Lower Serum Dipeptidyl peptidase-Ⅳ Level is Associated With 3 Types of Autoimmune Thyroid Diseases


 Backgroud:

Autoimmune thyroid diseases (AITD) are the most common organ specific autoimmune disorders. The reduction of serum dipeptidyl peptidase-IV (sDPPIV) levels have been reported in patients with autoimmune diseases. Few studies have analyzed the association between sDPPIV levels and AITD, especially in Graves’ disease (GD), Graves’ ophthalmopathy (GO) patients. So the aim of this study was to evaluate the association between sDPP-IV levels and 3 types of AITD, that is Graves’ disease (GD), Graves’ ophthalmopathy (GO), Hashimoto’s thyroiditis (HT).
Methods

65 newly diagnosed GD ,22 GO, 27 HT patients and 30 healthy individuals were recruited for this study. Clinical characteristics and thyroid function data were collected for all participants. sDPP-IV was measured by enzyme-linked immunosorbent assay.
Results

Compared with the controls, GD patients and GO patients had significantly lower sDPP-IV levels(662.2 ± 38.81 and 438.4 ± 31.78 vs.786.3 ± 46.95, P = 0.01 or P < 0.001). It was also found that in GO individuals, sDPP-IV was lower than in GD subjects(P = 0.002). The lower the sDPP-IV level is, the higher the risk for developing GD or GD will be. In addition, sDPP-IV levels have negative association with the antithyroid peroxidase antibody(TGab)(r =-0.20, p = 0.02) and antithyroglobulin antibody(TPOab)(r =-0.19, p = 0.03). But there was no significant relationship between thyroid hormone and sDPP-IV levels.GO parients were groups by proptosis with and without muscle thicken,the sDPP-IV levels in proptosis with muscle thicken were lower than proptosis without muscle thicken(P < 0.05).Logistic regression analysis showed that sDPP4 were negatively correlated with GO and GD.
Conclusions

Take together, the present study showed for the first time that sDPP-IV concentrations are aberrant in GD and GO patients and that the reduced sDPP-IV expression may be involved in the progression of GO and GD diseases.


Introduction
Autoimmune thyroid disease (AITD) are the most common organ speci c autoimmune disorder [1,2]. Graves' disease (GD) and Hashimoto's thyroiditis (HT) are the 2 main clinical presentations of AITD and are both characterized by lymphocytic in ltration of the thyroid parenchyma. Graves' ophthalmopathy Page 3/14 (GO) belong to the special subtype of GD which accounts for 30-50% [3,4]. GO has the clinical characteristics of high incidence, lesions involving multiple tissues of the eye, and symptoms and signs are relatively complex. The pathogenesis of GO and GD disease share the same pathogenesis basis.
Abnormal immune regulation plays an important role. However, the pathogenesis mechanism is not fully understood. Dipeptidyl peptidase-IV (DPP-IV) is a type transmembrane glycoprotein that having serine protease activity ,and which selectively cleaves an N terminal dipeptide from peptides with a proline or alanine residue in the penultimate position. DPP-IV is expressed on the surface of epithelial cells in various tissues (liver, kidney, intestine, etc.), and also in endothelial cells, broblasts, and lymphocytes [5].
When expressed on the surface of lymphocytes, it is called CD26 and is involved in the maintenance of lymphocyte composition and function, activation and co-stimulation of T lymphocytes, also Involved in activation of B lymphocytes and cytotoxicity of NK cells [6,7]. Therefore, relevant studies have explored the multiple roles of DPP-IV in metabolism, immunity, endocrine and tumor biology, including diabetes, HT[8],rheumatoid arthritis [9], multiple sclerosis [10], in ammatory bowel disease and thyroid cancer [11].
Up to now, the leaves of DPP-IV in GD and GO have remained unknown. Therefore, the purpose of the current study was to evaluate DPP-IV levels in patients with GD, GO and HT and to investigate the role of women with pregnancy or lactation period; (7) complicated with malignant tumors; (8) Patients with mental illness are receiving radiation, chemotherapy, antidepressant or immunosuppressive therapy; (9) abnormal liver function, with transaminase level 2 times higher than the upper normal limit; (10)Hyperthyroidism crisis or combined with myasthenia gravis. According to Werner standard, Graves' ophthalmopathy are classi ed as none (0-1) or presence (2-6) [12].The healthy controls were negative for thyroid antibodies, and they had no relevant medical history and no family history of thyroid diseases.
None of the subjects had any infectious diseases or other autoimmune diseases, including human immunode ciency virus (HIV), hepatitis B virus (HBV), T1DM, MS, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The patients of Hashimoto's thyroiditis (HT) inclusion criteria were as follows: increased TPOAb and/or TGAb, diffuse lesion in thyroid via ultrasound, normal thyroid function. The study was approved by The Luhe Hospital Ethics Committee and all participants provided signed written consent.

Sample Collection
Five-milliliter whole-blood samples were collected in EDTA vacutainers on empty stomach at early morning from patients and healthy controls. After centrifugation, blood sample stored at − 80°C until use.
sDPP-IV Expression sDPP-IV levels were measured using a human DPP-IV ELISA kit (RD, Systems, USA). The experiments were quanti ed in complied with the manufacture's instruction. The intra-assay and the interassay coe cient of variation was 5.8% and 8.6% respectively.

