| Autoimmune thyroid diseases (AITD) can be classified into two major types:over functioning (hyperthyroidism:Graves’ disease (GD)) and under functioning (hypothyroidism:Hashimoto thyroiditis (HT)) of the thyroid gland. Both diseases involve specific auto-antibodies (aAbs) against thyroid auto-antigens, such as thyroperoxidase (TPO), thyroglobulin (TG), and/or the TSH receptor (TSHR). Hallmarks of HT are TPOAb and antiTG(ATG) while most patients will remain euthyroid, and hallmark of GD is thyrotropin receptor antibodies (TSHRAbs). The hyper-/hypo thyroidism of GD and HT can be viewed as the opposite ends of a continuous spectrum of AITD with a substantial overlap. TPOAb and/or ATG present not only in almost all HT patients, but also in up to 70% of GD patients. Hypothyroidism due to chronic autoimmune thyroiditis develops eventually in up to 20% of patients with Graves’hyperthyroidism who have entered remission after a course of antithyroid drugs; blocking TSHRAbs contributes to the hypothyroidism in one third of cases, and chronic autoimmune thyroiditis in the remaining two thirds[3].The thyroid gland can produce, in addition to hormones, a variety of immunologically active factors such as adhesion molecules, growth factors, and inflammatory mediators (nitric oxide and prostaglandins), as well as cytokines. In addition to playing critical roles in growth and differentiation of target cells, these molecules also regulate the susceptibility to many thyroid diseases. Many cytokines have been reported to be synthesized by thyroid cells, including IL-1, IL-6, IL-8, IL-12, IL-13, and IL-15 , among which IL-1 has the ability to stimulate thyroid cell proliferation and inhibit several steps in the synthesis and release of thyroid hormones. It also stimulates the thyroidal production of other cytokines such as IL-6 and IL-8, disturbs the thyroid epithelial barrier, and influences the function of the thyroid cells. In fact, IL-1 downregulates the expression of thyroid-specific proteins such as Tg and TPO, inhibits iodide organification and the Na+/I- symporter(NIS), and reduces the delivery of thyroid hormone to the circulation. These inhibitory effects are demonstrated both in apparently normal human thyrocytes adjacent to adenomas or cancers, and in thyrotoxic cells. The IL-1 family consists of IL-lα, IL-1β, and IL-1 receptor antagonist (IL-IRα). IL-1β, which is produced primarily by monocytes and macrophages [15], features a similar structure and biological activity to IL-1α and is the predominant species of the human IL-1 class.To date, the physiopathological and diagnostic role of IL-1β in AITD has not been clearly established. In the present study, we examined the serum IL-1β level in HT and GD patients and in normal controls and found no significant difference between them. However, the IL-1β mRNA and protein levels in peripheral blood mononuclear cells (PBMC) were significantly higher in HT than in GD or in normal controls. Moreover, the IL-1β mRNA level in HT thyroid gland tissue was also higher than that in GD patients, which probably correlated with increased local infiltration of monocytes into the thyroid tissues of HT patients. Correlation analysis of the clinical samples validated the association of high IL-1β levels with HT pathogenesis. These results indicate that IL-1β may be an active etiologic factor in HT pathogenesis and could be a promising new diagnostic/treatment target.Materials and methods:PatientsTwelve female patients newly diagnosed with HT were enrolled in this study between June 2013 and November 2014 from the First Affiliated Hospital of Anhui Medical University. These patients ranged from 20 to 52 years of age (average age 37.58 ± 9.29). The diagnosis of HT was based on commonly accepted clinical and laboratory criteria:positivity for anti-thyroglobulin and anti-peroxidase (ATG and TPOAb, respectively) at high titers; negativity for TSHRAbs; thyroid ultrasound imaging suggestive of a chronic thyroiditis; the existence of a firm goiter, thyroid dysfunction. Twelve female GD patients ranging from 25 to 51 years of age (average age 37.92 ± 9.89) were also enrolled in this study, while twenty female volunteers were studied as the normal control group, ranging from 18 to 50 years of age (average age 34.35 ± 9.32).Blood samples and thyroid tissue specimensPeripheral blood samples were collected from patients and volunteers. The blood samples were centrifuged at 200 × g,4℃ for 5 min and the supernatant was collected and stored at -80℃ for IL-1β assay. PBMC were separated by standard Ficoll-Hypaque density centrifugation and stored at -80℃ with Trizol for extracting total RNA. The specimens of normal thyroid tissue adjacent to thyroid adenoma, and thyroid tissue samples from HT and GD patients were stored in liquid nitrogen.Immunohis to chemistryParaffin-embedded thyroid tissues were cut into 5-μm-thick sections and deparaffinized in xylene. Antigen retrieval was performed on the cleared slides by microwaving for 30 min, after which non-specific binding was blocked by incubating with diluted normal serum, then endogenous peroxidase activity was quenched with 0.3% H2O2 in methanol. Target epitopes were detected by incubating with 1:100 anti-CD14(Abcam, Cambridge, MA, USA) or 1:150 anti-CD64 (Abcam) overnight.next day the slides were incubated with the corresponding Horseradish Peroxidase(HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by color development with 3,3’-diaminobenzidine tetrahydrochloride (Life Technologies, Carlsbad, CA,USA). Sections were counterstained with hematoxylin, then cleared and mounted.Design of primers and response conditionsUsing Genbank sequences, the primers were designed by Primer Premier 5.0 software and synthesized by Shanghai Sangon Biological Engineering Technology & Service Company (Shanghai, China). All primer sequences are listed in Table 1.RNA extraction and cDNA synthesisTotal RNA was extracted from PBMC and thyroid tissues using Trizol (Ambion, Carlsbad, CA, USA) following the manufacturer’s instructions. cDNA was synthesized using reverse transcription kits (Invitrogen, Life Technologies, Carlsbad, CA, USA). All RNA samples were heated at 65℃ for 10 min to denature the secondary structure with the template then cooled immediately on ice for 5 min. Total RNA was reverse transcribed as previously described[17].Real-time PCR analysisTotal RNA was extracted from PBMC or thyroid tissues using Trizol. cDNA was synthesized from 1μg total RNA, and the mRNA expression level was determined using Applied Biosystems StepOnePlus Real-Time PCR System (Life Technologies) with the appropriate primers using the iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, Hemel Hempstead, UK) according to the manufacturer’s instructions. The cycling parameters were 95℃ for 10 s followed by 40 cycles of 95℃ for 5 s and 60℃ for 30 s. Gene expression was analyzed by relative quantification with the 2-△△Ct method. The target gene levels were normalized to β-actin, and the results are expressed as fold changes in threshold cycle(Ct) values relative to controls.Quantiglo ELISA for serum IL-1βSerum levels of IL-1β were measured by Quantiglo ELISA, following the manufacturer’s protocols (R&D Systems, Catalog Number QLBOOB, USA). All samples were measured in triplicate.Chemiluminescence for serum T3, T4, TSH, ATG and TPOAbSerum levels of T3,T4,TSH,ATG and TPOAb were measured using a Chemiluminescence, following the manufacturer’s protocol (SIEMENS ADVIA Centaur XP Immunoassay System, USA). All samples were measured in triplicate.Radioimmunoasssay for serum TSHRAbSerum levels of TSHRAb were measured using a Radioimmunoasssay, following the manufacturer’s protocol (Union Medical Science & Technology Co., Ltd., Tianjin,China). All samples were measured in triplicate.Flow cytometric analysisA BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (BD Biosciences, Oxford, UK) was used for cell staining. Following the manufacturer’s instructions, the cells were stained with anti-CD64-fluorescein isothiocyanate (FITC) (BD Biosciences) and intracellular staining was performed with anti-IL-1β-phycoerythrin (PE). Cells were analyzed in a Beckman FC500 flow cytometer (Cytomics FC500, Beckman Coulter, Brea, CA, USA), followed by data analysis using Beckman FC500CXP software. Statistical analysis.All statistical analyses were performed using SPSS17.0 (SPSS, Chicago, IL, USA). Data are expressed as the mean ± standard deviation (S.D.) in text and figures. Comparisons between paired or unpaired groups were made using the appropriate Student’s t-test. For non-parametric data, differences between two groups were analyzed by the Mann-Whitney test. Pearson correlation analysis was used to test the correlation between two continuous variables. P<0.05 was considered statistically significant.ResultsIL-1β is more highly expressed in PBMCs in HT than in GD or normal