Increased YKL-40 levels are linked with disease severity of initially diagnosed Graves’ disease

ABSTRACT

Background: YKL-40, which is also known as Chitiniase 3-like 1, has been found to be up-regulated in many autoimmune diseases including asthma, systemic sclerosis and systemic lupus, etc. However, the relationship between serum levels of YKL-40 and one another common autoinmmue thyroid disease – Graves’ disease (GD) has not yet been investigated.

Objective: The current study was performed to investigate the correlation of serum YKL-40 levels with disease severity of initially diagnosed GD.

Methods: A total of 142 newly diagnosed active GD and 137 healthy individuals were enrolled in the study. Methimazole was given to 55 GD patients and then 2-month study of follow-up was performed. A commercial ELISA kit was applied for the detection of YKL-40 in serum. Degree of goiter was assessed according to Pérez’s Grade. Receiver operating characteristic (ROC) curve analysis was carried out to detect the diagnostic value of serum YKL-40 with regard to goiter degree. The velocity of the peak systolic blood-flow and the thyroid tissue blood flow (TBF) were examined using Color Flow Doppler ultrasonography (CFDU).

Results: The patients with GD exhibited dramatically higher YKL-40 in serum compared to those of healthy controls (606.1 ± 149.8 pg/mL vs. 397.4 ± 95.1 pg/mL, P < 0.001). Positive associations of YKL-40 with free T3 (FT3) and T4 (FT4), as well as the negative correlation of YKL-40 with TSH in serum, were observed. Additionally, the YKL-40 in serum was dramatically reduced after methimazole intervention, and the correlation of the decline with the reduced FT3 and FT4 was also found (all P < 0.001). Serum YKL-40 levels were positively correlated with goiter degree. ROC curve analysis demonstrated that serum YKL-40 concentration may act as a decent marker for goiter degree. The positive correlations of YKL-40 in serum with the average superior thyroid artery velocity (STV) and thyroid tissue blood flow (TBF) were also observed.

Conclusion: Our findings implicated that YKL-40 may be closely connected to the pathogenesis of GD. Increased YKL-40 levels are linked with disease severity of initially diagnosed GD.

KEYWORDS:

• Graves’ disease
• YKL-40
• disease severity
• color flow Doppler ultrasonography
• hyperthyroidism

1. Introduction

As a dysfunction of thyroid, when excessive thyroid hormone was synthesized and secreted by thyroid gland, hyperthyroidism (HT) is induced [1]. The hypermetabolic state, including enhanced hepatic gluconeogenesis, accelerated lipolysis, excessive expenditure of energy, and loss of weight, was considered as the common feature of HT [2]. Almost ninety percent of the HT was caused or induced by Graves’ disease (GD) [3]. GD is one of the autoimmune thyroid diseases resulted from the production of IgG autoantibodies directed against the thyrotropin receptor [4]. These antibodies bind to and activate the receptor, causing the autonomous production of thyroid hormones (TH), thus inducing thyroid hyperfunction [5].

The inflammatory marker, YKL-40, also called human chitinase-3-like-1 or 40-kDa mammary gland protein (MGP-40), has been confirmed as an inflammatory glycoprotein associated with many inflammatory and autoimmune diseases [6]. YKL-40 is a 40 kDa heparin-and-chitin binding glycoprotein, the name YKL-40 was derived from the three N-terminal amino acids – tyrosine (Y), lysine (K), and leucine (L) - present on the secreted form [7].

