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Abstract

Breakdown of tolerance and abnormal activation in B cells is an important mechanism in the pathogenesis of Graves’ disease (GD) and high levels of thyroid hormones (THs) can drive the progression of GD. However, the interactions between THs and abnormal activation of B cells in the context of GD are not well understood. The aim of this study was to investigate B cell-activating factor (BAFF) mediating the cross talk between THs and B cells and the possible underlying mechanisms. A high-level triiodothyronine (T3) mouse model was used to verify T3-mediated induction of overexpression of BAFF and B cell abnormal differentiation. The possible promotion of BAFF overexpression in the mice spleen macrophages during polarization to M1 by T3 was also studied. We showed that high levels of T3 can induce BAFF overexpression and lead to abnormal differentiation of B cells in the mice. While the overexpression of BAFF was observed across many tissue types in the mice, high levels of T3 could induce M1 macrophages polarization by IFN (interferon-gamma)-γ in the spleen of the mice, which in turn generated BAFF overexpression. Our findings provide a novel insight into the interactions between the endocrine and immune systems, as well as provide insight into the role of TH in the pathogenesis of GD.

Keywords

thyroid hormone B cell-activating factor (BAFF)B cells Graves’ disease macrophages

Introduction

B cells are the source of autoantibodies against the thyroid-stimulating hormone receptor in Graves’ disease (GD), and the over production of the thyroid-stimulating hormone receptor antibody by activated B cells is the main mechanism of pathogenesis¹. B cell-activating factor (BAFF; CD257) belongs to the tumor necrosis factor–ligand family, which binds to the BAFF receptor of B cells and activates several downstream pathways that regulate basic survival functions. These survival functions include protein synthesis and energy metabolism required to extend the half-life of immature, transitional, and mature B cells, which results in increased serum immunoglobulin levels^2,3. BAFF is expressed by monocytes, macrophages, dendritic cells (DCs), bone marrow (BM) stroma cells, and activated T cells⁴, which play an important role in the immune homeostasis of B cells⁵. Abnormal BAFF expression is involved with the breakdown of B cell tolerance and autoantibody production, resulting in various autoimmune diseases^6,7. BAFF overexpression has also been found in patients with GD and is seemingly associated with thyroid autoantibody production^8,9. Unfortunately, apart from BAFF genetic variants, it is not known what induces BAFF overexpression in most of the autoimmune diseases patients as only a fraction of the patients have the genetic variants^10
–12.

In our previous study, we have found that the serum BAFF levels in patients with GD were in significantly positive correlation with thyroid hormone (TH) levels¹³. The main pathological characteristic of Graves’ hyperthyroidism is THs overproduction, including triiodothyronine (T3) and tetraiodothyronine. THs overproduction not only affects various organ systems and induces many symptoms, but also involves in pathological progression of GD¹⁴. The main role of antithyroid drug (ATD) is to inhibit thyroid peroxidase that blocks THs synthesis. The aim of ATD treatment is to reduce THs levels to the normal range in patients with hyperthyroidism. Hypothyroidism during ATD treatment is a favorable indicator for long-term remission and is independent of multiple other factors^15,16. Excess THs levels also influence B cell activation, differentiation, and proliferation. The high circulating levels of T3 stimulate B cells to form plasma cells (PCs) and increase the proportion of plasma PCs in the BM, which is a long-lived PCs (LLPCs) survival niche in mice^17,18. It is found that overproduction of the LLPCs is involved in the development of autoimmune disease¹⁹. We speculate that high circulating levels of T3 leading to abnormal B cell differentiation may be through BAFF overexpression in GD patients.

In this study, we employed a high-level T3 mouse model to clarify the roles of THs on BAFF expression, its source, and B cell immune function. It was found that a high-level of T3 could elevate the circulating concentrations of BAFF and induce abnormal differentiation of B cell induced in the mice. Furthermore, high T3 levels induced the macrophage polarized to M1. In the spleen of these mice, high T3 levels induced through interferon-gamma (IFN-γ) led to BAFF overexpression. The results of this study might provide novel evidence of endocrine and immune system cross talk and suggest a possible underlying molecular mechanism.

