Replicating dynamic immune responses at single-cell resolution within a microfluidic human skin equivalent

Abstract

The intricate immunological functions of human skin involve the interplay between multiple different cell types as well as dynamic trafficking of leukocytes in and out the tissue, both of which are extremely challenging to replicate in vitro. To enable in vitro investigation of human skin immunology, we developed a microfluidic human skin equivalent (HSE) that supports the delivery of circulating immune cells via a vascular microchannel embedded within the dermis of a full-thickness construct. We demonstrated that stimulation of keratinocyte inflammation with lipopolysaccharide and nigericin promoted rapid monocyte recruitment out of the vascular channel and into the epidermal layer within 24 hours, followed by a second wave of monocyte migration into the dermis over a period of six days. Single-cell transcriptomic analysis of the tissue-resident and recruited cell populations revealed dynamic and cell-specific patterns of gene expression that were characteristic of acute activation and resolution of an inflammatory immune response. Moreover, comparison of the gene signatures of the monocyte-derived cells to in vivo populations provided molecular level validation of the model and indicated a differentiation trajectory of the monocytes through to mature dermal macrophages. To extend the microfluidic platform to additional applications, we also modelled age-associated immune dysfunction by the inclusion of senescent fibroblasts, which promoted increased monocyte recruitment into the HSE, replicating previous in vivo human studies. Thus, the microfluidic HSE presented here replicates key aspects of dynamic inflammatory immune responses within the skin and represents a tractable experimental tool for interrogating mechanisms of human skin immunology.