Cellular immunity in subacute thyroiditis: a new perspective through neopterin

Abstract

Subacute thyroiditis (SAT) is an inflammatory disorder of the thyroid gland. Although its etiology is not fully understood, it is believed to occur shortly after viral infections and is mostly associated with human leukocyte antigen (HLA)-B*35. Cellular immunity is prominent in SAT. Neopterin is produced by activated monocytes/macrophages and is a marker of cellular immunity. Its production is stimulated by interferon gamma (IFN-γ), provided mainly by activated helper T lymphocytes type 1 (Th1) in the adaptive immune system. Therefore, with these cells’ activation, an increase in serum neopterin levels is expected. We aimed to evaluate neopterin levels in demonstrating cellular immunity in SAT and compared 15 SAT patients with 16 healthy controls. Since all SAT patients were in the active thyrotoxic phase, we found a significant difference in thyroid functions. Classical inflammatory markers, erythrocyte sedimentation rate, and C-reactive protein were markedly elevated in the patient group. Although we expected to find an increase considering that cellular immunity is at the forefront in the pathogenesis of SAT, we found serum neopterin levels significantly lower in the patient group than in the control group. There is an increase in CD8+ T cells in the thyroid tissue in SAT. The possible relationship with HLA-B*35- major histocompatibility complex class I in SAT, and the antigen presentation to CD8+ T cells may be the reason why we observed low serum neopterin levels in patients due to the cytokine imbalance. Neopterin provides unique and independent data from classical acute phase response indicators.

Subacute thyroiditis (SAT) is an inflammatory disorder of the thyroid gland in which cellular immunity is prominent, and it is associated with human leukocyte antigen (HLA)-B*35, which belongs to major histocompatibility complex (MHC) class I. Neopterin is a marker of cellular immunity, and it is produced by activated monocytes/macrophages with the stimulation of interferon gamma (IFN-γ) secreted from the CD4+ T helper cells. In this study, we found low neopterin levels in SAT patients, in contrast to the high levels of classical inflammation markers, and we explained this by CD8+ T cells dominating the cellular immunity with antigen presentation by HLA-B*35 to them and causing possible cytokine imbalance that affects the production of neopterin.

Graphical Abstract

Graphical Abstract.

Introduction

Subacute thyroiditis (SAT) is a non-suppurative inflammatory disorder of the thyroid gland. It is usually painful, and the pain may be unilateral or bilateral in the thyroid lodge in the neck, sometimes spreading to the chin, ears, and sternum. In some cases, there may be hoarseness and dysphagia. As a result of the destruction of the thyroid gland, a temporary thyrotoxicosis occurs with the release of the thyroid hormones stored in the follicle to the peripheral circulation. As systemic inflammation is also present, symptoms of fatigue and weakness are also evident. Like other thyroid diseases, SAT is more common in women than men. Antithyroid drugs do not have a place in the treatment since it is self-limiting and the thyrotoxicosis is transient. Non-steroidal anti-inflammatory drugs, acetylsalicylic acid, and corticosteroids are used for symptomatic and anti-inflammatory treatment, and beta-blocker drugs are used for thyrotoxicosis symptoms [1].

Although the etiology of SAT is not fully understood, both genetic and environmental factors are thought to play a role. It is believed to occur shortly after viral upper respiratory tract infections in genetically predisposed individuals. Various viruses, including mumps, measles, influenza virus, coxsackievirus, and adenoviruses, may play a role in the etiology [1]. These days when the world is trying to cope with the Coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also taken its place among the viruses that involve in etiology [2].

In SAT, there is a granulomatous reaction with the destruction of the thyroid follicles and mononuclear inflammatory cell infiltration histopathologically [1]. The self-limiting nature of the inflammatory reaction without the existence of an inflammatory trigger like an acute infectious pathogen and the complete recovery usually seen in prognosis are unique features of SAT. In this respect, it differs from autoimmune thyroid diseases in which chronic antigenic stimuli are involved.

