Genetic ablation of IRAK4 kinase activity inhibits vascular lesion formation

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Biochemical and Biophysical Research Communications 367 (2008) 642–648 www.elsevier.com/locate/ybbrc

Genetic ablation of IRAK4 kinase activity inhibits vascular lesion formation Mark Rekhter, Kirk Staschke, Thomas Estridge, Pamela Rutherford, Nancy Jackson, Donetta Gifford-Moore, Patricia Foxworthy, Charles Reidy, Xiao-di Huang, Michael Kalbfleisch, Kwan Hui, Ming-Shang Kuo, Raymond Gilmour, Chris J. Vlahos * Lilly Research Laboratories, Lilly Corporate Center DC1705, Indianapolis, IN 46285, USA Received 19 December 2007 Available online 9 January 2008

Abstract Inflammation is critically involved in atherogenesis. Signaling from innate immunity receptors TLR2 and 4, IL-1 and IL-18 is mediated by MyD88 and further by interleukin-1 receptor activated kinases (IRAK) 4 and 1. We hypothesized that IRAK4 kinase activity is critical for development of atherosclerosis. IRAK4 kinase-inactive knock-in mouse was crossed with the ApoE / mouse. Lesion development was stimulated by carotid ligation. IRAK4 functional deficiency was associated with down-regulation of several pro-inflammatory genes, inhibition of macrophage infiltration, smooth muscle cell and lipid accumulation in vascular lesions. Reduction of plaque size and inhibition of outward remodeling were also observed. Similar effects were observed when ApoE / mice subjected to carotid ligation were treated with recombinant IL-1 receptor antagonist thereby validating the model in the relevant pathway context. Thus, IRAK4 functional deficiency inhibits vascular lesion formation in ApoE / mice, which further unravels mechanisms of vascular inflammation and identifies IRAK4 as a potential therapeutic target. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Atherosclerosis; Inflammation; Signal transduction; Macrophage; Remodeling

Involvement of several critical inflammatory mediators, including interleukin (IL)-1 [1–3] and -18 [4] receptors as well as toll-like receptors (TLR)-2 [5] and -4 [6], has been demonstrated to play a central role in the development and progression of atherosclerotic lesions [7]. Signaling from these receptors is mediated by the adaptor protein MyD88 and further by interleukin-1 receptor activated kinases (IRAK) 4 and 1 [8]. Genetic knockout of MyD88 was associated with profound anti-atherosclerotic effects [6,9]. Since different combinations of upstream signals are likely to contribute at different stages of lesion development or complications, the inhibition of IRAK4, the convergence point in several inflammatory signaling path-

*

Corresponding author. Fax: +1 317 433 2815. E-mail address: [email protected] (C.J. Vlahos).

0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.12.186

ways, might be considered as a potential intervention for treating atherosclerosis. Also, IRAK4 is the most proximal kinase mediating IL-1, IL-18, and TLR signaling that makes it attractive as a potential therapeutic target [10]. We [11] and others [12,13] have recently generated IRAK4 kinase-inactive knock-in mice (IRAK4-ki) that revealed a critical role of IRAK4 kinase activity in TLR- and IL-1mediated inflammation. However, it is unknown whether IRAK4, and in particular its kinase activity, is critical for development of atherosclerotic plaques. To evaluate the role of IRAK4 in atherosclerosis, we crossed the IRAK4- ki mouse with the ApoE-deficient mouse. Development of accelerated atherosclerotic lesions was stimulated by carotid ligation [14]. IRAK4 functional deficiency was associated with down-regulation of several key pro-inflammatory genes, as well as inhibition of macrophage infiltration, smooth muscle cell accumulation and lipid accumulation in vascular lesions. In addition,

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reduction of plaque size and inhibition of outward remodeling were also observed. Similar effects were observed when ApoE / mice subjected to carotid ligation were treated with recombinant IL-1 receptor antagonist.

Results

Methods

In order to validate a model of accelerated atherosclerosis in the context of the relevant IL-1 mediated inflammatory pathway, we treated ApoE / mice on a high fat diet with recombinant IL-1 RA for 14 days following carotid ligation. Treatment with IL-1 RA significantly reduced accumulation of cholesterol ester (by 70%, p < 0.05) and free cholesterol by (55%, p < 0.05) in ligated arteries, associated with substantial reduction of the lesion size (by 98%, p < 0.01). This inhibition was achieved without any change in plasma cholesterol and triglycerides (data not shown) suggesting direct vascular effects of IL-1 RA. In a second model, the same dose of IL-1 RA effectively inhibited IL1b induced elevation of human CRP and mouse IL-6 in hCRP transgenic mice (Table 1). In this model, IL-1b rapidly induces production of IL-6 (maximum at 2 h), whereas maximal levels of hCRP are not reached until 9–15 h. However, IL-1 RA treatment completely eliminates the production of both IL-6 and hCRP across the entire study. Taken together, these data demonstrate that vascular lesion development in the flow cessation model is IL-1dependent, and upstream inhibition of this signaling pathway using a clinically meaningful dose of IL-1 RA can be sensitively detected.

