Correlation between Interleukin-23, Autoantibodies and Thyroid Profile in a Sample of Iraqi Patients with Hashimoto’s Thyroiditis

Hashimoto’s thyroiditis (HT) is the most common autoimmune condition characterized by hypothyroidism and thyroid cell death by leukocytes and antibody-mediated immunological mechanisms. The current paper set out to assess a number of inflammatory and metabolic potential indicators of Hashimoto’s thyroiditis. Fifty-one patients with Hashimoto’s thyroiditis took part in the current investigation. Ages ranged from 20 to 75 for them. Enzyme-linked immunosorbent tests were used to quantify the anti-thyroperoxidase antibody (anti-TPO Ab), anti-thyroglobulin antibody (anti-Tg Ab), T4, T3, and TSH at the beginning and conclusion of the trial. The result showed a significant relationship between the gender according to HT patient’s and healthy groups which showed that female effected more than male and there was no effected according to the age between these groups, In HT patients with greater TSH concentrations compared to healthy individuals, serum FT3 and FT4 concentrations were lower while IL-23 concentrations were higher. TSH levels were positively correlated with IL-23 levels. As a result, IL-23 can be useful for detecting disease activity in HT patients. However, more thorough, extensive, longitudinal investigations are required to evaluate whether IL-23 can be used in therapeutic settings. Our results showed that thyroid profile, serum IL-23, and autoantibodies were strong indicators of Hashimoto’s thyroiditis. To better understand these correlations and underlying pathogenic mechanisms, additional research is necessary.


Introduction
In Hashimoto's thyroiditis, the accumulation of lymphocytes in the thyroid gland occurs which eventually leads to thyroid fibrosis and gradual tissue destruction [1,2].Women are five to ten times more likely to be affected than men, and the condition affects 2% of the overall population on average [3,4].Additionally; it is one of the main factors contributing to dyslipidemia, cardiovascular disease, and high blood pressure [5].Thyroid cells deteriorate when thyroid auto-antibodies, such as anti-thyroglobulin (TG-Ab) and anti-thyroid peroxidase (TPO-Ab), are present [1,6].Hashimoto's thyroiditis causes goiter and thyroid failure, which in turn causes papillary thyroid cancer and thyroid carcinoma [7,8].Anti-thyroglobulin antibodies (TGAb) and anti-thyroid peroxidase antibodies (TPOAb) are produced in HT.About 90% of HT patients have circulating TPOAb, but TGAb are less sensitive (positive in 60-80% of individuals) and less specific than TPOAb [9,10].
Women are almost eight times more likely than males to get HT.Additionally; it affects Whites and Asians more frequently than African-Americans [11].With a higher incidence in the middle-aged, HT primarily affects females of all ages [12].
In numerous autoimmune disease models, the inflammatory cytokine IL-23 has been identified to be crucial for illness development [13].A heterodimeric cytokine called interleukin-23 (p40/p19) is made up of the p19 subunit of IL-12p70 and the p40 subunit of IL-12.According to studies, elevated serum IL-23 levels in HT patients contribute to the development of HT by promoting Th17 cell differentiation and IL-17 release, which restrict autophagy and raise neopterin levels [14,15].

Literature review
The literature review of this research article focuses on two aspects.The first aspect is to identify the effects of Hashimoto's thyroiditis in a sample of Iraqi patients; the other aspect was showed the association between Hashimoto's thyroiditis and the effects of Interleukin-23 on patients.

The Endocrine System
The endocrine system is a control system of ductless glands that secrete hormones within special organs.Hormones act as "messengers" and are carried by the bloodstream to different cells in the body, which interpret these messages and act on them [16,17].The endocrine system provides an electrochemical connection from the hypothalamus of the brain to all the organs that regulate the growth, development, body metabolism and reproduction [18].

Thyroid gland
The thyroid gland is a vital hormone gland; it plays a major role in the metabolism, growth and development of the human body.Additionally, it helps to regulate many body functions by continually releasing a stable amount of thyroid hormones into the bloodstream, when the body is under certain conditions, such as when it is growing, when it is cold, or during pregnancy, it creates extra hormones to meet the increased need for energy [19].In addition to having a crucial function in calcium metabolism; it is also an important factor in  [20].

Thyroid gland disorders
Thyroid disorder is a medical condition which affects the function of the thyroid gland, which refers to an inadequate, either lowered or elevated supply of thyroid hormones and an inappropriate reaction to those hormones [21].It is possible for pathophysiological illnesses or diseases to manifest themselves at a variety of levels, such as insufficiencies in the supply of hormones, abnormalities in regulation, or resistance to the hormones' effects on cellular responses [22].

Hyperthyroidism
Hyperthyroidism describes an overactive thyroid that has a persistently increased production of the hormones T3 and T4 [21].This condition, which is also known as thyrotoxicosis, manifests itself when the tissues of the body are subjected to high concentrations of T3 and T4.The main effects are due to increased basal metabolic rate.
Graves' disease is the auto-immune condition that causes the majority of cases of hyperthyroidism; in the United Kingdom, this condition affects approximately 2% of women and 0.2% of men.Graves' illness is distinguished by the development of autoantibodies that bind to receptors that are normally occupied by the TSH.

