EVALUATION OF APRIL IN SERUM OF PATIENTS WITH HASHIMOTO'S THYROIDITIS AVALIAÇÃO DE APRIL NO SORO DE PACIENTES COM

Hashimoto's thyroidi s (HT) is an autoimmune and inflammatory disease in which an bodies are directed against the thyroid gland leading to chronic i n fl a m m a o n a n d h y p o t h y r o i d i s m . T h e autoimmunity against thyroid an gens can be a s s o c i a t e d t o g e n e c b a c k g r o u n d a n d environmental factors. Thyroid peroxidase (TPO) and thyroglobulin (TG) are the major autoan gens for characterizing the disease. HT is related to the ac va on of autoreac ve CD4+ T cells, CD8+ cytotoxic T cells and an thyroid an body producing-B cells. Among several cytokines related to the pathogenesis of HT, a prolifera on-inducing ligand (APRIL) has been studied in the context of the establishment and/or maintenance of autoimmune diseases. The role of APRIL in the pathogenesis of HT is s ll poorly understood. Therefore, the present study aimed to compare APRIL serum concentra on in HT pa ents and healthy donors by ELISA. We observed a s ignificant decrease in APRIL concentra on in HT pa ents when compared to the control group, and a posi ve correla on between APRIL level and age. Our results suggest that the APRIL molecule can compose the cytokine profile along the inflammatory response in HT, however, other inves ga ons should be proposed to understand its molecular mechanisms via specific RESUMO A Tireoidite de Hashimoto (TH) é uma doença autoimune e inflamatória na qual os an corpos são direcionados contra a glândula reoide, levando à inflamação crônica e ao hipo reoidismo. A reóide peroxidase (TPO) e a reoglobulina (TG) são os principais autoan genos e esta autoimunidade pode estar relacionada a fatores gené cos e ambientais. A TH é caracterizada pela a vação de células T CD4+ autorea vas, células T citotóxicas CD8+ e células B produtoras de an corpos an reoidianos. Entre várias citocinas relacionadas à patogênese da TH, um ligante indutor de proliferação (APRIL) tem sido estudado no contexto do estabelecimento e/ou manutenção de doenças autoimunes. O papel da citocina APRIL na patogênese da TH ainda é pouco compreendido, portanto, o presente estudo teve como obje vo dosar a concentração sérica de APRIL em pacientes com TH e indivíduos saudáveis pela técnica de ELISA. Observamos uma diminuição significa va na concentração de APRIL em pacientes com TH quando comparados ao grupo controle, além de uma correlação posi va entre concentração de APRIL e a idade dos pacientes. Nossos resultados sugerem que a molécula de APRIL pode compor o perfil de citocinas ao longo da resposta inflamatória na TH, no entanto, outras inves gações devem ser Revista Cien fica da FMC. Vol. 16, no 1, 2021 ARTIGO ORIGINAL DOI 10.29184/1980-7813.rcfmc.269.vol.16.n1.2021

