Construction of a keratinocyte–<i>staphylococci</i> direct contact coculture model for analysis of inflammatory and defense responses regulated by bacterial ratio

Common skin microbiota contribute to host defense and inflammation control, with their effects depending on bacterial species and load along with cell-to-bacteria ratio. However, studies comparing these effects across species in a ratio-dependent manner under identical conditions are limited. This study aimed to establish a HaCaT–bacteria coculture system operable without CO2 and systematically evaluate the effects of Staphylococcus aureus, S. epidermidis, and S. capitis on inflammatory cytokine and antimicrobial peptide expression in keratinocytes under varying cell-to-bacteria ratios. HaCaT cells were cultured in five different media formulations without CO2 and with strains (ratio range: 1:0.01 to 1:3000), followed by viability assessment. Inflammatory markers were analyzed by quantitative reverse transcription-PCR, with 18S rRNA as housekeeping gene. Notably, the HaCaT cell viability reached approximately 80% without CO2 using 50:50 DMEM–Leibovitz's L-15 medium. S. aureus elevated inflammatory cytokine levels at cell:bacteria ratios of 1:30 and higher and triggered strong inflammation at ratios of 1:300–3000, with significant antimicrobial peptide upregulation. S. epidermidis showed protective effects at low to intermediate ratios (1:0.01–1) but increased inflammatory cytokines at 1:10 and above. S. capitis induced inflammation at low ratios (1:0.15), which was aggravated at high ratios (1:150), reducing cell viability. Keratinocyte responses depended on a species-specific “contact ratio threshold” rather than absolute bacterial abundance. Overall, these findings provide novel insights for developing treatment strategies, with bacterial ratios as indicators, for diseases associated with the disruption of normal skin flora, including atopic dermatitis and seborrheic dermatitis.