Introduction

Chronic pouchitis is a common complication following ileal pouch-anal anastomosis (IPAA), with limited effective treatment options.¹ Antibiotics remain the first-line therapy; however, in cases of chronic pouchitis, prolonged antibiotic use contributes to increased antimicrobial resistance within the ileal pouch microbiome.² Fecal microbiota transplantation (FMT) has been proposed as a means of restoring microbial balance. Nevertheless, randomized placebo-controlled trials have demonstrated that, although FMT can alter the microbiome, it does not confer clinical benefits compared to placebo.^3–5

Aims & Methods

Given the lack of efficacy of FMT using stool from healthy donors, the present study aims to characterize the microbiome of a healthy ileal pouch and to evaluate the impact of high antibiotic exposure in patients with chronic pouchitis.
In this cohort study, stool samples were collected from four groups: patients with a normally functioning IPAA, patients with chronic pouchitis, patients with ulcerative colitis, and healthy controls. Microbiome composition and antibiotic resistance genes were analyzed across these groups. Fecal samples underwent metagenomic sequencing, and metagenome-assembled genomes (MAGs) were reconstructed. For Escherichia coli MAGs, the antibiotic resistance gene (ARG) profiles and plasmid content were specifically examined.

Results

This study included 197 participants. A clear pattern emerged in the microbial composition, revealing that reduced alpha diversity is directly associated with declining gut function and clinical health. Both species richness and Shannon diversity were lowest in patients with chronic pouchitis and highest in healthy individuals, with intermediate levels observed in the other groups. Notably, the ileal pouch microbiome of many chronic pouchitis patients was dominated by E. coli. Given the potential clinical implications of high E. coli abundance, we further investigated the ARG profiles and plasmid content of E. coli MAGs. E. coli MAGs from chronic pouchitis patients exhibited a significantly higher frequency of single nucleotide polymorphisms (SNPs) associated with resistance to fluoroquinolones—an antibiotic uniformly used among this patient group—compared to MAGs from the normally functioning IPAA and healthy control cohorts. Furthermore, E. coli MAGs from chronic pouchitis patients harbored a greater number of plasmids, with an increased presence of plasmid-borne ARGs conferring resistance to antibiotics commonly administered in this group, relative to the other cohorts.

Conclusion

These findings demonstrate that the composition of the ileal pouch microbiome—both in chronic pouchitis and normally functioning IPAA—differs significantly from that of individuals with normal gut physiology. This suggests that the microbiome of a normally functioning IPAA may serve as a more appropriate baseline for comparative studies than the healthy gut microbiome. Additionally, our results indicate that antibiotic use exerts increased selective pressure on E. coli strains carrying antibiotic resistance genes, directly correlating with the types of antibiotics administered.

References

1. Hata K, Ishihara S, Nozawa H, et al. Pouchitis after ileal pouch-anal anastomosis in ulcerative colitis: Diagnosis, management, risk factors, and incidence. Dig Endosc 2017;29:26–34.
2. Shen B, Kochhar GS, Rubin DT, et al. Treatment of pouchitis, Crohn’s disease, cuffitis, and other inflammatory disorders of the pouch: consensus guidelines from the International Ileal Pouch Consortium. Lancet Gastroenterol Hepatol 2021;7:69–95.
3. Herfarth H, Barnes EL, Long MD, et al. Combined Endoscopic and Oral Fecal Microbiota Transplantation in Patients with Antibiotic-Dependent Pouchitis: Low Clinical Efficacy due to Low Donor Microbial Engraftment. Inflamm Intest Dis 2019;4:1–6.
4. Karjalainen EK, Renkonen-Sinisalo L, Satokari R, et al. Fecal Microbiota Transplantation in Chronic Pouchitis: A Randomized, Parallel, Double-Blinded Clinical Trial. Inflamm Bowel Dis 2021;27:1766–1772.
5. Kousgaard SJ, Cold F, Halkjær SI, et al. The effect of non-pooled multi-donor faecal microbiota transplantation for inducing clinical remission in patients with chronic pouchitis: Results from a multicentre randomised double-blinded placebo-controlled trial (MicroPouch). J Crohn’s Colitis 2024;18:1753–1766.

