198 Patients with fibrostenotic CD exhibit a Lachnoclostridium-associated gene network involved in immune regulation In pairwise comparative analyses, patients with fibrostenotic CD (Montreal B2, n=107) and patients using TNF-α-antagonists (n=113) exhibited several differentially abundant microbial taxa. We therefore analyzed microbiota-associated host mucosal gene interactions in these phenotypes (Figure 5). Pairwise comparisons between patients with non-stricturing, non-penetrating disease vs. fibrostenotic CD revealed 2,639 differentially abundant genes that were enriched in cellular energy metabolism and immune system pathways (FDR<0.05, Supplementary Table S20). When comparing microbial taxa, abundances of mucosal Faecalibacterium, Erysipelotrichaceae_UCG- 003 and Coprococcus_3 were lower in fibrostenotic CD, whereas abundances of Lachnoclostridium and Flavonifractor were elevated in these patients (FDR<0.05). We hypothesized that these altered bacterial abundances and gene expression patterns may also translate into altered microbiota– gene networks relating to fibrostenotic CD. In patients with non-stricturing, non-penetrating CD, we observed 1,508 individual gene–bacteria associations (corresponding to 84 different pathway–bacteria associations), whereas we found 541 individual associations (corresponding to 40 different pathway–bacteria associations) in patients with fibrostenotic CD. Comparing each bacteria-associated gene cluster between patients with non-stricturing, non-penetrating and fibrostenotic CD (FDR <0.05, Methods, Supplementary Table S21) we identified four distinct networks represented by mucosal Lachnoclostridium, Coprococcus, Erysipelotrichaceae and Flavonifractor. The most significantly altered connections were associated with Lachnoclostridium, which was associated with 955 genes in patients with non-stricturing, non-penetrating CD, and these connections were mainly involved in cell activation pathways such as vesicle-mediated cellular transport and membrane trafficking (Figure 5A). In total, 148 genes were associated with Lachnoclostridium in patients with fibrostenotic CD (FDR<0.05), and these genes were involved in cellular immunoregulatory interactions and adaptive immune system pathways (e.g. CD8A, CLEC2B and CXCR5), tyrosine kinase signaling (e.g. FGF16), opioid signaling and G alpha (s) signaling events (mediated via cAMP-dependent protein kinases, e.g. POMC, GNG7 and GNG11) and vesicle-mediated transport (e.g. APOE, COLEC12 and KIF3B) (Figures 5A-B). Earlier studies had shown that Lachnoclostridium bacteria are generally increased in patients with (complicated) CD, e.g. postoperative CD,61 ASCA-positive CD62 and active granulomatous colitis.63 Recently, Lachnoclostridium was also associated with the non-invasive diagnosis of colorectal adenoma and colorectal cancer.64,65 These associations may potentially explain associations with genes involved in cellular proliferation and activation pathways. Increased abundances of Lachnoclostridium have been observed in relation to pulmonary fibrosis and its progression66 but not in relation to intestinal fibrosis. In contrast, reduced abundances of Faecalibacterium and Eubacterium species (belonging to the Erysipelotrichaceae family) have previously been associated with luminal narrowing in patients with pediatric ileal CD.67 Our results suggest that it is not only increased Lachnoclostridium abundances that may play a role in fibrostenotic CD, host immuneregulatory expression patterns may also vary along with these bacterial shifts. Notably, as the tissues investigated in our study were not derived from fibrotic regions, our findings show that these gene expression signatures are already present in non-stenotic intestinal tissue. Chapter 6
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