Advertisement

Caspase-14-Deficient Mice Are More Prone to the Development of Parakeratosis

      Caspase-14 is an important protease in the proper formation of a fully functional skin barrier. Newborn mice that are deficient in caspase-14 exhibit increased transepidermal water loss and are highly sensitive to UVB-induced photodamage. Decreased caspase-14 expression and incomplete caspase-14 processing in lesional psoriatic parakeratotic stratum corneum has been reported previously. In this study, we show that caspase-14-deficient skin frequently displays incompletely cornified cells in the transitional zone between the granular and the cornified layers, pointing to a delay in cornification. We also demonstrate that after challenge of epidermal permeability barrier function by repetitive acetone treatment, a higher incidence of large parakeratotic plaques was observed in caspase-14-deficient skin. Furthermore, caspase-14-deficient mice are more prone than control mice to the development of parakeratosis upon induction of psoriasis-like dermatitis by imiquimod treatment. These results show that lack of caspase-14 expression predisposes to the development of parakeratosis and that caspase-14 has an important role in keratinocyte terminal differentiation and the maintenance of normal stratum corneum, especially in conditions causing epidermal hyperproliferation.

      Abbreviations

      CE
      cornified envelope
      IMQ
      imiquimod
      KO
      knockout
      SC
      stratum corneum
      TEWL
      transepidermal water loss
      WT
      wild type

      Introduction

      Skin barrier function is crucial for terrestrial mammalian life, as it protects the body against water loss. The epidermis mainly consists of keratinocytes, which differentiate to produce a functional skin barrier. An impaired balance between keratinocyte proliferation and differentiation causes a variety of skin disorders, including psoriasis, dermatitis, and skin cancer. Recently, it has become clear that defects in the epidermal barrier can underlie the development of inflammatory skin diseases (
      • Proksch E.
      • Brandner J.M.
      • Jensen J.M.
      The skin: an indispensable barrier.
      ). The epidermal permeability barrier function is pivotally maintained by two components of the stratum corneum (SC), namely the cornified envelopes (CEs), which are the end products of keratinocytes undergoing terminal differentiation, and the lipid-enriched extracellular matrix enshrouding these CEs.
      Caspases are cysteinyl aspartate–specific proteases that exert their functions during apoptosis and inflammation (
      • Lamkanfi M.
      • Declercq W.
      • Depuydt B.
      • et al.
      ). Recently, it has become clear that caspases also have important functions in maintaining epidermal homeostasis. Caspase-14 is the sole caspase that is fully processed during physiological keratinocyte terminal differentiation (
      • Fischer H.
      • Stichenwirth M.
      • Dockal M.
      • et al.
      Stratum corneum-derived caspase-14 is catalytically active.
      ;
      • Raymond A.A.
      • Mechin M.C.
      • Nachat R.
      • et al.
      Nine procaspases are expressed in normal human epidermis, but only caspase-14 is fully processed.
      ). Its expression is constrained to the suprabasal layers of the epidermis and the thymic Hassall’s bodies in mice and humans (
      • Eckhart L.
      • Declercq W.
      • Ban J.
      • et al.
      Terminal differentiation of human keratinocytes and stratum corneum formation is associated with caspase-14 activation.
      ;
      • Lippens S.
      • Kockx M.
      • Knaapen M.
      • et al.
      Epidermal differentiation does not involve the pro-apoptotic executioner caspases, but is associated with caspase-14 induction and processing.
      ), and to the forestomach of rodents (
      • Lippens S.
      • VandenBroecke C.
      • Van Damme E.
      • et al.
      Caspase-14 is expressed in the epidermis, the choroid plexus, the retinal pigment epithelium and thymic Hassall’s bodies.
      ;
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ). These tissues contain cornified epithelial cells and express additional terminal differentiation markers such as filaggrin and loricrin (
      • Jarnik M.
      • Kartasova T.
      • Steinert P.M.
      • et al.
      Differential expression and cell envelope incorporation of small proline-rich protein 1 in different cornified epithelia.
      ). Caspase-14-deficient mice show defects in skin barrier function reflected in a higher transepidermal water loss (TEWL) and increased sensitivity to UVB-induced photodamage. Identification of (pro)filaggrin as the first, and currently only, in vivo substrate of caspase-14 provides further evidence that caspase-14 has a crucial role in epidermal barrier function (
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ;
      • Hoste E.
      • Kemperman P.
      • Devos M.
      • et al.
      Caspase-14 is required for filaggrin degradation to natural moisturizing factors in the skin.
      ). We have previously shown that caspase-14 expression is significantly downregulated in parakeratotic plaques of psoriatic patients (
      • Lippens S.
      • Kockx M.
      • Knaapen M.
      • et al.
      Epidermal differentiation does not involve the pro-apoptotic executioner caspases, but is associated with caspase-14 induction and processing.
      ). Treatment of these patients with vitamin D3 analogs or with epigallocatechin-3-gallate, two inducers of caspase-14 expression, resulted in clinical improvement accompanied by increased caspase-14 expression levels (
      • Lippens S.
      • Kockx M.
      • Denecker G.
      • et al.
      Vitamin D3 induces caspase-14 expression in psoriatic lesions and enhances caspase-14 processing in organotypic skin cultures.
      ;
      • Hsu S.
      • Dickinson D.
      • Borke J.
      • et al.
      Green tea polyphenol induces caspase 14 in epidermal keratinocytes via MAPK pathways and reduces psoriasiform lesions in the flaky skin mouse model.
      ). Furthermore, in parakeratotic human SC, caspase-14 is present partly in its zymogen form (
      • Fischer H.
      • Stichenwirth M.
      • Dockal M.
      • et al.
      Stratum corneum-derived caspase-14 is catalytically active.
      ;
      • Raymond A.A.
      • Mechin M.C.
      • Nachat R.
      • et al.
      Nine procaspases are expressed in normal human epidermis, but only caspase-14 is fully processed.
      ), whereas in normal SC, caspase-14 is completely processed and uniquely present in its activated state. The importance of caspases in skin homeostasis has been reinforced by the finding that mice lacking epidermal caspase-8 exhibit a strong inflammatory skin phenotype (
      • Kovalenko A.
      • Kim J.C.
      • Kang T.B.
      • et al.
      Caspase-8 deficiency in epidermal keratinocytes triggers an inflammatory skin disease.
      ;
      • Lee P.
      • Lee D.J.
      • Chan C.
      • et al.
      Dynamic expression of epidermal caspase 8 simulates a wound healing response.
      ). However, mechanistically, caspase-8 and caspase-14 are acting at different levels. A low level of caspase-8 activity is required to prevent RIPK1 (receptor interacting kinase)- and RIK3-mediated necrotic cell death that leads to skin inflammation (
      • Kaiser W.J.
      • Upton J.W.
      • Long A.B.
      • et al.
      RIP3 mediates the embryonic lethality of caspase-8-deficient mice.
      ), whereas caspase-14 is required for filaggrin degradation during keratinocyte cornification (
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ;
      • Hoste E.
      • Kemperman P.
      • Devos M.
      • et al.
      Caspase-14 is required for filaggrin degradation to natural moisturizing factors in the skin.
      ).
      Psoriasis is a common inflammatory skin disorder manifested in a fine, silvery scaling on the affected skin regions and characterized by excessive keratinocyte growth, decreased barrier function (
      • Ghadially R.
      • Reed J.T.
      • Elias P.M.
      Stratum corneum structure and function correlates with phenotype in psoriasis.
      ), infiltration of neutrophils and mononuclear leukocytes (T cells and dendritic cells), and defective differentiation in the lesional epidermis, resulting in parakeratotic plaque formation (reviewed in
      • Bowcock A.M.
      • Krueger J.G.
      Getting under the skin: the immunogenetics of psoriasis.
      ;
      • Lowes M.A.
      • Bowcock A.M.
      • Krueger J.G.
      Pathogenesis and therapy of psoriasis.
      ). Parakeratosis is defined as incomplete cornification in which nuclei and DNA are still observed in the SC. It occurs in several dermatological disorders, but the molecular mechanisms leading to parakeratotic plaque formation are largely unknown (
      • Brady S.P.
      Parakeratosis.
      ). Although more insight into epidermal DNA catabolism has been gained during recent years, the process of nuclear degradation during cornification is still poorly understood (reviewed in
      • Eckhart L.
      • Fischer H.
      • Tschachler E.
      Mechanisms and emerging functions of DNA degradation in the epidermis.
      ). Recently, it has been shown that repeated topical application of imiquimod (IMQ) on mouse skin results in an inflammatory cutaneous response resembling psoriasis (
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ;
      • Swindell W.R.
      • Johnston A.
      • Carbajal S.
      • et al.
      Genome-wide expression profiling of five mouse models identifies similarities and differences with human psoriasis.
      ). Patients with a strong predisposition to psoriasis show exacerbation or relapse of psoriatic features upon IMQ treatment. IMQ binds Toll-like receptor-7 and -8, which are present on antigen-presenting cells, and upon binding of their natural or synthetic ligands they activate the innate immune system to produce several proinflammatory cytokines such as IFN-γ, tumor necrosis factor, IL-6, and IL-12 (
      • Wagner T.L.
      • Ahonen C.L.
      • Couture A.M.
      • et al.
      Modulation of TH1 and TH2 cytokine production with the immune response modifiers, R-848 and imiquimod.
      ). As in psoriasis patients, the IL-23/IL-17 axis is of key importance in IMQ-induced psoriasis-like dermatitis in mice (
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ).
      Here, we describe spontaneous ultrastructural abnormalities in caspase-14-deficient corneocytes pointing to defective or delayed keratinocyte differentiation. We also show that caspase-14−/− mice are more prone to the development of parakeratosis upon treatment with IMQ to induce psoriasis-like dermatitis. These data indicate the importance of caspase-14 for correct keratinocyte cornification, especially under challenging conditions that trigger epidermal hyperproliferation.