In mammals, several types of cells associated with inflammation, including chondrocytes, neutrophils, vascular smooth muscle cells, hepatic stellate cells, and macrophages, could express, synthesize, and release YKL-40 [8,9]. Previous studies have shown that YKL-40 as a potential and decent biomarker of both acute and chronic inflammatory progression that is correlated with multiple statuses of many diseases. For example, in neurodegenerative disease, the association of the enhanced YKL-40 concentration in serum with the other neuronal injury biomarkers, the disruption of synaptic, and the damage of large axonal, was observed by a recent study [10], thus considering it as a potential biomarker for neuroinflammation. In another study, YKL-40 concentration was characterized as a monitorial and diagnostic biomarker of chronic obstructive pulmonary disease (COPD), in that the researchers observed the close connections of serum YKL-40 with the remodeling and inflammation of bronchial in the patients with COPD [11]. Furthermore, elevated concentrations of YKL-40 were also found in the serum of type 1 and type 2 diabetic patients, and closely connected to the risk of cardiovascular diseases development [12]. Moreover, in early disease modifying antirheumatic drug (DMARD)-naive RA and during the process of the intensive treat-to-target therapy, significant association of elevated YKL-40 with the activity of disease was observed [13]. YKL-40 has also been implicated as a potential biomarker in various cancers including esophageal cancer [14], cervical cancer [15], bladder cancer [16], and breast cancer [17], etc. Moreover, YKL-40 has been found to be associated with thyroid cancer.Studies have shown that expression of YKL-40 was upregulated during thyroid cancer progression as well as structural recurrence in patients with differentiated thyroid cancer [18]. Another study conducted by Luo demonstrated that YKL-40 overexpression is associated with metastasis and is an indicator of poor prognosis in papillary thyroid carcinoma [19].

The thyroid-stimulating antibody is a hallmark of GD T helper type 2 (Th2) responses have been associated with the pathogenesis of GD [20]. YKL-40 has been shown to play important roles in the antigen-induced T-helper 2-response, antigen sensitization and IgE induction as well as activation of innate immune cells [21]. The participations of macrophages in the long stage inflammatory reactions and active/severe inflammations in the patients with the GD were observed by recent study, and simultaneously, these inflammatory reactions could significantly induce the fibrosis and tissue remodeling in the orbital tissue of Grave’s ophthalmopathy [22]. Another study demonstrated that YKL-40 drives allergic skin inflammation via Th2 immunity and M2 macrophage activation [23]. A growing number of studies have shown that YKL-40 has been involved in many autoimmune diseases including rheumatoid arthritis, asthma, systemic sclerosis, psoriasis, systemic lupus erythematosus, Behçet disease and inflammatory bowel disease [24], etc.

Based on the previous works above, we supposed that YKL-40 may play important roles in GD progression. However, the association of circulating YKL-40 with GD is not clearly explored. Herein, we compared the concentrations of YKL-40 in serum of GD patients before and after treatment, as well as clarified the relationships of YKL-40 concentration with the risk factors of metabolism and the function of thyroid in GD patients.

2. Patients and methods

2.1. Study participants

From August 2020 to August 2021, newly diagnosed patients with GD (n = 142, male = 50, female = 92) were enrolled in this study (the Department of Endocrinology, Jiangnan University Medical Centre). Based on the symptom, elevated thyroid hormone (TH) concentration, as well as the increased TSH receptor antibody and thyroid-stimulating hormone (TSH) secretion in serum, the state of HT and existence of TRAb of all the enrolled patients were confirmed. All the GD patients did not take any drugs before the study. Patients were excluded if they: having other reasons of hyperthyroidism (toxic nodular goitre,thyroiditis, iodine-induced and drug-induced thyroid dysfunction, etc.), younger than 18 years old, BMI>35 kg/m², diagnosed with cardiovascular disease, type I or II diabetes, tumor, impairment of renal function (creatinine≥120 μmol/L), and hypertension. Among these patients, methimazole medication was given to 55 Graves’ patients for two months. Meanwhile, 137 healthy volunteers who were used as the control group, were also subjected to medical examination routinely. This study protocol was approved by the Ethics Committee of the Jiangnan University Medical Center (Number: 20200023). Before the initiation of this study, a written informed consent was signed by all the participants. The content of the consent form insists of withdrawal, privacy and right, medical care, harm and benefit, study procedures, inclusion and exclusion criteria, and study aim. All procedures were conducted in accordance with the Helsinki Declaration.

2.2. Laboratory examination

All the participants were fasted overnight (12 h). Five milliliter venous blood samples were drawn from the participants at their clinical evaluation. After clotting, sampling tubes were centrifuged at 2000 rpm for 20 min. Serum FT3, FT4, TSH and TRAb were measured electrochemiluminescence immunoassay (Roche Diagnostics). For serum YKL-40 level investigation, after collected of the venous blood sample (5 mL) of each participant with a anticoagulant agent-free tube, a 15-minute centrifugation (3000 r/min) was performed obtain serum, and then the obtained serum was stored at−20°C before assay. Serum YKL-40 levels were investigated by a commercial ELISA kit (Abcam, Cambridge, UK, Cat No.ab255719). The detection limit of this assay was 3.9 pg/mL ranging from 23.44 pg/mL-1500 pg/mL. The precision CVs within-run was 2.4% and between-run was 1.7%.

