Effects of allergic diseases and age on the composition of serum IgG glycome in children

Acknowledgements Glycan analysis was partly supported by European Commission GlycoBioM (contract #259869), IBD-BIOM (contract #305479), HighGlycan (contract #278535), MIMOmics (contract #305280), HTP-GlycoMet (contract #324400) and IntegraLife (contract #315997) grants. The SEATON cohort was partly funded by the UK Medical Research Council (contract #80219) and Asthma UK (contract #00/011 and 02/017) grants.Peer reviewedPublisher PD

Minimal B Cell Extrinsic IgG Glycan Modifications of Pro- and Anti-Inflammatory IgG Preparations in vivo

Select residues in the biantennary sugar moiety attached to the fragment crystallizable of immunoglobulin G (IgG) antibodies can modulate IgG effector functions. Thus, afucosylated IgG glycovariants have enhanced cytotoxic activity, whereas IgG glycovariants rich in terminal sialic acid residues can trigger anti-inflammatory effects. More recent evidence suggests that terminal α2,6 linked sialic acids can be attached to antibodies post IgG secretion. These findings raise concerns for the use of therapeutic antibodies as they may change their glycosylation status in the patient and hence affect their activity. To investigate to what extent B cell extrinsic sialylation processes modify therapeutic IgG preparations in vivo, we analyzed changes in human intravenous IgG (IVIg) sialylation upon injection in mice deficient in B cells or in mice lacking the sialyltransferase 1, which catalyzes the addition of α2,6 linked sialic acid residues. By performing a time course of IgG glycan analysis with HILIC-UPLC-FLR (plus MS) and xCGE-LIF our study suggests that therapeutic IgG glycosylation is stable upon injection in vivo. Only a very small fraction of IgG molecules acquired sialic acid structures predominantly in the Fab- but not the Fc-portion upon injection in vivo, suggesting that therapeutic antibody glycosylation will remain stable upon injection in vivo

Periodic changes in the N-glycosylation of immunoglobulin G during the menstrual cycle

Immunoglobulin G (IgG) is the most abundant plasma glycoprotein and a prominent humoral immune mediator. Glycan composition affects the affinity of IgG to ligands and consequent immune responses. The modification of IgG N-glycosylation is considered to be one of the various mechanisms by which sex hormones modulate the immune system. Although the menstrual cycle is the central sex hormone-related physiological process in most women of reproductive age, IgG N-glycosylation dynamics during the menstrual cycle have not yet been investigated. To fill this gap, we profiled the plasma IgG N-glycans of 70 healthy premenopausal women at 12 time points during their menstrual cycles (every 7 days for 3 months) using hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC). We observed cyclic periodic changes in the N-glycosylation of IgG in association with the menstrual cycle phase and sex hormone concentration in plasma. On the integrated cohort level, the modeled average menstrual cycle effect on the abundance of IgG N-glycosylation traits was low for each trait, with the highest being 1.1% for agalactosylated N-glycans. However, intrapersonal changes were relatively high in some cases; for example, the largest difference between the minimum and maximum values during the menstrual cycle was up to 21% for sialylated N-glycans. Across all measurements, the menstrual cycle phase could explain up to 0.72% of the variation in the abundance of a single IgG glycosylation trait of monogalactosylation. In contrast, up to 99% of the variation in the abundance of digalactosylation could be attributed to interpersonal differences in IgG N-glycosylation. In conclusion, the average extent of changes in the IgG N-glycopattern that occur during the menstrual cycle is small; thus, the IgG N-glycoprofiling of women in large sample-size studies can be performed regardless of menstrual cycle phase

Dysregulated Antibody, Natural Killer Cell and Immune Mediator Profiles in Autoimmune Thyroid Diseases.