Transgenic mice. IRAK4-ki knock-in mice were generated at InGenKo (Vic., Australia) using C57Bl/6 ES cells. Ablation of kinase activity was accomplished by changing Lys213 and Lys214 in the ATP binding pocket of the kinase domain to methionines. A targeting construct containing these amino acid changes was generated. The complete nucleotide sequence of the targeting construct is available upon request. IRAK4-ki mice were crossbred with ApoE / mice (Taconic), and the resulting F1 heterozygotes were interbred to obtain double homozygous mutant mice. These mice were fertile and showed no abnormalities in growth or water/ food consumption. A detailed description of the phenotype of IRAK4-ki mice has been recently been published [11]. Flow cessation model of accelerated atherosclerosis. Experimental procedures using animals were approved by the Institutional Animal Care and Use Committee and performed in accordance with the Guide for the Care and Use of Laboratory Animals. Eight-week-old ApoE / mice and IRAK4-ki ApoE / mice were obtained from Taconic (Hudson, NY). The animals (n = 10 per group) were pre-fed with Western diet containing 0.21% cholesterol and 21% fat for 14 days prior to surgery. Plasma lipids were analyzed on a Hitachi 912 clinical chemistry analyzer, and total cholesterol was used for animal randomization. To accelerate lesion formation, left common carotid artery was ligated under isofluorane anesthesia as described previously [14]. Mice were kept on the same diet following the ligation surgery and were euthanized either 3 days or 14 days after ligation. For model validation, a subset of ApoE / mice were treated with the recombinant IL-1 receptor antagonist (IL-1 RA) Anakinra (Amgen; Thousand Oaks, CA) administered via implanted osmotic pumps at 100 mg/kg; PBS was administered in an identical manner for the vehicle control. Implantation of pumps was performed on the same day as ligation. Atherosclerotic lesion characterization. Gene expression was analyzed by reverse polymerase chain reaction using TaqMan. Forward and reverse probe/primer sets were purchased from ABI. Macrophage accumulation was determined by quantitative Western blot using MAC-2 antibody (clone M3/38, Cedarlane Laboratories; Burlington, Ont., Canada). Tissue cholesterol ester and free cholesterol were analyzed in chloroform–methanol extracts using liquid chromatography and mass spectroscopy. For histology, 10 equally spaced (200 lm) paraffin cross sections were stained using modified Masson’s trichrome procedure that included elasin staining. Macrophages and SMC were visualized immunohistochemically using MAC-2 and anti-a-smooth muscle actin antibody (DAKO, Carpinteria, CA) respectively. Trichrome-stained sections (3 per carotid artery) were used for morphometric analysis. Lesion area was calculated using ImagePro Plus Version 5.0.1 using sections spanning the entire length of the artery. The lesion area was defined as the region between the lumen and the internal elastic lamina (IEL), and the arterial size was defined as the area inside the external elastic lamina (EEL). Analysis of systemic inflammation. Male transgenic CRP mice expressing human C-reactive protein gene [15] (9–12 weeks old) were injected subcutaneously with PBS or Anakinra (100 mg/kg) at a dose volume of 10 ml/kg. Thirty minutes later, the mice were injected IP with IL-1b (107 U/kg) (PeproTech; Rocky Hill, NJ). Mice were bled by orbital puncture under isofluorane anesthesia at 2, 8, 12, and 15 h post-IL-1b dose. Plasma hCRP levels were determined for all time points on a Hitachi 912 clinical chemistry analyzer and mouse IL-6 levels were determined for the two hour time point by ELISA (R&D Systems; Minneapolis, MN). Statistics. The results are shown as means ± SEM. Differences between groups were determined using one-way ANOVA with post hoc Dunnett’s t-test. A value of p < 0.05 was regarded as a significant difference. The authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the manuscript as written.

