IgG Fc is desialylated in RA patients

Human and murine IgG of all subclasses have a conserved N-glycosylation site at Asn297 on their Fc regions (Fig. 1a). N-glycans on IgG Fc have been classified into three subgroups: the first ending with N-acetylglucosamine (GlcNAc) (G0F), the second with galactose on one arm (G1F) and the third with two galactose on both arms (G2F) (Fig. 1a). A minor fraction of galactosylated glycan can be further sialylated with either one (G1FS1 or G2FS1) or two sialic acid residues (G2FS2) (Fig. 1a). Agalactosylated glycoforms (G0F) are frequently observed in serum IgG of RA patients13,16. Likewise, agalactosylated glycoforms are observed in murine IgG in autoimmune-prone mouse strains such as the MRL/lpr strain25,26; however, the glycosylation profiles of RA-associated IgG have not been directly compared in RA patients and mouse models. We assessed RA-associated glycosylation profiles on IgG ACPA and total IgG in ACPA+ RA patients. ACPA+ RA patients showed high amounts of cyclic citrullinated peptide (CCP)-binding IgG (ACPA) but normal total IgG levels in their serum (Fig. 1b). Three types of IgG fractions were purified and subjected to liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MSI) analysis: serum ACPA purified by the binding ability to CCP from ACPA+ RA patients (ACPA/RA), total serum IgG deprived of ACPA from the same RA patients (FT/RA), and total serum IgG from healthy donors (total/HD) (Fig. 1c; Supplementary Fig. 1). To assess the antigen specificity of purified ACPA, the binding of purified ACPA to CCP and arginine-control peptide was compared in parallel (Supplementary Fig. 1). We observed that the binding of purified ACPA to the control peptide was 100-fold lower than that to CCP. Moreover, a competitive ELISA (enzyme-linked immunosorbent assay) confirmed the lack of binding to the control peptide because CCP binding of the purified ACPA was blocked by the addition of CCP but not by control peptide. However, low recovery of antibodies from ACPA-negative RA patients suggests that small amounts of non-ACPA is included in ACPA fraction through the purification process as previously noted27 (Supplementary Fig. 2).

Figure 1: IgG Fc in RA patients is desialylated because of lack of galactosylation. (a) Structure of N-glycosylation forms attached to Asn297 in the IgG Fc portion. Sugar chains of IgG comprise bi-antennary chains, and are further modified by binding bisecting N-acetylglucosamine (GlcNAc), core-fucosylation and terminal α2,6-linkage of sialic acid to galactose. Sia, sialic acid; Gal, galactose; Man, mannose; Fuc, fucose. (b) CCP-binding IgG (ACPA) titres and total IgG levels from RA patients (n=17) or healthy donors (n=12) are presented. Each symbol represents the data from an individual donor. Mean values were presented as bars. ND, not detectable. (c) LC-ESI-MS analysis of IgG1 Fc glycans in total IgG from HDs (Total/HD), and those in the flow-through (FT) of CCP columns (FT/RA) and CCP-binding IgG ACPA (ACPA/RA) from RA patients. Glycoforms of human IgG1 Fc are shown in Supplementary Table 1. pep, peptide moiety (EEQYNSTYR). (d,e) Ratios of sialylated (d) and galactosylated (e) IgG Fc glycans to agalactosylated IgG Fc glycan (G0F) in each group of serum were calculated and plotted. Each circle represents the result from an individual donor. Mean values were presented as bars. Data were analysed by Steel–Dwass non-parametric test (**P<0.01; ***P<0.001). Full size image