      Results

      Electron microscopic analysis reveals ultrastructural abnormalities in caspase-14−/− skin

      Caspase-14−/− mice exhibit a defect in the epidermal permeability barrier, and neonates display a shiny and hyperlichenified skin (
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ). As this phenotype is most prominent in caspase-14 neonates between P1.5 and P5.5, a detailed electron microscopic analysis was performed on the skin of newborn wild-type (WT) and caspase-14−/− mice at P3.5 and on adult skin at the age of 9 weeks. At P3.5, caspase-14-deficient skin frequently contains cells resembling transitional cells, characterized by disorganized nonhomogeneous intracellular content at the stratum granulosum/SC interface (Figure 1a). In some cases, such cells are found up to the fourth cornified layer (SC4). Similar but less frequent incompletely cornified cells were observed in adult caspase-14-deficient epidermis (Figure 1a). These cells were far less abundant in WT counterparts. The occurrence of such cells points to a delay in cornification, although no structural differences between CEs in WT versus caspase-14−/− skin were apparent (Figure 1b). We assessed whether the observed structural differences were due to defects in DNA degradation in caspase-14−/− skin by performing TUNEL staining, but TUNEL-positive cells were scarce in both WT and caspase-14−/− epidermis (data not shown). In addition, by using Hoechst staining in combination with differential interference contrast microscopy, we were unable to detect the remaining DNA in the cornifying layers in both genotypes (Supplementary Figure S1 online). No differences were observed in extracellular lamellar structures between WT and caspase-14−/− epidermis (data not shown). The structural differences observed in caspase-14-deficient skin did not affect the resistance of the skin to tape strip–induced physical damage compared with WT mice, as evaluated by measuring TEWL after repeated tape stripping (Supplementary Figure S2 online).
      Figure thumbnail gr1
      Figure 1Cornification defects in caspase-14−/−newborns and adults. Skin sections of caspase-14+/+ and caspase-14−/− mice at P3.5 days and at the age of 9 weeks were examined by electron microscopy for stratum corneum (SC) abnormalities. (a) General view of the transitional and cornifying layers in wild-type (WT) and caspase-14−/− skin. Upper panels, bar=2μm; lower panels, bar=1μm. (b) Detailed view on the SC1 layer in WT and caspase-14−/− skin at P3.5 days. Arrowheads indicate the cornified envelope. SG, stratum granulosum; SCx, xth layer of cornified cells. Bar=500nm.

