IL-25 enhances HSV-1 replication by inhibiting filaggrin expression, and acts synergistically with Th2 cytokines to enhance HSV-1 replication

Abstract

Atopic dermatitis (AD) is characterized by epidermal barrier defects and recurrent microbial skin infections. AD patients with a history of eczema herpeticum (ADEH+) have more severe skin disease and more highly T helper type 2 (Th2)-polarized immune responses as compared with uncomplicated AD (ADEH-). However, the mechanisms linking epidermal barrier defects and viral skin infection are not well understood. Recently, it has been reported that interleukin-25 may play a role in augmenting Th2 responses. We examined protein expression of IL-25 in the skin biopsies from normal subjects (n=10), ADEH- (n=18), ADEH+ (n=7), and psoriasis (n=9). IL-25 expression was increased in the skin from ADEH-, ADEH+, and psoriasis as compared with normal skin, and was significantly greater in lesional ADEH+ skin than in lesional ADEH- skin. Importantly, we demonstrated that IL-25 enhances herpes simplex virus (HSV)-1 and vaccinia virus replication by inhibiting filaggrin expression, and IL-25 acts synergistically with IL-4 and IL-13 to enhance HSV-1 replication in vitro. In contrast, IFN-γ inhibited HSV-1 replication in vitro. In addition, we demonstrate that filaggrin is a critical protein to inhibit HSV-1 replication because filaggrin small interfering RNA knockdown enhances HSV-1 replication in vitro. Filaggrin breakdown products, however, inhibited HSV-1 replication in vitro.

Figure 1. The expression of IL-25 in human skin

(a) Representative paraffin embedded skin biopsies from normal subjects (n=10) and patients with ADEH− (n=18), ADEH+ (n=7) and psoriasis (n=9) stained for IL-25 (red) are shown. Wheat germ agglutinin-conjugated fluorescein isothiocyanate (green) stained the cytoskeleton. Images were collected at x 400 magnification. Arrows point to IL-25 expression. Bar=50 μm. (b) The mean fluorescent intensity of IL-25 is shown in the epidermis of each biopsy. *P < 0.05, **P < 0.01.

Figure 2. IL-25 downregulates filaggrin expression, and acts synergistically with T_H2 cytokines to inhibit filaggrin expression

KCs were stimulated in the various concentrations of IL-25, T_H2 cytokines (50 ng/mL of IL-4 and 50 ng/mL of IL-13), a combination of IL-25 (50 ng/mL) and T_H2 cytokines or IFN-γ (20 ng/mL) for 5 days. (a) The gene expression of FLG was examined using real-time RT-PCR. (b) The protein expression of filaggrin was evaluated by western blot analysis. (c) Relative expression of profilaggrin in 250 KD and bigger profilaggrin bands. Data from 1 representative experiment of 3 independent experiments performed are shown. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. IL-25 enhances viral replications, and acts synergistically with T_H2 cytokines to enhance viral replication

KCs were differentiated in the absence or presence of IL-25 (50 ng/mL), T_H2 cytokines (50 ng/mL of IL-4 and 50 ng/mL of IL-13), a combination of IL-25 and T_H2 cytokines or IFN-γ (20 ng/mL) for 5 days. Then, the cells were incubated with HSV-1 (MOI, 0.1) for an additional 24 hours. The gene expression of HSV-1 (a), LL-37 (c), and HBD-3 (d) was examined using real-time RT-PCR. (b) Organotypic skin sections were stained for vaccinia virus (red) and the cytoskeleton (green). Arrows point to vaccinia virus. Bar=50 μm. Data from 1 representative experiment of 3 independent experiments performed are shown. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. FLG silencing enhances HSV-1 replication

KCs were transfected with scrambled siRNA or FLG siRNA, and the cells were differentiated for 5 days. Then, the cells were incubated with HSV-1 (MOI, 0.1) for an additional 24 hours. (a) The gene expression of FLG in the KCs was examined using real-time RT-PCR. (b) The protein expression of filaggrin and profilaggrin was evaluated by western blot technique. (c) The gene expression of HSV-1 was examined using real-time RT-PCR. (d) Viral plaque formation was determined by using the viral plaque assay. Data from 1 representative experiment of 3 independent experiments performed are shown. *P < 0.05, **P < 0.01.

Figure 5. Filaggrin breakdown products and an acidic environment inhibit HSV-1 replication

KCs were incubated with HSV-1 (MOI, 0.1) in the presence of various concentrations of UCA and PCA (a) or hydrochloric acid (b) for 24 hours. The gene expression of HSV-1 was examined using real-time RT-PCR. Data from 1 representative experiment of 3 independent experiments performed are shown. **P < 0.01, ***P < 0.001.