Understanding Dynamic Immune Responses within 3D in vitro Human Skin Models

Abstract

Dynamic communication between tissue-resident cells and circulating immune cells, such as monocytes, orchestrates the skin’s responses to infection and plays a role in tissue repair and regeneration. However, the factors that drive monocyte recruitment into human skin during inflammatory events and the specific signals that direct monocyte fate are poorly understood. Current 3D in vitro models recapitulate the structure of skin by fabricating dermal and epidermal-like layers. However, only a limited number of 3D in vitro skin models have incorporated immune cell populations and these do not effectively model the complex and dynamic interactions between the tissue and the immune system. Therefore, the aim of this research was to develop a novel immune-responsive 3D in vitro human skin model that contained a microchannel for the fluidic delivery of monocytes, which can be used to investigate the recruitment and differentiation of monocytes in response to inflammatory stimuli. Isolated CD14+ monocytes were successfully incorporated within the dermal compartment of fibrin human skin equivalents. Monocyte-derived populations could be visualised, using immunofluorescence staining in both the dermal and epidermal compartments on day 14. Dermal populations co-expressed the macrophage markers CD68 and CD163. Epidermal monocyte-derived cells expressed the Langerhans cell marker CD207 (langerin). A microfluidic model of human skin was fabricated by 3D printing a sacrificial gelatin microchannel template within a fibroblast embedded fibrin hydrogel. The microchannel template was then selectively removed by melting the gelatin at 37 ⁰C. The hollow microchannel was then lined with human endothelial cells, mimicking a vascularised dermis. Human keratinocytes were cultured on the surface of the construct to mimic the epidermis. Dynamic immune responses in the microfluidic model were investigated by exposing the epidermal layer to lipopolysaccharide and nigericin, activating the inflammasome and inducing the secretion of cytokines, including IL1β and IL18. The vascular microchannel was then employed as a conduit for the delivery of primary CD14+ monocytes, and monocyte trafficking into the tissue was investigated using live confocal microscopy. Monocyte fate within the microfluidic human skin model was first investigated using whole mount immunofluorescence staining. CD68+ and CD163+ cells could be identified within the dermal and epidermal compartments in both control and treated day 1 and day 6 conditions, with significantly increased numbers of recruited cells observed in the treated conditions when compared to untreated controls. Single cell transcriptomic analysis of the microfluidic model revealed dynamic responses activated by inflammation in resident skin cell populations and monocyte-derived cells on day 1 and evidence of resolution by day 6. Three distinct monocyte-derived clusters were identified in the day 6 samples and results highlighted the plasticity and differential potential of recruited monocytes. Furthermore, the gene signatures of these populations closely aligned with the profiles of immune cell populations identified within in vivo human skin datasets. Analysis of putative intercellular signalling networks (using CellPhoneDB) identified signalling interactions from resident cell populations that may direct monocyte recruitment and fate within the model. Key interactions identified on day 1 included tumour necrosis factor superfamily (TNFSF) and chemokine signalling, which may have contributed to monocyte activation and recruitment. By day 6, identified interactions included semaphorin and adenosine signalling, suggesting their potential role in directing monocyte fate within the microfluidic model. Furthermore, potentially novel nectin-nectin interactions were identified between keratinocytes and monocyte-derived cells. Overall, the microfluidic human skin model accurately replicated dynamic immune responses in human skin and could be a powerful tool during drug discovery and development.