      Epidermal caspase-14 deficiency facilitates the formation of parakeratotic plaques upon topical acetone treatment

      Upon repetitive topical treatment with acetone, the epidermis tries to compensate for the loss in the cutaneous permeability barrier by increasing the formation of corneocytes (hyperkeratosis) (
      • Denda M.
      • Wood L.C.
      • Emami S.
      • et al.
      The epidermal hyperplasia associated with repeated barrier disruption by acetone treatment or tape stripping cannot be attributed to increased water loss.
      ). It has been shown that repeated topical application of acetone removes all stainable neutral lipids from the cornified layers, thereby disrupting the skin barrier. This results in an increase in TEWL and causes a hyperproliferative response (
      • Menon G.K.
      • Feingold K.R.
      • Moser A.H.
      • et al.
      De novo sterologenesis in the skin. II. Regulation by cutaneous barrier requirements.
      ). We challenged the skin of WT and caspase-14−/− mice by repetitive acetone wipes two times a day for 5 consecutive days to test whether more pronounced cornification defects would be present in caspase-14-deficient skin compared with WT skin. Analysis of hematoxylin–eosin-stained skin sections demonstrated that repeated acetone treatment induced parakeratotic plaque formation in the skin of both WT and caspase-14−/− mice (Figure 2). A similar rise of TEWL and hyperproliferation was observed in the two genotypes upon acetone treatment (Figure 2b, Supplementary Figure S3 online). Interestingly, the percentage of parakeratotic SC induced by acetone treatment was higher in caspase-14−/− skin compared with WT skin (Figure 2c). The same trend was observed when counting parakeratotic plaques larger than 100μm (Figure 2d). Our results indicate that caspase-14 is important for the maintenance of normal keratinocyte terminal differentiation and SC architecture under skin barrier–challenging conditions.
      Figure thumbnail gr2
      Figure 2Caspase-14−/− mice develop more and larger parakeratotic plaques upon acetone-induced barrier disruption. (a) Hematoxylin and eosin (H&E)-stained skin sections of wild-tpe (WT) and caspase-14−/− back skin that was left untreated or subjected to repetitive treatment two times daily with saline or acetone wipes for 5 days. Acetone treatment results in parakeratotic plaque formation in WT and caspase-14−/− mice. A representative image of saline (NaCl)- and acetone-treated skin is shown for both genotypes. Bar=200μm. (b) Transepidermal water loss (TEWL) measurements were recorded 24hours after the last treatment. Values of the two genotypes were compared using two-way analysis of variance (ANOVA) testing with Bonferroni post-tests; P-values are indicated with * (<0.05). (c) Parakeratosis was quantified in all experimental groups on H&E-stained skin sections. Values depicted in the graph are the % of parakeratotic stratum corneum (SC) surface. These data were statistically analyzed by a logistic regression model as explained in Materials and Methods. Bars represent mean values±SEM. WT untreated: n=10; knockout (KO) untreated: n=10; WT NaCl: n=10; KO NaCl: n=10; WT acetone: n=16; KO acetone: n=16. The total length of skin analyzed was 107.75mm in WT untreated; 95.25mm in KO untreated; 78.5mm in WT NaCl; 111mm in KO NaCl; 252.25mm in WT acetone; and 351.75mm in KO acetone. (d) Mice treated as in c were analyzed for the number of parakeratotic plaques larger than 100μm over a total section length of 10cm. Only statistically significant differences are indicated by asterisks (b and c).

      Caspase-14-deficient mice are more prone to the development of parakeratosis upon induction of psoriasis-like dermatitis

