Loss of tolerance to gut immunity protein, glycoprotein 2 (GP2) is associated with progressive disease course in primary sclerosing cholangitis

Glycoprotein 2[GP2] is a specific target of pancreatic autoantibodies[PAbs] in Crohn’s disease(CD) and is involved in gut innate immunity processes. Our aim was to evaluate the prevalence and prognostic potential of PAbs in primary sclerosing cholangitis(PSC). Sixty-five PSC patients were tested for PAbs by indirect immunofluorescence and compared with healthy (n = 100) and chronic liver disease controls(CLD, n = 488). Additionally, a panel of anti-microbial antibodies and secretory (s)IgA levels were measured, as markers of bacterial translocation and immune dysregulation. PAbs were more frequent in PSC(46.2%) compared to controls(healthy:0% and CLD:4.5%), [P < 0.001, for each]. Occurrence of anti-GP2 antibody was 30.8% (20/65) and was exclusively of IgA isotype. Anti-GP2 IgA positive patients had higher sIgA levels (P = 0.021). With flow-cytometry, 68.4% (13/19) of anti-GP2 IgA antibodies were bound with secretory component, suggesting an active retro-transportation of anti-GP2 from the gut lumen to the mucosa. Anti-GP2 IgA was associated with shorter transplant-free survival [PLogRank < 0.01] during the prospective follow-up (median, IQR: 87 [9–99] months) and remained an independent predictor after adjusting for Mayo risk score(HR: 4.69 [1.05–21.04], P = 0.043). These results highlight the significance of gut-liver interactions in PSC. Anti-GP2 IgA might be a valuable tool for risk stratification in PSC and considered as a potential therapeutic target.


Detection of target specific anti-pancreatic antibodies (PAbs)
IgA and IgG type anti-GP2 and anti-CUZD1 antibodies, and also atypical P-ANCA were detected in sera using cell-based IIFT according to the test instructions [CIBD Mosaic, EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany]. The slides contained a biochip mosaic consisting of transfected HEK293 cells expressing CUZD1 or GP2 1 separately and mocktransfected HEK293 control cells, and also ethanol-and formalin-fixed human granulocytes. The slides were incubated with 25 μL pre-diluted sera [1:10, 1:100, 1:1000 and 1:32, 1:320 and 1:3200] in phosphate-buffered saline (PBS) per incubation field for 30 minutes at room temperature. Unbounded sample was then washed away with PBS-Tween and the slide was immersed in PBS-Tween for 5 minutes. In the second step, fluorescein isothiocyanatelabeled goat anti-human IgG or IgA (EUROIMMUN) were used to visualize bound antibodies of the patients' sera. Incubation time was 30 minutes at room temperature followed by another washing step as above. Slides were embedded in PBS-buffered glycerol (approximately 10 μL per field).
Evaluation was performed using a Eurostar Plus microscope with Bluelight LED [EUROIMMUN Medizinische Labordiagnostika AG]. Positive and negative controls were included in each run. A specific fluorescence at 1:10 or higher for anti-CUZD1 and anti-GP2 antibodies, and a dilution of 1:32 or higher was considered positive for atypical P-ANCA. The interpretation of ANCA pattern was based on the behavior of the specimens on ethanol-and formalin-fixed slides according to previously reported. 2

Detection of secretory subtype of total IgA (sIgA)
Secretory component (SC) on the total IgA pool (sIgA) was detected by inhouse developed double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) systems. Microtiter plates (flat bottom, high binding capacity, Greiner Bio-One, Mosonmagyarovar, Hungary) were coated by overnight incubation at 4C with 3 µg/mL polyclonal sheep anti-human sIgA (Antibodies-Online GmbH, Aachen, Germany) diluted in carbonate/bicarbonate buffer (pH 9.6) Thereafter, wells were blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich, Mo, USA) in PBS with 0.02% Tween-20. Duplicates of prediluted sera (1:128) were then incubated for 60 min at room temperature (RT) together with a serial dilution of purified sIgA of human colostrum (Athens Research & Technology, USA) with known concentrations as a reference.
After washing five times, specific horseradish peroxidise (HRP) conjugated anti-human IgA (α-chain specific) antibody (Sigma-Aldrich, Mo, USA) diluted 1:5000 in PBS containing 1% BSA at pH 7.4 was added and incubated for another 60 min at RT. Color was developed with tetramethyl-benzidine dihydrochloride (TMB, Sigma-Aldrich, Schnelldorf, Germany), stopped with 2M H 2 SO 4 , and read immediately at 450 nm in a Labsystem Multiscan MS plate reader (Thermo Scientific, Budapest, Hungary). The calculation of results was performed using the Genesis software program with four parametric curve fitting. Within-run, coefficients of variation (CV) was 4.6%, while between-run CV was 15.2%. The limit of detection was 0.03 μg/mL.

Characterization of IgA type anti-GP2 antibodies
The IgA subtype analysis was performed in the case of anti-GP2 IgA-positive sera of patients with PSC (n=18) and Crohn's disease (n=12) that were verified by ELISA test earlier (anti-GP2 IgA, GA Generic Assays, Dahlewitz/Berlin, Germany). Anti-GP2 IgA-negative sera of healthy subjects (n=20) served as the controls.
We used a GP2-coated bead-based in-house flow cytometric immunoassay to subtype anti-GP2 IgA in sera, namely IgA1, IgA2 and sIgA.