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. 2016 Apr 5:7:11205.
doi: 10.1038/ncomms11205.

Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis

Yuhsuke Ohmi  1   2 , Wataru Ise  3 , Akira Harazono  4 , Daisuke Takakura  5 , Hidehiro Fukuyama  6 , Yoshihiro Baba  3 , Masashi Narazaki  7 , Hirofumi Shoda  8 , Nobunori Takahashi  9 , Yuki Ohkawa  1   10 , Shuting Ji  1 , Fumihiro Sugiyama  11 , Keishi Fujio  8 , Atsushi Kumanogoh  7 , Kazuhiko Yamamoto  8 , Nana Kawasaki  4   5 , Tomohiro Kurosaki  3   6 , Yoshimasa Takahashi  2 , Koichi Furukawa  1   10
Affiliations

Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis

Yuhsuke Ohmi et al. Nat Commun. .

Abstract

Rheumatoid arthritis (RA)-associated IgG antibodies such as anti-citrullinated protein antibodies (ACPAs) have diverse glycosylation variants; however, key sugar chains modulating the arthritogenic activity of IgG remain to be clarified. Here, we show that reduced sialylation is a common feature of RA-associated IgG in humans and in mouse models of arthritis. Genetically blocking sialylation in activated B cells results in exacerbation of joint inflammation in a collagen-induced arthritis (CIA) model. On the other hand, artificial sialylation of anti-type II collagen antibodies, including ACPAs, not only attenuates arthritogenic activity, but also suppresses the development of CIA in the antibody-infused mice, whereas sialylation of other IgG does not prevent CIA. Thus, our data demonstrate that sialylation levels control the arthritogenicity of RA-associated IgG, presenting a potential target for antigen-specific immunotherapy.

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Conflict of interest statement

K.Y. received financial support or fees from AbbVie, Asahi Kasei, Astellas, BMS, Boehringer Ingelheim, Daiichi-Sankyo, ImmunoFuture, Janssen, MitsubishiTanabe, Pfizer, Sanofi, Santen, Takeda, Teijin, Chugai, Eisai, Ono Taisho Toyama and UCB. K.F. received financial support or fees from Astellas, BMS, Chugai, Daiichi-Sankyo, Eisai, Janssen, MitsubishiTanabe, Pfizer, Santen, Takeda, Taisho Toyama and UCB. The remaining authors declare no competing financial interests.

Figures

Figure 1
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).
Figure 2
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).
Figure 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).
Figure 4
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.
Figure 5
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).
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