      The reported reduction of caspase-14 in psoriatic plaques (
      • Lippens S.
      • Kockx M.
      • Denecker G.
      • et al.
      Vitamin D3 induces caspase-14 expression in psoriatic lesions and enhances caspase-14 processing in organotypic skin cultures.
      ;
      • Hsu S.
      • Dickinson D.
      • Borke J.
      • et al.
      Green tea polyphenol induces caspase 14 in epidermal keratinocytes via MAPK pathways and reduces psoriasiform lesions in the flaky skin mouse model.
      ), the observed delay in cornification in caspase-14−/− epidermis (Figure 1), together with the observation that caspase-14−/− mice develop larger parakeratotic plaques upon acetone treatment (Figure 2), led us to trigger a psoriasis-like inflammatory response in caspase-14-deficient skin to investigate whether the lack of caspase-14 facilitates the development of parakeratotic plaques (
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ). We topically treated the back skin of WT and caspase-14−/− mice repeatedly with IMQ to induce a psoriasis-like skin inflammation. Epidermal scaling was scored daily by two independent researchers. Scaling of back skin was apparent in both genotypes after 2 days of IMQ application and increased as treatment continued (Figure 3). After 8 days of IMQ treatment, caspase-14−/− skin showed marked scaling compared with the moderate scaling observed in control skin. Vehicle-treated WT mice exhibited very slight scaling compared with untreated mice. In contrast, caspase-14−/− skin treated with vehicle cream showed moderate scaling. The observed increased scaling in caspase-14−/− skin upon topical vehicle or IMQ treatment could point to a cornification defect in the absence of caspase-14. Indeed, a larger percentage of SC was parakeratotic in vehicle- or IMQ-treated caspase-14−/− skin relative to WT skin as observed on hematoxylin–eosin-stained sections (Figure 4a and b). Notably, vehicle-treated caspase-14−/− skin exhibited marked epidermal thickening, due to hyperproliferation (Supplementary Figure S4 online), in contrast to vehicle-treated WT skin (Figure 4c). IMQ-treated caspase-14−/− mice had a minor, but statistically significant, increase in skin thickness compared with WT counterparts after 8 days of IMQ treatment (Figure 4c). Whether this minor difference is of biological significance is not clear.
      Figure thumbnail gr3
      Figure 3Caspase-14−/− mice are more sensitive to vehicle- and imiquimod (IMQ)-induced epidermal scaling than wild-type (WT) mice. Scaling of the epidermis was scored independently by two researchers on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; and 4, very marked. WT untreated: n=4; knockout (KO) untreated: n=4; WT vehicle: n=6; KO vehicle: n=6; WT IMQ: n=11; and KO IMQ: n=12. All values from both genotypes were compared using two-way analysis of variance (ANOVA) testing with Bonferroni post-tests. Bars represent mean values±SEM. P-values are indicated with *** (<0.001). Only statistically significant differences are indicated by asterisks.
      Figure thumbnail gr4
      Figure 4Caspase-14−/− mice are more prone to the development of parakeratosis than their wild-type (WT) counterparts upon topical imiquimod (IMQ) treatment. (a) Hematoxylin and eosin (H&E) staining of back skin of caspase-14+/+ and caspase-14−/− mice left untreated, treated with vehicle cream, or treated with IMQ-containing cream for 8 consecutive days, after which they were killed and samples obtained. A representative image is shown for each condition. Bar=20μm. (b) Parakeratosis was quantified in all experimental groups on H&E-stained skin sections. Values depicted in the graph represent the percentage of parakeratotic stratum corneum (SC) out of the total SC. These data were statistically analyzed by a logistic regression model as explained in Materials and Methods. Three sections per mouse sample were analyzed. WT untreated: n=8; knockout (KO) untreated: n=8; WT vehicle: n=11; KO vehicle: n=11; WT IMQ: n=20; and KO IMQ: n=21 (c) Epidermal thickness was measured using the ImageJ software—four measurements were performed on each H&E-stained image; three images per mouse were analyzed. The thickness of the epidermis was determined by measuring the distance from the basement membrane to the upper granular layer, excluding the cornified layers. All values from the two genotypes were compared using two-way analysis of variance (ANOVA) testing with Bonferroni post-tests. Bars represent mean values±SEM. P-values are indicated with * (<0.05) or *** (<0.001). (b and c) Only statistically significant differences are indicated by asterisks.
      To exclude the possibility that the observed differential effects are due to a difference in IMQ penetration through the skin, we analyzed the IMQ concentration in the sera of IMQ-treated mice of both genotypes. Serum IMQ levels were similar in WT and caspase-14−/− mice (Supplementary Figure S5a online), indicating that IMQ penetrated equally through WT and caspase-14−/− skin. Consequently, IMQ-treated caspase-14 WT and caspase-14−/− mice showed a similar degree of splenomegaly (Supplementary Figure S5b online). It has been shown previously that topical IMQ treatment results in pronounced splenomegaly in mice (
      • Palamara F.
      • Meindl S.
      • Holcmann M.
      • et al.
      Identification and characterization of pDC-like cells in normal mouse skin and melanomas treated with imiquimod.
      ;
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ). Topical treatment with vehicle cream did not result in a significant increase in spleen mass, indicating that the splenomegaly was solely due to the proinflammatory systemic effects of IMQ.
      The greater susceptibility of caspase-14−/− skin to the development of parakeratosis upon IMQ treatment could be due to differences in the inflammatory status of the skin in both genotypes. Therefore, we analyzed skin immune cell infiltrates in both genotypes after topical IMQ treatment. CD45 leukocytes and CD3 T cells were increased in dermal and epidermal compartments after 8 days of topical IMQ treatment (Figure 5a). The area of the surface showing positive staining was quantified, and no significant difference was found in the amount of CD45+ cells in WT and caspase-14−/− skin recruited upon IMQ treatment (Figure 5b). In addition, CD3 staining did not reveal significant differences in T-cell skin infiltrates between IMQ-treated WT and caspase-14−/− mice (data not shown). Taken together, all these data indicate that caspase-14−/− keratinocytes are less able to complete the normal cornification program in hyperproliferative conditions.
      Figure thumbnail gr5
      Figure 5Imiquimod (IMQ)-treated wild-type (WT) and caspase-14−/− skin have similar numbers of infiltrating leukocytes. (a) Back skin sections of untreated, vehicle-treated, and IMQ-treated caspase-14+/+ and caspase-14−/− mice were probed with anti-CD45 antibody. A representative image is shown for each condition. Bar=40μm. (b) Quantification of diaminobenzidine (DAB) positivity in stained skin sections is represented as DAB-positive area as a percent of total area (epidermis and dermis). Three sections per mouse sample were analyzed. WT untreated: n=12; knockout (KO) untreated: n=12; WT vehicle: n=18; KO vehicle: n=18; WT IMQ: n=31; and KO IMQ: n=34. Values of the two genotypes were compared using a logistic regression model as explained in Materials and Methods. Bars represent mean values±SEM. We could not observe statistically significant differences between the two genotypes.

      Topical IMQ treatment leads to more severe skin barrier perturbation in caspase-14−/− mice

      Scaling of skin can be caused by abnormal stacking of corneocytes and leads to impairment of the epidermal permeability barrier (
      • Elias P.M.
      • Crumrine D.
      • Rassner U.
      • et al.
      Basis for abnormal desquamation and permeability barrier dysfunction in RXLI.
      ). We show that topical IMQ treatment initially led to an increased TEWL in both genotypes, observed from day 4 of treatment (Figure 6). After 8 days of IMQ treatment, a significant difference in TEWL between WT and caspase-14−/− mice was observed, pointing to a more severe skin barrier perturbation in the absence of epidermal caspase-14. Notably, TEWL measurements were also significantly higher in caspase-14−/− mice treated with vehicle cream compared with vehicle-treated WT mice or untreated mice (Figure 6). This observation shows that inflamed caspase-14-deficient skin is more susceptible to barrier perturbation. As mentioned in the previous section, the same trend was observed for epidermal thickness and parakeratotic lesions (Figure 4b and c).
      Figure thumbnail gr6
      Figure 6Barrier disruption induced by imiquimod (IMQ) treatment is more pronounced in caspase-14−/− mice relative to controls. Transepidermal water loss (TEWL) measurements were recorded on caspase-14+/+ and caspase-14−/− shaved back skin on days 0, 4, 6, and 8 of treatment. Bars represent mean values±SD. Wild-type (WT) untreated: n=4; knockout (KO) untreated: n=4; WT vehicle: n=6; KO vehicle: n=6; WT IMQ: n=11; and KO IMQ: n=12. Values of the two genotypes were compared using two-way analysis of variance (ANOVA) testing with Bonferroni post-tests; P-values are indicated with * (<0.05), ** (0.05–0.001), or *** (<0.001). Only statistically significant differences are indicated by asterisks.

