The immune and endocrine systems remain functionally connected and the relations between numerous cell types and humoral mediators are complex and not yet fully understood (1). The influence of the immune system on thyroid function has been investigated in various animal experimental models as well as in human thyroid disorders (2, 3). Accumulating evidence points at dendritic cells (DCs) as crucial players in the regulation of inflammatory events associated with thyroid pathology [reviewed in (4)]. DCs are considered as the most potent antigen presenting cells with strong ability to acquire and process exogenous and endogenous antigens and present them to naïve T lymphocytes. The broad expression of co-stimulatory molecules and secretion profile allows DCs to direct the differentiation and activation processes of these lymphocytes. DCs are involved not only in the activation of naïve T lymphocytes but also in a variety of cross-interactions with other immune cells. The extraordinary regulatory properties of DCs depend strongly on their maturity and activation state and differ considerably between particular DC subpopulations. In human settings, two main subpopulations of DCs—conventional/myeloid and plasmacytoid DCs (cDCs and pDCs, respectively) are commonly recognized (5–7). It is of great importance that the immune system and thyroid interactions may be also mediated by thyroid hormones (THs). The expression of thyroid hormone receptor (TR) was described in various immune cell types e.g. monocytes, macrophages, natural killer cells (NK cells), lymphocytes and DCs (8). An analysis of molecular mechanisms underlying the action of THs on murine bone marrow derived DCs suggested an involvement of signal transduction pathways associated with Akt and the NF-κB transcription factors (Nuclear Factor kappa-light-chain-enhancer of activated B cells) (9). Not only the direct influence of THs on the elements of the immune system, but also aberrations in the hypothalamic–pituitary–thyroid axis function including changes of thyrotropin (Thyroid-Stimulating Hormone, TSH) levels may be of significance. TSH receptors (TSHR) are present on the surface of B and T lymphocytes, NK cells, monocytes, and are at high levels in DCs (8). It was shown that TSH increased the phagocytic activity of murine DCs and selectively enhanced the secretion of interleukin (IL)-1β and IL-12 by DCs stimulated with an inducer of phagocytic activity (10). In combination with IL-2, TSH exerted also costimulatory activity on murine NK cells (11).

The high expression of TSHR on murine DCs may suggest the involvement of those cells in the immunoregulatory processes associated with the changes of thyrometabolic state. The majority of TSHR + DCs were found to exhibit myeloid subpopulation characteristics based on the expression of MHC class II and co-expression of CD11c and CD11b molecules (10). However, due to the complexity of thyrometabolic state regulation and methodological difficulties, very little is known about the effects exerted by TH and TSH on human immune system.

In our study we employed a unique clinical model for the assessment of TSH biological function in humans independently of TH influence. The study was performed in patients after therapy (total thyroidectomy or total thyroidectomy and ¹³¹I radioiodine ablation therapy) because of differentiated thyroid carcinoma (DTC) and qualified for recombinant human TSH (rhTSH) administration from standard indications. In our previous, preliminary experiments using this clinical model, we showed that the systemic administration of rhTSH did not influence the quantitative or phenotypic parameters of the peripheral blood DC subpopulations (12). In the current study we extended both the number of the patients and the spectrum of the analysis in order to assess the influence of TSH on multiple immune cell populations (including peripheral blood monocyte and DC subpopulations, B lymphocytes, T lymphocytes, NKT cells, and NK cell subpopulations) in humans under in vivo conditions allowing for the exclusion of effects of TH level fluctuations.

The direct effects of TSH are mediated by TSHR, transmembrane receptor highly expressed by thyrocytes. The expression of this receptor can be also detected in the cells of various tissue types including the cells of the immune system. Despite the low TSHR expression outside the thyroid, the very high binding affinity for TSH allows the hormone to activate a cellular response even in the case of low density of TSHR on cell surface (14).

