Serum IL-27 levels increase in subjects with hypothyroidism and are negatively correlated with the occurrence of nonalcoholic fatty liver disease

Background The level of serum interleukin-27 (IL-27) was significantly decreased in the obesity group. After injection of IL-27, obese mice showed significant weight loss,reduced fat accumulation, improved insulin resistance and hepatic steatosis.IL-27 plays a key role in the regulation of metabolic processes, but there are scarce data on circulating IL-27 levels in hypothyroidism. The purpose of this study was to assess the serum levels of IL-27 in patients with hypothyroidism and its relationship with NAFLD. Methods 185 participants were included in this cross-sectional survey. According to thyroid function, the subjects were classified into three groups: euthyroidism (n = 55), subclinical hypothyroidism (n = 53), and hypothyroidism (n = 77). Serum IL-27 concentrations were measured by ELISA. Results Serum IL27 levels were significantly higher in subclinical hypothyroidism and hypothyroidism groups than in the euthyroidism group. Serum IL27 levels had a negative correlation with HOMA-IR,FBG,TG, subcutaneous fat,and visceral fat, and had a positive correlation with HDL-C (P< 0.05). Furthermore, logistic regression analysis indicated that IL-27 levels, HOMA-IR, and visceral fat showed significant associations with NAFLD after complete adjustment (P< 0.05). ROC curves showed that theoptimal cut-off value of serum IL-27 for discriminating NAFLD was 95.87pg/mL. The area under the ROC curve was 77.3% (95% CI = 0.694-0.851, p < 0.001). Conclusions Serum IL-27 levels demonstrated a compensatory increase in patients with subclinical hypothyroidism or hypothyroidism and showed an independent association with NAFLD. Circulating IL-27 levels could predict the occurrence of NAFLD in hypothyroidism. These results suggested that altering the circulating levels of IL-27 may be a potential therapeutic target for NAFLD.


Introduction
Hypothyroidism has an association with an increased risk of developing metabolic syndromecomponents, including obesity, insulin resistance and nonalcoholic fatty liver disease, which is widely prevalent in the common population (1). Clinical and subclinical hypothyroidism is an independent risk factor for NAFLD (2). The underlying mechanisms between NAFLD and hypothyroidism include oxidative stress, insulin resistance, dyslipidemia,metabolic syndrome, and direct action of TSH on hepatocytes (2, 3). Thyroid hormones play a key role in hepatic lipid metabolism, stimulating hepatic lipogenesis and causing hepatic fat accumulation through the thyroid hormone b receptor (4). Certain adipocytokines, such as interleukin-1, tumor necrosis factor-a (TNF-a), visfatin and leptin, and increased oxidative stress that occurs in some cases of hypothyroidism, may coincide with the development of insulin resistance (5). However,the biological mechanisms underlying the development and progression of NAFLD in patients with hypothyroidism are not fully understood (6).
NAFLD is a continuous process from simple steatosis to NASH and eventually to cirrhosis, where there is no effective treatment other than lifestyle modification and regular physical activity (7,8).
In overweight/obese NAFLD, a 7-10% weight loss is the target of most lifestyle interventions, and results in improvement of liver enzymes and histology. Dietary recommendations should consider energy restriction and exclusion of NAFLD-promoting components, macronutrient composition should be adjusted according to the Mediterranean diet, Both aerobic exercise and resistance training effectively reduce liver fat (9). Therefore, early identification of patients at a high risk of NAFLD is extremely important.Liver biopsy is the gold standard for the diagnosis of NADLD, but it has not been widely accepted due to its aggressiveness and high cost (10). At present,non invasive testing is used as a risk assessment tool for steatosis and fibrosis,clinicians mainly use ultrasound to detect fatty liver. Nevertheless, it depends on the experience and skill level of the operator (11). In addition, magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) accurately determine the content of liver fat and the degree of fibrosis, but they have not been widely used in clinical practice due to the high cost (12). Therefore,noninvasive methods are urgently needed to identify NAFLD, monitor treatment effects, andtrack disease processes (13).
As a member of the IL-6/IL-12 cytokine family, IL-27 consists of two subunits, EBI3 and P28 (14,15). IL-6 production has an association with decreased energy expenditure and increased body fat mass (16). The IL-27-IL-27Ra signaling pathway plays a key role in enhancing insulin resistance, promoting thermogenesis, and combating diet-induced obesity, which is a promising target for anti-obesity immunotherapy (17). IL-27 may be a novel target for the clinical treatment of metabolic diseases in the future. Currently, there are few inconsistent data on circulating IL-27 levels in patients with hypothyroidism. Hence, the purpose of this study was to assess the serum levels of IL-27 in patients with hypothyroidism and its relationship with NAFLD. Furthermore, the diagnostic accuracy of IL-27 was measured in the detection of NAFLD in hypothyroidism.