Funder: FP7 Ideas: European Research Council; Grant(s): 278535, 305280, 324400, 315997The pathogenesis of autoimmune thyroid diseases (AITD) is poorly understood and the association between different immune features and the germline variants involved in AITD are yet unclear. We previously observed systemic depletion of IgG core fucosylation and antennary α1,2 fucosylation in peripheral blood mononuclear cells in AITD, correlated with anti-thyroid peroxidase antibody (TPOAb) levels. Fucose depletion is known to potentiate strong antibody-mediated NK cell activation and enhanced target antigen-expressing cell killing. In autoimmunity, this may translate to autoantibody-mediated immune cell recruitment and attack of self-antigen expressing normal tissues. Hence, we investigated the crosstalk between immune cell traits, secreted proteins, genetic variants and the glycosylation patterns of serum IgG, in a multi-omic and cross-sectional study of 622 individuals from the TwinsUK cohort, 172 of whom were diagnosed with AITD. We observed associations between two genetic variants (rs505922 and rs687621), AITD status, the secretion of Desmoglein-2 protein, and the profile of two IgG N-glycan traits in AITD, but further studies need to be performed to better understand their crosstalk in AITD. On the other side, enhanced afucosylated IgG was positively associated with activatory CD335- CD314+ CD158b+ NK cell subsets. Increased levels of the apoptosis and inflammation markers Caspase-2 and Interleukin-1α positively associated with AITD. Two genetic variants associated with AITD, rs1521 and rs3094228, were also associated with altered expression of the thyrocyte-expressed ligands known to recognize the NK cell immunoreceptors CD314 and CD158b. Our analyses reveal a combination of heightened Fc-active IgG antibodies, effector cells, cytokines and apoptotic signals in AITD, and AITD genetic variants associated with altered expression of thyrocyte-expressed ligands to NK cell immunoreceptors. Together, TPOAb responses, dysregulated immune features, germline variants associated with immunoactivity profiles, are consistent with a positive autoreactive antibody-dependent NK cell-mediated immune response likely drawn to the thyroid gland in AITD

Association of systemic lupus erythematosus associates with decreased immunosuppressive potential of the IgG glycome

Objective: Glycans attached to the Fc portion of IgG are important modulators of IgG effector functions. Interindividual differences in IgG glycome composition are large and they associate strongly with different inflammatory and autoimmune diseases. IKZF1, HLA–DQ2A/B, and BACH2 genetic loci that affect IgG glycome composition show pleiotropy with systemic lupus erythematosus (SLE), indicating a potentially causative role of aberrant IgG glycosylation in SLE. We undertook this large multicenter case–control study to determine whether SLE is associated with altered IgG glycosylation. Methods: Using ultra-performance liquid chromatography analysis of released glycans, we analyzed the composition of the IgG glycome in 261 SLE patients and 247 matched controls of Latin American Mestizo origin (the discovery cohort) and in 2 independent replication cohorts of different ethnicity (108 SLE patients and 193 controls from Trinidad, and 106 SLE patients and 105 controls from China). Results: Multiple statistically significant differences in IgG glycome composition were observed between patients and controls. The most significant changes included decreased galactosylation and sialylation of IgG (which regulate proinflammatory and antiinflammatory actions of IgG) as well as decreased core fucose and increased bisecting N-acetylglucosamine (which affect antibody-dependent cell-mediated cytotoxicity). Conclusion: The IgG glycome in SLE patients is significantly altered in a way that decreases immunosuppressive action of circulating immunoglobulins. The magnitude of observed changes is associated with the intensity of the disease, indicating that aberrant IgG glycome composition or changes in IgG glycosylation may be an important molecular mechanism in SLE

Association of Systemic Lupus Erythematosus With Decreased Immunosuppressive Potential of the IgG Glycome

OBJECTIVE: Glycans attached to the Fc portion of IgG are important modulators of IgG effector functions. Interindividual differences in IgG glycome composition are large and they associate strongly with different inflammatory and autoimmune diseases. IKZF1, HLA–DQ2A/B, and BACH2 genetic loci that affect IgG glycome composition show pleiotropy with systemic lupus erythematosus (SLE), indicating a potentially causative role of aberrant IgG glycosylation in SLE. We undertook this large multicenter case–control study to determine whether SLE is associated with altered IgG glycosylation. METHODS: Using ultra‐performance liquid chromatography analysis of released glycans, we analyzed the composition of the IgG glycome in 261 SLE patients and 247 matched controls of Latin American Mestizo origin (the discovery cohort) and in 2 independent replication cohorts of different ethnicity (108 SLE patients and 193 controls from Trinidad, and 106 SLE patients and 105 controls from China). RESULTS: Multiple statistically significant differences in IgG glycome composition were observed between patients and controls. The most significant changes included decreased galactosylation and sialylation of IgG (which regulate proinflammatory and antiinflammatory actions of IgG) as well as decreased core fucose and increased bisecting N‐acetylglucosamine (which affect antibody‐dependent cell‐mediated cytotoxicity). CONCLUSION: The IgG glycome in SLE patients is significantly altered in a way that decreases immunosuppressive action of circulating immunoglobulins. The magnitude of observed changes is associated with the intensity of the disease, indicating that aberrant IgG glycome composition or changes in IgG glycosylation may be an important molecular mechanism in SLE

