Specific deletion of caspase-8 from epidermal keratinocytes results in a similar skin disorder

To examine whether the cutaneous abnormality caused by the expression of enzymatically inactive caspase-8 reflects a functional role of this enzyme in the epidermal cells themselves, we crossed mice carrying a conditional caspase-8 allele with mice expressing Cre under control of the keratin 5 (K5) promoter. In this way, we obtained mice in which caspase-8 expression was specifically eliminated in the keratinocytes (Fig. 2, A and B). The progeny (Casp-8^F/−K5-Cre) appeared normal at birth but, 3 d later (i.e., at postnatal day 3 [P3]), began to develop a skin disease similar to that of the Casp-8^{+/−/BAC-C362S} mice (Fig. 2 C). They failed to thrive and died by P7. Consistently with the known activity of the K5 promoter in the squamous mucosa of the stomach, some of the mice also exhibited severe gastric inflammation (Fig. S1). However, death of the mice did not correlate with the occurrence of gastritis or with decreased food uptake and thus was apparently the result of another cause.

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Figure 2.

Epidermis-specific KO of caspase-8. (A) Quantitative assessment of WT and deleted caspase-8 alleles in Casp-8^F/−K5-Cre mice by genomic PCR. (B) X-gal staining for β-galactosidase activity in the skins of ROSA26-reporter Casp-8^F/+K5-Cre (F/+) and ROSA26-reporter Casp-8^F/−K5-Cre (F/−) mice, demonstrating specific Cre-induced recombination in the superficial and follicular epidermis and hair follicles. Bars, 50 µm. (C) General appearance of Casp-8^F/+K5-Cre and Casp-8^F/−K5-Cre mice at P7. The mutant is much smaller than the WT. The skin is unevenly thickened with marked alopecia and hyperkeratosis. (D) Microscopy of the skin. At P0, the skin of the Casp-8^F/−K5-Cre mice is indistinguishable in appearance from the skin of the Casp-8^F/+K5-Cre mice. At P7 there is marked epidermal hyperplasia and widespread dermal inflammatory cellular infiltration in the Casp-8^F/−K5-Cre mouse. Arrows in B and D point to intraepidermal eosinophilic pustules. H&E; Bars, 50 µm. (E) The affected epidermis contains numerous dyskeratotic foci, consisting of maloriented keratinocytes often forming a whorl around a central keratinocyte which has become prematurely keratinized (arrows). H&E; Bars, 50 µm. The data presented in B, C, D, and E are representative of analyses of 4, 350, 30, and 30 mice, respectively.

The skin in affected sites of the Casp-8^F/−K5-Cre mice displayed pronounced epidermal hyperplasia, as well as orthokeratotic and milder parakeratotic hyperkeratosis with loss of the stratum granulosum. Intraepidermal pustules were not uncommon and numerous cells were dyskeratotic (Fig. 2, D and E).

Epidermal hyperplasia was restricted to sites of inflammation (Fig. 3 A). In the dermis these sites exhibited severe diffuse infiltration of inflammatory cells, including eosinophils, macrophages, and granulocytes, with no significant increase in B lymphocytes (indicated by staining with anti-CD45R antibody; unpublished data), and a marked decrease in epidermal CD3⁺ T lymphocytes (Fig. 3 B). Eosinophils also accumulated within intraepidermal pustules (Fig. 2, B and D; and Fig. 3 B). The superficial and follicular epidermis expressed K6 specifically in affected areas (Fig. 3 A).

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Figure 3.

Assessment of inflammation in the skin of Casp-8^F/−K5-Cre mice. (A) Multifocal cutaneous inflammation at P5. To allow continuous viewing of the transition area between inflamed and unaffected skin, two overlapping images of adjacent regions in a skin section covering such an area were obtained, and a panoramic image was then constructed using a conventional Photoshop (Adobe) Photomerge routine. The broken line marks the site at which the two pictures were merged. The line of dots in the top indicates the approximate location of the transition point. The micrographs demonstrate epidermal hyperplasia, as well as increased dermal cellularity in the inflamed skin (top, H&E) as a result of infiltration of eosinophils and F4/80-positive cells (mononuclear phagocytes, middle). Interfollicular expression of K6, which is indicative of epidermal hyperproliferation (bottom), is restricted to the thickened epidermal region. Bars, 100 µm. (B) Staining for macrophages (anti-F4/80 antibody), eosinophils (phenol red uptake), granulocytes (anti–Gr-1 antibody), and T lymphocytes (anti-CD3 antibody) in skin sections from Casp-8^F/+K5-Cre and Casp-8^F/−K5-Cre mice at P7. Note that eosinophils also accumulate within pustules in the epidermis of the Casp-8^F/−K5-Cre mice (also marked by arrows in Fig. 2, B and D). Bars, 100 µM. (C) TUNEL staining (top) for assessing apoptosis and Ki67 staining (bottom) for determining cell proliferation rates in the Casp-8^F/+K5-Cre and Casp-8^F/−K5-Cre epidermis at P7. Fine broken lines delineate approximate contours of the epidermis. Scale bar, 50 µm. The data presented in A, B, and C are representative of analyses of 10, 20, and 4 mice, respectively.

