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.

Motugivatrep (SJP-0132) is a transient receptor potential cation channel subfamily V member 1 (TRPV1) antagonist and a potential therapeutic agent for dry eye disease. However, the possible carcinogenicity of TRPV1 antagonists has raised concerns. Therefore, this study aimed to evaluate the genotoxic and carcinogenic potential of motugivatrep by conducting standard genotoxicity assays and assessing carcinogenicity in Tg-rasH2 mice and rats following oral administration for 26 and 104 weeks, respectively. In the bacterial reverse mutation assay, no increase in the number of revertant colonies was observed at any concentration with or without metabolic activation. In the mouse lymphoma thymidine kinase assay, no increase in mutant fractions was observed under any treatment conditions, with or without metabolic activation. Oral administration of motugivatrep did not increase the number of micronucleated immature erythrocytes at doses up to 2000 mg/kg in the in vivo micronucleus assay in rats. In carcinogenicity studies in Tg-rasH2 mice and rats, no increase in tumor incidence in any organs/tissues of either sex was observed in the motugivatrep-treated groups at doses of up to 100 and 50 mg/kg/day, respectively, which correspond to systemic exposures exceeding the anticipated clinical levels. Overall, motugivatrep was negative for genotoxicity, and oral administration did not exhibit carcinogenic potential in rodents. These results suggest that ocular instillation of the motugivatrep ophthalmic formulation is unlikely to pose a carcinogenic risk to systemic organs and tissues.