Introduction
The precise aetiology of inflammatory bowel disease (IBD) remains unclear with excess intestinal cell death implicated yet vastly understudied. Necroptosis, a recently described lytic cell death pathway, is emerging as a key player in intestinal inflammation in mouse models of colitis (1, 2), however clinical data are sparse. This non-apoptotic regulated cell death can be initiated by tumour necrosis factor (TNF) receptor activation and is executed by a core axis of proteins, including receptor-interacting protein kinase (RIPK) -1 and -3, and mixed lineage domain-like pseudokinase (MLKL) independent of caspases (3). The pathway is usually repressed by cellular inhibitor of apoptosis protein (cIAP) -1 and -2, and cellular FLICE-like inhibitory (cFLIPL).
This project proposes the first study to comprehensively assess programmed cell death in intestinal tissue of adult patients with IBD using a novel toolbox of affinity reagents (4). By comparing this with non-inflamed control tissue, we have uncovered a new molecular defect in IBD: loss of essential cellular death brakes, even in tissue with apparent mucosal and histologic healing. This novel finding may underpin the vicious cycle of remission and disease relapse and poses a step towards molecular healing through restoration of these brakes.
Aims & Methods
Aims:
- Assess cell death prevalence in IBD
- Determine why cell death brakes disappear in intestinal IBD tissue, regardless of inflammatory status
- Determine whether IBD phenotypes can be uniquely profiled according to cell death signals
Methods: Matched intestinal biopsies are retrieved endoscopically from patients without IBD (healthy controls) and those with IBD. Patients requiring endoscopy as part of standard of care are screened for eligibility: exclusion criteria comprise active malignancy, non-IBD inflammation, infection, use of non-steroidal anti-inflammatory drugs, and polyposis syndromes. Biopsies from participants with IBD are taken from macroscopically non-inflamed, margin and inflamed areas. Control specimen are taken from healthy-appearing bowel. Specimen are collected fresh for investigation of pertinent targets (
Table 1). Clinical/endoscopy activity indices, biochemical markers (C-reactive protein, faecal calprotectin) and blinded histologic (Robarts Histopathology Index) scoring are correlated with laboratory data in a multi-parametric manner.
MEDIUM
| TECHNIQUE | PURPOSE |
Phosphate buffer system + protease + phosphatase inhibitors
| Immunoblotting | Protein quantification of 26 cell death-related proteins |
| 10% formalin | Immunohistochemistry
| Visualisation of 12 cell death-related markers |
Methanol + 5% acetic acid
| Immunofluorescence
| Optic microscopy of necroptosis hot-spots |
| RNA later | RNA sequencing
| Shotgun transcriptomics of host microbiome, immune and inflammatory infiltrates |
Dulbecco's Modified Eagle Medium: Nutrient Mixture F12 (DMEM F12) + Primocin
| Organoid development
| Gut organoid challenges to identify factors impacting cell death |
Results
Intestinal biopsies from 28 healthy controls and 52 patients with IBD: 25 UC; 25 CD; and 2 pouchitis have been collected. Interim analysis demonstrates suppression of cell death inhibitory proteins- cIAP-1, -2 and cFLIPL even in non-inflamed tissue in patients with IBD, unleashing apoptosis (hallmarked by a statistically significant increase in cleaved caspase-3) and/or necroptosis (hallmarked by statistically significant increases in phosphorylated RIPK-3) in nearby tissue.
Conclusion
For the first time, we demonstrate that cellular death brakes are removed in intestinal tissue in IBD, despite histologic remission. Our findings challenge current dogma that necroptosis occurs in the absence of apoptosis and that necroptosis is universally increased in all forms of IBD. Disinhibition of programmed cell death is likely intrinsic to the inflammatory sequence underlying disease recurrence and progression.
References
- Günther C, Martini E, Wittkopf al. Caspase-8 regulates TNF-α-induced epithelial necroptosis and terminal ileitis. Nature. 2011 Sep 14;477(7364):335-9
- Patankar JV, Müller TM, Kantham S et al. E-type prostanoid receptor 4 drives resolution of intestinal inflammation by blocking epithelial necroptosis. Nature Cell Biology. 2021 Jul;23(7):796-807
- Horne CR, Samson AL, Murphy JM. The web of death: the expanding complexity of necroptotic signaling. Trends in Cell Biology. 2023 Feb;33(2):162-174
- Samson AL,Fitzgibbon C, Patel KM et al. A toolbox for imaging RIPK1, RIPK3, and MLKL in mouse and human cells. Cell Death & Differentiation. 2021 Jul;28(7):2126-2144
Disclosure
AHA is supported by scholarships from the University of Melbourne/Australian Commonwealth Government, Crohn's and Colitis Australia, Avant Foundation, and a grant from the Gastroenterological Society of Australia and Celltrion Healthcare. ALS has received funding from the National Health and Medical Research Council (Grant 2002965). JMM has received funding from the National Health and Medical Research Council (Grants 1172929, 9000719). ALS and JMM contribute to a project developing necroptosis inhibitors with Anaxis Pharma Pty.
