Development of atopic dermatitis-like skin disease from the chronic loss of epidermal caspase-8

Abstract

Atopic dermatitis is an inflammatory skin disease that affects approximately 20% of children worldwide. Left untreated, the barrier function of the skin is compromised, increasing susceptibility to dehydration and infection. Despite its prevalence, its multifactorial nature has complicated the unraveling of its etiology. We found that chronic loss of epidermal caspase-8 recapitulates many aspects of atopic dermatitis, including a spongiotic phenotype whereby intercellular adhesion between epidermal keratinocytes is disrupted, adversely affecting tissue architecture and function. Although spongiosis is generally thought to be secondary to edema, we found that suppression of matrix metalloproteinase-2 activity is sufficient to abrogate this defect. p38 MAPK induces matrix metalloproteinase-2 expression to cleave E-cadherin, which mediates keratinocyte cohesion in the epidermis. Thus, the conditional loss of caspase-8, which we previously found to mimic a wound response, can be used to gain insights into how these same wound-healing processes are commandeered in inflammatory skin diseases.

Fig. 1.

AD characteristics in C8 cKO mice. (A) Expression of caspase-8 in normal and human AD skin measured by quantitative PCR. (B) Appearance of WT and C8 cKO at postnatal day 10. (C) Appearance of ears and paws in WT and KO animals. (D) Blood vessel population denoted by CD31 staining (green) in the WT and KO skin stained with keratin 5 (K5; red). (E) H&E of caspase-8–null skin treated with vehicle control (KO+ vehicle) or clobetasol (KO + clobetasol) twice daily for 10 d. (Scale bars, 30 μm.)

Fig. 2.

Immune cell profile in C8 CKO skin. Representative quantitative RT-PCR on three sets of skin samples from WT (blue) and KO (red) 10-d- or 150-d-old mice wherein WT samples were normalized to one. Error bars are SEM of experiments performed in triplicate. (A) T_H2 signature genes. (B) T_H2 chemokines. (C) T_H1 signature genes. (D) T_H1 chemokines. (E) Toluidine blue staining of 10-d- or 150-d-old skin from WT and KO mice. Epidermis (epi) and hair follicle (hf) are shown in blue and mast cells in the dermis (der) are shown in purple. Arrowhead indicates mast cells. (Scale bars, 30 μm.)

Fig. 3.

Serum levels of Ig from WT (blue) and C8 cKO (red) were determined by ELISA. (A) IgG1 levels and (B) IgE levels in 10-d- and 150-d-old mice. Results are representative of three sets of mice performed in triplicate and error bars indicate SEM.

Fig. 4.

C8 cKO mice have altered epidermal structure and function. (A) H&E staining of WT and KO skin from 5-mo-old mice. Right: Magnified views of boxed areas (Left). (B) Staining for E-cadherin (Ecad; green), laminin (lam; red) and nuclei (DAPI; blue) in WT and KO skin sections. Epi, epidermis; der, dermis; hf, hair follicle. Right: Magnified views of the boxed area (Left). (Scale bars, 30 μm.) Western blots using actin as a control on 5 mo WT and KO skin to detect intercellular adhesion proteins (C) and filaggrin (D). (E) TSLP levels secreted from the epidermis or in the serum of young (15 d) or adult (150 d) mice treated with vehicle control (veh) of clobetasol (clo). (F) Measurement of TEWL under similar conditions as E. Results are representative of three readings taken on different regions of the back from three sets of animals. Error bars indicate SEM.

Fig. 5.

MMP-2 expression and function. (A–C) RT-PCR using GAPDH as a loading control of five animals of each genotype performed in triplicate. (A) Expression of MMP-2, MMP-9, MMP-13, and keratin 1 and vimentin in postnatal day 10 WT versus KO skin. (B) Effect of p38 MAPK inhibitor on MMP-2 expression in postnatal day 10 skin. (C) Ability of the p38 MAPK chemical activator anisomycin to induce MMP-2 expression in WT epidermis. (D) Western blot of MMP-2 protein under conditions listed in B and C in young (10 d) and adult (150 d) mouse. (E) Effect of MMP-2 activity on adherens junctions in keratinocytes in vitro. E-cadherin is labeled in green and the nuclei labeled with DAPI is in blue. Keratinocytes were treated with buffer, recombinant MMP-2, or MMP-2 plus MMP-2 inhibitor (Inh). (Scale bars, 30 μm.) (F) Western blot of lysates made from cells treated with MMP-2 inhibitor using an antibody that recognizes Ecad FL and Ecad CTF following cleavage of the protein. Tubulin was used as a loading control. (G) Immunohistochemistry of phosphorylated p38 MAPK in KO skin from young mice treated with vehicle control or clobetasol. Dotted line denotes the basement membrane separating the epidermis (epi) from the dermis (der). (H) Western blot of Ecad FL, MMP-2, and tubulin of adult WT and KO skin treated with vehicle control of clobetasol.

Fig. 6.

Role of MMP-2 in the spongiotic phenotype (A) H&E staining of skin sections from adult KO mice injected with DMSO (KO + DMSO) vehicle control or MMP-2 inhibitor (KO + MMP2I). (Right) Magnified views of the boxed area (Left). (Scale bars, 30 μm.) (B) Western blot of proteins extracted from skin samples in A. Ecad FL and Ecad CTF were detected with an antibody recognizing both forms of the protein, and GAPDH was used as a loading control. (C) TEWL on adult WT and KO mice and KO mice treated with DMSO, MMP-2 inhibitor (MMP2I), vehicle control (veh), or clobetasol (clob). Results are representative of three independent experiments with three readings taken on the back skin per mouse. Error bars indicate SEM.