Model validation: anti-inflammatory and anti-atherogenic effects of IL-1 RA

Anti-atherogenic effects of genetic functional IRAK4 deficiency To evaluate if downstream manipulation of inflammatory signaling could effect development of accelerated atherosclerotic lesions, IRAK4-ki mice were crossed with ApoE / mice. Response to carotid ligation in these mice was compared with regular ApoE / mice possessing intact functional IRAK4. Plasma cholesterol and triglyceride levels in IRAK4-ki transgenic (1417 ± 66 mg/dl and 190 ± 22.8 mg/dl, respectively) and regular ApoE / mice (1422 ± 66 mg/dl and 180 ± 25.3 mg/dl, respectively) did not significantly differ suggesting that genetic ablation of IRAK signaling does not affect blood lipids.

Table 1 Systemic anti-inflammatory effects of Anakinra in hCRP transgenic mice stimulated with IL-1 (n = 8 per group) Vehicle Human CRP (ng/L) Mouse IL-6 (pg/ml)

19.8 ± 3.1 31.1 ± 7.6

IL-1 + vehicle *

133.0 ± 7.9 45572.7 ± 7926.6*

IL-1 + Anakinra 18.6 ± 1.4 170.4 ± 127.9

Human CRP transgenic mice were treated with either saline (vehicle control) or Anakinra and then challenged with IL-1b as described under Methods. IL-1 challenge shows an elevation in IL-6 production (2 h time point shown) that is completely absent in the presence of Anakinra. IL-1 also causes an elevation of hCRP production that again is completely absent in the presence of Anakinra. Values are means ± SEM. * p < 0.01.

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Table 2 Gene expression in carotid arteries 3 days after ligation (n = 10 per group) Gene MCP1 IL-6 Lcn2 p22phox p40phox p47phox Gp-91

% Down-regulation in IRAK4-ki ApoE / mice compared to ApoE /

p Value mice

52 22 94 43 34 29 53

<0.05 <0.05 <0.01 <0.001 <0.01 <0.01 <0.001

Three days after ligation surgery, the IRAK4-ki/ApoE / mice showed down-regulation of MCP-1, IL-6, and lipocalin 2 gene expression as well as expression of several NADPH oxidase subunits in ligated carotid arteries (Table 2) compared to ApoE / mice, suggesting that IRAK4 kinase functional deficiency modulated the inflammation associated with lesion formation. All these genes were significantly up-regulated in ligated carotid arteries of ApoE / mice compared to the non-ligated arteries (data not shown). Down-regulation of pro-inflammatory genes in the IRAK4-ki/ApoE / mice were associated with inhibition of macrophage accumulation in ligated arteries as detected using Mac-2 immunoblotting (Fig. 1). Causal relationships between gene expression and macrophage accumulation need to be further investigated. It is clear, however, that anti-inflammatory effects of IRAK4 genetic

ablation can be detected very early in the development of accelerated vascular lesions. Fourteen days after ligation, IRAK4-ki/ApoE / mice accumulated significantly less cholesterol ester and free cholesterol in ligated carotid arteries compared to ApoE / mice (Fig. 2). Vascular lesions were 89% smaller (Figs. 3 and 4). In the instances in which small lesions were present in the IRAK4-ki/ApoE / mice, they consisted primarily of macrophages with occasional SMC and very little extracellular matrix (Fig. 3). EEL area of ligated arteries in IRAK4-ki/ApoE / mice were also 42% smaller (Fig. 4) suggesting inhibition of outward arterial remodeling. Taken together, these data suggest that functional deficiency of IRAK4 inhibited formation of both early and advanced vascular lesions in a mouse model of accelerated atherosclerosis. Discussion We have shown that IL-1 RA prevented formation of vascular lesions in a mouse model of accelerated atherosclerosis. In this same accelerated model, genetic functional deficiency of IRAK4 (obtained by crossing mice expressing kinase-inactive IRAK4 with ApoE-deficient mice) exerted profound anti-atherogenic effects mediated by local down-regulation of pro-inflammatory genes as well as reduction of macrophage and SMC accumulation.

A G3PDH Mac2

IRAK4-ki ApoE-/-IRAK4-ki ApoE-/Ligated Nonligated

Mac2/G3PDH, optical density

B

ApoE-/Ligated

ApoE-/Nonligated

25

20

15

* 10

5

0 IRAK4ki ApoE-/Ligated

IRAK4ki ApoE-/Nonligated

ApoE-/- Ligated

ApoE-/- Nonligated

Fig. 1. Inhibition of macrophage accumulation in IRAK4-ki ApoE / mice 3 days after carotid ligation. Mac-2, a protein specific to macrophages, was measured in carotid tissue extracts as a surrogate marker for macrophage accumulation using Western blot (A). Quantification of Western blot data (B) indicates that ApoE / mice on high fat diet show significant levels of Mac-2 in ligated coronary arteries, whereas ApoE / mice in which IRAK4 is not functionally active are devoid of Mac-2. As a control, the non-ligated right carotid artery was also analyzed and found to be absent of Mac-2 levels. The asterisk indicates significant reduction of macrophage accumulation in ligated arteries of IRAK4-ki ApoE / mice compared to ligated arteries of ApoE / mice.

