126 Models discriminating patients with CD from healthy controls demonstrate a high degree of external generalizability To test the external validity of our classification results, we applied our classification models to an external, publicly available dataset from a population-based cohort featuring data on 1,874 PhIP-Seq-based antibody-bound peptides from 997 healthy individuals derived from the same microbiota phage library employed in the present study.12 The classificationmodels discriminating patients with CD from healthy controls showed fairly accurate performance on the external dataset (using only the negative side of the classification as there were no patients with IBD in this dataset), whereas this was not observed for the classification models discriminating between patients with UC and healthy controls (Table S9). For instance, antibody epitope repertoires demonstrated a highly accurate discrimination between patients with CD and healthy controls from the external dataset (accuracy = 0.82 vs. 0.80 in our own test set), which was not the case for the discrimination between patients with UC and these healthy controls (accuracy = 0.29 vs. 0.70 in our own test set). Similarly, when prioritizing the top-five and top-ten antibody-bound peptides using the RFE-optimized models classifying patients with CD from the external controls, we obtained high accuracies (top five: accuracy = 0.91 vs. 0.72 in our own test set; top ten: accuracy = 0.98 vs. 0.76 in our own test set), which was again not the case when discriminating patients with UC from external controls (top five: accuracy = 0.00 vs. 0.68; top ten: accuracy = 0.12 vs. 0.66) (Table S11). Overall, this analysis shows that both the full set and the top-five and top-ten selected antibody-bound peptides are able to accurately classify patients with CD from healthy controls and that this generalizes well to healthy controls from an independent external control cohort. Patients with ileal and stricturing CD show distinct antibody responses against specific Lachnospiraceae flagellins Many of the flagellin-derived antigen peptides that were significantly overrepresented in CD (Figure 3) were particularly enriched in patients with CD with ileal involvement (n = 187) as compared to patients with purely colonic CD or UC (n = 299). In total, 59 antibody-bound peptides were differentially abundant between patients with ileal CD vs. colonic IBD, of which 95% was overrepresented in ileal CD (FDR < 0.05, Figure 6A, Table S14). Most of these differentially abundant flagellin antigen peptides were derived from bacteria belonging to the order Clostridiales, especially to Clostridium clusters XIVa and IV, of which most were in the family Lachnospiraceae, including Roseburia spp. (26%), Lachnospira spp. (21%), Clostridium spp. (19%) and Eubacterium spp. (11%), as well as flagellin antigens from Agathobacter rectalis and Butyrivibrio crossotus. A few significantly enriched flagellin-derived antigen peptides belonged to pathogenic species, including Legionella pneumophila, Borrelia burgdorferi, Burkholderia pseudomallei and Yersinia enterocolitica. Furthermore, a few antibody-bound peptides represented ATP-ase and permease components of ATP-binding cassette (ABC)-type transport systems of Anaerostipes hadrus and Coprococcus catus, both belonging to the family Lachnospiraceae and both wellknown butyrate producers. Together, these 59 antibody-bound peptides demonstrated high accuracy in differentiating between patients with ileal CD vs. patients with colonic IBD (AUC = 0.82, Figure 6A). Chapter 4
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