To investigate the cellular function of caspase-8 in the cutaneous epidermal keratinocytes, we first examined whether its deficiency affects cell death, the most thoroughly documented function of caspase-8. Cornification seemed unharmed. Moreover, outside-in dye penetration tests consistently failed to reveal any defect in epidermal barrier function, even as late as P7 (unpublished data). No conspicuous aberration was observed in the expression or localization of E-cadherin (Fig. S2), the major mediator of intracellular adhesion in epithelial tissues and an essential contributor to epidermal barrier function in vivo (Tunggal et al., 2005). Terminal transferase-mediated dUTP nick-end labeling (TUNEL) revealed no decrease and, at a late stage of the pathology, a substantial increase of death in the caspase-8–deficient epidermis, mainly in its suprabasal layers (Fig. 3 C, top). Assessment of the cell proliferation rate by staining for the Ki67 antigen (Fig. 3 C, bottom) or by bromodeoxyuridine incorporation (not depicted) disclosed considerably enhanced proliferation of keratinocytes in the basal layer. Ki67-positive cells were also found in abundance suprabasally.

Immunohistological analysis of the caspase-8–deficient skin revealed aberrations of the expression patterns of proteins that characterize distinct steps in keratinocyte differentiation, a common observation in acute cutaneous inflammation. Whereas normally the expression of K14 is restricted to the basal layer, in our mutant mice it was also expressed suprabasally (Fig. 4 A). Moreover, the expression of proteins that mark the intermediate (K1) and late differentiation stages (loricrin and filaggrin) was considerably reduced. The reduction was progressive to the extent that in the lesions of P7 mice those proteins were barely discernible (Fig. 4 A). The deficiency of filaggrin in the epidermis of these mice seemed consistent with the scaly skin and loss of the granular layer, two characteristic features of human skin pathologies resulting from loss-of-function mutations of the filaggrin gene (McGrath, 2008).

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Figure 4.

Spatial analysis of the effects of caspase-8 deficiency on protein and gene expression in the epidermis. (A) Immunohistochemical characterization of the caspase-8–deficient epidermis. At P7 (right), immunostaining shows expression of the basal layer marker K14 throughout the Casp-8^F/−K5-Cre epidermis. Staining for the intermediate (K1 and involucrin [Inv]) and late (filaggrin [Fila] and loricrin [Lori]) differentiation markers shows either their disorganized localization or their almost complete absence in the Casp-8^F/−K5-Cre lesion areas. Also shown is the ubiquitous expression of K6, a general marker for epidermal hyperproliferation. None of these changes was discernible at P0 (left) or P1 (not depicted). Bars, 100 µm. (B and C) In situ hybridization for spatial analysis of up-regulated inflammatory genes in the caspase-8–deficient epidermis. Skin samples of Caspase-8^F/− K5-Cre and Caspase-8^F/+K5-Cre littermates at P0 and P7 were hybridized with DIG-labeled antisense RNA probe for mRNA of S100a9 (B) or IL33 (C) and then subjected to fluorescence detection (Cy2–anti-DIG, green). Nuclei were counterstained with Hoechst 342 (blue). Rabbit anti-laminin was used to visualize the basal membrane (red), highlighted in the magnified presentation of the insets by dotted lines and detected with Cy3-anti–rabbit Ig. Bars, 50 µm. The data presented in A, B, and C are representative of analyses of 10, 5, and 5 mice, respectively.

