Adipose-derived stem cells ameliorate atopic dermatitis by suppressing the IL-17 expression of Th17 cells in an ovalbumin-induced mouse model

Abstract

Background: Mesenchymal stem cells (MSCs) have therapeutic potential for atopic dermatitis (AD) owing to their immunoregulatory effects. However, the underlying mechanisms associated with the therapeutic efficacy of MSCs on AD are diverse and related to both cell type and delivery method.

Objectives: This study investigated the therapeutic effect and mechanisms of adipose-derived stem cells (ADSCs) on AD using an ovalbumin (OVA)-induced AD mouse model.

Methods: AD mice were subcutaneously injected with mouse ADSCs, cortisone, or PBS, and the therapeutic effects were determined by gross and histological examinations and serum IgE levels. Additionally, qPCR, RNA-sequencing analyses of skin samples and co-culture of ADSCs and Th17 cells were conducted to explore the underlying therapeutic mechanisms.

Results: ADSCs treatment attenuated the AD pathology, decreased the serum IgE levels, and decreased mast cells infiltration in the skin of the model mice. Moreover, tissue levels of IL-4R and Th17-relevant products (IL-17A, CCL20, and MMP12) were suppressed in the ADSC- and cortisone-treated groups. Genomics and bioinformatics analyses demonstrated significant enrichment of inflammation-related pathways in the downregulated genes of the ADSC- and cortisone-treated groups, specifically the IL-17 signaling pathway. Co-culture experiments revealed that ADSCs significantly suppressed the proliferation of Th17 cells and the expression of proinflammatory cytokines (IL-17A and RORγT). Furthermore, expression levels of PD-L1, TGF-β, and PGE2 were significantly upregulated in co-cultured ADSCs relative to those in monocultured ADSCs.

Conclusion: ADSCs ameliorate OVA-induced AD in mice mainly by downregulating IL-17 secretion of Th17 cells.

Keywords: Adipose-derived stem cells; Atopic dermatitis; IL-17 signaling pathway; Mouse model; Ovalbumin; Th17 cells.

Fig. 1

ADSCs alleviate the gross symptoms of OVA-induced AD in a murine model. a Schematic of OVA sensitization and ADSC application on the AD mouse model. × represents the injection site. b Representative macroscopic photographs and enlarged images of mice dorsal skin lesions on day 50. Red dotted boxes represent the areas covered by OVA patches. White dotted boxes represent the areas of the enlarged images. Scale bar = 1 mm. c Clinical severity of mice dorsal skin lesions on day 50. n = 6 per group. ***p < 0.001; ****p < 0.0001. AD, atopic dermatitis; OVA, ovalbumin; NC, normal control; PBS, phosphate-buffered saline; ADSC, adipose-derived stem cell; COR, cortisone

Fig. 2

ADSCs improve histological features of lesion sites and decrease serum IgE levels in mice with OVA-induced AD. a Representative H&E staining photomicrographs of dorsal skin samples from the four groups. The epidermis and dermis are distinguished by white dotted lines, with the parts above the white dotted line representing the epidermal layer. Scale bar = 100 μm. b Representative TB staining photomicrographs of dorsal skin samples from the four groups. Red arrows show infiltrated mast cells. Scale bar = 100 μm. c Measurement of epidermal thickness in skin samples; n = 10 per group. d Measurement of infiltrated mast cells in skin samples; n = 10 per group. e Serum IgE level on day 50; n = 6 per group. *p < 0.05; ****p < 0.0001. Scale bar = 100 μm. NC, normal control; PBS, phosphate-buffered saline; ADSC, adipose-derived stem cell; COR, cortisone; IgE, immunoglobulin E; AD, atopic dermatitis; OVA, ovalbumin; TB, Toluidine Blue

Fig. 3

Effect of ADSCs on gene expression level of proinflammatory cytokines in OVA-induced AD mice. Relative gene expression levels of a IL-13, b IL-4, c IL-4R, d IL-17A, e CCL20, f MMP12, g IFN-γ and h TNF-α in the skin lesions measured by qPCR. n = 6 per group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. NC, normal control; PBS, phosphate-buffered saline; ADSC, adipose-derived stem cell; AD, atopic dermatitis; OVA, ovalbumin; qPCR, quantitative real-time polymerase chain reaction; COR, cortisone; IL, interleukin; IL-4R, IL-4 receptor; CCL20, C–C motif chemokine ligand 20; MMP12, matrix metallopeptidase 12; IFN-γ, interferon-gamma; TNF-α, tumor necrosis factor-alpha

Fig. 4

RNA sequencing of mice dorsal skin samples. a PCA of dorsal skin samples in the NC, PBS, ADSC, and COR groups. b Heat maps of DEGs between the NC, PBS, ADSC, and COR groups. c Volcano plots of DEGs between the PBS and NC groups (left), the ADSC and PBS groups (middle), and the COR and PBS groups (right); n = 3 per group. NC, normal control; PBS, phosphate-buffered saline; ADSC, adipose-derived stem cell; COR, cortisone; PCA, principal component analysis; padj, adjusted p value

Fig. 5

GO and KEGG analyses based on DEGs between the NC, PBS, ADSC, and COR groups. a Top 10 GO terms for BP, CC, and MF of the upregulated DEGs of PBS versus the NC group. b Top 20 enriched KEGG signaling pathways of the upregulated DEGs of PBS versus the NC group. c Top 10 GO terms for BP, CC, and MF of the downregulated DEGs of the ADSC versus PBS and COR versus PBS groups. d Top 20 enriched KEGG pathways of the downregulated DEGs of the ADSC versus PBS and COR versus PBS groups; n = 3 per group. NC, normal control; PBS, phosphate-buffered saline; ADSC, adipose-derived stem cell; COR, cortisone; BP, biological process; CC, cellular component; MF, molecular function; padj, adjusted p-value; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Gene and Genome; DEGs, differentially expressed genes

Fig. 6

Effect of ADSCs on proliferation and activation of Th17 cells. a Representative photomicrographs of the monocultured (left) and co-cultured (right) Th17 cells after 72 h of incubation. Scale bar = 500 μm. b Cell count of the monocultured and co-cultured Th17 cells after 72 h of incubation. Relative gene expression levels of c IL-17A, d IL-17F, and e RORγT in the monocultured and co-cultured Th17 cells determined by qPCR. n = 5 per group. *p < 0.05; **p < 0.01; ****p < 0.0001. ADSCs, Adipose-derived stem cells; interleukin, IL; RORγT, RAR-related orphan receptor gamma T; qPCR, quantitative real-time polymerase chain reaction

Fig. 7

Gene expression level of PD-L1, TGF-β, and PGE2 in ADSCs. a Representative photomicrographs of the monocultured (left) and co-cultured (right) ADSCs after 72 h of incubation. Scale bar = 200 μm. Relative gene expression levels of b PD-L1, c TGF-β, and d PGE2 in the monocultured and co-cultured Th17 cells determined by qPCR. n = 5 per group. *p < 0.05. ADSCs, Adipose-derived stem cells; PD-L1, programed death ligand 1; TGF-β, transforming growth factor-beta; PGE2, prostaglandin E2; qPCR, quantitative real-time polymerase chain reaction