Introduction

Artificial Intelligence (AI)- enabled endoscopy and histology offer accurate, objective, and rapid assessment of disease activity in Ulcerative Colitis (UC). Emerging multi-source AI models integrating diverse datasets may enhance standardised disease evaluation and outcome prediction.

Aims & Methods

This study aimed to develop a novel AI model fusing endoscopic and histological findings to improve the assessment of disease remission and predict early response to therapy in UC clinical trials. A novel multimodal AI fusion algorithm was developed by integrating paired endoscopic videos and histological whole-slide images (WSIs) from the phase 2 clinical trial of Mirikizumab in UC (NCT02589665). The endoscopy branch of the model was trained with 291 white-light videos, using a convolutional neural network to select informative frames and the BioMedCLIP foundational model to extract features. The histology branch utilised the CONCH foundational model and was fine-tuned on 291 WSIs to obtain patch-level features. Features from both modalities were aggregated and fused using multi-head self-attention. The model’s ability to assess histological remission and response to therapy at weeks 12 and 52 was evaluated. Histological remission was defined as Geboes ≤2B.0, while a response to therapy at different time points was based on histological remission or improvement (Geboes <3.1).

Results

The fusion model outperformed single-modality assessments for histological remission, achieving a sensitivity of 89.72% (95% CI: 82.35–94.76), specificity of 89.67% (95% CI: 84.34–93.67), and accuracy of 89.69% (95% CI: 85.61–92.94). It demonstrated remarkable performance in assessing response to therapy at 12 and 52 weeks, with sensitivity of 97.96% (95% CI: 89.15–99.95), specificity of 86.84% (95% CI: 71.91–95.59) and accuracy of 93.10% (95% CI 85.59 – 97.43) for histological remission at week 52. Substantial agreement was observed between the AI fusion model and central readout.

Conclusion

This innovative multimodal fusion AI model enhances the assessment of histological remission and accurately predicts response to therapy. By potentially standardising central readouts and enabling automated disease assessment, this novel tool marks a significant advancement towards precision medicine in clinical trials.

Disclosure

Marietta Iacucci received research grants and consultant fees from Olympus, Pentax, Eli Lilly, and Janssen. All other authors declare no competing interests.

Introduction

Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, are characterized by dysregulated T-cell responses, causing chronic intestinal inflammation.¹ While anti-TNF therapy is established in IBD, targeting additional pathways may improve outcomes for patients who do not respond adequately to current treatments.² OX40L, an immune co-stimulator, has been implicated in the pathogenesis of several inflammatory conditions.³ Our study provides a comprehensive analysis of the effects of combined TNF and OX40L blockade in a murine adoptive T cell transfer (ATC) model of colitis, examining body weight, detailed histopathological parameters, and a broad range of inflammatory cytokines in colon tissue.

Aims & Methods

Colitis was induced in severe combined immunodeficiency (SCID) mice by intraperitoneal transfer of CD4⁺CD45RB^high T cells isolated from healthy BALB/c donor mice. We established five treatment groups (n=8-10): 1) CD45RB^low transfer “healthy control”, 2) CD45RB^high transfer with isotype antibodies “disease control group”, 3) CD45RB^high transfer with anti-TNF antibody (XT3.11, 250 μg/mouse), 4) CD45RB^high transfer with anti-OX40L antibody (RM134L, 250 μg/mouse), and 5) CD45RB^high transfer with both antibodies at 250 µg/mouse, each. Body weight was monitored for 46 days as a measure of disease progression and normalized to baseline weight at day 1. At study termination, we collected colon tissue for analysis of inflammatory cytokines using multiplex immunoassays and conducted histopathological examination. Histopathological scoring was performed blinded using a validated scoring system for colonic inflammation.⁴

Results

Overall, combined anti-TNF/anti-OX40L therapy was most effective in preventing body weight loss compared to the isotype disease control group, followed by anti-TNF monotherapy and anti-OX40L monotherapy (p<0.0001, p=0.0009, and p=0.03, respectively). Mean normalized body weight (MNBW) of the CD45RB^low healthy control group at termination on day 46 was 115.2 %, while CD45RB^high cell transfer in the disease control group triggered disease induction and body weight loss, with a MNBW of 93.8 % at day 46. The CD45RB^high groups treated with anti-TNF or anti OX40L monotherapy reached a MNBW of 107.6 % and 102.9%, respectively. The CD45RB^high group treated with combination therapy reached 112.6%, closest to the CD45RB^low healthy control group. Notably, only combination therapy led to a significant improvement in histopathological scoring (p=0.0176) compared to the isotype disease control. Similarly, combination treatment consistently resulted in the strongest downregulation of several inflammatory cytokines including IFNg, IL-4, or TNFa, which are increased in colon tissue in the CD45RB^high disease group compared to the CD45RB^low healthy control group.