Hypothyroidism
Hypothyroidism is a common disorder characterized by low levels of circulating thyroid hormones T3 and T4, which can be easily recognized and managed, but in severe cases, it can be fatal if treatment is not sought.The clinical presentation can vary depending on age, gender and a number of other factors [21].

Thyroiditis
Inflammation of the thyroid gland is called "thyroiditis," which is a generic term.
Thyroiditis can be categorized as acute, subacute, or chronic depending on the progression of the patient's symptoms [23].It encompasses a collection of distinct conditions, each of which can cause inflammation of the thyroid but manifests in its own unique way.Hashimoto's thyroiditis is the condition that leads to hypothyroidism more frequently than any other.The clinical environment, the quickness of the development of symptoms, the family history, as well as the presence or absence of prodromal symptoms and neck discomfort are the primary factors that are considered when differentiating thyroiditis [24].

Hashimoto's thyroiditis
As one of the autoimmune thyroid diseases, Hashimoto's thyroiditis (HT) is a common autoimmune disease that is characterized by lymphoid infiltration of the thyroid gland, including both T and B cells [25].Hashimoto's thyroiditis is named after Japanese physician Hakaru Hashimoto of Kyushu University's medical school, who was the first to describe the symptoms of people with Struma lymphomatosa, a severe lymphocyte infiltration of the thyroid gland.In addition, all of the data in his first study came from four women.
Researchers from the Middlesex Hospital in London were able to conduct human studies on individuals with comparable symptoms later that year.They were able to determine that these un-well patients have an immunological reaction to human thyroglobulin after purifying antithyroglobulin antibodies from their serum.Based on this information, it was hypothesized that Hashimoto's struma is an autoimmune thyroid disease [26].The etiology of HT involves excessively stimulated CD4+ T cells [27], and a decreased sensitivity of CD4+ T cells to the inhibitory effects of transforming growth factor beta (TGFb) may be another potential mechanism [28].

Thyroid peroxidase antibodies (TPO-Ab)
Thyroid peroxidase antibodies are more common than Tg antibodies and more indicative for thyroid disease [30].It has been termed microsomal antigen based on its intracellular localization.Antibodies react against conformational epitopes at the surface of the molecules and against linear epitopes [31].On the other hand, correlations of anti-thyroid antibody production with decreased fertility is suspected but was not confirmed in a recent study [32], which was performed in a highly selected study collective of predominantly very healthy and well educated women, and might not be representative for all women [33].

Thyroglobulin antibodies (TG-Ab)
Thyroglobulin is a large (600 kDa) glycoprotein consisting of dimers and contains an average of 2-3 molecules of T4 with 3 molecules of T3.The molecule is heterogeneous regarding hormone content, glycosylation, and size [34].

Research Methodology
The samples of this study were collected from The National Diabetes Center, Al-Mustansiriyah University/Baghdad/Iraq.The study covered the period from January 2022 to July 2022.The samples of study included (51) Hashimoto's thyroiditis patients in addition to 50 healthy people as control) with the age range (20-75) year.Seven milliliters of whole blood were extracted from hypothyroidism patient's vein.After centrifuging, serum was separated by using gel tube and stored at -20 • C until measuring the concentration of the hormonal tests (FT3, FT4 and TSH) which were estimated to sure that patients were having a thyroid disorder and determined its type, in addition to immunological tests (anti-TG and anti-TPO).Also measuring the level of interleukin-22 in serum of HT patients.

Ethics approval
All study samples were treated and handled in accordance with the necessary biosafety and

Biochemical diagnosing test by Cobas system
The Cobas analyzer is an apparatus that ideal for laboratories that process a large number of samples per day.After centrifuge the sample and took the sufficient size of the serum (20µl) from the gel tube that put in the cup then the device begin started working.Eighteen minutes photometry, Each and every reagent needed for the analysis contained in a single cassette, which was immediately recognized during loading by attached barcode labels with assayspecific information [35].Serum concentrations of IL-23 was measured by commercial ELISA kits.

Statistical Analysis
The statistical software SPSSTM (SPSS Inc., Chicago, IL, USA) was used to conduct the analysis.Quantitative and qualitative data were presented using mean, standard deviation (SD), frequency, and percent, respectively.The Kolmogorov-Smirnov test was used to determine whether the data were normal.ANOVA and ANCOVA were used to compare continuous variables between groups while accounting for confounders like age and gender.
The Pearson correlation coefficient was used to determine the association between the variables.Statistics were considered significant for P-values under 0.05.

Distribution of thyroiditis according to age and gender
As shown in the below thyroid distribution disorders patients (Table 3-1), the number of samples is 101, including 51 patients and 50 healthy controls.The patients' group's mean age was (38.20±2.32)while in the control group was (34.95±2.32).As long as the P-value is 0.340, there was no statistically significant difference between the groups.This outcome was consistent with that of Geng and colleagues, who found no difference between the HT and non-HT groups at P>0.05 [36].
Results in table  describe the relationship between the gender according to HT patient's which showed that female effected more than male, this may be in part due to sex hormone influences as shown in the P-value 0.0001 that consider a significant differences between gender which acceptable with the study that had a major frequency in women than in men (4-10 times) [37].