The functional meaning of TSH outside the thyroid gland is not fully determined. It has been suggested that TSH may act as a modulator, operating at multiple different systems and organs (15–24). Under clinical conditions similar to the ones applied in our study, rhTSH was shown to influence serum ghrelin levels (19), as well as serum levels of total cholesterol and triglycerides without any significant changes of high and low lipoproteins (20). Another study demonstrated that the serum level of platelet microparticles in athyroid patients rose fivefold after rhTSH stimulation (21). Exogenous rhTSH was also shown to have an impact on the cardiovascular system, where systolic and diastolic blood pressure increased 5 days after administration (22). What is more, rhTSH administration was associated with significant deterioration of renal perfusion and reduction in glomerular filtration (23). On the other hand, the protective effect of rhTSH on the coronary endothelium, resulting in an increase of coronary blood flow was observed in a small group of athyroid patients undergoing echo-Doppler examination with evaluation of the coronary flow reserve (24).

The effects exerted by TSH on immune cells are not sufficiently described, and the available data were obtained mainly with the use of animal models or in in vitro assays with human PBMCs. TSHR expression was detected in murine DCs and lymphocytes. Interestingly the level of TSHR expression differed substantially between particular lymphoid organs. Approximately 2–3% of murine splenic CD4⁺ or CD8⁺ T lymphocytes and CD19⁺ B lymphocytes and 10–20% of lymph node lymphocytes were shown to express TSHR. The receptor density was very low in splenic T lymphocytes, whereas, in lymph nodes significant proportion of T lymphocytes expressed TSHR at high level (10). In human PBMCs, binding of biotinylated TSH was shown mainly to monocytes and NK cells (25). In animal models, TSH significantly stimulated secretory and phagocytic activity of DCs (8, 10). Animal experiments indicated also that at least some of the immunomodulatory effects of thyrotropin-releasing hormone (TRH) may be mediated by TSH. In rats, the increase of splenocyte proliferative response after TRH administration in vivo was abolished by anti-TSH antibodies (26). Most importantly, multiple cell populations of hematopoietic lineage, including lymphocytes and DCs, were shown to produce TSH either spontaneously or in response to proinflammatory stimuli or TRH (27–29).

In our study we demonstrated for the first time the influence of systemic rhTSH administration on the structure of peripheral blood immune cells in vivo, encompassing selective increase of the percentage of NKT cells and B lymphocytes in athyroid patients. Little is known about the possible TSH–NKT cells’ interactions. The available data pertaining to the influence of thyreometabolic state on NKT consist of an evaluation of the number of NKT cells in 11 patients with Graves’ disease (GD). In those patients no link was found between thyreotoxicosis (low TSH, high HTs) and the amount of NKT cells in the peripheral blood (30). However, in an aforementioned study the influence of TH level fluctuations cannot be excluded and separated from the direct effects of TSH. To the contrary, our results were obtained in a large group of prospectively assessed athyroid patients, allowing analysis of TSH activity on immune system. Although the observed quantitative changes in NKT cells and B lymphocyte populations may still be dependent on the indirect, cumulative effect of other humoral, cellular, or hormonal factors, direct effects of acute rhTSH administration are more plausible in such clinical model.

As indicated earlier, more is known about TSHR expression on particular immune cell subsets in lymphoid organs of laboratory animals (10). Till now, TSHR expression has not been studied in NKT cells, but it was documented on particular subsets of B lymphocytes (10). Interestingly, a study performed with an animal model revealed the impact of hypothalamus pituitary thyroid axis on the frequency and absolute number of pro-B, pre-B cells, and B lymphocytes in the bone marrow of hypothyroid mice (31). The (hyt/hyt) hypothyroid strain of mice is characterized by reduced serum levels of THs and up to 100-fold elevated serum levels of TSH (32). In these mice the percentage of B lymphocytes and their precursors was significantly reduced, as compared to normal mice, and administration of THs resulted in a specific increase in the frequency and total number of pro-B cells in peripheral circulation (31). Although in our study the population of peripheral blood B lymphocytes increased after rhTSH administration in patients with stable TH levels, both observations underline the importance of thyrometabolic parameters in the lymphocyte biology. The differences may be dependent on species characteristics and significantly diverse experimental conditions—i.e. lack of changes in TH levels in our model and longitudinal versus short term elevation of serum TSH concentration as well as other sources of investigated immune cells. Complex and dependent on microenvironment, interactions between particular cell subsets as a result of TSH stimulation should be also taken into consideration. Invariant subset of NKT cells is known to take part in the regulation of the B lymphocyte function on multiple levels, especially in the context of autoimmune and anti-tumor reaction (33, 34).