Conclusion

Combined blockade of TNF and OX40L demonstrated significantly greater efficacy in the ATC preclinical model of IBD in prevention of weight loss, downregulation of key inflammatory cytokines, and reduction of histological damage compared to either monotherapy. These data suggest potential additive efficacy for TNF and OX40L inhibition in controlling intestinal inflammation, and warrant further exploration of dual TNF/OX40L inhibition in clinical trials, particularly among those with inadequate response to anti-TNF monotherapy.

References

1. Kaser A, Zeissig S, Blumberg RS. Inflammatory bowel disease. Annu Rev Immunol. 2010;28:573-621.
2. Vermeire S. Combination biologic therapy for ulcerative colitis. Lancet Gastroenterol Hepatol. 2023;8:288-90.
3. Fu Y, Lin Q, Zhang Z, Zhang L. Therapeutic strategies for the costimulatory molecule OX40 in T-cell-mediated immunity. Acta Pharm Sin B. 2020;10:414-33.
4. Erben U, Loddenkemper C, Doerfel K, Spieckermann S, Haller D, Heimesaat MM, Zeitz M, Siegmund B, Kühl AA. A guide to histomorphological evaluation of intestinal inflammation in mouse models. Int J Clin Exp Pathol. 2014;7:4557-76.

Disclosure

Thomas Leeuw, Rolf Keiffer, Danping Ding-Pfennigdorff, Christian Asbrand, Raaj Mehta, Matthias Herrmann are employees of Sanofi and may hold stock or equity options in Sanofi

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

1. Fiorucci, S. et al. Bile Acids-Based Therapies for Primary Sclerosing Cholangitis: Current Landscape and Future Developments. Cells 13, (2024).
2. Di Giorgio, C. et al. Liver GPBAR1 Associates With Immune Dysfunction in Primary Sclerosing Cholangitis and Its Activation Attenuates Cholestasis in Abcb4-/- Mice. Liver Int. Off. J. Int. Assoc. Study Liver 45, e16235 (2025).
3. Reich, M. et al. Downregulation of TGR5 (GPBAR1) in biliary epithelial cells contributes to the pathogenesis of sclerosing cholangitis. J. Hepatol. 75, 634–646 (2021).
4. Biagioli, M. et al. Development of dual GPBAR1 agonist and RORγt inverse agonist for the treatment of inflammatory bowel diseases. Pharmacol. Res. 208, 107403 (2024).
5. Garcia Moreno, A. S. et al. IL-17 signaling in primary sclerosing cholangitis patient-derived organoids. Hepatol. Commun. 8, (2024).
6. Louis, E. et al. Risankizumab for Ulcerative Colitis: Two Randomized Clinical Trials. JAMA 332, 881–897 (2024).
7. D’Haens, G. et al. Mirikizumab as Induction and Maintenance Therapy for Ulcerative Colitis. N. Engl. J. Med. 388, 2444–2455 (2023).
8. Andrews, T. S. et al. Single-Cell, Single-Nucleus, and Spatial RNA Sequencing of the Human Liver Identifies Cholangiocyte and Mesenchymal Heterogeneity. Hepatol. Commun. 6, 821–840 (2022).

Introduction

Treatment response in inflammatory bowel disease (IBD) is highly variable, with an urgent need for biomarkers to guide therapeutic decisions. Circulating monocytes, key players in intestinal immunity, act as precursors to tissue macrophages and contribute to inflammation and mucosal healing. The transcriptional programs that govern monocyte fate and their association with treatment outcomes remain poorly defined. Ikaros family proteins regulate gene expression through distinct mechanisms in lymphocytes, but little is known about their role in monocyte-macrophage function in IBD. We aimed to investigate the role of Ikaros family (Ikaros, Helios, Aiolos, Eos, Pegasus) in shaping monocyte function linked to advanced therapies response.

Aims & Methods

Peripheral blood samples were collected at baseline (W0) and after 8-12 weeks of induction therapy (W8/12) from IBD patients who initiated or switched advanced therapy [biologics, oral synthetics] and from healthy controls (HC) (n=8). We included patients achieving clinical remission after induction [Super-Responders] SR (n=18) and those showing no response [Non-Responders] NR (n=16). Immune phenotype and expression of Ikaros proteins were assessed via multiparametric flow cytometry (FACS) and correlated with clinical outcomes. To assess functional roles of Ikaros proteins, monocyte-derived macrophages were generated in vitro from buffy coat (n=6). Cells were polarised using cytokine cocktails to induce pro-inflammatory (LPS+IFNγ or TNF+PGE2+Pam3CSK4; TPP), alternative (IL1β+LPS), or regulatory (IL10) phenotypes. siRNA-loaded nanoparticles were used to silence Ikaros or Helios prior to polarisation. Phenotypic changes were analysed by FACS; cytokines secretion by ELISA; genes expression by qPCR; and phagocytosis by fluorescent beads uptake.

