Oral dosing with multi-antigenic construct induces atheroprotective immune tolerance to individual peptides in mice Lakshmi Mundkur a, ⁎, Thiruvelselvan Ponnusamy a , Sheena Philip a , Lakshmi Narasimha Rao a , Suryakant Biradar a , Vrushali Deshpande a , Ramesh Kumar a , Xinjie Lu b , Vijay V. Kakkar a,b, ⁎ a Mary and Gary Western and Tata Molecular Immunology Unit, Thrombosis Research Institute, Bangalore, India b Molecular Immunology Unit, Thrombosis Research Institute, London, United Kingdom abstract article info Article history: Received 26 February 2014 Received in revised form 26 April 2014 Accepted 1 June 2014 Available online 10 June 2014 Keywords: Atherosclerosis Inflammation Immune tolerance Dendritic cells Vaccine Inflammatory immune response to self-antigens plays an important role in the development of atherosclerosis. Restoring immune tolerance to self-proteins reduces the pro-inflammatory response. We previously showed that oral tolerance to a combination of two peptides is atheroprotective. In the present study we expressed epi- topes from apolipoprotein B 100 (ApoB), human heat shock protein (HSP60) and Chlamydia pneumonia outer membrane protein (Cpn) in a single protein scaffold and used this multi-antigenic construct to induce tolerance to individual peptides by oral route in ApoB tm2Sgy /Ldlr tm1Her/J mice. Antigen specific tolerance to individual peptides was observed in treated animals as seen by an increase in regulatory T cells. Tolerance to the peptides resulted in a 46.5% (p = 0.002) reduction in the development of atherosclerosis compared with control. Atheroprotection was associated with a significant (p b 0.05) decrease in plaque inflammation and an increase in the expression of immune regulatory markers in the aorta. CD11c + cells coexpressing CD11b and CD103 in- creased in lymphoid organs and were found to activate regulatory T cells and reduce effector T-cell response. Adoptive transfer of CD11c + cells was atheroprotective. Our results suggest that atheroprotection by oral toler- ance to a multi-antigenic construct is mediated by antigen specific regulatory T cells and CD11c + cells with im- mune regulatory properties. © 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Atherosclerosis is a chronic inflammatory disease that develops in response to lipid accumulation, immune response and inflammation [1]. Recent evidence suggests that inflammation is mediated by an auto- immune response to self-antigens such as oxidized low-density lipopro- tein (ox-LDL) and heat shock proteins (HSPs) in the vascular wall [2,3]. Immunotherapy for atherosclerosis is directed towards inducing toler- ance to self-antigens by increasing the number of antigen-specific regulatory T cells (Tregs), which can suppress the pro-atherogenic im- mune response [4,5]. Oral administration of antigen leads to systemic unresponsiveness and represents a powerful tool for treating autoimmune and inflamma- tory diseases. Repeated administration of a low dose of antigen induces regulatory T cells, secreting transforming growth factor β (TGF-β) or in- terleukin (IL)-10, and CD4 + CD25 + Foxp3 + Tregs [6]. Dendritic cells (DCs) play a major role in the development of antigen specific immune regulation to orally administered antigens [7]. Antigen-induced, tolerogenic CD11c + , CD11b + DCs are shown to increase in Peyer's patches and confer protection against experimental arthritis by induc- ing regulatory T cells in mice [8]. Recent studies have shown that muco- sal DCs expressing CD103 were able to induce Foxp3 + Tregs in the presence of TGF-β [9]. Several studies have demonstrated an effective early reduction of ath- erosclerosis in mouse models by inducing tolerance to peptides derived from apolipoprotein B (ApoB) 100, HSPs 60/65, and β2-glycoprotein [10–15]. We have earlier shown that the combination of ApoB and HSP60 peptides confer better protection compared with either of the in- dividual peptides [15] and oral tolerance to this combination could pre- vent development and progression of atherosclerosis in mice [16]. International Journal of Cardiology 175 (2014) 340–351 Abbreviations: Ox-LDL, Oxidized low-density lipoprotein; HSP, Heat shock proteins; Tregs, Regulatory T cells; TGF-β, Transforming growth factor β; IL, Interleukin; ApoB, Apolipoprotein B; Cpn, Chlamydia pneumonia; DSP, Dendroaspin; GST, Glutathione S-transferase; IFN, Interferon; MMP, Matrix metalloproteinase; MRP8/14, Myeloid related protein complex; TNF, Tumor necrosis factor; OMP, Outer membrane protein. ⁎ Corresponding authors at: Thrombosis Research Institute (Bangalore), Narayana Hrudayalaya, 258/A, Bommasandra Industrial Area, Anekal Taluk, Bangalore 560099, India. Tel.: +91 80 27835303; fax: +91 80 27835302. E-mail addresses: lakshmi.mundkur@triindia.org.in (L. Mundkur), president@tri-kakkar.ch (V.V. Kakkar). http://dx.doi.org/10.1016/j.ijcard.2014.06.001 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved. 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