Gene expression analysis reveals that visible pathological changes in the skin are preceded by prenatally initiated generation of inflammatory mediators in the epidermis

In seeking clues to the molecular changes underlying the inflammatory process in the skin and its trigger, we compared patterns of gene expression in the skin of Casp-8^F/−K5-Cre and WT mice at different times before and after birth. Numerous genes were found to be up-regulated in the Casp-8^F/−K5-Cre skin, most of them in the epidermis. The up-regulated genes included several groups encoding proteins that play a role in inflammation (Fig. 5 A), and nearly half were genes encoding proteins shown to be induced during epidermal wound healing in neonatal mice (WHA; Cooper et al., 2004). A rather large subgroup of the up-regulated genes were encoders of proteins known to participate in the IFN response (Fig. 5 A and Fig. S3) and to depend on activation of the transcription factor IRF3 (Grandvaux et al., 2002; Andersen et al., 2008).

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Figure 5.

Expression-profiling analysis of the Casp-8^F/−K5-Cre skin. (A) Functional groups of genes overexpressed in caspase-8 KO skin. Total RNA was extracted from the dermis (Der) or epidermis (Epi) or from cultured keratinocytes at the first and third passages (KC_E and KC_L, respectively) derived from Casp-8^F/−K5-Cre and Casp-8^F/+K5-Cre mice at P3. Epidermal or dermal total RNA from two or three mice of each genotype was pooled for genome-wide expression analysis using Agilent microarrays (see Materials and methods). Ratios of mRNA expression in Casp-8^F/−K5-Cre and Casp-8^F/+K5-Cre samples are presented as heat maps with the corresponding values recorded in the cells. Depicted ratios from keratinocyte samples (KC_E and KC_L) represent geometric mean values from two microarray experiments. The 100 most strongly up-regulated genes in the caspase-8–deficient mouse epidermis at P3 were depicted and categorized into functional groups according to published references. In addition to the flagged spots, defined as described in Materials and methods, we also excluded Y-chromosomal and poorly annotated transcripts from the list. WHA, genes previously found to be induced during epidermal wound healing in neonatal mice (Cooper et al., 2004). (B and C) Kinetic analysis of gene-expression changes in the caspase-8–null epidermis and assessment of their dependence on TNF. (B) RNA isolated from the epidermal tissues of 22 pairs of Casp-8^F/+K5-Cre and Casp-8^F/−K5-Cre mice (from eight litters) and from the epidermal tissues of 22 pairs of TNF^−/−Casp-8^F/+K5-Cre/TNF^−/−Casp-8^F/−K5-Cre mice (from seven litters) was used for real-time RT-PCR determination of the expression of 15 selected genes representative of most of the functional groups identified in A. For heat map visualization, the log2-transformed expression ratios were ordered according to increasing consistency of changes in mRNA expression in the TNF^+/+ mice (horizontal axis). Percentages of Casp-8^F/−K5-Cre mice at a given age with a significantly up-regulated transcript relative to its mean expression in all Casp-8^F/+K5-Cre age-matched mice are recorded in the heat map boxes. n, number of experimental/control mice pairs analyzed for the given age (also see Materials and methods). (C) RNA was isolated from the epidermis of Casp-8^F/−K5-Cre mice (empty circles, values for individual mice born on different dates; broken lines, mean values for all individuals analyzed at a particular age) and of Casp-8^F+K5-Cre mice (filled circles and solid lines) 1 d before birth (D-1) and at different times postnatally. For each mouse, mRNA expression of the indicated four genes was analyzed by real-time PCR. Samples from pairs of Casp-8^F/−K5-Cre and Casp-8^F+K5-Cre mice were tested simultaneously; these pairs are depicted at identical horizontal positions. (In the case of Isg15, four additional D-1 WT samples were analyzed.) Shown are the −Δ cycle-threshold (Ct) values obtained by subtraction of the Ct value of each PCR product from the Ct value of the housekeeping gene Hprt1. Red arrows point to values for two Casp-8^F/−K5-Cre mice that showed significant up-regulation of Isg15 expression before birth.

Kinetic analysis disclosed that although initial signs of the pathological change in the skin could be discerned only at about P3, expression of many of the induced genes was enhanced in the Casp-8^F/−K5-Cre epidermis before that time. Some were found to be elevated prenatally, 1 d (Fig. 5, B and C [D-1]) and even 2 d (not depicted) before birth.

