Study On Exosome-induced T Lymphocyte Imbalance In The Pathogenesis Of Autoimmune Thyroiditis

Objective:Autoimmune thyroiditis（AIT）is a common autoimmune disease,Hashimoto thyroiditis（HT）is the main type of AIT,mainly manifested by infiltration of lymphocytes in the thyroid gland and thyroid follicle cells damage and the presence of thyroid-specific antibodies in the serum,such as antithyroid peroxidase antibodies（TPOAb）and thyroglobulin antibodies（TgAb）.The occurrence of HT is related to genetic susceptibility,environment,immune imbalance and many other factors,but the specific pathogenesis is not clear.Abnormal changes in the thyroid gland itself or the immune system may lead to the breakdown of immune tolerance,and then start the autoimmune response.In recent years,the innate immune system is considered to be the intersection of genetic susceptibility and environmental risk factors.The outcome of the innate immune response determines whether to develop further into autoimmune damage dominated by adaptive immune response.The results of previous research in AIT patients and iodine-induced AIT animal models NOD.H-2h4 mice and experimental autoimmune thyroiditis（EAT）models support Th1/Th2 shift,abnormal proportions and decreased function of Treg and Th17 cells are also involved in the pathogenesis of AIT.Exosomes are a kind of extracellular vesicles secreted by cells with a diameter of30-100nm.The contents of the exosomes include proteins,lipids,and RNA derived from mother cells,which can be passed by exosomes to other cells as signal molecules and change the function of other cells.In recent years,a large number of studies have found that exosomes are involved in a variety of autoimmune diseases,including rheumatoid arthritis（RA）,multiple systemic sclerosis（MS）,systemic lupus erythematosus（SLE）,inflammatory bowel disease（IBD）,and primary biliary cirrhosis（PBC）and type 1diabetes（T1DM）,and play an important role in the occurrence,development and outcome of these diseases.This study was performed in HT patients and healthy people,and with or without IFN-γstimulated thyroid follicular cell lines.The purpose of this study was to investigate whether serum exosomes from HT patients and IFN-γstimulated-thyroid cell-exosomes contain thyroid-specific antigens（thyroid peroxidase（TPO）and thyroglobulin（Tg））,inflammatory molecules（High mobility group box 1（HMGB1）and heat shock protein 60（HSP60））,and antigen-presenting molecules（MHC-II and intercellular adhesion molecule 1（ICAM-1））,have antigen-presenting functions and pro-inflammatory effects.whether serum exosomes from HT patients and IFN-γstimulated-thyroid cell-exosomes can activate immune cells,such as dendritic cells（DCs）and CD4+T lymphocytes,and participate in the occurrence of HT.Methods:The first part---Exosomes derived from serum:This study collected 30 HT patients and 30 age-and sex-matched healthy controls（HCs）.Serum exosomes extracted from HT patients and HCs by ultracentrifugation.Exosomes were identified by transmission electron microscopy（TEM）,nanoparticle tracking analysis（NTA）,and Western blot.Western blot was used to detect the expression levels of TPO,Tg,HMGB1,HSP60,MHC-II and ICAM-1 in HT-exosomes and HC-exosomes.Peripheral blood mononuclear cells（PBMCs）or sorted and cultured DCs were co-cultured with PKH-67-labeled serum exosomes for 24 hours,and immunofluorescence was used to detect whether HT-exosomes and HC-exosomes can be uptaked by PBMCs from healthy people,and whether they can bind to Toll-like receptors（TLR）2/3 on DCs,flow cytometry further detected which types of cells in PBMCs mainly take up exosomes.After co-culturing PBMCs from healthy people with HT-exosomes or HC-exosomes for24 hours,the percentage of CD11c+TLR2+,CD11c+TLR3+,and CD11c+TLR4+cells was detected by flow cytometry.In the presence or