Abstract
Background
To explore whether IgG4 is involved in the pathogenesis of IgG4 HT.
Methods
Serum TgAb IgG4 and TPOAb IgG4 were measured in IgG4 HT and non-IgG4 HT. C1q, mannose-binding lectin (MBL), Bb, C3d, C4d, and membrane attack complex (MAC) in thyroid tissues from IgG4 HT, non-IgG4 HT, and controls were examined by immunohistochemistry. We assessed IgG4 and MAC deposition in mouse thyroid by immunohistochemistry after injecting purified IgG4 into mice. The glycosylation patterns of TgAb IgG4 from IgG4 HT were identified by MALDI-TOF-MS. The ability of IgG4 to bind to MBL before and after deglycosylation was assessed by ELISA. MBL and MAC fluorescence were detected in thyrocytes after the addition of IgG4 or deglycosylated IgG4.
Results
Serum TgAb IgG4 and TPOAb IgG4 levels were significantly higher in the IgG4 HT group. MBL, Bb, C3d, C4d, and MAC levels were significantly higher in the thyroid tissues of IgG4 HT than in non-IgG4 HT (all P < 0.001). IgG4 colocalized with MBL by immunofluorescence. In mice, follicular cell structure disruption was observed after the injection of IgG4 from IgG4 HT, as well as the colocalization of IgG4 with MAC. High levels of TgAb IgG4 glycosylation patterns, including monogalactose glycan (G1F), galactose-deficient glycan (G0F), and high-mannose glycan (M5), were detected in IgG4 HT. After deglycosylation, IgG4 reduced its ability to bind to MBL, and there was low MBL and MAC activation in thyrocytes.
Conclusion
High levels of IgG4 glycosylation patterns, including G1F, G0F, and M5, may activate the complement lectin pathway, thereby participating in the pathogenesis of IgG4 HT.
Introduction
Hashimoto’s thyroiditis (HT) is an organ-specific autoimmune disease characterized by increased levels of thyroglobulin antibody (TgAb) and thyroid peroxidase antibody (TPOAb), which primarily belong to the IgG class (1). Immunoglobulin (Ig) G4 HT is a subtype of HT, first reported by Li et al. (2) in 2009. It is characterized by an abundance of IgG4-positive plasma cells and fibrosis in the thyroid, similar to the pathological characteristics of IgG4-related disease (IgG4-RD) (3). IgG4 HT exhibits rapid progression, a tendency towards subclinical hypothyroidism, and a predisposition to co-occur with papillary thyroid carcinoma (4). Therefore, IgG4 HT is an important subtype of HT. It is essential to explore its pathogenesis.
The abundance of IgG4-positive plasma cells in the thyroid of IgG4 HT patients suggests that IgG4 antibodies may play an important role in its pathogenesis. IgG4 is traditionally known as a poor activator of immune responses, mainly attributed to its reduced binding to C1q and a process termed Fab-arm exchange (5, 6). Nevertheless, in certain diseases, such as pemphigus vulgaris (7), idiopathic membranous glomerulonephritis (8), and muscle-specific kinase myasthenia gravis (9), IgG4 antibodies function as tissue-destructive autoantibodies. Similar tissue damage in the pancreas was induced in experimental mice after the passive transfer of IgG4 from patients with IgG4-RD (10), but the pathogenic mechanism of IgG4 has not been thoroughly studied, especially in IgG4 HT.
IgG is a type of glycoprotein, and N-glycosylation presents in the Fab and Fc fragments of all IgG subclasses (11, 12, 13). Notably, the attached glycans regulate the stability of IgG and its effector functions (14, 15). Our previous study demonstrated that the glycosylation level of TgAb IgG in HT patients was higher than that in healthy individuals (16). However, there are no studies on IgG4 glycosylation in IgG4 HT patients.
Moreover, altered N-glycans of IgG4 are associated with hypocomplementemia in patients with IgG4-RD (17). It is common to find deposits of both IgG4 antibodies and complement components in tissue biopsy samples from patients with IgG4-RD (18, 19, 20). Although IgG4 cannot activate the complement classical pathway (21), it might activate the lectin pathway and the alternative pathway (22, 23, 24), leading to the formation of the membrane attack complex (MAC) to induce target cell lysis or activation (25, 26). We previously published our findings on the activation of the three complement pathways in HT (27). However, the role of IgG4 antibodies in complement activation in IgG4 HT and the related mechanisms remain unclear.