Immunoglobulin glycosylation in atopic children

Imunoglobulin G (IgG) smatra se jednim od modulatora imunog odgovora u atopijskim bolestima, najčešće putem interakcija s Fcγ receptorima (FcγR) na površini imunoloških efektorskih stanica, a naročito inhibitornog receptora FcγRIIB. Diferencijalna N-glikozilacija Fc regija IgG utječe na afinitet IgG za FcγR, te stoga možda predstavlja jedan od mehanizama regulacije imunog odgovora u atopijskim bolestima. Ukupni N-glikom plazme (engl. total plasma N-glycome, TPNG) i N-glikom IgG djece po sastavu i dobnoj dinamici razlikuju se od onog u odraslih, što zahtijeva odvojene analize N-glikoma ove dvije populacije. Kako bi se utvrdilo postoji li povezanost između specifičnog N-glikoprofila IgG i atopijskih bolesti u pedijatrijskoj populaciji, ova studija ispitala je TPNG i N-glikoprofil IgG u djece koja pate od atopijskih bolesti i zdrave djece. Prikupljena je periferna krv 61 pedijatrijskog pacijenta i 74 zdrave djece oba spola starosti od 5 do 18 godina. Plazma je odvojena centrifugiranjem, te IgG izoliran afinitetnom kromatografijom pomoću proteina G. TPNG je ispitan tekućinskom kromatografijom visoke učinkovitosti s hidrofilnim interakcijama, a N-glikom IgG tekućinskom kromatografijom ultravisoke učinkovitosti s hidrofilnim interakcijama. N-glikozilacijski profil IgG kod djece s atopijom pokazao se specifičnim za ovaj protein, te različitim od onog kod zdrave djece. Udio većine jednostavnih, manjih, nesijaliniziranih struktura u ukupnim N-glikanima IgG manji je, a udio većih, većinom sijaliniziranih složenih struktura uglavnom veći kod djece s atopijom. Udio sijaliniziranih (naročito disijaliniziranih), digalaktoziliranih i sržno fukoziliranih (većinom bez presijecajućeg GlcNAc) struktura veći je, dok je udio agalaktoziliranih struktura i udio struktura s presijecajućim GlcNAc manji nego kod zdrave djece, što je u načelu suprotno od glikoprofila IgG i drugih proteina koji uobičajeno karakteriziraju upalna stanja. Analiza N-glikozilacije IgG ima zbog toga potencijal da se razvije u predispozicijski, dijagnostički i/ili prognostički marker za atopijske bolesti kod djece, te pruža temelj za istraživanje ovog područja s ciljem razvoja novih vrsta bioloških terapeutika za liječenje atopijskih bolesti.Immunoglobulin G (IgG) is considered one of modulators of immune response in atopic diseases, most often through interactions with Fcγ receptors (FcγR) on the surface of immune effector cells, and especially with the inhibitory receptor FcγRIIB. Differential N-glycosylation of IgG Fc regions affects the IgG affinity for FcγR, and thus maybe represents one of regulatory mechanisms of immune response in atopic diseases. Children's total plasma N-glycome (TPNG) and IgG N-glycome differ in composition and age dynamics from those found in adults, requiring independent N-glycome analyses of the two populations. To verify the existence of an association between a specific IgG N-glycoprofile and atopic diseases in pediatric population, this study examined TPNG and IgG N-glycoprofile in atopic vs. healthy children. Peripheral blood was collected from 61 patients and 74 healthy controls of both sexes aged 5-18 years. Plasma was separated by centrifugation and IgG isolated by affinity chromatography using protein G. TPNG was examined by hydrophilic interaction high performance liquid chromatography, and IgG N-glycome by hydrophilic interaction ultra performance liquid chromatography. A protein specific IgG N-glycosylation profile different from the one in healthy children was found. The content of majority of simple, smaller, asialylated structures in total IgG N-glycome was smaller and the content of greater, mainly sialylated complex structures was mostly greater in children with atopy. The content of sialylated (especially disialylated), digalactosilated and core fucosylated (mostly without GlcNAc) structures was greater, while the content of agalactosylated and the content of structures with bisecting GlcNAc was smaller in comparison with healthy children, displaying a glycoprofile in principle opposite from the one usually observed in IgG and other proteins in inflammatory conditions. The analysis of IgG N-glycoslyation thus has the potential to be developed into a predisposition, diagnostic and/or prognostic biomarker for atopic diseases in children, and provides a ground for further research in the area, aiming at the development of novel class of biological therapeutics for the treatment of atopic diseases