Introduction

We recently described an interleukin(IL)-23-inducible pathogenic subset of CCR6⁺CXCR3^-CCR5⁺ mucosal T helper (Th)17 cells (pTh17) infiltrating Crohn’s disease (CD) inflamed mucosa and specifically activated in response to Adherent-Invasive Escherichia Coli. pTh17 cells are functionally and phenotypically distinct from CCR6⁺CXCR3^-CCR5^- Th17 cells (cTh17) and from IL-12-induced CCR6⁺CXCR3⁺ Th1/17 subset. Here, we investigated pTh17 cells' recirculation into the bloodstream, localization into the intraepithelial lymphocyte (IEL) compartment, and in-vivo modulation by different biological therapies.

Aims & Methods

Adult patients with active CD initiating either vedolizumab as per clinical practice or ustekinumab and risankizumab during the SEQUENCE trial were prospectively recruited at Fondazione IRCCS Cà Granda, Ospedale Policlinico di Milano. In vedolizumab-treated patients, peripheral blood and intestinal biopsies were collected at baseline and at the end of induction. In ustekinumab- and risankizumab-treated patients, intestinal biopsies were collected outside study protocol assessments at baseline and week 24. Endoscopic response was defined as a greater than 50% decrease in the Simple Endoscopic Score from baseline. Peripheral blood mononuclear cells, lamina propria mononuclear cells (LPMC), and IEL were isolated and analyzed through flow cytometry.

Results

Overall, 34 CD patients were assessed. Demographic and baseline disease characteristics are summarized in Table 1. At baseline, as compared with cTh17, pTh17 cells: 1. are rarely found in the peripheral blood and express low levels of CCR9 and α4β7, 2. express high levels of tissue-resident markers CD69⁺CD103⁺ in the mucosa, and 3. infiltrate the IEL layer upon binding of CD103 with E-cadherin. Baseline pTh17 cells were upregulated in patients not achieving vedolizumab-induced endoscopic response. Upon vedolizumab treatment, a selective downregulation of mucosal cTh17 cells but not pTh17 was observed. Remarkably, risankizumab induced a specific downregulation of pTh17 cells, but not cTh17, in both LPMC and IEL layers. Conversely, ustekinumab reduced Th1/17 mucosal subsets, but not pTh17 cells (Figure 1).

Conclusion

pTh17 cells are largely unable to recirculate through the bloodstream, express markers of gut-tissue residency, and infiltrate the IEL compartment. IL-23 neutralization by risankizumab specifically downregulates mucosal pTh17 cells, as compared with vedolizumab and ustekinumab. Our immunologic results might provide mechanistic clues to vedolizumab latency effect in CD, and potentially explain early signs of risankizumab superiority over ustekinumab in the SEQUENCE study in terms of endoscopic response at 24 weeks.

Disclosure

DN received research grant from Pfizer, consulting fees from Dr. Falk, lecturer fees from Ferring. VP is employee of AbbVie. MV served as a consultant for Abbvie, MSD, Takeda, Janssen-Cilag, and Celgene. He received lecturer fees from Abbvie, Ferring, Takeda, MSD, Janssen-Cilag, and Zambon. FCa has served as consultant for Mundipharma, AbbVie, MSD, Takeda, Janssen, Roche, and Celgene; received lecture fees from AbbVie, Ferring, Takeda, Allergy Therapeutics, and Janssen; and received unrestricted research grants from Giuliani, SOFAR, MS&D, Takeda, Pfizer, and AbbVie.

Introduction

Faecal microbiota transplantation (FMT) has shown promise as treatment in inducing remission in patients with ulcerative colitis (UC), however effectiveness varies (1). Optimizing FMT protocols involving anaerobic stool processing to improve bacterial viability, dual-route administration and donor selection may improve efficacy.