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A

B 70

*

30 25

50

cholesterol, nM

cholesterol ester, nM

60

40 30 20

645

*

20 15 10 5

10 0

ApoE-/-

IRAK4-ki ApoE-/-

0

ApoE-/-

IRAK4-ki ApoE-/-

Fig. 2. Reduced carotid lesion cholesterol ester (A) and free cholesterol (B) accumulation in IRAK4-ki ApoE / mice 14 days after carotid ligation. ApoE / mice were pre-fed a diet high in cholesterol and fat, and were then subjected to left common carotid artery ligation. ApoE / mice show evidence of vascular lesion development (elevated deposition of cholesterol ester and free cholesterol in the carotid tissue) whereas IRAK4-ki ApoE / mice showed no evidence of vascular lesions. Lesion free cholesterol and cholesterol ester were determined by LC-MS of vascular extracts as described under Methods. The data are presented as concentration of cholesterol esters or free cholesterol from extracts of carotid artery that was cut at the standard length (7 mm). Values are means ± SEM, *p < 0.01.

Fig. 3. Inhibition of vascular lesion formation in IRAK4-ki/ApoE / mice 14 days after carotid ligation. (A, C, and E) Carotid lesions of Apo E / mice; (B, D, and F) carotid lesions of IRAK4-ki ApoE / mice. Cross-sections were stained with Masson’s trichrome (A,B), immunostained with MAC2 antibody for macrophage visualization (C,D) or an anti-actin antibody to visualize SMC (E,F). Arrow denotes IEL, arrowhead denotes EEL. 10 objective.

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A

B

50000 45000

*

160000 140000

*

40000 120000

EEL area, μm2

lesion area, μm2

35000 30000 25000 20000 15000

100000 80000 60000 40000

10000

20000

5000 0

0 ApoE-/-

IRAK4-ki ApoE-/-

ApoE-/-

IRAK4-ki ApoE-/-

Fig. 4. IRAK4-ki ApoE / mice show reduced lesion area and arterial size reduction 14 days after carotid ligation compared to ApoE / Lesion area; (B) EEL area. Morphometric analysis performed on Masson’s stained slides as described under Methods.

Our IL-1 RA data are consistent with earlier demonstrations that pharmacological inhibition of IL-1 receptor reduced spontaneous fatty streak formation in ApoE / mice [3], along with numerous studies utilizing genetic deficiency of IL-1b, IL-1 receptor or endogenous IL-1 RA [1,2,16–18] in various mouse models of atherosclerosis and vascular inflammation. Using human CRP transgenic mice, we were also able to demonstrate that the same dose of IL-1 RA effectively inhibited IL-1b induced elevation of human CRP. Similar effects of IL-1 RA on CRP were documented in the clinic [19]. Taken together, these data generate a relevant reference for comparison with effects of IRAK4 genetic functional deficiency. Marked anti-atherogenic effects of IRAK4 functional deficiency demonstrated in this paper are in agreement with previously reported effects of MyD88 deficiency in ApoE / mice [6,9]. MyD88, an adaptor protein located upstream of IRAK4, functions as part of the signaling pathways that include IL-1 receptor, IL-18 receptor as well as toll-like receptors [8]. Consistent with observations in MyD88 / mice, we detected down-regulation of the chemokine MCP-1 with corresponding inhibition of macrophage recruitment into early lesions [6,9]. However, our data suggest broader anti-inflammatory effects of IRAK4 functional deficiency, including down-regulation of lipocalin 2, a pivotal gene of the innate host response [20], IL-6, a cytokine implicated in atherosclerotic lesion development [21], and various subunits of NADPH oxidase involved in the local generation of reactive oxygen species. In addition, Bjorkbacka et al. [9] reported isolated effects of MyD88 deficiency on macrophage recruitment without any changes in lipid accumulation. Our data on IRAK4 functional deficiency demonstrated profound inhibition of cholesterol and cholesterol ester accumulation in the accelerated lesions that is in agreement with MyD88-null data of Michelsen et al. [6]. It is unclear whether these differences reflect the nature of the lesions in various mouse models

mice. (A)