Cultured keratinocytes derived from the Casp-8^F/−K5-Cre mouse epidermis generate inflammatory mediators, but the generation gradually abates

To examine the mechanism underlying the up-regulation of inflammatory genes in the Casp-8^F/−K5-Cre epidermis, we tried to establish whether this up-regulation occurs autonomously in the caspase-8–deficient keratinocytes or depends on extracellular stimuli. By applying gene array analysis, we found that some of the inflammatory genes that were up-regulated in the Casp-8^F/−K5-Cre epidermis were still activated in keratinocytes derived from that epidermis shortly after their culture was initiated. However, in the course of passaging of the cells this increased expression gradually abated (Fig. 5 A, KC_E versus KC_L). These findings suggested that in addition to the caspase-8 deficiency in the keratinocytes, some other molecular determinants that occur in the skin and dissipate under our cell culturing conditions contribute to activation of inflammatory genes in these cells.

The disease caused by caspase-8 deficiency in keratinocytes is triggered independently of TNF, IL-1, the TLR adapter proteins, or macrophages

Cells of the immune system and mediators generated by them play crucial roles in various inflammatory skin disorders of humans and in their experimental models (Kupper and Fuhlbrigge, 2004). This is true for an inflammatory skin disorder that was described in mice with mutations preventing the expression or activation of NF-κB transcription factors in keratinocytes (Pasparakis et al., 2002; Gugasyan et al., 2004; van Hogerlinden et al., 2004; Omori et al., 2006; Sayama et al., 2006) and that closely resembles the one seen here in Casp-8^F/−K5-Cre mice. The former disorder was found to be fully dependent on accumulation of macrophages in the dermis, as well as on TNF whose expression in the macrophages is high (Pasparakis et al., 2002; Omori et al., 2006). To determine whether TNF or some other macrophage-produced mediator is also responsible for triggering the skin disease in the Casp-8^F/−K5-Cre mice, we first assessed the effect of deletion of the TNF or the TNFR1 gene from these mice by crossing them with the corresponding KO mouse strains. Such deletion dramatically increased survival of the offspring (Fig. 6 B and not depicted). Their skin disease was not prevented, however, but only delayed for a few days. In fact, because these mice survived and their skin condition kept worsening, disease severity eventually exceeded that seen in the TNF-proficient Casp-8^F/−K5-Cre mice (Fig. 6 A and not depicted). Histologically, late-stage disease in TNF^−/−Casp-8^F/−K5-Cre mice was characterized by widespread and severe dermal fibrosis, adnexal loss, and persistence of melanin pigment in the dermis, either in melanocytes or in melanophages, after adnexal destruction (pigmentary incontinence; Fig. 6 C). Cutaneous scarring was of such a magnitude that it led to severe contraction and rigidity of the skin, causing the mice to be in permanent kyphosis (Fig. S4). Deletion of the TNFR2 gene in addition to TNFR1 caused no further delay in onset of the skin disorder (unpublished data).

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Figure 6.

Limited effect of deficiency of TNF, IL-1, or macrophage depletion on cutaneous disease development in the caspase-8–null skin. (A) Gross appearance of TNF^−/−Casp-8^F/+K5-Cre and TNF^−/−Casp-8^F/−K5-Cre mice at different ages. (Arrows point to skin lesions at P7 and P30.) There is progressive hyperkeratosis, scaliness, and hair loss, with ulceration in some cases (middle). (B) Survival curves for colonies of Casp-8^F/−K5-Cre (green; 62 mice), IL1α^−/−IL1β^−/−Casp-8^F/−K5-Cre (yellow; 55 mice), TNF^−/−Casp-8^F/−K5-Cre (red; 37 mice), and IL1α^−/−IL1β^−/−TNF^−/−Casp-8^F/−K5-Cre mice (blue; 42 mice). (C) Microscopic features of late-stage disease in a TNF^−/−Casp-8^F/−K5-Cre mouse at P60. There is diffuse epidermal hyperplasia, dermal fibrosis, adnexal loss, and accumulation of melanocytes and melanophages (pigmentary incontinence). H&E; Bar, 100 µm. (D) Microscopic appearance of the skin of IL1α^−/−IL1β^−/−TNF^−/−Casp-8^F/+K5-Cre and IL1α^−/−IL1β^−/−TNF^−/−Casp-8^F/−K5-Cre mice at P7. H&E; Bars, 100 µm. (E) Macrophage content (anti-F4/80 staining, left) and microscopic appearance (H&E, right) of the skin of Casp-8^F/−K5-Cre mice injected with control liposomes (top) and clodronate-containing liposomes (bottom) at P5. Bars, 100 µM. (F) Effect of macrophage depletion on gene expression changes in the Casp-8^F/−K5-Cre epidermis. To deplete dermal macrophages, mice were injected subcutaneously every other day, starting at P0, with clodronate-loaded liposomes or PBS-loaded liposomes (control). RNA was isolated from the epidermis at P5 and analyzed for expression of CSF1, a macrophage marker (left), and of four inflammation markers found to be increased in Casp-8^F/−K5-Cre mice (right). Mean values ± SD for three experimental/control mice pairs are shown. The data presented in A, C, D, E, and F are representative of analyses of 10, 4, 7, 15, and 5 mice, respectively. The data in E and F are the results of three independent experiments in both cases.

