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

Portal hypertension (PHT) most commonly due to cirrhosis, but non-cirrhotic portal hypertension (NCPH) related to metabolic dysfunction, specifically metabolic dysfunction-associated steatotic liver disease (MASLD), necessitates further clarification of its mechanisms and identification of therapeutic targets.

PHT may precede the development of hepatic fibrosis in MASLD, and even serve as a driving factor in the pathogenesis of MASLD. Consequently, metabolic dysfunction-associated PHT might not depend on structural alterations such as hepatic fibrosis or pseudolobule formation, but rather correlate with other factors such as endothelial dysfunction, which has been demonstrated to play a pivotal role in NCPH. Endothelial dysfunction encompasses endothelial injury and endothelial-to-mesenchymal transition (EndMT), the latter being a specific type of epithelial-to-mesenchymal transition (EMT). As reported, the JAK/STAT3 signaling pathway plays a pivotal role in the initiation and progression of EMT. Similarly, patients with NCPH induced by myeloproliferative neoplasms (MPN) often lack pathological features such as hepatic fibrosis or pseudolobule formation. The Janus kinase 2 (JAK2) V617F mutation is highly prevalent in MPN patients. Therefore, targeted inhibition of JAK2 serves as an effective therapeutic strategy for MPN.

Given the shared pathological features between MPN-associated portal hypertension and MASLD-associated portal hypertension, as well as the vital​ role of JAK2 in both disease, the application of JAK2 inhibitor holds significant therapeutic potential for metabolic dysfunction-associated portal hypertension.

Aims & Methods

This study aims to verify the therapeutic efficacy of JAK2 inhibition in attenuating MASLD-associated PHT, with particular focus on its modulation of EndMT. Mice were fed with a high-fat diet (HFD) diet for 12 weeks to induce MASLD-associated PHT and divided into two groups, the treatment group (Rux) and control (Ctr) group. Ruxolitinib (JAK2 inhibitor) was given as a suspension by gavage (60 mg/kg in 0.2 ml) every two days for 3 weeks, while controls received the same volume of saline, from week 10-12 of feeding. Portal pressure was measured, serological analysis and histopathological staining were performed. RT-PCR and Western Blot were conducted to measure EndMT-related genes expression.

Results

The treatment group (Rux) demonstrated a significant reduction in directly measured portal pressure, with concordant changes observed in the spleen-to-body weight ratio, which served as an indirect indicator of portal pressure. Concurrent improvement was noted in ALT, AST and TC. H&E, Masson and Sirius red staining showed decreased perisinusoidal and perivascular fibrosis in the Rux group. Electron microscopy revealed restoration of endothelial fenestration. Additionally, ruxolitinib treatment led to downregulation of endothelial damage markers, mesenchymal cell markers as well as EndMT-related regulatory factors, indicating an alleviation of EndMT and endothelial dysfunction.

Conclusion

The JAK2 inhibitor demonstrates therapeutic potential for MASLD-associated PHT through modulation of endothelial dysfunction and EndMT. These mechanistic insights position ruxolitinib as a promising candidate for clinical management of NCPH, particularly in the MASLD-associated PHT.

References

[1] European Association for the Study of the Liver; European Association for the Study of Diabetes; European Association for the Study of Obesity. EASL-EASD-EASO Clinical Practice Guidelines on the management of metabolic dysfunction-associated steatotic liver disease (MASLD): Executive Summary. Diabetologia. 2024 Jun 13. [2] Song Z, Liu X, Zhang W, et al. Ruxolitinib suppresses liver fibrosis progression and accelerates fibrosis reversal via selectively targeting Janus kinase 1/2. Journal of Translational Medicine. 2022, 20(1): 157.
[3] Zhang XL, Zhang XY, Ge XQ, et al. Mangiferin prevents hepatocyte epithelial-mesenchymal transition in liver fibrosis via targeting HSP27-mediated JAK2/STAT3 and TGF-β1/Smad pathway. Phytotherapy Research. 2022, 36(11): 4167-4182.
[4] Jin W. Role of JAK/STAT3 Signaling in the Regulation of Metastasis, the Transition of Cancer Stem Cells, and Chemoresistance of Cancer by Epithelial-Mesenchymal Transition. Cells. 2020;9(1):217.
[5] Hammoutene A, Rautou PE. Role of liver sinusoidal endothelial cells in non-alcoholic fatty liver disease. Journal of Hepatology. 2019, 70(6): 1278–1291.

