Heat-stressed Holstein and Brown Swiss cows' small extracellular vesicles impair polymorphonuclear leukocyte immunity

Climate change and rising global temperatures pose significant challenges to dairy cattle, particularly through heat stress (HS), which compromises immune function and overall productivity. Small extracellular vesicles (sEV) play a crucial role in intercellular communication by transferring bioactive molecules, including proteins, lipids, metabolites, and nucleic acids. Environmental stressors can modify sEV cargo, potentially altering cell function. The effects of sEV isolated from the blood of heat-stressed cows on bovine PMN cells remain unexplored. To investigate this, sEV were isolated from the blood of Holstein (HO) and Brown Swiss (BS) lactating cows fed the same diet and kept in the same barn, under thermoneutral (NT) and HS conditions during a 4-d natural heat wave. Their effects on the viability, chemotaxis, phagocytosis, and reactive oxygen species (ROS) production of PMN isolated from Holstein cows in NT were evaluated. Due to the lack of standardized sEV concentrations in bovine immunology, a range of sEV protein concentrations from 1 to 625 µg/mL was initially tested, with 5 µg/mL selected for further assays. Blood HO sEV at high doses exerted a cytotoxic effect on PMN, whereas BS sEV did not affect viability. Chemotaxis was significantly reduced after challenge with sEV isolated from the 2 breeds, with a greater decline induced by HO sEV compared with BS sEV. Challenging PMN with HS sEV from both HO and BS cows impaired phagocytosis; however, this inhibitory effect was more pronounced in PMN treated with HO-derived HS sEV. Although HO sEV significantly suppressed ROS production, BS sEV had no significant effect on PMN oxidative activity. These findings indicate that HS sEV alter immune cell function, suggesting a role for heat stress-induced sEV in modulating bovine immunity. Our findings demonstrate for the first time that HS sEV impaired PMN activity. Although both breeds' sEV affected function, those from HO more significantly diminished chemotaxis, phagocytosis, and ROS production compared with BS sEV in PMN isolated from Holstein cows.