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Monomeric serum immunoglobulin A (IgA) can contribute to the development of various autoimmune diseases, but the regulation of serum IgA effector functions is not well defined. Here, we show that the two IgA subclasses (IgA1 and IgA2) differ in their effect on immune cells due to distinct binding and signaling properties. Whereas IgA2 acts pro-inflammatory on neutrophils and macrophages, IgA1 does not have pronounced effects. Moreover, IgA1 and IgA2 have different glycosylation profiles, with IgA1 possessing more sialic acid than IgA2. Removal of sialic acid increases the pro-inflammatory capacity of IgA1, making it comparable to IgA2. Of note, disease-specific autoantibodies in patients with rheumatoid arthritis display a shift toward the pro-inflammatory IgA2 subclass, which is associated with higher disease activity. Taken together, these data demonstrate that IgA effector functions depend on subclass and glycosylation, and that disturbances in subclass balance are associated with autoimmune disease. Immunoglobulin A (IgA) has two subclasses, IgA1 and IgA2, but differential effects on inflammation are unclear. Here the authors show that IgA2, when compared with IgA1, has stronger pro-inflammatory functions associated with changed glycosylation and higher disease scores in patients with rheumatoid arthritis.

Krönke and colleagues show that the cytokine IL-23 controls the glycosylation profile and inflammatory activity of autoantibodies through control of sialyltransferase activity in plasma cells mediated by the T H 17 subset of helper T cells. The checkpoints and mechanisms that contribute to autoantibody-driven disease are as yet incompletely understood. Here we identified the axis of interleukin 23 (IL-23) and the T H 17 subset of helper T cells as a decisive factor that controlled the intrinsic inflammatory activity of autoantibodies and triggered the clinical onset of autoimmune arthritis. By instructing B cells in an IL-22- and IL-21-dependent manner, T H 17 cells regulated the expression of β-galactoside α2,6-sialyltransferase 1 in newly differentiating antibody-producing cells and determined the glycosylation profile and activity of immunoglobulin G (IgG) produced by the plasma cells that subsequently emerged. Asymptomatic humans with rheumatoid arthritis (RA)-specific autoantibodies showed identical changes in the activity and glycosylation of autoreactive IgG antibodies before shifting to the inflammatory phase of RA; thus, our results identify an IL-23–T H 17 cell–dependent pathway that controls autoantibody activity and unmasks a preexisting breach in immunotolerance.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family members mediate receptor- and tissue-specific sequestration of infected erythrocytes (IEs) in malaria. Antibody responses are a central component of naturally acquired malaria immunity. PfEMP1-specific IgG likely protects by inhibiting IE sequestration and through IgG-Fc Receptor (FcγR) mediated phagocytosis and killing of antibody-opsonized IEs. The affinity of afucosylated IgG to FcγRIIIa is up to 40-fold higher than fucosylated IgG, resulting in enhanced antibody-dependent cellular cytotoxicity. Most IgG in plasma is fully fucosylated, but afucosylated IgG is elicited in response to enveloped viruses and to paternal alloantigens during pregnancy. Here we show that naturally acquired PfEMP1-specific IgG is strongly afucosylated in a stable and exposure-dependent manner, and efficiently induces FcγRIIIa-dependent natural killer (NK) cell degranulation. In contrast, immunization with a subunit PfEMP1 (VAR2CSA) vaccine results in fully fucosylated specific IgG. These results have implications for understanding protective natural- and vaccine-induced immunity to malaria. Here, Larsen et al. describe differences in Fc fucosylation of P. falciparum PfEMP1-specific IgG produced in response to natural infection versus VAR2CSA-type subunit vaccination, which leads to differences in the ability to induce FcγRIIIa-dependent natural killer cell degranulation.

Immunglobulin G (IgG) sialylation represents a key checkpoint that determines the engagement of pro- or anti-inflammatory Fcγ receptors (FcγR) and the direction of the immune response. Whether IgG sialylation influences osteoclast differentiation and subsequently bone architecture has not been determined yet, but may represent an important link between immune activation and bone loss. Here we demonstrate that desialylated, but not sialylated, immune complexes enhance osteoclastogenesis in vitro and in vivo . Furthermore, we find that the Fc sialylation state of random IgG and specific IgG autoantibodies determines bone architecture in patients with rheumatoid arthritis. In accordance with these findings, mice treated with the sialic acid precursor N-acetylmannosamine (ManNAc), which results in increased IgG sialylation, are less susceptible to inflammatory bone loss. Taken together, our findings provide a novel mechanism by which immune responses influence the human skeleton and an innovative treatment approach to inhibit immune-mediated bone loss. The IgG sugar moiety modulates the binding of immune complexes to their Fcγ receptors resulting in pro- or anti-inflammatory response. This study shows that IgG sialylation also affects osteoclastogenesis and bone mass in mice and humans, identifying a new link between bone and the immune system.

