Characterization of human monocytic cell line THP-1 associated with human 3D skin models under UVA exposure
Tissue resident macrophages play important roles in tissue homeostasis and acute response to external stimuli. Human skin equivalents (HSEs) incorporating human monocytic cell line THP-1, were fabricated to generate immunocompetent human skin models. These HSEs were used to investigate the influence of the skin microenvironment and UVA exposure on the phenotypes of macrophages. THP-1 in HSEs exhibited mixed M1 and M2 macrophage phenotypes. Transcriptomic analysis demonstrated that THP-1 in HSEs enriched extracellular matrix interaction while downregulated a DNA replication hallmark. Upon UVA exposure, immunocompetent HSEs exhibited epidermal distortion and increased DNA double-strand breaks (DSB). THP-1 isolated from UVA-exposed HSEs revealed significant upregulation of genes associated with oxidative stress, antioxidant regulation, inflammatory and UV response. A photoprotective agent, mycosporine-2-glycine (M2G), derived from cyanobacteria was applied on to immunocompetent HSEs and the responses of THP-1 cell line was investigated after UVA exposure. The result showed that UVA-induced DSB was significantly lower in M2G-treated HSEs. In addition, the inflammatory and UV response hallmarks were downregulated while the oxidative phosphorylation hallmark was upregulated in THP-1 from M2G-treated UVA exposed HSEs. Taken together, this study provides an immunocompetent 3D skin model that can be used to interrogate responses of skin resident innate immune cells to microenvironment and external stimuli such as UVA irradiation. Overall design: Human skin equivalents (HSEs) fabrication: NHDFs were mixed with fibrinogen and thrombin, and seeded immediately into the transwell insert treated with plasma ion generated from plasma generator device for 2 mins. For immunocompetent HSEs, NHDFs and THP-1 cells were co-cultured in fibrin hydrogel. After gelation, the dermal layer was submerged in DMEM for 24 h. NHEKs were seeded on the dermal layer coated with collagen type IV, then submerged in the mixed medium and incubated for 24 h. Epidermal differentiation was initiated by replacing the medium with L-ascorbic acid, followed by air-liquid interface cultivation for 7 days. UVA irradiation and procedure: HSEs were exposed to UVA irradiation by using a BlueWave® MX-250 LED lamp, RediCure™ at a single dose of 80 J/cm2. The UVA lamp was mounted on a stable support allowing an exposure distance of 6 cm from the sample surface. Before irradiation, the culture medium was replaced with 1?PBS to prevent phototoxic effects. PBS solution was removed, and a fresh differentiated medium was added after UVA exposure. The UVA-induced damage was evaluated 24 h post-UVA exposure. Applying Mycosporine-2-Glycine (M2G) on HSEs: M2G was diluted in Milli-Q water was applied for 2 h before, during, and 30 min after UVA irradiation. Afterward, M2G was removed, and HSEs were subsequently incubated. Harvesting and tissue histology, along with gene expression analysis, were performed 24 h post-UVA exposure. CD45+ cell isolation, RNA extraction, and Library preparation: HSEs were digested with 0.05% trypsin/ 0.01% EDTA in 1?PBS. Labeled immune cells with Alexa Fluor® 488 anti-human CD45 Antibody were isolated using SH800S Cell Sorter. Harvested CD45+ cells underwent total RNA extraction with PicoPureTM RNA Isolation Kit. Total RNA was subjected to RNA-sequencing.