Extracellular vesicles from IFN-γ-primed mesenchymal stem cells repress atopic dermatitis in mice

Abstract

Background: Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by immune dysregulation, pruritus, and abnormal epidermal barrier function. Compared with conventional mesenchymal stem cell (MSC), induced pluripotent stem cell (iPSC)-derived mesenchymal stem cell (iMSC) is recognized as a unique source for producing extracellular vesicles (EVs) because it can be obtained in a scalable manner with an enhanced homogeneity. Stimulation of iMSCs with inflammatory cytokines can improve the immune-regulatory, anti-inflammatory, and tissue-repairing potential of iMSC-derived EVs.

Results: Proteome analysis showed that IFN-γ-iMSC-EVs are enriched with protein sets that are involved in regulating interferon responses and inflammatory pathways. In AD mice, expression of interleukin receptors for Th2 cytokines (IL-4Rα/13Rα1/31Rα) and activation of their corresponding intracellular signaling molecules was reduced. IFN-γ-iMSC-EVs decreased itching, which was supported by reduced inflammatory cell infiltration and mast cells in AD mouse skin; reduced IgE receptor expression and thymic stromal lymphopoietin and NF-kB activation; and recovered impaired skin barrier, as evidenced by upregulation of key genes of epidermal differentiation and lipid synthesis.

Conclusions: IFN-γ-iMSC-EVs inhibit Th2-induced immune responses, suppress inflammation, and facilitate skin barrier restoration, contributing to AD improvement.

Keywords: Atopic dermatitis; Extracellular vesicles; IFN-γ; iPSC-derived MSC.

Fig. 1

Characterization of IFN-γ-iMSCs and IFN-γ-iMSC-EVs. A Flow cytometry analysis of IFN-γ-iMSCs. The reactivities of IFN-γ-iMSCs against positive (CD90, CD105, and CD73) or negative (CD45 and CD34) markers of MSCs were tested. The IgG isotype was used as a non-specific control (black peaks). B qPCR analysis of IDO1 mRNA in iMSCs and IFN-γ-iMSCs. n = 3. Data are presented as the mean ± SE. *p < 0.05. C Morphology of IFN-γ-iMSC-EVs under cryo-TEM. Scale bar = 200 nm. D Size distribution of IFN-γ-iMSC-EVs shown by NTA and DLS. The surface charge and polydispersity index (PDI) was also measured by DLS. E Western blot analyses for markers of extracellular vesicles (CD63, CD81, and TSG101) or cellular organelles (GM130 and calnexin) in IFN-γ-iMSCs and IFN-γ-iMSC-EVs. F Expression analysis of EV markers (CD9, CD63, and CD81) in IFN-γ-iMSC-EVs by flow cytometry. G Biodistribution of IFN-γ-iMSC-EVs. DNCB-induced AD mice were subcutaneously injected with DiR only or DiR-labeled IFN-γ-iMSC-EVs. After 8 h, the localization of DiR or DiR-labeled IFN-γ-iMSC-EVs in whole body and major internal organs was observed by in vivo imaging

Fig. 2

Immune system disease association with IFN-γ-iMSC-EVs. A IFN-γ-induced differentially expressed genes (DEGs) in iMSCs. CPM means count per million of transcripts. B Gene Set Enrichment Analysis (GSEA) of IFN-γ-induced up-regulated genes with hallmark collection. Red spots highlight the representative enriched functions of DEGs. C GSEA of IFN-γ-induced up-regulated genes with the immunologic signature gene collection. Gene ratio means the number of genes overlapping with the total number of genes in a gene set. D Differentially expressed proteins (DEPs) in IFN-γ-iMSC-EVs. Red circles indicate up-regulated EV proteins, green indicates down-regulated EV proteins, gray indicates non-differentially expressed EV proteins, and black indicates canonical EV markers. E GSEA of IFN-γ-induced up-regulated EV proteins with the hallmark collection. Red spots highlight the representative enriched functions of DEPs. F GSEA of IFN-γ-induced up-regulated EV proteins with the immunologic signature gene collection. G Comparison between the enriched functions of IFN-γ-induced DEGs and EV DEPs. Blue bars indicate the enriched functions of DEGs and red bars indicate DEPs. H Clustering coefficient with the IFN-γ-induced EV DEPs and immune system disease gene set. The horizontal axis indicates the local clustering coefficient and vertical axis indicates the representative immune system diseases associated with DEPs of IFN-γ-iMSC-EVs over iMSC-EVs. I Core protein–protein interaction network constructed with the atopic dermatitis gene set and DEPs of IFN-γ-iMSC-EVs over iMSC-EVs. Green arrows indicate transcriptional activation and gray unspecified effects