Like the limited effect of TNF deficiency on development of the skin disease, the absence of TNF had little effect on gene expression in the epidermis. It resulted only in moderation of gene induction, with no evident change in the pattern of the induced genes (Fig. 5 B, compare bottom and top).

An increase in IL-1, which, like TNF, is known to play a major role in the induction of inflammation, was reported to accompany the cutaneous inflammation that results from the arrest of NF-κB activation in the epidermis (Pasparakis et al., 2002). We found that the messenger RNAs (mRNAs) of IL-1α and IL-1β were also increased in the epidermis of our Casp-8^F/−K5-Cre mice (Fig. 5 B and not depicted). However, simultaneous deletion of the genes for IL-1α and IL-1β had no effect on the skin pathology in these mice (not depicted), nor did it affect their survival (Fig. 6 B). Deletion of these genes from Casp-8^F/−K5-Cre mice that were also deficient in TNF led to a slight further increase in their survival (Fig. 6 B) but did not alleviate the cutaneous inflammation (Fig. 6 D and not depicted).

To determine whether signaling by TLRs contributes to the initiation of the skin disease in the Casp-8^F/−K5-Cre mice, we crossed these mice with KO mice for MyD88 and for TRIF, which encode two adapter proteins that mediate TLR-dependent signaling. Neither the rate nor the extent of development of cutaneous inflammation in the Casp-8^F/−K5-Cre mice nor the onset of their death was affected by deficiency of either of these proteins (unpublished data).

In mice with an inflammatory skin disease resulting from IKK2 deficiency in the keratinocytes, depletion of macrophages from the dermis by subcutaneous injection of clodronate-containing liposomes abolished the symptoms (Stratis et al., 2006). However, no such effect on the kinetics or extent of development of cutaneous inflammation was detectable in our Casp-8^F/−K5-Cre mice (Fig. 6 E and not depicted). Moreover, such treatment did not significantly affect either the pattern or the extent of changes in gene expression in the epidermis (Fig. 6 F and not depicted).

Thus, unlike in the case of the inflammatory skin disease that results from deficiency of the NF-κB activation pathway in keratinocytes, the findings described in this section indicated that triggering of the cutaneous inflammation that results from caspase-8 deficiency in the Casp-8^F/−K5-Cre mice occurs independently of TNF or of any other mediator produced by the dermal macrophages. It also does not depend on IL-1–induced or TLR-induced signaling.

The skin disease caused by caspase-8 deficiency in keratinocytes is associated with constitutive signaling for IRF3 activation

Western analysis of IRF3 in the Casp-8^F/−K5-Cre mouse epidermis revealed that this transcription factor is phosphorylated as early as P1 (Fig. 7, A and B). Moreover, the Casp-8^F/−K5-Cre epidermis was found to contain the phosphorylated form of TANK-binding kinase (TBK; TBK1), which is known to activate IRF3 (Fig. 7 A), and to have heightened expression of Isg15 (Fig. 7 A), an IRF3-dependent ubiquitin-related polypeptide. This was consistent with our finding that the Isg15 gene was activated in the Casp-8^F/−K5-Cre mice epidermis even before birth (Fig. 5, A–C). The analysis also revealed enhanced conjugation of this polypeptide to cellular proteins, a hallmark of the antiviral response (Sadler and Williams, 2008). Although expression of the IRF7 transcript was also enhanced in the Casp-8^F/−K5-Cre epidermis, Western analysis of these epidermal cells failed to show any expression of the IRF7 protein in them (unpublished data).

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Figure 7.