of atherosclerosis, different methods of analyzing tissue lipid content, or yet unknown biological consequences of blocking the same pathway at different levels. Interestingly, vascular lesions in IRAK4-ki/ApoE / mice, while substantially reduced in size, were preferentially comprised of macrophages and almost devoid of SMC and extracellular matrix. It is unknown whether this is indicative of inhibition of lesion development (early fatty streak-like phenotype as opposed to a more advanced fibrous plaque-like phenotype) or, alternatively, whether IRAK-4 kinase activity is specifically involved in regulation of SMC migration, proliferation, and collagen synthesis. The latter can be also interpreted as features of the less stable lesions. Current data, however, are not sufficient for any far-reaching interpretation. Along with inhibition of atherosclerotic plaque formation, we found that the size of carotid artery (defined as EEL area) was significantly smaller in IRAK4-ki/ ApoE / mice. According to Ivan et al. [14], expansive arterial remodeling is associated with increased neointimal macrophage foam cell content in the model of accelerated atherosclerosis that was used in the current study. Therefore, it appears that IRAK4 might be involved in outward arterial remodeling. Since IRAK4 is critical for TLR signaling, this interpretation corroborates the data of Hollestele et al. [22] that demonstrated inhibition of outward arterial remodeling in TLR4-deficient mice. Exact mechanisms of IRAK4 involvement, i.e. direct effect on remodeling vs. indirect effect via inhibition of plaque formation, require further clarification. Regardless, these effects might confer clinical benefits since, although outward remodeling can temporarily preserve lumen diameter, it is associated with a vulnerable plaque phenotype [23]. It is unknown what combination of specific pro-atherogenic endogenous stimuli drives lesion development and which downstream effectors are blocked as a result of IRAK4 functional deficiency. Based on IL-1 RA efficacy,

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demonstrated in this model, it is highly likely that blockade of IL-1 signaling is a key component. Furthermore, it is possible that IL-18 [4], TLR4 [6], and/or TLR2 [5] signaling could be disrupted since IRAK4 is a central component in these pathways as well. Since different combinations of upstream signals are likely to contribute at different stages of lesion development or complications in various animal models and clinical situations, the inhibition of IRAK4 could be considered as a potential intervention for treating atherosclerosis. Feasibility of IRAK4 as a therapeutic target should be rigorously tested though given potential side effects associated with inhibition of innate immune defense [24–26]. This study has several limitations. First, carotid ligation model was chosen since it generates vascular lesions in an accelerated manner. In this case, it provided an opportunity to compare effects of functional IRAK4 deficiency and the reference anti-inflammatory drug. IL-1RA was delivered by osmotic pump that is consistent with a short-term study only. However, it is yet unknown whether this model accurately represents all the mechanisms of chronic atherosclerosis. Further studies elucidating the role of IRAK4 kinase activity in the models of spontaneous atherosclerosis are necessary. Second, potential role of adaptive immune component cannot be excluded without detailed analysis of T cell response. This important question requires separate study. Overall, the data presented in our study demonstrate anti-atherosclerotic effects of IRAK4 functional deficiency in ApoE / mice, which provides further evidence of the involvement of innate immunity in atherosclerotic lesion development and identifies IRAK4 as a potential therapeutic target. Acknowledgments The authors thank Bob Gill and Bonita Jones for their excellent help with the mouse surgery. References [1] K. Isoda, S. Sawada, N. Ishigami, T. Matsuki, K. Miyazaki, M. Kusuhara, Y. Iwakura, F. Ohsuzu, Lack of interleukin-1 receptor antagonist modulates plaque composition in apolipoprotein Edeficient mice, Arterioscler. Thromb. Vasc. Biol. 24 (2004) 1068– 1073. [2] C.M. Devlin, G. Kuriakose, E. Hirsch, I. Tabas, Genetic alterations of IL-1 receptor antagonist in mice affect plasma cholesterol level and foam cell lesion size, PNAS 99 (2002) 6280–6285. [3] R. Elhage, A. Maret, M.T. Pieraggi, J.C. Thiers, J.F. Arnal, F. Bayard, Differential effects of interleukin-1 receptor antagonist and tumor necrosis factor binding protein on fatty-streak formation in apolipoprotein E-deficient mice, Circulation 97 (1998) 242–244. [4] Z. Mallat, A. Corbaz, A. Scoazec, P. Graber, S. Alouani, B. Esposito, Y. Humbert, Y. Chvatchko, A. Tedgui, Interleukin-18/interleukin-18 binding protein signaling modulates atherosclerotic lesion development and stability, Circ. Res. 89 (2001) 41e–45e. [5] A.E. Mullick, P.S. Tobias, L.K. Curtiss, Modulation of atherosclerosis in mice by Toll-like receptor 2, J. Clin. Invest. 115 (2005) 3149– 3156.

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