Material and Methods

Mice

C57BL/6J mice at 6 to 8 weeks of age, with an initial weight of 16 to 22 g, were purchased from T CYK Co., Ltd. and reared under specific pathogen-free conditions (Experimental Animal Center of Nanjing First Hospital, Nanjing Medical University). All animal procedures were reviewed and approved by the Ethics Committee of Laboratory Animals of Nanjing First Hospital, the First Affiliated Hospital of Nanjing Medical University (Authorization number: DWSY-2001741).

Animal Experimental Design and Animal Model Preparation

The mice were randomly assigned to one of six groups: control, T3, T3+negative control shRNA (T3 + NCs), T3+BAFF shRNA (T3 + Bs), T3+negative control liposomes (T3 + NCCLs), or T3+clodronate liposomes (T3 + CLs) groups. The tail veins of the mice in the T3 + Bs and T3 + NCs groups were intravenously injected with BAFF shRNA or negative shRNA, respectively. Four weeks later, the T3, T3 + NCs, and T3 + Bs groups were injected subcutaneously with T3 (5 μg/10 g; M0818A, Meilun Biotech) every day for 6 weeks while the control group was subcutaneously injected with the same volume of saline. The T3 + NCCLs and T3 + CLs groups were intravenously injected in their tails with PBS or CLs (CP-005-005; Liposoma B.V.). Two days later, the mice were injected subcutaneously with T3 (5 μg/10 g) every day for 6 weeks, while the control mice received intravenous injections in the tail with PBS or CLs once a week. This procedure ensures that macrophage subsets are eliminated²⁰.

Lentivirus Production and Infection

Lentivirus construction encompassing BAFF was generated using the AdMax (Microbix) and pSilencer adeno 1.0-CMV (Ambion) systems. The viruses were packaged, amplified in 293T cells, and purified. Titering was performed on 293T cells using the Adeno-X Rapid Titer kit, according to the manufacturer’s instructions. The anti-BAFF shRNA sequence was 3′-CGGGAGAATGCACAGATTT-5′ (Genechem). For in vivo infection, lentiviruses were suspended in 100 µl of PBS, containing 7.6 × 107 IFUs of loaded lentivirus per mouse, and intravenously injected through the tail as described previously²¹. The shRNA dose was 10 mg/kg per mouse.

Sample Collection, Cell Isolation, and Histological Staining

BM cells from mice were obtained by flushing the femurs of animals with culture medium injected through a 21-gauge needle. The peripheral blood mononuclear cells (PBMCs) of mice were prepared from fresh peripheral whole blood, using Ficoll-Hypaque density gradient centrifugation (LTS1077; TBD Science). CD11c+ DCs, F4/80 macrophages, and CD4+ T cells were isolated from mouse spleen MCs, using CD11c+ DC cell isolation kits, F4/80 macrophage isolation kits, and CD4+ T cell isolation kits (130-125-835, 130-110-443, and 130-090-319; Miltenyi Biotec).

For histological analysis, the spleen was sliced into small pieces and maintained in 4% formalin overnight. The material was dehydrated in graded ethanol baths for 15 min and washed in xylene thrice for 15 min before paraffin embedding. Paraffin blocks with spleens were sliced and 5 mm tissue sections were stained with Harris’s hematoxylin and eosin (H&E staining).

Immunohistochemical Staining

The spleen sample was preserved in 10% formalin, dehydrated, and embedded in paraffin following routine methods. Paraffin sections were routinely dewaxed, incubated at room temperature with 3% H₂O₂, and repaired with 0.01 mol/l citric acid solution at high temperature; anti-BAFF antibody (ab16081; Abcam) was used for immunohistochemical staining and the DAB-H₂O₂ colorant was added for color rendering. Finally, hematoxylin was used for dehydration and sealed with neutral gum for observation under a light microscope. The cytoplasm of BAFF-positive cells was brownish yellow. A pathological image analysis system was used in this study. The antibody signal was quantified using the Imageproplus 6.0.

