Inflammasomes And Their Pathway Induced Thyroid Immunal Microenvironment Imbalance In Patients With Hashimoto Thyroiditis And Mouse Model

Objective:Hashimoto thyroiditis?HT?is the most common subtype of autoimmune thyroiditis?AIT?in human.It is reported that HT is associated with multiple factors,such as environmental,genetic and immunological factors.Known as an organ specific autoimmune disease,HT is characterized by immune cell infiltration and thyroid follicles destructions in thyroid tissue.But the etiology and mechanisms remain unknown.Since there are various kinds of cells in the thyroid gland,we aim to explore the thyroid microenvironment in HT patients by 10X genomics single-celll RNA sequencing.Then we further compare the difference of RNA expression in each cluster between HT patients and controls and analyze cell-to-cell communication among clusters.NOD.H-2h4 mice,a canonical model of spontaneous autoimmune thyroiditis?SAT?,have the same pathological appearance in thyroid glands and elevated serum TgAb level as human HT when fed with high iodine water.The inflammasomes–cytosolic multimeric innate immune signaling complex–are composed of pattern recognition receptors?PRRs?,apoptosis-associated speck-like protein containing a CARD?ASC?and the effector cysteine protease caspase-1.Our previous studies have found that thyroid tissues from HT patients express higher NLRP1,NLRP3,NLRC4 and AIM2inflammasome than controls.In mouse experiments,we explored the dynamic changes of inflammasomes and their downstream pathway.We also examined dynamic changes of Th17?T helper 17?,Th1?T helper 1?,Tfh?T follicular helper cells?and Tfr?T follicular regulatory cells?lymphocytes in splenic mononuclear cells.Then we further examine the severity of HT symptoms through inhibiting or knockdown of NLRP3 to explore the role and mechanisms of inflammasomes in the initiation and development of HT.Methods:In this study,we selected 3 female HT patients and 3 baseline-matched normal controls?Con?.Thyroid speciments and serum were collected.Thyroid tissue samples were dissected and minced with scissors into small pieces and digested with type II collagenase to obtain qualified single cell samples for 10X genomics single-celll RNA sequencing.Detailed steps are as follows:Single-cell capture,cDNA library preparation,barcoding and sequencing,Bioinformatic analysis by Cell Ranger and Seurat software programme.In animal study,a total of 168 NOD.H-2^h4 mice were randomly divided into high iodine supplement?HT?group and normal iodine group?Con??half male and half female in each group?at 6 weeks of age.The H groups were given drinking water containing extra0.05%NaI while the N groups were given regular drinking water for the same time period.Animals were sacrificed at the time point of 0,2,4,6,8,12,16 weeks after high iodine solutions,with o week as an initial time of the experiment.We collected thyroid tissues in each group,weighed the thyroid weight and observed severity of lymphocytic infiltration by HE-staining.The serum thyroglubulin antibody?TgAb?levels were measured by Enzyme Linked Immunosorbent Assay?ELISA?.NLRP3,NLRC4,NLRP1A,AIM2,IL-1?,IL-18 and caspase-1 mRNA expression in thyroid tissues were detected via Real-Time PCR at different time point.The percentage of Th17,Th1,Tfh and Tfr lymphocytes in splenic mononuclear cells was measured by multicolor flow cytometry.We also examined specific NLRP3 inhibitor effect in NOD.H-2^h4 mouse model.A total of36 NOD.H-2^h4mice were randomly divided into 3 groups?n=12 per group,half male and half female?as follows:normal water group?N?,High iodine drinking water group?H?and intragastric injection administration of MCC950 at the time of iodine supplement group?H+MCC950?.After 8 weeks,the severity of lymphocytic infiltration in thyroid glands was evaluated by histopathological study.The serum titiers of TgAb was measured by ELISA.The Th1/Th17/Tfh/Tfr percentages were assessed using multicolor flow