Aims & Methods

Our aim was to evaluate the effectiveness of an optimized FMT protocol using anaerobically prepared stool from pre-selected donors in a randomized controlled multi-centre trial using dual-route administration in patients with mild-to-moderately active UC. A total of 85 patients were randomized to either receive donor FMT (n = 44) or autologous FMT (n = 41) via four weekly FMTs comprising two dual-route administrations, nasoduodenal administration combined with enema, and two single enemas. The study was performed at the Amsterdam University Medical Centre and University Medical Centre Groningen from June 2020 till January 2025. Follow-up time is 12 months and ongoing. Donors were selected based on their microbiota profile, informed by our previous TURN trial (2, 3). The primary outcome was steroid-free clinical remission at week 8 defined as a total adapted Mayo score of ≤2, with a stool frequency subscore of ≤1, a rectal bleeding subscore of 0, and an endoscopic subscore of ≤1. Open-label extension was offered to patients in the autologous FMT group who did not reach clinical remission.

Results

Eighty-five patients were randomized (mean age 43.6 years, 48% female), with 76 (89%) patients completing all four treatments. In the modified intention-to-treat analysis, meaning all patients that received at least one FMT treatment (n = 82), primary endpoint was reached in 10 of 44 (23%) patients receiving donor FMT and in 2 of 38 (5%) patients receiving autologous FMT, (two-sided χ², p = 0.026). A further total of 26 patients received donor FMT in open-label extension of which 21 (81%) received all four treatments with 7/26 (27%) reaching the primary endpoint. Serious adverse events occurred in 6/44 (14%) in the donor FMT group vs 4/41 (10%) the autologous group. Two events were deemed related to the FMT treatment (pneumothorax after nasoduodenal placement and overnight hospitalization for abdominal pain one-day post FMT).

Conclusion

In this multi-centre randomized controlled trial, anaerobically processed, donor-selected, dual-route FMT demonstrated a higher likelihood of inducing clinical remission with donor FMT compared to autologous FMT at 8 weeks. The protocol proved feasible and generally safe. Larger trials are warranted to confirm efficacy and to optimize donor and patient selection strategies. ClinicalTrial.gov. Number: NCT05998213

References

1. Imdad A, Pandit NG, Zaman M, Minkoff NZ, Tanner-Smith EE, Gomez-Duarte OG, et al. Fecal transplantation for treatment of inflammatory bowel disease. Cochrane Database Syst Rev. 2023;4(4):Cd012774.
2. Fuentes S, Rossen NG, van der Spek MJ, Hartman JH, Huuskonen L, Korpela K, et al. Microbial shifts and signatures of long-term remission in ulcerative colitis after faecal microbiota transplantation. Isme j. 2017;11(8):1877-89.
3. Rossen NG, Fuentes S, van der Spek MJ, Tijssen JG, Hartman JH, Duflou A, et al. Findings From a Randomized Controlled Trial of Fecal Transplantation for Patients With Ulcerative Colitis. Gastroenterology. 2015;149(1):110-8.e4.

Disclosure

The authors report no COI with regard to this study

Introduction

Although anti-TNF therapy is a cornerstone treatment for moderate to severe ulcerative colitis (UC)¹, up to 40% of patients are primary non-responders². The molecular basis for non-response in colonic mucosa remains poorly understood.

Aims & Methods

We aimed to identify compartment-specific molecular signatures in colonic epithelium and lamina propria that distinguish anti-TNF non-responders from responders with similar disease activity before treatment. UC patients with unambiguous endoscopic response (n = 6) or non-response (n = 6) after 6 months anti-TNF therapy were included. Pre-treatment inflamed and uninflamed colonic histological biopsies were laser microdissected into epithelial and lamina propria samples for total RNA sequencing. We analysed differentially expressed genes (DEGs), gene enrichment and weighted gene co-expression networks (WGCNA). Cellular deconvolution and pseudobulk validation were performed using a publicly available single-cell atlas³, and findings were further validated by immunohistochemistry.

