Aplicación práctica: Recursos naturales ··················································································

ABSTRACT

Atherosclerosis is an inflammatory disease, which is illustrated by the influx of macrophages and T cells in the sub-endothelial layer. IL-17 is an important cytokine, which bridges the innate and adaptive immune response and moreover IL-17 is involved in the transition from an acute into a chronic inflammation.

In this study we investigated the role of IL-17 receptor signaling in atherosclerosis. We therefore performed a bone marrow transplantation with IL-17 receptor deficient donor bone marrow into LDL receptor deficient recipient mice. After full bone marrow reconstitution a Western-type diet feeding was started and atherosclerotic lesions were quantified after 12 weeks.

A 46% reduction in lesion size in the aortic root was observed (P<0.05). Furthermore, a decrease in auto-antibodies against oxLDL was detected. The inflammatory status of IL-17 deficient bone marrow recipients was changed as indicated by the reduced IL-6 production by the spleen and increased IL-10 production within the HLN and PBMCs.

In conclusion, the IL-17 signaling is involved in the aggravation of atherosclerosis. This is probably mediated by a decrease in IgG anti-oxLDL antibodies and a change in the inflammatory status. Therefore, interfering in the IL-17 receptor pathway could provide a new therapeutic approach for inhibiting atherosclerosis lesion development.

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INTRODUCTION

Atherosclerosis is an inflammatory disease, which involves both components of the immune system, the adaptive and innate immune system1_{. The inflammatory}

response is tightly regulated by several mechanisms for example via interleukins (IL).

IL-17 is involved in the early activation of the immune system and plays an important role in bridging the innate immune response with the adaptive immune response.2 _{IL-17 is mainly produced by T cells, especially CD4}+ _{effector memory T}

cells and has more recently been linked to a new class of T helper cells, the Th17 cells.3, 4 _{IL-17 has a protective role, since IL-17 protects against infectious}

microorganisms such as Klebsiella pneumoniae , Candida albicans and Toxoplasma gondii.5,6 _{On the other hand an elevated concentration of IL-17 is associated with}

different autoimmune diseases such as, rheumatoid arthritis and multiple sclerosis, where IL-17 plays a pathogenic role. 7, 8

The IL-17 family comprises six members (IL-17A, B, C, D, E and F) and the best characterized member is IL-17A, which is also designated as IL-17 as it is the founding member of the IL-17 family. The receptor for IL-17 (IL-17R) is a type I transmembrane protein consisting of a 293 amino acids long extracellular domain and a relatively long intra cellular domain consisting of 525 amino acids.9, 10 _{The IL-}

17 receptor is widely expressed with a prominent mRNA expression in lung, kidney, liver, spleen and also in isolated fibroblasts, endothelial cells, mesothelial cells and myeloid cells from mice.9 _{This wide expression is also seen in humans,}

were the IL-17R for example is found on peripheral blood T cells and vascular endothelial cells. 11,12 _{The pathogenic role of IL-17 in autoimmunity and the parallel}

in function with proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and IL-1β makes IL-17 an interesting target for studying its role in atherosclerosis.13

IL-17 induces the expression of a wide range of proinflammatory cytokines and chemokines in various cell types as a consequence of the broad expression of IL-17R.14 _{IL-17 stimulates the expression of IL-6 and CXCL8 (IL-8) by stromal cells}

and ICAM-1 by fibroblasts and keratinocytes.9, 10, 15 _{Even more interesting is the}

effect of IL-17 on macrophages, which produce IL-1β, IL-1Ra, IL-6, IL-10, TNF-α and prostaglandin E2 (PGE2) in response to IL-17.Matrix metalloproteinase (MMP)-

3 and MMP-9 are also induced by IL-17.16, 17 _{These proteinases and}

proinflammatory interleukins and chemokines have already been implicated in atherosclerotic lesion growth and destabilization of the plaque.18-20

The role of the IL-17R signaling pathway, although extensively studied in other autoimmune diseases, is not yet established in the process of atherosclerosis. Therefore we transplanted bone marrow of IL-17R-/- _{mice to LDLr}-/- _{mice, which}

resulted in a 46% reduction in atherosclerotic lesion size. These data indicate an aggravating role of IL-17 in the process of atherosclerosis and establish a new target to beneficially influence atherosclerosis lesion development.

