Experimental Research On Effects Of ?-enolase Specific Autoimmune Response On Thyroid And Brain

Objective:Autoimmune thyroiditis(AIT)is one of the common endocrine diseases.Inaddition to the destruction of thyroid tissue,it can also damage to other vital tissues organs,in which steroid responsive encephalopathy associated with AIT is gradually being concerned by multidisciplinaries.Patients usually have high levels of anti-thyroid peroxidase(thyroid peroxidase,TPO)and/or anti-thyroglobulin((thyroglobulin,Tg)antibodies,and the level of anti-?-enolase(ENO1)antibodies in serum(ENO1Ab)is higher than that of healthy controls.Our previous studies have found that the level of ENO1Ab in AIT patients'serum was significantly higher than that in non-AIT patients.Through autoantigen screening by proteome,we also found ENO1 coexisted in thyroid and brain and could be identified by the specific autoantibodies in autoimmune thyroiditis mice serum.ENO1 is a multifunctional protein,which is widely expressed in eukaryotic cells.It has been confirmed in rheumatoid arthritis and other autoimmune diseases,where ENO1Ab can mediate autoimmune damages to target organ.However,it is still unclear whether ENO1 is the target antigen in thyroid and brain tissue during the development of AIT,and whether it has some effects on mediating the autoimmune injury in thyroid and brain functions.In this study,Tg and ENO1 recombinant protein were respectively used to establish the experimental animal model to investigate above mentioned questions,and provide new targets for clinical prevention and treatment of AIT-related injuries,especially the brain damage.Methods:8-week-old CBA/J female mice were randomly divided into EAT group and CON group.Each of mice in EAT group was given murine Tg(m Tg)200?g and Freund adjuvant,then challenged 2 weeks later to establish the experimental autoimmune thyroiditis(EAT)animal model.CON group were treated with sterile distilled water and Freund's adjuvant.Mice were sacrificed at 4 weeks after the last immunization.The serum levels of TgAb and ENO1Ab were detected by ELISA.HE staining was used to observe the inflammatory infiltration degree in thyroid tissues.Western Blot,immunohistochemistry and immunofluorescence staining were used to determine the expression and localization of ENO1 in normal thyroid and brain tissues of adult mice.Western Blot was used to analyze the changes of ENO1 expression in the thyroid and brain tissues of mice at different growth stages.5-week-old CBA/J female mice were randomly divided into ENO1 group and CON group.As also given m Tg immunization to establish the EAT model,each of mice in ENO1 group was given ENO1 recombinant protein 100?g and Freund adjuvant,then challenged 2 weeks later to establish the experimental animal model.Correlation tests were performed at 4 weeks,6 weeks,10 weeks,and 14 weeks after the last immunization.Serum levels of ENO1Ab and TgAb were detected by ELISA.HE staining and inflammatory score to assess the degree of inflammatory infiltration in thyroid tissues.Immunofluorescence staining was used to analyze the distributions of CD4~+,CD8~+,CD19~+and CD16~+cells in the area of inflammatory infiltration and the presence of IgG or complement and CD16~+cells aggregation.Serum detection of TT4 and TSH to evaluate thyroid function in mice.RT-PCR analysis of the mRNA expressions of IFN-,IL-4,IL-17 and TGF-?mRNA in spleens.Methods of brain function research include:Morris water maze and long-term potentiation(LTP)tests to evaluate the learning and memory ability of mice.The microvascular ultrastructure of cerebral cortex and hippocampus was observed by transmission electron microscope.Immunofluorescence staining was used to observe the expressionsofIgG,complement,Iba-1,GFAP,Tauandphosphorylated Tau.Expressions of cytokines and chemokines in brain were detected by proteomic microarray(Microarray).Western Blot were used to analyze the protein expression levels of tight junction proteins(Claudin-5 and Occludin),Iba-1(microglia),GFAP(astrocytes),Tau and phosphorylated Tau(Ser396),cyclin dependent-5(CDK-5),glycogen synthase kinase-3?