absence of TLR2/3 inhibitors,DCs sorted from healthy people were co-cultured with HT-exosomes or HC-exosomes for 24hours.Western blot detected the expression levels of MyD88,TRIF and p-P65 in DCs.The average fluorescence intensity of CD40,CD80 and CD83 was detected by flow cytometry,and the expression level of IL-6 in the cell supernatant was detected by enzyme-linked immunosorbent assay（ELISA）.In the presence or absence of TLR2/3inhibitors,PBMCs from healthy people were co-cultured with HT-exosomes or HC-exosomes for 24 hours,the percentage of CD4+IFN-γ+Th1,CD4+IL-17A+Th17A and CD4+CD25+Foxp3+Treg cells in PBMCs were detected by flow cytometry,and the expression levels of IFN-γ,IL-17A and IL-10 in serum were detected by ELISA.The second part---Exosomes derived from thyroid follicular cells:In this study,the supernatant of thyroid follicular cell line（Nthy-roi 3-1 cells）with or without IFN-γstimulation was collected and centrifuged by ultra high speed.The identification method was the same as the first part.Western blot was used to detect the expression of proteins in exosomes derived from thyroid follicular cells（Exo）and exosomes derived from thyroid follicular cells stimulated by IFN-γ（IFN-γ-Exo）.The indicators are the same as the first part.DCs sorted and cultured in healthy people were co-cultured with Exo or IFN-γ-Exo for 24 hours.The average fluorescence intensity of CD40,CD80 and CD83was detected by flow cytometry,and the mRNA expression levels of IL-6 and TNF were detected by RT-PCR.With or without GW4869 inhibiting the release of exosomes from thyroid follicular cells,thyroid follicular cells were co-cultured with DCs for 24 hours.Flow cytometry was used to detect the average fluorescence intensity of CD40,CD80and CD83,and RT-PCR was used to detect the mRNA expression levels of IL-6 and TNF.After DCs were co-cultured with Exo or IFN-γ-Exo for 24 hours,the cell supernatant was removed by centrifugation,and then co-cultured with autologous CD4+T lymphocytes for 24 hours.RT-PCR was used to detect the mRNA expression levels of IFN-γ,IL-17A,IL-22,IL-4,IL-10 and TGF-β1,and the expression levels of IFN-γ,IL-17A and IL-10 in the supernatant were detected by ELISA.CD4+T lymphocytes sorted by healthy people were co-cultured with Exo or IFN-γ-Exo for 24hours,and the expression levels of the above cytokines were detected by RT-PCR and ELISA.The effects of IFN-γ-Exo and IFN-γ-Exo-stimulated DCs(DC_IFN-γ_-Exo)on the mRNA expression levels of the above cytokines in CD4+T lymphocytes and the protein expression levels of the above cytokines in the supernatant were compared.Results:The first part---Exosomes derived from serum:1.TEM results show that HT-exosomes are"cup-shaped"vesicles with a diameter of 30-100 nm.NTA results showed that HT-exosomes were uniformly distributed,with the main peak at 95±5 nm.Western blot confirmed that HC-exosomes and HT-exosomes highly expressed exosome marker CD63.2.Western blot confirmed that the protein expression of TPO,HSP60 and MHC-II in HT-exosomes was significantly higher than that in HC-exosomes（P=0.002,0.012 and 0.016）,and the expression levels of TPO and HSP60 in serum exosomes were positively correlated with the titers of TPOAb and TgAb in serum（TPO and TPOAb:P=0.004;TPO and TgAb:P=0.022;HSP60 and TPOAb:P<0.001;HSP60 and TgAb:P<0.001）.3.Immunofluorescence results showed that both HT-exosomes and HC-exosomes can be taken up by PBMCs.Flow cytometry results further confirmed that in PBMCs,CD14+monocytes and CD11c+DCs mainly take up exosomes（P<0.001 and<0.001）.4.Immunofluorescence results showed that both HT-exosomes and HC-exosomes can bind to TLR2/3 on DCs.Flow cytometry further confirmed that HT-exosomes can increase the percentage of CD11c+TLR2+and CD11c+TLR3+cells in PBMCs（P=0.009 and 0.014）.5.Western blot,flow cytometry,and ELISA