Atopic dermatitis (AD) is a multifactorial, heterogeneous disease associated with epidermal barrier disruption and intense systemic inflammation. Previously, we showed that exosomes derived from human adipose tissue-derived mesenchymal stem cells (ASC-exosomes) attenuate AD-like symptoms by reducing multiple inflammatory cytokine levels. Here, we investigated ASC-exosomes’ effects on skin barrier restoration by analyzing protein and lipid contents. We found that subcutaneous injection of ASC-exosomes in an oxazolone-induced dermatitis model remarkably reduced trans-epidermal water loss, while enhancing stratum corneum (SC) hydration and markedly decreasing the levels of inflammatory cytokines such as IL-4, IL-5, IL-13, TNF-α, IFN-γ, IL-17, and TSLP, all in a dose-dependent manner. Interestingly, ASC-exosomes induced the production of ceramides and dihydroceramides. Electron microscopic analysis revealed enhanced epidermal lamellar bodies and formation of lamellar layer at the interface of the SC and stratum granulosum with ASC-exosomes treatment. Deep RNA sequencing analysis of skin lesions demonstrated that ASC-exosomes restores the expression of genes involved in skin barrier, lipid metabolism, cell cycle, and inflammatory response in the diseased area. Collectively, our results suggest that ASC-exosomes effectively restore epidermal barrier functions in AD by facilitating the de novo synthesis of ceramides, resulting in a promising cell-free therapeutic option for treating AD.

Stratum corneum (SC) pH regulates skin barrier functions and elevated SC pH is an important factor in various inflammatory skin diseases. Acidic topical formulas have emerged as treatments for impaired skin barriers. Sodium proton exchanger 1 (NHE1) is an important factor in SC acidification. We investigated whether topical applications containing an NHE1 activator could improve skin barrier functions. We screened plant extracts to identify NHE1 activators in vitro and found Melissa officinalis leaf extract. Rosmarinic acid, a component of Melissa officinalis leaf extract, significantly increased NHE1 mRNA expression levels and NHE1 production. Immunofluorescence staining of NHE1 in 3D-cultured skin revealed greater upregulation of NHE1 expression by NHE1 activator cream, compared to vehicle cream. Epidermal lipid analysis revealed that the ceramide level was significantly higher upon application of the NHE1 activator cream on 3D-cultured skin, compared to application of a vehicle cream. In a clinical study of 50–60-year-old adult females (n = 21), application of the NHE1 activator-containing cream significantly improved skin barrier functions by reducing skin surface pH and transepidermal water loss and increasing skin hydration, compared to patients who applied vehicle cream and those receiving no treatment. Thus, creams containing NHE1 activators, such as rosmarinic acid, could help maintain or recover skin barrier functions.

Both intrinsic (i.e., an individual’s body clock) and extrinsic factors (i.e., air pollutants and ultraviolet irradiation) accelerate premature aging. Epidemiological studies have shown a correlation between pollutant levels and aging skin symptoms. Diesel particle matter in particular leads to some diseases, including in the skin. Our recent study demonstrates that diesel particulate extract (DPE) increases apoptosis via increases in an anti-mitogenic/pro-apoptotic lipid mediator, ceramide in epidermal keratinocytes. Here, we investigated whether and how DPE accelerates premature skin aging using cultured normal human dermal fibroblasts (HDF). We first demonstrated that DPE increases cell senescence marker β-galactosidase activity in HDF. We then found increases in mRNA and protein levels, along with activity of matrix metalloprotease (MMP)-1 and MMP-3, which are associated with skin aging following DPE exposure. We confirmed increases in collagen degradation in HDF treated with DPE. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is activated by DPE and results in increased ceramide production by sphingomyelinase activation in HDF. We identified that ceramide-1-phosphate (C1P) (produced from ceramide by ceramide kinase activation) activates MMP-1 and MMP-3 through activation of arachidonate cascade, followed by STAT 1- and STAT 3-dependent transcriptional activation.

Since ceramide is a key epidermal barrier constituent and its deficiency causes barrier-compromised skin, several molecular types of ceramides are formulated in commercial topical agents to improve barrier function. Topical ceramide localizes on the skin surface and in the stratum corneum, but certain amounts of ceramide penetrate the stratum granulosum, becoming precursors to endogenous ceramide synthesis following molecular modification. Moreover, exogenous ceramide as a lipid mediator could modulate keratinocyte proliferation/differentiation. We here investigated the biological roles of exogenous NP (non-hydroxy ceramide containing 4-hydroxy dihydrosphingosine) and NDS (non-hydroxy ceramide containing dihydrosphingosine), both widely used as topical ceramide agents, in differentiated-cultured human keratinocytes. NDS, but not NP, becomes a precursor for diverse ceramide species that are required for a vital permeability barrier. Loricrin (late differentiation marker) production is increased in keratinocytes treated with both NDS and NP vs. control, while bigger increases in involucrin (an early differentiation marker) synthesis were observed in keratinocytes treated with NDS vs. NP and control. NDS increases levels of a key antimicrobial peptide (an innate immune component), cathelicidin antimicrobial peptide (CAMP/LL-37), that is upregulated by a ceramide metabolite, sphingosine-1-phosphate. Our studies demonstrate that NDS could be a multi-potent ceramide species, forming heterogenous ceramide molecules and a lipid mediator to enhance differentiation and innate immunity.

Inactive cortisone is converted into active cortisol by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Excessive levels of active glucocorticoids could deteriorate skin barrier function; barrier impairment is also observed in aged skin. In this study, we aimed to determine whether permeability barrier impairment in the aged skin could be related to increased 11β-HSD1 expression. Aged humans (n = 10) showed increased cortisol in the stratum corneum (SC) and oral epithelium, compared to young subjects (n = 10). 11β-HSD1 expression (as assessed via immunohistochemical staining) was higher in the aged murine skin. Aged hairless mice (56-week-old, n = 5) manifested greater transepidermal water loss, lower SC hydration, and higher levels of serum inflammatory cytokines than the young mice (8-week-old, n = 5). Aged 11β-HSD1 knockout mice (n = 11), 11β-HSD1 inhibitor (INHI)-treated aged wild type (WT) mice (n = 5) and young WT mice (n = 10) exhibited reduced SC corticosterone level. Corneodesmosome density was low in WT aged mice (n = 5), but high in aged 11β-HSD1 knockout and aged INHI-treated WT mice. Aged mice exhibited lower SC lipid levels; this effect was reversed by INHI treatment. Therefore, upregulation of 11β-HSD1 in the aged skin increases the active-glucocorticoid levels; this suppresses SC lipid biosynthesis, leading to impaired epidermal permeability barrier.

Summary: Decline in tissue NAD levels during aging is linked to aging and its associated diseases. However, the mechanism for aging-associated NAD decline remains unclear. Here we report that pro-inflammatory M1-like macrophages, but not naïve or M2 macrophages, accumulate in metabolic tissues including visceral white adipose tissue and the liver during aging. Remarkably, these M1-like macrophages highly express the NAD consuming enzyme CD38 and have enhanced CD38-dependent NADase activity. We also find that senescent cells progressively accumulate in visceral white adipose tissue during aging and that inflammatory cytokines found in the supernatant from senescent cells (Senescence associated secretory proteins, SASP) induce macrophages to proliferate and to express CD38. These results highlight a new causal link between visceral tissue senescence and tissue NAD decline during aging and represent a novel therapeutic opportunity targeting maintenance of NAD levels during aging.