Consistent with previous reports16, the frequencies of terminally sialylated glycoforms, G1FS1 (peak 1), G2FS1 (peak 2), and G2FS2 (peak 4) were equivalently reduced in both IgG1 ACPA and total IgG1 from RA patients compared with total IgG1 from healthy donors; no difference was found between ACPA and total IgG1 from RA patients (Fig. 1d). As sialic acid is covalently attached to galactose, reduced sialylation may represent either a lack of sialic acid itself (Fig. 1a, arrow 1) or a lack of galactosylation (Fig. 1a, arrow 2). To discriminate between these possibilities, we analysed the ratios of galactosylated G1F (peak 11) and G2F (peak 15) glycoforms per G0F backbone (peak 8). The frequency of galactosylation was reduced in both ACPA and total IgG1 in RA patients to levels comparable to those with terminal sialylation (Fig. 1e). In fact, galactose-containing glycoforms were equivalently sialylated in all three types of IgG1 (Supplementary Fig. 3). Therefore, these results indicate that the reduced sialylation of Fc primarily reflects a lack of galactose rather than defective terminal sialylation. Glycosylation profiles of IgG2 subclass were consistent with those observed for IgG1, indicating that this is a common event in all IgG classes (Supplementary Fig. 4). It is unlikely that the contaminating non-ACPA antibodies affect the results, because the removal of non-specific antibodies using an arginine-control-peptide-coated column did not demonstrate any difference in the glycan profiles (Supplementary Fig. 5).

IgG Fc is desialylated in arthritis mouse models

The glycosylation profiles of antigen-specific and total IgG1 were assessed in a CIA mouse model after immunization with adjuvanted type II collagens (Col II). We used highly susceptible DBA/1 strains, all of which developed joint inflammation after booster immunization (Fig. 2a). Although the CIA mouse model is widely used to mimic the antibody-dependent process of RA pathogenesis, conflicting reports on ACPA induction in this mouse model exist10,28. Indeed, at least in the experimental conditions defined by us, ACPAs were produced at insufficient levels for glycosylation profiling by mass spectrometry (Fig. 2b). Hence, anti-Col II IgG antibodies, which have arthritogenic activity29,30,31, were purified for glycosylation profiling (Supplementary Fig. 6). We prepared four types of serum IgG fractions for LC-ESI-MS analysis: anti-Col II antibody from CIA mice (αCol II/CIA), total IgG deprived of anti-Col II antibody from the same mice (FT/CIA) and total serum IgG from CFA primed (total/CFA) or naive mice (total/naive) (Fig. 2c; Supplementary Fig. 6b,c). The ratios of sialylated glycoforms were significantly reduced in anti-Col II and total IgG1 of CIA mice relative to naive mice; the reduction was significantly exaggerated in bi-galactosylated forms of anti-Col II IgG1 (Fig. 2d). Sialylation was also reduced in total IgG1 from CFA-injected mice, albeit at an insignificant level. The glycoforms containing terminal galactose residues were comparable among the four groups in non-CIA and CIA mice (Fig. 2e) and similar results were obtained for IgG2a/b subclasses (Supplementary Fig. 7). Thus, these results indicate that poor sialylation of IgG Fc is a feature common to both RA patients and CIA mice, although the underlying mechanisms may not be identical.

Figure 2: Sialylation, but not galactosylation, levels on IgG Fc decreased in CIA mice. (a) An experimental scheme for collagen-induced arthritis (CIA). DBA/1 mice were primed with either bovine Col II (bCol II) in complete Freund’s adjuvant (CFA) or CFA alone on −4 weeks. At day 0, DBA/1 mice were boosted with the same antigens and sera were collected at day 14. (b) CCP2-binding ACPA and anti-Col II antibodies were collected from 50 μl (ACPA) and 1 μl (Col II) of the serum from naive and CIA mice, and then subjected to western blotting using purified mouse IgG (10, 100 and 1,000 ng per each lane) as reference. (c) LS-ESI-MS analysis of IgG1 Fc glycans in the total IgG from naive (total/naive) or CFA-immunized mice (total/CFA) and those in the flow-through of Col II column (FT/CIA) or anti-Col II IgG1 (αCol II/CIA) from CIA-induced mice. Glycoforms of DBA/1 mouse IgG1 Fc are shown in Supplementary Table 2. (d,e) Ratios of sialylated (d) or galactosylated (e) IgG Fc glycans to the agalactosylated IgG Fc glycan (G0F) in the indicated groups were calculated and plotted. Each circle represents the result from an individual mouse. Mean values were presented as bars. Data were analysed by Steel–Dwass non-parametric test (*P<0.05). Full size image

