Roles And Mechanism Of Circadian Clock And Bmal1 Gene In The Pathogenesis Of Autoimmune Thyroiditis

Objective:Autoimmune thyroiditis（AIT）,also known as Hashimoto thyroiditis（HT）,is a common autoimmune thyroid disease.AIT is characterized by lymphocytes infiltration and destruction of follicular structure of the thyroid gland,accompanied by elevated serum thyroid autoantibodies and inflammatory cytokines.AIT can be caused by multiple pathogenic factors ranging from genetic,environmental to immune-related influence.Aberrant expression of HLA-DR,cytokines and chemokines on thyroid follicular cells can induce the breakdown of immune tolerance towards thyroid autoantigen,leading to the imbalanced differentiation of Th1/Th17/Treg lymphocytes and autoimmune responses targeting the thyroid gland.However,the exact pathogenesis and risk factors of AIT remained to be elucidated.Recent evidence suggested that the circadian clock and core clock gene Bmal1 provides a novel insight for understanding the pathogenesis of autoimmune diseases.At the molecular level,the circadian clock is orchestrated by 3 transcription-translation feedback loops（TTLs）.The Bmal1 is the core regulator of TTLs,other clock genes include Clock,Per,Cry,Rev-erb and Ror.Bmal1 and its downstream clock gene and immune-related genes contributes to the rhythmic changes of immune function.The desynchronization between the environmental clock and the internal clock caused by night shift or sleep disorders is closely associated with the prevalence and aggravation of chronic inflammatory diseases.In addition,local clock dysfunction has been observed in multiple autoimmune diseases including rheumatoid arthritis,systemic lupus erythematosus and multiple sclerosis.The interaction between immune cells and thyrocytes is a key initiating factor of AIT,and both of which showed e rhythmic oscillations in clock gene expression.However,few studies investigated the pathogenesis and treatment target of AIT from the insights of“chrono-immunology”.Our group have previously found the oscillated expression of Bmal1 and Per2 in mouse spleen,and the level of Per2 was negatively related to the severity of AIT.Methods:Thyroid tissues were collected from patients underwent thyroidectomy for benign thyroid nodules,and divided into autoimmune thyroiditis group（AIT group,n=30）and control group（Control group,n=30）according to their postoperative pathological diagnosis and thyroid function.Total RNA from thyroid tissues was extracted and q PCR was applied to detect the expression of clock genes Bmal1,Clock,Per2,Cry1,Rev-erb and Ror,and to analyse the association of their expression levels with local and serum inflammatory biomarkers.In animal studies,6-week-old C57BL/6J mice were randomly divided into normal control（NC）and experimental autoimmune thyroiditis（EAT）groups,and the EAT group was administered with porcine thyroglobulin（p Tg）emulsified by adjuvants at weeks 10 and12,respectively,while the NC group were injected with adjuvant+PBS at the same time.Both EAT and NC mice were sacrificed at four time points,ZT0,ZT6,ZT12 and ZT18,respectively.In addition,NC and EAT mice were random Ly divided into light shift（LS）and normal light groups:LS,NC,LS+EAT and EAT groups（n=6/group）;mice in the EAT group were immunized with adjuvant+p Tg at weeks 10 and 12,and mice in the NC and LS groups were given adjuvant+PBS injections;LS and LS+EAT mice were housed in light shifted cages（lights on 6 hours earlier every 4 days）.Mice in all groups were sacrificed at 16 weeks for the collection of serum and thyroid tissue.Inflammation was assessed by HE staining of frozen thyroid sections and serum levels of thyroglobulin antibodies（Tg Ab）.Total RNA was extracted from the thyroid tissue and the relative m RNA expression levels of the clock genes Bmal1,Clock,Per2,Cry1,Rev-erb and Ror in the thyroid tissue were measured by q PCR.The protein expression of BMAL1,CRY1and PER2 was detected by immunofluorescence staining of frozen thyroid sections.Serum levels of IL-17,TNF-α,IFN-γ,TGF-β,IL-6,IL-10 and cortisol were detected by Elisa assay.The Spontaneous autoimmune thyroiditis（SAT）model of NOD.H-2h4 mice fed by0.05%sodium