IRF3 is constitutively activated in the Casp-8^F/−K5-Cre epidermis. (A) Western blot analysis of lysates of the epidermis of two Casp-8^F/+K5-Cre and two Casp-8^F/−K5-Cre littermates at P3 for phosphorylation of TBK1 (ph-TBK1, enriched by immunoprecipitation with anti-TBK antibody) and of IRF3 (ph-IRF3), as well as for expression of IRF3 and of the IRF3-induced protein Isg15, and conjugation of Isg15 to the cellular proteins. (B) Western analysis of ph-IRF3, as in A, for mouse littermates at P1. As illustrated, the small amount of ph-IRF3 in the lysates of the Casp-8^F/−K5-Cre epidermis at that date could be discerned only when the cells were lysed in the presence of the sulphhydryl blocking agent N-ethylmaleimide. (C and D) Activation of inflammatory genes in early passage Casp-8^F/−K5-Cre keratinocytes is dependent on the presence of IRF3. (C) Relative expression (means ± SD of the indicated gene transcripts in Casp-8^F/−K5-Cre mRNA to the same transcripts in Casp-8^F/+K5-Cre mRNA) assayed in keratinocytes from three pairs of P1 littermates that were transfected, 1 d after plating, with either control siRNA (blue) or IRF3 siRNA (maroon). (D) Western analysis of the effect of IRF3 siRNA transfection on IRF3 expression and on expression of Isg15 in two of the Casp-8^F/−K5-Cre keratinocyte samples used in C. Analysis of the expression of β-actin served as a loading control. The data presented in panels A, B, C, and D are representative of findings obtained in three, two, four, and four independent experiments, respectively.

Knockdown of IRF3 in cultured Casp-8^F/−K5-Cre keratinocytes abolished the up-regulation of Isg15, as well as the activation of three out of five other tested genes chosen from those which were still up-regulated in these cells after they were plated for culturing (Fig. 7, C and D). These findings indicated that the constitutive activation of IRF3 in the Casp-8^F/−K5-Cre epidermis contributes to the up-regulation of inflammatory mediators in the epidermal cells.

Generation of inflammatory mediators in the epidermis occurs mainly in its suprabasal layer

In seeking clues to the nature of the endogenous stimulus that triggers gene up-regulation in the epidermis, we applied in-situ hybridization to determine the epidermal site at which two of the most strongly up-regulated genes, those encoding the alarmin S100a9 and the cytokine IL33, are activated. As shown in Fig. 4 (B and C), although caspase-8 deletion is induced in the basal cell layer of the epidermis, neither of these genes was expressed at that site but rather was expressed in the higher layers. Expression of S100a9 could also be discerned in some distinct cells in the dermis. These findings suggested that activation of inflammatory genes in the epidermis of the Casp-8^F/−K5-Cre mice is triggered by molecular changes that occur suprabasally.

Antibodies

Rabbit anti-cytokeratin 1, 6, and 14, as well as involucrin, filaggrin, and loricrin antibodies, were purchased from Covance. Rat anti-F4/80 and anti-CD3 were obtained from AbD Serotec, rat anti-Ki67 from Dako, rat anti–mouse Ly6G (Gr1) from eBioscience, rabbit anti-IRF3, rabbit anti–mouse Isg15, and anti–caspase-14 from Santa Cruz Biotechnology, Inc., rabbit anti-TBK1 and rabbit phospho (ph)-IRF3 from Cell Signaling Technology, mouse monoclonal anti–ph-TBK1 from BD, goat anti-TNF from R&D Systems, anti–mouse caspase-8 from Enzo Biochem, Inc., anti–β-actin and anti-laminin from Sigma Aldrich, and Cy2–anti-DIG and Cy3-anti–rabbit Ig from Jackson ImmunoResearch Laboratories. Immunoperoxidase polymer anti–rat was obtained from Nichirei.

Depletion of macrophages from the skin

To deplete skin macrophages, we injected the mice subcutaneously, at 10 sites over the back, with clodronate-loaded liposomes or, as a control, with PBS-loaded liposomes (100 µl/mouse; gift of N. van Rooijen, Vrije Universiteit, Amsterdam, Netherlands) every second day, starting from the day of birth (P0).