      Discussion

      Generation of caspase-14-deficient mice led to the identification of caspase-14 as an important factor in proper SC formation. A basal increase in TEWL was observed in newborn caspase-14−/− mice concomitant with a decreased SC hydration level (
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ). Here, we performed detailed electron microscopic analysis revealing ultrastructural defects in caspase-14−/− newborn and adult epidermis (Figure 1). The higher incidence of incompletely cornified cells in the transitional layer of caspase-14−/− epidermis points to a delay in terminal keratinocyte differentiation, although the CEs appeared normal in caspase-14−/− skin. These transitional cells resemble those observed during prolonged occlusion after tape stripping (
      • Ahn S.K.
      • Hwang S.M.
      • Jiang S.J.
      • et al.
      The changes of epidermal calcium gradient and transitional cells after prolonged occlusion following tape stripping in the murine epidermis.
      ), or after tape stripping of caspase-14−/− mice (
      • Demerjian M.
      • Hachem J.P.
      • Tschachler E.
      • et al.
      Acute modulations in permeability barrier function regulate epidermal cornification: role of caspase-14 and the protease-activated receptor type 2.
      ). In contrast to these previous studies, we did not observe clear remnants of the degrading nucleus or other cellular organelles in these cells. In spite of the fact that
      • Hibino T.
      • Fujita E.
      • Tsuji Y.
      • et al.
      Purification and characterization of active caspase-14 from human epidermis and development of the cleavage site-directed antibody.
      reported that activated caspase-14 colocalizes with TUNEL-positive cells in human epidermis, we did not observe obvious differences in TUNEL-positive cell numbers when comparing WT and caspase-14−/− epidermis (data not shown). We previously showed that caspase-14 is needed to degrade (pro)filaggrin into natural moisturizing factors, such as urocanic acid and 2-pyrrolidone-5-carboxylic acid (
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ;
      • Hoste E.
      • Kemperman P.
      • Devos M.
      • et al.
      Caspase-14 is required for filaggrin degradation to natural moisturizing factors in the skin.
      ). Flaky tail mice, which are filaggrin deficient, and therefore have reduced natural moisturizing factor levels, exhibit impaired lamellar body secretion and have abnormal extracellular lamellae (
      • Scharschmidt T.C.
      • Man M.Q.
      • Hatano Y.
      • et al.
      Filaggrin deficiency confers a paracellular barrier abnormality that reduces inflammatory thresholds to irritants and haptens.
      ). However, differences in the incidence of transitional cells were not reported in these mice, nor in filaggrin-deficient patients. This indicates that the delay in cornification observed in caspase-14−/− skin is probably due to unknown caspase-14 substrates other than (pro)filaggrin, contributing to this phenotype. Why incompletely cornified cells were observed less frequent in adult caspase-14 deficient mice is currently not clear. However, this could be due to higher epidermal proliferation rates in neonatal mice than in adult mice. The ultrastructural differences observed in caspase-14-deficient skin further illustrate the important role for caspase-14 in proper keratinocyte cornification.
      Epidermal barrier disruption can induce a new wave of cornification to compensate for the loss of permeability barrier function (
      • Demerjian M.
      • Hachem J.P.
      • Tschachler E.
      • et al.
      Acute modulations in permeability barrier function regulate epidermal cornification: role of caspase-14 and the protease-activated receptor type 2.
      ). To test the effect of caspase-14 ablation on this process, we disrupted the epidermal barrier by repetitive topical acetone application. Compared with WT skin, the percentage of parakeratotic skin was significantly increased in caspase-14−/− mice (Figure 2c). In addition, caspase-14−/− mice showed a marked increase in large parakeratotic plaques (Figure 2d). This is in accordance with the observation that correct keratinocyte cornification was highly attenuated in transitional cells in tape-stripped caspase-14−/− skin (
      • Demerjian M.
      • Hachem J.P.
      • Tschachler E.
      • et al.
      Acute modulations in permeability barrier function regulate epidermal cornification: role of caspase-14 and the protease-activated receptor type 2.
      ).
      Psoriatic skin contains a large number of infiltrating immune cells that produce many different inflammatory molecules. This results in epidermal hyperproliferation, parakeratosis, and a defective epidermal barrier (reviewed in
      • Roberson E.D.
      • Bowcock A.M.
      Psoriasis genetics: breaking the barrier.
      ). Clinical topical use of IMQ can elicit psoriasis in patients predisposed to the disease. When it is applied repetitively on murine back skin, it induces a psoriasis-like phenotype, fulfilling many criteria for a valid psoriasis mouse model (
      • Conrad C.
      • Nestle F.O.
      Animal models of psoriasis and psoriatic arthritis: an update.
      ;
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ;
      • Wagner E.F.
      • Schonthaler H.B.
      • Guinea-Viniegra J.
      • et al.
      Psoriasis: what we have learned from mouse models.
      ;
      • Swindell W.R.
      • Johnston A.
      • Carbajal S.
      • et al.
      Genome-wide expression profiling of five mouse models identifies similarities and differences with human psoriasis.
      ). Nevertheless, no single mouse model for psosiaris recapitulates all human pathology features of the disease (
      • Wagner E.F.
      • Schonthaler H.B.
      • Guinea-Viniegra J.
      • et al.
      Psoriasis: what we have learned from mouse models.
      ;
      • Swindell W.R.
      • Johnston A.
      • Carbajal S.
      • et al.
      Genome-wide expression profiling of five mouse models identifies similarities and differences with human psoriasis.
      ). Repeated topical IMQ treatment of WT and caspase-14−/− mice resulted in a higher degree of scaling, TEWL, and parakeratotic SC in caspase-14−/− mice relative to controls (Figures 3, 4, 5, 6). The more severe barrier perturbation in caspase-14−/− mice can be due to the higher degree of parakeratosis observed in these mice. How the absence of caspase-14 facilitates parakeratosis upon epidermal barrier challenge is currently not clear. One possibility is that caspase-14 has a role in the enucleation process or DNA degradation during terminal keratinocyte differentiation in inflammatory conditions, but not under homeostatic conditions (cfr. absence of a spontaneous phenotype in caspase-14−/− mice). The interaction of the keratinocytes with mononuclear leukocytes is of key importance for the development of psoriatic lesions (reviewed in
      • Armstrong A.W.
      • Voyles S.V.
      • Armstrong E.J.
      • et al.
      A tale of two plaques: convergent mechanisms of T-cell-mediated inflammation in psoriasis and atherosclerosis.
      ), but no difference was observed in infiltrating leukocyte or T-cell numbers between IMQ-treated WT and caspase-14−/− skin. The difference in parakeratotic plaque formation in caspase-14-deficient skin was not due solely to the IMQ treatment, because an increase, although less extensive, in epidermal thickness, parakeratosis, and TEWL was also observed upon topical treatment of these mice with vehicle cream, which was not the case in control mice (Figure 6). The exact reason for this differential effect of control cream on WT and caspase-14−/− mice is currently not clear, but could be due to a higher sensitivity to friction-induced stress (cfr. repetitive treatment) or to an altered penetration efficacy of the control cream in caspase-14−/− mice compared with WT mice.
      Hyperproliferating skin requires an enhanced differentiation rate to keep the epidermis in check. Apparently, in such conditions, caspase-14-deficient skin encounters more difficulties in achieving proper SC formation and barrier maintenance compared with normal skin. This suggests that the low levels of caspase-14 expression or activation observed in psoriatic parakeratotic lesions (
      • Lippens S.
      • Kockx M.
      • Knaapen M.
      • et al.
      Epidermal differentiation does not involve the pro-apoptotic executioner caspases, but is associated with caspase-14 induction and processing.
      ,
      • Lippens S.
      • Kockx M.
      • Denecker G.
      • et al.
      Vitamin D3 induces caspase-14 expression in psoriatic lesions and enhances caspase-14 processing in organotypic skin cultures.
      ;
      • Fischer H.
      • Stichenwirth M.
      • Dockal M.
      • et al.
      Stratum corneum-derived caspase-14 is catalytically active.
      ;
      • Raymond A.A.
      • Mechin M.C.
      • Nachat R.
      • et al.
      Nine procaspases are expressed in normal human epidermis, but only caspase-14 is fully processed.
      ) were a cause rather than a consequence of parakeratosis. The lowered caspase-14 expression levels in psoriatic skin could be due to the action of T-helper type 1 cytokines, which were shown to downregulate caspase-14 expression (
      • Hvid M.
      • Johansen C.
      • Deleuran B.
      • et al.
      Regulation of caspase 14 expression in keratinocytes by inflammatory cytokines—a possible link between reduced skin barrier function and inflammation?.
      ). Moreover, treatment of lesional psoriatic skin with vitamin D3 analogs does ameliorate psoriatic plaque morphology and re-establishes normal caspase-14 expression levels (
      • Lippens S.
      • Kockx M.
      • Denecker G.
      • et al.
      Vitamin D3 induces caspase-14 expression in psoriatic lesions and enhances caspase-14 processing in organotypic skin cultures.
      ). When comparing IMQ-induced psoriasis-like conditions in murine skin with psoriatic lesions in patients, it is important to keep in mind that multiple differences exist between murine and human skin (
      • Swindell W.R.
      • Johnston A.
      • Carbajal S.
      • et al.
      Genome-wide expression profiling of five mouse models identifies similarities and differences with human psoriasis.
      ). It should also be noted that the psoriasis-like phenotype induced by topical IMQ application shares features with other inflammatory skin disorders, such as atopic eczema. Psoriatic lesions share several characteristics with wound healing responses, and hence it would be interesting to investigate whether ablation of epidermal caspase-14 affects atopic dermatitis or wound healing dynamics. Interestingly, caspase-14 activity levels are reduced in atopic dermatitis skin (
      • Yamamoto M.
      • Kamata Y.
      • Iida T.
      • et al.
      Quantification of activated and total caspase-14 with newly developed ELISA systems in normal and atopic skin.
      ).
      In summary, we show that caspase-14 deficiency leads to a delay in keratinocyte cornification in homeostatic conditions, which may affect epidermal barrier function under basal conditions. In addition, we clearly demonstrate that caspase-14 activity protects against loss of skin permeability upon challenging of the cutaneous barrier and against development of parakeratotic plaques in IMQ-induced psoriasis-like epidermal dermatitis. Therefore, our results underscore the important role of caspase-14 in terminal keratinocyte differentiation and identify caspase-14 downregulation as an important facilitating factor in developing parakeratotic plaques in inflammatory skin conditions. These findings point to the putative beneficial effects of therapies that could re-establish caspase-14 expression in conditions of parakeratosis or barrier disruption.