T3 Treatment in Vitro

PBMCs and MCs from spleen were isolated and treated with T3 (CAS 5817-39-0; Meilun Biotech) 10³, 10⁴, 10⁵, and 10⁶ pmol/l for 72 h in a 5% CO2 incubator at 37°C.

Measurement of Serum T3 and BAFF Levels

Blood was collected from the mouse heart and centrifuged at 250 g for 5 min. Serum was stored at −80°C until use. T3 and BAFF levels in mouse plasma were analyzed using a T3 (CEA453Ge; Cloud-Clone Corp) or BAFF (mouse: EK0664; Boster. Human: SEB686Hu; Cloud-Clone Corp) enzyme-linked immunosorbent assay kit, which was operated strictly according to the manufacturer’s instructions.

Western Blotting

Total tissues were homogenized and total proteins were extracted using a tissue and cell total protein extraction kit (KGP902; KeyGen Biotech), which was used to analyze the expression of BAFF. Equal amounts of protein were separated by 15% SDS-PAGE and then transferred to polyvinylidene fluoride membranes. Subsequently, the membranes were blocked with 5% nonfat milk for 2 h, then incubated with anti-BAFF, anti-IFN-γ, anti-β-actin, anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase; ab16081, ab8226, ab171081, and ab8245; Abcam), and anti-IL-4 (Clone No. 1G2C5; Proteintech Group), respectively, at 4°C overnight. β-actin or GAPDH was used as a reference. The membranes were washed three times with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. Detection was visualized using an enhanced chemiluminescence system. The protein signal was quantified using the ImageJ software.

Quantitative Real-Time Transcription Polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted from the BM, spleen, and blood using the conventional TRIzol method (Life Technologies, USA). The RNA precipitate was diluted 100 times with deionized water and then quantitatively analyzed using a spectrophotometer. Reverse transcription was performed using PrimeScript RT Reagent Kit (Takara Bio Inc., Japan) with gDNA Eraser, in accordance with the manufacturer’s instructions for two-step reverse transcription polymerase chain reaction: first synthesis cDNA, and then PCR amplification was performed. Reverse transcriptase was omitted from the negative control to determine whether genomic DNA remained in the cDNA. PCR reaction system: SYBR Premix Ex Taq (2 x) 10 ul, cDNA 2 ul, 0.8 ul either upstream or downstream primers, ROX Reference Dye II (50 x) 0.4 ul, and steam sterilization double water 6 ul. Cycle parameters for 95°C pre-degeneration 30 s, into the PCR cycle at 95°C degeneration 5 s, and 60°C annealing/extension in 34 s, for 40 cycles. The normalized expression values for each transcript were calculated as the quantity of target gene mRNA relative to the quantity of β-actin mRNA using the 2^−ΔΔCt method. All reactions were performed independently at least three times. The primer sequences used are listed in the Table 1.

Table 1.

The Primer Sequences for Reverse Transcription Polymerase Chain Reaction in Mice.

Gene(mice)	Forward (5′-3′)	Reverse (5′-3′)
β-actin	AGAGGGAAATCGTGCGTGAC	CAATAGTGATGACCTGGCCGT
GAPDH	TGATGCTGGTGCTGAGTATGTCGT	TCTCGTGGTTCACACCCATCACAA
BAFF	CCACCGTGCCTCTGTTTTTG	CTTCTGCGGAGTGATGGGAT
IL-4R	GGCTTTGGTATTGTGTACTCGTC	AGGGATGGTGATCTGTCATCG
INF-γR1	CGTGCTTGTGGATGTTGGG	GCCTCGGGAGACCTTAGGA

BAFF: B cell-activating factor; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Flow Cytometry

