The Potential Effect of Simvastatin on Regulatory T cells in Experimentally Induced Autoimmune Thyroiditis in Female Rats

: Autoimmune thyroiditis also named Hashimoto's thyroiditis: is an inflammatory disorder of the thyroid gland. It is characterized by circulating antibodies to thyroid antigens, and enlargement of the gland with lymphocytic infiltration. Regulatory T cells (Tregs), a particular subset of CD4+ T cells that express CD25 at a markedly increased level. they can regulate immune response in order to preserve homeostasis and self-tolerance. T cell expansion and cytokine production are restricted by Tregs. The development of this autoimmune disease is influenced by TGF-ß1 shortage. Management of HT is usually a symptomatic therapy that concentrates on HT's symptoms rather than its underlying cause. Statins, the lipid lowering medications have pleotropic effects. they can alter immunological reactions. Twenty-four female rats were used for this experiment divided into four groups (n=6), the disease was induced in all groups except group1 (control) where rats only received phosphate buffer saline, group 2 (induction group), group 3 received Prednisolone 2mg\kg orally for 30 days and group 4 received Simvastatin (4mg\kg) orally for 30 days too. Hashimoto’s thyroiditis was induced experimentally by subcutaneous injection of porcine thyroglobulin in Freund’s adjuvant emulsion (4mg/ml). Results showed an increase in Tregs and serum TGF-ß level in rats treated with simvastatin. In conclusion, simvastatin improves the number of functioning Tregs and increases TGF-ß signaling to suppress the autoimmune reaction. Therefore, simvastatin can be a promising approach in the treatment of this disease.


Introduction
Autoimmune thyroiditis also named Hashimoto's thyroiditis (HT) is an inflammatory disorder of the thyroid gland that is characterized by circulating antibodies to thyroid antigens, enlargement of the gland with lymphocytic infiltration, and varying degrees of thyroid dysregulation.At the beginning, MHC class II-positive antigen-presenting cells (dendritic cells and other subclasses of macrophages), cluster in the thyroid.These cells expose naive CD4+ T lymphocytes to thyroid-specific autoantigens, which causes the latter to become activated and clonally expanded (1) .T helper type 1 (TH1) cells are specifically involved in cell mediated cytotoxicity by secreting regulatory cytokines as interleukin-12, interferon, and tumor necrosis factor α (2) .Thyroid autoantibodies are produced as a result of interactions between antigen presenting cells, autoreactive T cells (that persist as a result of immunological tolerance breakdown or dysregulation), and B cells.Through the proliferation of lymphocyte clones and lymphoid tissue within the thyroid gland, antigenproducing B lymphocytes, cytotoxic T cells, and macrophages invade and cluster in the thyroid.Eventually, a significant loss of thyrocytes occurs (3) .A large number of cells have immunosuppressive properties.Among them, regulatory T cells (Tregs), a particular subset of CD4+ T cells that express CD25 at a markedly increased level.They represent just 5-10% of Th lymphocytes.Tregs are able to regulate immune response in order to preserve homeostasis and self-tolerance (4) .T cell expansion and cytokine production are restricted by Tregs.By releasing immunosuppressive cytokines like IL-10 and TGF-ß, they suppress the immunological response.The development of autoimmune disease is influenced by TGF-ß1 shortage.There is a strong correlation between the development of autoimmune thyroiditis and the decrease in the number or dysfunction of regulatory T cells.The reduced proportions of CD4+CD25+ T cells in autoimmune thyroiditis may be responsible for the autoimmune process and lack of immunological tolerance (3) TGF-ß, a pleiotropic cytokine, is probably involved in a variety of physiological processes, including cell growth, differentiation, cell death, and migration (5) .This cytokine plays a variable role in the pathogenesis of Hashimoto's thyroiditis, it regulates the interaction between Th cells and thyroid-specific autoantibodies.TGF-β1 is thought to convert CD4 + CD25 -T cells to CD4 + CD25 + forkhead box P3 + (FoxP3 + ) regulatory T (Treg) cells.TGF-β1 deficiency, hinders the suppressive action of Treg and this leads to autoimmunity (6) .TGF-β1 deficit (or likely decreased production) also affects T-cell activation, differentiation, and apoptosis, and the expression of cytokines, MHC, and cell adhesion molecules, which all have a role in the development of autoimmune illness (5)   .Multiple ways exist through which TGF-β1 may inhibit the activation/expansion of autoreactive cells.For instance, TGF-β1 can inhibit the differentiation of naive CD4+ T-cells into their Th1 or Th2 effector cells by blocking key transcription factors necessary for polarization, namely T-bet and GATA-3.TGF-β1 can also inhibit autoaggressive Tcell responses by preventing the maturation of antigen presenting cells.Management of Hashimoto's thyroiditis is usually limited to reducing the symptoms of hypothyroidism and restoring normal TSH, T4 and T3 levels by levothyroxine.It is therefore a symptomatic therapy that concentrates on Hashimoto's disease symptoms rather than its underlying cause .(7)   .
Statins, lipid lowering agents.are potent HMG-CoA reductase (3-hydroxy-3methylglutaryl coenzyme A) inhibitors.This enzyme catalyzes the process of turning mevalonate from HMG-CoA (8,9) .Since Mevalonate, a byproduct of the HMG CoA reductase activity, serves as a precursor for several other nonsteroidal isoprenoids in addition to cholesterol, inhibiting this crucial enzyme may have pleiotropic consequences (10) .There is a fair amount of evidence that statins can alter immunological reactions.These include impacts on a variety of immune cells' intimal recruitment, differentiation, proliferation, and secretory activities, primarily T cells and monocyte/macrophages (11) .

