PSVI-6 Effect of cow neutrophil activation on expression of T cell immunoglobulin and Mucin-Domain-Containing-Protein 3 (TIM-3) and its ligand Galectin 9 gene

The objective of this study was to assess the effect of neutrophil activation on the expression of T cell immunoglobulin and mucin domain-containing protein-3 (TIM-3) and its ligand Galectin 9. T cell immunoglobulin and Mucin-Domain-Containing-Protein 3 is a type I transmembrane protein involved in the regulation of cell-mediated immunity. Galectin-9 is a β-galactoside-binding lectin, that can function as an alarmin in response to diverse pathogens. Binding of Gal-9 to TIM-3 and stimulation with phorbol 12-myristate 13-acetate (PMA) induced activation of NADPH oxidase on human neutrophils. Increased understanding of Gal-9 to TIM-3 gene expression and modulation in cow neutrophils is needed. Blood was collected from the jugular vein of clinically healthy Holstein-Friesian cows (n = 3) from North Carolina A&T dairy unit. Neutrophils were isolated by differential centrifugation and incubated with either 10ng/ml of PMA or maintained in Phosphate buffer saline for 30 minutes at 370 C. Total RNA was isolated from neutrophils reverse transcribed to cDNA. Specific primers for TIM-3 and LGALS -9 were designed using the NCBI Primer 3 tool. Pooled cDNA samples were used for real-time PCR. Housekeeping genes RPLP0 and UCHL5 served as internal controls. Fold change in transcript abundance was calculated using the Livak method (Fold change >2 were considered as significant). Both LGAS9 and TIM-3 were expressed in resting neutrophils. Treatment with PMA upregulated transcription of TIM-3 (5.55 fold). Whereas, LGALS -9 transcription remained unchanged. Activation of cow neutrophils using PMA changes in the expression of TIM-3. Further studies are needed to define the signaling pathways involved and functional implications for animal health and production.