Neutralizing Anti-Granulocyte Macrophage-Colony Stimulating Factor Autoantibodies Recognize Post-Translational Glycosylations on Granulocyte Macrophage-Colony Stimulating Factor Years Before Diagnosis and Predict Complicated Crohn's Disease

Abstract

Background & aims: Anti-granulocyte macrophage-colony stimulating factor autoantibodies (aGMAbs) are detected in patients with ileal Crohn's disease (CD). Their induction and mode of action during or before disease are not well understood. We aimed to investigate the underlying mechanisms associated with aGMAb induction, from functional orientation to recognized epitopes, for their impact on intestinal immune homeostasis and use as a predictive biomarker for complicated CD.

Methods: We characterized using enzyme-linked immunosorbent assay naturally occurring aGMAbs in longitudinal serum samples from patients archived before the diagnosis of CD (n = 220) as well as from 400 healthy individuals (matched controls) as part of the US Defense Medical Surveillance System. We used biochemical, cellular, and transcriptional analysis to uncover a mechanism that governs the impaired immune balance in CD mucosa after diagnosis.

Results: Neutralizing aGMAbs were found to be specific for post-translational glycosylation on granulocyte macrophage-colony stimulating factor (GM-CSF), detectable years before diagnosis, and associated with complicated CD at presentation. Glycosylation of GM-CSF was altered in patients with CD, and aGMAb affected myeloid homeostasis and promoted group 1 innate lymphoid cells. Perturbations in immune homeostasis preceded the diagnosis in the serum of patients with CD presenting with aGMAb and were detectable in the noninflamed CD mucosa.

Conclusions: Anti-GMAbs predict the diagnosis of complicated CD long before the diagnosis of disease, recognize uniquely glycosylated epitopes, and impair myeloid cell and innate lymphoid cell balance associated with altered intestinal immune homeostasis.

Keywords: Autoantibodies; Crohn’s Disease; GM-CSF; Innate Lymphoid Cells; Macrophages.

Figure 1.

Characterization of aGMAb in patients with CD. (A) Reciprocal titers for total serum IgG aGMAb in sera of HD, patients with PAP, patients with CD, and patients with UC. (B) Isotype profiles of aGMAb in patients with PAP and patients with CD. Horizontal rows represent patients and vertical rows indicate isotypes. (C) Western blots probed with polyclonal sera from patients with CD and PAP show binding of anti-GM-CSF IgG and IgA to glycosylated (post-translationally-modified) and stripped GM-CSF. (D) Levels of GNA, AAL, and L-PHA binding to GM-CSF from HDs (black) and patients with CD (red), normalized for the total levels of GM-CSF in each sample. Schematic representation of N-glycan highlighting lectin recognition. (E) Native polyacrylamide gel electrophoresis of GM-CSF (sargramostim) and stripped GM-CSF stained with Coomassie Brilliant Blue. (F) Heat maps of pSTAT5 signal in peripheral blood mononuclear cells after stimulation with glycosylated (top) or deglycosylated GM-CSF in the presence of PAP serum, aGMAb⁻ CD serum, or aGMAb⁺ CD serum. Heat maps show signal intensity of anti-pSTAT5 staining in yellow code for individual patients (lanes) within the indicated cell populations identified using mass cytometry (rows). (G) Plots show quantification of pSTAT5 signal in DCs, monocytes, and plasmacytoid DCs either unstimulated or stimulated with GM-CSF preincubated with serum from the indicated patient groups. (H) Loss in pSTAT5 correlates with aGMAb titers. (I) Quantification of pSTAT signal for monocytes and DC shown in (F). One-way analysis of variance (ANOVA) Bonferroni’s multiple comparison test was performed. Mann-Whitney test. *P-value <.05.

Figure 2.

CD-specific aGMAbs precede the onset of disease by years. (A) and (B) show reciprocal titers of aGMAbs (IgG and IgA) in combined serum samples (training and validation cohort) at 2 time points before and 1 time point after diagnosis. (C) Trajectory of aGMAb titers in patients with CD. Blue lines indicate aGMAb⁺, black lines aGMAb⁻ patients, and red lines indicate sero-converters. (D) Kaplan-Meier analysis with hazard ratio for developing complications after diagnosis in aGMAb⁺ (red) and aGMAb⁻ patients 6 years before disease. (E) Correlations of aGMAb with anti-saccharomyces cerevisiae antibodies (ASCA) IgG and ASCA IgA antibodies at different time points before diagnosis. (F) Clusters of SOMAmers correlating with aGMAbs before (d-2175) and after (d+100) diagnosis of CD.

Figure 3.

The inflamed CD mucosa shows impaired homeostatic functions in GM-CSF–responsive myeloid cells. Lamina propria leukocytes from NI and INF ileal mucosa were analyzed. (A) Visualized stochastic neighbor embedding analysis shows distribution of leukocyte populations in NI and INF tissues indicating supervised annotation of populations. (B) Stimulation of cells in (A) with GM-CSF, followed by pSTAT5 measurement. Signal intensity is visualized in t-distributed stochastic neighbor embedding plots (blue = low, red = high pSTAT5). (C) Representative histograms show pSTAT5 levels in the indicated myeloid cell populations. (D) Heat map shows pSTAT5 intensity across myeloid populations from (A) and (B). (E) DC subset and macrophage identification by flow cytometry. (F) Mean fluorescence intensity (MFI) of ALDEFLOUR staining in CD45⁺CD11c⁺HLA-DR⁺ cells. ALDEFLUOR staining in CD45⁺CD11⁺HLA-DR⁺ cells in CD control and one CSF2RB^MUT carrier from (G) NI and (H) INF tissue biopsies. (I) Heat map shows gene expression for genes associated with retinol metabolism in NI ileal tissues of aGMAb+ or aGMAb⁻ patients with CD.

Figure 4.

Innate and adaptive sources of intestinal GM-CSF in patients with CD. (A) This shows GM-CSF⁺CD45⁺ cells. Adjacent dot plot shows frequency in NI and INF CD mucosa. (B) GM-CSF⁺CD45⁺ cells were analyzed for CD3 and NKp44 expression in NI and INF CD mucosa. Numbers in gate represents percentages. (C) Plots show percentages of NKp44⁺GM-CSF⁺ and CD3⁺GM-CSF⁺ cells. (D) This shows identification of NKp44⁺ CD117⁺ cells and (E) expression of CD127, CD161, RORγt, and CD69 on NKp44⁺ CD117⁺ cells. (F) GM-CSF production by NCR⁺ILC3 (CD45⁺CD3⁻CD4⁻CD127⁺CD161⁺NKp44⁺CD117⁺), (G) NCR⁺ILC3 frequencies, and (H) interferon-γ-producing ILC1 were quantified. Numbers in gates represent percentages. (I) This image and (J) this image show gene expression analysis of glycosyltransferase genes in the indicated population from INF and NI patients with CD. (K) This image and (L) this image show scatter plots of PC1 values of ILC1-PC and ILC3-PC derived from principal component analysis of signature ILC1 and ILC3 genes against a sub-dataset (195 NI ileal CD and 121 NI ileal non-IBD samples) of the bulk messenger RNAseq samples from the Mount Sinai Crohn’s and Colitis Registry stratified by the presence of aGMAb.