Neopterin is produced by activated monocytes/macrophages and is a marker of cellular immunity. Neopterin production in monocytes/macrophages is stimulated by interferon gamma (IFN-γ) [3]. IFN-γ production is provided by natural killer cells in the innate immune system and by activated helper T lymphocytes type 1 (Th1) in the adaptive immune system [4]. Therefore, in cases where these cells are activated, an increase in serum neopterin levels is expected. In addition to IFN-γ, tumor necrosis factor alpha (TNF-α) is another important cytokine that plays a role in stimulating neopterin production. Increased neopterin levels have been demonstrated in viral infections (including COVID-19), malignancies, allograft rejection, and autoimmune diseases in which these cytokines are increased [5–9]. Serum neopterin concentrations tended to be higher in the elderly, but no gender-related differences were detected [3]. Due to all these features of it, we aimed to reveal whether neopterin could be a useful marker in demonstrating cellular immunity in SAT while designing our study.

Materials and methods

Study population

The current study is an observational study with a prospective design. Fifteen patients newly diagnosed with SAT in the Internal Medicine and Endocrinology outpatient clinics and 16 healthy control subjects were included in the study. The study was approved by the Eskisehir Osmangazi University Ethics Committee (Approval No: 28, dated 25 May 2021). The study was carried out in accordance with the statement of Helsinki Declaration. Informed consent was obtained from each participant.

Laboratory measurements

Blood samples for hematological and biochemical parameters were obtained from the participants after a 12-h fasting. Samples were centrifuged at 3500 rpm for 10 min; serums were separated and stored at –80°C for thyrotropin receptor antibodies (TRAb) and neopterin measurements. Other parameters were measured on the day of sampling.

Complete blood count parameters were determined on a Sysmex XN 9100 (Sysmex Corporation, Kobe, Japan) hematology analyzer. Erythrocyte sedimentation rate (ESR) was studied in a fully automated Vacuplus ESR-120 (Ankara, Turkey) analyzer by the Westergren method. Serum C-reactive protein (CRP) levels were measured by immunoturbidimetric method, thyrotropin (TSH), free triiodothyronine (fT3), free thyroxine (fT4), anti-thyroglobulin antibodies (TgAb), and anti-thyroid peroxidase antibodies (TPOAb) were analyzed by electrochemiluminescence immunoassay (ECLIA) in a Cobas 8000 (c702 and e801) autoanalyzer (Roche Diagnostics, Mannheim, Germany). Serum TRAb and neopterin levels were measured by the enzyme-linked immunosorbent assay method. Ready commercial kits from Medipan GMBH (Ref No: 3505, Berlin, Germany) were used for TRAb, and Bioassay Technology Laboratory (Cat. No: E3155Hu, Shanghai, China) for neopterin measurements.

Statistical analysis

Statistical analyses of the data were performed with the IBM SPSS Statistics 21.0 package program. Shapiro–Wilk test was used to examine whether variables were normally distributed or not. Continuous variables that were distributed normally or not were expressed as mean ± standard deviation or median (25th–75th quartiles), respectively. The nominal variable was expressed as number and percentage. For the nominal variable, Fisher’s exact test was used. Between-group comparisons of normally distributed and non-normally distributed continuous variables were made with Student’s t-test or Mann-Whitney U test, as appropriate. P-values lower than 0.05 were considered statistically significant.

Results

The mean age was 50.13 (±11.48) in the patient group and 43.44 (±9.04) in the control group. There were 11 females and four males in the patient group and 12 females and four males in the control group. There was no difference between the patient and control groups regarding age and gender. The mean height, weight, and body mass indexes of the groups were also similar. The P values of the demographic characteristics of the groups are shown in Table 1. While one person in the control group was a smoker, the entire patient group was non-smoker.

