| Purpose.Thyroid associated ophthalmopathy(TAO)is also known as Graves disease,hyperthyroidism exophthalmos and endocrine ophthalmopathy.TAO belongs to the organ specific autoimmune disease and is considered as the manifestation of Graves' disease outside the thyroid gland.A large number of studies suggest that thyroid stimulating hormone receptor(TSHR)and insulin-like growth factor 1 receptor(IGF-1R)as TAO autoantibodies.They form as a structural and functional complex,which plays a critical role in the molecular pathogenesis of TAO.It is still not clear that whether there exist other molecules,which may also play important roles in the course of TAO.In the first part of this paper,we plan to perform the second generation high-throughput sequencing to grasp and analyze the transcriptome information on the orbital adipose connective tissue,which comes from thyroid associated ophthalmopathy patients and healthy control patients.This step is in order to have a better understanding of other molecules and signaling pathways involved in the occurrence and development of TAO,except for TSHR and IGF-1R.Based on transcriptome sequencing analysis mentioned above,the expression of pentraxin-3(PTX3)in orbital adipose connective tissue of patients with thyroid associated ophthalmopathy is higher than that of healthy control group.PTX3 belongs to the long chain penetrant family.This family also includes C-reactive protein(CRP)and serum amyloid P component(SAP).In systemic lupus erythematosus,multiple sclerosis and other autoimmune inflammatory diseases,PTX3 has a high concentration of expression in the very early phase of disease,whose sensitivity is higher than CRP and SAP.Moreover,the expression of PTX3 in serum is positively correlated with the severity of illness.These clues suggest that PTX3 may demonstrate important implications on the prognosis of the disease.Found in a previous study of our group,thyroid stimulating hormone(TSH)can be applied to fibrocytes(Fc),which stimulated the high expression of PTX3.This effect was achieved by increasing the stability of PTX3 mRNA,and can be blocked by anti-inflammatory drugs and anti IGF-1R drugs.Based on transcriptome sequencing,in the second part of this study,we plan to collect the orbital adipose connective tissue and serum samples taken from TAO patients and healthy people,and then compare the differential expression of PTX3 in tissue and serum of different sources to explore the possibility of PTX3 as a potential biomarker for TAO.Recently,a multi-center,double blind,randomized controlled clinical trial confirmed that anti IGF-1R antibody,teprotumumab has high safety and efficacy for TAO treatment.This finding changed the current limited situation of TAO treatment from symptomatic therapy to etiological treatment.However,the molecular mechanism of how teprotumumab acts needs further exploration.In the third part of this study,we plan to culture fibrocytes and Th1 cells from patient's peripheral venous blood,treated with teprotumumab.Then we stimulate the cells with PMA accompanied by Ionomycin antigen to observe the expression of human major histocompatibility class II(MHC?)and interferon gamma(IFN-gamma).This experiment may shed a light to the understanding of whether teprotumumab affects the antigen presentation ability of fibrocytes and the activation of Th1 cells.We can even go further to explore the mechanism of how teprotumumab acts.Methods.1.The mRNA quality of orbital adipose connective tissue was detected in the control group and TAO case group.Five samples of each group,which were qualified for the quality requirements,were further explored by RNA-seq technology.The original data,Rawdata,was grasped from these samples.2.The gene expression gained from Rawdata was quantified and corrected.The differentially expressed genes,Dif-gene,between the two groups were obtained.3.Gene Ontology Analysis(GO)and pathway analysis were performed on Dif-gene.4.Function tree analysis(GO-tree)was obtained based on the significant GO entries.The signal path network(Path-Act-Network)was constructed according to the significant signal path.5.Four genes statistically differential expressed between two groups with high abundance were selected.Real-time PCR was performed on orbital adipose connective tissue to verify the differential expression of dif-gene in TAO group and healthy controls.6.The orbital adipose connective tissue from TAO patients and healthy controls was collected.Fibroblasts were cultured from these tissues.The expression of PTX3 on mRNA and protein level was verified by Real-time PCR and immunohistochemistry(IHC).7.The peripheral venous blood samples from the TAO patients and the healthy controls were collected respectively.The concentration of PTX3 protein in the samples was detected by ELISA.8.The peripheral venous blood samples from patients with TAO and healthy controls were collected.fibrocytes were cultured with or without teprotumumab.The expression of MHC? on fibrocytes was