Therefore, the aim of our study was to explore whether IgG4 glycosylation promotes the activation of the complement system in thyroid tissues and contributes to the pathogenesis of IgG4 HT.
Materials and methods
Patient selection
Thyroid tissues and preoperative serum samples from 35 patients with HT at Peking University First Hospital between 2011 and 2013 were collected. The patients were diagnosed with HT after bilateral total or subtotal thyroidectomy by pathological analyses. Three of the 35 patients had HT alone, and the remaining 32 were diagnosed with HT with PTC. In our previous study (28), these patients were classified as having IgG4 HT (n = 18) and non-IgG4 HT (n = 17) based on immunohistochemical staining of IgG4 and IgG. Normal thyroid tissues contralateral to or distant from the tumors from 18 patients diagnosed with microscopic PTC alone were used as the control (CON) group. Serum samples were also obtained from healthy donors (HD) (TgAb- and TPOAb-negative, with normal thyroid function and ultrasound results). All serum samples were stored at −80°C for further analysis.
This study complied with the Helsinki Declaration and was approved by the Ethics Committee of Peking University First Hospital (2019-095). Written consent has been obtained from each patient or subject after a full explanation of the purpose and nature of all procedures used.
Detection of serum TgAb IgG4 and TPOAb IgG4
The procedures were similar to those used in our previous study (4). Briefly, 96-well plates were coated with different antigens (4 μg/mL thyroglobulin; Calbiochem Merck KGaA; 2 μg/mL thyroid peroxidase; Diarect AG). Serum samples were diluted and incubated for 30 min. Horseradish peroxidase (HRP)-labeled anti-human IgG4 (HP6025; Southern Biotech; 1:5000 for TgAb IgG4 and 1:1000 for TPOAb IgG4) was added.
Immunohistochemistry and immunofluorescence of thyroid tissues from IgG4 HT patients, non-IgG4 HT patients and controls
To investigate complement pathway activation and the IgG4-complement interaction, immunohistochemical and immunofluorescence experiments were conducted on thyroid tissues from IgG4 HT patients, non-IgG4 HT patients, and controls. C1q and mannose binding lectin (MBL) are the major components that trigger the activation of the classical pathway and lectin pathway, respectively. Bb represents alternative pathway activation (29). C4d represents the activation of the classical pathway and lectin pathway. C3d is generated from all three pathways. Additionally, MAC is the terminal product of complement activation. Primary antibodies against C1q (1:100, ab216979, Abcam), Bb (1:30, A227, Quidel, San Diego, CA, USA), MBL (1:100, ab203303, Abcam), C3d (1:100, ab136916, Abcam), C4d (1:100, ab87424, Abcam), and MAC (1:250, ab55811, Abcam) were used. Immunostaining for each slide was scored according to the staining intensity and percentage of positive cells according to our previous study (27). The sections were assessed by two pathologists who were blinded to each other’s assessments. If there was any inconsistency, it was resolved through negotiation.
Preparation of purified IgG4 and TgAb IgG4
A CaptureSelect™ IgG4 affinity matrix (#2942902005, Thermo Fisher Scientific) was used for IgG4 purification of the serum samples from IgG4 HT patients and healthy donors. After verifying the purity of IgG4 (Supplemental Figure 1, see section on supplementary materials given at the end of this article), we prepared a thyroglobulin (Tg) affinity chromatography column, as described in a previous study, to purify TgAb IgG4 from the total IgG4 of IgG4 HT patients (16). All the purified IgG4 and TgAb IgG4 samples were stored at −80°C.
Animal model
Neonatal C57/6J mice (<24 h old) were obtained from Beijing Vital River Lab Animal Technology Co., Ltd. Neonatal mice are immunocompromised and seem to be immunologically incapable of clearing exogenous antibodies (30). Purified pooled IgG4 from IgG4 HT patients or HD was intraperitoneally injected into neonatal mice (n = 5 in each group) via an insulin needle at a single dose of 10 mg human purified IgG4 per gram body weight in 50 µL of PBS, and another five mice were injected with 50 μL of PBS as controls. All the mice were killed 24 h after injection, and the thyroid tissues were obtained and stained with hematoxylin and eosin (H&E) to observe their histopathological changes. Animal experiments were performed with the approval of the institutional animal ethics committee of Peking University First Hospital (J2023035).