Introduction

Up to 40% of patients with acute severe ulcerative colitis (ASUC) do not respond to infliximab (IFX)(1) presumably due to insufficient drug exposure (1, 2). We investigated whether personalised IFX induction using predefined serum concentrations was superior to standard dosing.

Aims & Methods

In this prospective, multi-centre trial, adult IFX-naive steroid-refractory ASUC patients were randomised 1:1 to standard (SD) or personalised IFX dosing (PD). After an initial 5 mg/kg IFX infusion, SD was 5 mg/kg IFX at week 2 and 6. In PD, additional 5 mg/kg IFX infusions were administered guided by a Bayesian pharmacokinetic algorithm (iDose™) aiming at IFX serum concentrations >28 ug/mL day 0-28 and >15 ug/mL day 29-42 (3). The primary composite endpoint was clinical and endoscopic response at day 42 (Lichtiger score <10 and ≥3 points drop and UCEIS ≥2 points drop from baseline). Video-endoscopies were performed at baseline, day 42 and 182 and scored by 2 expert readers (XR) with adjudication by a third XR and post hoc also by the DovaVision UC AI tool (AI-R)(4). Key secondary endpoints were day 42 clinical response, day 42 endoscopic response, day 182 clinical remission (Lichtiger ≤3), day 182 endoscopic remission (UCEIS ≤1 on all components), and safety.

Results

48 patients were randomised (23 PD/25 SD) and 31 completed the 26-week study (19 PD/12 SD). Patient characteristics were comparable. Median cumulative IFX dose until day 42 was 18.41 mg/kg [1.77, 20.27] for PD vs 13.79 mg/kg [10.38, 14.82] for SD. The primary composite endpoint at day 42 was not met with XR (57% in PD vs 44% in SD; p=0.564). However, AI-R showed a significantly higher composite response rate in PD (74% in PD vs 32% in SD; p=0.0047). PD showed higher day 42 clinical response vs SD (91% vs 64%; p=0.039). Day 42 endoscopic response was observed in 57% in PD vs 44% in SD (p=0.564) with XR and 74% in PD vs 32% in SD with AI-R (p=0.0047). The proportion of patients in clinical and endoscopic remission at day 182 was higher in PD than in SD (65% vs 36%; p=0.082). Day 182 endoscopic remission with AI-R was higher in PD (52% in PD and 20% in SD; p=0.0337). SAEs occurred in 9% of patients on PD vs 20% of patients on SD. Following an interim analysis, the trial was discontinued based on futility.

Table 1. Baseline characteristics

Conclusion

Personalised IFX dosing was superior to standard dosing in ASUC when endoscopies were read by AI. This is the first IBD study where the primary endpoint, missed by expert assessment, was overturned and met by AI assessment instead. This demonstrates the potential of AI to fundamentally improve clinical trial methodology.

References

1. Seow CH, Newman A, Irwin SP, Steinhart AH, Silverberg MS, Greenberg GR. Trough serum infliximab: a predictive factor of clinical outcome for infliximab treatment in acute ulcerative colitis. Gut. 2010;59(1):49-54.
2. Papamichael K, Van Stappen T, Vande Casteele N, Gils A, Billiet T, Tops S, et al. Infliximab Concentration Thresholds During Induction Therapy Are Associated With Short-term Mucosal Healing in Patients With Ulcerative Colitis. Clin Gastroenterol Hepatol. 2016;14(4):543-9.
3. Ungar B, Mazor Y, Weisshof R, Yanai H, Ron Y, Goren I, et al. Induction infliximab levels among patients with acute severe ulcerative colitis compared with patients with moderately severe ulcerative colitis. Aliment Pharmacol Ther. 2016;43(12):1293-9.
4. Byrne M, Requa J, Panaccione R, Panes J, Bressler B, East JE, et al. Development and Validation of a novel AI-based computer vision solution for Ulcerative Colitis severity scoring on video for real world using high-volume expert-annotated video frames. United European Gastroenterol J. 2025 Oct.

Disclosure

This project received grant support from Pfizer Inc. and research support from Baysient LLC, Buhlmann Laboratories and Dova Health Intelligence.