Functional and transcriptomic profiling of bovine circulating neutrophils during in vitro lipopolysaccharide challenge across different energetic states.

Polymorphonuclear leukocytes are pivotal players in the innate immune response of dairy cows. Their functionality (phagocytosis [PC] and oxidative burst [OB]) and gene expression patterns, however, can be modulated by varying energetic conditions. Energetic fluctuations are particularly evident in periparturient dairy cows, when distinct levels of negative energy balance can trigger PMN dysfunction. This study aimed to assess how different energetic conditions affect PMN functionality and to examine their transcriptomic profile following an in vitro LPS challenge. Over 4 biological replicates, circulating PMN isolated from 4 pubertal nulliparous heifers were suspended in cell culture medium and supplemented with glucose (1.5 and 3 mM to mimic hypo- and normoglycemia, respectively) and with 1 mM nonesterified fatty acids (NEFA). Control PMN suspensions with no glucose or NEFA supplementation were incubated alongside. After 3 h of incubation under the aforementioned conditions, PMN were challenged with 50 μg/mL LPS for 1 h to mimic infection. At 0 and 3 h of incubation, the PMN viability (viable, apoptotic, or necrotic) and functionalities (PC and OB) were measured via flow cytometry. After the LPS challenge, the total RNA of PMN was extracted and sequenced on an Illumina platform. The effects of glucose and NEFA in the culture medium on PMN viability and functional parameters were fitted in mixed linear regression models, using the replicate as a random effect. The analyses of differentially expressed genes (DEG) were performed by edgeR. Regardless of NEFA, supplementation with 3 mM glucose increased PMN OB compared with PMN without glucose and NEFA supplementation. Transcriptomic analysis showed that PMN exposed to 3 mM glucose plus NEFA exhibited upregulation of oxidative e phosphorylation and reactive oxygen species pathways compared with 3 mM glucose alone (no-NEFA), suggesting that NEFA promotes an excessive PMN response. Under NEFA supplementation, PMN exposed to 1.5 mM glucose showed downregulation of energy metabolism and immune signaling pathways when compared with 3 mM glucose, indicating immune dysfunction related to metabolic stress (presence of NEFA) when glucose is low. These results provide insights into how different metabolic conditions during the transition period induce innate immune (dys)function in dairy cows.