Chronic skin inflammation accelerates macrophage cholesterol crystal formation and atherosclerosis

Abstract

Inflammation is critical to atherogenesis. Psoriasis is a chronic inflammatory skin disease that accelerates atherosclerosis in humans and provides a compelling model to understand potential pathways linking these diseases. A murine model capturing the vascular and metabolic diseases in psoriasis would accelerate our understanding and provide a platform to test emerging therapies. We aimed to characterize a new murine model of skin inflammation (Rac1V12) from a cardiovascular standpoint to identify novel atherosclerotic signaling pathways modulated in chronic skin inflammation. The RacV12 psoriasis mouse resembled the human disease state, including presence of systemic inflammation, dyslipidemia, and cardiometabolic dysfunction. Psoriasis macrophages had a proatherosclerotic phenotype with increased lipid uptake and foam cell formation, and also showed a 6-fold increase in cholesterol crystal formation. We generated a triple-genetic K14-RacV12-/+/Srb1-/-/ApoER61H/H mouse and confirmed psoriasis accelerates atherogenesis (~7-fold increase). Finally, we noted a 60% reduction in superoxide dismutase 2 (SOD2) expression in human psoriasis macrophages. When SOD2 activity was restored in macrophages, their proatherogenic phenotype reversed. We demonstrate that the K14-RacV12 murine model captures the cardiometabolic dysfunction and accelerates vascular disease observed in chronic inflammation and that skin inflammation induces a proatherosclerotic macrophage phenotype with impaired SOD2 function, which associated with accelerated atherogenesis.

Keywords: Atherosclerosis; Cardiology; Cardiovascular disease; Inflammation; Macrophages.

Figure 1. Overexpression of constitutive active Rac1 V12 in keratinocytes induces psoriasis accompanied by systemic chronic inflammation.

(A) Severity of psoriasis phenotype based on visible symptoms was determined on a 0–5 scale. Severity levels of 2 and 3 accounted for the majority of all animals (n = 117). (B) Blood cell composition differences were determined by flow cytometry. Decreased levels of B cells, T cells, classical (Ly6C^hi), and nonclassical (Ly6C^lo) monocytes were observed, while the neutrophil population increased (n = 8/9). (C) Chronic inflammation was detected by measuring plasma cytokine levels in adolescent (younger than 10 weeks) and adult (older than 12 weeks) K14-Rac1V12^–/+ mice and their LMC. Increased levels of all proinflammatory cytokines were detected (n > 5). (D) Transmission electron microscopy of mouse aortas showed infiltration of myeloid cells into the subendothelial space (n = 3/3) and the tunica media, which was confirmed by flow cytometry (E) of Liberase-digested aortas (n = 10/13). Neutrophils, resident (res) DCs and especially macrophages are showing the most significant changes. (F) Plasma from adult LMC and K14-Rac1V12^–/+ mice were used to determine MCP-1 and RANTES chemokine levels (n = 27/38). (Data are expressed as mean ± SEM, n = LMC/K14-Rac1V12^–/+; Mann-Whitney test P < 0.05) (LMC, littermate control; EC, endothelial cell; E, elastin layer; C, collagen; SMC, smooth muscle cell; L/M/MØ, lymphoid or myeloid cell; MØ, macrophages; conv, conventional; res, resident; myl, myeloid; Monos, monocytes; M0, macrophages; MCP-1 (CCL2), monocyte chemoattractant protein 1. Scale bar: 5µm. *P < 0.05, **P < 0.0005.

Figure 2. Psoriatic mouse macrophages display proatherosclerotic characteristics.

