In vivo enzymatic modulation of IgG glycosylation inhibits autoimmune disease in an IgG subclass-dependent manner

Abstract

IgG antibodies are potent inducers of proinflammatory responses. During autoimmune diseases such as arthritis and systemic lupus erythematosus, IgG autoantibodies are responsible for the chronic inflammation and destruction of healthy tissues by cross-linking Fc receptors on innate immune effector cells. The sugar moiety attached to the asparagine-297 residue in the constant domain of the antibody is critical for the overall structure and function of the molecule. Removal of this sugar domain leads to the loss of the proinflammatory activity, suggesting that in vivo modulation of antibody glycosylation might be a strategy to interfere with autoimmune processes. In this work, we investigated whether removal of the majority of the IgG-associated sugar domain by endoglycosidase S (EndoS) from Streptococcus pyogenes is able to interfere with autoimmune inflammation. We demonstrate that EndoS injection efficiently removes the IgG-associated sugar domain in vivo and interferes with autoantibody-mediated proinflammatory processes in a variety of autoimmune models. Importantly, however, we observed a differential impact of EndoS-mediated sugar side chain hydrolysis on IgG activity depending on the individual IgG subclass.

Fig. 1.

EndoS-mediated hydrolysis of the IgG-associated glycan moiety in mice. (A) Shown is the fully processed Asn-297 attached sugar moiety of IgG. EndoS cleaves after the first GlcNAc, resulting in the generation of a minimal sugar moiety containing only one GlcNAc with or without a branching fucose residue. (B) Recombinant EndoS was injected i.v. at the indicated amounts followed by detection of the sugar moiety by blotting with LCA. (C) Mice were injected once with 10 μg of purified EndoS, and serum IgG was purified at the indicated time points and analyzed for the presence of the intact sugar moiety by lectin blotting with LCA.

Fig. 2.

IgG subclass-specific effects of EndoS-mediated hydrolysis of the IgG-associated sugar side chain. (A) IgG subclass switch variants of the platelet-specific 6A6 antibody were digested with EndoS and analyzed for the removal of the IgG-associated sugar moiety by lectin blotting with LCA and Coomassie staining. (B) Equal amounts of untreated or EndoS-treated 6A6-IgG subclass switch variants were injected into C57BL/6 mice, and platelet counts were determined before and 4 h after antibody injection. *, P < 0.05. (C) EndoS-treated or untreated 6A6-IgG2a was injected into C57BL/6 (B6) or FcR common γ-chain (g−/−)-deficient mice, and platelet counts were determined as before. Shown is one representative result of two experiments with at least five mice per group; n.s., no significant differences.

Fig. 3.

Effect of EndoS treatment on serum transfer arthritis. (A) Serum from K/BxN mice was incubated with EndoS, and the efficiency of EndoS-mediated removal of the IgG-associated sugar moiety was analyzed by lectin blotting with LCA after purification of IgG from total serum. (B) Groups of five mice were injected with 200 μl of untreated or EndoS-treated K/BxN serum and followed for the development of clinical signs of arthritis. (C) Representative ankle sections of mice at day 6 after injection with untreated or EndoS-treated K/BxN serum. Sections were stained with hematoxylin/eosin to visualize the inflammatory infiltrate. (D) Quantification of the infiltrate area of mice at day 6 after injection with untreated or EndoS-treated K/BxN serum. *, P < 0.05 between experimental groups.

Fig. 4.

Appearance of autoreactive IgG subclasses in BXSB mice. (A) Serum of BXSB mice at 16 or 24 weeks of age was analyzed for the presence of autoantibodies with specificity for dsDNA. (B) The presence of anti-nuclear antibodies of the IgG1 and IgG2b subclasses in the serum of BXSB mice at the indicated age was investigated by an ANA assay using Hep2 cells as described in Materials and Methods.

Fig. 5.

Treatment of autoimmune disease in BXSB mice. (A) Serum IgG and IgM of BXSB mice before (T0) and 24 h after (T24) injection with 10 μg of EndoS was quantified for the presence of the intact IgG sugar moiety by lectin blotting with LCA. (B and C) Serum of untreated and EndoS-treated animals was analyzed for the presence of autoantibodies specific for nuclear antigens (B) and dsDNA (C). Shown are representative results of groups of untreated or EndoS-treated animals (n = 10). Animals that died in the course of the experiment are indicated (#). (D) Survival of untreated mice (n = 10) or animals that received two injections of 10 μg of EndoS at 18 and 26 weeks of age (n = 10).