Fig. 3

Blockade of AD progression by IFN-γ-iMSC-EVs. AD was induced by DNCB in NC/Nga mice. AD mice were subcutaneously administered with PBS or IFN-γ-iMSC-EVs (50 or 500 μg) for negative control and test groups, respectively. A Gross appearance of the skin lesions of AD animals that received IFN-γ-iMSC-EVs. B Analysis of dermatitis severity score. n = 5. Data are presented as mean ± SE. ***p < 0.001; ^#p < 0.05. C Immunoblot analysis of IL-4Rα and IL-13Rα1 in skin tissues from AD mice that received IFN-γ-iMSC-EVs. n = 5. Data are presented as mean ± SE. *p < 0.05; ^##p < 0.01. D Immunoblot analysis of phosphorylated JAK1 in skin tissues from AD mice that received IFN-γ-iMSC-EVs. The density of phosphorylated JAK1 was normalized to that of total JAK1. n = 5. Data are presented as the mean ± SE. *p < 0.05; ^#p < 0.05. E Immunoblot analysis of phosphorylated STAT6 in skin tissues from AD mice that received IFN-γ-iMSC-EVs. The density of phosphorylated STAT6 was normalized to that of total STAT6. n = 5. Data are presented as mean ± SE. **p < 0.01; ^##p < 0.01

Fig. 4

Attenuation of inflammation by IFN-γ-iMSC-EVs in AD mice. AD mice were subcutaneously administered with PBS or IFN-γ-iMSC-EVs (50 or 500 μg) for negative control and test groups, respectively. A Distribution and number of mast cells in skin tissues. Mast cells in the skin layer were examined by staining with Toluidine blue. Scale bar: 100 μm. n = 5. Data are presented as mean ± SE. ***p < 0.001; ^###p < 0.001. B Analysis of inflammatory cell number in the skin layer of AD mice that received PBS or IFN-γ-iMSC-EVs. n = 5. Data are presented as mean ± SE. ***p < 0.001; ^###p < 0.001. C Immunoblot analysis of TSLP in the skin tissues collected from AD mice that received PBS or IFN-γ-iMSC-EVs. n = 5. Data are presented as mean ± SE. **p < 0.01; ^###p < 0.001. D Immunoblot analysis of CD23 and FcεRI in skin tissues of AD mice that received PBS or IFN-γ-iMSC-EVs. n = 5. Data are presented as mean ± SE. *p < 0.05; ^##p < 0.01; ^###p < 0.001. E Immunoblot analysis of phosphorylated p65 in skin tissues from AD mice that received PBS or IFN-γ-iMSC-EVs. The density of phosphorylated p65 was normalized to that of total p65. n = 5. Data are presented as mean ± SE. ***p < 0.001; ^##p < 0.01

Fig. 5

Reduction of pruritus by IFN-γ-iMSC-EVs in AD mice. AD mice were subcutaneously administered with PBS or IFN-γ-iMSC-EVs (50 or 500 μg) for the negative control and test groups, respectively. A Analysis of transepidermal water loss (TEWL) levels. n = 5. Data are presented as mean ± SE. *p < 0.05; **p < 0.01. B The effect of IFN-γ-iMSC-EVs on itching number in AD mice. n = 5. Data are presented as mean ± SE. *p < 0.05. C Immunoblotting of IL-31Rα and OSMRβ in the skin tissue of AD mice that received IFN-γ-iMSC-EVs. n = 5. Data are presented as mean ± SE. *p < 0.05; ^#P < 0.05; ^##p < 0.01. D Immunoblot analysis of phosphorylated STAT1 and STAT5 expression in skin tissues of AD mice that received IFN-γ-iMSC-EVs. Densities of phosphorylated STAT1 and STAT5 were normalized to those of total STAT1 and STAT5, respectively. n = 5. Data are presented as mean ± SE. *p < 0.05; ^#P < 0.05; ^##p < 0.01

Fig. 6

Restoration of skin barrier and lipid synthesis by IFN-γ-iMSC-EVs in the epidermis of AD mice. AD mice were subcutaneously administered with PBS or IFN-γ-iMSC-EVs (50 or 500 μg) for negative control and test groups, respectively. A Microscopic images of the skin tissues collected from IFN-γ-iMSC-EVs-injected AD mice. Tissues were stained with hematoxylin and eosin. The thickness of the epithelium was compared. Scale bar: 100 μm. n = 5. Data are presented as mean ± SE. ***p < 0.001; ^##p < 0.01. B Immunoblot analysis of skin barrier-related proteins in the epidermis of AD mice that received PBS or IFN-γ-iMSC-EVs. n = 5. Data are presented as mean ± SE. **p < 0.01; ^#p < 0.05; ^##p < 0.01. C Immunoblot analysis of proteins involved in lipid synthesis in the epidermal tissue of AD mice that received PBS or IFN-γ-iMSC-EVs. n = 5. Data are presented as mean ± SE. ^##p < 0.01; ^###p < 0.001