Oral Immune therapy to Atherosclerosis in mice model induces alternate activation of macrophages and reduces markers of plaque destabilization and inflammation by oral tolerance Lakshmi NarasimhaThota, Lakshmi Mundkur Thrombosis Research Institute, Molecular Immunology Unit, Bangalore, India ABSTRACT: Background and aims: Atherosclerosis is the leading cause for cardiovascular mortality. We determined the effect of multi-antigenic construct expressing three peptides AHC (ApoB100, HSP60 and outer membrane protein of chlamydia pneumonia) in stabilizing advanced atherosclerosis in Apobtm2Sgy/Ldlrtm1Her/J mice. Methods: Apobtm2SgyLdlrtm1Her/J mice (5-6 weeks) were orally dosed with multi antigenic construct (AHC) molecule on alternate days, followed by high-fat diet feeding to initiate atherosclerosis. Results: Treated animals showed an efficient reduction in plaque growth and lipid accumulation at 6 weeks (49%, p<0.01) and 12 weeks (42.3%, p<0.01) which reduced to 29% (p=0.0001) at 18 weeks and at later time points. Global gene expression analysis revealed decrease in inflammatory chemokine response genes (cxcl9, cxcl10, EGF, IRF1) and higher levels of anti-inflammatory genes (Ltbp1). Macrophage accumulation was significantly reduced and increased Tregs in both spleen and aortic sinus could lead to alternate activation of M2 macrophages. We observed an increase in antibody levels to LDL and oxidized LDL at later stages. Conclusions: Our results suggest that immune tolerance to AHC protein by oral administration is able to provide efficient atheroprotection up to 18 weeks and moderately at later stages. Apart from immune regulatory cells, protective antibodies seem to play a role in controlling atherosclerosis. AHC could stabilize plaque by reducing the inflammatory immune response through regulated T cells and alternate activation of M1 macrophages to M2 macrophages. METHODS: A) Groups of 6 mice (5-6 weeks, 3M+3F) were given a diet rich in cholesterol (HFD) for 10 weeks to induce atherosclerosis and orally dosed with 1 ug of the purified recombinant multi antigenic molecule (AHC) for 5 alternate days after 10 weeks of HFD. HFD was continued for another 10 weeks B) Groups of 6 mice (5-6 weeks) were orally dosed 5 times with 1 ug of the purified recombinant multi antigenic molecule (AHC) or PBS on alternate days and fed on HFD for 6,12,18,24 and 30 weeks C). Both treated AHC and control HFD group of mice fed on a high-fat diet for a period 0, 6, 18 and 30 weeks. At the end of the experiment, mice were humanely sacrificed with a high dose of isoflurane and ascending aorta was collected for microarray experiments. AHC group: Mice aged for 5-6 weeks were orally dosed with 1µg/ml of AHC protein for 5 alternate doses and fed on a high-fat rich diet. HFD group: a group of mice fed on a high-fat diet alone. RESULTS 1
Multi-antigenic AHC Molecule: (A) Wild type Dendroaspin Molecule: (B) Multi-antigenic AHC molecule in dendroaspin backbone with three peptides
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Conclusion
References
1) Mundkur L, et al. (2013) Comparison of Oral Tolerance to ApoB and HSP60 Peptides in Preventing Atherosclerosis Lesion Formation in Apob48-/Ldlr- Mice. Journal of Vaccines 2013: 13 2) Lu X, Chen D, Endresz V, Xia M, Faludi I, et al. (2010) Immunization with a combination of ApoB and HSP60 epitopes significantly reduces early atherosclerotic lesion in Apobtm2SgyLdlrtm1Her/J mice. Atherosclerosis 212: 472-480 3) Lakshmi Narasimha Thota, et al. Immune regulation by oral tolerance induces alternate activation of macrophages and reduces markers of plaque vulnerability in apobtm2sgy/ldlrtm1her/j mice. Nature Scientific Reports 2017 | 7: 3997 | DOI:10.1038/s41598-017-04183-w
Efficient reduction in plaque growth and lipid accumulation in early time points which reduced by 18 weeks Oral tolerance is maintained efficiently up to 12 weeks beyond which the overwhelming autoimmune response is not very efficiently balanced by the tolerance mechanism suggesting a booster dose before 18 weeks Protective antibodies to LDL and Ox-LDL may have a role in controlling the lesion especially in the later stages of the disease Reduction in inflammation by lower chemokine and cytokines and higher Regulatory T cells in treated animals AHC molecule can stabilize the plaque by higher collagen content and, lesser no of breaks in plaque cap and decreased expression of TF, MMP9 and TUNEL markers in the plaque AHC therapy could induce the M2 macrophages phenotype in the disease which correlates to gene expression changes by lower expression of HDAC9, EDC4, Arginase II, IL-23, inos, TNFα and higher TGF-b, Arginase I and IL-10