      Materials and Methods

      Mice

      Caspase-14+/+ and caspase-14−/− mice have been described previously (
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ). Mice were kept under specific pathogen-free conditions, and all procedures were approved by the institutional ethics committee.

      Scoring of epidermal scaling

      Arbitrary scores were given for skin scaling, as described previously (
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ). Epidermal scaling was scored independently by two researchers on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; and 4, very marked.

      Tissue preparation for electron microscopy

      Back skin from WT and caspase-14−/− mice at the ages of 3.5 days and 9 weeks was immersed in a fixative solution of 2.5% glutaraldehyde, 3% formaldehyde, and 0.02% CaCl2 in 0.1M Na-cacodylate buffer, placed in a vacuum oven for 30minutes and then left rotating for 3hours at room temperature. This solution was later replaced with fresh fixative, and samples were left rotating overnight at 4°C. After washing, samples were postfixed in 1% OsO4 with K3Fe(CN)6 in 0.1M Na-cacodylate buffer, pH 7.2. Samples were dehydrated through a graded ethanol series, including a bulk staining with 2% uranyl acetate at the 50% ethanol step, followed by embedding in Spurr’s resin. To have a larger overview of the phenotype, semithin sections were first cut at 0.5mm and stained with toluidine blue. Ultrathin sections of a gold interference color were cut using an ultramicrotome (Leica EM UC6, Wetzlar, Germany), followed by poststaining with uranyl acetate and lead citrate in a Leica EM AC20 and collected on formvar-coated copper slot grids. They were viewed with a transmission electron microscope JEOL 1010 (JEOL, Tokyo, Japan).

