Keratinocyte but not endothelial cell-specific overexpression of Tie2 leads to the development of psoriasis - PubMed (original) (raw)

Keratinocyte but not endothelial cell-specific overexpression of Tie2 leads to the development of psoriasis

Julie A Wolfram et al. Am J Pathol. 2009 Apr.

Abstract

Psoriasis is initiated and maintained through a multifaceted interplay between keratinocytes, blood vessels, gene expression, and the immune system. One previous psoriasis model demonstrated that overexpression of the angiopoietin receptor Tie2 in endothelial cells and keratinocytes led to the development of a psoriasiform phenotype; however, the etiological significance of overexpression in each cell type alone was unclear. We have now engineered two new mouse models whereby Tie2 expression is confined to either endothelial cells or keratinocytes. Both lines of mice have significant increases in dermal vasculature but only the KC-Tie2-overexpressing mice developed a cutaneous psoriasiform phenotype. These mice spontaneously developed characteristic hallmarks of human psoriasis, including extensive acanthosis, increases in dermal CD4(+) T cells, infiltrating epidermal CD8(+) T cells, dermal dendritic cells and macrophages, and increased expression of cytokines and chemokines associated with psoriasis, including interferon-gamma, tumor necrosis factor-alpha, and interleukins 1alpha, 6, 12, 22, 23, and 17. Host-defense molecules, cathelicidin, beta-defensin, and S100A8/A9, were also up-regulated in the hyperproliferative skin. All of the phenotypic traits were completely reversed without any scarring following repression of the transgene and were significantly improved following treatment with the anti-psoriasis systemic therapeutic, cyclosporin A. Therefore, confining Tie2 overexpression solely to keratinocytes results in a mouse model that meets the clinical, histological, immunophenotypic, biochemical, and pharmacological criteria required for an animal model of human psoriasis.

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Figures

Figure 1

Figure 1

EC-Tie2 and KC-Tie2 mice have distinct phenotypes. Real time RT-PCR and Western blot analysis confirmed increases in Tie2 RNA and protein in ear and back skin of KC-Tie2 (A) and EC-Tie2 (B) double transgenic mice (DT) compared with littermate controls (WT)(n = 6–8 for KC-Tie and littermate controls; n = 4 for EC-Tie2 and littermate controls). KC-Tie2 (C) and EC-Tie2 (D) mice showed increased angiogenesis (arrows) in the ear and superficial fascia compared with control littermates; only KC-Tie2 animals had erythematous ears (C). Endothelial cells of blood vessels in ear and back skin were stained using an antibody specific for MECA-32 (E). KC-Tie2 and EC-Tie2 mice have increased dermal angiogenesis compared with littermate controls (E; n = 4–6 each). KC-Tie2 but not EC-Tie2 mice have increased expression of VEGF protein compared with littermate controls as measured using ELISA (F) (KC-Tie2 n = 10–11; EC-Tie2 n = 7). *P < 0.05 compared with littermate controls. Scale bar = 100 μm.

Figure 2

Figure 2

KC-Tie2 mice spontaneously develop a psoriasis phenotype. KC-Tie2 mice develop raised plaques on their torso (A, B) and bilaterally flakey, scaly skin on their elbows (C) and surrounding their genitals (D). H&E stained sections of ear (E, F) and back (G, H) skin demonstrate acanthosis, loss of the granular cell layer, thickening of the interfollicular epidermal layers, confluent parakeratotic scale (closed arrows), increased dermal angiogenesis (open arrows), and extensive inflammatory infiltrate (asterisks) in KC-Tie2 mice (F, H) compared with control mice (E, G). Increases in Ki-67 staining were evident in ear (I, J) and back (K, L) skin from KC-Tie2 mice (J, L) compared with littermate controls (I, K). Decreases in Loricrin staining in ear (M, N) and back (O, P) skin were found in KC-Tie2 mice (N, P), as compared with control mice (M ,O). Scale bar = 100 μm.

Figure 3

Figure 3

Inflammatory cell infiltrate in KC-Tie2 mice contains T cells, dendritic cells, macrophages and neutrophils. Skin from control ear (A, E, I, M, Q) and back skin (C, G, K, O, S), and KC-Tie2 ear (B, F, J, N, R) and back skin (D, H, L, P, T) was stained using immunohistochemical methods with antibodies against CD4+ T cells (A–D), CD8+ T cells (E–H), CD11c+ dendritic cells (I–L), F4/80+ macrophages (M–P), and Gr-1+ granulocytes (Q–T). Higher magnification insets correspond to boxed areas. Scale bar = 100 μm, 10 μm in insets. Arrow in R indicates a micro abscess.