Because CIA mouse model do not completely replicate RA in humans, we decided to analyse another RA model mice which express human HLA-DR4 known to be associated with RA32,33. Immunization of this mouse strain with citrullinated fibrinogen (cFib) induces joint inflammation concomitant with the induction of cFib-specific B- and T-cell responses, mimicking clinical and immunological features of RA patients (Supplementary Fig. 8a). However, similar to that observed with the previous CIA model, ACPAs were again produced at insufficient levels for glycosylation profiling. Therefore, following RA induction, we purified three types of serum IgG fractions for LC-ESI-MS analysis: anti-Fib antibody from cFib-immunized mice (Fib/cFib), total IgG deprived of anti-Fib antibody in the same mice (FT/cFib) and total IgG from naïve mice (total/naive) (Supplementary Figs 8 and 9). As observed for the CIA model, sialylated IgG1 was significantly reduced in cFib-immunized transgenic mice compared with naive transgenic mice, but the galactosylation level was comparable (Supplementary Figs 9 and 10). Thus, the comparative glycosylation profiling of RA patients and two independent mouse models indicates that desialylation of IgG Fc is associated with RA disease in both species, although the changes in Fc-sialylation are also observed in other inflammatory conditions34.

Loss of sialylation exacerbates joint inflammation in CIA

The elevated levels of desialylated IgG observed in RA patients and mouse models led us to investigate the direct link between desialylated IgG and the progression of RA pathogenesis. While degalactosylated IgG has been suggested to exacerbate the pathogenesis of RA17, the role of desialylated IgG in the RA mouse model has not been addressed. Here, we utilized a gene-targeting approach to block the sialylation of endogenously produced IgG. Sialylation of IgG Fc is carried out by the glycosyltransferases, namely, ST6Gal1 and/or ST6Gal2, that catalyse the transfer of a sialic acid to galactose with α2,6-linkage. The expression level of mSt6gal2 gene was much lower than mSt6gal1 in all tissues examined (Supplementary Fig. 11). Therefore, we crossed ST6Gal1f/f with mice expressing Cre recombinase under the control of activation-induced cytidine deaminase gene promoter (AID-Cre) (ref. 35) (Supplementary Fig. 12), which is selectively activated in stimulated B cells. Both homozygous (ST6Gal1f/f AID-Cre+) and control AID-Cre (ST6Gal1+/+ AID-Cre+) mice were immunized with adjuvanted chicken Col II twice to induce CIA (Fig. 3a). To assess the sialylation levels of serum IgG in these mice, we purified both anti-Col II IgG and total IgG from each genotype of mice after CIA induction. Sambucus nigra (SNA) lectin blot and MALDI-TOF-MS analysis revealed the reduced sialylation of total IgG from ST6Gal1−/− and ST6Gal1f/f AID-Cre mice, confirming the major contribution of ST6Gal1 to IgG Fc-sialylation (Fig. 3b; Supplementary Figs 13 and 20). We then analysed anti-Col II IgG titres in CIA mice and observed that homozygous mice produced comparable levels of anti-Col II IgG after priming and boosting of Col II, although total IgG levels were slightly but significantly reduced in homozygous mice (Fig. 3c; Supplementary Fig. 14). Therefore, deficient ST6Gal1 expression in activated B cells did not cause severe defects in B-cell pathways for arthritis-associated IgG production. However, despite equivalent production of arthritis-associated IgG, homozygous mice developed CIA at earlier time points and exhibited a >2-fold higher incidence of CIA compared with control mice (Fig. 3d). More severe joint swellings or ankylosis of the limb were also observed in homozygous mice (Fig. 3e). These data clearly demonstrate that sialylation levels of activated B-cell-intrinsic molecules regulate joint inflammation. Although there are several other candidate molecules (i.e., cytokines) that may be involved in this process, here we focused on sialylation levels of IgG Fc as the reduced sialylation of IgG Fc was common feature in RA patients and mouse models (Figs 1, 2, 3).