Study group
A total of 185 patients (20-70 years) were included in this crosssectional study. 77 patients were newly or previously diagnosed with clinical hypothyroidism, and 53 patients were newly or previously diagnosed with subclinical hypothyroidism, but had not received thyroid hormone replacement therapy within 3 months. 55 patients had normal thyroid functions. Exclusion criteria were: acute or chronic virus hepatitis, hepatolenticular degeneration, total parenteral nutrition, drug-induced liver disease, previous chronic heavy drinking, liver or kidney disease, heart failure,myocardial infarction breastfeeding or pregnancy, chronic inflammation, acute infection, and other specific diseases that cause fatty liver. Participants were recruited from the Endocrine Clinic at Beijing Chaoyang Hospital, Affiliated to Capital Medical University. All patients signed the informed consent, andthis study was approved by the Ethics Committee (2019-science-363) of Beijing Chaoyang Hospital, Capital Medical University.

Statistical analysis
Data were analyzed with SPSS 26.0(IBM Corporation, New York) and GraphPad Prism9.0(Inc,CA,USA). The normal distribution of data was represented by the mean ± standard. Nonnormally distributed data were represented bythe median and interquartile range. One-way ANOVA or Kruskal-Wallis test and Bonferroni post hoc test were used for comparison between groups. In addition, covariance (ANCOVA) and Bonferroni post hoc tests were used to compare the potential confounders of the adjusted differences between groups. The association between serum IL-27 levels and NAFLD was investigated using Pearson analysis. To further explore the association between serum IL-27 levels and dyslipidemia, linear regression was conducted.ROC curves were used to define the cut-off value of IL-27. The significance was indicated as P < 0.05, and all tests were two-tailed.

Characteristics of study groups
The clinical characteristics of all the participants are shown in Table 1. The subjects were classified into three groups. Among the three groups, sex, age, ALT, AST, GGT, TG, LDL-C, HOMA-IR, fasting glucose, and subcutaneous fat showed no significant differences. Compared with the euthyroidism group,patients with hypothyroidism tended to have higher levels of BMI, TC, and visceral fat (P <0.05). The cytokine level of IL-27showed a higher serum concentration in patients with hypothyroidism compared to the euthyroidism group at 119.13(16.78,131.35) versus 8.94 (4.65,32.07) pg/mL. Serum IL-27 levels were higher in the subclinical hypothyroidismgroup than in the euthyroidism group. There was no statistically significant difference in IL-27 between hypothyroidism and subclinical hypothyroidism patients ( Figure 1).

Independent relationship between serum IL-27 levels and NAFLD in hypothyroidism
According to the diagnosis of NAFLD, patients with hypothyroidism or subclinical hypothyroidism were classified into two groups.There was no significant difference in TC, LDL-C, sTSH, FT3, and FT4 between the two groups. Patients with NAFLD had higher age, BMI, AST, ALT, GGT, TG, FBG, HOMA-IR, subcutaneous fat, visceral fat and lower HDL-C,IL-27 compared to those without NAFLD (Table 2). To identify whether serum IL-27 levels had an independent correlation with NAFLD,    (Figure 3). The optimal cut-off value of serum IL-27 for discrimination of NAFLD was 95.87pg/mL (AUC = 0.773, P < 0.001).