2.3. Definition of degree distribution of goiter

The degree of goiter was defined according to Pérez [25]. Grade 0: The untouchable or palpable lobe, but smaller than or equal to the distal phalanx of the thumb; Grade 1 : Goiters could be palpable but larger than the distal phalanx of the individual’s thumb being explored, or goiters could be visible only when the neck is fully extended; Grade 2: Visible goiter with neck in normal position; Grade 3: goiter can be seen from 10 meters away.

2.4. Measurement of vascularity by color flow Doppler ultrasonography

All the healthy individuals and patients were subjected a Color flow Doppler ultrasonography (CFDU) measurement (Toshiba, Japan) with a 10-MHz linear transducer. After confirming the location of the superior thyroid artery (STV) on each side of the neck, the angles between the vessel diameter and ultrasound beam, as well as the vessel axis and ultrasound beam, vessel diameter, and the time-averaged blood velocity, were measured. And then all the obtained information were input in the computer to calculate the rate of blood flow in the STV (mL/min). The sagittal section images generated using CFDU of the thyroid gland were applied to evaluate the TBF of thyroid on both sides.

2.5. Statistical analysis

GraphPad 8.0 software was applied to analyze the data and show the results (USA). Data normality was examined by Kolmogorox-Smirnov test, normal distributed data were expressed as mean ± SD. Data that were not normally distributed were expressed as median with interquartile range (IQR). χ2 test was carried out to compare the difference between different categorical variables. The data collected from the patients with or without treatment were compared using Student’s paired t test. The Student’s t test or Mann-Whitney U test was used for examining different indices between and healthy controls. The correlations were obtained by the correlation coefficient of the Spearman or Pearson. The variables, which were independently correlated with the YKL-40 in serum, were obtained by multiple stepwise regression. The P-values less than 0.05 (two-sided) was set as significant difference. Kappa values were generated by setting the observed proportion of agreement expected by chance with regard to the goiter degree evaluation. The Kappa value more than 0.85 was set as significant difference.

3. Results

3.1. Demographic statistics

The clinical characteristics of the studied subjects are shown in Table 1. There were no significantly differences of age, sex distribution and systolic blood pressure (SBP) between patients and controls. GD patients had significantly reduced BMI and higher diastolic blood pressure (DBP) when compared with GD patients. In comparison to the healthy control individuals, the GD patients exhibited dramatically increased TRAb, FT3, and FT4, concentrations and reduced TSH (all P < 0.001) (Table 1).

Table 1. Demographic characteristics of enrolled participants.

Variables	Graves’ disease Patients(n=142)	Healthy Controls(n=137)	P value
Age(Y)	40.4±10.1	41.3±9.8	0.315
Gender(M/F)	50/92	53/84	0.548
BMI(kg/m^2)	21.4±2.6	23.2±2.4	<0.001
SBP(mmHg)	125±12	123±10	0.078
DBP(mmHg)	78±7	72±6	<0.001
FT3(pmol/L)	14.53(9.70–19.19)	4.67(4.00–5.69)	<0.001
FT4(pmol/L)	44.82(30.29–55.12)	16.29(14.27–18.41)	<0.001
TSH(μIU/mL)	0.024(0.005–0.044)	2.541(1.560–3.388)	<0.001
TRAb(IU/mL)	29.29(21.32–65.55)	3.12(0.71–5.69)	<0.001
YKL-40(pg/mL)	606.1±149.8	397.4±95.1	<0.001

3.2. Correlations of YKL-40 in serum with thyroid hormone

In comparison to that of healthy individuals, the concentration of YKL-40 in serum of the GD patients was dramatically elevated (606.1 ± 149.8 pg/mL vs. 397.4 ± 95.1 pg/mL, P < 0.001) (Figure 1A). Next, the relationships of the concentration of YKL-40 in serum with the other parameters of GD were also explored. The positive associations of the concentration of YKL-40 in serum with the concentrations of FT3 (r = 0.440, P < 0.001) (Figure 1B) and FT4 (r = 0.468, P < 0.001) (Figure 1C) in serum, as well as the negative correlation with the concentration of TSH (r = −0.469, P < 0.001)(Figure 1D) in serum were observed. These results were consistent with the analysis after the adjustment for BMI, gender, and age, and the correlation coefficient was 0.375 (P = 0.001), 0.366 (P = 0.001) and −0.381 (P = 0.001), respectively.