      Topical IMQ application

      Female mice at the age of 8 to 12 weeks were shaved with clippers (Wella contoura, Bio-services, The Netherlands) and a close shaving device (EpiLady, Philips, Belgium). Topical IMQ treatment (125mg Aldara (3M Pharmaceuticals, Diegem, Belgium), containing 5% of IMQ) was started 48hours after shaving and was repeated daily for 8 consecutive days (modified from
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ). The IMQ dose causing the most reproducible skin inflammation in WT and caspase-14−/− mice was empirically determined (data not shown). Vehicle cream contained the same ingredients as IMQ-containing cream. On the days indicated, mice were killed by cervical dislocation and back skin was isolated for further processing.

      Acetone-induced skin barrier disruption

      Acetone or control saline treatment was performed two times a day (morning and evening). Acetone was applied by wiping shaved back skin five times with a paper cloth soaked in acetone or 1M NaCl.

      Analysis of parakeratosis

      Paraffin sections of skin samples were stained with hematoxylin and eosin. Parakeratosis was independently assessed by two researchers using a light microscope. The length of parakeratotic plaques was measured using a ruler ocular. For parakerotosis data, expressed as % of affected SC as determined on skin sections, a logistic regression model of the Yijk=μ+genotypej+treatmentk+errorijk was fitted to the data, partitioning the parakerototic cell number variation into fixed genotype and treatment effects, and random error effects. The regression line was assessed by comparing its deviance with χ2. All analyses were performed by means of GenStat v.14 (
      • Payne R.W.
      Genstat Release 14 Reference Manual.
      .

      Immunohistochemical analysis of skin sections

      Monoclonal rat anti-mouse Ki-67 antibody (Dako Cytomation, Heverlee, Belgium; Clone TEC-3) was used at a 1/30 dilution to analyze the proliferative status of the epidermal keratinocytes. Ki-67-positive keratinocytes in the interfollicular epidermis were manually counted or automated analysis was performed by determining the percentage of diaminobenzidine (DAB)-stained area as explained below. Rat anti-mouse CD45 antibody (Becton Dickinson Benelux, Erembodegem, Belgium; cat. no. 550539) and rabbit anti-mouse CD3 antibodies (Abcam, Cambridge, UJ; cat. no. ab5640) were used at a dilution of 1:100. The number of mice that were analyzed were as follows: WT untreated, n=4; caspase-14−/− (knockout (KO)) untreated, n=4; WT vehicle day 8, n=6; KO vehicle day 8, n=5; WT IMQ day 8, n=9; and KO IMQ day 8, n=10. Three sections per mouse were analyzed. To quantify the amount of CD45-positive cells, we determined the percentage of DAB-stained area in each image. The area of the section was manually segmented (epidermis+dermis) with Volocity image analysis software (Perkin-Elmer, Waltham, MA). Within this segment, the DAB-positive cells were identified using a RGB threshold. The percentages of DAB-stained pixel count area (100 × DAB-stained pixel area divided by the section pixel area) were calculated for all images, and the results were plotted and statistically analyzed with the GraphPad software using two-way analysis of variance (ANOVA) with Bonferroni post-testing.

      Measurement of TEWL

      Mice were anesthetized by intraperitoneal injection of a ketamine/xylazine mixture. TEWL was measured using a TEWA meter (Courage and Khazaka, Cologne, Germany; TM 210). Data were analyzed for statistical significance with the GraphPad Prism 5.0 software using two-way ANOVA with Bonferroni post-testing.

      ACKNOWLEDGMENTS

      We thank Tugba Mehmetoglu for experimental assistance and A Bredan for editing the manuscript. This research has been supported by the Flanders Institute for Biotechnology (VIB), Ghent University, and several grants: European grants, FP6 Integrated Project Epistem LSHB-CT-2005-019067 and COST action SKINBAD BM0903; Belgian grants, Interuniversity Attraction Poles, IAP 6/18; Flemish grants, Fonds Wetenschappelijke Onderzoek Vlaanderen, G.0226.09 and 1.5.169.08; and Ghent University grants, BOF-GOA–12.0505.02. PV holds a Methusalem grant (BOF09/01M00709) from the Flemish Government. We also thank M Vuylsteke (PSB, UGent, Gent, Belgium) for advice on statistical analysis.

