Study On The Role And Mechanism Of Immune Tolerance Induced By Nasal Oxidized Low-density Lipoprotein In Atherosclerosis

PartⅠ. Establishment and identification of ApoE-/- mice model of immune tolerance through intranasal treatment with oxLDLObjective:To establish ApoE-/- mice model of immune tolerance through nasal administration of oxLDL.Methods:ApoE-/- mice were divided into three groups:experimental groups, immunized control groups, and blank control groups. The mice in the experimental and immunized control groups were nasally administrated oxLDL and PBS daily for 5 consective days. Three days after the final nasal oxLDL administration, mice were immunized by intraperitoneal injection of 100μg of oxLDL, and 2 weeks later mice were sacrificed by removing the eyeballs. The state of immuno-tolerance to oxLDL in ApoE-/-mice was evaluated using 3H-thymidine incorporation assay, ELISA, Real-Time RT-PCR, and flow cytometry to assess the lymphocyte proliferation, the content of cytokines such as IFN-y, IL-4, IL-10, IL-17, and TGF-βand the levels of serum oxLDL-Ab, the cytokines mRNA expression and the amount of CD4+CD25+Tregs, respectively.Results:Splenocytes proliferative response to oxLDL were significantly reduced in experimental groups compared to control groups (p<0.05). The TGF-βproduction and mRNA levels were significantly increased in experimental groups compared to control groups (p<0.05), meanwhile, the mRNA levels of IFN-y, IL-4, and IL-17 were markedly decreased in experimental groups compared with control groups (p<0.05). Interestingly, the IL-10 mRNA levels were similar in three groups (p>0.05). A significant increase in CD4+CD25+Tregs in spleens were found in experimental groups than in control groups (p<0.05). In addition, the serum anti-oxLDL-Ab levels were significantly down-regulated in experimental groups compared to control groups (p<0.05).Conclusion:Intranasal administration with oxLDL can establish ApoE-/-mice model of immune tolerance successfully. Part II. CD4+LAP+and CD4+CD25+Foxp3+Regulatory T Cells Induced by Nasal Oxidized Low-Density Lipoprotein Suppress Effector T Cells Response and Attenuate Atherosclerosis in ApoE-/- MiceObjective:Increasing studies have demonstrated that atherosclerosis (AS) is a chronic immunoinflammatory disease, and that oxidized low-density lipoprotein (oxLDL)-specific T cells contribute to the autoimmune process in atherosclerosis. Oral administration of oxLDL, which was identified as a candidate autoantigen in atherosclerosis, was shown to induce tolerance and suppress atherogenesis. However, the precise mechanisms of mucosal tolerance induction, in particular nasal tolerance, remain unknown.In this study, we explored the effect of nasal oxLDL on atherosclerosis as well as the cellular and molecular mechanisms leading to atheroprotective responses.Methods and Results:Six-week-old or sixteen-week-old male ApoE-/- mice were nasally given oxLDL at 3μg/day on 5 consecutive days, the mice were given a high-fat diet, which induces atherosclerotic plaque formation. Nasal administered with phosphate buffered saline (PBS) served as control. The mice were euthanized at age 16 or 24 weeks. Atherosclerotic plaque was assessed by oil red O and Hematoxylin staining. The number of CD4+LAP+Tregs, CD4+CD25+Foxp3+Tregs, T-helper cells type (Th) 1, Th2, and Th17 present in spleens and CLNs were detected on the 4th and 14th days after the last nasal treatment using FACS analysis. The cytokine levels and mRNA expression of IFN-γ,IL-4,IL-10,IL-17 and TGF-βin splenocytes were analyzed by ELISA kits and Real-Time RT-PCR. We analyzed mRNA expression of TGF-β, CD25, Foxp3, IFN-γ, MCP-1, and VCAM-1 in the atherosclerotic lesions in the aortas. We found a significant 47.6% reduction in atherosclerotic lesion size in the aortic root was found that nasal oxLDL can markedly attenuated the initiation (47.6%, p<0.001) and progression (21.1%, p=0.001) of atherosclerosis, especially a significant 35.8% reduction of the progression of atherosclerosis was observed in the enhanced immunization group (p<0.001). These effects were accompanied by a significant increase in CD4+latency-associated peptide (LAP)+ regulatory T cells (Tregs) and CD4+CD25+Foxp3+Tregs in spleens and cervical lymph nodes, together with increased transforming growth factor (TGF)-βproduction and suppressed T-helper cells type 1,2, and 17 immune responses. Surprisingly, neutralization of TGF-P in vivo partially counteracted the protective effect of nasal oxLDL treatment.Conclusions:Our data suggested that nasal oxLDL treatment suppresses atherosclerosis development via inducing CD4+CD25+Foxp3+ and CD4+LAP+ Tregs, which secrete a large number of TGF-β. We conclude that the induction of nasal tolerance to oxLDL may be an effective approach for anti-atherosclerosis.