Introduction

Perianal fistulas occur in ~30-40% of patients with Crohn’s disease (CD) associated with high morbidity and an impaired quality of life. The management of perianal fistulizing CD (PCD) is a major clinical challenge, where its underlying etiology remains elusive. The development of disease-specific treatment for PCD requires better understanding of its pathogenesis.

Aims & Methods

To define the single-cell landscape of PCD, we recruited patients with 1) PCD (n = 25); 2) CD without perianal involvement (NPCD; n = 10); 3) idiopathic perianal fistulas (IPF, or cryptoglandular perianal fistula; n = 28). Biopsies were taken from the fistula tracts, openings of the fistulas, and rectal mucosa during examination under anesthesia (EUA) or colonoscopy. Mucosal immune cells were analyzed using mass cytometry (or CyTOF). All cells from PCD and IPF fistula tracts (n = 11 or 8) were characterized using single-cell RNA-sequencing (scRNA-seq). Spatial transcriptomics, immunohistochemistry (IHC), and ex vivo cell stimulation were performed to validate the findings. Integrated analysis of published scRNA-seq datasets was performed to define rectal and ileal cell responses in PCD patients vs. those without perianal disease.

Results

CyTOF revealed a skewed mucosal immune landscape in PCD compared to NPCD and IPF. PCD expanded Th17 cells in the fistula tracts and IL17-producing CD8 T cells (Tc17) in the rectum. Altered exhaustion markers CD39 and CD127 were present in both CD4 and CD8 T cells from PCD fistula tracts, fistula openings, and rectum. Regulatory B cells, with immunomodulatory function in the gut, were dramatically diminished in PCD compared to IPF. In addition, PCD fistula tracts, fistula openings, and rectum all exhibited substantially higher CD172+TREM1+ macrophages, which are associated with anti-TNF resistance in luminal CD. ScRNA-seq unraveled immune and non-immune cell compartments in PCD and IPF fistula tracts. PCD fistulas showed hyperactivated pathogenic pathways including interferon (IFN)G response, TNF signaling, and IL6-JAK-STAT3 in myeloid cells. Similarly, stromal cells from PCD fistulas exhibited higher activities in IFNG and TNF response, TNF signaling, and epithelial-mesenchymal transition (EMT). Further analysis revealed cellular modules associated with anti-TNF therapy in PCD patients. In addition to fistula tracts, luminal (rectal and ileal) cells from PCD patients also expressed greater levels of IFNG-responsive and EMT genes compared to those without perianal disease. In addition, we showed that both fistula tracts and ileal mucosa from PCD patients harbored expanded IFNG+ Th17 cells, which expressed elevated inflammatory mediators including IL17A/F, TNF, GZMA, and CCL5. The findings were independently validated using spatial transcriptomics, IHC, and ex vivo stimulation of PCD fistula-associated cells.

Conclusion

Using multi-omics analysis of perianal fistula tracts and luminal tissues from extensive patient cohorts, we revealed previously unknown immune, stromal, and epithelial cell landscapes of PCD. In addition to IL17 signaling, T cell exhaustion, pathogenic macrophages, and pro-inflammatory/remodeling fibroblasts, our data highlight the pathogenic role of hyperactivated IFNG signaling in both fistula tracts and luminal mucosa of patients with PCD. This study identified IFNG as a potential therapeutic target for PCD.

Disclosure

None

Introduction

Immune checkpoint inhibitor (ICI) mediated enterocolitis (IMC) may lead to treatment discontinuation. Corticosteroids for IMC is associated with serious side effects and might affect cancer progression in addition to preclude continued ICI treatment. We hypothesized that vedolizumab, a gut selective α4β7 integrin inhibitor, is effective as a first-line treatment and investigated whether vedolizumab reduces corticosteroid exposure and facilitate ICI resumption.

Aims & Methods

We conducted a single center, open-label, explorative phase 2 trial including patients treated with ICI who developed IMC. Patients were randomized 1:1 ratio to receive vedolizumab (300 mg) or standard treatment with corticosteroids, with infliximab used as recue therapy for corticosteroid failure. The primary outcome was cumulative corticosteroid dose at week 30. Key secondary outcomes included cumulative corticosteroid dose at week 10, corticosteroid free remission at week 10 and ICI resumption rates.