Results

At W0, IBD patients exhibited a significant increase in circulating monocyte counts (vs HC, p<0.001). At W8/12, monocyte levels significantly decreased in the SR group, while remaining elevated in the NR group. FACS analysis revealed an increased proportion of non-classical monocytes (CD14⁻CD16⁺) in the IBD group at W0 (vs HC; p< 0.05), persisting in NR at W8/12 (vs SR; p<0.05). IBD monocytes also showed higher expression of CCR2 and CX3CR1 compared to HC. Helios expression was absent in monocytes from IBD patients at W0, while the mean expression of Helios was 47.31% in monocytes from the HC group. At W8/12, Helios expression re-emerged in 34.14% of monocytes derived from the SR patients but remained undetectable in the NR. In vitro, macrophages treated with LPS+IFNγ or TPP upregulated Ikaros, STAT1, and CXCL10 gene expression (p< 0.01), while Helios, TGM2, and MCR1 were upregulated in response to IL10 (p< 0.05). Helios expression was absent in TNF-stimulated macrophages. Ikaros silencing reduced pro-inflammatory markers, IL6 and IL1β cytokines secretion, and inhibited phagocytosis in response to LPS, whereas Helios silencing increased macrophages activation, and pro-inflammatory markers in response to IL10.

Conclusion

Monocyte subsets distribution and Helios, Ikaros expression dynamics clearly distinguished SR from NR in IBD. Sustained absence of Helios and persistence of non-classical monocytes in NR suggest a failure to transition toward a regulatory phenotype. This may further contribute to altered migratory capacity and influence their functional fate within intestinal tissue. Functional assays confirm opposing roles of Ikaros and Helios in the macrophages activation, underscoring their unique potential as biomarkers or therapeutic targets to modulate inflammation in IBD.

Introduction

Very early onset inflammatory bowel disease (VEO-IBD) is often associated with a severe disease course and unique characteristics including an underlying monogenic defect. In this study, we identified new pathogenic mutations in CGNL1 associated with VEO-IBD and other inflammatory GI disease. CGNL1 (Cingulin Like 1) encodes an epithelial junctional protein whose function was previously unknown in the gastrointestinal tract.

Aims & Methods

Whole exome sequencing (WES) was performed on the affected patient with VEO-IBD and all first-degree relatives with IBD and other inflammatory disease. Intestinal epithelial organoids (IEOs) were generated from the affected patient and were characterized by transepithelial electrical resistance (TEER), FITC-dextran permeability assay on epithelial cell monolayer, immunostaining, RNA sequencing, and transmission electron microscopy (TEM). In addition, we generated a new Cgnl1^-/- mouse model, which was characterized using FITC-dextran permeability assay, TEM, dextran-sodium sulfate (DSS)-induced colitis, and infections by Citrobacter rodentium and Clostridioides difficile. Single-cell datasets were analyzed to determine CGNL1 expression in IBD, primary sclerosing cholangitis (PSC), and autoimmune hepatitis (AIH).

Results

In the affected patient with VEO-IBD who required early total colectomy and pouch formation with coexistent PSC and celiac disease, WES identified two rare, new, pathogenic variants in the CGNL1 gene. Both pathogenic variants were also present in the patient’s younger sister, who has a similar history including early-onset, fulminant ulcerative colitis requiring total colectomy with ileostomy and PSC-AIH overlap syndrome. Furthermore, multiple relatives of the patient suffer from IBD and other inflammatory conditions including celiac disease, eosinophilic esophagitis, and type-1 diabetes. The RNA and protein levels of CGNL1 were significantly diminished in the patient’s intestinal epithelial cells compared to that of healthy individuals and other IBD patients by RNA-seq, immunoblotting, and immunostaining. The patient’s IEOs from pouch body and pre-pouch ileum formed smaller 3D spheroids with altered cell composition and significantly impaired barrier function compared to those from healthy controls and other IBD patients. Immunofluorescence and TEM demonstrated that CGNL1 is required for the assembly of junctional complexes in human IEOs and an epithelial cell line. Similarly to findings from the patient, impaired junctional complexes in intestinal epithelial cells and elevated intestinal permeability were present in Cgnl1^-/- mice at steady state. Cgnl1^-/- mice showed substantial vulnerability to DSS-induced colitis and bacterial infections that directly compromise the epithelial barrier. In addition, 6-month-old Cgnl1^-/- mice exhibited spontaneous inflammation in the colon and terminal ileum due to disrupted intestinal barrier, which was microbiome-dependent. Furthermore, single-cell analysis revealed diminished expression of CGNL1 in the intestinal and hepatic epithelial cells from patients with IBD or PSC/AIH, respectively.

Conclusion

The pathogenic CGNL1 variants contribute to VEO-IBD by disrupting gut barrier integrity, as demonstrated by patient’s cells and novel murine models. This study expands our understanding of epithelial barrier impairment in the pathogenesis of VEO-IBD, and highlights intestinal epithelium as a therapeutic target in some IBD patients.