Abstract:

【Abstract】Objective This study aimed to elucidate whether Med1 is involved in the development and progression of atopic dermatitis (AD) by regulating the expression of key inflammatory factors in keratinocytes. Methods Six-week-old female wildtype C57BL/6J mice, Med1^fl/fl mice, and K14Cre-Med1^fl/fl mice were used. Each genotype was divided into a control group (ethanol) and a model group (MC903). Mice in the model group received daily topical application of MC903 in ethanol on both ears for 13 consecutive days to establish an AD-like dermatitis model, whereas the control group received an equal volume of ethanol. First, immunohistochemical staining was performed to detect Med1 expression levels in lesional skin from patients with AD and in ear skin from MC903-treated wildtype mice. Next, AD-like dermatitis models were established in Med1^fl/fl and K14Cre-Med1^fl/fl mice to investigate the effect of Med1 in keratinocytes on the progression of AD-like inflammation. H&E staining was performed to evaluate epidermal thickness and inflammatory cell infiltration. Quantitative real-time PCR (qRT-PCR) was used to measure mRNA expression of inflammatory factors (IL-4, IL-6, S100A8/9), chemokines (CXCL1/2/3/5, CCL6/8), and receptors (CXCR2, ST2, CCR3) in whole skin. Histopathological staining was employed to evaluate the infiltration of neutrophils, eosinophils, mast cells, and T cells. Flow cytometry was further performed to assess the immune cell infiltration in ear skin. Mouse epidermis was isolated, and qRT-PCR was performed to detect the expression of key inflammation?related genes (IL-1β, S100A8, S100A9, CXCL2, CXCL3, CCL11), while immunohistochemical staining was used to detect the expression of IL-1β, CCR3, and CXCL1. Finally, primary mouse keratinocytes were isolated, cultured, and stimulated with MC903 to detect changes in the expression of key inflammatory factors. For comparisons of Med1 expression between two groups, an unpaired t-test was used. For comparisons among multiple groups, homogeneity of variance was tested first, followed by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. Results Compared with normal skin, Med1 expression was significantly upregulated in the epidermis of AD patient lesions and MC903-treated mouse ears (P < 0.001). Compared with Med1^fl/fl mice, K14Cre-Med1^fl/fl mice exhibited significantly reduced ear thickness after MC903 treatment on day 14, the mean ± SD was 398.70 ± 24.17 μm for Med1^fl/fl mice compared with 238.06 ± 59.87 μm for K14Cre-Med1^fl/fl mice (P < 0.001). H&E staining showed decreased epidermal thickness (54.32±6.99 μm vs. 32.17±4.101 μm, P < 0.001). qRT-PCR revealed that the expression of cytokines (IL-4, IL-6), chemokines (CCL6, CCL8), and key receptors (ST2, CCR3) in whole skin of K14Cre-Med1^fl/fl mice was significantly reduced compared to that in Med1^fl/fl mice (all P < 0.01). The expression of neutrophil?associated mediators (S100A8, S100A9), chemokines (CXCL1, CXCL2, CXCL3, and CXCL5) and receptors (CXCR2) was also significantly downregulated (all P < 0.01). Flow cytometry further confirmed that the most prominent change in skin lesions of Med1-knockout mice was in neutrophils. The proportion of neutrophils in K14Cre-Med1^fl/fl mice was significantly lower than that in Med1^fl/fl mice (5.88 ± 0.79% vs. 15.77 ± 1.22%, P < 0.001), and both the magnitude of the decrease and the cell proportion were the largest among the immune cells examined. Furthermore, qRT-PCR showed that epidermal Med1 deficiency inhibited the expression of multiple inflammatory mediators (IL-1β, S100A8, S100A9) and chemokines (CXCL3, CCL11) (all P < 0.01), and immunohistochemical staining confirmed that Med1 deficiency suppressed the expression of IL-1β, CCR3, and CXCL1 in the epidermis (all P < 0.01). In vitro experiments further verified that after MC903 stimulation of primary keratinocytes, Med1 deficiency inhibited the mRNA expression of IL-1α, CXCL1, and CXCL2 (all P < 0.01). Conclusion Med1 in keratinocytes promotes neutrophil infiltration by regulating the expression of multiple key inflammatory cytokines and chemokines, thereby participating in the development and progression of AD-like dermatitis.

Key words: Mediator complex subunit 1 (Med1), Keratinocytes (KCs), Atopic dermatitis (AD), Calcipotriol (MC903), Inflammation