Glycosylation of the immunoglobulin G (IgG)-Fc tail is required for binding to Fc-gamma receptors (FcγRs) and complement-component C1q. A variety of IgG1-glycoforms is detected in human sera. Several groups have found global or antigen-specific skewing of IgG glycosylation, for example in autoimmune diseases, viral infections, and alloimmune reactions. The IgG glycoprofiles seem to correlate with disease outcome. Additionally, IgG-glycan composition contributes significantly to Ig-based therapies, as for example IVIg in autoimmune diseases and therapeutic antibodies for cancer treatment. The effect of the different glycan modifications, especially of fucosylation, has been studied before. However, the contribution of the 20 individual IgG glycoforms, in which the combined effect of all 4 modifications, to the IgG function has never been investigated. Here, we combined six glyco-engineering methods to generate all 20 major human IgG1-glycoforms and screened their functional capacity for FcγR and complement activity. Bisection had no effect on FcγR or C1q-binding, and sialylation had no- or little effect on FcγR binding. We confirmed that hypo-fucosylation of IgG1 increased binding to FcγRIIIa and FcγRIIIb by ~17-fold, but in addition we showed that this effect could be further increased to ~40-fold for FcγRIIIa upon simultaneous hypo-fucosylation and hyper-galactosylation, resulting in enhanced NK cell-mediated antibody-dependent cellular cytotoxicity. Moreover, elevated galactosylation and sialylation significantly increased (independent of fucosylation) C1q-binding, downstream complement deposition, and cytotoxicity. In conclusion, fucosylation and galactosylation are primary mediators of functional changes in IgG for FcγR- and complement-mediated effector functions, respectively, with galactose having an auxiliary role for FcγRIII-mediated functions. This knowledge could be used not only for glycan profiling of clinically important (antigen-specific) IgG but also to optimize therapeutic antibody applications.

Markers for longevity that reflect the health condition and predict healthy aging are extremely scarce. Such markers are, however, valuable in aging research. It has been shown previously that the N-glycosylation pattern of human immunoglobulin G (IgG) is age-dependent. Here we investigate whether N-linked glycans reflect early features of human longevity. The Leiden Longevity Study (LLS) consists of nonagenarian sibling pairs, their offspring, and partners of the offspring serving as control. IgG subclass specific glycosylation patterns were obtained from 1967 participants in the LLS by MALDI-TOF-MS analysis of tryptic IgG Fc glycopeptides. Several regression strategies were applied to evaluate the association of IgG glycosylation with age, sex, and longevity. The degree of galactosylation of IgG decreased with increasing age. For the galactosylated glycoforms the incidence of bisecting GlcNAc increased as a function of age. Sex-related differences were observed at ages below 60 years. Compared to males, younger females had higher galactosylation, which decreased stronger with increasing age, resulting in similar galactosylation for both sexes from 60 onwards. In younger participants (<60 years of age), but not in the older age group (>60 years), decreased levels of non-galactosylated glycoforms containing a bisecting GlcNAc reflected early features of longevity. We here describe IgG glycoforms associated with calendar age at all ages and the propensity for longevity before middle age. As modulation of IgG effector functions has been described for various IgG glycosylation features, a modulatory effect may be expected for the longevity marker described in this study.

Sepsis is a life-threatening condition with a rising disease burden worldwide. It is a multifactorial disease and is defined as a dysregulated host response to infection. Neutrophils have been shown to be involved in the pathogenesis of sepsis by exacerbating inflammation. However, the exact effector mechanism of action still remains a mystery. Changes in the glycosylation pattern of the immunoglobulin G (IgG) Fc region are described for several diseases including meningococcal sepsis. In this study, we investigated the possible contribution of neutrophils and neutrophil implication, potentially related to degranulation or neutrophil extracellular trap (NET) formation in changing the IgG Fc N-glycosylation pattern in a murine sepsis model. We have measured the serum level of cytokines/chemokines and immunoglobulins, the serum activity of neutrophil elastase (NE), and analyzed the IgG Fc glycosylation pattern by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) and Lectin enzyme-linked immunosorbent assay (ELISA). We observed an increased activity of NE- and neutrophil-associated cytokines such as keratinocyte chemoattractant (KC) with the development of sepsis. Regarding the IgG Fc N-glycosylation, we observed an increase in fucosylation and α1,3-galactosylation and a decrease for sialyation. Interestingly, these changes were not uniform for all IgG subclasses. After depletion of neutrophils, we saw a change in the exposure of fucose and α2,6-linked sialic acid during the time course of our experimental sepsis model. In conclusion, neutrophils can influence changes in the IgG glycosylation pattern in experimental sepsis.