The cell suspensions were submitted to flow cytometry analyses. Mouse BM cells were incubated with APC-anti-B220, PE-anti-CD138, and PE-Cy7-anti-IgM antibodies (catalog nos. 553092, 553714, and 552867; BD BioSciences). Spleen cells from the mice were stained with combinations of MABs specific for APC-anti-B220, PE-anti-CD138, PE-Cy7-anti-IgM, and PE-anti-IgD (catalog no. 558597; BD BioSciences). Spleen macrophages from mice were stained with APC-anti-F4/80, FITC-anti-CD11b, PE/Cy7-anti-CD206, and PE-anti-CD86 (catalog nos. 557396, 141720, and 105007; BD BioSciences). Blood cells from the mice were incubated with APC-anti-B220, PE-Cy7-anti-IgG (catalog no. 560667, BD BioSciences), and PE-anti-CD138. The samples were acquired on a BD Canton-II flow cytometer and analyzed using FlowJo software. Dead cell exclusion was performed using scatter profiles and 7-AAD staining during all the flow cytometric analyses.

Immunofluorescence Staining

The spleen sections were fixed in 4% paraformaldehyde for 30 min and then placed in a cassette filled with EDTA antigen buffer (pH 8.0) in a microwave oven for antigen repair. After shaking dry, the sections were placed in 3% hydrogen peroxide solution for closure and were then incubated with monoclonal rabbit anti-CD86 (GB13585; Servicebio Technology), monoclonal rabbit anti-CD206 (GB13438; Servicebio Technology), and monoclonal rat anti-BAFF (ab16081; Abcam), followed by incubation with goat anti-rabbit/mouse corresponding species immunoglobulin antibodies (GB23303, GB23301, and GB22302; Servicebio Technolog). DAPI (4(,6-diamidino-2-phenylindole; G1012; Servicebio Technology) was used to label nuclear DNA.

Statistical Analysis

To examine the significance of the mean differences between groups, we used two-tailed unpaired or paired Student t tests for normally distributed data and the nonparametric Mann–Whitney U test for non-normally distributed data. The relationship between the two features of interest was determined using the Spearman’s rank correlation test. All statistical tests were performed using GraphPad Prism V8.0. Unless otherwise specified, all P values presented were two-sided, and statistical significance was set at P < 0.05.

Results

T3 Induced BAFF Overexpression in T3-Treated Mice

To evaluate the effects of T3 on BAFF expression, we used 5 µg/10 g of T3 to create a high-level T3 mouse model, BAFF shRNA (Bs) to interfere with BAFF expression, and CLs to eliminate macrophages in the spleens of mice (Fig. 1A). After 6 weeks of continuous subcutaneous injection of T3, the average gain of body weight decreased, whereas the average food and water consumption increased in all three T3 groups compared with those in the control groups (Fig. 1B–D). The serum T3 levels were markedly higher, more than a twofold increase, in the T3, T3 + Bs, and T3 + NCs groups of mice than that in the control group (Fig. 1E). Meanwhile, the serum BAFF levels and BAFF mRNA expression in BM and spleen significantly increased in the T3 and T3 + NCs groups compared with those in the control and T3 + Bs groups, respectively (Fig. 1F–H). Furthermore, BAFF protein expression in the spleen also significantly increased in the T3 and T3 + NCs groups compared with that in the control and T3 + Bs groups (Fig. 1I).

Figure 1.