Results

Inflammation elicited mucosal compartment-specific transcriptional changes, with more alteration in the epithelium (2,654 DEGs, log2 fold change (FC) > 0.3, padj < 0.05) enriched for interferon signaling and antigen presentation, compared to heightened cell-cycle and adaptive immune responses in the lamina propria (1,525 DEGs). We found no underlying differences in treatment response in either uninflamed or inflamed epithelium, nor in gene expression of TNF and its receptors in any compartment. The main difference between non-responders and responders was in uninflamed lamina propria where we identified distinct inflammatory signatures more overlapping with inflamed tissue (395 DEGs vs 1,112 in responders) and with high expression of cell cycle genes (e.g. TOP2A, MKI67, CDC20, CDKN1A, FZR1). A co-expression gene network enriched for humoral activity (log2 FC > 0.3, padj < 0.01) correlated strongly with non-response (r = 0.6, padj = 0.06) and contained numerous immunoglobulin genes (IGHA2, JCHAIN), including antigen-binding variable segments of heavy (IGHV4-34, IGHV3-7, IGHV3-73) and light chains (IGKV1-6, IGKV1D-17, IGLV2-11, IGLV2-14), in addition to other B cell-associated genes (BMI1, POU2AF1, TPD52). The cell cycle and humoral genes were systematically expressed higher in uninflamed lamina propria of non-responders compared to responders, with the differences disappearing in inflamed lamina propria. Cellular deconvolution³, showed that the humoral gene network was expressed in plasma cell populations (mainly IgA), which were confirmed increased in non-responder uninflamed lamina propria through tissue staining and gene expression. Pseudobulk analyses of uninflamed lamina propria samples from UC non-responders (n = 6) and responders (n = 3) filtered form the independent single-cell dataset³, validated the association of both cell cycle (14 of 18) and humoral (50 of 61) genes with uninflamed lamina propria in non-responders. Also, DEGs in uninflamed lamina propria IgA plasma cells from non-responders compared to responders in the validation dataset, were confirmed (15 of 18) in our data.

Conclusion

Our findings suggest that anti-TNF non-response in UC involves pre-existing humoral immune activation in macroscopically uninflamed colon mucosa. This spatially resolved signature offers a potential biomarker for treatment stratification and highlights the need for personalized therapeutic approaches in UC.

References

1 Raine, T. et al. ECCO Guidelines on Therapeutics in Ulcerative Colitis: Medical Treatment. J Crohn's Colitis 16, 2-17 (2021). https://doi.org/10.1093/ecco-jcc/jjab178
2 Ben-Horin, S., Kopylov, U. & Chowers, Y. Optimizing anti-TNF treatments in inflammatory bowel disease. Autoimmunity Reviews 13, 24-30 (2014). https://doi.org/https://doi.org/10.1016/j.autrev.2013.06.002
3 Thomas, T. et al. A longitudinal single-cell atlas of anti-tumour necrosis factor treatment in inflammatory bowel disease. Nature Immunology 25, 1-14 (2024). https://doi.org/10.1038/s41590-024-01994-8

Introduction

While trans-perineal ultrasound (TPUS) is promising, its accuracy compared to trans-abdominal ultrasound (TAS) and endoscopic ultrasound (EUS) in predicting rectal histoendoscopic activity in ulcerative colitis (UC) is unclear.

Aims & Methods

We aimed to evaluate the reliability of TPUS in predicting rectal histoendoscopic activity in UC using EUS as the reference standard. This prospective study included adult UC patients who underwent TAS and TPUS, followed by EUS. Rectal total wall thickness (TWT) and vascularity (Modified Limberg Score, MLS) were measured on all modalities. Additionally, mucosal thickness (MT) and submucosal thickness (SMT) were assessed on EUS. Receiver operating characteristic (ROC) analysis was performed to determine the area under the curve (AUC) for predicting endoscopic activity (Ulcerative Colitis Endoscopic Index of Severity, UCEIS >1) and histologic activity (Nancy Index >1).

