period, a state of negative energy balance in the cow increases plasma concentrations of fatty acids (FA), which are associated with inflammation. Among immune cells, CD4⁺ T are able to function under high-FA conditions, but the underlying mechanisms regulating these events remain unclear. The objective of this study was to clarify the functional mechanisms of CD4⁺ T cells under high-FA conditions. The effects of glycolysis and calcium release-activated calcium modulator 1 (ORAI1) on migration of CD4⁺ T cells exposed to high FA were investigated in vivo and in vitro. The CD4⁺ T cells were isolated from peripheral blood of healthy (n = 9) and high-FA (n = 9) Holstein cows (average 2.5 ± 0.2 lactations [SE], 12.3 ± 0.8 DIM). In the first experiment, real-time quantitative PCR was used to assess chemokine receptors in isolated CD4⁺ T cells and migration capacity. The relative mRNA measurements results revealed downregulation of CCR1 and CXCR2, and upregulation of CCR2, CCR4, CCR5, CCR7, CCR8, CCR10, CXCR1, CXCR3, CXCR4, and CX3CR1. Among them, the expression of CXCR4 was relatively high. Therefore, CXCL12, a ligand chemokine of CXCR4, was an inducer of CD4⁺ T cell migration. The CD4⁺ T cells were inoculated in the upper chamber and CXCL12 (100 ng/mL, Peprotech) in RPMI1640 was added to the lower chamber and transmigrated for 3 h at 37°C and 5% CO₂. The cell migration assay revealed that the migration capacity of CD4⁺ T cells from high-FA cows was greater. Real-time-qPCR indicated greater abundance of the glycolysis-related targets HIF1A, HK2, PKM2, Glut1, GAPDH, and LDHA and Western blotting indicated greater abundance of the glycolysis-related targets HIF1A, HK2, PKM2, Glut1, GAPDH, and LDHA in CD4⁺ T cells of high-FA cows. To characterize specific mechanisms of CD4⁺ T cell migration in vitro, cells from the spleens of 3 newborn (1 d old, 40–50 kg) healthy female Holstein calves were isolated after euthanasia. Inhibition of glycolysis attenuated the migration ability of cells, but had no effect on the protein and mRNA abundance of store-operated Ca²⁺ entry (SOCE)-associated calcium release-activated calcium modulator 1 (ORAI1) and stromal interaction molecule 1 (STIM1). In contrast, ORAI1 was upregulated in CD4⁺ T cells of cows exposed to high FA. To explore the potential mechanisms whereby an active glycolytic metabolism affects CD4⁺ T cells under high-FA conditions, we knocked down ORAI1 using small interfering RNA (siORAI1). Isolated CD4⁺ T cells from high-FA cows with the siORAI1 had an attenuated glycolytic metabolism and migration capacity. Taken together, these data suggested that calcium ions in CD4⁺ T cells from cows with high FA regulate glycolytic metabolism and influence cell migration at least in part by modulating ORAI1. Thus, these studies identified a novel mechanism of Ca²⁺ regulation of CD4⁺ T cell glycolytic metabolism affecting their migration through the SOCE pathway.