iodide in drinking water was repeated for validation.Thyroid cell-specific Bmal1 knockout mice(Bmal1^thyroid-/-,c KO group)were established,with Cre-negative,flox-flox mice as control mice(Bmal1^thyroid+/+,Ctrl group).The light was switched on at ZT0 and c KO and Ctrl mice were sacrificed at four time points,ZT0,ZT6,ZT12 and ZT18,respectively.Thyroid tissues were collected,and q PCR was used to detect the expression of clock genes.The mice were subjected to p Tg immunization at ZT6and ZT18,and control group were administered with adjuvant+PBS.Mouse serum,thyroid tissues and spleens were collected.Magnetic bead sorting method was performed to isolate CD4+T cells from splenocytes,and was analyzed by RNA sequencing.Flow cytometry was used to analyze the differentiation of Th1/Th17/Treg subsets.Thyroid tissues from each group of mice were collected for q PCR and immunohistochemistry analysis.Results.:1.q PCR analysis revealed the expression of Bmal1 and Per2 in AIT thyroid tissue were significantly decreased compared with the control group（P<0.05）,while the expression of Clock,Cry1,Rev-erb and Ror did not show significant change;the expression of Bmal1 and Per2 in thyroid tissues were negatively correlated with thyroid autoantibodies,serum cytokines and inflammatory genes in thyroid（P<0.05）.2.Consistently,rhythmic changes in thyroid clock gene expression were observed in mice and were disturbed in the EAT mouse model;no significant rhythmic changes in serum Tg Ab were found in EAT mice,but serum IL-10,IFN-γand TNF-αlevels were significantly different between ZT6 and ZT18.Light shift did not induce the inflammatory phenotype,but aggravated inflammatory infiltration of the thyroid gland and disruption of thyroid follicular structures caused by p Tg immunization.serum Tg Ab and TNF-αlevels were significantly higher in EAT mice under light shift than in EAT mice raised in a normal light environment（P<0.05）.Consistently,inflammation of the thyroid gland,levels of serum Tg Ab and IL-17 in SAT under light shift were increased compared with SAT mice under normal light.q PCR and immunofluorescence staining showed that light disorder reduced Bmal1 and Cry1 expression in thyroid tissues of normal and EAT mice,and the expression of Bmal1 and Cry1 were negatively correlated with Tg Ab titers.3.Thyrocyte-specific Bmal1 knock out led to persistent reduced expression of Bmal1 and Clock in thyroid tissues,accompanied by arhythmic expression of other downstream clock genes,while no significant thyroid inflammatory phenotype was observed.In the EAT mouse model experiments,this study found that p Tg immunization at ZT6 led to more severe EAT than ZT18,which was consistent in c KO and Ctrl mice;compared to Ctrl-EAT mice,c KO-EAT mice showed higher inflammatory scores and significantly increased serum Tg Ab titers（P<0.05）.Transcriptomic analysis revealed 1114 differentially expressed genes in CD4+T cells from the c KO-EAT and Ctrl-EAT groups,which were enriched in immune and inflammation-related pathways.Flow cytometry analysis further confirmed that the increased proportion of Th1 and Th17 cells and reduced proportion of Treg in c KO-EAT mice compared to Ctrl-EAT mice（P<0.05）.q PCR experiments revealed that the expression of Tnf-α,Cxcr3 and Cxcl10 was significantly higher in thyroid tissues of c KO-EAT mice,and immunostaining experiments showed significant infiltration of CD4positive cells in the thyroid tissue of c KO-EAT mice.Conclusion:1.The expression of Bmal1 and Per2 in thyroid tissues of AIT patients was significantly reduced and negatively correlated with AIT-related inflammatory markers,suggesting that disruption of the local thyroid biological clock may be involved in the development of AIT.2.Animal models further validated the disruption of thyroid clock in AIT mice,and found light disruption was a risk factor for the progression of AIT.3.Thyrocyte-specific Bmal1 knock out exacerbated p Tg immunization-induced thyroid inflammation and imbalanced T cell differentiation,in which the alterations in immune microenvironment in transgenic mice could be potential mechanisms mediating the aggregation of AIT posed by clock disruption.