Introduction

Crohn’s disease (CD) is associated both (i) with polymorphisms in the card15 gene, coding for the cytosolic NOD2 innate immunity receptor, recognizing bacterial peptidoglycan (PG), and (ii) with a decrease in Faecalibacterium, an anti-inflammatory intestinal bacterium¹. Oral administration of Faecalibacterium could represent a novel therapeutic strategy for CD patients, which is currently under investigation with F. prausnitzii EXL01 strain (human clinical trial #NCT05542355). Indeed, Faecalibacterium exhibits anti-inflammatory effects both in vitro and in vivo in colitis models^1-6 and we recently discovered that it directly induced the production of Interleukin-10 (IL-10) in CD14+ human monocytes⁷. However, the bacterial molecules, the host receptors and signaling pathways involved are still poorly understood. Therefore, the aim of the current study was to decipher the role of the interaction between Faecalibacterium, its PG and NOD2 in health and CD.

Aims & Methods

The PG structure of F. prausnitzii EXL01 was determined by ultra high-performance liquid chromatography and mass spectrometry. NOD2 activation was evaluated using HEK-human NOD2 reporter cell line. Following stimulation with either bacteria or PG, secretion of IL-10 and TNF-a by i) WT and NOD2 KO THP1 human monocytic cell line and ii) by dendritic cells (DC) from WT and NOD2^-/- mice and from human patients with CD harboring 0 or 2 NOD2 loss-of-function frameshift mutations were evaluated by ELISA.

Results

Faecalibacterium PG presents an amino acid substitution in the minimal motif recognized by NOD2 (data patented). PG of F. prausnitzii activates NOD2, but not NOD1, in a dose-dependent manner and has a higher ability to activate NOD2 than the one from other Gram positive bacteria. We showed that the production of IL-10 by THP1 monocytes and murine DC upon stimulation either with F. prausnitzii or its PG was decreased in absence of NOD2, indicating that secretion of IL-10 by human and murine immune cells upon Faecalibacterium stimulation is dependent on NOD2. Interestingly, no significant effect was observed for TNF-a secretion, leading to a significant decrease of the IL-10/TNF- a ratio in NOD2 KO cells. In addition, secretion of IL-10 upon stimulation with F. prausnitzii or its PG was lower in DC from CD patients with 2 NOD2 loss-of-function frameshift mutations compared to DC from CD patients without NOD2 mutation. Levels of secreted TNF-a were higher in DC from CD patients with 2 NOD2 loss-of-function frameshift mutations compared to DC from CD patients without NOD2 mutation, leading to decreased IL-10/TNF- a ratio in absence of functional NOD2. The pathways downstream of NOD2 as well as the effects of Faecalibacterium and its PG on NOD2 in vivo are currently under investigation.

Conclusion

Our study showed that Faecalibacterium and its PG activate NOD2, and therefore the production of IL-10, in a different way than other Gram-positive bacteria. These results provide new insight into the molecular pathways underlying the immunomodulatory effects of F. prausnitzii, and highlight NOD engagement as a key immunological feature. Identification of PG as a molecule responsible for the therapeutic effects paves the way for the next generation of Faecalibacterium-based drugs, whether whole-cell or component-driven, in inflammatory diseases such as CD.

References

1. Sokol H, Pigneur B, Watterlot L, et al. Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc Natl Acad Sci U S A. 2008; 105: 16731-6.
2. Lenoir M, Martin R, Torres-Maravilla E, et al. Butyrate mediates anti-inflammatory effects of Faecalibacterium prausnitzii in intestinal epithelial cells through Dact3. Gut Microbes. 2020; 12: 1-16.
3. Quevrain E, Maubert MA, Michon C, et al. Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn's disease. Gut. 2016; 65: 415-25.
4. Rossi O, van Berkel LA, Chain F, et al. Faecalibacterium prausnitzii A2-165 has a high capacity to induce IL-10 in human and murine dendritic cells and modulates T cell responses. Sci Rep. 2016; 6: 18507.
5. Sarrabayrouse G, Bossard C, Chauvin JM, et al. CD4CD8 lymphocytes, a novel human regulatory T cell subset induced by colonic bacteria and deficient in patients with inflammatory bowel disease. PLoS Biol. 2014; 12: e1001833.
6. Touch S, Godefroy E, Rolhion N, et al. Human CD4+CD8alpha+ Tregs induced by Faecalibacterium prausnitzii protect against intestinal inflammation. JCI Insight. 2022; 7.
7. https://www.biorxiv.org/content/10.1101/2024.10.06.616495v1