Histology and immunostaining

Skin and other organs were fixed in 10% phosphate-buffered formalin, pH 7.4, embedded in paraffin, cut into 4-µm sections, and stained with H&E. Adjacent sections were used for immunostaining with the indicated antibodies according to the manufacturers' instructions. For fluorescent detection we used Cy-2– or Cy-3–coupled secondary antibodies (Jackson ImmunoResearch Laboratories). To visualize nuclei, we further counterstained sections with Hoechst 33342 (Sigma-Aldrich). For chromogenic detection, sections were stained with immunoperoxidase polymer anti–rat antibodies and developed with 3,3′-diaminobenzidine (Sigma-Aldrich) for 10 min. A TUNEL staining kit (Roche Diagnostics) was used to detect apoptotic cells. Frozen sections (10-µm thick) were stained with X-Gal (5-bromo-4-chloro-3-indolyl-d-galactoside) or with phenol red (Ain et al., 2002). All histological and immunohistological analyses shown are representative of at least five sections examined.

In situ hybridization

Probe synthesis and in situ RNA hybridization were performed essentially as described (Darby and Hewitson, 2006). In brief, 6-µm skin sections were prepared from paraffin-embedded tissue blocks. After deparaffinization, slides were treated with 10 µg/ml proteinase-K for 15 min, fixed in 4% paraformaldehyde, and acetylated. The slides were then hybridized with sense or antisense DIG-labeled RNA probe overnight at 65°C. The next day, the slides were washed in saline-sodium citrate buffer and incubated with Cy2-labeled anti-DIG antibody for 1 h at room temperature. The data shown are representative of analysis of at least five different sections.

Western analysis of epidermal proteins

The epidermis and dermis were pulled apart after incubation for 2–3 s at 65°C. Epidermis samples were lysed in RIPA buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5% NP-40, 4 mM EDTA, 0.1% SDS, and 0.5% sodium deoxycholate) supplemented with 1 mM PMSF, 50 mM NaF, 1 mM sodium vanadate, 80 mM β-glycerophosphate, and a cocktail of protease inhibitors (Roche). For detection of ph-IRF3 in the epidermis at P1, we further supplemented the lysis buffer with 1 mM N-ethylmaleimide (Sigma-Aldrich). This greatly facilitated detection, probably by blocking the function of some potent protein phosphatases which had remained active in spite of the presence of phosphatase inhibitors in the lysis buffer.

Keratinocyte culture

Skin was removed from the mice and incubated overnight at 4°C in 2.4 U/ml Dispase (Roche) to separate the epidermis from the dermis. The keratinocytes were then dissociated by incubation of the epidermis for 10 min in 0.1% trypsin, recovered by centrifugation, and plated on tissue culture dishes in keratinocyte serum-free medium containing 50 µg/ml bovine pituitary extract and 5 ng/ml of epidermal growth factor (Invitrogen) supplemented with antibiotics. For passaging, cells were detached by incubation in PBS containing 0.1% trypsin, 0.5 mM EDTA, and 0.1% glucose and replated in keratinocyte serum-free medium.

Genotyping of mice

Deletion of caspase-8 from the skins of Casp-8^F/−K5-Cre and Casp-8^F/+K5-Cre mice was quantitatively assessed by PCR, as previously described (Kang et al., 2004).

RNA interference

Keratinocytes were plated in 12-well plates at 50,000 cells/well and transfected 1 d later with 500 nmol of small interfering RNA (siRNA) duplex per well (Accell; Thermo Fisher Scientific). After 72 h the cells were processed for analysis.

DNA transfection

Keratinocytes were transfected either with 1 µg/ml poly(deoxyadenylic-thymidylic) (poly(dA-dT); Sigma-Aldrich) or calf thymus DNA (Sigma-Aldrich) using Lipofectamine 2000 (Invitrogen) as specified by the manufacturer.

Real-time PCR

We isolated RNA from epidermis and dermis samples (peeled apart after brief heating as described in Western analysis of epidermal proteins) using the RNeasy Fibrous Tissue mini kit, and from cultured keratinocytes using the RNeasy kit (QIAGEN). 1 µg of total RNA was reverse transcribed into complementary DNA (cDNA) using 50 U BioScript RNase H Low (Bioline). Aliquots (20 ng, 1:50 dilution) of this reaction mixture were used to amplify cDNAs with Assays-on-Demand (Applied Biosystems) on an ABI7500 real-time PCR system. The Ct value for each individual PCR product was calculated from duplicate measurements by means of the instrument's software, and the Ct values obtained were normalized by subtracting the Ct values obtained for Hprt1 as a housekeeping gene. The resulting mean −ΔCt values from WT or KO mice were then used to calculate relative changes of mRNA expression as the ratio (R) of mRNA expression, according to the following equation: R(log2) = (mean − ΔCt(caspase-8^−/−)) − (mean − ΔCt(WT)). The increase in gene expression for individual KO mice was defined as significant if the −ΔCt value obtained for a given gene and a given KO mouse was greater than the mean −ΔCt + 2 SD of all WT mice of corresponding ages. Heat maps were visualized by importing log2-transformed ratio data into the Mayday program (http://www.zbit.uni-tuebingen.de/pas/mayday/mayday.html).

