Effect of γ-secretase inhibitor on Th17 cell differentiation and function of mouse psoriasis-like skin inflammation

Abstract

Background: Th17 cells and its effective cytokine IL-17A play an important role in the pathogenesis of abnormal immune responses in psoriasis. Notch1 signaling has been implicated in Th17 cell differentiation and function. In this study, our aim was to evaluate the possible inhibitory effect of Notch1 signaling inhibitor, γ-secretase inhibitor DAPT, on psoriatic Th17 cell differentiation and function in a mouse model of psoriasis-like skin inflammation.

Methods: Mouse psoriasis-like skin inflammation model was established by topical 5% imiquimod (IMQ) application, and experimental mice were divided into control group, IMQ-treated group and IM + DAPT-treated group. DAPT and the equivalent amount of Dimethyl sulfoxide was intraperitoneally injected in IMQ + DAPT-treated group and the other two experimental groups respectively. Skin tissues of the three experimental groups were acquired and stained with haematoxylin and eosin (HE). Splenic single-cells and serum were collected to detect the percentage of Th17 cells, the mRNA expression levels of Notch1 and its target gene Hes-1, Th17-specific transcription factor RORγt and its effective cytokines IL-17A, as well as IL-17A serum concentration. In addition, splenic CD4⁺ T cells from IMQ-treated mice were isolated and treated by DAPT to further measure the inhibitory effect of DAPT on the Th17 cell differentiation and IL-17A secretion in vitro.

Results: DAPT treatment alleviated the severity of IMQ-induced mouse psoriasis-like skin inflammation and decreased the scores of erythema, scaling and thickening. HE stain reveals obviously reduced epidermal hyperplasia and dermal inflammatory cells infiltration in IMQ + DAPT-treated mice. The increased expression of splenic Th17 cell percentage, along with Notch1, Hes-1, RORγt and IL-17A mRNA and IL-17A serum concentration in IMQ-treated mice were significantly decreased when experimental mice were treated by IMQ and DAPT combinedly. Data obtained from in vitro study in IMQ-treated mice also demonstrated that blocking Notch1 signaling by DAPT can result in a dose-dependent decrease of Th17 cell proportion, mRNA expression of Notch1, Hes-1, RORγt and IL-17A as well as IL-17A secretion in splenic CD4⁺ T cells.

Conclusion: These data suggest that Notch1 inhibition by DAPT can effectively alleviate the severity of mouse psoriasis-like skin inflammation by regulating the differentiation and function of Th17 cells, indicating that DAPT might be a potential therapeutic candidate for the treatment of psoriatic inflammation.

Keywords: Notch1 signaling; Psoriasis; Th17 cells; γ-secretase inhibitor.

Fig. 1

γ-secretase inhibitor DAPT Alleviates the Severity of IMQ-Induced Psoriasis-like Skin Inflammation BALB/c mice were treated daily with IMQ cream (IMQ-treated and IMQ + DAPT-treated groups) or control cream (vaseline group) applied on their shaved back skin. In IMQ + DAPT-treated group, in addition to the topical IMQ application, the mice were co-treated daily with intraperitoneal injection of γ-secretase inhibitor DAPT (10 mg/kg/day). In control and IMQ-treated mice, they received an equal amount of DMSO. After 7 consecutive days of experimental process, mice were euthanized. A Phenotypical presentation of mouse back skin after 7 days of treatment. Control mice (a) did not present any sign of inflammation. IMQ-treated mice (b) presented significant inflammation changes of erythema, scaling and thickening. The severity of inflammation was obviously alleviated in IMQ + DAPT-treated mice (c). B Erythema, scale, and thickness were scored daily on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. The cumulative score was calculated by scores of erythema plus scale plus thickness (scale 0–12). Each score of IMQ-treated mice was higher than that of control mice (^#P < 0.01), while, after DAPT and IMQ co-treatment, all of these scores were reduced (^#P < 0.01). C Pathological examination of mouse back skin after 7 days of treatment observed by haematoxylin and eosin (HE) stain. The epidermis layer of control mice (a) was thin and composed by only 1–2 layers of epidermal cells. IMQ-treated mice (b) presented obviously epidermal hyperplasia with hyperkeratosis, parakeratosis, Munro microabscess and elongation of trochanterellus as well as massive dermal infiltration of inflammatory cells with dilated capillaries. The severity of epidermal hyperplasia and dermal inflammatory cells infiltration were significantly reduced in IMQ + DAPT-treated mice (c)

Fig. 2

DAPT reduces IMQ-induced splenomegaly mice were euthanized after 7 consecutive days of experimental process, and the spleen mass (a) and spleen index (b) were observed. Compared to control mice, the spleen mass and spleen index of IMQ-treated mice were significant increased (^#P < 0.01), while combined treatment of DAPT and IMQ led to the reduction of spleen mass and spleen index to a great degree (^#P < 0.01)

Fig. 3

γ-secretase inhibitor DAPT decrease the mRNA expression of Notch1 and its target gene Hes-1. Mice were euthanized after 7 consecutive days of experimental process, and RNA samples from splenic single-cells was used for detection. Both the mRNA expression of Notch1 (a) and Hes-1 (b) was obviously enhanced in IMQ-treated mice compared to control mice (^#P < 0.01). In IMQ + DAPT-treated mice, Notch1 and Hes-1 mRNA expression was significant decrease (^#P < 0.01). Splenic CD4⁺ T cells from 8 IMQ-treated mice were collected and treated with DAPT, Notch1 (c) and Hes-1 (d) mRNA expression presented a dramatically reduced trend with the increased concentration of DAPT (^#P < 0.01)

Fig. 4

γ-secretase inhibitor DAPT down-regulates the percentage of Th17 cells. Mice were euthanized after 7 consecutive days of experimental process, and percentages of splenic Th17 cells were determined. a Representative graphs of flow cytometric analysis of Th17 cell percentage (CD4 was labeled by FITC and IL-17A was labeled by PE) in control mice, IMQ-treated mice and IMQ + DAPT-treated mice. b Th17 cell percentage of IMQ-treated mice was obviously enhanced compared to control mice (^#P < 0.01), while the percentage was significantly reduced in IMQ and DAPT combinedly treated experimental mice (^#P < 0.01). c After interruption by DAPT in vitro, splenic CD4⁺ T cells of IMQ-treated mice was polarized and detected the change of Th17 cell percentage, which was gradually down-regulated in a dose-dependent matter of DAPT (^#P < 0.01)

Fig. 5

γ-secretase inhibitor DAPT reduces the expression and secretion of IL-17A. Both IL-17A mRNA expression level (a) and serum concentration (b) in IMQ-treated mice were significantly elevated in comparison with control mice (^#P < 0.01), while the IL-17A expression levels were reduced in IMQ + DAPT-treated mice (^#P < 0.01). After DAPT treatment and Th17-cell polarization in vitro, IL-17A mRNA expression in splenic CD4⁺ T cells (c) and its concentration in cell-free supernatant (d) of IMQ-treated mice were decreased in DAPT treated groups compared to control group in a dose-dependent way (^#P < 0.01)

Fig. 6

γ-secretase inhibitor DAPT inhibits the expression of RORγt mRNA. a RORγt mRNA expression in splenic single-cell suspension was significantly elevated in IMQ-treated mice compared to control mice (^#P < 0.01), while it was obviously decreased in IMQ + DAPT-treated mice (^#P < 0.01). b After DAPT treatment and Th17-cell polarization in vitro, RORγt mRNA expression was significantly decreased with the increased dose of DAPT in splenic CD4⁺ T cells of IMQ-treated mice (^#P < 0.01)