BAFF overexpression induced by TH and the results of inhibiting BAFF expression by shRNA in mice. (A) Schematic representation of the experimental protocol. The mice were divided into six groups: Control, T3, T3+negative control shRNA (T3 + NCs), T3+BAFF shRNA (T3 + Bs), T3+negative clodronate liposome control (PBS; T3 + NCCls), and T3+clodronate liposome (T3 + CLs) groups. The mouse of T3 group was treated with 5 μg/10 g T3 for 6 weeks. The mice of T3 + Bs and T3 + NCs groups were treated with shRNA and negative control shRNA for 4 weeks before 6 weeks of T3 treatment. (B to D) Representative of the changes of body weight (B), food consumption (C), and water consumption (D) in mice of Control, T3, T3 + NCs and T3 + Bs groups during the 6 weeks. (E to H) Represents the changes of serum T3 level (E), serum BAFF level (F), BAFF mRNA expression in BM (G), and BAFF mRNA expression in spleen (H) for the four groups (n = 8 biological replicates for each group of the mice). (I) Represents the changes of BAFF protein expression in mice spleen of the four groups. Data are presented as mean ± SD. Statistical significance is assessed by two-tailed unpaired t test. (J) The spleen pathological sections stained with hematoxylin and eosin (top panels) and immunohistochemically stained with anti-BAFF antibody (lower panels) for the four groups. (K) Represents the fraction of BAFF positive cells in the four groups. The cytoplasm of BAFF-positive cells was visualized as a brownish yellow stain using a pathological image analysis system. Five visual fields were randomly selected from each section. Integral absorbance was calculated as positive BAFF expression (n = 5 biological replicates). Mean density was calculated by mean IOD/mean area. Scale bar = 100 um. Data are presented as mean ± SD. Statistical significance is assessed by two-tailed unpaired t test. BAFF: B cell-activating factor; TH: thyroid hormone; CL: clodronate liposome; BM: bone marrow; PBS: phosphate-buffered saline; Bs: BAFF shRNA; IOD: Integrated optical density.

We noted that the white pulp compartment of the spleen was expanded and fused in T3-treated mice, as detected by H&E staining (Fig. 1J). Immunohistochemical staining confirmed that BAFF expression increased in the spleen of T3-treated mice compared with that in control mice. When shRNA was used to inhibit BAFF expression, however, the degree of fusion in the white pulp areas of the spleen was reduced in the T3 + Bs group, as detected by H&E staining, compared with the T3 + NCs group (Fig. 1J). Immunohistochemical staining showed that BAFF expression was also suppressed in the T3 + Bs group compared with the T3 + NCs group (Fig. 1K).

Elevated BAFF Levels Induce Abnormal B Cell Differentiation

B cell development in human BM proceeds in successive stages to form transitional B cells with low avidity to self-antigens. Immature B cells are formed with the cell surface expression of IgM^22,23; they exit the BM, enter circulation, and migrate to the spleen, completing the early steps of B cell development²⁴. The resulting mature B cells express IgM and IgD. BAFF is an essential survival factor for splenic B cells and is required for the maintenance of normal splenic B cell numbers²⁵. Excess of BAFF, however, leads to severe B cell hyperplasia²⁶ and promotes the development of autoreactive B cells and autoantibody production^27,28.

We assessed whether T3-induced BAFF overexpression affected B cell differentiation in the BM and spleen. The flow cytometry results showed that the proportion of B220+IgM+ B cells and PCs in the BM (Fig. 2A–C), and B220+IgD+IgM+ B cells and PCs in the spleen (Fig. 2A, D, E) were markedly increased in the mice of T3-treated groups compared with those in the mice of the control group. When shRNA was used to interfere with BAFF expression, the proportions of spleen B220+IgM+ B cells and PCs in the BM (Fig. 2A–C), and B220+IgM+IgD+B cells and PCs in the spleen (Fig. 2A, D, E) were all significantly reduced in the mice of the T3 + Bs group compared with those in the mice of the T3 + NCs group.

Figure 2.

B cells differentiation in BM and spleen of the mice after T3 treatment. (A) Represents expressions of CD138 and IgM on B220+ B cells in BM, CD138, IgM and IgD on B220+ B cells in spleen of the control, T3, T3 + NCs, and T3 + Bs groups. (B to E) Represents the average fraction of B220+ B cells positive for the CD138 and IgM in BM (B and C), and CD138, IgM, and IgD in spleen (D and E) of the four groups. Data are presented as mean ± SD (n = 8 independent biological experiments). Statistical significance is assessed by two-sided independent t test. BM: bone marrow; NC: negative control; PE: P-phycoeythrin; IgM: immunoglobulin G; IgD: Immunoglobulin D.