Immunohistochemistry and immunofluorescence of mouse thyroid tissues
To investigate the infiltration of IgG4, MAC, and CD45-positive lymphocytes in the thyroid tissues of mice following IgG4 injection, an immunohistochemical study was performed, similar to the Methods section 3. Moreover, an immunofluorescence study was then performed to demonstrate the interaction between IgG4 and the complement system in thyroid tissues. The primary antibodies used were anti-TPO (1:5000, ab278525, Abcam), anti-human IgG4 (1:500, HP6025, Origene, Rockville, MD, USA), anti-MAC (1:500, bs-2673R, Bioss, Beijing, China), and anti-CD45 (1:5000, ab208022, Abcam).
Glycosylation analysis of TgAb IgG4
Our previous study found the level of TgAb IgG4 was significantly elevated in IgG4 HT (4, 31). To clarify the glycosylation patterns of TgAb IgG4 rather than total IgG4 from IgG4 HT patients, MALDI-TOF-MS was applied according to our prior study (16). In brief, TgAb IgG4 underwent reduction and carboxymethylation, followed by dialysis in Ambic buffer. After incubation with trypsin (Promega), the resulting glycopeptides were purified and underwent sequential elution. After lyophilization, the glycopeptides were digested with PNGase F (New England BioLabs, Ipswich, MA, USA) for 24 h at 37°C. N-glycans were isolated and purified with a Sep-Pak C18 96-well plate. The structures of the N-glycans were analyzed using an Axima MALDI Resonance mass spectrometer (Shimadzu, Kyoto, Japan). Serum samples from all 18 IgG4 HT patients were mixed for analysis due to the low concentration of antigen-specific IgG4 in each patient.
IgG4 deglycosylation
Purified IgG4 antibodies (1 μg/μL) from IgG4 HT patients were treated with PNGase F (P0704S, New England BioLabs) for 2 h at 37°C, and the volume ratio of IgG4 to PNGase F was maintained at 20:1. Then, the resulting mixture was separated from PNGase F using a protein A centrifugal column (89952, Thermo Fisher Scientific) to obtain the purified deglycosylated IgG4.
MBL-IgG4 ELISA
To assess the binding capacity of IgG4 and deglycosylated IgG4 to MBL, commercial MBL protein (10405-HNASL, Beijing, China) was coated (4 μg/mL) onto an ELISA plate. IgG4 and deglycosylated IgG4 were added (5 μg/mL) in the presence of TBST/Ca2+ (0.05% Tween 20, 10 mM CaCl2). The binding of IgG4 was subsequently detected with a rabbit anti-IgG4 antibody (1:2000, ab109493, Abcam) and an HRP-conjugated anti-rabbit IgG antibody (1:10000, ZB-2301, ZSGB-BIO).
Cell immunofluorescence after IgG4 stimulation
The conditionally immortalized human thyroid cell line Nthy-ori 3-1 (CL-0817, Procell, Wuhan, China) was cultivated and then seeded on coverslips (Thermo Scientific) after being passaged four times. IgG4, deglycosylated or not, from IgG4 HT patients was added at a concentration of 100 μg/mL in RPMI-1640 supplemented with 5% normal human serum (NHS) or 5% inactivated NHS for 1 h at 37°C. PBS was added as a control. The cells were fixed and permeabilized. IgG4, MBL, or MAC were examined by immunofluorescence as described in Methods section 3.
Statistical analysis
The results are presented as the mean ± S.D. for normally distributed data or the median and interquartile range (IQR) for nonnormally distributed data. For normally distributed data, unpaired t tests for two groups and one-way ANOVA for multiple groups were used, while nonparametric tests (Mann‒Whitney U test and Kruskal‒Wallis test) were used for nonnormally distributed data. Enumeration data were compared using a χ2 test or Fisher’s exact test. Analyses were performed using the SPSS version 26 (SPSS, Inc.) statistical package, and P < 0.05 was considered to indicate statistical significance.
Results
General information of the participants
As shown in Table 1, the thyroid function, levels of TgAb, TPOAb, TgAb IgG4, and TPOAb IgG4, and ultrasound features were significantly different among IgG4 HT, non-IgG4 HT, and control groups. Furthermore, compared with non-IgG4 HT patients, more IgG4 HT patients developed hypothyroidism (3/18 vs 0/17, P < 0.05). In IgG4 HT group, the levels of TgAb, TPOAb, TgAb IgG4, and TPOAb IgG4 were significantly higher than those in the non-IgG4 HT group (P < 0.05). Ultrasound examinations revealed that IgG4 HT was less likely to exhibit equal echogenicity compared to non-IgG4 HT (1/18 vs 8/17, P < 0.05). However, there was no discernible difference in the presence of pseudonodules. The patients with IgG4 HT didn’t suffer from other autoimmune diseases and had no evidence of extra-thyroid IgG4-RD-related organ damage.
Demographic data of IgG4 HT, non-IgG4 HT, and control groups. Age, disease duration, and levels of TgAb, TPOAb, TgAb IgG4, and TPOAb IgG4 levels are presented as median (Q1, Q3) values. The levels of TgAb IgG4 and TPOAb IgG4 are shown by the OD values.
| Groups | IgG4 HT (n = 18) | Non-IgG4 HT (n = 17) | Control (n = 18) | P |
|---|---|---|---|---|
| Age (years) | 35 (30, 47) | 47 (39, 54) | 45 (36, 54) | 0.038 |
| Gender (F/M) | 16/2 | 15/2 | 10/7 | 0.056 |
| Disease duration (month) | 2 (1, 9) | 12 (1, 48) | 0.456 | |
| Goiter (−/I°/II°) | 3/14/1 | 6/11/0 | 0.154 | |
| Other autoimmune disease (+/−) | 0/18 | 0/17 | >0.999 | |
| Extra-thyroid IgG4-RD (+/−) | 0/18 | 0/17 | >0.999 | |
| Ultrasound (diffuse low echogenicity/equal echogenicity/mixed echogenicity | 2/1/15 | 0/8/9 | 0.012 | |
| Presence of pseudonodules (+/−) | 11/7 | 8/9 | 0.404 | |
| Thyroid function (normal/hypothyroidism/ hyperthyroidism) | 11/3/4a, b | 17/0/0 | 18/0/0 | 0.016 |
| TPOAb (IU/mL) | 110.6 (26.4, 209.4)a,b | 43.89 (12.3, 166.7)a | 10 (10, 10) | 0.005 |
| TgAb (IU/mL) | 421.3 (229.4, 896.2)a,b | 181.8 (137.8, 416.4)a | 10.6 (9.3, 12.3) | 0.008 |
| TPOAb IgG4 | 1.01 (0.17, 1.60)b | 0.10 (0.10, 0.21) | 0.003 | |
| TgAb IgG4 | 0.54 (0.23, 1.03)b | 0.14 (0.10, 0.24) | 0.004 |
aP < 0.05 compared with CON; bP < 0.05 compared with non-IgG4 HT.
HT, Hashimoto’s thyroiditis; IgG4-RD, IgG4 related disease; TgAb, thyroglobulin antibody; TPOAb, thyroid peroxidase antibody.
Deposition of complement components in thyroid tissues
In the thyroid tissues of both IgG4 HT patients and non-IgG4 HT patients, positive staining for MAC, C3d, C4d, C1q, MBL, and Bb was observed, which suggested that all three complement pathways were activated. No positive staining for any complement component except C4d was detected in the controls (Fig. 1). As shown in Fig. 2, the immunostaining scores of all complement components in the IgG4 HT group were significantly higher than those in the control group (all P < 0.001), while the staining intensities of MBL, Bb, C3d, C4d, and MAC in IgG4 HT group were also higher than those in the non-IgG4 HT group (all P < 0.001). These findings suggested that the lectin pathway and the alternative pathway were more strongly activated in IgG4 HT patients than in non-IgG4 HT patients. Colocalization of IgG4 with MBL was detected in the thyroid follicular epithelial cells of the IgG4 HT patients by immunofluorescence (Fig. 3).
Immunohistochemical staining of the complement components MAC, C3d, C4d, C1q, MBL, and Bb in thyroid tissues from IgG4 HT patients. All complement components were identified in thyroid tissues from IgG4 HT patients and non-IgG4 HT patients. Only C4d was detected in thyroid tissues from normal controls without other complements. Bars = 50 μm. Representative photos are shown.