Figure 1. A. Comparison of serum YKL-40 levels between GD patients and healthy control B. Correlation of serum YKL-40 levels with serum FT3 levels C. Correlation of serum YKL-40 levels with serum FT4 levels D. Correlation of serum YKL-40 levels with serum TSH levels.

4. Correlations of serum YKL-40 levels with goiter degree

After the evaluation of goiter degree, we obtained the Kappa value (Kappa-value = 0.88). Based on the palpation, 40 patients were grade 0, 47 patients were grade 1,34 were grade 2 and 21 were very grade 3. In comparison to the patients with degree 0 goiter, the patients with degree 1 goiter had dramatically enhanced YKL-40 secretion in serum (580.4 ± 152.1 pg/mL vs 503.3 ± 96.2 pg/mL, P = 0.003). The patients with degree 2 goiter showed significantly elevated YKL-40 levels in serum compared with degree 1 (646.4 ± 138.1 pg/mL vs 580.4 ± 133.0 pg/mL, P = 0.033). Patients with goiter degree 3 had significantly higher serum concentrations of YKL-40 compared with patients with degree 2 goiter (794.1 ± 74.4 pg/mL vs 646.4 ± 138.1 pg/mL, P < 0.001) (Figure 2A). Serum YKL-40 levels were positively correlated with goiter degree (r = 0.616, P < 0.001) (Figure 2B). ROC curve analysis indicated that serum YKL-40 may act as a decent marker with diagnostic value with regard to degree of Goiter at any stage (Figure 3A-C) (0 vs 1: AUC = 0.664, P = 0.008; 1 vs 2: AUC = 0.647, P < 0.025; 2 vs 3 AUC = 0.850, P < 0.001).

Figure 2. A. Comparison of serum YKL-40 levels between among patients with different goiter degrees B. Correlation of serum YKL-40 levels with goiter degree.

Figure 3. A. ROC curve analysis of serum YKL-40 levels with regard to goiter degree 0 v 1 B. ROC curve analysis of serum YKL-40 levels with regard to goiter degree 1 vs 2 C. ROC curve analysis of serum YKL-40 levels with regard to goiter degree 2 vs 3.

5. Correlation of YKL-40 levels with blood flow velocity of the STV

The velocity of peak systolic blood-flow in the STV and the thyroid TBF was measured by CFDU. In comparison to the healthy controls, the GD patients exhibited dramatically increased blood flow in the their STV (left side: 63.6 ± 11.4 mL/min vs 21.2 ± 6.0 mL/min; right side: 64.8 ± 10.3 mL/min vs 20.3 ± 5.8 mL/min; Average: 64.1 ± 10.4 mL/min vs 20.5 ± 5.5 mL/min,all P < 0.001). In addition, TBF in GD patients was also drastically increased in comparison with healthy controls (left side: 6.6 ± 2.0%) vs 1.5 ± 0.4%); right side: 6.8 ± 1.7%) vs 1.4 ± 0.4%); Average: 6.7 ± 1.9%), all P < 0.001). Serum YKL-40 levels were significantly and positively related to average STV (r = 0.401, P < 0.001) (Figure 4A) and TBF (r = 0.405, P < 0.001) (Figure 4B) in GD patients.

Figure 4. A. Correlation of serum YKL-40 levels with average STV in GD patients B. Correlation of serum YKL-40 levels with average TBF in GD patients.

6. Effect of methimazole medication on metabolic parameters and serum YKL-40 concentrations

No serious adverse events were observed after methimazole treatment. The dramatically reduced concentrations of FT3 and FT4 (P < 0.001) from baseline were found, however, obviously elevated TSH concentrations in serum (P < 0.001) were also observed. Serum YKL-40 levels were decreased from 593.1 ± 123.8 pg/mL to 412.5 ± 100.2 pg/ml after methimazole treatment (P < 0.001) (Table 2).

