Caspase-1 Deficiency Decreases Atherosclerosis in Apolipoprotein E-Null Mice

Canadian Journal of Cardiology 28 (2012) 222–229

Basic Research

Caspase-1 Deficiency Decreases Atherosclerosis in Apolipoprotein E-Null Mice Jessica Gage, MSc,a Mirela Hasu, BSc,b Mohamed Thabet, MSc,a and Stewart C. Whitman, PhDa,b,c,ⴱ a

Vascular Biology and Atherosclerosis Laboratory, University of Ottawa Heart Institute, Ottawa, Ontario, Canada b

Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ontario, Canada c

Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada

ABSTRACT

RÉSUMÉ

Background: Caspase-1 is a cysteine protease that contributes to mammalian immunity through proteolytic activation of the proinflammatory cytokines, interleukin (IL)-1␤ and IL-18. Methods: To determine if caspase-1 deficiency can protect apolipoprotein E-null (Apoe⫺/⫺) mice from atherosclerosis, gender-matched, paired-littermate Apoe⫺/⫺ mice with (Casp1⫹/⫹Apoe⫺/⫺) or without (Casp1⫺/⫺Apoe⫺/⫺) a functional caspase-1 (Casp1) gene were fed either a low fat diet for 26 weeks, or a saturated fat and cholesterolenriched diet for 8 weeks. Plasma lipids and lipoproteins were determined and atherosclerosis was quantified in the aortic sinus and aortic arch. Results: On either diet, caspase-1 deficiency did not affect total serum cholesterol concentrations and lipoprotein-cholesterol distributions. However, caspase-1 deficiency significantly decreased atherosclerosis in the ascending aorta by 35%-45% in both sexes of mice fed either diet. We further examined atherosclerotic lesions for 2 indices of immune cell activation: Major Histocompatibility Complex (MHC) class II and interferon (IFN)-␥ expression. There was a 40%-50% reduction in the number of lesion-associated cells expressing MHC class II from both sexes of Casp1⫺/⫺Apoe⫺/⫺ mice compared with Casp1⫹/ ⫺/⫺ ⫹Apoe mice and, a significant reduction in lesion-associated IFN-␥ in female Casp1⫺/⫺Apoe⫺/⫺ compared with their Casp1⫹/⫹Apoe⫺/⫺ counterparts.

Introduction : La caspase 1 est une cystéine protéase qui contribue à l’immunité des mammifères par l’activation protéolytique des cytokines pro-inflammatoires, de l’interleukine (IL) 1␤ et de l’IL 18. Méthodes : Pour déterminer si le déficit en caspase 1 peut protéger les souris déficientes en apolipoprotéine E (apoE⫺/⫺) contre l’athérosclérose, les souris apoE⫺/⫺ de même sexe, de même portée, avec ou sans gène fonctionnel de la caspase 1 (Casp-1⫹/⫹ apoE⫺/⫺ ou Casp-1⫺/⫺ apoE⫺/⫺) ont été nourries soit par une diète faible en gras durant 26 semaines, soit par une diète riche en gras saturé et en cholestérol durant 8 semaines. Les liquides plasmatiques et les lipoprotéines ont été déterminés, et l’athérosclérose a été quantifiée dans le sinus de l’aorte et l’arc de l’aorte. Résultats : Dans l’une ou l’autre des diètes, le déficit en caspase 1 n’a pas modifié les concentrations de cholestérol sérique total et les répartitions de cholestérol dans les lipoprotéines. Cependant, le déficit en caspase 1 a considérablement diminué l’athérosclérose dans l’aorte ascendante de 35 % à 45 % chez les deux sexes de souris nourries par l’une ou l’autre des diètes. Nous avons examiné de manière plus approfondie les lésions athéroscléreuses selon 2 indices d’activation de la cellule immunitaire : la classification du complexe majeur d’histocompatibilité (CMH) II et l’expression de l’interféron (INF)-␥. Il y a eu une réduction de 40 % à 50 % dans le nombre de cellules associées aux lésions exprimant la classification CMH II des

