Loss of epidermal Evi/Wls results in a phenotype resembling psoriasiform dermatitis

Abstract

Cells of the epidermis renew constantly from germinal layer stem cells. Although epithelial cell differentiation has been studied in great detail and the role of Wnt signaling in this process is well described, the contribution of epidermal Wnt secretion in epithelial cell homeostasis remains poorly understood. To analyze the role of Wnt proteins in this process, we created a conditional knockout allele of the Wnt cargo receptor Evi/Gpr177/Wntless and studied mice that lacked Evi expression in the epidermis. We found that K14-Cre, Evi-LOF mice lost their hair during the first hair cycle, showing a reddish skin with impaired skin barrier function. Expression profiling of mutant and wild-type skin revealed up-regulation of inflammation-associated genes. Furthermore, we found that Evi expression in psoriatic skin biopsies is down-regulated, suggesting that Evi-deficient mice developed skin lesions that resemble human psoriasis. Immune cell infiltration was detected in Evi-LOF skin. Interestingly, an age-dependent depletion of dendritic epidermal T cells (DETCs) and an infiltration of γδ(low) T cells in Evi mutant epidermis was observed. Collectively, the described inflammatory skin phenotype in Evi-deficient mice revealed an essential role of Wnt secretion in maintaining normal skin homeostasis by enabling a balanced epidermal-dermal cross talk, which affects immune cell recruitment and DETC survival.

Figure 1.

Generation of floxed Evi mice and conditional depletion in squamous epithelium. (A) Targeting strategy for the conditional Evi allele with loxP sites flanking exon3. Thin lines, mouse genomic DNA; gray boxes, sequences included in the targeting construct; triangles, LoxP/FRT sites; black boxes, probes for Southern blot analysis; arrows, PCR primer. (B) Southern blot analysis of genomic ESC DNA digested with BamHI and Bmt1 confirmed homologous recombination with a 5′-primed and 3′-primed probe. (C) PCR analysis of genomic DNA amplified a 340-bp recombined/floxed and a 210-bp wild-type fragment. (D) Crossing of Evi^fl/fl with general Cre deleter generated embryonic lethal Evi knockout mice with defects in gastrulation (embryonic day 8.5 [E8.5]). (E) Evi-LOF and wild-type littermate at age P7 showing the red nose phenotype. (F) Skin phenotyping of Evi^fl/fl, K14-Cre mice (P50 mutant mice [right] and normal littermate [left]). (G) Ears. (H) Legs, exposed blood vessels are marked with arrows. (I) Eye. (J) Immunohistochemistry against Evi in the skin. A representative of three independent experiments is shown. Bars, 200 µm.

Figure 2.

Hyperproliferation of basal cells in Evi-LOF epidermis. (A) H&E-stained sections of Evi-LOF and control skin at different ages. SG, sebaceous gland; Bu, bulge; DP, dermal papilla. Bars, 200 µm. (B) Phospho-H3 stainings of skin sections of Evi-LOF and wild-type mice. Bars, 200 µm. (C) Quantitation of phospho-H3⁺–labeled basal cells/mm. Error bars represent SD. *, P < 0.05; **, P < 0.001, n = 3–4 per age and group. Results are combined from two independent experiments.

Figure 3.

Impaired keratin expression in Evi-LOF indicates ongoing inflammation. (A) Immunohistochemistry against β-catenin and keratinocyte differentiation marker (loricrin, K5, K8, K16, and K14). Bar, 200 µm. (B) Expression pattern of differentially expressed keratins in Evi-LOF epidermis based on deep sequencing data. Values represent normalized log₂ fold changes (n = 3, P < 0.05). (C) Heatmap showing cytokines differentially regulated in Evi-LOF epidermis. Values represent normalized log₂ fold changes (n = 3 per group, P < 0.05). (D) H&E-stained section of Evi-LOF and control skin. Vessels are marked with arrowheads. Bar, 200 µm. (E) Comparative analysis of dermal vessel density (n = 4 per group, error bars represent SD; *, P < 0.05). (F) Ultrastructural characterization of epon-embedded thin sections. D, dermis; E, epidermis (n = 3 per group). Results are combined from two (A–C and F) and three (D) independent experiments.

Figure 4.

Evi-LOF mice develop skin barrier defects. (A) Lucifer yellow penetration analyzed in newborns and adult mice (bar, 200 µm; n = 4 per group). (B) TEWL analysis of pups and adult mice. Error bars represent SD. (C) Skin surface temperature on shaved skin and body temperature (n = 5 per group; **, P < 0.001). (D) Log₂ fold changes of EDC gene expression in the epidermis of Evi-LOF skin. Genes with adjusted p-values <0.05 were labeled in dark gray and genes with adjusted p-values >0.05 were labeled in light gray. Results are combined from three (A–C) and two (D) independent experiments.

Figure 5.

Wnt signaling affects neutrophil recruitment in the skin. (A) Immunohistochemistry against S100A9 in Evi-LOF skin. (B) Quantitation of S100A9-labeled cells in the dermis of Evi-LOF and wild-type littermate skin in differently aged mice (n = 4 per age and group). (C) Quantitation of S100A9-labeled cells after treatment of Evi-LOF mice and control littermates with or without ciprofloxacin (Cipro; n = 3 per group). (D) Stainings of mast cell (Toluidine blue) and macrophage (CD68) infiltration (n = 4 per group). (E) Quantitation of S100A9-labeled cells in β-catenin, K14-Cre LOF (P60) mice (n = 4 per group). (F) Normalized mRNA expression levels of S100A9 in Evi-LOF and β-cat-LOF mice. Horizontal bars show mean values (n = 4 per group). (G) H&E-stained sections of ROSA26:Evi-YFP (Evi-GOF) knockin and control mice. (H) Expression of Evi-YFP protein in the skin. (I) Immunohistochemistry against S100A9 in skin sections of Evi-GOF and control mice 24 h after TPA application (n = 5 per group and time point). (J) Time-dependent quantitative analysis of dermal S100A9 infiltration after TPA application in Evi-GOF skin compared with control littermates (n = 5 per group and time point). Results are combined from three (A–C and E–I) and two (D) independent experiments. Error bars reflect SD (B–E) or SEM (J). *, P < 0.05; **, P < 0.001. Bars, 200 µm.