Figure 3: Loss of sialylated IgG Fc by gene targeting exacerbates joint inflammation in CIA model. (a) An experimental scheme for CIA induction is presented. Mice of different genotypes were immunized with chicken Col II (cColI II)/CFA at 4-week intervals. Mice were monitored daily for the incidence and scores of arthritis for 14 days. (b) Sialylation levels of anti-Col II IgG and FT from the indicated genotypes were assessed by lectin blot analysis. The locations of heavy (H) and light chains (L) are indicated by arrows. (c) Anti-Col II IgG and total IgG titres in serum were determined by ELISA after first and second immunization. Each circle represents the result from an individual mouse. Mean values were presented as bars. ND, not detectable. The data are representative of three independent experiments. Data were analysed by two-tailed Student’s t test (*P<0.05; ***P<0.001). (d,e) Arthritis frequency (d) and swelling score or ankylosis score (e) in ST6Gal1f/f AID-Cre (n=31) and AID-Cre (n=33) mice. Each circle represents the result from an individual mouse. Mean values were presented as bars. The data are representative of two independent experiments. Arthritis frequency (d) was analysed by two-tailed Fisher’s exact test (*P<0.05; **P<0.01) and swelling scores (e) were analysed by Mann–Whitney non-parametric test (*P<0.05; **P<0.01). Full size image

Sialylation diminishes the arthritogenic activity of ACPAs

ACC4 monoclonal antibody is a mouse ACPA (IgG1) that binds citrullinated Col II, and infusion of this antibody frequently induces collagen antibody-induced arthritis (CAIA) in the presence of the anti-Col II monoclonal antibody M2139 (IgG2b)11. To produce sialylated forms of these antibodies, both mSt6gal1 and mB4galt1 cDNAs were transfected into ACC4 and M2139 hybridomas (Fig. 4a). The majority of terminal sugars of IgG Fc from double-transfected ACC4 and M2139 hybridoma cells were found to extend to α2,6 sialic acid (G2FS2), whereas those from non-transfected ACC4 and M2139 hybridoma cells were ended with terminal galactose or GlcNAc (G0F or G1F) (Fig. 4b; Supplementary Fig. 15). SNA lectin blot analysis also supported increased Fc-sialylation in the transfectants, whereas SNA binding of the Fab fragment remained unaffected (Supplementary Fig. 16). SNA lectin recognizes Neu5Ac(α2–6)Gal/GalNAc residue found in N-linked and O-linked glycans36,37; however, the removal of N-linked glycans by PNGase F treatment selectively abolished SNA binding to the Fc fragment, but not to the Fab fragment even under SDS denaturation (Supplementary Fig. 17). The lack of sequence motif for N-linked glycosylation site (N-X-S/T, X is any residues except proline) in the ACC4 Fab domain further supported that SNA binding to the Fab fragment is mediated by either O-linked glycans or other unidentified molecules, rather than by N-linked glycans. Thus, the overexpression of mSt6gal1 and mB4galt1 produced IgG antibodies with increased sialylation in an Fc-specific manner. Sialylation of both monoclonal antibodies showed no significant impact on the binding ability to antigens (Fig. 4c). To elucidate the arthritogenic activity of sialylated antibodies, we administered ACC4/M2139 IgG-Sia (+) or ACC4/M2139 IgG into DBA/1 mice to induce CAIA (Fig. 4d). As previously reported, the infusion of ACC4/M2139 IgG induced CAIA in approximately half of DBA/1 mice11. However, ACC4/M2139 IgG-Sia (+) failed to induce CAIA in all mice (Fig. 4e). Likewise, histopathological analysis of the joints revealed no signs of inflammation in mice infused with ACC4/M2139 IgG-Sia (+), whereas those with ACC4/M2139 IgG showed the expected arthritis phenotype, i.e., infiltration of monocytes, lymphocytes and granulocytes (Fig. 4f). These results clearly demonstrate that sialylation reduces the pathogenicity of anti-Col II antibodies, including ACPAs.

Figure 4: Enforced sialylation reduces the arthritogenic activity of ACPAs. (a) An experimental scheme for enforced sialylation. (b) LC-ESI-MS analysis of ACC4 and M2139 was performed to detect desialylated glycoforms (G0F, G1F and G2F) and sialylated glycoforms (G1FS1, G2FS1 and G2FS2) of IgG Fc glycans. To normalize the variability, summation of peak areas of all complex type N-glycans were deliberately set at 100%. (c) Antigen-binding ability of Sia (+) and control ACC4/M2139 was compared by ELISA. (d) An experimental scheme for CAIA. (e) Frequency (upper panel) and score (lower panel) of arthritis is plotted. Data for arthritis scores are shown as mean±s.d. (n=13 for ACC4/M2139 and n=10 for ACC4/M2139 Sia (+)). The data are representative of two independent experiments. (f) Histological analysis of joint inflammation at day 14 post-LPS injection. Paraffin sections of the limb were stained by HE (upper panel) and Safranin O/Fast green for cartilage (red) staining (lower panel). Scale bar, 60 μm. Full size image