Caspases are a family of cysteine proteases that play critical roles in mammalian apoptosis and the proteolytic activation of cytokines.1,2 Caspase-1, also known as interleukin (IL)-1␤-con-

verting enzyme, is a member of the inflammatory caspase subfamily and is constitutively expressed in various cells, including monocyte-derived macrophages as a proenzyme that requires 2 internal cleavages before becoming an enzymatically active heterodimer. Caspase-1 was originally identified as the enzyme responsible for the maturation of both IL-1␤ 3,4 and IL-18.5 Both IL-1␤ and IL-18 are major mediators of inflammation and a number of laboratories have shown that both cytokines promote atherosclerosis in the apolipoprotein E-null (Apoe⫺/⫺) mouse.6-8 IL-33 is also a substrate for caspase-1 but, unlike IL-1␤ and IL-18, caspase-1 cleavage of IL-33 results in its inactivation rather than its activation.9 Caspase-1 is expressed in human ath-

Received for publication September 15, 2011. Accepted October 19, 2011. *The manuscript was communicated by Ross W. Milne and Yves L. Marcel, on behalf of the senior author, Dr Stewart C. Whitman, who passed away on February 19, 2010. Corresponding author: On behalf of Dr Stewart C. Whitman, Drs Ross W. Milne and Yves L. Marcel, University of Ottawa Heart Institute, 40 Ruskin St, Ottawa, Ontario K1Y 4W7, Canada. Tel.: ⫹1-613-761-5254; fax: ⫹1613-761-5281. E-mail addresses: [email protected] and [email protected] See page 228 for disclosure information.

0828-282X/$ – see front matter © 2012 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.cjca.2011.10.013

Gage et al. Caspase-1 and Atherosclerosis Conclusions: We conclude that caspase-1 promotes atherosclerosis by enhancing the inflammatory status of the lesion through a mechanism likely involving activation of lesion-associated immune cells and IFN-␥ expression.

erosclerotic lesions, but not in nondiseased vessels10 and it has been recently demonstrated that cholesterol crystals within atherosclerotic plaques can trigger nucleotide-binding domain leucinerich repeat pyrin domain-containing 3 (NLRP3) inflammasomes leading to caspase-1-mediated activation of IL-1␤,11 a pathway that appears to be important in early atherogenesis.11 Together, these observations merit further examination as to whether caspase-1 activity affects the process of atherogenesis. IL-1␤ is a proinflammatory cytokine that has a variety of activities, including the induction of cyclooxygenase type 2,12 cytosolic phospholipase A2,13 and inducible nitric oxide synthase14 expression. IL-18 plays an important role in cell-mediated immune responses via the activation of macrophages15 and natural killer cells.16 IL-18 is thought to enhance cellmediated immune responses by promoting the synthesis and release of interferon (IFN)-␥.15,16 IFN-␥ has a range of biological properties in cultured cells that could influence development of atherosclerotic lesions. These include the promotion of lipoprotein oxidation, macrophage-derived foam cell formation, and immune cell activation.17 Animal models that combine genetic risks for atherosclerosis with an altered immune system have been invaluable in demonstrating a link between atherosclerosis and immunity.18 Deficiency of IL-1␤ has been shown to prevent atherosclerosis in Apoe⫺/⫺ mice.8 Furthermore, we and others have shown that IL-18 promotes atherosclerosis in Apoe⫺/⫺ mice6,7 and that IFN-␥ is a potent proatherogenic cytokine in vivo.19-21 Although caspase-1 is primarily responsible for the maturation of IL-1␤ and IL-18, other proteases such as proteinase-322 may contribute to the activation of these 2 cytokines. By examining the effect of caspase-1 deficiency on lesion development in both sexes of Apoe⫺/⫺ mice, we show in this current study that functionally active caspase-1 contributes to the development of atherosclerotic lesions. Furthermore, we provide evidence that caspase-1 activity promotes atherosclerosis by enhancing the inflammatory status of the lesion through a mechanism most likely involving the production of IFN-␥. Methods Animals Caspase-1-null (Casp1⫺/⫺) mice generated within and maintained on the C57BL/6 strain3 were a generous gift from Abbott Bioresearch Center, Worcester, MA. Apoe⫺/⫺ mice had been backcrossed for 10 generations to the C57BL/6 background originally obtained from The Jackson Laboratory (Bar Harbor, ME), and maintained in the specific pathogen-free Animal Care Facility at the University of Ottawa Heart Institute. Casp1⫺/⫺ and Apoe⫺/⫺ mice were crossed to eventually obtain the Casp1⫹/⫹Apoe⫺/⫺ and Casp1⫺/⫺Apoe⫺/⫺ mice that constituted the experimental groups. Genetic screening for the Apoe and Casp1 genes was carried out by polymerase chain reaction on DNA from mouse tail samples. The Apoe gene was screened using the primers and the reaction conditions recommended by the Jackson Laboratory. The Casp1 gene was