Figure 4

Figure 4

KC-Tie2 mouse skin has increased Stat3 activity and a cytokine profile consistent with human psoriasis. Stat3 (A–D) and phospho-Stat3 (E–H) are up-regulated in KC-Tie2 ear (B, F) and back skin (D, H), as compared with control ear (A, E) and back skin (C, G). ELISA analyses of a panel of cytokines shows significant increases in Th1-related cytokines (I), including IFNγ, IL-23p19p40, and IL-12p70. Th17-derived cytokines, IL-17 and IL-22, were also up-regulated in ear and back skin of KC-Tie2 mice as compared with controls. The Th2-related cytokine, IL-4 remained unchanged in both ear and back skin (I) (n = 6 to 8). Real time RT-PCR results show significant upregulation of IL-1α, IL-6, TNFα, cathelicidin, β-defensin, S100A8, and S100A9 in KC-Tie2 back skin compared with littermate controls (J)(n = 3–4). Higher magnification insets correspond to boxed areas. *P < 0.05 compared with littermate controls. Scale bar = 100 μm, 10 μm in insets.

Figure 5

Figure 5

Doxycycline and CsA treatments lead to reversal and improvement in the psoriatic phenotype, respectively. Real time RT-PCR (A) and Western blotting (B) analyses of Tie2 expression following doxycycline (Dox) or cyclosporin A (CsA) treatment (n = 4 each). Tie2 expression returns to control levels following Dox-mediated gene repression at both RNA and protein levels, whereas CsA treatment leads to decreases in Tie2 RNA in both ear and back skin, but no change in Tie2 protein in back skin only. Western blot (B) displays representative samples from littermate control (ST, n = 1), KC-Tie2 (DT, n = 1), KC-Tie2 + Dox (DT+Dox, n = 2), KC-Tie2 + CsA (DT+CsA, n = 2). Western blot (B) and ELISA (C) analyses of VEGF protein show return to baseline levels in ear and back skin of Dox treated KC-Tie2 mice. CsA treatment lead to reductions in VEGF compared with untreated animals, however these levels remained significantly higher than control mice. Representative H&E stained ear and back skin (E) and epidermal thickness quantification of control mice post treatment, KC-Tie2 mice before treatment and KC-Tie2 mice following 4 weeks of Dox exposure or CsA administration (D). Quantification of MECA-32-stained endothelial cells from ear and back skin (G) with corresponding analysis of blood vessel number for control mice post treatment, KC-Tie2 mice pretreatment and KC-Tie2 mice following 4 weeks of Dox or CsA exposure (F). *P < 0.05 compared with littermate controls. +P < 0.01 compared with KC-Tie2 mice before treatment. Scale bar = 100 μm.

Figure 6

Figure 6

Immunocyte profile changes in back skin of KC-Tie2 mice treated with doxyclycline or CsA. CD4+ T cell (A–D), CD8+ T cell (E–H), Cd11c+ dendritic cell (I–L), and F4/80+ macrophage (M–P) staining in back skin of control animals (A, E, I, M), KC-Tie2 mice before treatment (B, F, J, N), and KC-Tie2 mice treated with doxycycline (C, G, K, O) or CsA (D, H, L, P). Higher magnification insets correspond to boxed areas. Scale bar = 100 μm, 10 μm in insets.

Figure 7

Figure 7

Immunocyte profile changes in ear skin of KC-Tie2 mice treated with doxycycline or CsA. CD4+ T cell (A–D), CD8+ T cell (E–H), Cd11c+ dendritic cell (I–L), and F4/80+ macrophage (M–P) staining in ear skin of control animals (A, E, I, M), KC-Tie2 mice before treatment (B, F, J, N), and KC-Tie2 mice treated with doxycycline (C, G, K, O) or CsA (D, H, L, P). Higher magnification insets correspond to boxed areas. Scale bar = 100 μm, 10 μm in insets.

Figure 8

Figure 8

Cytokine regulation following treatment with doxycycline or CsA. IFNγ, IL-17, and IL-12p70 protein expression in ear and back skin measured using ELISA returns to control levels following doxycycline (n = 4 each) or CsA treatment (n = 4 each) (A). IL-23p19p40 levels significantly decreased in back skin following doxycycline and CsA treatment but remained elevated as compared with littermate controls. In contrast, IL-23p19p40 expression in ear skin increased from that of KC-Tie2 levels following doxycycline treatment. IL-22 expression in ear and back skin decreases to baseline levels following gene repression using doxycycline but not CsA treatment (A). Real-time RT-PCR analysis of IL-6, TNFα, cathelicidin, β-defensin, and S100A9 expression in back skin harvested from KC-Tie2 mice treated with doxycycline or CsA for 4 weeks showed a complete return to baseline expression levels (B). *P < 0.05 compared with littermate controls, +P < 0.05 compared with KC-Tie2 pretreatment.

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