Discussion
In this study, a compensatory increase in the concentration of serum IL-27 was observed in patients with hypothyroidism or subclinical hypothyroidism. Serum IL-27 levels were negatively associated with FBG, HOMAIR, subcutaneous fat, visceral fat, and TG, and positively associated with HDL-C in patients with hypothyroidism. More notably, lower circulating IL-27 levels in patients with hypothyroidism were independently related to NAFLD. These results suggested that IL-27 could be a promising therapeutic target for NAFLD in patients with hypothyroidism.
Hypothyroidism is a common endocrine disorder characterized by increased sensitivity to cold and unexpected weight gain, suggesting a change in response to cold and energy expenditure (20). Thyroid hormones play an important role in the normal function and coldinduced thermogenesis of brown adipose tissues in rodents (21,22). Wang et al. reported that IL-27Ra-deficient mice were cold-intolerant because of impaired adaptive thermogenesis, and IL-27 improved thermogenesis and directly targeted adipocytes to counteract obesity (17). Other studies have shown that IL-27 exerts its beneficial effects by the upregulation of adaptive thermogenesis in brown adipose tissues (23). Consistent with their findings, a compensatory increase in the concentration of serum IL-27 was observed in patients with hypothyroidism or subclinical hypothyroidism. IL-27 may combat hypothermia in patients with hypothyroidism through its febrile effects.  Comparison of IL-27 levels among the three groups.
NAFLD is a worldwide health problem and has an increased risk of diabetes, CVD and CKD (24). NAFLD is a complex and heterogeneous disease that is inaccurately diagnosed by liver biopsy (25). Current therapeutic strategies mainly focus on lifestyle interventions, and there are still limited appropriate drugs specifically available for NAFLD (26). Therefore, the discovery of non-invasive detection, novel therapeutic targets and strategies shall be required. Patients with hypothyroidism are at an increased risk of NAFLD. Hypothyroidism plays an important role in the development and progression of NAFLD (27).
Dyslipidemia related to hypothyroidism leads to intrahepatic fat accumulation, resulting in NAFLD and thus leading to the development of hepatic insulin resistance (1). Thyroid hormones increase the expression of HMG-CoA reductase in the liver to increase cholesterol synthesis (28). Plasma cholesteryl ester transfer proteins (CETPs) are decreased in a hypothyroid state, which shift cholesterol from HDL-C to LDL-C and VLDL (29). Consistently, data showed that NAFLD patients with hypothyroidism had higher BMI, LDL-C, FBG, HOMA-IR, subcutaneous fat, visceral fat, and lower HLD-C than non-NAFLD patients. It was found that IL-27 exerted a protective effect against diabetes by ameliorating STZinduced hyperglycemia and islet inflammation. The hypertrophy of chronic inflammation and white adipocytes in white adipose tissues was blocked and HFD-induced liver steatosis was suppressed by Il-27 gene transfer (23). This study showed that serum IL-27 levels were negatively associated with FBG, HOMAIR, subcutaneous fat, visceral fat, and TG, and positively associated with HDL-C. Therefore, the findings support previous observations that IL-27 could improve insulin resistance and reduce fat accumulation. Meanwhile, data suggested that IL-27 could be involved in lipid metabolism. Thus, it was hypothesized that IL-27 may improve fatty liver by improving insulin resistance and reducing blood lipids. Besides, NAFLD patients with hypothyroidism had lower IL-27 compared to those without NAFLD. It can be speculated that the increase of IL-27 content may improve NAFLD. IL-27 levels may be a potential therapeutic target for dyslipidemia and NAFLD. The role of circulating IL-27 in improving NAFLD in hypothyroidism needs further investigation.
Multiple studies have demonstrated variables for predicting NAFLD (30). The noninvasive screening mode facilitates the timely identification and intervention of NAFLD and its complications in high-risk patients (31). Baseline HOMA-IR and weight gain were used as predictors of NAFLD incidence in a 7-year prospective study (32). Logistic regression analysis displayed that circulating IL-27 levels,HOMA-IR, and visceral fat were independently related to an increased risk of NAFLD. It was demonstrated that IL-27 could also predict the occurrence of NAFLD. In this study,IL-27 could predict NAFLD with an AUC of 0.897 (95% CI 0.757-0.859). In conclusion, the exact mechanism by which IL-27 regulates NAFLD metabolism needs to be further studied.
This study had a number of limitations.First, the sample size was small and the cross-sectional study failed to construct a causal relationship between IL-27 and disease. Second, other potential confounders were not eliminated, particularly cold exposure and exercise. Finally, only the Chinese population was included in this study, thus making the generalizability of the findings an issue.
In summary, serum IL-27 levels had a compensatory increase in patients with hypothyroidism or subclinical hypothyroidism and had an independent association with NAFLD. This study manifests that altering circulating IL-27 levels could predict the occurrence of NAFLD in hypothyroidism.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Ethics statement
The studies involving human participants were reviewed and approved by Ethics Committee of Beijing Chaoyang Hospital, Capital Medical University. The patients/participants provided their written informed consent to participate in this study.  Receiver operative characteristics curves and cut off values of serum IL-27 levels for NAFLD.