Figure 6.

Epidermal depletion of Evi led to loss of DETC and increase in CD3^low γδ^low subpopulation in the skin. (A) Expression of CD3 on frozen skin sections of neonatal mice and paraffin skin section of P40 mice. Bar, 200 µm. (B) FACS profile of CD3⁺ cells in skin and lymphatic tissue (n = 5 per group). (C) Quantification of immunohistochemically labeled DETC at different ages (n = 4 per group). (D) A representative FACS profile showing the frequency of CD3 and γδ TCR⁺ T cell subsets of neonatal and P60-old skin. (E) Age-dependent quantitation of CD3⁺, γδ⁺ cells in the epidermis of Evi-LOF and wild-type littermates. (F) Representative FACS profiles showing CD3⁺ and γδ TCR⁺ T cell subsets (P40) and further analysis regarding Vγ3 TCR expression. Numbers in columns represent the number of analyzed animals. Results were pooled from three (A–C and F) and four (D–E) independent experiments. Error bars represent SD (B and C) or SEM (E). *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Figure 7.

Altered proliferation and activation of DETC in Evi-LOF mice. (A) FACS-based analysis of CD3^highγδ^high cells in dermis, lymph nodes (LNs), and spleen. (B) EdU incorporation in epidermal and dermal CD3^high cells. FACS plots on the right illustrate the gated populations. (C) Morphological analysis of the activation status of DETC in epidermal ear sheets. Activation status was determined based on number of spines. Bar, 200 µm. (D) CD3 staining of epidermal ear sheets of Evi, K14-Cre and β-cat, K14-Cre in K14-β-cat-LOF mice. Bar, 400 µm (n = 4 per group). Numbers in columns represent the number of analyzed animals. Results were pooled from three (A and C) and two (B) independent experiments. Error bars represent SD. *, P < 0.05; **, P < 0.001.

Figure 8.

Thymic and thymocyte development in Evi-LOF mice and immunophenotyping of blood and lymphoid tissue. (A) Thymus weight normalized to body weight in mice of different ages (P4, P21, and P65). (B) H&E-stained sections of Evi-LOF and control thymi at different ages. Dashed lines indicate transition between cortex and medulla. Bar, 500 µm. (C) Total thymus cellularity in mice of different age. (D) Frequencies of CD4⁻CD8⁻ double-negative (DN), CD4⁺ single-positive (CD4SP), CD8⁺ single-positive (CD8SP), and CD4⁺CD8⁺ double-positive (DP) thymocytes of P4 and P40 mice derived from either females or males. Representative FACS plots were shown on the right. (E) Whole blood from adults mice was analyzed by FACS with the indicated antigens for T cells (CD3), B cells (CD45R), monocytes (CD11b⁺, Gr1⁻), and granulocytes (CD11b⁺, Gr1⁺). (F) FACS-based analysis of naive (CD62L expressing) CD4⁺ and CD8⁺ T cells in secondary lymphatic tissue. Evi-LOF tissue had less naive CD4⁺ cells in the lymph node, indicating activation of cell-mediated immunity probably based on chronic skin inflammation and stress. (G) αβ and γδ T cells as well as CD4⁺ and CD8⁺ populations of the indicated tissues were analyzed via FACS profiling. Diagrams illustrate ratios of the populations and indicate impaired T cell homeostasis. Results were pooled from three (A, C, and D) from two (B and E–G) independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001. Error bars represent SEM (A–D) or SD (E–G). (H) Skin sections of FoxN1-Evi-LOF (P90), K14-Evi-LOF (P40), and K14-βcat-LOF (P60) were stained with CD3. Only K14-Evi-LOF and β-cat-LOF mice revealed enhanced T cell recruitment. FoxN1-Evi-LOF skin had well developed hair follicles and no hyperproliferative epidermis. Bar, 250 µm (n = 4 per group). Representative pictures of two independent experiments were shown.

Figure 9.

Evi was down-regulated in human psoriasis. (A) A representative analysis out of 12 psoriasis samples and control tissue stained against Evi. Bar, 100 µm. (B) Quantitation of Evi expression in skin biopsies based on immunohistochemistry (Error bars represent SD, n = 12; **, P < 0.001). Results were pooled from two independent experiments. (C) Enrichment plot showing a significant correlation between the 50 most up-regulated genes in human psoriasis compared with the expression profile of Evi-LOF epidermal sheets.

Figure 10.

STAT3 signaling is activated in Evi-LOF skin. (A) Immunohistochemistry against P-STAT3. Bar, 200 µm (n = 4 per group). (B) Quantitative Western blot analysis of P-STAT3 expression in the epidermis. (n = 8 per group; Error bars represent SD; *, P < 0.05). (C) Heatmap showing STAT3 signaling–related genes, which are differentially regulated in the epidermis of Evi-LOF and control mice. Values represent normalized log₂ fold changes (n = 3 per group, P < 0.01). (D) Immunohistochemistry against P-STAT3 in β-catenin-LOF epidermis. Bar, 200 µm (n = 4 per group). Results were pooled form three (A and B) and two (C and D) experiments.