a s s o c i a t e d t o g e n e c b a c k g r o u n d a n d environmental factors. Thyroid peroxidase (TPO) and thyroglobulin (TG) are the major autoan gens for characterizing the disease. HT is related to the ac va on of autoreac ve CD4+ T cells, CD8+ cytotoxic T cells and an thyroid an body producing-B cells. Among several cytokines related to the pathogenesis of HT, a prolifera on-inducing ligand (APRIL) has been studied in the context of the establishment and/or maintenance of autoimmune diseases. The role of APRIL in the pathogenesis of HT is s ll poorly understood. Therefore, the present study aimed to compare APRIL serum concentra on in HT pa ents and healthy donors by ELISA. We obser ved a significant decrease in APRIL concentra on in HT pa ents when compared to the control group, and a posi ve correla on between APRIL level and age. Our results suggest that the APRIL molecule can compose the cytokine profile along the inflammatory response in HT, however, other inves ga ons should be proposed to understand its molecular mechanisms via specific RESUMO A Tireoidite de Hashimoto (TH) é uma doença autoimune e inflamatória na qual os an corpos são direcionados contra a glândula reoide, levando à inflamação crônica e ao hipo reoidismo. A reóide peroxidase (TPO) e a reoglobulina (TG) são os principais autoan genos e esta autoimunidade pode estar relacionada a fatores gené cos e ambientais. A TH é caracterizada pela a vação de células T CD4+ autorea vas, células T citotóxicas CD8+ e células B produtoras de an corpos an reoidianos. Entre várias citocinas relacionadas à patogênese da TH, um ligante indutor de proliferação (APRIL) tem sido estudado no contexto do estabelecimento e/ou manutenção de doenças autoimunes. O papel da citocina APRIL na patogênese da TH ainda é pouco compreendido, portanto, o presente estudo teve como obje vo dosar a concentração sérica de APRIL em pacientes com TH e indivíduos saudáveis pela técnica de ELISA. Observamos uma diminuição significa va na concentração de APRIL em pacientes com TH quando comparados ao grupo controle, além de uma correlação posi va entre concentração de APRIL e a idade dos pacientes. Nossos resultados sugerem que a molécula de APRIL pode compor o perfil de citocinas ao longo da resposta inflamatória na TH, no entanto, outras inves gações devem ser receptors and other regulatory loops. Key words: Hashimoto´s Thyroidi s, APRIL, inflammatory response.
propostas para entender seus mecanismos moleculares por meio de receptores específicos e outras alças regulatórias. Palavras-chave: Tireoidite de Hashimoto, APRIL, resposta inflamatória, Introduc on Autoimmune thyroid diseases (AITD) cons tute 30% of all autoimmune condi ons and a re c l a s s i fi e d a s o rga n s p e c i fi c d i s e a s e s . Hashimoto's thyroidi s (HT), described in 1912 by Hakaru Hashimoto, is considered a mul factorial disease in which an bodies are directed against the thyroid gland leading to chronic inflamma on and hypothyroidism. The autoimmunity against thyroid a n g e n s c a n b e r e l a t e d t o g e n e c a n d environmental factors and the disorder is prevalent in individuals aged > 60 years, with a female/male ra o ranging from 5:1 to 10:1 (1,2). The diagnosis of HT is based on the increase of an -thyroid peroxidase (an -TPO) and an -thyroglobulin (an -TG) serum levels and the typical clinical signs associated to the change in the ultrasonographic aspects of the thyroid gland (3,4). Although definite diagnos c criteria are not yet available, >20 IgG4posi ve plasma cells per high-power field and >30% IgG4-posi ve/IgG-posi ve plasma cells have been proposed as addi onal diagnos c criteria for HT (5). The disease is a result of a Th1 immune response which triggers cell mediated immunity and thyroid follicular cell death by apoptosis. Ac vated an genspecific T-helper CD4+ cells par cipate in the ac va on of intra-thyroid cytotoxic CD8+ T effector cells, B cells, which differen ate and produce autoan bodies (6-8). A prominent role of Th17 ( C D 4 + I L -1 7 + ) a n d T r e g l y m p h o c y t e s (CD4+CD25+highFoxP3+) respec vely in the induc on and modula on of autoimmune reac ons was demonstrated (9).
Several studies indicate that tumor necrosis factors alpha (TNF-alpha) superfamily cytokines such as B cell ac va on factor (BAFF; also known as BlyS) and a prolifera on-inducing ligand (APRIL) play an important role in B cell biology. It has been shown that APRIL has an important role in T-cell independent an body produc on, immunoglobulin isotype switching, B cell selec on, matura on and survival, as well as IL-10 secre on (10-12). APRIL is produced by a variety of cell types, par cularly leukocytes and binds to two surface receptors expressed on B cells: the transmembrane ac vator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell matura on an gen (BCMA) (13). APRIL, one of the last cloned members of the TNF superfamily, has been shown to s mulate tumor cells and B lymphocyte prolifera on in vitro (14). Several lines of evidence suggest that APRIL is important in the establishment and/or maintenance of autoimmune diseases including systemic lupus erythematosus (SLE), rheumatoid arthri s (RA) and . Fernandez and collaborators suggested that APRIL can be a nega ve regulator of inflammatory diseases mainly due to CD5+ B1 cells ac va on (17). APRIL has also been inves gated as a possible therapeu c strategy for chronic inflammatory diseases. Jagessar and collaborators recently demonstrated that treatment with an -BLyS and an -APRIL an bodies was effec ve in Experimental Allergic Encephalomyeli s (EAE), a model for mul ple sclerosis (20). In the present work we aimed to es mate the circula ng levels of APRIL in HT pa ents.

