TSG-6 is an anti-inflammatory hyaluronan-binding protein involved in extracellular matrix remodelling whose functions have never been studied in skin. Characterization of TSG-6 was carried out in vivo in healthy human epidermis and in vitro using keratinocyte monolayers and reconstructed human epidermis (RHE). Distribution of TSG-6 protein was highlighted by immunofluorescence and levels of relative mRNA and protein expression were respectively measured by RT-qPCR and ELISA. The immunofluorescent signal of TSG-6 protein was located in the living layers of epidermis in vivo and in vitro, mostly in basal keratinocytes, and was restricted to the intracellular compartment. Because the localization of hyaluronan (HA) is in the extracellular matrix, no colocalization of TSG-6 and HA could thus be detected. ELISA assay shows that TSG-6 is abundant in the intracellular compartment, and is present in weak amounts in pericellular spaces, as well as in the culture medium. In vitro, TSG-6 mRNA level did not change during epidermal differentiation induced by confluence and increased cell density. The expression of TSG-6 was then analysed in RHE subjected to inflammatory conditions using two different models. In the first one, RHE were infected with arthrospores of Trichophyton rubrum dermatophyte, allowing adhesion and invasion of cornified layer by fungal elements. The second one corresponds to an atopic dermatitis-like model where RHE were first deprived from plasma membrane cholesterol using methyl--cyclodextrin ahead of exposure to interleukins (IL-4, IL-13, IL-25). In both inflammatory models, despite TSG-6 mRNA and protein levels were increased, simultaneously with an increase in HA fluorescent detection no colocalization of TSG-6 with HA was ever detected. In summary, these results demonstrate that TSG-6 is present intracellularly in epidermal keratinocytes in physiological conditions, and its expression is induced by inflammatory conditions. Only low concentration of this protein is detectable extracellularly.

Characterization of TSG-6 protein in healthy and inflammatory models of reconstructed human epidermis