Table 1.

demographic characteristics of the subjects

	SAT patients (n = 15)	Healthy subjects (n = 16)	P
Age (year)	50 ± 11.5	43 ± 9.0	0.093
Gender (female)	11 (73,3%)	12 (75%)	1.0
Height (m)	1.64 ± 0.05	1.67 ± 0.1	0.495
Weight (kg)	69 ± 11.5	72 ± 15.6	0.682
Body mass indexes (kg/m2)	25.8 ± 3.69	26.0 ± 5.01	0.861

In the thyroid function tests, TSH was found to be significantly lower in the patient group than in the control group; fT3 and fT4 were found to be higher than in the control group (P < 0.01 for all). Of the thyroid autoantibodies, there was no difference between the two groups for the levels of TRAb. Titers of TgAb and TPOAb were found to be significantly higher in the patient group than in the control group (P < 0.01 for both). The thyroid function tests and the levels of autoantibodies are shown in Table 2.

Table 2.

thyroid function tests and autoantibody levels of the groups

	SAT patients	Healthy subjects	P
TSH (μIU/mL)	0.02 (0.01–0.053)	1.66 (1.29–2.11)	<0.0001
fT3 (pg/mL)	4.60 (3.38–7.32)	3.03 (2.74–3.28)	<0.0001
fT4 (ng/dL)	2.08 (1.65–2.93)	1.06 (0.91–1.36)	<0.0001
TRAb (U/L)	0.25 (0.2–0.4)	0.3 (0.2–0.43)	0.861
TgAb (IU/mL)	18.3 (11.9–33.9)	11.3 (1.49–12.33)	0.003
TPOAb (IU/mL)	9 (9–13.9)	0.9 (0.20–9)	<0.0001

In the hemogram evaluation, no difference was found in hemoglobin, leukocyte, neutrophil, and lymphocyte counts, but monocyte and thrombocyte counts were found to be significantly higher in the patient group than in the control group (P < 0.05 for both). While ESR and CRP, the classical indicators of inflammation, were found to be significantly higher in the patient group (P < 0.01 for both), a statistically significant difference was found between the two groups, with serum neopterin levels being lower in the patient group (0.91 nmol/L (0.7–1.085) in the patient group, 2.68 nmol/L (1.88–3.64) in the control group, P: 0.002). The hemogram parameters and inflammatory markers of the groups, including neopterin levels, are shown in Table 3.

Table 3.

hemogram parameters and inflammatory markers of the groups

	SAT patients	Healthy subjects	P
Hemoglobin (g/dL)	14.0 ± 1.37	14.2 ± 1.61	0.740
White blood cells (10³/μL)	8.01 (6.71–9.63)	6.72 (6.27–9.31)	0.318
Neutrophil (10³/μL)	4.57 (3.5–5.92)	3.65 (3.11–4.18)	0.093
Lymphocyte (10³/μL)	2.38 (1.78–3.29)	2.5 (2.09–2.93)	0.711
Monocyte (10³/μL)	0.65 (0.61–0.83)	0.57 (0.46–0.71)	0.041
Platelet (10³/μL)	346 (256–426)	270.5 (227.7–294)	0.041
ESR (mm/h)	45 (20–74)	10 (6–14.5)	<0.0001
CRP (mg/L)	53.7 (28.8–109.7)	0.6 (0.52–0.8)	<0.0001
Neopterin (nmol/L)	0.91 (0.7–1.085)	2.68 (1.88–3.64)	0.002

Discussion

In this study, we compared 15 SAT patients with 16 healthy controls. Since all SAT patients were in the active thyrotoxic phase, we found a significant difference in thyroid functions compared to the control group, as expected. While TRAb levels were similar in both groups, mean TgAb and TPOAb levels were found to be higher in the patient group, although they were within the reference ranges in both groups. We think that the reason for this is the temporary increase in antibody response due to the release of intracellular antigens into the circulation because of the destruction of the thyroid follicles in SAT [10].