(A) BMDM were analyzed for the presence of Rac1 and the attached Myc tag. No differences in expression of Rac1 or Myc were detected. (B) Equal amounts of BM cells were plated for differentiation. Cell count after differentiation was determined. Resulting cell counts are increased in psoriatic mice, possibly due to differences in adhesion and proliferation. (C) Gene expression of macrophage polarization was determined using qPCR and is altered in psoriatic BMDMs as compared with LMC BMDMs. (D and E) Scanning electron microscopy analysis of BMDM phenotype with subsequent quantification of cells presenting a round phenotype (n = 6, each with 5 fields imaged and quantified). (F) Phenotypically, BMDMs were characterized using scanning electron microscopy and immunofluorescence analysis for CD206 (red) and CD68 (green) (nuclei: DAPI, blue) (representative images of n = 3). (G and H) Modified lipoprotein uptake by BMDM was determined after treatment with 20 μg/ml DiI-labeled AcLDL or OxLDL for 2h and 4h of incubation. Five images per condition were taken at equal exposure and MFI of 100 individual cells per image analyzed using ImageJ. (I) Differences in modified LDL receptors between LMC and K14-Rac1V12^–/+ BMDM were detected using qPCR. (J) Macrophage foam cell formation upon 24h 50μg/ml AcLDL or OxLDL treatment was detected after subsequent Bodipy493/503 labeling (green, nuclei: DAPI, blue) and ImageJ analysis as described in E and F. (K and L) Presence of cholesterol crystals was visualized using polarized light microscopy of mouse (K) and human (L) macrophages, displaying increased cholesterol crystal presence under baseline and lipid treated conditions (50 μg/ml) (BMDM n = 6/6, HMDM n = 5/10). (Data are expressed as mean ± SEM; 2-way ANOVA with Bonferroni correction [H and J] with P < 0.007; duplicates for H and J; Mann-Whitney test [B, C, E, and I] with P < 0.05; n ≥ 6/6, *P < 0.05, **P < 0.005, ***P < 0.0005 indicates significance to LMC control; ^P < 0.05, ^^P < 0.005, ^^^P < 0.0005 indicates significance between indicated groups; scale bars: 50 μm [F and G],100 μm [J]) (LMC, littermate control; AcLDL, acetylated low-density lipoprotein; OxLDL, oxidized LDL)

Figure 3. Psoriasis-induced chronic inflammation accelerates atherogenesis.

K14-Rac1V12^–/+ mice were backcrossed into Srb1^–/–/ApoeR61^H/H mice and an atherosclerosis study conducted. (A) Psoriasis severity of Srb1^–/–/ApoeR61^H/H/K14-Rac1V12^–/+ mice increased due to HFD for 2 weeks. After 2 weeks of HFD, detection of atherosclerotic plaques in whole aorta (B, images presented of the aortic arch) and aortic root sections (C) using Oil Red O staining showed a significant increase in atherosclerosis development in psoriatic mice vs. LMC mice. (D) Macrophage (MOMA-2), smooth muscle cell (SM22α) and cholesterol crystal content, necrotic core area, general collagen and elastin (Movat) deposition, and calcification (Van Kossa) in the aortic root of animals presenting plaque formation in C are shown to compare the anatomy and quality of the developed atherosclerotic lesions. Quantifications (E) were done using ImageJ software and are displayed as percentage of total plaque area. (F) Sirius Red staining was performed and visualized using bright field and polarized light microscopy. Content of different collagen fiber thicknesses was determined using ImageJ. Green/yellow indicates thinner collagen fibers, while orange/red indicates thick collagen fibers. (Data are expressed as mean ±SEM, Mann-Whitney test with *P < 0.05, **P < 0.005, n = 9/ 4 for Oil Red O staining, n = 6/4 for plaque characterization staining, scale bar: 500 μm) (PL, polarized light; CC, cholesterol crystals; HFD, high-fat diet; PSO, psoriasis; rVSMC, resident vascular smooth muscle cells [indicated by the arrows]. Arrow heads indicate plaque/infiltrated VSMC).

Figure 4. Manganese-dependent superoxide dismutase 2 (SOD2) function is impaired in psoriatic macrophages.

