CARD14 Expression in Dermal Endothelial Cells in Psoriasis

<div><p>Mutations in the <i>caspase recruitment domain, family member 14</i> (<i>CARD14</i>) gene have recently been described in psoriasis patients, and explain the <i>psoriasis susceptibility locus 2</i> (<i>PSORS2</i>). CARD14 is a scaffolding protein that regulates NF-κB activation, and psoriasis-associated <i>CARD14</i> mutations lead to enhanced NF-κB signaling. CARD14 is expressed mainly in epidermal keratinocytes, but also in unidentified dermal cells. In this manuscript, the identity of the dermal cell types expressing CARD14, as well the potential functional consequence of overactive CARD14 in these dermal cell types, was determined. Using two-color immunofluorescence, dermal CARD14 did not co-localize with T-cells, dendritic cells, or macrophages. However, dermal CARD14 did highly co-localize with CD31⁺ endothelial cells (ECs). CARD14 was also expressed non-dermal endothelial cells, such as aortic endothelial cells, which may indicate a role of CARD14⁺ECs in the systemic inflammation and cardiovascular comorbidities associated with psoriasis. Additionally, phosphorylated NF-κB was found in psoriatic CARD14⁺ CD31⁺ ECs, demonstrating this pathway is active in dermal ECs in psoriasis. Transfection of dermal ECs with psoriasis-associated <i>CARD14</i> mutations resulted in increased expression of several chemokines, including CXCL10, IL-8, and CCL2. These results provide preliminary evidence that CARD14 expression in ECs may contribute to psoriasis through increased expression of chemokines and facilitating recruitment of immune cells into skin.</p></div

Patient with a confirmed <i>CARD14</i> mutation expressed CARD14 and activated NF-κB in dermal endothelial cells.

<p>(a) Two color immunofluorescence of lesional skin for patient GEN001 (harbors an activating <i>CARD14</i> mutation) for CARD14 (red) versus CD3⁺ T-cells, CD11c⁺ dendritic cells, CD163⁺ macrophages, and CD31⁺ endothelial cells (green). (b) Immunohistochemistry of phosphorylated NF-κB (pNF-κB) in non-lesional (middle) and lesional (right) frozen skin sections from patient GEN001 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111255#pone.0111255-Jordan1" target="_blank">[2]</a>. Negative-control immunohistochemistry staining is shown in lesional psoriatic skin (left). (c) Two-color immunofluorescence staining of CD31 (green) and pNF-κB (red) in non-lesional (left) and lesional (right) frozen skin sections from patient GEN001. Enlarged images are shown to the left. Bar = 10 µm.</p

CARD14 was expressed in dermal endothelial cells.

<p>Two color immunofluorescence on frozen sections of psoriatic skin for CARD14 (red) versus (a) CD3⁺T-cells, CD11c⁺ dendritic cells, CD163⁺ macrophages, and CD31+ endothelial cells (green); as well as versus (b) PAL-E+ blood endothelial cells and LYVE-1 lymphatic endothelial cells (green). Representative images; bar = 10 µm. (c) Quantitative RT-PCR analysis of various CARDs in keratinocytes (green), endothelial cells (red), T-cells (blue), and monocytes (yellow).</p

Phosphorylated NF-κB (pNF-κB) was upregulated in psoriatic skin and dermal pNF-κB co-localized with CARD14⁺ ECs.

<p>(a) Immunohistochemistry of pNF-κB in normal, non-lesional, and lesional frozen skin sections. (b) Two-color immunofluorescence of CD31 (green) and pNF-κB (red) in normal, non-lesional, and lesional skin. Representative images; bar = 10 µm. (c) Triple-color immunofluorescence staining of CD31 (green), CARD14 (red), and pNF-κB (blue) in lesional skin. Representative images at 63X magnification; enlarged images are shown to the left.</p

Transfection of psoriasis-associated <i>CARD14</i> mutations into dermal endothelial cells resulted in increased expression and of several chemokines.

<p>Protein expression of various chemokines in cell culture media of endothelial cells transfected with wild-type (fl and sh) or psoriasis-associated <i>CARD14</i> mutation constructs (G117S and E138A) and stimulated overnight with TNFα (25 ng/mL). N = 3 per group. * P<0.05, ** P<0.01.</p

Clinical photographs of two representative patients with disease relapse upon cessation of efalizumab treatment.

<p>(<b>A</b>) Baseline lesions (left), response to efalizumab (center) and a marked erythematous psoriatic reaction 5 weeks after ceasing treatment (right). (<b>B</b>) Baseline photographs showing upper leg psoriatic plaques (left) which responded to efalizumab at week 12 (center) and a relapse in the same location (right). Note the inflammatory nature of the lesions during relapse.</p

GSEA analysis of published gene sets.

a<p>Size, number of genes in gene set.</p>b<p>ES, enrichment score.</p>c<p>NES, normalized enrichment score.</p>d<p>CS, connectivity score.</p>e<p>REF, reference for gene set.</p

Enrollment flowchart.

<p>Enrollment flowchart.</p

Clinical and histological response to efalizumab and during relapse.

<p>(<b>A</b>) Mean PASI scores, (<b>B</b>) circulating absolute lymphocyte count, (<b>C</b>) epidermal thickness (µm), (all n = 8) and (<b>D</b>) normalized keratin 16 (K16) mRNA expression (n = 4), throughout the trial and at time of relapse. Non-lesional (NL) and lesional skin (LS), error bars represent the standard error of the mean. ^* p<0.05; ^** p<0.01; ^*** p<0.001. Representative (<b>E</b>) haematoxylin and eosin (H&E), (<b>F</b>) K16 protein, (<b>G</b>) neutrophil elastase, (<b>H</b>) CD3⁺ T cell immunohistochemistry, and (<b>I</b>) CD3⁺ T cell counts, showing the psoriasiform nature of the relapse lesion, with characteristic K16 staining, neutrophils and CD3⁺ T cells. Bar is 100 µm.</p

Genomic characterization of relapsed psoriasis lesions.

<p>(<b>A</b>) Venn diagram showing DEGs for different comparisons, week 12 versus LS; week 12 versus relapse; relapse versus LS. There were no unique DEGs in relapse compared to LS skin. (<b>B</b>) Scatter plot showing an excellent correlation between the “treatment effect” and “relapse effect”. (<b>C</b>) Number of genes improved by treatment and reversed by relapse. Red blocks were increased DEGs, green blocks were decreased DEGs.</p