We found higher monocyte and platelet counts in our patient group compared with the control group. We think that platelet elevation occurs due to the systemic acute phase response. We believe that the monocyte elevation is correlated with the monocyte/macrophage stimulation seen in SAT. Similarly, the monocyte-eosinophil ratio (Mo/Eo) combined with fT3 to fT4 ratio (fT4/fT3) has been suggested as a potentially useful parameter in SAT [11]. As expected, we found the classical inflammatory markers, ESR and CRP, higher in our patients than in the control group. Although we expected to see an increase considering that cellular immunity is at the forefront in the pathogenesis, we found serum neopterin levels significantly lower in the patient group than in the control group.

Neopterin is produced by activated monocytes/macrophages. It is known that the hormones released from the hypothalamo–pituitary–adrenal axis with the effect of stress cause suppression in macrophage activities [12]. It can be thought that some central mechanisms may be activated with TSH suppression in thyrotoxicosis and may suppress neopterin release via the hypothalamo–pituitary axis. However, we rejected this hypothesis because we found neopterin levels high in Graves’ disease patients by measuring with the same method used in the current study [unpublished data]. Although there is no study in the literature evaluating neopterin levels in SAT patients, studies in Graves’ disease patients have shown high levels of neopterin [13]. This has led us away from the hypothalamo-pituitary axis as the mechanism that determines the different neopterin levels between the two conditions with thyrotoxicosis.

Although SAT is not a common thyroid disease in the population, it is the leading cause of painful thyrotoxicosis. Even though its etiology is not fully understood, studies have shown that genetic predisposition and environmental factors play a role together. No autoimmune basis for SAT has been demonstrated so far. The fact that it causes a pronounced acute inflammatory response in the active phase shows that it is of immunological origin. Still, its features of self-limitation and mostly resulting in complete recovery suggest that it differs from autoimmune thyroid diseases in which there are chronic antigenic stimuli.

T-cell-mediated immunity against thyroid antigens is thought to play a role in SAT. Histopathologically, patchy lesions are seen in the thyroid tissue, and follicular epithelium and basement membrane deterioration occur with the infiltration of the follicles by mononuclear inflammatory cells. As a result, multinucleated giant cells are formed by lymphocytes and histiocytes that congregate around the colloid material, and the characteristic epithelioid granuloma appearance occurs. For this reason, it is also called granulomatous thyroiditis. In addition to granuloma formation, interstitial edema and lymphocytic infiltration in the thyroid gland are also prominent histopathological features of SAT [1].

Neopterin released from activated monocytes/macrophages is a good indicator of cellular-mediated immunity. Stimulation of neopterin secretion occurs with the activation of CD4+ Th1 cells and IFN-γ released from these cells. Neopterin has been shown to increase in viral infections [5–7]. We believe that neopterin levels were probably high during the viral infection period before the emergence of SAT. However, we think that we found neopterin levels to be low since the active viral infection process ends and inflammation occurs with a different immunological mechanism.

In the cellular immune response, the presentation of peptide antigens to receptors on the cell surface occurs by major histocompatibility complex (MHC) molecules. MHC is a gene region encoded in the 6th chromosome divided into class I and II. MHC class I molecules are expressed on all nucleated cells and are involved in antigen presentation to CD8+ T cells, and consist of human leukocyte antigens (HLA)-A, HLA-B, and HLA-C. MHC class II is expressed on professional antigen presenting cells such as dendritic cells, macrophages, and B lymphocytes, and is involved in antigen presentation to CD4+ T cells and consists of HLA-DP, HLA-DR, and HLA-DQ [14]. The HLA molecule most associated with SAT is HLA-B*35, and it was first reported by Nyulassy et al [15]. Not only HLA-B*35 but also HLA-B*67 was found to be associated with SAT by Ohsako et al [16]. It has been reported by Stasiak et al. that HLA-B18*01, HLA-C*04:01, and HLA-DRB1*01 are also seen in SAT [17]. HLA-B*35, which is the most well-known antigen associated with SAT, belongs to MHC class I and is known to be involved in antigen presentation to CD8+ T cells.

