Introduction

Antibiotic-associated colitis due to Clostridioides difficile infection is an increasing health problem worldwide. The currently recommended therapy for primary infection is antibiotic treatment with either vancomycin or fidaxomicin. Fecal microbiota transplantation is recommended for recurrent C. difficile infection, but its benefits in primary C. difficile infection are unclear. Our objectives were to investigate if fecal microbiota transplantation is non-inferior to standard antibiotic treatment to achieve lasting clinical cure of primary C. difficile infection, and to investigate adverse effects related to fecal microbiota transplantation in primary C. difficile infection.

Aims & Methods

We performed a non-inferiority, multicenter randomized trial in adult patients in Norway with acute C. difficile infection (≥ 3 loose stools per day and positive C. difficile toxin stool test) and no previous C. difficile infection. Patients were randomized to either one rectal fecal microbiota transplantation, or 10 days oral vancomycin 125 mg four times a day. Each fecal microbiota transplant was made from 50 g feces from healthy donors aged 16-30 at University Hospital of North Norway (Harstad, Norway). Before administration, patients assigned to fecal microbiota transplantation were asked to empty their bladder and rectum. There were no other preparatory procedures, nor any sedation for the procedure. The primary endpoint was the proportion of patients with clinical cure (firm stool consistency or ≤3 bowel movements per day) at 14 days and no recurrence within 60 days in modified intention-to-treat analyses. The trial was registered at clinicaltrials.gov (ID: NCT03796650).

Results

Out of 104 randomized patients, 100 received fecal microbiota transplantation or the first dose of vancomycin and were eligible for analyses. Clinical cure and no disease recurrence was observed in 34 of 51 patients (66.7%; 95% CI: 52.6% to 78.3%) with fecal microbiota transplantation versus 31 of 49 (63.3%; 95% CI: 48.9% to 75.6%) with vancomycin (difference in treatment success 3.4%; 95% CI: -21.2% to 28.0%; p-value for non-inferiority of fecal microbiota transplantation versus vancomycin: 0.001). Clinical cure at day 14 and no recurrence with or without additional treatment was 40 out of 51 (78.4%; 95% CI: 64.9% to 87.7%) with fecal microbiota transplantation and 31 out of 49 (63.3%; 95% CI: 48.9% to 75.6%) with vancomycin (difference in treatment success 15.2%; 95% CI: -8.0 to 38.3; p-value for non-inferiority of fecal microbiota transplantation versus Vancomycin: <0.001. There were no differences in adverse events between the groups.

Conclusion

Our study shows that fecal microbiota transplantation is safe and non-inferior to Vancomycin in primary C. difficile infection.

Disclosure

F.E. Juul has talked about fecal microbiota transplantation, pro bono, at a scientific meeting organized by Tillotts Pharma. ​The other authors ​have no conflicts of interest to declare.

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.

Introduction

Primary sclerosing cholangitis (PSC) is a progressive autoimmune cholestatic liver disease often associated with inflammatory bowel disease (IBD), particularly ulcerative colitis [1]. In PSC, reduced expression and function of the bile acid receptor GPBAR1 (TGR5) in cholangiocytes is associated with a dysfunctional gut–liver immune axis and impaired resolution of inflammation [2,3]. Parallelly, the transcription factor RORγt in Th17 cells sustains IL-17 perpetuating IL-23-driven intestinal [4] and liver inflammation [5]. While GPBAR1 mediates anti-inflammatory responses to secondary bile acids, RORγt governs Th17 differentiation and cytokine secretion. Recent clinical studies have demonstrated that IL-23 inhibitors, such as risankizumab [6] and mirikizumab [7], are effective in treating IBD, highlighting the significance of the IL-23/IL-17 axis. GPBAR1/RORγt dual simultaneous modulation represents a novel strategy to counteract the fibrotic and inflammatory environment characteristic of PSC.

Aims & Methods

We evaluated the therapeutic efficacy of PBT002, a dual GPBAR1 agonist and RORγt inverse agonist, in modulating immune and fibrotic pathways in PSC. Transcriptomic distribution of target genes was assessed using publicly available single-cell RNA-sequencing datasets from healthy human livers [8]. Differential gene expression was validated in two independent cohorts of patients with PSC and PBC, and further confirmed in an external validation cohort (GSE159676). Functional studies were performed in vitro using primary human cholangiocytes (NHC), immortalized hepatic stellate cells (LX-2), primary monocyte-derived macrophages and CD4+ T lymphocytes. In vivo efficacy was assessed in Abcb4^⁻/⁻ mice treated with PBT002 (10 mg/kg/day) for 12 weeks.

Results

Single-cell transcriptomic analysis revealed that IL-17 and IL-23 cytokines are predominantly expressed by immune cells, whereas their receptors (IL23R, IL17RA, IL17RC, IL17RE) are enriched in epithelial cells, including hepatocytes and cholangiocytes. Deconvolution analysis of liver bulk transcriptomes from PSC and PBC cohorts showed features of immune exhaustion, with a mild upregulation of pro-inflammatory markers and IL-17 receptor subunits, along with prominent overexpression of PD-L1, a known pro-fibrotic mediator. In vitro, exposure of hepatic stellate cells, macrophages, and CD4+ T cells to supernatants from inflamed cholangiocytes induced strong activation, which was reversed by PBT002 treatment. In vivo, PBT002 significantly reduced hepatocellular damage, as shown by decreased serum ALT, AST, ALP, and bilirubin levels. Histological analyses revealed marked reductions in periductal inflammation and liver fibrosis. Gene expression profiling confirmed significant downregulation of M1 and Th17-associated genes (Rorc, Il17f, Il22, Il23a) in colon and liver. Bile acid profiling showed restoration of key GPBAR1 agonists and RORγt antagonists (DCA, 3-oxo-DCA, allo-LCA), while microbiota analysis demonstrated increased abundance of protective taxa (e.g., Blautia, Sutterellaceae), inversely associated with disease severity.

Conclusion

PBT002 exerts potent immunomodulatory effects in experimental PSC by rebalancing M1/M2 and Th17/Treg homeostasis through dual GPBAR1 activation and RORγt inhibition. These effects correlate with restoration of bile acid and microbiota profiles, confirming the key role of the microbiota–bile acid–immune axis in PSC and supporting PBT002’s clinical development for immune-mediated cholangiopathies, particularly in PSC-IBD overlap.

References

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