Additionally, the results of our earlier studies suggest that the influence of thyreometabolic status on the immune system may be modulated by factors associated with the ongoing pathological processes e.g. in the thyroid gland. Levothyroxine (L-T4) supplementation in patients after thyroidectomy because of DTC was associated with a significant increase in peripheral blood pDCs and cDCs and an increase in surface expression of CD86 on both DC subpopulations (35). It was also demonstrated that THs increased the ability of human peripheral blood DCs to stimulate the proliferation and secretion of IL-12 by PBMCs in vitro (35). However, the effect of L-T4 supplementation was different in patients with Hashimoto’s disease. In this group of patients, the level of pDCs in the PBMC fraction decreased during the L-T4 treatment, while the percentage of cDCs remained unchanged (36).

In our previous work (12) as well as in the current study—performed on a significantly enlarged group of patients, we did not observe any significant influence of systemic administration of rhTSH on the quantity and structure of human peripheral blood DC subpopulations nor on the expression of costimulatory molecule CD86—one of the main DC maturation and activation markers.

Although the expression of TSHR is significantly higher in DCs than in other immune cells from murine lymph node and spleen (10), little is known about the expression of TSHR in human peripheral blood DCs, and this area deserves definitely further research also in the light of our results. Beside the species-specific characteristics, also the difference in the source of examined cells may help in understanding the lack of TSHR effects on DCs in our study. It has been demonstrated that peripheral blood DCs represent mostly immature cells, and they change their phenotype and functional properties upon entering target tissues and organs [reviewed in (37)]. Furthermore, the microenvironmental factors influencing the process of DC activation and maturation may differ considerably between particular localizations. In our earlier analysis we observed e.g. differences in DC subsets between peripheral and portal blood are dependent additionally on the pancreatic pathology (38). Thus, it is possible that the high expression of TSHR on DCs in murine lymphatic organs does not reflect the situation in peripheral blood. Additionally, it is highly possible that the TSHR ligation without other inflammatory stimuli is not sufficient for DC activation.

Previous studies demonstrated that biotinylated TSH can bind to human peripheral blood CD14⁺ monocytes (25). It was also shown that in athyroid patients rhTSH administration increased the pro-inflammatory gene expression in monocytes isolated from peripheral blood leukocytes (39). Yet, in our experimental settings, administration of rhTSH in high doses did not impact the percentage of the investigated monocyte subpopulations. The current observation together with the results of the few earlier studies suggests that the influence of TSH on monocytes may be associated with functional but not overt quantitative changes in the main monocyte subpopulations.

The presented findings indicate that rhTSH may have a direct, THs independent, impact on particular populations of peripheral blood immune cells in humans. Still, the role of indirect effects associated with changes of the complex humoral and cellular networks cannot be excluded. Interestingly, in one of the studies rhTSH was suggested as a potential trigger of autoimmune response in GD (40). However, in the large group of patients with DTC remaining in a long-term follow-up in our Department, Graves’ orbitopathy has never occurred. Nevertheless, further investigations seem to be mandatory to resolve the exact short- and long-term effects exerted by rhTSH administration on human immune system. Beside the importance of this observation in the basic knowledge of immunoregulatory mechanisms, better understanding of the immune properties of high systemic levels of TSH can be of special meaning for patients with inflammatory and malignant diseases of the thyroid.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Local Ethics Committee, the Polish Mother’s Memorial Hospital—Research Institute, Lodz, Poland. The patients/participants provided their written informed consent to participate in this study.

The conceptualization of the study was done by ZA and MS. The data curation was made by PŚ, KW-S, ZA, and AZ. The formal analysis was done by MS, ZA, PŚ, and AL. The investigation was done by ZA, MS, and PŚ. The methodology was made by MS, PŚ, KW-S, and AL. MS, ZA, and AL were in charge of the project administration. MS was in charge of the resources. MS and AL supervised the study. MS and PŚ validated the study. The visualization was done by PŚ. MS, PŚ, and KW-S wrote the original draft. MS, ZA, AZ, and AL wrote, reviewed, and edited the manuscript. All authors contributed to the article and approved the submitted version.

This study was supported by statutory funds from the Medical University of Lodz, Poland (no. 503/1-107-03/503-11-001-19-00).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.