results showed that compared with HC-exosomes,HT-exosomes increased the protein expression levels of MyD88,TRIF,and p-P65 in DCs（P=0.003,0.006,and 0.003）and the mean fluorescence intensity of CD40 and CD83（P=0.004 and 0.028）and the protein expression level of IL-6 in the supernatant（P=0.026）.Most of these results can be reduced by TLR2 or TLR3 inhibitors.6.Flow cytometry and ELISA results demonstrated that compared with HC-exosomes,HT-exosomes increased the percentage of CD4+IFN-γ+Th1 and CD4+IL-17A+Th17A cells in PBMCs（P=0.027 and 0.039）,and the protein expression levels of IFN-γand IL-17A in the supernatant（P=0.035 and0.008）,while reducing the percentage of CD4+CD25+Foxp3+Treg cells（P=0.014）and the protein expression level of IL-10 in the supernatant（P=0.024）.Most of these results can be reversed by TLR2 or TLR3 inhibitors.The second part---Exosomes derived from thyroid follicular cells:1.TEM,NTA and Western blot results show that we successfully extracted exosomes from thyroid follicular cell supernatant.2.Western blot results showed that the protein expression levels of TPO,HSP60 and MHC-II were significantly higher in IFN-γ-Exo than Exo（P=0.015,0.025 and 0.026）.3.Flow cytometry and RT-PCR confirmed that compared with Exo,IFN-γ-Exo significantly increased the expression levels of co-stimulatory factors CD40 and CD80 and mature molecule CD83 on DCs（P=0.013,0.009,and 0.036）,significantly increased the mRNA expression levels of IL-6 and TNF in DCs（P<0.001and 0.026）.4.After GW4869 inhibited the release of IFN-γ-Exo,it reduced the expression levels of co-stimulatory factors CD40 and CD80 and mature molecule CD83on DCs（P<0.001,0.017 and 0.045）and the mRNAs expression levels of IL-6 and TNF in DCs（P=0.007 and 0.01）.5.RT-PCR and ELISA results showed that compared with DC_Exo,DC_IFN-γ_-Exo significantly increased the mRNA expression levels of IFN-γand IL-17A in CD4+T lymphocytes（P<0.001 and 0.002）,and increased the protein expression levels of IFN-γand IL-17A in the supernatant（P<0.001 and 0.03）.In contrast,compared with DC_Exo,DC_IFN-γ_-Exo significantly reduced the mRNA expression levels of IL-10 and TGF-β1 in CD4+T lymphocytes（P=0.029 and 0.036）and reduced the protein expression level of IL-10 in the supernatant（P=0.047）.6.The results of RT-PCR and ELISA showed that compared with Exo,IFN-γ-Exo has the tendency to promote the expression and release of pro-inflammatory factors in CD4+T lymphocytes and in the supernatant,and inhibit the expression and release of anti-inflammatory factors.There was no difference in mRNA expression levels in CD4+T lymphocytes and protein expression levels in the supernatant of all inflammation indicators between the two groups.7.The results of RT-PCR and ELISA showed that the mRNA expression levels of IFN-γ,IL-17A,IL-22,IL-4,IL-10 and TGF-β1（P=0.002,0.001,0.025,0.011,0.01,and 0.011）,and and the protein expression levels of IFN-γ,IL-17A,and IL-10 in the supernatant（P=0.004,0.02,and 0.008）were significantly higher in CD4+T lymphocytes co-cultured with DC_IFN-γ_-Exo than those in CD4+T lymphocytes co-cultured with IFN-γ-Exo.Conclusion:1.HT-exosomes and IFN-γ-Exo both highly express thyroid-specific antigen TPO,antigen-presenting molecule MHC-II,and inflammatory factor HSP60.2.HT-exosomes can bind to TLR2/3 on DCs through TLR2/MyD88/NF-κB and TLR3/TRIF/NF-κB signaling pathways,activated DCs cause imbalance in the differentiation of CD4+T lymphocytes.TLR2/3 inhibitors can partially reverse these results.3.IFN-γ-Exo cannot directly cause the expression and release of cytokines of CD4+T lymphocytes,and DCs need to be activated first.4.Exosome inhibitor GW4869inhibits the activation of DCs after inhibiting exosomes released by IFN-γstimulated thyroid follicular cells,suggesting that exosomes play a key role in antigen-presenting inflammatory response.