170 the intestinal barrier (as oxygen levels increase towards the intestinal mucosa), and, in turn, their potential exposure to the immune system.33 Of note, we observed a surprisingly low relative abundance and low IgG immune response against Bacteroides, whereas this genus is usually one of the most dominant bacterial groups detected in fecal samples.34 Therefore, the abundance of Bacteroides as observed in the present study (<1%) greatly underestimates its actual prevalence. One potential explanation for a relatively low IgG immune response could be that IgG responses to other bacterial genera were strongly enhanced, which could make the response to Bacteroides seem relatively lower since we have worked with relative bacterial quantifications. Another possible explanation could be that the specific primers that were used (341F/806R) may have failed to adequately amplify Bacteroides, but variations in sample storage conditions and DNA extraction procedures may also be responsible for such decreased abundance.35 The observed enrichment after IgG-coating of several types of Lachnospiraceae (e.g. Dorea and Coprococcus) that belong to Clostridium cluster XIVa, but also that of many other bacteria like Clostridium, Lactococcus, Lactobacillus, and Enterococcus species could be explained by the fact that at least some species (e.g., Enterocloster bolteae and Enterocloster clostridioforme, or Ruminococcus gnavus belonging to the family of Lachnospiraceae) belonging to these bacterial genera are flagellated.10,36,37 Bacterial flagellae or flagellins are highly immunogenic proteins and dominant antigens in the context of IBD.36 Recently, a study demonstrated strong IgG immune responses against Lachnospiraceae flagellins in patients with CD, particularly those that were localized within the small intestine.11 This anti-flagellin antibody signature has been reported to be indicative of a more complicated disease course in CD, characterized by small intestinal disease involvement, frequent exacerbations and increased surgery rates.38,39 Well-established flagellin antigens include CBir1, Fla-X, and A4-Fla2 (associated with Lachnospiraceae bacteria). 40,41 Circulating anti-flagellin or antimicrobial antibodies are able to identify individuals who will develop CD years before the actual diagnosis, serving as serological predictors of the disease. 42-45 A recent study showed that pre-existent anti-flagellin antibodies associated with future development of CD independently of subclinical inflammation, genetic susceptibility and intestinal barrier function.45 This suggests that the formation of adaptive immune responses against microbiota flagellins occurs very early in disease pathogenesis. Apart from the highly immunogenic properties of flagellins, bacterial flagellamay facilitate transport across the epithelial mucus layer and enhance contact with a disrupted epithelial barrier, which may translate into an increased propensity to be exposed to the mucosal immune system.41,46,47 An important observation of our study was that many of the enriched IgG-coated bacterial genera are consistently among those reported to be increased in abundance in patients with IBD.5,6,33 For example, this includes the previously mentioned (potentially pathogenic) small intestinal-type bacteria, but also Enterobacteriaceae, certain Lachnospiraceae (e.g., Ruminococcus gnavus), and Clostridium species. Similarly, patients with IBD consistently have lower numbers of Faecalibacterium, Roseburia, Blautia and Bifidobacterium, which were also not significantly enriched within the IgG-coated fractions in this study.48-50 Thus, bacterial groups that are commonly reported to be more abundant in patients with IBD compared with healthy individuals also largely overlap with those suggested to be more immunogenic based on findings from the present study. Chapter 5
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