Defined polymicrobial communities elicit distinct AHR, HBD2, and cytokine responses in reconstructed human skin models

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Abstract
Reconstructed human skin microbiome models represent human-relevant New Approach Methodologies (NAMs) for investigating host–microorganism interactions and evaluating microbiome-modulating ingredients. However, microbiome-compatible reconstructed skin models remain poorly standardized and require further characterization to support their broader application. In this study, we developed reconstructed human skin models colonized with defined combinations of Malassezia furfur, Staphylococcus epidermidis, and Cutibacterium acnes to establish experimental conditions representative of preserved and disrupted tissue homeostasis associated with seborrheic dermatitis. The models were characterized by histology, scanning electron microscopy, Grocott–Gomori staining, coproporphyrin III-associated autofluorescence, host gene expression, and cytokine secretion. Among the tested microbial combinations, the M. furfur + S. epidermidis model preserved tissue morphology and exhibited host-response markers comparable to those of uncolonized skin, whereas the M. furfur + C. acnes model induced moderate epidermal alterations together with increased expression of the aryl hydrocarbon receptor (AHR) and human β-defensin 2 (HBD2), and was selected as the challenge model for treatment evaluation. The platform was subsequently applied to assess the effects of α-glucan oligosaccharide (AGO), an antimicrobial preservative, and empty or AGO-loaded cupuaçu butter nanoparticles. AGO-loaded nanoparticles significantly reduced AHR expression, whereas all treatments reduced HBD2 expression. Among the cytokines evaluated, only the antimicrobial preservative significantly decreased IL-6 secretion. Collectively, these findings demonstrate that the reconstructed human skin microbiome platform can discriminate among distinct host responses to defined microbial communities and detect treatment-specific biological effects, supporting its application as a human-relevant in vitro model for mechanistic studies and preclinical evaluation of microbiome-modulating cosmetic ingredients.