CARD14 expression in dermal endothelial cells in psoriasis

Abstract

Mutations in the caspase recruitment domain, family member 14 (CARD14) gene have recently been described in psoriasis patients, and explain the psoriasis susceptibility locus 2 (PSORS2). CARD14 is a scaffolding protein that regulates NF-κB activation, and psoriasis-associated CARD14 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 CARD14 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.

Figure 1. CARD14 was expressed in dermal endothelial cells.

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).

Figure 2. Phosphorylated NF-κB (pNF-κB) was upregulated in psoriatic skin and dermal pNF-κB co-localized with CARD14⁺ ECs.

(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.

Figure 3. Patient with a confirmed CARD14 mutation expressed CARD14 and activated NF-κB in dermal endothelial cells.

(a) Two color immunofluorescence of lesional skin for patient GEN001 (harbors an activating CARD14 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 . 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.

Figure 4. Transfection of psoriasis-associated CARD14 mutations into dermal endothelial cells resulted in increased expression and of several chemokines.

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

Figure 5. Increased expression of CARD14-modulated chemokines in psoriatic skin co-localized with dermal endothelial cells.

(a) Immunohistochemistry of CXCL1, CCL2, and CCL5 in non-lesional and lesional skin of both classical psoriasis (left, representative images) and patient GEN001 (right). (b) Two-color immunofluorescence of these chemokines (red) and CD31+ endothelial cells (green) in classical psoriasis lesional skin. Representative images; bar = 10 µm.