A unique serum IgG glycosylation signature predicts development of Crohn’s disease and is associated with pathogenic antibodies to mannose glycan

Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the gut. There is growing evidence in Crohn’s disease (CD) of the existence of a preclinical period characterized by immunological changes preceding symptom onset that starts years before diagnosis. Gaining insight into this preclinical phase will allow disease prediction and prevention. Analysis of preclinical serum samples, up to 6 years before IBD diagnosis (from the PREDICTS cohort), revealed the identification of a unique glycosylation signature on circulating antibodies (IgGs) characterized by lower galactosylation levels of the IgG fragment crystallizable (Fc) domain that remained stable until disease diagnosis. This specific IgG2 Fc glycan trait correlated with increased levels of antimicrobial antibodies, specifically anti-Saccharomyces cerevisiae (ASCA), pinpointing a glycome–ASCA hub detected in serum that predates by years the development of CD. Mechanistically, we demonstrated that this agalactosylated glycoform of ASCA IgG, detected in the preclinical phase, elicits a proinflammatory immune pathway through the activation and reprogramming of innate immune cells, such as dendritic cells and natural killer cells, via an FcγR-dependent mechanism, triggering NF-κB and CARD9 signaling and leading to inflammasome activation. This proinflammatory role of ASCA was demonstrated to be dependent on mannose glycan recognition and galactosylation levels in the IgG Fc domain. The pathogenic properties of (anti-mannose) ASCA IgG were validated in vivo. Adoptive transfer of antibodies to mannan (ASCA) to recipient wild-type mice resulted in increased susceptibility to intestinal inflammation that was recovered in recipient FcγR-deficient mice. Here we identify a glycosylation signature in circulating IgGs that precedes CD onset and pinpoint a specific glycome–ASCA pathway as a central player in the initiation of inflammation many years before CD diagnosis. This pathogenic glyco-hub may constitute a promising new serum biomarker for CD prediction and a potential target for disease prevention.


Glycopeptide preparation
To isolated human and murine IgGs, 0.2 μg of TPCK-treated trypsin (Promega, Madison, WI) was added, followed by an overnight incubation at 37°C.Tryptic digests were purified using a solid phase extraction (SPE) on C18 beads (Chromabond, Macherey-Nagel, Duren, Germany).10 ug of C18 beads was applied to each well of an OF1100 96-well polypropylene filter plate (Orochem, Naperville, IL).The RP stationary phase was activated 3 times with 200 μl of 80 % ACN and conditioned with 3 times with 200 μl 0.1 % TFA (Sigma-Aldrich/Merck, Darmstadt, Germany).The IgG digests were diluted with 0.1 % TFA (Sigma-Aldrich/Merck) loaded onto the C18 beads and washed 3 times 200 μl 0.1 % TFA.The entire procedure was performed on a vacuum manifold under pressure reduction of 2 inches of mercury.IgG glycopeptides were eluted into a PCR 96 well plate with 200 μl of 20 % ACN by centrifugation at 105 g for 5 min.Eluates were dried by vacuum centrifugation and stored at -20 °C until analysis by MS.
Human IgA enrichment and digestion 20 µL of plasma samples were transferred to wells of PCR plate and diluted with 110 µL of 1xPBS, pH 7.4.Diluted plasma samples were transferred into corresponding wells of Orochem filter plate containing 40 µL of IgA beads slurry (Thermo Scientific, IgA Affinity Matrix).Following 1 hour of incubation and several steps of washing with 1x PBS and MQ water, captured IgA was eluted with 100 mM formic acid.The collected eluate was dried in a speedvac at 60°C for 2.5 hours and resuspended in 25 mM ammonium bicarbonate for the subsequent reduction and alkylation with 200 mM DTT (Sigma-Aldrich) and 400 mM IAA (Sigma-Aldrich).Finally, to the reduced and alkylated sample, 1 µL of 0.2 µg/ µL TPCK-treated trypsin stock (Promega, Madison, WI) was added, followed by an overnight incubation at 37°C.Samples were stored at -20°C until the analysis by LC-MS.