cytometry.Additionally,we generated the NLRP3-/-NOD.H-2^h4 mice and their NLRP3-/+and NLRP3+/+couples using the existing NOD.H-2^h4 mice and NLRP3 knockout NLRP3+/-C57BL/6 mice.According to their genotypes,mice were divided into 3 groups:NLRP3-/-,NLRP3+/-and NLRP3+/+,each group was given high iodide?0.05%sodium?drinking water with NLRP3+/+group given normal drinking group?N?as controls.Methods for assessment of severity of lymphocytic infiltration in thyroid gland and serum TgAb levels were conducted as described above.Results:1.We found that immune cells especially T/B lymphocytes,thyrocytes and endothelial cells in thyroid microenvironment were more diversity in patients with HT than controls using 10X Genomics RNA sequencing analysis.2.By comparing the differences of m RNA expression between HT patients and healthy controls,we found that IL-1?mRNA expressed in epithelial cells was significantly elevated in HT patients compared with healthy controls.3.Through analyzing the receptor-ligand pair,we also found that there exists complex cell-cell communication in thyroid microenvironment in patients with HT.The results of inflammasomes expression in SAT mice:4.The relative weight of thyroid,serum TgAb levels and lymphocytic infiltration in thyroid were increased significantly in H group,compared with N group from 4 weeks high iodine supplementation till 16weeks,while no significant changes at 2 weeks.5.The m RNA expression of NLRP3,NLRC4,IL-18,caspase-1 and IL-1?was higher in H group than N group from 2 week to16 week.While NLRP1A mRNA started to increase in H groups at 4 week till 16 week.6.Compared the N group,in H group:the percentage of CD4⁺IFN?⁺Th1 and CD4⁺IL17A⁺Th17 lymphocyts were increased at 8,12,16 weeks;the percentage of CD4⁺CXCR5⁺ICOS⁺Foxp3^-Tfh lymphocyts?ICOS⁺Tfh?was increased at 8,12,16weeks;the percentage of CD4⁺CXCR5⁺ICOS⁺Foxp3⁺Tfr lymphocyts?ICOS⁺Tfr?lymphocyts was decreased at 8,12,16 weeks;the ICOS⁺Tfh/ICOS⁺Tfr ratio were increased at 8,12,16 weeks;the percentage of CD4⁺CXCR5⁺PD-1⁺Foxp3^-Tfh lymphocyts?PD-1⁺Tfh?was increased at 12,16 weeks,while the percentage of CD4⁺CXCR5⁺PD-1⁺Foxp3⁺Tfr lymphocyts?PD-1⁺Tfr?lymphocyts was not changed,the PD-1⁺Tfh/PD-1⁺Tfr ratio was increased at 8,12,16 weeks.7.We used MCC950 to specifically inhibit NLRP3 inflammasome:Compared with the N group,serum TgAb levels and lymphocytic infiltration in thyroid were increased significantly,while the H+MCC950 group were decreased in contrast with the H group.And the imbalance of Th1,Th17,ICOS⁺Tfh/ICOS⁺Tfr,PD-1⁺Tfh/PD-1⁺Tfr lymphocytes were improved in H+MCC950 group compared with H group.But the relative weight of thyroids in H+MCC950 group was similar to that in H group.8.After fed with iodine water,NLRP3+/+group?H?suffer severer lymphocytic infiltration in thyroid and higher serum TgAb titers.However,serum level of TgAb and incidence and severity of thyroiditis in NLRP3-/-?H?and NLRP3+/-group?H?were lower than NLRP3+/+group?H?,but there were no deiffernces in The relative weight of thyroids among the three groups.Conclusion:1.Immune cells and cytokines in thyroid microenvironment were more diversity in patients with HT than controls and there exists complex cell-cell communication in thyroid microenvironment in patients with HT.2.IL-1?mRNA expressed in epithelial cells was significantly elevated in HT patients compared with healthy controls.3.In high iodine induced mouse models,the time of NLRP3,NLRP4and their downstream pathway caspase-1/IL-18/IL-1?mRNA overexpression was ahead of that of Th1,Th17,ICOS+Tfh/ICOS+Tfr?PD-1+Tfh/PD-1+Tfh imbalance.4.Specific inhibition or knockdown of NLRP3 can alleviate clinical symptoms of HT and T lymphocytes imbalance including Th1,Th17,ICOS⁺Tfh/ICOS⁺Tfr,PD-1⁺Tfh/PD-1⁺Tfr lymphocytes,but could not block the enlargement of thyroid follicular cells in vivo in high iodine induced mouse models.These above results imply that inflammasomes and its pathway played part role in the occurrence and development of AIT...