Distribution of thyroiditis according to thyroid hormones
The hormonal tests for thyroid function (FT3, FT4, and TSH) and autoimmunity functions (Anti TPO and Anti TG) showed that people suffer from thyroid disorder, and depending on the level of hormones, whether it is high or low, From hypothyroidism samples, it was found that 51 people suffering from Hashimoto's thyroiditis, this increase or decrease was  [38] that there were highly significant differences between the autoimmunity functions of HT patients and the healthy controls, the statistical results of thyroid autoantibodies (Anti TG and Anti TPO) in this study are consistent with those findings.This indicates that there was a clear association between the occurrence of these tests and the risk of thyroid and its complications.
However, this evidence has been strengthened by two recent research.In the first, there was no relationship between autoantibody levels and thyroid function tests, although there was a link between thyroglobulin and TPO antibody levels and quality of life scores in HT patients [39].In the second, both autoantibodies correlated with a score of 16 hypothyroidism symptoms, but thyroid hormone levels were not correlated with either; rather, thyroglobulin antibodies were linked to specific symptoms in a logistic regression model [40].
The study of hypothyroid HT patients who continued to experience symptoms after euthyroidism was restored with standard replacement therapy provides perhaps the most compelling evidence that the autoimmune process contributes to the burden of symptoms in HT.The medically treated control group did not improve, but total thyroidectomy followed by thyroxine replacement was related with an increase in general health score, a drop in tiredness score, and a decrease in the frequency of chronic fatigue from 82 to 35% [41].TPO antibody levels decreased significantly after thyroidectomy, however there are other conceivable mechanisms by which autoimmunity could induce such consequences besides one that is antibody-mediated, most notably via cytokines.[43].
The main objective of our investigation is to provide data for IL-23 axis involvement in the HT development by measuring IL-23 serum levels in patients and healthy groups.Horie et al. [44] were the first that reported on the possible role of Th17 for the induction of an autoimmune thyroiditis on animal model.Subsequently, Figueroa-Vega et al. found that HT patients have higher amounts of Th17 cells in their thyroid tissue and peripheral blood, as well as better in vitro T cell differentiation into Th17 lymphocytes produced by IL-6/IL-23 [45].
Our findings in euthyroid stage of HT consists of data from studies of Ruggeri et al. and Liu et al.where they found that untreated HT patients have higher serum levels of IL-23 [46,47].In these studies most of patients were euthyroid and they did not receive any treatment.
The authors concluded that their findings concerning IL-23 could be an early stage in the development of HT naturally.Bossowski et al. [48]  To our knowledge we are the first investigators to evaluate the serum levels of IL-23 in HT patients.We found a significant increase of serum levels of IL-23 in HT patients; it would suggest that IL-23 plays a function in maintaining autoimmune inflammation in addition to initiating pathogenic processes.Additionally, IL-23 stimulates activated T cells like Th17 to produce the proinflammatory cytokine IL-17 [49].It takes exposure to IL-23 to keep Th17 cells that are involved in inflammation active.The confirmed association between these two cytokines and their participation in illness development for euthyroid patients between IL-17 and IL-23 is good.

Conclusion
It have shown that increased serum IL-23 may be a significant factor in the development of Hashimoto's thyroiditis.

Acknowledgements
Very appreciative of my department's (biotechnology department) and my university's (Al-Nahrain University) support in providing the tools and materials needed to complete this work.

References
Autoimmune thyroid diseases are usually accompanied by the presence of TPO-Ab, Tg-Ab TSHR-Ab [29], In addition to Antinuclear antibodies and double strand antibodies which attack thyroid gland and destruct tissues as shown in figure (1).
security protocols.Before beginning this study, the Ethics Committee of the College of Biotechnology, Al-Nahrain University,, Ministry of Higher Education and Scientific Research, Iraq (Number 312/4031 A.P. on November 2, 2021) accepted the guidelines for the care and use of health laboratory of WHO guide.

Table ( 2
): Levels serum of the thyroid hormones and autoimmunity functions in thyroid disorder patients and healthy groups.

. Effect of Hashimoto's thyroiditis on IL-23 serum level
When it comes to thyroid cell damage, lymphocytic infiltrate-derived cytokines are crucial because they can encourage thyroid cells to generate proinflammatory mediators, which amplifies and maintains the autoimmune response[42].In the current study, the level of IL-23 serum was determined to assess its role in the response of Iraqi patients with Hashimoto's thyroiditis.The recorded data in the tabledemonstrated that the levels of IL-23 in the patient's serum samples were greater than expected based on the normal range (14.727 - described elevated serum levels of IL-23

Table ( 3
): Levels serum of the Interleukin-23 (IL-23) in thyroid disorder patients and healthy groups.