nanoLC-ESI-MS of human IgG, human IgA and murine IgG N-glycopeptides
ACQUITY M Class UPLC system (Waters, Milford, MA), consisting of binary pump, auxiliary pump, trap valve manager and autosampler maintained at 10 °C with column compartment set to 30°C. 10 μL of sample was applied to a PepMap 100 C18 (5 mm × 300 μm i.d., 5μm, Thermo) SPE trap column conditioned with 0.1 % TFA (mobile phase A) for 1 min at 40 μl/min.For human IgGs: after sample loading, the trap column was switched in-line with the gradient and C18 nano-481 LC column (150 mm x 100 μm i.d., 2.7 μm HALO fused core particles; Advanced Materials Technology, Wilmington, DE) for 9.5 minutes.Trap column was cleaned with two full loop injections containing 20 μl of 95 % ACN and 50% ACN in IPA respectively.C18 nano-LC column was equilibrated with 100 % mobile phase A (0.1% TFA), for 2 minutes.IgG glycopeptides were reconstituted in 100 μl of ultrapure water before injection.Separation was achieved at 1 ml/min using the following gradient of mobile phase A and mobile phase B (80 % ACN and 20 % 0.1 % TFA respectively): 0.5 min 12 % B, 0.5 -4 min 12 % B -17 % B, 4 -5 min 17 % B. The ACQUITY M-Class system was coupled with Compact mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with Captive Spray ion source and nano Booster for introduction of acetonitrile vapor into the source.Nitrogen was used as a drying gas (4 l/min) and nebulizer (0.4 bars).Quadrupole and collision energies were set to 4 eV.Spectra were recorded from m/z 800 to 2000 with 2 averaged scans at a frequency of 0.5 Hz.Acquity M-class UPLC system was operated under MassLynx software 4.1 while Bruker Compact Q-TOF-MS was operated under HyStar software version 4.1.For human IgA, and murine IgGs: after sample loading, the trap column was switched in-line with the gradient and C18 nano LC column (150 mm x 100 μm i.d., 2.6 μm SunShell core shell particles; Chromanik Technologies Inc.) for 9 minutes.Trap column was cleaned with two full loop injections containing 20 μl of 95 % ACN and 50% ACN in IPA respectively.C18 nano-LC column was equilibrated with 100 % mobile phase A (0.1% TFA), for 2 minutes.IgG glycopeptides were reconstituted in 30 μl of ultrapure water before injection.Separation was achieved at 1 ml/min using the following gradient of mobile phase A and mobile phase B (80 % ACN and 20 % 0.1 % TFA respectively): 1 -1.5 min 0 % B -18.5 % B, 1.5 -5 min 18.5 % B -26 % B, 5 -9 min 26 % B. The ACQUITY M-Class system was coupled with Compact mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with Captive Spray ion source and nano Booster for introduction of acetonitrile vapor into the source.Nitrogen was used as a drying gas (4 l/min) and nebulizer (0.2 bars).Quadrupole and collision energies were set to 4 eV.Spectra were recorded from m/z 600 to 2500 with 2 averaged scans at a frequency of 0.5 Hz.Acquity M-class UPLC system was operated under MassLynx software 4.1 while Bruker Compact Q-TOF-MS was operated under HyStar software version 4.1.The detected glycopeptides are presented in Supplementary Table 2, and in accordance with previous reports 1, 2, 3 .

IgG aggregation assay
To assess the degree of IgG aggregation, a subset of IgG samples from healthy controls and CD patients (preclinical phase, at diagnosis, or full-blown disease) were analyzed using a protein aggregation assay kit (Abcam), according to manufacturer's instructions.Besides the standards from the kit, a commercial IgG antibody was incubated at 65ºC for 45 minutes to aggregate (positive control).Fluorescence was measured at Ex/Em 440/500 nm and compared with the standard curve of the kit.

Isolation of Natural Killer (NK) cells
NK cells were purified by negative selection from freshly PBMCs using EasySep™ Human NK Cell Isolation Kit, according to the manufacturer's instructions (Stem Cell Technologies).The purity (>94%) of NK cells was determined by flow cytometry analysis on FACSCanto™ II system (BD Biosciences, San Jose, CA).Monoclonal antibodies used for staining of NK cells are shown in Supplementary Table 6.Cells were cryopreserved in freeze medium (90% of fetal bovine serum (FBS) and 10% of dimethyl sulfoxide (DMSO)) at -80°C.After defrosting, cryo-preserved PBMCs and cryo-preserved NK cells were maintained in complete culture medium, RPMI 1640 Medium, GlutaMAX™ Supplement, HEPES (Thermo Fisher Scientific), supplemented with 10% of FBS, 1% penicillin/streptomycin and 0,1% gentamicin overnight at 37°C in a humidified incubator with 5% of CO2.

