Rhythmic Features Of T Lymphocytes Subsets And Core Clock Gene Expression In AIT Animal Models

Objective: The occurrence of autoimmune thyroiditis is mainly affected by environment and genetics.One of the most important pathogenesis is immune disturbance,in which immune imbalance among T lymphocyte subtypes play a dominant role,such as the increasing number of Th1 and Th17 lymphocytes as well as decreasing number of Tregs.Regular circadian rhythm could maintain physiological homeostasis,while disruption of circadian rhythm possibly participates in dysfunction of differentiation and function of lymphocytes,resulting in immune imbalance,consequently,leading to autoimmune diseases.Conversely,the occurrence of inflammation could affect day-night cycle of clock gene and its expression.Previous epidemiological studies have shown that disruption of oscillation was possibly associated with the increasing incidence of thyroid dysfunction.However,the mechanism of immune imbalance in AIT still remains unclear.Therefore,we infer that the occurrence of AIT may affect the rhythmic characteristics of T lymphocytes,leading to further exacerbation of immune imbalance.Methods: 5-week-old NOD.H-2h4 mice and Kunming mice were randomly divided into4 groups: normal Kunming mice group(CON1): fed with sterile deionized water;Kunming mice group(CON2)with high iodine: 0.05% sodium iodide Water feeding;normal NOD.H-2h4 mice group(CON3): sterile deionized water feeding;NOD.H-2h4 mice group(AIT)with high iodine: 0.05% sodium iodide water feeding.All mice were raised and bred under 12-h dark and 12-h light conditions in the specific pathogen-free facility in the Experimental Animal Center at China Medical University.The light-on time is Zeitgeber 0(ZT0)(7:30 am),and light-off time is ZT12(7:30 pm).4-8 mice in each group were sacrificed at 6hrs interval when all mice were at 12 weeks.The serum was collected to evaluate levels of Tg Ab,IL-10,TGF-β and CXCL12.The spleen was taken out under aseptic conditions to analyze the proportion of T lymphocytes subsets by flow cytometry.Total RNA was extracted from the spleen to detect core clock genes Per2,Bmal1,and Treg(Foxp3)m RNA expression by real-time quantitative RT-PCR.Results: 1.The characteristics of clock gene expression: 1)the extent of thyroiditis using HE staining and serum Tg Ab titers by ELISA,the severity of thyroiditis and levels Tg Ab were higher in AIT group than CON3(p<0.05),while there was no significant difference between CON1 and CON2.2)T lymphocyte subgroups were detected by Flow cytometry,there was no significant difference in the ratio of Th2 lymphocytes among the4 groups.The proportion of Th1 and Th17 lymphocytes in the AIT group was significantly higher than that in the CON3 group(Th1%: p<0.001;Th17%: p<0.05).The percentage of Th17 and Th1 lymphocytes in the CON1 and CON2 groups showed no difference.The proportion of Treg lymphocytes in the AIT group was significantly lower than that in the CON3 group(p<0.05),while this was almost similar between CON1 and CON2 groups;3)The expression of core clock genes Per2 and Bmal1 in the spleen by real-time quantitative PCR was detected.Per2 expression in monocytes in the AIT group was significantly lower than that in CON3 group(p<0.05),while the expression of Per2 in the CON1 and CON2 groups was nearly similar.However,Bmal1 expression showed no significant difference between the AIT group and CON3 group,and there was no significant difference between CON1 group and CON2 group.2.Rhythm characteristics of T lymphocyte subsets and related factors: 1)The rhythm of the ratio of T lymphocyte subsets at different time points in each group of mice was determined by flow cytometry.The percentage of Th2 cells revealed no obvious rhythmic change among 4 groups,while the ratio of Th1 lymphocytes showed obvious rhythmic features in all groups,peaking at ZT7(p<0.05).The ratio of Th17 lymphocytes had no obvious day-night variation between CON1 and CON2 groups.However,in both the AIT group and the CON3 group,the ratio of Th17 cells showed rhythmic features,reaching its highest level at ZT7(p<0.05).The proportion of Treg lymphocytes was highest at ZT13 among CON1,CON2 and CON3 mice,while the percentage of Tregs in the AIT group lost its rhythm.2)Rhythmic gene expression was evaluated by real-time quantitative PCR.In CON1,CON2 and CON3 mice,Per2 expression showed rhythmic characteristics and reached the highest level in ZT13,while in the AIT group,the rhythm of Per2 expression disappeared;Bmal1 expression showed rhythmic tendency but no significant difference in CON1,CON2 and CON3 groups;3)The levels of serum cytokines was detected by ELISA.IL-10 presented obviously rhythmicity in CON3 mice,peaking at ZT13.The serum titers of CXCL12 and TGF-β in CON3 group still remain day-night variation,being the highest at ZT19;Interestingly,the rhythm of IL-10 and TGF-β disappeared in the AIT group and serum level of CXCL12 showed forward shift,peaking at ZT7.Conclusion: 1.Per2 expression is negatively correlated with the severity of AIT.2.The rhythmic feature of Per2 expression disappears after the occurrence of AIT.3.AIT does not change the rhythmic characteristics of Th1 lymphocytes ratio.The percentage of Tregs loses its regular rhythm in AIT group,and cytokines and chemokines associated with Tregs,such as IL-10,TGF-β and CXCL12,present similar change.The day-night variation of Th17 lymphocytes shows species differences.AIT does not affect the circadian rhythm of Th17 cells in NOD.H-2h4 mice,while in Kunming mice,they showed no day-night cycle of Th17 cells.