High T3 Levels Induce Macrophages to Express BAFF and Polarize Them to M1 Cells

To further clarify the sources of the T3-induced overexpression of BAFF in the peripheral blood of both humans and mice, the thyroid, spleen, muscle, liver, small intestine, lung, brain, kidney, stomach, heart, and pancreas tissues were harvested from the mice after 6 weeks of T3 treatment and subsequently sacrificed. BAFF protein expression was significantly increased in most tissues, except for the muscular and stomach tissues, compared with that in the control group (Fig. 3A).

Figure 3.

BAFF expression in some tissues and spleen macrophages after T3 stimulation. (A) Represents BAFF protein expressions in various tissues of the mice treated with T3 for 6 weeks. The average BAFF protein expressions in various tissues are shown at the bottom panel. Data are presented as mean ± SD and P value between T3 and control groups is calculated by two-sided independent t test (n = 8 independent biological experiments). (B and C) Represents BAFF mRNA expression in PBMCs (B) and spleen MCs (C) of the normal mice after various concentrations of T3 stimulation. Data are presented as mean ± SD and P value between 0 and various concentrations of T3 was calculated by two-sided independent t test (n = 8 independent biological experiments). (D to G) Represents BAFF mRNA expression of spleen parenchyma cells (D), CD4+ T cells (E), CD11c+ DC cells (F) and macrophages (G) in the mice treated with T3 for 6 weeks and control. Data are presented as mean ± SD and P value between control and T3 groups is calculated by two-sided independent t test (n = 8 independent biological experiments). (H) Represents expressions of CD86 and CD206 on spleen F4/80+ CD11b+ macrophages in the mice spleen after 6 weeks T3 treatment. The average fraction of spleen F4/80 CD11b macrophages positive for CD86 and CD206 are shown at the right panel. Data are presented as mean ± SD and P value is assessed by two-sided independent t test. (n = 8 independent biological experiments). (I and J) Represents mRNA expression of IFN-γR1 (I) and IL-4 (J) in MΦ macrophages. (K and L) Represents mRNA expression BAFF mRNA expression in M1 macrophages (K) and M2 macrophages (L). Data are presented as mean ± SD and P value is assessed by two-sided independent t test (n = 8 independent biological experiments). BAFF: B cell-activating factor; PBMC: peripheral blood mononuclear cell; MC: mononuclear cell; DC: dendritic cell; PE: P-phycoeythrin.

To investigate what cells can be induced by T3 to overexpress BAFF, we used various concentrations of T3 to stimulate the PBMCs and spleen mononuclear cells (MCs) for 72 h. We found that following the concentrations of T3 elevation (10⁴, 10⁵, and 10⁶ pmol/l), the BAFF mRNA expression significantly increased in spleen MCs, but T3 did not affect BAFF expression in PBMCs (Fig. 3B, C). Then, we exposed mice to 5 μg/10 g of T3 regularly for 6 weeks and isolated spleen parenchyma cells, DCs, T cells, and macrophages. The results showed that only macrophages highly expressed BAFF mRNA after T3 stimulation (Fig. 3D–G). The heterogeneity of macrophage phenotypes is commonly referred to as polarization, which conventionally subdivides macrophages into three groups: naive (Mφ; also called M0), which readily differentiates into pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes^29,30. The M1 macrophages are activated by toll-like receptor ligands, such as lipopolysaccharide and interferon-gamma (IFN-γ), which express proinflammatory cytokines, mediate immune defense of the host, and exhibit antitumor immunity. The M2 macrophages are stimulated by interleukin IL-4 or IL-13; they have anti-inflammatory and pro-tumoral properties and play a role in wound healing^31,32.

