Heat Stress Small Extracellular Vesicles Impair PMNs' Immunity in Holstein and Brown Swiss Dairy Cows

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 (sEVs) play a crucial role in intercellular communication, transferring bioactive molecules, including proteins, lipids, metabolites, and nucleic acids. Environmental stressors can modify sEVs cargo, potentially altering cell function. The effects of sEVs isolated from the blood of heat-stressed cows on bovine polymorphonuclear cells (PMNs) remain unexplored. To investigate this, sEVs were isolated from the blood of Holstein (H) 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 sEVs 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-sEVs had no cytotoxic effects on PMNs, as viability remained unchanged. Chemotaxis was significantly reduced after challenge with sEV isolated from the 2 breeds, with a greater decline induced by Holstein-sEVs compared with Brown Swiss- sEVs. Challenging PMNs with HS-sEVs from both Holstein and Brown Swiss cows impaired phagocytosis; however, this inhibitory effect was more pronounced in PMNs treated with Holstein-derived HS-EVs. While Holstein-sEVs significantly suppressed ROS production, Brown Swiss-sEVs had no significant impact on PMN oxidative activity. These findings indicate that HS-sEVs alter immune cell function, suggesting a role for heat stress-induced sEVs in modulating bovine immunity. Our findings demonstrate for the first time that HS-sEVs impaired PMN activity. While both breeds' EVs impacted function, those from Holsteins more significantly diminished chemotaxis, phagocytosis, and ROS production compared with Brown Swiss-EVs in PMN isolated from Holstein cows.