(A) Mouse BM cell SOD2–derived ROS were labeled using MitoSox and examined by flow cytometry (n = 12/15). (B) qPCR analysis of BMDMs revealed changes in mitochondria-related gene expression (n ≥ 4). (C) Mitochondrial ROS of differentiated macrophages are increased in K14-Rac1V12^–/+ BMDM. A representative histogram is shown (n = 12/15). (D) Differentiation levels of BMDMs in the presence of MnCl₂, a SOD2 activator, was determined and significantly reduced (n ≥ 11). (E) mRNA expression profile was compared with pre-MnCl₂ differentiation conditions using qPCR (n ≥ 5). (F) Since mitochondrial ROS staining does not qualify as an SOD2 activation measurement, SOD2 activity was detected using the SOD assay kit in the presence of potassium cyanide to inhibit SOD1 and SOD3 activity, displaying decreased activity in psoriatic BMDMs, which is rescued to LMC levels when differentiated with MnCl₂ and treated with MnTBAP (n ≥ 5). (G and H) Mitochondrial ROS were labeled using MitoSox after treatment with the SOD2 mimetic MnTBAP (12.5 μM) in MnCl₂ and control-differentiated LMC and psoriatic BMDM. Mitochondrial ROS were visualized using microscopy after PFA fixation and DAPI nuclei labeling (n = 3) and detected using flow cytometry (n ≥ 5). A representative histogram is shown. (Data are expressed as mean ± SEM; *P < 0.05, **P < 0.005, ***P < 0.0005 indicates significance to LMC control; ^#P < 0.05 indicates significance to K14-Rac1V12^–/+ control; ^{^}P < 0.05 indicates significance between indicated groups; [A, B, C, D, and F] Mann-Whitney test, [E and G] 2-way ANOVA with Bonferroni correction P = 0.01, scale bar: 50 μm) (LMC, littermate control; MnTBAP, Manganese [III] Tetrakis [4-Benzoic Acid] Porphyrin chloride).

Figure 5. Manganese-dependent superoxide dismutase 2 (SOD2) is a key regulator in psoriasis-induced proatherosclerotic macrophage reprogramming.

BMDM were isolated from LMC and K14-Rac1V12^–/+ mice. Cells were differentiated in the absence or presence of 5 μM MnCl₂. Additionally, sets of BMDMs were pretreated with 12.5 μM MnTBAP, a SOD2 mimetic, before conducting the experiment. (A) Apoptosis and necrosis in BMDMs was determined using flow cytometry. Results of early apoptotic events under baseline and 50 μg/ml AcLDL-treated conditions (24h) displays a significant reduction in K14-Rac1V12^–/+ BMDMs differentiated in the presence of Manganese (n ≥ 4). Modified lipid uptake (B), foam cell formation (C), and cholesterol efflux (D) experiments were performed in the presence of MnCl₂ or MnTBAP using control and MnCl₂ differentiated BMDMs (n ≥ 5). (E) BMDMs were treated with 50 μg/ml DiI-AcLDL (red) for 24h and nuclei labeled with DAPI (blue) to visualize AcLDL cholesterol crystal formation (arrows indicated CC, arrowheads indicate large densely lipid packed intracellular areas, representative images of total n = 3; scale bar: 50 µm). (F) CC were visualized with a polarized light microscope and quantified using ImageJ under baseline and 50 μg/ml AcLDL treatment conditions (n ≥ 5). (Data are expressed as mean ± SEM; 2-way ANOVA with Bonferroni correction [A, D, and F] P < 0.007, [B and C] ^{^/#}P < 0.01, ^{^^/##}P < 0.005, ^{^^^/###}P < 0.0005) (MnCl₂, Manganese chloride; LMC, littermate control; MnTBAP, Manganese [III] Tetrakis [4-Benzoic Acid] Porphyrin chloride; ROS, reactive oxygen species; FC, foam cell formation; Ac, acetylated; LDL, low-density lipoprotein; MFI, median fluorescence intensity).