Materials and methods
Table 1 shows the chemicals and medications used for this study, their companies and origin.

Methods
Induction of Hashimoto's thyroiditis: On day 0, healthy Wistar rats were subcutaneously injected with 0.4 milliliters of porcine thyroglobulin (pTg) emulsion (4 mg/ml) at various locations on their hind legs.To make the emulsion, Porcine thyroglobulin powder was dissolved in phosphate buffer saline (2 mg/ml) Porcine thyroglobulin in phosphate buffer saline solution was emulsified with an equal volume of complete Freund's adjuvant (CFA) (1:1), it was then mixed by vortex for ten minutes until a proper emulsion was formed.A booster dose was given on days 7, 14, and 21 following the initial immunization, using the same dosage but emulsifying the pTg solution with in incomplete Freund's adjuvant (IFA) rather than CFA . (12,13).

Animal grouping:
Twenty-four female Wistar rats weighing between 200 and 250 g and aging between 12 and 16 weeks were used.Rats were purchased from the Iraqi Center for Cancer Research and Medical Genetics and housed in suitable cages with free access to food and water at the College of Pharmacy, Mustansiriyiah University.In addition to excluding pregnant rats, they were permitted to adapt for 14 days in a cycle of 12

Biochemical assessment:
Rats were given an intra-peritoneal injection of 50mg/kg ketamine and 10 mg/kg Xylazine at the end of the investigation to make them fully unconscious (14) .Rat cardiac blood was drawn and allowed to stand at room temperature for 20 minutes, then placed in tubes that promote clotting and centrifuged for 15 minutes at a speed of 3000x to collect serum for the ELISA procedure to measure TGF-ß.Thyroid of each rats was extracted and kept in buffered formalin 10% for histopathological study and immunohistopathological procedure.

Histopathological assessment
In this study, "the paraffin embedded method" was used to prepare thyroid tissue for microscopic examination (15) .This procedure required the following steps: it was incubated at 4 ºC for an overnight duration.8-Repeating step 4. 9-A second wash is performed using a TBS solution with 0.3 percent peroxidase for 15 minutes.10-After adding the secondary antibody, the sections were incubated for an hour at room temperature.11-Repeating step 4. 12-Another incubation was done using hematoxylin as the counterstain.13-With tap water, the slides were cleaned.14-The slides were covered and kept secure for subsequent examination.
B. Assessment of the staining signal: toachieve this, a light microscope equipped with a digital camera was utilized to repeatedly take random shots of slides at a 400x magnification.The following equation was used to evaluate the outcome using the IHC staining score system: (Number of positive stained cells / total number of cells) * 100 C. Immunostaining Score (IS): the immunostaining was scored, according to the percentages of positive cells, as the following: While the intensity reads were as the followings: • 1, weak • 2, moderate • 3, strong, and they were multiplied to get an immunostaining score (IS).

Results
Table 2 shows the mean serum TGF-ß level of the four groups measured by ELISA technique 0.285±0.024abThe data is expressed as mean ± SD. Results with small, non-identical letters (a and b) differ significantly (p < 0.05).
Results revealed that group 2 was decreased significantly as compared with group 1 (control group).However, mean serum level of TGF-ß was increased in group 3 and 4.There was non-significant difference between group 3 and 4 as seen in figure 1.As for group 3 in which female Wistar rats were treated with prednisolone, less lymphocytic infiltration was seen compared to group 2. Yet, most of the thyroid follicles are smaller in size and contain less colloid as compared to control group.More congested blood vessels in the interstitial connective tissue were noticed.
In group 4, where rats received simvastatin treatment, different histological alterations were seen.Thyroid follicles resemble those in group 1 in appearance.However, there were fewer follicles overall.Although, as can be seen in the picture below, there was a little decline in the number of thyroid follicles.

Immunohistochemistry staining for CD4, CD25
The following results are aligned as marker/antibody-based.
Expression of CD25: different expressional patterns were noticed, as summarized in the table below.In the group 1, a small number of CD4 cells with weak cytoplasmic intensity were seen.In contrast, a very high number of positive CD4 cells with strong intensity were noticed among group 2 (induction group), which started to reduce to moderate in Group 3 then lowered to moderate in group 4 as seen in Figure 7.

Discussion
Experimental autoimmune thyroiditis was induced in female rats by using an emulsion of porcine thyroglobulin in Freund's adjuvant.Autoantibodies against thyroid antigen were detected by ELISA technique.
CD4+ cells are known to perform the most significant role in the pathogenesis of Hashimoto thyroiditis (16) .In our current study, group 2 (the induction group) had a very high number of strongly positive CD4 cells, which began to decline to a moderate level in group 3 and group 4. As seen in figure 7.

Figure 1 :
Figure 1: The effects of simvastatin on serum TGF-ß level in experimental autoimmune thyroiditis induced in female rats.

Figure 2 :
Figure 2: photomicrograph for a transverse section for group 1. Displaying thyroid follicles of various sizes, some of which had little follicular vacuoles (400 X)

Figure 3 :
Figure 3: photomicrograph for a transverse section for group 2. Displaying decrease of the thyroid follicles and the replacement with lymphocytes in the parenchymal tissue (400 X)

Table 3 : Immunostaining scores for CD25 expression in thyroid tissue of female rat.
Very low expression of CD25 was seen among group 2. Prednisolone (group 3) and Simvastatin group (group 4) both showed an elevation of the expression according to the score mentioned.Examining a transverse section of the thyroid gland from the four groups stained with CD25 monoclonal Antibody, showed different expressional patterns.Expression of CD4: different expressional patterns were noticed, as summarized in the table below: