Translational implications of Th17-skewed inflammation due to genetic deficiency of a cadherin stress sensor

Desmoglein 1 (Dsg1) is a cadherin restricted to stratified tissues of terrestrial vertebrates, which serve as essential physical and immune barriers. Dsg1 loss-of-function mutations in humans result in skin lesions and multiple allergies, and isolated patient keratinocytes exhibit increased proallergic cytokine expression. However, the mechanism by which genetic deficiency of Dsg1 causes chronic inflammation is unknown. To determine the systemic response to Dsg1 loss, we deleted the 3 tandem Dsg1 genes in mice. Whole transcriptome analysis of embryonic Dsg1–/– skin showed a delay in expression of adhesion/differentiation/keratinization genes at E17.5, a subset of which recovered or increased by E18.5. Comparing epidermal transcriptomes from Dsg1-deficient mice and humans revealed a shared IL-17–skewed inflammatory signature. Although the impaired intercellular adhesion observed in Dsg1–/– mice resembles that resulting from anti-Dsg1 pemphigus foliaceus antibodies, pemphigus skin lesions exhibit a weaker IL-17 signature. Consistent with the clinical importance of these findings, treatment of 2 Dsg1-deficient patients with an IL-12/IL-23 antagonist originally developed for psoriasis resulted in improvement of skin lesions. Thus, beyond impairing the physical barrier, loss of Dsg1 function through gene mutation results in a psoriatic-like inflammatory signature before birth, and treatment with a targeted therapy significantly improved skin lesions in patients.

3 immunofluorescence studies. Immunoblot analyses include use of peroxidase-conjugated anti-mouse andrabbit secondary antibodies (SeraCare Life Sciences). The following antibodies were a gift from J Segre (National Human Genome Research Institute, National Institutes of Health): rabbit anti-loricrin, rabbit antiinvolucrin.

Immunoblot analysis of proteins.
Immunoblots for exon 2 knockout mice were performed as follows. Skin samples were lysed with lysis buffer (6% SDS, 0.125M Tris-HCl pH 6.8, 1x Protease Inhibitor, 1x Phosphatase Inhibitor and 1x PMSF) supplemented with 20% β-mercaptoethanol and separated by SDS-PAGE. Transferred blots were then incubated with the Dsg1 B-11 antibody and the anti-actin antibody for 2 hours at room temperature, then incubated with HRP conjugated secondary for 1 hour at room temperature.
All other immunoblots were performed as follows. Lysates were collected from E18.5 dorsal skin by manual homogenization using the Tissue Squisher (Zymo Research) in urea sample buffer (8 M urea, 1% SDS, 60 mM Tris (pH 6.8), 5% β-mercaptoethanol, 10% glycerol), sonicated and centrifuged. Samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk in PBS, incubated with primary antibody overnight at 4°C, and secondary antibody conjugated to HRP for 1 hour at room temperature. Proteins were imaged using chemiluminescence on the Odyssey FC imaging system (Licor) or exposed to film. Densitometry values were analyzed using ImageStudio software (Licor) and normalized to GAPDH or actin.

Whole mount imaging and analysis.
Dorsal skin was harvested from E18.5 mice, and either fixed immediately for 2 hours in 4% paraformaldehyde or incubated with 2.4 U/mL dispase in PBS at 37°C for 1 hour. The epidermis was peeled from the dermis and fixed for 15 minutes with 4% paraformaldehyde. Samples were blocked in blocking solution (5% normal goat serum, 1% Triton X-100, in PBS) overnight at 37°C. Samples were then incubated with Phalloidin-647 (ThermoFisher Scientific) diluted in blocking solution overnight at 37°C and then mounted onto glass slides with Prolong Gold (Life Technologies). Z-stack images (z-step size of 1.5 µm) were taken on a Nikon A1R confocal laser scanning microscope with two PMT detectors and two GaAsP detectors using a 40x objective (1.0 NA, Plan Apochromat, Oil), controlled by NIS Elements software (Nikon). Analysis of cell circularity was performed using ImageJ software on phalloidin stained images.

