DOP27 Systemic antibody responses against gut microbiota flagellins implicate shared and divergent immune reactivity in Crohn's Disease and chronic fatigue syndrome

Abstract

Abstract Background Patients with Crohn’s disease (CD) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) exhibit elevated antibody responses against gut microbiota flagellins. However, flagellin-specific antibody repertoires and functional roles in the diseases remain incompletely understood. Bacterial flagellins can be categorized into three types depending on their interaction with toll-like receptor 5 (TLR5): (1) "stimulator" and (2) "silent" flagellins, binding TLR5 through a conserved N-terminal motif, with only stimulators activating TLR5 due to a specific C-terminal domain; (3) "evader" flagellins of pathogens, which circumvent TLR5 activation via mutated N-terminal TLR5 binding motifs. Here we studied the characteristics, epitope binding, and sequence (dis)similarity of anti-flagellin antibody responses in CD and ME/CFS. Methods Since conventional antibody profiling methods like enzyme-linked immunosorbent assays [ELISAs] do not allow for large-scale measurements of antibody repertoires, we leveraged phage-display immunoprecipitation sequencing (PhIP-Seq) to characterize 344,000 rationally selected peptide antigens in 256 patients with CD, 40 patients with ME/CFS and in two equally sized groups of age- and sex-matched healthy controls from population-based cohorts in the Netherlands and U.K., respectively. Different sequence alignment strategies were employed to compare flagellin peptide structures with observed antibody-bound flagellin peptide reactivity. Results Both patients with CD and ME/CFS exhibited elevated antibody responses against distinct regions of flagellin peptides compared to healthy individuals (P<0.001). N-terminal binding to Lachnospiraceae flagellins was comparable in both diseases, while C-terminal binding was more prevalent in CD. N-terminal antibody-bound flagellin sequences were similar across CD and ME/CFS, resembling ‘stimulator’ and ‘silent’ flagellins more than evaders. However, C-terminal antibody-bound flagellins showed higher resemblance to stimulator than to silent flagellins in CD, but not in ME/CFS. This group of antibody-bound flagellins was exclusively identified in a subset (10-20%) of patients with CD and characterized by its strong overrepresentation (exceeding 20-fold), underscoring its potential significance in distinguishing pathophysiologic subtypes of CD. Conclusion Antibody binding to the N-terminal domain of stimulator and silent flagellins may impact TLR5 activation in both CD and ME/CFS patients. Furthermore, elevated antibody binding to the C-terminal domain of stimulator flagellins in CD may explain pathophysiological differences between diseases. Our results highlight the diagnostic potential of these antibody responses and their impact on innate/adaptive immunity balance.