Citation: European Thyroid Journal 13, 5; 10.1530/ETJ-24-0156
Immunohistochemical staining scores of complement components in the thyroid tissues of IgG4 HT, non-IgG4 HT, and control groups. Each dot represents one patient. Significance was determined by a nonparametric test with the post hoc Bonferroni correction. The data are presented as the median (Q1, Q3) values. *P<0.05.
Citation: European Thyroid Journal 13, 5; 10.1530/ETJ-24-0156
Colocalization of MBL and IgG4 in the thyroid tissues of IgG4 HT patients. IgG4 colocalized with MBL on thyroid follicular epithelial cells. The IgG4 stain was green, and the MBL stain was red. Representative images were captured using a Leica DMi8 confocal microscope. Bars = 10 μm.
Citation: European Thyroid Journal 13, 5; 10.1530/ETJ-24-0156
IgG4 deposition and colocalization with MAC in the mouse thyroid after IgG4 injection
To confirm that complement activation was induced by IgG4, we injected purified IgG4 from IgG4 HT patients or HD into neonatal mice. Edematous changes in the interfollicular space, disruption of follicular cell structure, and hemorrhage were observed in thyroid tissues in the IgG4 HT group but not in the HD group or PBS group (Fig. 4). IgG4 deposition and CD45-positive lymphocyte infiltration in thyroid tissues were detected in 3 out of 5 mice in the IgG4 HT group and one out of 5 mice in the HD group. However, mice injected with PBS did not exhibit such changes (Supplementary Table 1). Additionally, MAC deposition was detected in 2 out of 5 mice in the IgG4 HT group, and MAC colocalization with IgG4 was detected (Fig. 5). However, such deposition and colocalization were not found in mice injected with IgG4 from HD or PBS. These findings suggested that IgG4 from IgG4 HT may be involved in thyroid destruction by interacting with the complement system.
H&E and immunohistochemical staining of human IgG4, MAC, and CD45 in thyroid tissues. (A) Thyroid tissues from mice injected with IgG4 from IgG4 HT patients; (B) Thyroid tissues from mice injected with IgG4 from healthy controls; (C) Thyroid tissues from mice injected with PBS. Edematous changes in the interfollicular space, follicular cell structure disruption, and hemorrhage were observed in A, while thyroid structure integrity was maintained in B and C. IgG4 and MAC deposited on scattered thyroid follicular epithelial cells, along with dispersed CD45-positive lymphocytes in the surrounding area, are visualized in A. Scale bars: 20 μm. Representative photos are shown.
Citation: European Thyroid Journal 13, 5; 10.1530/ETJ-24-0156
IgG4 and MAC colocalization in mouse thyroid tissues injected with IgG4 from IgG4 HT patients. (A) mouse thyroid tissues injected with IgG4 from IgG4 HT patients, (B) mouse thyroid tissues injected with IgG4 from healthy controls, (C) mouse thyroid tissues injected with PBS. The IgG4 stain was green, and the MAC was red. IgG4 colocalized with MBL on thyroid follicular epithelial cells in A. Representative images were captured using a Leica DMi8 confocal microscope and a 63X objective lens.
Citation: European Thyroid Journal 13, 5; 10.1530/ETJ-24-0156
TgAb IgG4 from IgG4 HT patients exhibited high levels of glycosylation pattern that could activate the lectin pathway
Our previous study found that thyroid-specific IgG4 antibodies were significantly elevated in IgG4 HT (4, 31). To explore the effect of glycosylation on the pathogenic role of TgAb IgG4, we next analyzed the glycosylation pattern of TgAb IgG4, rather than total IgG4, isolated from mixed serum samples of IgG4 HT patients. Ten glycosylation patterns were discovered (Fig. 6), and the mass-to-charge ratios corresponding to the three glycosylation patterns with the highest peaks were 2040.025, 1835.925, and 1579.783, which were G1F (monogalactose glycan), G0F (galactose-deficient glycan), and M5 (high-mannose type glycan), respectively (Fig. 7). The lectin pathway can be activated by fucose, mannose, and GlcNAc-terminated glycans (32, 33). Consequently, the above glycosylation patterns of TgAb IgG4 may facilitate its binding to MBL and initiate the activation of the lectin pathway.