We used flow cytometry and RT-PCR to identify the polarization of macrophages and the respective macrophage subsets in the spleen that can be induced to overexpress BAFF with high T3 levels. The results showed that the proportion of spleen F4/80+CD11b+CD86+ cells (M1) significantly increased, but there was no significant difference in F4/80+CD11b+CD206+ cells (M2) in T3 treated mice compared with those in the control (Fig. 3H). The IFN-γR1 and BAFF mRNA expression in spleen F4/80+ cells (Mφ) and M1 cells significantly increased (Fig. 3I, K), but IL-4 and BAFF mRNA expression were not significantly different in F4/80+ cells (Mφ) and M2 cells in T3 treated mice, compared with those in the control (Fig. 3J, L).

The Elimination of Macrophages Reduces BAFF Expression Induced by T3

To further verify the effect of T3 on BAFF expression in spleen macrophages, we used CLs to eliminate macrophages in T3-treated mice. The results showed that the spleen weight was similar between the T3 and T3 + CLs groups, but all larger than control group (Fig. 4A). Compared with the T3 + NCCLs group, the serum T3 level was not significantly different from the T3 + CLs group, but the BAFF in serum, BAFF mRNA, and BAFF protein expression in the spleen tissue were all significantly decreased in the T3 + CLs group (Fig. 4B–E). Furthermore, the spleen IFN-γR1 protein expression was significantly increased in the T3 + CLs group, but there was no significant difference in IL-4 protein expression between the T3 + CLs group and T3 + NCCLs group (Fig. 4F). The immunohistochemical staining results showed that the spleen BAFF protein expression was significantly suppressed in the T3 + CLs group compared with that in the T3 + NCCLs group (Fig. 4G). The immunofluorescence staining results suggested that, compared with the control group, the expression of BAFF was significantly reduced after the reduction of M1 macrophages and M2 macrophages (Fig. 4H).

Figure 4.

Polarization of spleen MΦ macrophages to M1 macrophages by TH stimulation and effects of eliminating macrophages BAFF expression. (A) Represents spleen weight changes among the control, T3, T3 + NCCLs, and T3 + CLs groups of mice. The T3 + NCCLs and T3 + CLs groups were intravenously injected PBS or CLs through tail veins, respectively. Two days later, the mice were injected subcutaneously with T3 (5 μg/10 g) every day for 6 weeks; meanwhile, the mice were intravenously injected PBS or CLs through tail veins once a week. The spleen volumes in the three groups are showed in the left panel and the average spleen weights of the three groups of mice are shown in the right panel. Data are presented as mean ± SD and P value is assessed by two-sided independent t test (n = 8 independent biological experiments). (B to D) Represents the difference of serum T3 (B) and serum BAFF levels (C), and BAFF mRNA expression (D) in PBMC between the T3 + NCCLs and T3 + CLs groups. Data are presented as mean ± SD and P value is assessed by two-sided independent t test (n = 8 independent biological experiments). (E and F) Represents the difference of BAFF (E), IFN-γR1, and IL-4 (F) protein expression between the T3 + NCCLs and T3 + CLs groups. Data are presented as mean ± SD and P value is assessed by two-sided independent t test (n = 8 independent biological experiments). (G) Represents the fraction of BAFF positive cells in T3 + NCCLs and T3 + CLs groups. The cytoplasm of BAFF-positive cells was visualized as a brownish yellow stain using a pathological image analysis system. Five visual fields were randomly selected from each section. Integral absorbance was calculated as positive BAFF expression (n = 5 biological replicates). Mean density was calculated by mean IOD/mean area. Scale bar = 100 um. Data presented are mean ± SD. Statistical significance is assessed by two-tailed unpaired t test. (H) Comparison of co-immunostaining for anti-BAFF (green, white arrows), anti-CD86 (red), anti-CD206 (pink), and DAPI (blue) in spleen between the T3 + CLs and T3 + NCCLs groups. TH: thyroid hormone; BAFF: B cell-activating factor; NCCLs: negative control liposomes; CL: clodronate liposome; PBMC: peripheral blood mononuclear cell; DAPI: 4l,6-diamidino-2-phenylindole; IL: interleukin; IOD: Integrated optical density.

