Loss of IL10RA rewires epithelial immune responses to intracellular Staphylococcus aureus small colony variants in bovine mammary epithelial cells

Introduction Staphylococcus aureus small colony variants (SCVs) represent a persistence-adapted phenotype linked to chronic and recurrent bovine mastitis. Interleukin-10 receptor ⍺ (IL10RA) is a central regulator of anti-inflammatory signalling in the mammary gland, but its role in shaping transcriptional responses to intracellular staphylococcal challenge has not been characterized. Methods CRISPR/Cas9-generated IL10RA knockout (KO) MAC-T bovine mammary epithelial cells (MECs), alongside wild-type (WT) controls, were infected with either S. aureus SCV strain Heba3231 or its isogenic parental strain (PS). Transcriptional responses were profiled by RNA sequencing and compared across genotype and infection conditions. Results WT cells mounted markedly divergent responses to PS (8 DEGs) versus SCV (461 DEGs) infection, with no overlap between the two gene sets, indicating strain-specific engagement of distinct molecular programmes. SCV infection of WT cells up-regulated lipid and sterol biosynthetic pathways while suppressing epithelial barrier integrity genes ( S100A8 , S100A9 , TGM3 , KRTDAP ). IL10RA disruption substantially amplified transcriptional dysregulation: IL10RA -KO cells infected with PS yielded 619 DEGs (a 77.4-fold increase over WT-PS), while IL10RA -KO cells infected with SCV yielded 1,297 DEGs (a 2.8-fold increase over WT-SCV). Approximately 32 to 36% of IL10RA-regulated genes formed a shared core across infection conditions. Loss of IL10RA derepressed pro-inflammatory cytokine pathways (TNF, IL-17), enhanced pro-apoptotic programmes during SCV infection, and suppressed phagosomal maturation and lipid metabolic gene programmes. Candidate IL10RA-responsive genes, including CD79B, WFDC2, AMN and LY6E , were identified as targets for further validation. Discussion These findings establish IL10RA as a broad transcriptional homeostasis regulator in bovine MECs during staphylococcal infection, with disproportionate effects on inflammatory and barrier-related gene programmes under SCV challenge. The identified candidate genes warrant validation in primary bovine mammary cells, milk somatic cells, or clinical mastitis samples to confirm their relevance to chronic and recurrent disease pathogenesis.