Effect Of Induction Of Oral Tolerance To Heat Shock Protein60on Atherosclerosis

OBJECTIVE: The aim of this study was to discover the efficacy of oral tolerance withheat shock protein (HSP)6O in two non-overlapping prototypes of murine atherosclerosisand also to found out the level of atherosclerosis associated cytokines (interleukin-10,transforming growth factor-β, and interferon-γ) in blood.BACKGROUND: Atherosclerosis is the most common pathologic course leading tocardiovascular disease (CVD), including myocardial infarction (MI) and cerebral infarction,the number one cause of death in western societies. Although many factors such as smoking,hypertension, diabetes mellitus, hyperlipidemia are thought to be the risk factor foratherosclerosis, but the precise mechanism is not mentioned, It is broadly accepted thatinflammation plays an main role during atherosclerotic lesion formation. Recentlyautoimmune mechanisms have been shown to impact atherogenesis in experimental animalmodels.Mucosal tolerance is a specific suppression of cellular and/or humoral immuneresponses to an antigen by administration of the antigen via a mucosal surface. It is a form ofperipheral tolerance that evolved to treat external agents that gain access to the body via anatural route and usually achieve by oral route. It is of unique immunologic importance,because it is a continuous natural immunologic event driven by exogenous antigens. Oraltolerance is a simple, effective, non-toxic method that has been already used as a treatmentin animal models for autoimmune diseases such as multiple sclerosis, rheumatoid arthritisand type I diabetes. As autoimmune processes have been shown to participate inatherosclerosis, with abundant evidence pointing to HSP60as a possible antigenic candidate;we therefore reasoned that tolerization of the immune response to this antigen would resultin reducing atherosclerosis.The effects of oral tolerance induction are mainly determined by the dose of the antigen.Low dose feeding results in regulatory T cell activation in the gut. Regulatory T cells, whichmainly producing interleukin-10(IL-10) and transforming growth factor-β (TGF-β), maymigrate to lymphoid organs and target organs to suppress the disease both in an antigen-specific and an antigen-non-specific way. Many studies have shown that the deficiency ofIL-10and/or TGF-beta or their receptors cause increase in size of atherosclerosis indicating anti-atherosclerotic role of these cytokines. Furthermore, studies have shown that interferon-γ (IFN-γ), which is produced by T helper1cells, causes increase in atherosclerosis.Our study tries to detect the effect of oral tolerance with HSP60on atherosclerosis andalso to find its effects on serum level TGF-β1, IL-10, and changes in IFN–γ.METHODS: Apo-lipoprotein E knockout (ApoE-/-) mice were randomly divided intohigh fat group and HSP60group. Out of14mice,7were in high fat group and remaining7were in HSP60group. All mice were7weeks old male and weighted18to20grams.HSP60group: All mice in this group were fed by a tube,4doses (every other day) of5μg of HSP60in0.25ml normal saline and0.25ml long chain fatty acid.7days after the lastfeed, each mice were immunized via intra-peritoneal injection with5μg of HSp60. All micewere fed normal diet during this period.1day after intra-peritoneal injection all mice weregiven high fat diet containing1%cholesterol,10%lard and0.2%cholic acid.High fat group: All mice of this group were fed with distilled water instead of HSP60along with0.25ml long chain fatty acid,4times on alternative days.7days after the lastfeeding all mice were immunized via intra-peritoneal injection of5μg of HSP60. All micewere fed normal diet during this period.1day after intra-peritoneal injection all mice weregiven high fat diet containing1%cholesterol,10%lard and0.2%cholic acid.All mice were euthanized10weeks after high fat diet. The aortic arch along with aorticroot was taken for plaque area comparison and blood was collected for cytokines level (IL-10, TGF-β1, and IFN-γ) analysis by ELISA test.RESULTS:1. Aortic root plaque area: Pathological image analyzer is used to calculate the aorticplaque area of each group of ApoE-/-mice. Pathological examination showed that oraladministration of HSP60resulted in97.1%reduction in aortic atherosclerotic plaque areathan that of high-fat group (P<0.01).2. Serum cytokines level:a. The serum level of IL-10and TGF-β1is significantly higher in HSP60treated groupthan high fat group.(P<0.05and P<0.01respectively)b. The serum level of IFN-γ is significantly lower in HSP60group than high fat group.(P<0.05).CONCLUSION:1. Induction of oral tolerance to HSP60results in decrease of atherosclerotic plaque sizein ApoE-/-mice. 2. There is a significantly increase in serum IL-10and TGF-β1after oral tolerance withHSP60which reflects their protective role in atherosclerosis. Furthermore, as thesecytokines are produced by regulatory T cells, there may be the role of regulatory T cell afteroral tolerance with HSP60in decreasing atherosclerosis.3. As serum IFN-γ is decreased in HSP60treated group, suggesting the role of IL-10and TGF–β (or regulatory T cells) in down regulating Th1cytokine.We conclude that these beneficial results of oral tolerance induction to HSP60mayopen a further new therapeutic approach for the treatment of atherosclerosis.