detected by real-time PCR and flow cytometry.9.The peripheral venous blood samples from patients with TAO and healthy controls were collected.Mononuclear cells were cultured with or withour teprotumumab.At the time of maturation,cells were stimulated with or without P/I antibody.Flow cytometry was used to detect IFN-gamma production in Th1 cells.10.The supernatant from samples during culture process mentioned above was collected separately.The expression of IFN-gamma in the supernatant with / without teprotumumab was detected by ELISA.11.The peripheral venous blood samples from patients with TAO and healthy controls were collected.Mononuclear cells were cultured with or without teprotumumab.At the time of maturation,cells were treated with or without IFN-gamma neutralizing antibody.The expression of MHC? on fibrocytes surface was detected by flow cytometry.Results.1.The adipose connective tissue from TAO patients and healthy controls was obtained.The quality scores of Rawdata obtained from the transcriptome sequencing were all greater than 20.The lowest mappedrate was 0.895.The highest mappedrate was 0.931.Data was mainly coming from the exon region.A total number of 784 differentially expressed genes were detected from RNA-seq.2.Compared with the expression of healthy controls,the expression of 445 genes,including PTX3,were upregulated in the orbital adipose connective tissue of TAO patients.The expression of 339 genes,such as CTHRC1,were downregulated in the orbital adipose connective tissue of TAO patients.The difference was statistically significant.3.Compared with the expression of healthy controls,cytokine and cytokine receptor interaction,chemokines and other 19 pathways were significantly upregulated in orbital adipose connective tissue of TAO patients.Cell adhesion,extracellular matrix receptor and other 20 pathways were significantly downregulated in orbital adipose connective tissue of TAO patients.4.Differentially expressed genes and signaling pathways were mainly involved in the following functions: inflammatory response,immune response,scar repair,T cell regulation,etc.PTX3 gene is mainly involved in inflammatory processes,immune response,and other biological processes.5.The expression of PTX3 at the level of mRNA and protein was higher in the orbital adipose connective tissue of TAO patients,than that in the healthy control group.6.The expression of PTX3 in the blood samples from the TAO patients was higher than that in the healthy controls.There was no significant difference observed between active TAO and inactive TAO.7.Teprotumumab could reduce the expression of MHC? on the surface of peripheral blood fibrocytes on gene level and protein level,no matter whether there was antigenic stimulation or not.In addition,this effect could be observed both in the fibrocytes from the TAO patients and that from the healthy controls.8.Teprotumumab could significantly reduce the IFN-gamma production inside the Th1 cells,whether the cells were derived from TAO or healthy controls.9.Teprotumumab could significantly reduce the IFN-gamma protein level in cell culture supernatant,whether the cells were derived from TAO or healthy controls.10.When treated with IFN-gamma neutralization antibody,the expression of MHC? on the surface of peripheral blood fibrocytes was significantly reduced.Conclusions.1.Genes like PTX3 may be related in the occurrence and progression of TAO.Cytokine and cytokine receptor interaction and chemokine pathway may be involved in the pathological process of TAO.TAO patients' inflammatory processes,immune responses,scar repair,T cell immune activity and other biological processes were enhanced.The expression of multiple inflammatory related genes and autoimmune related genes in the orbital adipose connective tissue of TAO patients increased,which once again confirmed the correlation between TAO and autoimmune and inflammation diseases.2.The expression of inflammatory molecule PTX3 was increased in the orbital adipose tissue and blood of TAO patients,compared with that of healthy controls.It suggested that PTX3 might be involved in the inflammatory progression of TAO.PTX3 may be a potential biomarker for the diagnosis and prognosis of TAO.3.Th1 cells stimulated the expression of MHC? molecule on the surface of fibrocytes by secreting IFN-gamma.Teprotumumab could reduce IFN-gamma expression produced and secreted by Th1 cells and attenuated the expression of MHC? molecule on the surface of fibrocytes.4.Teprotumumab could attenuate the activation of Th1 cells and the antigen presentation ability of fibrocytes whether stimulating antigen was added or not.This effect could be observed both in the peripheral blood fibrocytes from TAO patients and healthy controls.This suggested that the mechanism of how teprotumumab was effective in the treatment of TAO might be at least partly by weakening the immune effects of Th1 cells and fibrocytes against to autoantibodies. |
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