In the study published by Kojima et al., the cellular content of SAT patients’ thyroid specimens was examined immunohistochemically, and it was determined that the small lymphocytes in the granulomas were CD3+, CD8+, CD45RO+ cytotoxic T cells and there were no CD3+ CD4+, CD45RO+ helper T cells. Histiocyte infiltration with CD8+ T-lymphocytes and plasmacytoid monocytes in the follicles in non-granulomatous lesions were also shown in this study [18]. This increase in CD8+ T cells, which has a possible relationship with HLA-B*35- MHC class I in SAT, may be the reason why we observed low serum neopterin levels in patients. Even though monocyte/macrophages are present in the thyroid tissue, it may result in insufficient neopterin production due to inadequate T helper cell stimulation. In our literature review, we did not find any other study on CD4 and CD8+ T-cell counts in thyroid tissue in SAT. However, as Kojima et al. showed, some of the CD8+ T lymphocytes are found to express T-cell intercellular antigen-1 (TIA-1) in another study, and it was defined as a unique immunohistochemical feature for SAT, unlike other types of thyroiditis [19]. In addition, in their comparative study, Yoshikawa et al. obtained mononuclear cells from peripheral blood of patients with SAT, Graves’ disease, and healthy subjects and engrafted them into mice with severe combined immunodeficiency to compare the production of IFN-γ. They found that IFN-γ levels were similar in the sera of mice in which cells from SAT and healthy subjects were engrafted, while IFN-γ levels were significantly higher in the sera of mice that have the cells from Graves’ patients [20]. This indirectly shows that there may be a difference in favor of CD8+ T cells in T-cell homeostasis in SAT.

Sarcoidosis is an inflammatory disease with granulomatous inflammation as in SAT. There is CD4+ T-cell accumulation in the affected organs. Serum neopterin levels were found to be significantly higher in patients with sarcoidosis [21, 22]. There is evidence for a strong genetic influence of the HLA class II alleles in sarcoidosis [23]. In the light of these data, we can say that which T-cell type will receive antigen presentation is determined according to the class of the genetically predisposing HLA allele in cases where cellular immune activation is involved, and the nature of the immunological event is shaped according to the antigen presented T-cell type. We think that in cases where CD8+ T cells are dominant in the inflammatory event, neopterin levels may be low due to the resulting cytokine imbalance.

On the contrary, although the CD4/CD8 T-cell ratio decreased in human immunodeficiency virus (HIV) infection, serum neopterin levels were found to increase [24]. The possible mechanism to explain this situation is pyroptosis. Pyroptosis is a highly inflammatory form of apoptosis, and there is a complete release of the cytoplasmic contents of the dying T cell, thus resulting in a drastic increase in proinflammatory cytokines, including IFN-γ. It has been shown that only 5% of CD4+ T-cell depletion in HIV infection occurs by apoptosis, and 95% is by pyroptosis [25, 26]. In this case, it can be said that despite the decrease in CD4+ T cells in HIV infection, excess IFN-γ levels stimulate neopterin production from macrophages.

There are a limited number of studies in the literature in which neopterin is found to be low. The neopterin tests which are routinely performed to increase the safety of blood donations in terms of infectious diseases in Austria were evaluated, and it was found that neopterin levels were significantly lower in smoker donors than non-smoker donors [27]. mRNA levels for interleukin-6, IFN-γ, and TNF-β have been shown to be significantly lowered in bronchoalveolar lavage cells of smokers compared with never-smokers [28]. Moreover, several studies have demonstrated that smokers have an increased CD8+ T-cell count, altering the CD4/CD8 ratio compared with non-smokers [29]. A number of studies have reported that chronic obstructive pulmonary disease patients who were free from clinical infection and were not on medication have CD8+ T cells as the dominant lymphocyte phenotype over CD4+ T cells in lung tissues [30]. Piaggeschi et al. found that active tobacco smoking was associated with increased frequencies of circulating CD8+ T cells expressing the CD25 activation marker [31]. Low neopterin levels in smokers may be associated to the dominance of CD8+ T lymphocytes and the related cytokine imbalance.