Statistical Analysis
All data were analyzed using the SPSS Statistics 20.0 software package (IBM, New York, NY, USA). Quantitative data were presented as the mean ± standard deviations (for normally distributed data) or as medians and quartiles (for non-normally distributed data), as appropriate. Between-group differences in quantitative parameters were assessed by Student's t-test in cases in which the data were normally distributed; otherwise, these differences were assed with the Mann-Whitney U test. Correlations were analyzed using Spearman's rank test. A P-value less than 0.05 was considered statistically signi cant.

Results
The Expression of sDPPIV in Serum among Different Groups.
To evaluate whether the changes in the sDPPIV re ect disease activity among different autoimmune thyroid diseases. We collected the clinical data from GD, GO, HT and healthy control. As shown in Table 1, there were no signi cant difference in gender, age among different groups. There were increased thyroid hormones and decreased TSH levels in GD and GO patients, whereas there were decreased thyroid hormones and increased TSH levels in HT patients. Then we examined the sDPP4 level among autoimmune thyroid diseases. Results shown that patients with GO patients had signi cantly lower levels of sDPP4 than GD patients (P = 0.002) and healthy controls (p < 0.001),but no signi cant differences were identi ed between HT group and the control(p = 0.24) (Fig. 1). To investigate the relationship between sDPPIV and other variables for all participants, we performed a Spearman correlation analysis. sDPPIV was negatively correlated with TPOAb (r =-0.19, p = 0.03) and TgAb (r =-0.20, p = 0.02). But there were no any correlations between sDPPIV and other variables( Table 2).

Relationship between sDPPIV and severity of GO
In order to explore the relationship between sDPPIV and the severity of GO,sDPPIV patients in different subgroups were compared between the control and GO group.GO parients were groups by proptosis with and without muscle thicken,the sDPP4 levels in proptosis with muscle thicken were lower than proptosis without muscle thicken(P < 0.05) (Fig. 3).
Correlations among sDPPIV and GO,GD.

Discussion
In the current study, we demonstrated that GD and GO patients had signi cantly lower sDPPIV compared with the controls and importantly the sDPPIV levels in GO were lower than in GD. These ndings provide an insight into clinical implication of sDPPIV in GD or GO patients and is de ned as early predictive biomarker in thyroid autoimmune disease.
Constantly growing literature data concerning DPPIV can be found not only involved in enzymatic activity that can cleaved and inactivated many regulatory peptides such as glucagon-like peptide-1(GLP-1),brain natriuretic peptide(BNP), glucose homeostasis, cancer progression [13], but also immunological functions [14].There are mounting evidences that demonstrate the role of DPPIV as a protein playing an important role in the development ,maturation, activation and differentiation of T-cells and regulating immune system [15,16]. So far DPPIV was widely studied in other immune disease like type 1 diabetes and multiple sclerosis [17,18]. A large body of evidence showed that DPPIV is also identi ed as a predictive biomarker in the other autoimmunity diseases.
To our knowledge, this is the rst study to report changes of sDPPIV levels in different autoimmune thyroid diseases. What the undering mechanism of the association between DPPIV and AITD? Immunoregulation should a more reasonable explanation for this phenomenon. Firstly, previous studies have con rmed that CD26 knock out mice can increase severity of the disease and enhanced type 1 cytokine production, suggesting that CD26 acts as a negative regulatory molecule in autoimmunity [19]. Secondly, extensive literature shows a Th1 immune-preponderance and Th1-chemokines (CXCL 9, CXCL10, CXCL11) and their (C-X-C) R3 receptor play a crucial role in the immunopathogenesis of GD and GO [20,21].Previous study had shown increased CXCL 10 levels were observed in GD and GO [22,23]. since DPPIV as a result of its N-terminal X-Pro cleaving activity regulates chemotactic responses to the in ammatory chemokines CCL3-5, 11 and 22, CXCL2, 9-12 [24], which to some extent explain lower sDPPIV were associated in patients of GD and GO. Importantly, we demonstrated that sDPPIV is lower in GO patients than in GD subjects, indicating a progressive increase of in ammatory state from GD to GO.
In subgroups analysis of GO, sDPPIV levels negative correlate with the progress of GO. Our study also did not show reduction of DPPIV levels in HT patients, which is consistent with Yalei Liu and colleagues [25].
There are several limitations in our current study. First, as we all known, DPPIV was expressed both as a soluble form in body uids such as serum, but also as a cell surface glycoproteins of various cell type including immune cells, so in future should be also evaluated membrane-bound CD26 levels on immune cells. Second, the sample size was relatively small and consisted entirely of Chinese people, which may have hampered the generalization of our ndings. Although there are some limitations, it seems likely that DPPIV may have a pathophysiological role in patients with GD and GO. Further detailed studies are still needed to better elucidate the underlying molecular mechanisms.

Conclusions
In conclusion, Our study showed for the rst time that sPPIV levels are signi cantly decreased in GO and GD patients and reduced sDPP-IV expression may be involved in the progression of GO and GD diseases.

Declarations Data Availability
The data that support the ndings of this study are available on request from the corresponding author Dong Zhao.
Authors' Contributions  sDPP4 levels between proptosis with and without muscle thicken in GO patients