MATERIALS AND METHODS SUBJECTS AND CONTROLS
The study was designed based on exclusion criteria previously determined for the forma on of two groups: pa ents with HT and healthy individuals as the control group (Table 1) According to the Brazilian Society of Endocrinology and Metabology (SBEM), HT pa ents were characterized by elevated serum thyroid s mula ng hormone (TSH) and decreased free thyroxine (FT4) levels, raised serum an -TPO and/or an -TG an bodies, and undetectable TSH receptor an body (an -TSHR) (21,22). In addi on to these laboratory findings, the diagnosis was confirmed by clinical signs and ultrasonographic images revealing diffuse swelling of the thyroid gland without any other cause. All pa ents have been followed clinically since the beginning of the study and were receiving different doses of levothyroxine (T4; 25-125 ng/day; median: 76.4 ng/day).

CLINICAL PARAMETERS
TSH, FT4, an -TPO and an -TG levels were measured in serum by electrochemiluminescence (ICMA, Roche Diagnos cs, Pla orm: Elycsys 2010). For dosage of ANA in serum, the indirect immunofluorescence in human epithelial cells (HEP2) was used, and the c-reac ve protein was measured in blood by the agglu na on technique. In addi on, laboratory tests for autoan bodies to an -TSHR, CRP; glucose intolerance test (TTG): lipid profile, blood count, liver func on, renal func ons were performed.

SERUM AND PBMCS ISOLATION
Blood was collected from all pa ents with Hashimoto's Thyroidi s and from previously selected healthy donors. Blood samples were kept at room temperature for 30 minutes ll they coagulated. A er centrifuga on at 400g for 10 minutes, the serum samples were collected and frozen at -80ºC un l use.

DETERMINATION OF SERUM LEVELS OF APRIL
APRIL levels were quan fied using Human APRIL Pla num ELISA Kit (eBioscience, Vienna, Austria). According to the manufacturer's instruc ons, calibra on curve, samples, blank and posi ve control were set in duplicate. APRIL concentra on was determined upon the standard curve and the results were expressed by mean ± SEM of APRIL (ng/mL).

STATISTICAL ANALYSIS
Sta s cal analysis of data was performed using GraphPad Prism 7.0 so ware and the results were expressed as mean ± SEM. The comparison between groups (pa ents and control) was performed by the non-parametric Mann-Whitney test and the correla ons were made through the Spearman correla on. Two-tailed significance levels of less than 0.05 were considered significant.

PATIENT'S CHARACTERISTICS
A total of twenty HT pa ents and twenty healthy donors were included in this study. Pa ents age (mean ± SEM), TSH (mIU/L), FT4 (ng/mL), an -TPO (27 IU/mL) and an -TG (IU/mL) values are described in table 2.

APRIL LEVELS IN THE SERUM OF HT PATIENTS
To evaluate the secre on of APRIL in the context of the inflammatory reac on related to HT, we measured circula ng levels of the cytokine. Our results revealed a significant decrease in APRIL in HT pa ents compared to controls. APRIL concentra on for pa ents with HT (n = 20) was 6,531 ± 0,6807 ng / mL, whereas the concentra on for healthy controls (n = 20) was 17,52 ± 2,41 ng / mL, (p = 0.0013, Figure  and control (CT). APRIL serum levels (ng/mL) of control (n=20, squares) and HT pa ents (n=20, circles) groups quan fied by ELISA. Difference of means was observed between groups HT (6,531 ± 0,6807) and CT (17,52 ± 2,41), p=0.0013. The results were expressed by mean ± SEM and, the comparison between each study group was done using non-parametric Mann-Whitney U-test. P < 0.05 was considered significant.

