O06 Exploring host–microbiome–immune interactions using singular and multispecies inoculations in optimized 3D skin models

Abstract The human microbiome has a fundamental role in skin homeostasis, barrier function and host immune networks. Specifically, a complex and multispecies microbiome population is required for healthy skin. Acute and chronic skin conditions are often associated with imbalances in host–microbiome–immune interactions. Despite recent advances in skin microbiome research, there are limited models for studying these interactions in healthy and diseased skin; therefore, we have developed a model to investigate how different microbiome profiles alter epidermal homeostasis and T-cell responses. Full-thickness three-dimensional (3D) skin models, developed using primary dermal fibroblasts and keratinocytes isolated from adult skin, were colonized with five human microbiome species: Staphylococcus epidermidis (SE), Staphylococcus capitis (SC), Cutibacterium acnes (CA), Malassezia restricta (MR) and Malassezia globosa (MG), in singular or combined inoculations (5M). Significant changes in epidermal thickness were observed in models colonized with single-species inoculations compared with sterile control models; however, no significant changes in epidermal thickness were observed in models colonised with 5M inoculations. The effect of colonization on epidermal differentiation and barrier formation was observed via the expression of K14, K10, E-cadherin and Ki67. Expression of E-cadherin significantly decreased in models colonized with SC, MR and MG compared with sterile control models. Despite significant changes in epidermal thickness, only MG showed a significant decrease in Ki67 expression compared with sterile control models. No significant changes were observed in K10 and K14 expression following microbial inoculation. Overall, 5M inoculation showed no significant changes in biomarker expression or epidermal thickness compared with sterile control models suggesting a combination of microbes supports epidermal homeostasis as observed in vivo. Commensal microbes have been shown to regulate epidermal barrier function via the aryl hydrocarbon receptor (AhR). Increased nuclear expression of AhR was observed in response to SC, CA and 5M, suggesting these microbes have a greater role in AhR activation. To investigate microbiome–immune interactions in the skin, the expression of CD69, cutaneous lymphocyte antigen (CLA) and HLA-DR on T cells was assessed in response to incubating peripheral blood mononuclear cells with conditioned media from inoculated models. SE caused a significant increase in CD3+/CLA+ cells and 5M significantly reduced CD3+/HLA-DR+ cells. RNA sequencing aims to support current analysis and elucidate any more targets to be explored. Our findings suggest different microbial profiles alter epidermal barrier formation and immune responses.