223 souris des deux sexes Casp-1⫺/⫺ apoE⫺/⫺ comparativement aux souris Casp-1⫹/⫹ apoE⫺/⫺ et une réduction significative dans l’ INF-␥ associée aux lésions chez la femelle Casp-1⫺/⫺ apoE⫺/⫺ comparativement à leurs homologues Casp-1⫹/⫹ apoE⫺/⫺. Conclusions : Nous concluons que la caspase 1 conduit à l’athérosclérose en augmentant l’état inflammatoire de la lésion par un mécanisme susceptible d’impliquer l’activation des cellules immunitaires associées à la lésion et à l’expression de l’INF-␥.

screened using the following primers: 5-CATGCCTGAATAATGATCACC-3 (specific for the targeted gene), 5-GAAGAGATGTTACAGAAGCC-3 (specific for the native Casp1 gene upstream of the targeting construct), and 5-GCGCCTCCCCTACCCGG-3 (specific for the neo resistance gene), and the following reaction conditions: 1 cycle (95oC, 30 seconds), 30 cycles (94oC, 30 seconds; 56oC, 30 seconds; 72oC, 3 minutes), and 1 cycle (72oC, 5 minutes), hold 4oC. Diet and study groups Male and female Apoe⫺/⫺ mice genotyped as Casp1⫹/⫹ or Casp1⫺/⫺ were terminated with an overdose of Somnotol (MTC Pharmaceuticals, Cambridge, ON) after being fed a normal rodent diet for 26 weeks from the time of weaning, or a diet supplemented with 1.5% (wt/wt) cholesterol and 16% (wt/wt) butter fat (TD 94059; Harlan Teklad, Madison, WI) for 8 weeks when the mice reached 8 weeks of age. Feeding Apoe⫺/⫺ mice a normal diet for 26 weeks would be anticipated to give early atherosclerotic lesions comprised mainly of macrophage foam cells, whereas Apoe⫺/⫺ mice fed a high-fat diet for 8 weeks would lead to more frequent advanced-stage lesions characterized by a well-defined necrotic core with or without a fibrous cap.23 All mice at the end of both studies appeared normal and were of average weight for their age and gender. However, if mice were left on the high fat, high cholesterol diet for more than 10 weeks a significant proportion of Casp1⫺/ ⫺/⫺ ⫺Apoe mice (3 times more prevalent in males than females) died suddenly due to interstitial pneumonitis accompanied by severe perivascular eosinophilia. None of the Casp1⫺/ ⫺/⫺ ⫺Apoe mice fed the atherogenic diet for 8 weeks displayed any form of histocytic pneumonitis after detailed histological analysis. All procedures involving animals were approved by the University of Ottawa Animal Care Committee. Plasma cholesterol and lipoprotein profiles Serum cholesterol concentrations were measured using commercial kits (Wako Bioproducts, Richmond, VA). To determine serum lipoprotein profiles, 60 ␮L serum samples from individual mice were subjected to Superose 6 (Pharmacia LKB Biotechnology, Uppsala, Sweden) gel exclusion chromatography and total cholesterol concentrations (Wako Bioproducts) were measured in 0.5 mL fractions. Tissue collection Hearts from Somnitol-anesthetized mice were perfused with phosphate-buffered saline via a cannula inserted into the left ventricle and the perfusate was collected from the right atrium. Hearts were separated from the base of the aorta, embedded in optimum cutting temperature medium (Fisher Scientific, Ottawa, ON) and snap frozen on a metal block immersed in liquid nitrogen and stored at ⫺80oC.