Results

Twenty-two patients received vedolizumab, and 19 patients received standard treatment. At week 10, the mean cumulative corticosteroid use was significantly lower in the vedolizumab arm (1157 mg vs. 2207 mg). By week 30, the mean corticosteroid use remained lower but not statistically significant (1378 mg vs. 2390 mg, estimated difference 1012 mg, 95% CI -229 to 2253, p=0.107), Among patients with less severe IMC, who did not need corticosteroids during screening, vedolizumab-treated patients used in average 1729 mg less corticosteroid (95% CI 631 to 2827). Corticosteroid-free remission at weeks 2 and 10 was more common with vedolizumab. Seven patients (33.3%) in the vedolizumab group resumed with ICI compared to three (16.7%) in the standard arm. In patients with severe colitis, who required upfront corticosteroid treatment, a trend towards longer hospitalization in the vedolizumab arm was observed.

Conclusion

For patients with less severe colitis, not needing corticosteroids during screening, vedolizumab may be a viable corticosteroid-sparing option, enabling faster remission and ICI resumption.

Disclosure

The study was funded by Aage og Johanne Louis-Hansens Fond, National Research grants. Takeda Pharma provided the study drug vedolizumab.

Introduction

Barrier healing (BH) has emerged as a novel treatment target in Inflammatory Bowel disease (IBD). Persistence of barrier impairment despite endoscopic and histological remission has been associated with worse long-term outcomes. However, accurate real-time assessment of intestinal barriers remains challenging in clinical practice. This study explores the role of advanced endoscopy (ultra-high magnification endocytoscopy [ECS] and confocal laser endomicroscopy [CLE]) for real-time, in vivo, structural and functional assessment of epithelial and vascular intestinal barrier. Further, we aim to investigate their correlation with automated barrier protein expression and clinical outcomes in IBD.

Aims & Methods

IBD patients undergoing endoscopic evaluation and healthy controls (HC) were recruited from two tertiary university centres.
The intestinal mucosa was assessed with ECS or CLE by novel scoring systems including epithelial and vascular features. BH was defined as ECS score ≤1 or CLE score ≤4, with preserved villi/crypt architecture, normal vessels, and, for CLE, absence of fluorescein leakage.
Biopsies were collected from both inflamed and non-inflamed areas. Epithelial (ZO-1, Claudin-2, E-cadherin) and vascular (PV-1 and CD-31) barrier markers were analysed through confocal microscopy, and their mean expression was automatically quantified using QuPath (v0.5.1). A biopsy-wise analysis assessed the correlation between endoscopic and histopathological evaluation. Moreover, occurrence of clinical outcomes at 6-month follow-up according to imaging-detected barrier impairment was evaluated.

Results

A total of 74 IBD patients (39 Crohn’s disease [CD], 35 Ulcerative colitis [UC]) and 9 HC) were considered. 46% (34/74) and 42% (30/74) IBD patients were in endoscopic and histological remission, respectively, while advanced imaging-defined BH was present in 24.3% (18/74) patients (Table 1). Barrier impairment was significantly associated with clinical outcomes at 6-month follow-up (p<0.01).
Mean expression of Claudin-2 was significantly higher in UC with ECS-based barrier impairment (85.5 vs 175.1, p 0.02), while PV1 expression was elevated in CD with CLE-based barrier dysfunction (362.1 vs 442.2, p 0.1). Significant correlations were found between ECS/CLE scores and epithelial and vascular barrier proteins.
In UC, Claudin-2 showed a moderate correlation with altered crypt architecture, goblet cell depletion and ECS total score (r 0.5), while PV-1 was moderately associated with altered vascular pattern and ECS total score (r -0.4).
In CD, Claudin-2 demonstrated a moderate correlation with crypt distortion, goblet cell depletion and total CLE scores (r 0.5), while PV-1 moderately correlated with fluorescein leakage, altered vessel architecture and total CLE score (r 0.4).

Conclusion

pCLE and ECS enable real-time intestinal barrier assessment in IBD, showing good correlation with epithelial and vascular barrier proteins and predicting clinical outcomes. This approach shows promise for targeting BH, guiding early and long-term disease management, and enabling flare-up prediction.