Results

A total of 142 UC patients (33.8% female, median age 40 years, BMI 22.5 kg/m²) were included. Disease extent was proctitis (12%), proctosigmoiditis (58%), and extensive colitis (30%). The median SCCAI score was 3 (range: 0-8). Most patients were on 5-ASA (90.8%), azathioprine (31%), and tofacitinib (10.6%).
The median rectal length visualized on TPUS was 3.9 cm (2.4-6.3 cm). Endoscopic remission (UCEIS ≤1) was observed in 49.3% of patients, and histologic remission (NHI ≤1) in 38.7%.
For endoscopic activity, EUS had the highest accuracy, with MT >0.95 mm (AUC: 0.96, sensitivity: 98.6%, specificity: 87%) and MLS >1 (AUC: 0.95, sensitivity: 81.7%, specificity: 94%). TPUS had moderate accuracy(TWT >5.4 mm, AUC: 0.73), while TAS performed poorly (TWT >5.8 mm, AUC: 0.63).
For histologic remission, EUS vascularity (MLS >1) was most predictive (AUC: 0.91, sensitivity: 66.3%, specificity: 98.1%), followed by rectal TWT ≥4 mm (AUC: 0.83) and MT ≥0.95 mm (AUC: 0.86). Among non-EUS techniques, TPUS (TWT ≥6.1 mm, AUC: 0.66) showed moderate accuracy, while TAS was the least predictive (AUC: 0.62).
The Milan Ultrasound Criteria (MUC) demonstrated highest accuracy for endoscopic remission with EUS MUC ≥8 (AUC: 0.94), followed by TPUS (AUC: 0.78) and TAS (AUC: 0.67). For histologic remission, EUS MUC >7 had the highest accuracy (AUC: 0.87), followed by TPUS (MUC ≥9.7, AUC: 0.70) and TAS (AUC: 0.64) (Table 1). BMI-based comparisons showed no significant differences in MUC scores.

Measure	Sensitivity	Specificity	AUC	p-value	95% CI
	Endoscopic Activity
TAS (MUC) (≥8.5)	67.6%	60.9%	0.67	<0.001	0.59-0.76
TPUS (MUC) (≥8)	86%	62.3%	0.78	<0.001	0.70-0.86
EUS (MUC) (≥8)	81.7%	81.2%	0.94	<0.001	0.85-0.95
	Histologic activity
TAS (MUC) (>8.6)	62.8%	66%	0.64	0.005	0.54-0.74
TPUS (MUC) (≥9.7)	59.3%	79.6%	0.70	<0.001	0.61-0.79
EUS (MUC) (>7)	83.7%	81.5%	0.87	<0.001	0.81-0.93

Correlation analysis showed a strong correlation between TPUS and EUS TWT (r=0.371, p<0.001). TPUS and TAS had moderate correlation (r=0.309, p<0.001), while TAS and EUS showed weaker correlation (r=0.264, p=0.002 for average TWT, r=0.234, p=0.005 for lower rectal TWT). MUC scores correlated best between EUS and TPUS (r=0.425, p<0.001).

Conclusion

While EUS remains the most accurate, TPUS shows moderate accuracy for assessing rectal inflammation in UC. However, TPUS is limited to the lower rectum and needs further validation for better reproducibility. Standardized cutoffs and integration into routine UC monitoring could enhance non-invasive assessment, reducing endoscopy reliance and improving patient management.(NCT06717906).

References

1. Sagami S, Kobayashi T, Aihara K, Umeda M, Morikubo H, Matsubayashi M, Kiyohara H, Nakano M, Ohbu M, Hibi T. Transperineal ultrasound predicts endoscopic and histological healing in ulcerative colitis. Aliment Pharmacol Ther. 2020;51(12):1373-1383. [10.1111/apt.15767]
2. Sagami S, Kobayashi T, Aihara K, Umeda M, Odajima K, Morikubo H, Asonuma K, Miyatani Y, Fukuda T, Matsubayashi M, Kiyohara H, Nakano M, Hibi T. Early improvement in bowel wall thickness on transperineal ultrasonography predicts treatment success in active ulcerative colitis. Aliment Pharmacol Ther. 2022;55(10):1320-1329. [10.1111/apt.16817]