Background Immunoglobulin (Ig) glycosylation modulates the immune response and plays a critical role in ageing and diseases. Studies have mainly focused on IgG glycosylation, and little is known about the genetics and epidemiology of IgA glycosylation. Methods We generated, using a novel liquid chromatography-mass spectrometry method, the first large-scale IgA glycomics dataset in serum from 2423 twins, encompassing 71 N- and O- glycan species. Results We showed that, despite the lack of a direct genetic template, glycosylation is highly heritable, and that glycopeptide structures are sex-specific, and undergo substantial changes with ageing. We observe extensive correlations between the IgA and IgG glycomes, and, exploiting the twin design, show that they are predominantly influenced by shared genetic factors. A genome-wide association study identified eight loci associated with both the IgA and IgG glycomes ( ST6GAL1 , ELL2 , B4GALT1 , ABCF2 , TMEM121 , SLC38A10 , SMARCB1 , and MGAT3 ) and two novel loci specifically modulating IgA O- glycosylation ( C1GALT1 and ST3GAL1 ). Validation of our findings in an independent cohort of 320 individuals from Qatar showed that the underlying genetic architecture is conserved across ancestries. Conclusions Our study delineates the genetic landscape of IgA glycosylation and provides novel potential functional links with the aetiology of complex immune diseases, including genetic factors involved in IgA nephropathy risk.

Background Glycosylation is an important posttranslational modification of proteins and in most cases indispensable for proper protein function. Like most soluble proteins, IgA, the second most prevalent antibody in human serum, contains several N- and O-glycosylation sites. While for IgG the impact of Fc glycosylation on effector functions and inflammatory potential has been studied intensively, only little is known for IgA. In addition, only glimpses exist regarding the regulation of IgA glycosylation. We have previously shown that IgA1 and IgA2 differ functionally and also show differences in their glycosylation pattern. The more pro-inflammatory IgA2 which is linked to autoimmune diseases displays decreased sialylation, galactosylation, fucosylation and bisection as compared to IgA1. In the present study, we aimed to investigate these differences in glycosylation in detail and to explore the mechanisms underlying them. Methods IgA1 and IgA2 was isolated from serum of 12 healthy donors. Site specific glycosylation was analyzed by mass spectrometry. In addition, human bone marrow plasma cells were investigated using single cell mRNA sequencing, flow cytometry and ELISpot. Results We found that certain glycoforms greatly differ in their abundance between IgA1 and IgA2 while others are equally abundant. Overall, the IgA2 glycans displayed a more immature phenotype with a higher prevalence of oligomannose and fewer fully processed glycans. Of note, these differences can’t be explained by differences in the glycosylation enzyme machinery as mRNA sequencing and flow cytometry analysis showed equal enzyme expression in IgA1 and IgA2 producing plasma cells. ELISpot analysis suggested a slightly increased antibody production rate in IgA2 producing plasma cells which might contribute to its lower glycan processing rates. But this difference was only minor, suggesting that further factors such as steric accessibility determine glycan processing. This is supported by the fact that glycans at different positions on the same IgA chain differ dramatically in fucosylation, sialylation and bisection. Conclusion In summary, our detailed overview of IgA1 and IgA2 glycosylation shows a class, subclass, and site-specific glycosylation fingerprint, most likely due to structural differences of the protein backbones.

Protein N-glycosylation patterns are known to show vast genetic as well as physiological and pathological variation and represent a large pool of potential biomarkers. Large-scale studies are needed for the identification and validation of biomarkers, and the analytical techniques required have recently been developed. Such methods have up to now mainly been applied to complex mixtures of glycoproteins in biofluids (e.g. plasma). Here, we analyzed N-glycosylation profiles of alpha1-antitrypsin (AAT) and immunoglobulin A (IgA) enriched fractions by 96-well microtitration plate based high-throughput immuno-affinity capturing and N-glycan analysis using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). Human plasma samples were from the Leiden Longevity Study comprising 2415 participants of different chronological and biological ages. Glycosylation patterns of AAT enriched fractions were found to be associated with chronological (calendar) age and they differed between females and males. Moreover, several glycans in the AAT enriched fraction were associated with physiological parameters marking cardiovascular and metabolic diseases. Pronounced differences were found between males and females in the glycosylation profiles of IgA enriched fractions. Our results demonstrate that large-scale immuno-affinity capturing of proteins from human plasma using a bead-based method combined with high-throughput N-glycan analysis is a powerful tool for the discovery of glycosylation-based biomarker candidates.