controlsAs previously reported, multiple proinflammatory cytokines have been suggested to play roles in the pathogenesis of AITD. We therefore examined the mRNA level of a series of Thl/Th2 cytokines and IL-1β in monocytes from PBMCs of AITD patients (HT; n=12, and GD; n=12) and normal controls (n=20),which included IL-4, INF-γ, TNF-α, IL-13 and IL-1β by qRT-PCR assay. The results showed no significant differences in the levels of IL-4, INF-y, TNF-a or IL-13 among these three groups, but an approximately 3-fold higher mRNA level of IL-1β in the PBMC of HT patients than GD patients. IL-1β level in the PBMC of GD patients was 7-fold that of samples from control subjects,which means the IL-1β mRNA level in the PBMCs of patients with both AITD diseases was higher than the normal controls.Furthermore, we verified the protein levels of IL-1β in PBMC of GD and HT patients as well as normal controls. PBMC were double-stained with CD64 and IL-1β antibodies and subjected to flow cytometric analysis. CD64-positive cells (the two right quadrants) were characterized as monocytes, and the proportion of IL-1β/CD64 double-positive cells (upper-right quadrant) within the CD64 positive cells was calculated for each sample. Thus there were 67.17-98.49% IL-1β positive cells among the PBMC of HT patients (n=9), much more than the 23.19-51.35% of GD patients (n=7) or the 5.22-18.42% of normal controls (n=8)(Fig.1C). Consistent with the previous qPCR data in PBMCs, these results showed that both mRNA and protein levels of IL-1β were significantly higher in the PBMCs of HT patients than in GD patients, while the level of IL-1β was lowest in PBMCs of normal controls.Serum IL-1β levels did not significantly differ among HT, GD and normal controls We further analyzed IL-1β protein levels in the serum of HT (n12) and GD (n=12) patients and in normal controls (n=20) by Quantiglo ELISA. In contrast to our data from PBMC, serum IL-1β levels from HT and GD patients and from normal controls were 0.47360 ± 0.21313 pg/mL,0.46241 ± 0.05170 pg/mL and 0.52672 ± 0.13024 pg/mL, respectively, which showed no significant difference among these three groups.IL-1β is significantly more highly expressed in HT thyroid tissue samples than in those from GD patients or normal controlsAs well as the levels in PBMCs, the expression level of IL-1β in thyroid tissues of HT and GD patients is of even greater concern and was further analyzed by qRT-PCR. In agreement with the data from PBMCs, our results showed that IL-1β was expressed in the thyroid tissues of GD patients at about half the level observed in HT thyroid tissues, while there was no significant difference in expression between normal controls and GD patients.This result is consistent with the data of Giordano et al. who identified a high level of IL-1β expression in HT thyroid tissue samples using an Immunohistochemistry(IHC) assay.In addition, we examined the infiltration of monocytes into thyroid tissues by IHC assay. Monocytes that are the main producer of IL-1β were immunostained with mAbs against CD64 or CD14. Sections from HT thyroid tissue samples showed markedly more positive stained cell infiltration than samples from GD patients or normal controls.IL-1β correlates with clinical features of HT and GDTo determine the potential role of different level of IL-1β in AITD patients,12 female patients with GD and 12 female patients with HT, as well as 20 normal female controls were recruited. There was no significant difference in the age distribution among the three groups. The significant differences in the levels of serum T3, T4, TSH, ATG and TPOAb among the groups (for each index mentioned,P<0.05)correlated and validated the diagnosis.These data demonstrated the difference of thyroid function and titer of ATG and TPOAb among these groups. When each group was analyzed separately, no association was found between either serum IL-1β level or PBMC IL-1β mRNA level with these serum markers. However, when the cases of all 3 groups were collected and analyzed together, PBMC IL-1β mRNA level was demonstrated to be positively associated with serum ATG (r=0.711,P<0.001) and TPOAb (r=0.713,P<0.001). In summary, our data indicate IL-1β level in PBMC positively correlates with ATG and TOPAb in the serum.ConclusionOur study suggests that IL-1β contributes to the Pathogenesis of autoimmune thyroid diseases,especially of Hashimoto Thyroiditis. |
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