Chronic, not acute, skin-specific inflammation promotes thrombosis in psoriasis murine models

Abstract

Background: Psoriasis patients exhibit an increased risk of atherothrombotic events, including myocardial infarction and stroke. Clinical evidence suggests that psoriasis patients with early onset and more severe disease have the highest risk for these co-morbidities, perhaps due to the extent of body surface involvement, subsequent levels of systemic inflammation, or chronicity of disease. We sought to determine whether acute or chronic skin-specific inflammation was sufficient to promote thrombosis.

Methods: We used two experimental mouse models of skin-specific inflammation generated in either an acute (topical Aldara application onto wild-type C57Bl/6 mice for 5 days) or chronic (a genetically engineered K5-IL-17C mouse model of psoriasiform skin inflammation) manner. Arterial thrombosis was induced using carotid artery photochemical injury (Rose Bengal-green light laser) and carotid artery diameters were measured post-clot formation. We also examined measures of clot formation including prothrombin (PT) and activated partial thromboplastin time (aPTT). Skin inflammation was examined histologically and we profiled plasma-derived lipids. The number of skin-draining lymph-node (SDLN) and splenic derived CD11b(+)Ly6C(high) pro-inflammatory monocytes and CD11b(+)Ly6G(+) neutrophils was quantified using multi-color flow cytometry.

Results: Mice treated with topical Aldara for 5 days had similar carotid artery thrombotic occlusion times to mice treated with vehicle cream (32.2 ± 3.0 vs. 31.4 ± 2.5 min, p = 0.97); in contrast, K5-IL-17C mice had accelerated occlusion times compared to littermate controls (15.7 ± 2.1 vs. 26.5 ± 3.5 min, p < 0.01) while carotid artery diameters were similar between all mice. Acanthosis, a surrogate measure of inflammation, was increased in both Aldara-treated and K5-IL-17C mice compared to their respective controls. Monocytosis, defined as elevated SDLN and/or splenic CD11b(+)Ly6C(high) cells, was significantly increased in both Aldara-treated (SDLN: 3.8-fold, p = 0.02; spleen: 2.0-fold, p < 0.01) and K5-IL-17C (SDLN: 3.4-fold, p = 0.02; spleen: 3.5-fold, p < 0.01) animals compared to controls while neutrophilia, defined as elevated SDLN and/or splenic CD11b(+)Ly6G(+) cells, was significantly increased in only the chronic K5-IL-17C model (SDLN: 11.6-fold, p = 0.02; spleen: 11.3-fold, p < 0.01). Plasma-derived lipid levels, PT and aPTT times showed no difference between the Aldara-treated mice or the K5-IL-17C mice and their respective controls.

Conclusions: Chronic, but not acute, skin-specific inflammation was associated with faster arterial thrombotic occlusion. Increased numbers of splenic and SDLN monocytes were observed in both acute and chronic skin-specific inflammation, however, increased splenic and SDLN neutrophils were observed only in the chronic skin-specific inflammation model. Understanding the cellular response to skin-specific inflammation may provide insights into the cellular participants mediating the pathophysiology of major adverse cardiovascular events associated with psoriasis.

Fig. 1

Chronic, but not acute, skin inflammation promotes shorter arterial clotting times in the Rose Bengal thrombosis assay. a Representative photographs of a C57Bl/6 WT mouse treated for 5 days with topical Aldara (left panel) and a K5-IL-17C mouse (right panel). b Representative images of H&E stained mouse back skin from a C57Bl/6 WT mouse treated for 5 days with topical Aldara and a K5-IL-17C mouse demonstrate increases in acanthosis compared to controls. c C57Bl/6 WT + Aldara clotting times (n = 25) are not different than C57Bl/6 WT + control cream (n = 21); however K5-IL-17C mice (n = 14) have significantly decreased clotting times compared to controls (n = 21), p < 0.01. d Representative photographs of cross sections of carotid arteries post-thrombus formation demonstrate no difference in diameter between the groups. Scale bar in b = 100 μm

Fig. 2

CD11b⁺Ly6C^high monocytes increase in both Aldara-treated and K5-IL-17C mice whereas CD11b⁺Ly6G⁺ neutrophils increase only in K5-IL-17C mice. a Representative flow cytometry dot plots of CD11b and Ly6C surface staining in skin-draining axillary and inguinal lymph nodes (LN). C57Bl/6 WT mice treated for 5 days with topical Aldara (n = 4 pooled samples) and K5-IL-17C mice (n = 3 pooled samples) have increases in skin-draining LN-CD11b⁺Ly6C^high cells compared to their respective controls (n = 4 and n = 6 pooled samples; p = 0.02 and p = 0.02, respectively). Each point represents lymph nodes pooled from 2 to 3 animals. b Increased splenic-CD11b⁺Ly6C^high are also observed in Aldara-treated WT mice (n = 5) and K5-IL-17C mice (n = 4) when compared to their respective controls (n = 5, n = 9; p < 0.01 and p < 0.01, respectively). Each point represents a single animal. c Aldara-treated mice have similar levels of SDLN-derived neutrophils (CD11b⁺Ly6G⁺) as control-cream treated mice (n = 4, n = 4) whereas SDLN CD11b⁺Ly6G⁺ cells are significantly increased in K5-IL-17C mice compared to littermate controls (n = 3 pooled samples, n = 6 pooled samples; p = 0.02, respectively). d Aldara-treated mice have similar levels of splenic CD11b⁺Ly6G⁺ cells as control-cream treated mice (n = 5, n = 5) and K5-IL-17C mice have significant increases in splenic CD11b⁺Ly6G⁺ cells compared to littermate controls (n = 4, n = 9 pooled samples; p < 0.01, respectively)