Glycosylation patterns of TgAb IgG4 and relative quantity. Ten glycosylation patterns of TgAb IgG4 from IgG4 HT patients were detected by MALDI-TOF-MSn. The relative quantity of each glycan varied. Blue square, N-acetylglucosamines; red triangle, fucose; green circle, mannose; yellow circle, galactose.
Citation: European Thyroid Journal 13, 5; 10.1530/ETJ-24-0156
The details of TgAb IgG4 N-glycans.
Citation: European Thyroid Journal 13, 5; 10.1530/ETJ-24-0156
The binding affinity of IgG4 for MBL was significantly reduced after deglycosylation
As shown in Fig. 8, the deglycosylation of IgG4 significantly weakened its binding affinity for MBL (P<0.01) in ELISA, suggesting that the binding of IgG4 to MBL is dependent on N-glycosylation.
Comparison of the binding affinity of IgG4 in different states with MBL. IgG4 deglycosylation significantly weakened its binding affinity for MBL. *P <0.01.
Citation: European Thyroid Journal 13, 5; 10.1530/ETJ-24-0156
MBL binding and complement activation by IgG4 is glycosylation-dependent
Subsequently, we performed in vitro experiments to elucidate the ability of IgG4 glycosylation to activate the complement system. After the cells were stimulated with purified IgG4 from IgG4 HT patients, IgG4 was found to bind to permeabilized thyroid cells and colocalize with MBL or MAC, as demonstrated by immunofluorescence (Fig. 9). In contrast, little deposition of MBL and MAC was detected on cells incubated with deglycosylated IgG4 in the presence of NHS serving as a source of complement. Additionally, weak MBL fluorescence and no MAC fluorescence were observed in the absence of complement (inactivated NHS).
Immunofluorescence analysis of complement deposition and its colocalization with IgG4 under various experimental conditions. The cells were treated with total IgG4 isolated from IgG4 HT patients (A) at a final concentration of 100 μg/mL with 5% NHS as a complement source, deglycosylated IgG4 from the same IgG4 HT patients with 5% NHS (B), IgG4 from the same IgG4 HT patients with 5% inactivated NHS (C), or PBS with 5% inactivated NHS (D). The cells were then fixed, permeabilized, and stained for IgG4 (green), MBL (red), or MAC (red). IgG4 was found to bind to cells and colocalize with MBL or MAC (A). Little MBL and MAC deposition was detected on cells (B). Weak MBL fluorescence and no MAC fluorescence were observed in the absence of complement (C). Representative images were captured using a Leica DMi8 confocal microscope and a 63× objective lens.
Citation: European Thyroid Journal 13, 5; 10.1530/ETJ-24-0156
Discussion
In this study, we first found that IgG4 antibodies might participate in the pathogenesis of IgG4 HT. High levels of TgAb IgG4 glycosylation patterns, such as G1F, G0F, and M5, may induce complement activation through the lectin pathway, thereby participating in the pathogenesis of IgG4 HT (Fig. 10).
Schematic of the role of IgG4 glycosylation mediated complement activation in the pathogenesis of IgG4 Hashimoto’s thyroiditis. MBL, mannose-binding lectin; MAC, membrane attack complex. IgG4 thyroid-autoantibodies bind complement MBL by its glycosylation, which in turn activates the complement system to form MAC and may ultimately damage thyrocytes.
Citation: European Thyroid Journal 13, 5; 10.1530/ETJ-24-0156
IgG4 HT is characterized by the infiltration of abundant IgG4-positive lymphocytes into thyroid tissues (2). The present study revealed higher levels of serum TgAb IgG4 and TPOAb IgG4 in IgG4 HT patients than in non-IgG4 HT patients, similar to previous studies (4, 31), therefore, we hypothesized that the IgG4 antibody may be involved in the pathogenesis of IgG4 HT. Furthermore, compared with non-IgG4 HT, the complement lectin pathway and the alternative pathway were significantly activated in IgG4 HT. The alternative pathway can be activated as long as any of the three pathways is activated to form C3b, without dependence on the antibody (34). Thus, complement activation, especially the lectin pathway, may participate in the pathogenesis of IgG4 HT.