Ozkan et al. found low serum neopterin levels in their study with Behçet’s disease patients. The authors explained this situation with low IFN-γ levels, which was attributed to the inhibitory effect of colchicine treatment on T cell activation and IFN-γ production [32]. Although not included in the discussion of this article, it has been shown that there is a decrease in serum CD4/CD8 ratios in Behçet’s disease and an increase in CD8+ T cells in the aqueous humor in those with uveitis [33, 34]. This may explain the low levels of neopterin in Behçet’s patients.

In a study comparing the neopterin levels of patients with Type 1 and Type 2 diabetes mellitus (DM), although it was predicted to be higher in Type 1 DM than Type 2 DM since it is an autoimmune disease, lower neopterin levels were found, and this was explained by the effect of insulin used by patients on T-cell activation [35]. Although CD4/CD8 T-cell ratios are also not mentioned in this article, it has been shown in the pathology specimens that CD8+ T lymphocytes are predominant in mononuclear cell infiltration in pancreatic islet cells in Type 1 DM [36]. A recent study showed an increase in the percentage and absolute CD8+ T-cell counts in the peripheral blood of patients with Type 1 DM [37].

In SAT, there is an inflammatory state that occurs after the end of active viral stimulation. Therefore, we think that we found neopterin levels to be low because CD8+ T cells play a prominent role in the cellular immune response, although the classic acute phase markers, which are the general indicators of inflammation, are elevated.

Insufficient sample size due to the low incidence of SAT and the inability to measure control neopterin levels after SAT remission are limitations of our study. However, our results are important in showing that neopterin provides unique and independent data from classical acute phase response indicators such as ESR and CRP in inflammatory conditions. Although SAT is not a potential field for developing costly treatments due to its self-limiting feature, it is worthy of investigation as it can be an immunological model for the development of targeted therapies in diseases CD8+ T cells play a role in the pathophysiology with similar immunological mechanisms.

Glossary

Abbreviations

CD

cluster of differentiation

CRP

C-reactive protein

COVID-19

Coronavirus disease 2019

DM

diabetes mellitus

ECLIA

electrochemiluminescence immunoassay

ELISA

enzyme-linked immunosorbent assay

ESR

erythrocyte sedimentation rate

fT3

free triiodothyronine

fT4

free thyroxine

HLA

human leukocyte antigens

HIV

human immunodeficiency virus

IFN-γ

interferon gamma

MHC

major histocompatibility complex

Mo/Eo

monocyte-eosinophil ratio

SARS-CoV-2

severe acute respiratory syndrome coronavirus 2

SAT

subacute thyroiditis

TgAb

anti-thyroglobulin antibodies

Th1

helper T lymphocytes type 1

TIA-1

T-cell intercellular antigen-1

TNF-α

tumor necrosis factor alpha

TPOAb

anti-thyroid peroxidase antibodies

TRAb

thyrotropin receptor antibodies

TSH

thyrotropin

Funding

None.

Conflicts of interest

The authors declare no conflicts of interest.

Author contributions

M.S.T. participated in the design and coordination of the study, contributed to data analysis, reviewed the literature, and wrote the manuscript. E.K. participated in the design and coordination of the study and carried out the laboratory studies. S.G. participated in the design and coordination of the study and contributed to data analysis and literature review. H.K. carried out the laboratory studies and performed the statistical analysis. B.D. participated in the coordination of the study. G.Y. conceived the study and contributed to data collection, analysis, and manuscript writing. M.S.T., B.D., and G.Y. were involved in the clinical care of the patients. All authors read and approved the final version of the manuscript.

Ethical approval

The study was approved by the Eskisehir Osmangazi University Ethics Committee (Approval No: 28, dated 25 May 2021). The study was carried out in accordance with the statement of Helsinki Declaration.

Patient consent

Informed consent was obtained from each participant.

Data availability

An anonymized data set is available upon a reasonable request from the corresponding author.