IgG-dependent NK cell degranulation
Purified NK cells were suspended in complete RPMI medium supplemented with IL-2 (100 U/mL, Pepotrech) and plated in wells previously coated with mannan, β-glucan or di-GlcNAc (as described above for DCs), at 1x10 5 cells/well in the presence of anti-human CD107a (LAMP-1; Supplementary Table 6).Following 1 hour, 10 μg/ml of Brefeldin A (Sigma-Aldrich) and GolgiStop™ Protein Transport Inhibitor were added and incubated 5 hours in similar conditions.After incubation, cells were stained for viability, as well as for surface markers (Supplementary Table 6).Cells were fixed and permeabilized with Transcription Factor Staining Buffer Set (eBioscience), followed by intracellular staining (Supplementary Table 6).Samples were acquired by flow cytometry on a FACSCanto™ II system (BD Biosciences) using the FACSDiva™ software (BD), and data were analyzed in FlowJo version 10.5.3 (Tree Star, Inc., Ashland, OR), using gating strategy shown in Supplementary Fig. 9b.

C1q (complement) binding assay
96-well Nunc® MaxiSorp™ high-binding plates were pre-coated with mannan as previously described.A blocking step with 5% of BSA was performed.6 µg/mL of total IgGs were added to plates previously coated with mannan (10 µg/mL), for 90 minutes at 37°C in a humidified incubator with 5% of CO2.After three washing steps with PBS 3% BSA 0.05% Tween20, complement component C1q was added (10 µg/mL) for 1 hour at room temperature.After washing (3x), HRP sheep anti-human C1q was added for incubation for 1 hour at room temperature.Plate was washed four times (as previously), and 3,3',5,5'-tetramethylbenzidine (substrate solution) was added for 15 min.Reaction was stopped with 1 N H2SO4 and absorbance was measured at 450 nm.

1.
Momčilović levels and titers in the serum from HC, preclinical and diagnosed CD, as well as in full-blown CD patients.In (d):HC, n=42; n=38; n=16; n=19; dx, n=40; n=20.In (e), HC, n=5; Pred_6y,   n=3; Pred_Dx, n=3; and (e), data presented were analyzed comparing each condition with the control (HC), by Kruskal-Wallis test with uncorrected Dunn's test and are shown as mean ± SD; p-values are shown in the graphs.Each data point represents the data from a single patient/subject (biological replicates).decrease in galactosylation of IgGs from HC treated with β-galactosidase was also confirmed.(f-g) CD107a expression on DCs co-cultured with diGlcNAc-specific IgGs (f) or β-glucan-specific IgGs from HC and CD patients (g) (normalized to HC); for (f), HC, n8; Pred_6y, n=8; Pred_Dx, n=8; full-blown CD, n=7.For (g), HC, n8; Pred_6y, n=8; Pred_Dx, n=8; full-blown CD, n=8.Data in (b), (c) and (d) were analyzed comparing each condition with the control (HC), by Kruskal-Wallis with uncorrected Dunn's test; data in (f) and (g) were analyzed comparing each condition with the control (HC), by one-way ANOVA with uncorrected Fisher's LSD.Each data point represents the data from a single patient/subject (biological replicates).

Table 1 .
Demographic and clinical characteristics of the samples from the PREDICTS cohort.

Table 2 .
Glycan traits of IgG and IgA characterized in serum samples.Cluster names for IgA are referring to the first three letters of the peptide sequence.

Table 3 .
Demographic and clinical characteristics of the samples from established CD patients.

Table 4 .
Demographic and clinical characteristics of the samples from inaugural CD patients, first-degree relatives (FDR) of CD patients and healthy controls.
a Age at sample collection.Supplementary

Table 5 .
Disease activity index (DAI).The DAI is obtained by the average score in each category.

Table 6 .
Antibodies and dyes used in flow cytometry.