Sialylated anti-Col II antibody exhibits regulatory activity

Sialylation has been shown to enhance the regulatory activity of IVIG in a K/BxN serum-mediated arthritis model22. This prompted us to assess the regulatory activity of sialylated ACPAs in the RA model. We utilized the CIA model because this model mimics both early autoimmune responses and later antibody-dependent effector phase that triggers the development of joint inflammation. In these experiments, some groups of mice were infused with a cocktail of ACC4/M2139 antibody with or without sialylation one day before boosting for CIA induction (Fig. 5a). Although the infusion of sialylated ACC4/M2139 antibodies reduced the incidence of CIA and that of control antibodies increased the incidence, the difference did not reach statistical significance (Fig. 5b). However, mice treated with sialylated antibodies showed significantly milder arthritis scores than untreated mice or those treated with control antibodies (Fig. 5c,d). To estimate whether disease severity correlates with sialylation levels of anti-Col II IgG antibodies in serum, we compared the sialylation levels of anti-Col II IgG from mice infused with ACC4/M2139 (Fig. 5) and ST6Gal1-deficient mice (Fig. 3) on day 7 after CIA induction (Supplementary Fig. 18). Infusion of control ACC4/M2139 did not alter the sialylation levels of anti-Col II IgG in serum; however, infusion of sialylated forms of ACC4/M2139 significantly increased the sialylation levels of anti-Col II IgG. Additionally, the sialylation levels of anti-Col II IgG from ST6Gal1-deficient mice were below the detection limit. The levels were even lower than those in the control mice without antibody infusion, consistent with the data depicted in Fig. 3b. Thus, sialylation not only reduces the arthritogenic activity of anti-Col II antibodies, including ACPAs, but also induces the regulatory activity in the CIA model.

Figure 5: Sialylated ACPAs exhibit antigen-specific regulatory activity in CIA model. (a) An experimental scheme to evaluate the regulatory activity of infused antibody is presented. (b,c) Frequency (b) and score (c) of arthritis are plotted. Each symbol represents the result from an individual mouse. Data are representative of three independent experiments; these were analysed by Steel–Dwass non-parametric test (*P<0.05). (d) Histological analysis of joint inflammation at day 14 after booster immunization. Paraffin sections of the limb were stained by HE (upper panel) and Safranin O/Fast green for cartilage (red) staining (lower panel). Scale bar, 60 μm. (e) An experimental scheme to evaluate the regulatory activity of infused antibody. (f) Arthritis score of each group of mice is presented. Each symbol represents the result from an individual mouse. Mean values were presented as bars. The data are representative of three independent experiments. Data were analysed by Steel–Dwass non-parametric test (*P<0.05). Full size image

Sialylated IVIG inhibits K/BxN serum-induced arthritis and other autoimmune diseases more potently than non-sialylated IVIG22,34,38,39. Thus, the observed regulatory effects of sialylated ACPAs might simply represent antigen-non-specific processes as provided by IVIG. To address this issue, we sialylated the isotype-matched mouse IgG1 antibody, namely, 1E11 (influenza hemagglutinin specific). IgG1 Fc of both ACC4 and 1E11 were heavily sialylated to similar levels after the introduction of mSt6gal1 and mB4galt1 genes into the hybridomas (Supplementary Fig. 19). Sialylated ACPA or irrelevant IgG (1E11) was co-infused with M2139 and the regulatory activity of these antibodies was examined (Fig. 5e). M2139 was used in both infusions so that the specific effect of the ACPA could be evaluated. Strikingly, a cocktail of sialylated 1E11/M2139 did not show any regulatory activity at all, while that of sialylated ACC4/M2139 did, as measured by the severity of the arthritis score (Fig. 5f). Thus, these data demonstrate that the regulatory activity shown by sialylated ACPA is based on an antigen-specific event.