Disclosure

HS reports lecture fees, board membership, or consultancy from Carenity, AbbVie, Astellas, Danone, Ferring, Mayoly Spindler, MSD, Novartis, Roche, Tillots, Enterome, BiomX, Takeda, Biocodex, has stocks from Enterome and is co-founder of Exeliom Biosciences. PL report lecture fee, board membership, or consultancy from Biose, Biostime, Boiron, Bonduelle, BMS, Bromatech, IPSEN, iTaK, Lallemand, Lesaffre, L’Oréal, Mayoli, Merck, Procter and Gamble, Second Genome, Therascience and URGO and is co-founder of Exeliom Biosciences. Other authors declare no competing interests. DS, LH, PR and BH are employees of Exeliom Biosciences.

Introduction

Crohn’s disease (CD), as a type of inflammatory bowel disease (IBD), is characterized by chronic inflammation in the intestinal tract and its incidence is rising globally. The factors involved in causing or triggering Crohn’s disease (CD) have yet to be confirmed. We recently demonstrated that the intestinal microbiome composition is associated with the risk of CD development; however, its potential causal effect remains unknown. Our study investigated whether stool microbiome might contribute to or trigger dysregulated immune response by transplanting stool into germ-free mice (GFM).

Aims & Methods

Fecal samples were collected from 12 discordant pairs of siblings (one who did not develop CD referred to as Healthy Matched Control (HMC) and another who subsequently developed CD, referred to as pre-CD). These subjects were recruited as part of the Crohn's and Colitis Canada Genetic, Environmental, Microbial Project (CCC-GEM) project, a cohort of prospectively followed healthy first-degree relatives. The stool was transplanted into RAG1-/- GFM before T-cell transfer-induced colitis (3 transplanted mice per donor). Body weight was monitored for 6 weeks after T-cell transfer. Fecal Lipocalin-2 (LCN-2) was measured by ELISA to assess gut inflammation. Intestinal tissue damage was assessed by a validated histology scoring. Pro-inflammatory cytokine gene profiles of intestinal tissue were measured by qPCR. The feces of colitic mice were collected after 6 weeks of T-cell transfer and used to investigate metabolic profile measured by UHPLC-MS/MS.

Results

At week 2 after T-cell transfer, mouse recipients of pre-CD stool experienced greater weight loss than mice receiving stool from HMC (p=0.004). LCN-2 of pre-CD mice was significantly higher after T-cell transfer at week 3. Intriguingly, pre-CD stool recipient mice had a higher histological damage score in the ileum (p=0.031). Mice receiving pre-CD stool had increased expression of selected pro-inflammatory cytokine genes in the ileum including Tnfα, Il-1β, Ifnγ, Il-10 and Tgfβ compared to mice that received HMC stool. Colonic tissue showed significant increases in TNFα and Tgfβ in the mice recipient of the pre-CD stool. Moreover, pre-CD stool led to significant changes in 40 fecal metabolites compared to HMC microbiome (0.01<p≤0.05). Spearman correlation between certain metabolites and colitis markers in mouse was observed. Fecal metabolomics in humans revealed two shared metabolic pathways associated with future onset of CD and murine inflammatory markers. Notably, there was a positive correlation between sphingolipids in humans and mice that was associated with increased colitis in mice. Sphingolipid metabolites also demonstrated positive correlation with mouse colitis marker and human CD subclinical markers.

Conclusion

These results showed that transplanted pre-CD stool worsened T cell transfer colitis in mice compared to HMC stool. This suggests that stool microbiome of individuals with CD is functionally altered years before diagnosis, exhibiting greater inflammatory potential when transferred to susceptible mice. Modulating pre-CD microbiome composition and/or function could lead to novel methods of CD prevention.