Microarray experiments

The Mouse Oligo Microarray V2 (G4121B, design ID 13326; Agilent Technologies) used in this study contained oligonucleotide probes directed against 19,307 endogenous mRNAs. RNA was used to prepare Cy3- or Cy5-labeled cRNA by oligo-(dT)-T7–primed double-stranded cDNA synthesis (cDNA Synthesis System; Roche), followed by in vitro transcription and labeling (Amino Allyl MessageAmp II kit; Applied Biosystems) as directed by the manufacturer. For the cRNA mixture, the fragment length, yield, and labeling efficiency were analyzed by a 2100 Bioanalyzer (Agilent Technologies) and a NanoDrop ND1000 spectrophotometer. cRNA populations (differentially labeled with 3 µg each of Cy3 and Cy5) were combined for cohybridization onto a single microarray. cRNA fragmentation, hybridization, and washing steps were performed according to Two-Color Microarray-Based Gene Expression Analysis Protocol V5.0.1 (Agilent Technologies; for details see http://www.agilent.com).

Hybridized and washed arrays were scanned on a 428 Array Scanner (Affymetrix). Data were extracted using of Imagene V5.0 software (BioDiscovery). Fluorescence intensity values were normalized according to LOWESS-like calculations. Data from qualitatively suboptimal spots were flagged and excluded from further analysis if CVsignal mean > 0.20 or if SMglobal background corrected/SMlocal background corrected > 1.3, where CVsignal mean is the coefficient of variance deduced from signal values for all pixels of a spot and SMglobal background corrected/SMlocal background corrected is the ratio of a global background-corrected signal mean value of a spot divided by its local background-corrected signal mean value.

mRNA expression analysis of cultured keratinocytes was performed using Mouse Whole Genome 4 × 44k Microarrays (G4122F, design ID 014868; Agilent Technologies). Sample processing, hybridization, data extraction, and normalization were performed exactly as recommended by the manufacturer. The microarray-based data discussed in this publication have been deposited at NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession no. GSE17365.

Modification of a BAC encompassing caspase-8 and generation of BAC-transgenic mice

We thank Drs. Nathaniel Heintz, Shiaoching Gong, Gérard Eberl, Nathalie Uyttersprot, and Yael Pewzner-Jung for their advice and help with BAC transgenesis, Karen Laviv for assistance in BAC DNA modification, Golda Damari for BAC DNA microinjection, Dr. Jose Jorcano for the K5-Cre transgenic mice, Dr. Shizuo Akira for the MyD88-deficient mice, Dr. Marina Gartsbein for advice on culturing of keratinocytes, Shoshana Grossfeld for maintenance of the mice, Jadi Natan, Tamara Berkutzki, and Calanit Raanan for assistance with histology, Inna Kolesnik, Tatiana Shalevich, and Dvir Mintz for genotyping the mice, Heike Schneider for performance of microarray and real-time PCR experiments, Kelly Jackson (University Health Network Microarray Centre, Toronto, Canada) for microarray hybridization and data analysis, and Dr. Nico van Rooijen for the generous gift of clodronate-loaded liposomes. Our thanks are also due to Drs. Cord Brakebusch, Uri Gat, Tamar Tennenbaum, and Dina Ron for helpful advice and discussions and to Shirley Smith for scientific editing. Special thanks to Professor Israel Vlodavsky for assistance. Microarray data analyses were performed using BRB ArrayTools developed by Dr. Richard Simon and Amy Peng Lam.

This work was supported in part by grants from Ares Trading SA (Switzerland), a Center of Excellence Grant from the Flight Attendant Medical Research Institute, and the Kekst Family Center for Medical Genetics and the Shapell Family Center for Genetic Disorders Research at The Weizmann Institute of Science.

The authors have no conflicting financial interests.