No abstract

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.

No abstract

Figure 6. RORα activates Alox12-dependent MaR1 synthesis. (A) Seven-week-old C57BL/6 mice were fed with either LFD or HFD for 12 weeks (n = 4) or fed with MCS or MCD for 4 weeks (n = 5) (first and second panels). The LFD-fed C57BL/6 mice were treated with 5 mg/kg BW SR1078 for 5 days (n = 5) (third panel). Seven-week-old LFD-fed floxed and RORα-MKO mice were sacrificed (n = 11) (fourth panel). (B) Liver samples were obtained from the floxed and RORα-MKO mice those described in Supplemental Figure 1 (n = 5). Levels of MaR1 and RvD1 in liver tissues were measured. *P < 0.05 and **P < 0.01; ## P < 0.01 for A and B. (C) DHA-treated peritoneal macrophages (PM) and Raw 264.7 cells were treated with 5 μM SR1078 for 24 hours, or the cells were infected by lenti-shGFP or lenti-shRORα for 48 hours. Intracellular amount of MaR1 were measured. *P < 0.05 (n = 3). (D) A scheme for biosynthesis of MaR1 by LOX family. (E) Expression levels of 12-LOX protein (Alox12 mRNA) and 12/15-LOX protein (Alox15 mRNA) in liver macrophages (LM), PM, Raw 264.7, bone marrow-derived macrophages (BMDM), and hepatocytes were measured by Western blotting and qRT-PCR. (F) mRNA levels of Alox genes in the isolated LMs from floxed and RORα-MKO mice as shown in A were measured by qRT-PCR. (G) LMs were treated with SR1078 or MaR1 (left). LMs were infected by AAV-GFP/AAV-RORα or lenti-shGFP/lenti-shRORα as indicated (right). The mRNA levels of Alox12 were measured by qRT-PCR. *P < 0.05 (n = 3) for F and G. (H) Schematic representation of the mouse Alox12 promoter with the putative ROREs shown as red boxes (top). Raw 264.7 cells were transfected with the deleted Alox12 promoter-Luc reporter with empty vector (EV) or Myc-RORα. Luciferase activity was measured and normalized by β-galactosidase activity. *P < 0.05 (n = 3) (middle). Raw 264.7 cells were transfected with Myc-RORα, or cells were treated with SR1078 or MaR1. DNA fragments that contain flanking region of the ROREs on the Alox12 promoter were immunoprecipitated with indicated antibodies and then amplified by PCR (bottom). (I) DHA-treated PMs were treated with 5 μM SR1078, 5 μM baicalein, or 10 μM NCTT-956. Intracellular MaR1 content was measured. (J) LMs were treated with baicalein, or NCTT-956 in the presence or absence of DHA. The mRNA levels of Rora were measured by qRT-PCR (left). The CD206 + /CD80 + ratio of F4/80 + cells was determined by flow cytometry (right). *P < 0.05 and # P < 0.05 (n = 3) for I and J. The data represent mean ± SD. Data were analyzed by Mann-Whitney U test for simple comparisons or Kruskal-Wallis test for multiple groups.