In vivo, thyrocyte destruction was induced after injection of IgG4 from IgG4 HT in mice in the current study, similar to the findings of a study on autoimmune pancreatitis (10). Additionally, the co-deposition of MAC and IgG4 in mouse thyroid tissues suggested that IgG4 may contribute to thyrocyte injury by activating the complement system. Finally, the colocalization of IgG4 with MBL in the thyroid of IgG4 HT patients confirmed our hypothesis. Similarly, George’s study showed that IgG4 from primary membranous nephropathy (pMN) patients facilitated complement activation through the lectin pathway (24). Thus, the involvement of IgG4 antibodies in the pathogenesis of IgG4 HT may be mediated through the interaction of IgG4 with MBL.
We further explored the ways in which IgG4 bound to MBL and activated the complement system. The interaction of IgG with MBL has been ascribed to terminal GlcNAc and mannose residues of the N-linked glycan in the IgG γ chain (32, 33). George et al. (24) demonstrated that IgG4 with galactose-deficient glycans (G0) activated the lectin pathway and induced podocyte injury in pMN. In rheumatoid arthritis, G0 glycan enabled IgG to activate complement via the lectin pathway (32). In IgG4 HT, Inomata et al. (35) reported that serum IgG4 antibodies from IgG4 HT patients recognized Tg and its isoforms as autoantigens mainly. As TgAb is produced by thyroid-derived lymphocytes, B cells at the site of chronic inflammation might be the major source of differentially glycosylated (auto)antibodies (36), thyroid-specific IgG4 antibodies rather than total IgG4 were obtained to elucidate their glycosylation patterns in the present study. TgAb IgG4 from IgG4 HT patients were confirmed to exhibit high levels of M5 and GlcNAc-terminated glycoforms (G0F and G1F), which are capable of activating the lectin pathway.
IgG4 antibodies vary in Fab segments recognizing different antigens, while Fc γ4 segments remain identical, where the majority of N-glycans are occupied (37). Therefore, purified total IgG4 was used to perform validation trials in vitro due to the limited amount of TgAb IgG4. It confirmed that the glycosylation of IgG4 played a key role in complement activation.
To our knowledge, this is the first report showing the important role of IgG4 isolated from IgG4 HT patients in the pathogenesis of IgG4 HT. Moreover, the glycosylation patterns of TgAb IgG4 in IgG4 HT were identified for the first time. Glycosylation promoted complement activation mediated by IgG4 antibodies. These findings contribute to enhancing our understanding of IgG4 HT pathogenesis and offer novel therapeutic targets for preventing the progression of HT.
There are several limitations in our research. First, the surgical thyroid samples were acquired between 2011 and 2013. Though it has been proved that most antigens, besides the nuclear antigens, in formalin-fixed, paraffin-embedded tissues are well preserved over several decades (38), the influence of long storage cannot be ruled out. Second, for the glycosylation analysis, starting material consisting of at least 100 μg of protein before enrichment was reasonable (39, 40). Since only a few or up to a dozen micrograms of TgAb IgG4 can be isolated from the serum sample of each patient, mixed sera were used for purification of TgAb IgG4 and subsequent glycosylation analysis. Also, there is much less TgAb IgG4 in the serum samples from healthy controls compared with IgG4 HT patients, so the evaluation of TgAb IgG4 glycosylation of healthy controls was not performed.
In conclusion, high levels of IgG4 glycosylation patterns, specifically G1F, G0F, and M5, activate the complement lectin pathway, thereby participating in the pathogenesis of IgG4 HT.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/ETJ-24-0156.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the study reported.
Funding
This work was supported by the National Natural Science Foundation of China (grant nos 8190032271 and 82170801).
Author contribution statement
CZ: conceptualization, methodology, formal analysis, investigation, resources, writing - original draft, and project administration. ZS:conceptualization, methodology, formal analysis, writing - review and editing. SW, methodology, formal analysis, and investigation. JZ, GL and LC:methodology. JL:methodology, resources. YY:conceptualization, methodology, resources, and investigation. YG:conceptualization, project administration, writing - review and editing, and funding acquisition.
Acknowledgements
We would like to thank Professor Yan Li and Professor Chuncui Huang from the Chinese Academy of Sciences for their help in performing glycosylation mass spectrometry of IgG4 antibodies.
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