Topical administration of the secretome derived from human amniotic epithelial cells ameliorates psoriasis-like skin lesions in mice

Abstract

Background: Psoriasis is a chronic inflammatory skin disease. Tissue stem cells have exhibited a therapeutic effect on psoriatic mice. However, the therapeutic effect of topical administration of the secretome derived from tissue stem cells on psoriasis has not been reported.

Methods: The secretome from human amniotic epithelial cells (AEC-SC) and human umbilical cord mesenchymal stem cells (UMSC-SC) was topically administrated on the back of imiquimod-induced psoriasis-like mice. Subsequently, we observed the skin lesions and skin inflammation of psoriasis-like mice. Next, we further analyzed the paracrine factors in AEC-SC and UMSC-SC by protein chips. Lastly, the effect of the crucial paracrine factor was investigated by imiquimod-induced psoriasis-like mice.

Results: We found that AEC-SC had a better therapeutic effect on attenuating psoriasis-like skin lesions including skin scales, skin redness and skin thickness than UMSC-SC, and it had a better regulatory effect on keratinocyte hyperproliferation and altered differentiation. Thus, we focused on AEC-SC. Further study showed that AEC-SC reduced the infiltration of neutrophils and interleukin-17-producing T cells. Next, the analysis of AEC-SC with protein chip revealed that the levels of anti-inflammatory factor interleukin-1 receptor antagonist (IL-1ra) were much higher in AEC-SC compared to that in UMSC-SC. More importantly, the beneficial effect of AEC-SC on psoriasis-like skin lesions and skin inflammation of mice were significantly impaired when neutralizing with IL-1ra antibody, while the recombinant human IL-1ra showed a less protective effect than AEC-SC.

Conclusions: The present study demonstrated that AEC-SC could efficiently ameliorate psoriasis-like skin lesions and skin inflammation and IL-1ra plays an essential role. Therefore, topical administration of AEC-SC may provide a novel strategy for treating psoriasis-like inflammatory skin diseases.

Keywords: AEC-SC; IL-1ra; Psoriasis; Skin inflammation; Skin lesions.

Fig. 1

AEC-SC attenuated IMQ-induced psoriasis-like skin lesions. a A schematic illustration of animal experiment design. b Representative phenotypic image of mouse back skin after 6-day IMQ administration and different medium treatment. CON represents normal control mice. c Representative images of H&E staining of the back skin of mice in different groups. Scale bar: 50 μm. d Quantification of the epidermal thickness of mice in different groups. The data are expressed as mean ± SEM. At least six representative sections of the skin of mice were counted. At least three mice were used in each group. **P < 0.01, ***P < 0.001. e Immunostaining of back skin tissue of mice with Ki67 antibody and the representative images are shown. Scale bar: 50 μm. f Qualification of the number of Ki67 positive cells per field of the back skin of mice in different groups. g The relative mRNA levels of FLG, HENR and IVL in the back skin of mice in different groups. The mRNA levels of genes in CON group were set as 1. Data were collected from at least 3 mice and the data are expressed as the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

AEC-SC reduced the inflammation of back skin in IMQ-induced psoriasis-like mice. a Immunostaining of back skin tissue with a Gr-1 antibody and the representative images are shown. Scale bar: 50 μm. b Qualification of the number of Gr-1 positive cells of lesion skin tissue per field in different groups. At least 6 fields for each group were counted and at least three mice were used in each group. **P < 0.01, ***P < 0.001. c Flow cytometry analysis of IL7-producing δγ T cells (IL-17⁺ γδ T cells) and IL-17-producing γδ TCR negative cells (Th17 cells). d Quantification of IL-17⁺ δγ T cells and Th17 cells. e The relative mRNA levels of IL-1β, IL-17A, IL-23, IL-6, TNFα, CXCL1, and CCL20 in the lesion skin of mice in each group. The mRNA levels of genes in CON group were set as 1 and the data are expressed as the mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Analysis of paracrine factors in the AEC-SC and UMSC-SC. a Soluble factors in the AEC-SC and UMSC-SC were analyzed by a human antibody array 507. HEF-SC was used as a control. The normalized array data of 507 proteins in the secretome from different cells were analyzed by SAM, and the relative concentrations of these factors were shown as a “heat map”. b The relative concentrations of anti-inflammatory meditors were shown as a heat map. c ELISA analysis of IL-1ra concentration in the groups of HEF-SC, UMSC-SC, AEC-SC, and AEC-SC neutralized with IL-1ra antibody

Fig. 4

Anti-inflammatory factor IL-1ra is necessary for AEC-SC-mediated amelioration on skin lesions of IMQ-induced psoriasis-like mice. a Representative phenotypic image of mouse back skin  after 6-day IMQ administration and different medium treatment. b Representative image of H&E staining of the back skin of mice in different groups. Scale bar: 50 μm. c Quantification of epidermal thickness of mice in different groups. d Immunostaining of lesion skin tissue with Ki67 antibody and the representative images are shown. Scale bar: 50 mm. e Qualification of the number of Ki67 positive cells for per field of the lesion skin tissue in different groups. f Relative mRNA levels of FLG, HENR and IVL in the lesion skin tissue of mice in different groups. The mRNA levels of genes in CON group were set as 1 and the data are expressed as the mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Anti-inflammatory factor IL-1ra is necessary for AEC-SC-mediated immunosuppression on skin inflammation of IMQ-induced psoriasis-like mice. a Immunostaining of back skin tissues with a Gr-1 antibody and the representative images are shown. Scale bar: 50 μm. b Qualification of the number of Gr-1 positive cells of back skin for per field in different groups. c Flow cytometry analysis of IL-17-producing γδ T cells and Th17 Cell. d Quantification of IL-17⁺ γδ T cells and Th17 cells. e, f The relative mRNA levels of IL-1β, IL-17A, IL-23, TNF-a, IL-6, CXCL1 and CCL20 in the lesion skin tissue of mice in each group by real-time PCR analysis. *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 6

AEC-SC inhibited the inflammation stimulated by TNFα in HaCaT karatinocytes and IL-1ra mediated the function of AEC-SC. a The relative mRNA levels of IL-1β, IL-6 and CCL20 in HaCaT cells in each group as indicated. Data was collected from at least 3 separate experiments and the data are expressed as the mean ± SEM.*P < 0.05, **P < 0.01 and ***P < 0.001. b Western blot analysis of the protein levels of NF-κb p-P65 and MAPK p-P38 in the cell lysates of HaCaT cells. HaCaT cells were pretreated with AEC-SC or AEC-SC + IL-1ra Ab or IL-1ra for 4 h, then stimulated with TNFα for 15 min. c Qualification of relative protein levels of p-P65 and p-P38 in the cell lysates