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 [2]. 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.