Table 2. Clinical parameters of the GD patients after methimazole administration.

Variables	Before treatment (n=55)	After treatment(n=55)
Age(Y)	40.4±10.1	40.4±10.1
Gender(M/F)	20/35	20/35
Weight	55.3±6.7	57.2±7.4**
BMI(kg/m^2)	21.5±2.3	22.2±2.4
SBP(mmHg)	122±9	120±8
DBP(mmHg)	77±8	75±7
FT3(pmol/L)	14.21(9.88–18.34)	9.65(5.08–13.23)***
FT4(pmol/L)	43.33(31.26–54.10)	25.33(20.27–31.11)***
TSH(μIU/mL)	0.015(0.005–0.029)	0.193(0.099–0.418)***
YKL-40(pg/mL)	593.1±123.8	412.5±100.2***

**P<0.01 ***P<0.001.

7. Discussion

Herein, we investigated the clinical utility of YKL-40 in initially diagnosed GD patients. We have demonstrated for the first time, to our knowledge, that serum YKL-40 concentrations were significantly increased in GD patients compared with the control subjects and were decreased after medication. Our data also showed that serum YKL-40 concentrations positively correlated with FT3, FT4 and negatively related to TSH. Serum YKL-40 levels were positively correlated with goiter degree. ROC curve analysis demonstrated that serum YKL-40 concentration may act as a decent marker for goiter degree. These findings implicated YKL-40 may be involved in the pathogenesis of GD. Increased YKL-40 levels are linked with disease severity of initially diagnosed GD.

The etiology of GD remains not completely clear, involving genes, diet, living habits, emotions, environment and other factors. At present, the mechanism is recognized to be partially related to autoimmunity, resulting in excessive thyroxine synthesis through immune pathway, leading to a series of hypermetabolic syndrome, such as weight loss, overeating, insomnia, emotional excitement, hand tremor, etc.

Circulation TRAb is the hallmark of GD. By binding to the TSH receptor, TRAb enhances the production of cAMP in thyrocytes, thereby promoting the release of TSH and the growth of thyrocytes, and eventually causing GD [26]. Therefore, the production of TRAb plays the key role in the onset and progression of GD. Dendritic cells (DCs) present antigens to CD4+ T cells which then stimulate the persistent activation and expansion of B cells to produce TRAb [27]. When subjected to invading pathogen and foreign antigen, immature DCs (iDCs) which are widely distributed among peripheral tissues can be activated. One study showed that YKL-40 is expressed during the process of differentiation and maturation of dendritic cells in time dependent manner [28]. In addition, YKL-40 is evenly distributed in cytoplasm and in the nucleus of both the immuture DCs and muture DCs [29]. These findings indicate that YKL-40 may play crucial role in the DCs immunoresponse. Hence, YKL-40 may participate in the onset and progress of GD by stimulating DCs cells to promote the production of TRAb. Serum YKL-40 levels were also positively associated with blood flow defined by average STV and TBF. We also detected that TBF and STV were strongly associated with serum FT3 and serum FT4 (data not shown), indicating TBF and STV could reflect disease severity of GD. One previous study by Cheng expression of YKL-40 was upregulated during thyroid cancer progression as well as structural recurrence in patients with differentiated thyroid cancer [18]. This finding suggests YKL-40 may also participate the thyroid tumor related HT.

There are several limitations that should be noted. First, this is a cross-sectional observational and simple non-controlled study, thus some bias might be caused. Second, due to the relatively small size of the samples, the statistical power of our study may be weak. Multicenter studies with larger samples are needed in the future. Thirdly, we only tested YKL-40 in the study, investigation of other inflammatory markers may provide much more valuable information. To better clarify the mechanism underlying the association of YKL-40 in the pathogenesis of GD, further studies should be designed and done.

8. Conclusions

Collectively, this was the first time to identify YKL-40 as a potential marker in GD. We also found that serum YKL-40 concentrations are increased in patients with GD and decreased after methimazole treatment. Our observations demonstrated the involvement of YKL-40 in the progression of GD. Increased YKL-40 levels are linked with disease severity of initially diagnosed GD.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.