Additional gene expression datasets.
The fold change signature of Dsg1 -/vs. Dsg1 +/+ skin was compared to 36 others generated from microarray experiments comparing psoriasis (PSO) or atopic dermatitis (AD) lesions to normal or uninvolved human skin (Figure 7). Genome-wide fold-change estimates were calculated from each PSO/AD vs. normal/uninvolved comparison as described previously (2). Mouse genes were paired with their human orthologues based on the Homologene database (https://www.ncbi.nlm.nih.gov/homologene), creating human-mouse orthologous gene pairs. Spearman's rho statistic was calculated for each comparison with p-values calculated based on the asymptotic t approximation. To identify genes robustly elevated by PSO and AD, we calculated meta-signatures by averaging fold-change estimates across the subset of comparisons that used the same Affymetrix Human Genome Plus 2.0 microarray platform (n = 11, PSO; n = 10, AD). Human genes without a mouse orthologue were excluded from this analysis. Based on the composite meta-signature, we identified the 100 genes most strongly increased by PSO and AD (i.e., highest average fold-change) and the 100 genes most strongly decreased by PSO and AD (i.e., lowest average foldchange). We then evaluated cumulative overlap between these 100 genes and the list of corresponding mouse genes ranked according to Dsg1 -/-/Dsg1 +/+ fold-change.

RNA-seq data processing and analysis.
Quality control and adaptor trimming were performed on sequence reads from the RNA-seq data. STAR alignment (3) was used to align reads to the reference (GRCh37 for human and mm10 for mouse samples) (4). HTSeq was used for gene quantification and DESeq2 (5) was used for normalization and differential expression analysis.
For the comparison between the different datasets (e.g. Dsg1 -/vs SAM), only genes that are common were considered in the analysis. Mouse genes were paired with their human orthologue based on the Homologene database (https://www.ncbi.nlm.nih.gov/homologene), creating human-mouse orthologous gene pairs. To identify the cytokine signature of each skin condition, we took the genes induced by cytokines in human keratinocytes, and computed the enrichment among the top 500 most significant genes upregulated in the corresponding skin condition using the hypergeometric test. Data are plotted as Observed/Expected ratio for enrichment in cytokine response as a function of the adjusted p value, with adj p value < 0.05 considered statistically significant. The data set for the keratinocytes treated with cytokines was previously published in Tsoi et al. 2019 (6). Functional enrichment analysis was performed using Metascape (metascape.org) using the Gene Ontology (GO) pathways, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Pathways were considered statistically significant with p < 0.05. To identify changes in gene expression of genes involved with specific stages of keratinocyte differentiation a single-cell RNA-seq data set was used to identify genes expressed in basal, differentiated, or keratinized keratinocytes in an unbiased manner. Expression levels of these genes from the different datasets (i.e., SAM Syndrome and PF) were graphed.

Transmission electron microscopy.
Dorsal skin from E18.5 embryos were processed for conventional electron microscopic analysis as described in (7). Briefly, dorsal skin was cut into pieces and fixed in 0.1 M cacodylate buffer pH 7.3 containing 2% PFA and 2.5% glutaraldehyde overnight. Tissues were postfixed in 2% osmium tetraoxide followed by 2% uranyl acetate. Tissues were dehydrated in ascending grades of ethanol, infiltrated with propylene oxide and embedded in Embed 812 resin, cured overnight at 60°C. Tissues were ultrathin sectioned with a Leica Ultracut UC6 ultramicrotome with a diamond knife and collected on formvar coated copper mesh grids. Sections on grids were stained in 3% uranyl acetate followed by Reynolds lead citrate solution. Grids were rinsed briefly in 0.02 M NaOH, followed by distilled water and air dried. Stained sections were viewed and photographed using an FEI Tecnai Spirit G2 transmission electron microscope.

Skin barrier toluidine blue assay.
For outside-in barrier testing, E18.5 embryos were sacrificed and rinsed in PBS followed by dehydration immersion steps of 25%, 50%, 75%, 100%, 75%, 50%, 25% methanol. After the dehydration steps the embryos were rehydrated in PBS and immersed in 1-5% toluidine blue for up to 10 minutes and washed several times with PBS.

Measuring transepidermal water loss (TEWL).
A Tewameter TM300 system (Courage + Khazaka electronic GmbH) fitted with a small animal adapter was used to measure water evaporation from the skin surface and to quantify epidermal permeability/barrier function of P1 pups over 5 hours on the dorsal back. Measurements were recorded when TEWL readings were stabilized at approximately 45 seconds after the probe was placed on the skin and readings were averaged from 5 readings per time point.