Characterization of inflammatory skin diseases using 3D skin models, patient samples and highly multiplexed microscopy

Abstract

Accounting for omnipresent diseases, inflammatory skin defects like Psoriasis and Atopic Dermatitis deserve close and thorough research to investigate pathological mechanisms and drivers of inflammation. Using CODEX to characterize the complex constructs of inflammation cascades and antimicrobial pathways describes a novel approach to better understand molecular interactions of inflammatory skin diseases. This work demonstrates a method aiming to visualize and quantitatively analyze the effects of inflammation on immune as well as DNA damage and epithelial markers. Suitability of 3D human inflammatory skin models for use in functional assays in vitro was successfully visually validated and proved histological accordance to inflammatory skin. However, quantification of marker expression evinced limitations due to small cell count and missing uniformity. CODEX analysis of patient samples revealed enhanced presence of Polymorphonuclear neutrophils, showing high expression of the neutrophil marker Neutrophil Elastase in skin of AD patients and an elevation of NET markers MPO and citH3, as well as a significant increase of HMGB1 expression in AD skin. This correlated with more severe S. aureus colonization, DNA damage by reactive oxygen species and activation of inflammatory cascades. The presented data hereby confirms preliminary findings in vitro that illustrated an enhancement of bacterial colonization in Atopic Dermatitis through NET formation and adds a possible explanation for phenotypical differences between PS and AD by revealing dissimilarities between the samples in reaction to ROS exposure. Furthermore, this work lays the foundation for more detailed exploration of CTCL progression in skin models and the prospects of viable reflection of CTCL in HSE models for testing potential therapeutics in vitro. Tailored analysis methods that account for marker-specific localization and expression patterns are essential for advancing our understanding of inflammatory skin diseases and identifying precise therapeutic strategies.