Discussion

In this study, we found that high levels of T3 could induce BAFF overexpression and promote B cell differentiation into IgM+ B cells and PCs in the BM, IgM+IgD+ B cells in the spleen of the mice. When shRNA was used to interfere with BAFF expression, the B cell activation and differentiation were markedly receded, which confirmed that high level of T3-induced B cell abnormal activation and differentiation mainly through BAFF overexpression.

BAFF promotes the activation and survival of B cells and has been linked to pathogenic B cell responses that underlie autoimmune reactions and lymphoid hyperplasia in humans and mice³³. Immature B cells in the BM are prone to apoptotic selection at two differentiation stages, associated with IgH chain gene rearrangement and B cell receptor expression, respectively, which indicates that selection by apoptosis is a primary mechanism for eliminating aberrant or autoreactive B cells³⁴. BAFF can suppress apoptosis of IgM+ B cells, and, in excess, BAFF promotes the survival and maturation of low-affinity self-reactive transitional B cells^2,4,35.

Transgenic mice with abnormally high expression of BAFF are known to develop autoimmune disorders characterized by B cell hyperplasia and autoantibody production, including anti-DNA and rheumatoid factors, and eventually succumb to an immune complex-mediated, lupus-like nephritis³⁶. B cells were targeted due to their key role as enhancers of the immune response in autoimmunity as they give rise to autoantibody-producing PCs³⁷. In addition, suppressing BAFF with the BAFF-specific receptor-Fc can lead to significant reductions in hyperthyroidism, as demonstrated in a GD murine model³⁸.

Although BAFF can be expressed in many different immune cells, including monocytes, macrophages, DCs, neutrophils, follicular DCs, epithelial cells, and stromal cells^39
–41, we confirmed that only macrophages were stimulated by T3 to overexpress BAFF. THs exert their genomic action mainly through T3 binding to its specific T3 nuclear receptors (TRs) α and β⁴². The macrophages, dendritic, T and B cells all express TRs^43
–45, so the role of T3 on BAFF expression in macrophages is apparently not through T3 binding to TRs. In this study, we also found that THs could promote IFN-γR1 upregulation in macrophages and increase IFN-γ expression from Th1 cells⁴⁶ that induce MΦ polarization to M1, similarly mechanistically to T3-induced BM MΦ polarization of BM to M1⁴⁷, which might be one of the reasons for the overexpression of BAFF in macrophages by T3 stimulation. The exact mechanism by which T3 promotes BAFF expression requires further investigation.

Conclusions

Taken together, our results suggest that THs can induce BAFF overexpression in macrophages to activate B cells and promote B cell differentiation into PCs, which may be involved in the development and progression of GD. Our study provides new evidence of cross-talk between the endocrine and immune systems in autoimmune diseases.

Footnotes

Author Contribution

S.L. and X.-M.M. contributed to conception and design of the study. S.L.,G.-Q.L.,Q.-W.G. and J.W. designed and performed experiments,and analyzed data. X.-M.M. acquired funding for the study. G.-Q.L. and X.-M.M. supervised the study. G.-Q.L. and J.W. provided critical feedback during manuscript preparation. S.L.,W.-S.G.,Q.S.,and Q.-W.G. provided assistance with experiments. S.L. and G.-Q.L. wrote the first draft of the manuscript. X.-M.M. reviewed and edited the manuscript. All authors read and approved the submitted version.

Ethical Approval

This report does not contain specific human subject information and therefore there was no need for Ethical Committee’s approval.

Statement of Human and Animal Rights

All of the experimental procedures used in this study were conducted in accordance with the Experimental Animal Ethics Committee of Nanjing Medical University,Jiangsu Province,China.

Statement of Informed Consent

This article did not involve human subject research and informed consent was not applicable.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research,authorship,and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research,authorship,and/or publication of this article: This work was supported by the National Natural Science Foundation of China (81570710).

ORCID iD

Xiao-Ming Mao

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