Bovine mastitis represents a major health and economic challenge in dairy production. Due to rising antibiotic resistance, research is increasingly focusing on understanding innate immunity and searching for alternative approaches for disease control. The immune response in the udder relies on mammary epithelial cells and immune cells, which recognize pathogen-associated molecular patterns, such as lipopolysaccharide (LPS), via TLR receptors. Activation of downstream signalling pathways triggers the release of inflammatory cytokines (TNF-α, IL-1β, IL-6) and the chemokine IL-8, leading to the recruitment of neutrophils to the site of infection and an increase in the somatic cell count in milk. Primary bovine mammary epithelial cells are widely used as in vitro models to study these mechanisms. Because excessive inflammation can cause significant damage to the glandular tissue, the immunomodulatory role of vitamin D (1,25(OH)₂D₃) is crucial, as it stimulates the synthesis of antimicrobial peptides and dampens the inflammatory response via the VDR receptor. In the experimental part of this study, we evaluated the effect of pretreatment with vitamin D at two different concentrations (10 nM and 100 nM) on the LPS induced inflammatory response in pbMEC. Using qPCR, we analysed changes in the differential gene expression of inflammatory genes (TNF α, IL 8, NF κB, MyD88, IL 6) and compared early (3 hours prior to RNA isolation) and late (24 hours prior to RNA isolation) immune responses. The results confirmed that LPS stimulation triggers an inflammatory response, whereas pretreatment with vitamin D demonstrated a potential immunomodulatory effect. We also demonstrated cell line specificity of the immune response, although a direct dependency on vitamin D concentration was not confirmed.
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