(GSK-3?)and IL-6 in cerebral cortex and hippocampus.Results:1.Serum levels of ENO1Ab IgG and its subclasses in EAT group were all significantly higher than those of control group(P<0.05),and the levels of IgG2a were higher in above 4 subclasses.2.ENO1 protein was both expressed in the thyroid and brain(neurons,microglia and astrocytes)tissues of normal CBA/J mice.Within growing weeks,the expression of ENO1 gradually decreased in thyroid tissues,but gradually increased in brain tissues.On the date of birth of 14th(P14),ENO1 protein was expressed with stable and high level in brain.3.ENO1Ab existed in serum of EAT mice and could specifically recognize the ENO1 antigen coexisting in the thyroid and brain tissues by western blot analysis.4.After ENO1 recombinant protein immunization,serum levels of ENO1Ab IgG and the four subclasses were significantly higher than those of control mice(P<0.05),and their high levels could be maintained to 18 weeks after the last immunization;the levels of IgG2a were higher in above 4 subclasses.5.In ENO1 group,the thyroid tissues were suffered from different levels of inflammatory infiltration.Both the incidence of thyroiditis and the inflammatory score for mononuclear cell infiltration in ENO1 group were on the up-trend,and the above two items were significantly higher than those in CON group at 10 and 14 weeks after the last immunization.Different levels of CD4~+,CD8~+,CD19~+and CD16~+cells were existed in the thyroid tissues,and most of the inflammatory infiltration belongs to CD4~+cells.6.Serum levels of TgAb total IgG in ENO1 group were significantly higher than those of CON group,and the rising trend showed statistically significant differences since 10weeks after the second immunization(P<0.05).7.In ENO1 group,there was significant IgG deposition of inflammatory cell infiltration in thyroid tissue in the local of thyroid follicular epithelial cells.It was revealed by confocal fluorescence microscope that the deposition of IgG might be ENO1Ab.CD16~+cells were gathered around the IgG deposition area.8.The expression level of Cleaved Caspase-3 in thyroid tissues of ENO1group was significantly higher than that in CON group.The serum level of TSH but not TT4 in ENO1 group was significantly higher than that in CON group(P<0.05).9.Morris water maze experiment showed that 14 weeks after the second immunization,ENO1 group mice gradually showed the decline in the spatial learning and memory capacity.The daily average escape latency was decreased although with the increase of training cycle,but the latency was still prolonged as compared with CON group.In addition,the frequency of crossing the platform was also decreased,and there was a statistically significant difference in ENO1 group as compared with CON group at 18weeks after the second immunization,(P<0.05).Results of LTP showed that the slopes of fEPSP were(158.86±11.80)%in ENO1 group and(274.85±15.56)%in CON group,respectively,at 14 weeks after the second immunization,P=0.003 between ENO1and CON groups.10.SEM observation revealed that there was significant edema in the cortex and hippocampus microvascular of ENO1 group mice at 10 weeks after the second immunization.In addition,there was IgG deposition in the microvascular endothelial cells,which had been demonstrated by confocal fluorescence microscope.10weeks after the second immunization,the expression levels of connexin proteins(Claudin-5 and Occludin)in ENO1 group were significantly lower than those in CON group(P<0.05).11.Immunofluorescence staining and western blot were shown that the expression levels of Iba-1 in the cortex and hippocampus in ENO1 group were significantly higher than those in CON group at 10 weeks after the second immunization.The increasing expression levels of GFAP appeared later than the change of Iba-1,which only showed a statistically significant difference in the hippocampus of mice in ENO1group(P<0.05).12.The expression levels of IL-6 in brain of ENO1 group mice were higher than that of CON group.13.The expressions of phosphorylated Tau protein in the cortex and hippocampus of ENO1 group mice were significantly higher than those of CON group at 10 weeks after the second immunization.14.We used the major kinases GSK-3?and CDK-5 to analyze the phosphorylation of Tau,and found only the expression of CDK-5 was significantly increased(P<0.05),which indicated that the abnormal phosphorylation of Tau protein in brain tissues of ENO1 group was mainly mediated by CDK-5.Conclusions:1.ENO1 was an autoantigen that coexists in the thyroid and brain tissues of normal mice and could be recognized by ENO1Ab,which was specifically present in serum of EAT mice.2.ENO1-immunization induced autoimmune response could stimulate the occurrence of autoimmune inflammation in the thyroid tissues of adult mice.It might also damage the thyroid follicular epithelial cells through ADCC,result in the reserve dysfunction of thyroids.3.ENO1-immunization induced autoimmune response could damage the functions of microvascular endothelial cells in the cerebral cortex and hippocampus,bring about changes in blood-brain barrier permeability,stimulate the hyperplasia and activation of glial cells,and increase the expression levels of IL-6 and other proinflammatory cytokines.Activated microglia and high levels IL-6 could further lead to impaired neuronal cell functions,result in the abnormal accumulation of phosphorylated Tau protein and cause neuronal dysfunction,which might have negative effects on the spatial learning and memory functions and even change the brain function.