Differential expression of TLR and CXCR genes in mammary HC11 cells challenged with Bacillus cereus and Bacillus subtilis: Implications for mastitis resistance.

Background and aim: Mastitis remains a major health challenge in dairy cattle, often caused by Gram-positive pathogens. Toll-like receptors (TLRs) and chemokine receptors (CXCRs) play essential roles in the innate immune response of mammary epithelial cells (MECs). However, the differential expression of these genes in response to specific mastitis-causing Bacillus spp. has not been comprehensively evaluated. This study aimed to characterize the temporal gene expression patterns of TLR and CXCR family members in murine mammary epithelial HC11 cells exposed to Bacillus cereus and Bacillus subtilis, thereby providing insights into their immunological roles in mastitis pathogenesis.

Materials and methods: HC11 cells were cultured and infected with B. cereus and B. subtilis (5 × 10⁷ colony-forming units/mL) and incubated at 37°C with 95% O₂ and 5% CO₂ for 48 h in RPMI 1640 medium supplemented with serum and antibiotics. Gene expression of interleukin (IL)-6, IL-8, TLR2, TLR4, IL-1 alpha (IL-1α), and CXCR1 was evaluated by quantitative real-time polymerase chain reaction at 0, 6, 12, 24, 48, and 72 h post-infection. Expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase and analyzed using ΔCt methods and Spearman correlation.

Results: TLR2 exhibited a biphasic expression pattern, with early upregulation followed by suppression, while TLR4 showed higher expression in response to B. subtilis than B. cereus. IL-6 displayed prolonged expression under B. subtilis challenge but was transient under B. cereus exposure. IL-1α showed consistent expression across both bacterial challenges, suggesting its potential as a stable biomarker for mastitis susceptibility. CXCR1 exhibited delayed but sustained expression, indicative of its role in secondary neutrophil recruitment. IL-8 expression correlated with early immune activation and chemotactic signaling.

Conclusion: The immune response of HC11 MECs to Gram-positive bacterial infection is gene- and pathogen-specific. TLR and CXCR genes show distinct temporal profiles, underscoring their utility in understanding epithelial-driven immune defense. These findings provide molecular insights into mastitis pathogenesis and identify IL-1α, IL-6, and CXCR1 as promising targets for genetic selection and therapeutic intervention.