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

      REFERENCES

        • Ahn S.K.
        • Hwang S.M.
        • Jiang S.J.
        • et al.
        The changes of epidermal calcium gradient and transitional cells after prolonged occlusion following tape stripping in the murine epidermis.
        J Invest Dermatol. 1999; 113: 189-195
        • Armstrong A.W.
        • Voyles S.V.
        • Armstrong E.J.
        • et al.
        A tale of two plaques: convergent mechanisms of T-cell-mediated inflammation in psoriasis and atherosclerosis.
        Exp Dermatol. 2011; 20: 544-549
        • Bowcock A.M.
        • Krueger J.G.
        Getting under the skin: the immunogenetics of psoriasis.
        Nat Rev Immunol. 2005; 5: 699-711
        • Brady S.P.
        Parakeratosis.
        J Am Acad Dermatol. 2004; 50: 77-84
        • Conrad C.
        • Nestle F.O.
        Animal models of psoriasis and psoriatic arthritis: an update.
        Curr Rheumatol Rep. 2006; 8: 342-347
        • Demerjian M.
        • Hachem J.P.
        • Tschachler E.
        • et al.
        Acute modulations in permeability barrier function regulate epidermal cornification: role of caspase-14 and the protease-activated receptor type 2.
        Am J Pathol. 2008; 172: 86-97
        • Denda M.
        • Wood L.C.
        • Emami S.
        • et al.
        The epidermal hyperplasia associated with repeated barrier disruption by acetone treatment or tape stripping cannot be attributed to increased water loss.
        Arch Dermatol Res. 1996; 288: 230-238
        • Denecker G.
        • Hoste E.
        • Gilbert B.
        • et al.
        Caspase-14 protects against epidermal UVB photodamage and water loss.
        Nat Cell Biol. 2007; 9: 666-674
        • Eckhart L.
        • Declercq W.
        • Ban J.
        • et al.
        Terminal differentiation of human keratinocytes and stratum corneum formation is associated with caspase-14 activation.
        J Invest Dermatol. 2000; 115: 1148-1151
        • Eckhart L.
        • Fischer H.
        • Tschachler E.
        Mechanisms and emerging functions of DNA degradation in the epidermis.
        Front Biosci. 2012; 17: 2461-2475
        • Elias P.M.
        • Crumrine D.
        • Rassner U.
        • et al.
        Basis for abnormal desquamation and permeability barrier dysfunction in RXLI.
        J Invest Dermatol. 2004; 122: 314-319
        • Fischer H.
        • Stichenwirth M.
        • Dockal M.
        • et al.
        Stratum corneum-derived caspase-14 is catalytically active.
        FEBS Lett. 2004; 577: 446-450
        • Ghadially R.
        • Reed J.T.
        • Elias P.M.
        Stratum corneum structure and function correlates with phenotype in psoriasis.
        J Invest Dermatol. 1996; 107: 558-564
        • Hibino T.
        • Fujita E.
        • Tsuji Y.
        • et al.
        Purification and characterization of active caspase-14 from human epidermis and development of the cleavage site-directed antibody.
        J Cell Biochem. 2010; 109: 487-497
        • Hoste E.
        • Kemperman P.
        • Devos M.
        • et al.
        Caspase-14 is required for filaggrin degradation to natural moisturizing factors in the skin.
        J Invest Dermatol. 2011; 131: 2233-2241
        • Hsu S.
        • Dickinson D.
        • Borke J.
        • et al.
        Green tea polyphenol induces caspase 14 in epidermal keratinocytes via MAPK pathways and reduces psoriasiform lesions in the flaky skin mouse model.
        Exp Dermatol. 2007; 16: 678-684
        • Hvid M.
        • Johansen C.
        • Deleuran B.
        • et al.
        Regulation of caspase 14 expression in keratinocytes by inflammatory cytokines—a possible link between reduced skin barrier function and inflammation?.
        Exp Dermatol. 2011; 20: 633-636
        • Jarnik M.
        • Kartasova T.
        • Steinert P.M.
        • et al.
        Differential expression and cell envelope incorporation of small proline-rich protein 1 in different cornified epithelia.
        J Cell Sci. 1996; 109: 1381-1391
        • Kaiser W.J.
        • Upton J.W.
        • Long A.B.
        • et al.
        RIP3 mediates the embryonic lethality of caspase-8-deficient mice.
        Nature. 2011; 471: 368-372
        • Kovalenko A.
        • Kim J.C.
        • Kang T.B.
        • et al.
        Caspase-8 deficiency in epidermal keratinocytes triggers an inflammatory skin disease.
        J Exp Med. 2009; 206: 2161-2177
        • Lamkanfi M.
        • Declercq W.
        • Depuydt B.
        • et al.
        The Caspase Family. Landes Bioscience/Kluwer Academic-Plenum Publishers, Georgetown/New York2003: 40
        • Lee P.
        • Lee D.J.
        • Chan C.
        • et al.
        Dynamic expression of epidermal caspase 8 simulates a wound healing response.
        Nature. 2009; 458: 519-523
        • Lippens S.
        • Kockx M.
        • Denecker G.
        • et al.
        Vitamin D3 induces caspase-14 expression in psoriatic lesions and enhances caspase-14 processing in organotypic skin cultures.
        Am J Pathol. 2004; 165: 833-841
        • Lippens S.
        • Kockx M.
        • Knaapen M.
        • et al.
        Epidermal differentiation does not involve the pro-apoptotic executioner caspases, but is associated with caspase-14 induction and processing.
        Cell Death Differ. 2000; 7: 1218-1224
        • Lippens S.
        • VandenBroecke C.
        • Van Damme E.
        • et al.
        Caspase-14 is expressed in the epidermis, the choroid plexus, the retinal pigment epithelium and thymic Hassall’s bodies.
        Cell Death Differ. 2003; 10: 257-259
        • Lowes M.A.
        • Bowcock A.M.
        • Krueger J.G.
        Pathogenesis and therapy of psoriasis.
        Nature. 2007; 445: 866-873
        • Menon G.K.
        • Feingold K.R.
        • Moser A.H.
        • et al.
        De novo sterologenesis in the skin. II. Regulation by cutaneous barrier requirements.
        J Lipid Res. 1985; 26: 418-427
        • Palamara F.
        • Meindl S.
        • Holcmann M.
        • et al.
        Identification and characterization of pDC-like cells in normal mouse skin and melanomas treated with imiquimod.
        J Immunol. 2004; 173: 3051-3061
        • Payne R.W.
        Genstat Release 14 Reference Manual.
        Part 3: Procedure library PL21. VSN International, Oxford2011
        • Proksch E.
        • Brandner J.M.
        • Jensen J.M.
        The skin: an indispensable barrier.
        Exp Dermatol. 2008; 17: 1063-1072
        • Raymond A.A.
        • Mechin M.C.
        • Nachat R.
        • et al.
        Nine procaspases are expressed in normal human epidermis, but only caspase-14 is fully processed.
        Br J Dermatol. 2007; 156: 420-427
        • Roberson E.D.
        • Bowcock A.M.
        Psoriasis genetics: breaking the barrier.
        Trends Genet. 2010; 26: 415-423
        • Scharschmidt T.C.
        • Man M.Q.
        • Hatano Y.
        • et al.
        Filaggrin deficiency confers a paracellular barrier abnormality that reduces inflammatory thresholds to irritants and haptens.
        J Allergy Clin Immunol. 2009; 124 (e1-6): 496-506
        • Swindell W.R.
        • Johnston A.
        • Carbajal S.
        • et al.
        Genome-wide expression profiling of five mouse models identifies similarities and differences with human psoriasis.
        PLoS One. 2011; 6: e18266
        • van der Fits L.
        • Mourits S.
        • Voerman J.S.
        • et al.
        Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
        J Immunol. 2009; 182: 5836-5845
        • Wagner E.F.
        • Schonthaler H.B.
        • Guinea-Viniegra J.
        • et al.
        Psoriasis: what we have learned from mouse models.
        Nat Rev Rheumatol. 2010; 6: 704-714
        • Wagner T.L.
        • Ahonen C.L.
        • Couture A.M.
        • et al.
        Modulation of TH1 and TH2 cytokine production with the immune response modifiers, R-848 and imiquimod.
        Cell Immunol. 1999; 191: 10-19
        • Yamamoto M.
        • Kamata Y.
        • Iida T.
        • et al.
        Quantification of activated and total caspase-14 with newly developed ELISA systems in normal and atopic skin.
        J Dermatol Sci. 2011; 61: 110-117