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Quantification of atherosclerotic lesions in tissue sections The size of atherosclerotic lesions in the ascending aorta was determined from 4 Sudan IV-stained serial sections, cut 10 ␮m thick and separated by 100 ␮m. Lesion analysis began with the first section of tissue that contained the ostia for the coronary arteries; the region defining the boundary between the aortic sinus and ascending aorta. Using the Sudan IV staining as a guide, lesion area defined as intimal tissue within the internal elastic lamina was determined using Image-Pro Plus software (V6.2; Media Cybernetics, Silver Springs, MD) on images that were created using a digital CoolSNAP cf camera (Roper Scientific Inc, Duluth, GA). The mean lesion area derived from the 4 serial sections was taken as the average lesion size for each animal as described previously.24 Quantification of atherosclerotic lesions on the intimal surface of the aorta The intimal surface of the aorta that had been fixed in 4% formaldehyde was exposed as described previously.24 The aorta was pinned to a dark surface, digitally photographed, and scanned. The aortic arch (comprising the superior 3 mm) as well as the individual lesions within this region were traced and the total surface and lesion areas were calculated using ImagePro software.24 Results are presented as % lesion area. Immunohistochemistry Methods for immunohistochemical detection of IFN-␥ (anti-mouse IFN-␥; BD Pharmagen, Mississauga, ON), IL-1␤ (anti-mouse IL-1␤; Rockland Immunochemicals, Gilbertsville, PA), IL-18 (anti-rat IL-18; R&D Systems, Minneapolis, MN), CD3-positive cells (anti-mouse CD3E; BD Pharmagen), Major Histocompatibility Complex (MHC) class II-positive cells (anti-mouse MHC class II; Biosource International, Camarillo, CA), and macrophages (anti-F4/80; BD Pharmagen) in frozen sections of mouse ascending aortas have been described in detail.20,25 As a negative control, sections were incubated with the omission of the primary antibodies.

Lesion staging classification Based on the staining patterns of lesions for neutral lipid (Sudan IV), collagen (Gomori Trichrome),20,25 and macrophages (see above), the 4 histological sections used per mouse to determine the mean lesion area in each mouse were classified using the human lesion classification format as being in either the early stages (I to III), consisting mainly of macrophagederived foam cells, or advanced stages (IV and V), which are characterized by a well-defined necrotic core either with or without a fibrous cap.26,27 Results are expressed as number of early or advanced lesions/total lesions ⫻ 100%. Statistics Data analyses were performed using SigmaStat 2.03 software (SPSS Inc, Chicago, IL). Statistical differences between groups were determined by Student t test after testing that the data complied with the constraints of parametric analysis (Kolmogorov-Smirnov normality test). P ⬍ 0.05 was considered statistically significant. Results Serum cholesterol concentrations (Table 1) and cholesterol distribution amongst the lipoprotein subfractions (Fig. 1) did not differ between Casp1⫹/⫹Apoe⫺/⫺ and Casp1⫺/⫺Apoe⫺/⫺ mice. When mice were fed a diet enriched in cholesterol (high-fat diet) serum total cholesterol concentrations were 2.5 to 3 times greater than those of mice fed a normal rodent diet (Table 1). In both strains of mice, cholesterol was principally in the lipoprotein subfractions consisting of chylomicrons, very low density lipoproteins, and their remnants (Fig. 1). Loss of endogenous caspase-1 expression significantly decreased atherosclerotic lesion size by approximately 35%-45 % in the ascending aorta (Fig. 2), but not in the aortic arch (Fig. 3) of both male and female mice fed either the low- or high-fat diets. Atherosclerotic lesions in the ascending aorta were classified, using the human lesion classification format,26,27 as being either in the early (I-III) or advanced (IV-V) stage of development. As shown in Table 2, no differences were observed between Casp1⫹/⫹Apoe⫺/⫺ and Casp1⫺/⫺Apoe⫺/⫺ mice in the

Table 1. Quantification of lesion-associated cells that express MHC class II, CD3, and IFN-␥ in the ascending aorta of Apoe⫺/⫺ mice fed a highfat diet for 8 weeks or a normal diet for 26 weeks Diet group 8-week high-fat diet

Sex Male Female

26-week normal diet

Male Female

Caspase-1 genotype

Total serum cholesterol (mg/dL)

Lesion-associated MHC class II-positive cells

Lesion-associated CD3-positive cells

Lesion-associated IFN-␥-positive cells

⫹/⫹ ⫺/⫺ ⫹/⫹ ⫺/⫺ ⫹/⫹ ⫺/⫺ ⫹/⫹ ⫺/⫺

2105.4 ⫾ 246 (10) 2321.0 ⫾ 528 (10) 1562.9 ⫾ 386 (14) 1589.3 ⫾ 296 (10) 675.0 ⫾ 239 (14) 796.7 ⫾ 237 (14) 622.6 ⫾ 146 (13) 649.3 ⫾ 165 (13)