AUTOANTIBODY ANTI-TPO AND ANTI-TG
APRIL levels showed a posi ve correla on with age (r² = 0.2804, p = 0.0002, Figure 2A). According to the Brazilian Society of Endocrinology and Metabology (SBEM) guidelines, an -TPO and an -TG serum levels are important for clinical classifica on of HT (22). We evaluated the correla on between these markers with serum levels of APRIL. Our data indicated that there is no significant correla on between circula ng levels of APRIL with an -TPO (r²=0.11126; p=0.5775) or an -TG (r² = 0.0195; p = 0.8847) serum levels in HT pa ents ( Figure 2B, C). DISCUSSION APRIL, a cytokine member of the TNF-α family, regulates B cell ac vity and can be an important molecule in the establishment and/or maintenance of chronic inflammatory autoimmune diseases (15,18,19,23,24). This cytokine can be produced by a variety of myeloid and nonmyeloid cells, and binds to BCMA and TACI receptors. Both are expressed on B lymphocytes, while TACI was also detected on a subset of ac vated T lymphocytes (25). Therefore, APRIL plays an important role in the modula on of B cell ac vity, such as T-cell independent an body secre on, immunoglobulin isotype switching and in the selec on, matura on and survival of B cells (26,27). Changes in the level of APRIL and its receptors have been described in many autoimmune disorders (28), but the role of APRIL in HT is s ll poorly understood. Our present study aimed to determine APRIL serum concentra on of pa ents with Hashimoto's Thyroidi s, evalua ng possible correla ons with pa ent's age, an -TPO and an -TG levels.
HT is one of the most prevalent human autoimmune diseases, responsible for high morbidity in women. The an thyroid inflammatory reac on in HT is enhanced by several mechanisms, coun ng on the par cipa on of autoreac ve CD4+ T cells, CD8+ cytotoxic T cells and an -thyroid an bodies producing-B cells. An influx of lymphoid cells, dendri c cells and macrophages into thyroid occurs as a consequence of inflammatory events (3).
The HT pa ents tested in the present study w e r e s e l e c t e d b a s e d o n s i m i l a r t h y r o i d histopathological score, TSH and FT4 levels.
The concentra on levels of APRIL in the HT pa ents showed a significant decrease when compared to control group (Fig 1). Likewise, similar findings were recently described by our group in pa ents with type 1 diabetes, another autoimmune disease (24). In contrast, pa ents with systemic lupus erythematosus were shown to have increased levels of APRIL, being related to the increased proinflammatory cytokines, IL-17 and .
Although several studies have related APRIL both to the regula on network and pathogenesis of different inflammatory diseases, it is not yet completely characterized how this cytokine is able to interfere with the immune response (12,18). A significant posi ve correla on between APRIL levels and age (Fig 2A), shows an interes ng profile of this cytokine. In our study, 50% of HT pa ents were over 55 years of age, all were treated with FT4 and had a good prognosis of the disease. Fabris and collaborators showed that APRIL was undetectable in the majority of HT pa ents with subclinical hypothyroidism, while the FT4-treated pa ents did not differ from healthy donors. They suggested that APRIL levels can be normalized under FT4-therapy (19). Our pa ents were also in treatment with T4, however, we found that APRIL levels were decreased considering healthy donors.
In this sense, several clinical trials have demonstrated the efficacy of the BAFF/APRIL blockade which showed considerable variability in the response to B cell-targeted therapy (29,30). APRIL promotes B cell survival and differen a on, which involves intricate regulatory mechanisms according to the varie es of the receptors (BCMA or TACI) and the differen a on stage of B cells. Further studies are required to be er understand the par cipa on of APRIL in the regulatory phenotype of these cells, which would favor the development of new therapies in pa ents in early stages of HT.