Mechanistic Effects of Long-Term Ultraviolet B Irradiation Induce Epidermal and Dermal Changes in Human Skin Xenografts

UVB irradiation has been reported to induce photoaging and suppress systemic immune function that could lead to photocarcinogenesis. However, because of the paucity of an UVB-induced photodamaged skin model, precise and temporal mechanism(s) underlying the deleterious effects of long-term UVB exposure on human skin have yet to be delineated. In this study, we established a model using human skin xenografted onto severe combined immunodeficient mice, which were subsequently challenged by repeated UVB irradiation for 6 weeks. Three-dimensional optical image analysis of skin replicas and noninvasive biophysical measurements illustrated a significant increase in skin surface roughness, similar to premature photoaging, and a significant loss of skin elasticity after long-term UVB exposure. Resembling authentically aged skin, UVB-exposed samples exhibited significant increases in epithelial keratins (K6, K16, K17), elastins, and matrix metalloproteinases (MMP-1, MMP-9, MMP-12) as well as degradation of collagens (I, IV, VII). The UVB-induced deterioration of fibrous keratin intermediate filaments was also observed in the stratum corneum. Additionally, similarities in gene expression patterns between our model and chronologically aged skin substantiated the plausible relationship between photodamage and chronological age. Furthermore, severe skin photodamage was observed when neutralizing antibodies against TIMP-1, an endogenous inhibitor of MMPs, were administered during the UVB exposure regimen. Taken together, these findings suggest that our skin xenograft model recapitulates premature photoaged skin and provides a comprehensive tool with which to assess the deleterious effects of UVB irradiation.Morphological changes in the skin, such as wrinkling and sagging, are often found in photoaged skin caused by chronic UV irradiation.^1,2,3 Skin photodamage is physiologically correlated with several alterations including the increase and disorganization of elastic fibers and the reduction of collagens in the dermal extracellular matrix^{4,5,6,7,8,9,10,11} and the increase of keratin contents and the deterioration of keratin intermediate filaments (KIFs) in the epidermis.^12,13,14Elastic fibers are age-dependent-decreasing dermal extracellular matrix that respond primarily to the fibrous mechanism controlling cutaneous elasticity.^4,5,15,16 The accumulation of dystrophic elastotic material in the reticular dermis, referred to as solar elastosis, is commonly observed in photoaged skin.^17,18,19 Reported in both in vivo and in vitro, UVB irradiation induces tropoelastin gene and protein expression in fibroblasts and keratinocytes, resulting in an aberrant accumulation of dermal elastic fibers and elastin content.^5,8,10,11,20 On the other hand, it has been reported that the degeneration of these fibers in chronologically and/or photoaged skin stems from an increase in matrix metalloproteinase-12 (MMP-12) and/or elastase, an elastin-degrading enzyme that is produced and secreted by dermal fibroblasts.^21,22,23,24 To date, the mechanisms underlying the alteration of elastin fibers in photoaged skin including their production, accumulation, and degradation are not fully understood.As the main constituent of the dermal matrix, collagen fibers are responsible in regulating the mechanical properties of the skin (ie, skin resilience and skin elasticity). In photoaged skin, the concomitant reductions of collagen I and III occur as procollagen synthesis subsides and the existed dermal collagen degrades because of the disproportionate expression of MMP-1.^25,26,27,28 The reduction of collagens VII and IV at the dermo-epidermal junction has been also reported in the pronounced wrinkling skin.²⁹ In hairless mouse, repetitive UVB exposure has been reported to escalate the degradation of collagens VII and IV by up-regulating MMP-2 and MMP-9 expression.^30,31In addition to the alterations in the dermal compartment, the changes in several epithelial keratins have been well documented in photoaged skin. In healthy skin, keratins compose up to 85% of a fully differentiated keratinocyte and orderly form the epithelial-specific intermediate filament composed of acidic type I (K9 to K20)-basic type II (K1 to K8) coiled-coil heterodimer in healthy skin.^{32,33,34,35,36,37} The existence of keratin fibers strengthening the viscoelastic properties in the stratum corneum (SC) provides a semi-impermeable membrane that is tough and resilient.^38,39,40,41 In hairless mice, a repeated low-dose UV exposure has been reported to deteriorate KIFs and induce the expression of epithelial keratins (K1, K5, and K10), resulting in the loss of skin elasticity and eventually initiating wrinkled skin, consistent with previous reports demonstrating no causal relationship between wrinkle formation and dermal changes.^{12,13,14,42,43}Many studies of skin photoaging have been conducted using animal models and human skin substitute. The results may be misleading because of inferior architecture, such as the relative thin epidermal layer and compromised barrier function.⁴⁴ The use of actual human skin or human skin xenografts to study skin photoaging is more appropriate.^45,46,47 The reversible hyperproliferation and differentiation of human epidermis after acute UVB exposure has been successfully demonstrated using human skin grafted onto congenitally athymic nude mice.⁴⁸ Although the lack of inflammatory cells, T and B cells, in athymic and severe combined immunodeficient (SCID) mice allows the implantation of human skin without rejection, it may pose a problematic interpretation of immune function and UV response. A result from a clinical study suggested that T lymphocytes are involved in collagen degradation by the activation of MMP-1.⁴⁹ To date, the detail mechanisms of premature photoaging because of long-term UVB exposure on actual human skin had not been assessed. Therefore, it is of particular interest to establish a chimeric model with human skin xenograft on SCID mice to examine the mechanism(s) of photodamaged skin after long-term UVB irradiation. Here we report for the first time that repeated UVB irradiation in combination with the repetitive motion of the human skin xenograft represents a photodamaged skin model reflecting the documented changes in both the epidermis and the dermis. In addition, we also suggest new insights for the involvement of K6 and its partners in this process.