35.6 ⫾ 6.4 (7) 20.0 ⫾ 3.2 (7)* 17.6 ⫾ 2.6 (8) 8.5 ⫾ 1.6 (7)† 43.2 ⫾ 7.6 (9) 24.9 ⫾ 2.7 (10)§ 42.6 ⫾ 7.0 (10) 22.2 ⫾ 5.7 (9)¶

7.9 ⫾ 2.0 (8) 8.3 ⫾ 2.4 (7) 8.6 ⫾ 2.3 (11) 6.6 ⫾ 2.3 (10) 13.7 ⫾ 3.9 (9) 8.3 ⫾ 1.4 (11) 16.6 ⫾ 3.7 (9) 15.8 ⫾ 4.6 (9)

15.4 ⫾ 0.2 (3) 12.6 ⫾ 0.9 (3) 23.8 ⫾ 0.5 (3) 17.9 ⫾ 1.0 (3)‡ 16.7 ⫾ 1.7 (3) 13.2 ⫾ 0.8 (3) 16.9 ⫾ 1.0 (3) 12.4 ⫾ 0.6 (3)储

Results are presented as mean ⫾ standard error of the mean. number in parentheses indicate number of mice per group. IFN, interferon; MHC, Major Histocompatibility Complex. * P ⫽ 0.050 vs caspase-1 ⫹/⫹ males of the 8-week high-fat diet group. † P ⫽ 0.013 vs caspase-1 ⫹/⫹ females of the 8-week high-fat diet group. ‡ P ⫽ 0.01 vs caspase-1 ⫹/⫹ females of the 8-week high-fat group. § P ⫽ 0.030 vs caspase-1 ⫹/⫹ males of the 26-week normal diet group. ¶ P ⫽ 0.039 vs caspase-1 ⫹/⫹ females of the 26-week normal diet group. 储 P ⫽ 0.02 vs caspase-1 ⫹/⫹ females of the 26-week normal diet group.

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Figure 1. Characterization of serum cholesterol concentrations in lipoproteins. Serum (60 ␮L) from male (A, C) and female (B, D) Casp1⫹/⫹Apoe⫺/⫺ (circular symbol) and Casp1⫺/⫺Apoe⫺/⫺ (square symbol) mice was resolved by size exclusion chromatography using a Superose 6 column and cholesterol concentrations were determined in 0.5 mL fractions. Symbols represent the means and error bars the standard error of the mean of values obtained from the serum of 5 mice per curve.

percentage of complex lesions when the mice were fed either the low- or high-fat diets. Representative images of the ascending aorta of male Casp1⫹/⫹Apoe⫺/⫺ and Casp1⫺/⫺Apoe⫺/⫺ mice fed the high-fat diet, are shown in Figure 4. The sections which illustrate type III-IV atherosclerotic lesions were stained to detect neutral lipid, macrophages, and collagen. Similarly stained sections from

the ascending aorta of mice fed the normal rodent diet for 26 weeks are presented in Supplemental Figure S1. Atherosclerotic lesions from Casp1⫹/⫹Apoe⫺/⫺ and Casp1⫺/⫺Apoe⫺/⫺ mice on either the low- or high-fat diets were also analyzed for the status of immune activation as defined by the expression of MHC class II, CD3, (Fig. 4 and

Figure 2. Quantification of atherosclerosis within the ascending aorta. The extent of atherosclerotic lesion development in the ascending aorta of male and female Casp1⫺/⫺Apoe⫺/⫺ and Casp1⫹/⫹Apoe⫺/⫺ was determined as described in the Methods section. Atherosclerotic lesion area was quantified after the mice had been fed a high-fat diet for 8 weeks (A) or a normal diet for 26 weeks (B). Values of individual mice are represented as circles (Casp1⫹/⫹Apoe⫺/⫺) and triangles (Casp1⫺/⫺Apoe⫺/⫺) for both males (solid symbols) and females (open symbols). The mean lesion area of each group of mice is presented as a single horizontal line to the right of each grouping of symbols, with error bars denoting standard error of the mean.