METHODS ANIMALS

All animal work was approved by Leiden University and was in compliance with the Dutch government guidelines. LDL receptor deficient (LDLr-/-_{) mice were}

purchased from Jackson Laboratories. IL-17 receptor knockout mice were a kind

gift from J. Peschon (Amgen, Seattle, WA) and created as described by Ye et al.6

The mice were kept under standard laboratory conditions and food and water were provided ad libitum.

BONE MARROW TRANSPLANTATION (BMT) AND INDUCTION OF HYPER-

CHOLESTEROLEMIA

To induce bone marrow aplasia, male LDLr–/– _{micewere exposed to a single dose}

of 9 Gy (0.19 Gy/min, 200 kV,4 mA) total body irradiation, using an Andrex Smart 225 Röntgensource (YXLON Int, Copenhagen, Denmark) with a 6-mm aluminum filter. Bone marrow was isolated by flushing the femurs andtibias from mice with phosphate-buffered saline (PBS). Single-cellsuspensions were prepared by passing the cells through a 30 µmnylon gauze. Irradiated recipients received 0.5x107 _bone

marrowcells by intravenous injection into the tail vein. After a recoveryof 8 weeks animals received a Western-type diet ad libitum containing 15% cocoa butter and 0.25% cholesterol (Special Diet Services, Witham, Essex, UK) for 12 weeks. During the experiment the mice were weighted every week and checked for well-being.

SERUM LIPID LEVELS

Every 3 weeks the serum cholesterol levels were determined to assess the effect of the Western-type diet. Blood samples were collected by tail bleeding from non- fasting animals. Total cholesterol levels were quantified spectrophotometrically using an enzymatic procedure (Roche Diagnostics, Germany). Precipath standardized serum (Boehringer, Germany) was used as an internal standard.

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HISTOLOGY AND IMMUNOHISTOCHEMISTRY

Mice were anaesthetized with ketamine-hypnorm and perfused with PBS and subsequently with FormalFixx. The heart and complete aorta were removed. The heart was embedded in OCT compound (TissueTek; Sakura Finetek, The Netherlands) and cryosections of 10 µm were made of the aortic root containing the aortic valves. Cryosections were routinely stained with Oil-Red-O and hematoxylin (Sigma Diagnostics, MO). Corresponding sectionson separate slides were stained immunohistochemically for macrophages using an antibodyagainst a macrophage-specific antigen (MoMa-2, Research Diagnostics Inc.) and for collagen using Masson trichrome staining according to manufacturer's protocol (Sigma Diagnostics). Neutrophils were stained by specific esterase staining (Naphthol AS- D chloroacetate, Sigma). Mast cells were stained by Toluidin Blue Staining (Sigma). The different histological stains were quantified using a Leica DM-RE microscope and LeicaQwin Imaging software (Leica Ltd., Germany).

FACS ANALYSIS OF LEUKOCYTES

Peripheral Blood Mononuclear Cells (PBMC) were isolated via orbital bleeding and erythrocytes were removed by incubating the cells with erythrocyte lysis buffer (0.15 M NH4Cl, 10 mN NaHCO3, 0.1 mM EDTA, pH 7.3). Spleens, Heart lymph

nodes (HLN) and Mesenteric lymph nodes (MNL) were dissected from the mice and single cell suspension was obtained by passing the organs through a 70 µm cell strainer (Falcon, The Netherlands). Cells were stained with surface markers (0.20 µg antibody/300.000 cells) and subsequently analyzed by flow cytometric analysis. The F4/80-FITC and CD19-FITC antibody were used for the detection of macrophages and B cells, respectively (Immunosource, Belgium). The unlabeled antibody for IL-17R was purchased from R & D systems and as a secondary antibody anti goat-IgG-PE (Abcam, UK) was used according manufacturers protocol. All data were acquired on a FACScalibur and analyzed with CELLQuest software (BD Biosciences, The Netherlands).