DOP109 A necroptotic-to-apoptotic signaling axis underlies inflammatory bowel disease
Authors: Al-Ani A , Pang J , Patel K , Young S N , Kong I , Chen J J , Barrios M , Rickard J , Chen S , Foroughi S , Cawthorne W , Jacobsen A , Jois A , Weir A , Whitehead L , Rajasekhar P , Horne C , Azeez I , Zhang Y , Sieber O , Spelman T , Giulino-Roth L , Hawkins E , Rogers K , Bowden R , Nicholson S , Lawlor K , Samson A L , Vince J E , Murphy J M , Christensen B 
Published: January 2025

Abstract

Background
Dysregulated programmed cell death plays a key role in IBD pathogenesis, with clinical trials exploring anti-necroptotic therapies (1,2). While apoptosis and necroptosis are well-studied in murine colitis models, their prevalence, mechanisms, and therapeutic relevance in human IBD remains unclear. We examined cell death pathways in patients on advanced therapies, uncovering unique signalling mechanisms that could inform future treatments.

Methods
Over 900 paired intestinal biopsies were analysed from 52 IBD patients (25 Crohn’s disease, 27 ulcerative colitis) and 28 non-IBD controls. Biopsies were taken from inflamed, marginal, and non-inflamed areas. Cell death processes, including apoptosis (e.g. cleaved caspase-3) and necroptosis (e.g. RIPK3, MLKL), were assessed using histology, immunoblotting and RNA sequencing. The effects of IBD-associated inflammatory cytokines (TNF, IFNγ) on epithelial death were tested on human-derived intestinal organoids(Fig 1). Data was correlated with clinical parameters like disease indices and treatments, with tissue quality validated using S100A8/S100A9 expression and blinded histology scoring.

Results
Increased necroptotic and apoptotic signalling were prominent in IBD, correlating with intestinal inflammation, regardless of treatment. Necroptotic signalling was detected in non-inflamed IBD tissue, suggesting early activation in disease progression, while apoptotic signalling was primarily in inflamed tissue. Gene set enrichment analysis identified dysregulated TNF and IFNγ-related pathways linked to cell death signalling, exacerbated by inflammation. Bulk RNA sequencing demonstrated that inflammation-induced transcriptional reprogramming of epithelial cells triggered a macrophage-like phenotype, promoting RIPK1-independent necroptotic signalling(Fig1). In vitro, exposure to TNF and IFNγ in intestinal organoids synergistically induced cell death, primarily through mitochondrial apoptosis. IFNγ enhanced epithelial cell reprogramming, amplifying cell death signalling, while both cytokines together induced mitochondrial apoptosis. In intestinal stem cells, apoptosis was driven by increased PUMA, while upregulated iNOS expression mediated epithelial cell death.

Conclusion
Early IBD inflammation reprograms epithelial cells into a macrophage-like state, promoting RIPK1-independent necroptotic signalling. IFNγ and TNF synergise to induce iNOS- and PUMA-mediated mitochondrial apoptosis in epithelial and stem cells. This aberrant epithelial cell death signalling persists despite histologic remission or treatment. Necroptotic signalling occurs upstream of apoptotic cell death, suggesting that targeting the necroptotic-to-apoptotic axis could facilitate molecular healing and sustained deep remission in IBD.

References
1.Pang J, Al-Ani AH et al. A necroptotic-to-apoptotic signaling axis underlies inflammatory bowel disease. bioRxiv 2024.11.13.623307;doi: https://doi.org/10.1101/2024.11.13.623307
2.C. g. I. #NCT05570006, Study of ABBV-668 Oral Capsules to Assess Adverse Events and Change in Disease Activity in Adult Participants With Moderate to Severe Ulcerative Colitis. (2023).
3.A. B. Keenan et al., ChEA3: transcription factor enrichment analysis by orthogonalomics integration. Nucleic Acids Res 47, W212-W224 (2019).
4.K. Parikh et al., Colonic epithelial cell diversity in health and inflammatory bowel disease. Nature 567, 49-55 (2019)