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Figure 3. Quantification of atherosclerosis within the aortic arch. The extent of atherosclerotic lesion development in the aortic arch of male and female Casp1⫹/⫹Apoe⫺/⫺ and Casp1⫺/⫺Apoe⫺/⫺ mice was determined as described in the Methods section. Atherosclerotic lesion area as a percent of total area was quantified after the mice had been fed an atherogenic diet for 8 weeks (A) or a normal diet for 26 weeks (B). Values of individual mice are represented as circles (Casp1⫹/⫹Apoe⫺/⫺) and triangles (Casp1⫺/⫺Apoe⫺/⫺) for both males (solid symbols) and females (open symbols). The mean percent lesion area for each group of mice is presented as a single horizontal line to the right of each grouping of symbols, with error bars denoting standard error of the mean.

Supplemental Fig. S1) and the inflammatory cytokines IL-18, IL-1␤, and IFN-␥ (Fig. 5). Caspase-1 deficiency in both male and female mice, regardless of diet, resulted in significantly fewer cells that stained for MHC class II (Table 1, Fig. 4, and Supplemental Fig. S1). In both male and female mice, the lesion area that stained positive for IL-18, IL-1␤, and IFN-␥ appeared to be less in Casp1⫺/⫺Apoe⫺/⫺ mice (Fig. 5) and quantification of lesion-associated IFN-␥-positive cells confirmed this for females on both diets (Table 1). Although no significant difference was seen for IFN-␥-positive cells between Casp1⫺/⫺Apoe⫺/⫺ and Casp1⫹/⫹Apoe⫺/⫺ males, this may reflect the small number of mice that were analyzed. Discussion By superimposing caspase-1 deficiency onto mice that are already susceptible to atherosclerosis, we have shown that loss of caspase-1 activity reduced atherosclerotic lesion formation in both male and female mice. Analysis of the activation status of immune cells revealed a significant reduction in MHC class II-positive cells in atherosclerotic lesions from the Casp1⫺/⫺Apoe⫺/⫺ vs Casp1⫹/⫹Apoe⫺/⫺ mice fed either a low- or high-fat diet and this was accompanied by an apparent decrease in staining for IL-18, IL-1␤, and IFN-␥. Previous studies have shown that

deficiency of IL-1␤8 and IL-187 decreased atherosclerosis in male Apoe⫺/⫺ mice. Although parallel pathways for the proteolytic activation of IL-18 and IL-1␤ have been reported,22 our results demonstrate an important role of caspase-1. The findings of this study, along with the now well established primary role of caspase-1 activity in the process of modulating an inflammatory process, suggests that caspase-1 activity contributes to in vivo atherosclerotic lesion formation by promoting the production of the inflammatory cytokines, IL-18 and IL-1␤, and the downstream formation of IFN-␥. Given our current study’s findings, it would appear that casapase-1 activity and subsequent downstream maturation of IL-1␤ and IL-18 may promote atherosclerosis via multiple mechanisms. We have previously shown that: (1) administration of either exogenous IL-186 or IFN-␥19 will accelerate early-stage atherosclerotic lesion development in Apoe⫺/⫺ mice; (2) that exogenous IL-18 accelerates early-stage atherosclerosis via a pathway that requires endogenous production of IFN-␥6; and finally (3) that endogenous IFN-␥ promotes atherosclerotic lesion development in Apoe⫺/⫺ mice.20 Signalling via the IL-18 receptor scaffold protein, MyD88, may be key to the proatherogenic nature of IL-18, as inactivation of MyD88 has been shown to reduce atherosclerosis in Apoe⫺/⫺ mice through

Table 2. Classification of lesion development in Apoe⫺/⫺ mice fed a high-fat diet for 8 weeks or a normal diet for 26 weeks Diet group 8-week high fat diet

Sex Male Female

26-week normal diet

Male Female

Caspase-1 genotype (n)

Early-stage (I-III) lesions, %

Advanced-stage (IV-VI) lesions, %

⫹/⫹ (10) ⫺/⫺ (10) ⫹/⫹ (14) ⫺/⫺ (10) ⫹/⫹ (14) ⫺/⫺ (14) ⫹/⫹ (13) ⫺/⫺ (13)

90.98 87.76 93.91 94.57 96.65 96.10 94.23 96.23

9.02 12.24 6.09 5.43 3.35 3.90 5.77 3.77

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Figure 4. Histological characterization of the atherosclerotic lesions. Representative histological sections from a region where the aortic sinus becomes the ascending aorta of a male Casp1⫹/⫹Apoe⫺/⫺ mouse (A, C, E, G, I, K) and a male Casp1⫺/⫺Apoe⫺/⫺ mouse (B, D, F, H, J, L). Mice were fed a high-fat diet for 8 weeks. Sections were stained with (A, B) Sudan IV for neutral lipids, (C, D) anti-mouse CD3⫹E molecular complex antibody, (E, F) anti-mouse Major Histocompatibility Complex (MHC) class II. (G, H) anti-mouse macrophages (F4/80), (I, J) anti-mouse smooth muscle cell ␣-actin, and (K, L) and Gomori trichrome to detect collagen. Magnification: (A, B) ⫻40; (C-L) ⫻200. Sequential sections from the region shown in the insets of (A) and (B) are presented in (C, E, G, I, K) and (D, F, H, J, L), respectively. Arrows indicate individual cells that are staining positive for: (C, D) CD3⫹ and (E, F) MHC class II.

a mechanism partially explained by a decrease in MHC class II cell recruitment to the artery wall.28,29 In the context of this current study, one of the protective mechanisms, complementary to a reduction in IL-18 and IL-1␤, and subsequent

downstream IFN-␥ production as a result of caspase-1 deficiency, would be a reduction in the recruitment to, and retention of, antigen-presenting cells at sites of lesion development. Casp1⫺/⫺Apoe⫺/⫺ and Casp1⫹/⫹Apoe⫺/⫺ mice developed ath-

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lease of mature IL-1␤ and IL-18 as well as IFN-␥ would then promote lesion development by altering the recruitment of monocytes to the site of lesion development, by altering the activation status of lesion-associated macrophages and the ability of these cells to undergo foam cell formation and by possibly altering the activation status of other immune cells known to be capable of promoting atherosclerosis such as T lymphocytes, natural killer cells, and natural killer T cells.30 Acknowledgements The authors thank Anna Galerkin for her help in maintaining the mouse colony and Drs Yves Marcel and Ross Milne for their critical reading of this manuscript. Funding Sources This work was supported by a Canadian Institutes of Health Research Grant MOP-53344 (S.C.W.). S.C.W. was the recipient of a Great-West Life and London Life New Investigator Award from the Heart and Stroke Foundation of Canada. Disclosures The authors have no conflicts of interest to disclose. References 1. Boatright KM, Renatus M, Scott FL, et al. A unified model for apical caspase activation. Mol Cell 2003;11:529-41. 2. Shi Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell 2002;9:459-70. 3. Li P, Allen H, Banerjee S, Seshadri T. Characterization of mice deficient in interleukin-1 beta converting enzyme. J Cell Biochem 1997;64:27-32. 4. Thornberry NA, Bull HG, Calaycay JR, et al. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 1992;356:768-74. Figure 5. Histological detection of interferon (IFN)-␥ within the atherosclerotic lesions. Representative histological sections from a region where the aortic sinus becomes the ascending aorta of a Casp1⫹/⫹Apoe⫺/⫺ mouse (A, B, E, F, I, J) and a Casp1⫺/ ⫺/⫺ ⫺Apoe mouse (C, D, G, H, K, L). Mouse atherosclerotic lesions depicted in (A, C, E, G, I, K) were from mice fed a high-fat diet for 8 weeks whereas the mouse atherosclerotic lesions depicted in (B, D, F, H , J, L) were from mice fed a normal diet for 26 weeks. Sections were stained for immunoreactive interleukin (IL)-1␤ (A-D), IL-18 (E-H) and IFN-␥ (I-L) as described in the Methods section.

erosclerotic lesions of similar complexity with the only noted morphological difference being a reduced lesion size in caspasedeficient mice. This may suggest that caspase-1 deficiency may decrease the number of macrophages in the developing plaque, rather than retard the atherogenesis process itself. How this process occurs and the intricate roles of IL-1␤, IL-18, and IFN-␥ will need to be examined in more detail in subsequent studies. This study shows that caspase-1 activity is proatherogenic and likely promotes lesion development by mediating the maturation of IL-1␤, IL-18, and in turn IFN-␥. As has recently been demonstrated11 the process may be initiated by crystalline cholesterol within the artery wall that triggers NLRP3 inflammasome-mediated activation of caspase-1. The subsequent re-

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Supplementary Material To access the supplementary material accompanying this article, visit the online version of the Canadian Journal of Cardiology at www.onlinecjc.ca, and at doi: 10.1016/j.cjca.2011.10.013.