Restriction lift date: 2025-10-31
Investigation of the TNF superfamily of ligands as potential therapeutic targets in inflammatory bowel diseases
University College Cork
Inflammatory bowel diseases (IBD) are chronic diseases of the gut affecting a significant proportion of the population in the western world with increasing incidence globally. They are multifactorial diseases with susceptibility mediated by a combination of host genetics and environmental factors leading to altered gut barrier function with ongoing non-resolving innate and adaptive inflammatory responses to components of the gut microbiota. There are two major forms - Crohn's disease (CD) and Ulcerative colitis (UC) differing in their location along the gastrointestinal tract, their pathophysiology, and characteristics of the ongoing innate and adaptive immune responses. While CD is largely characterised by a mixed Th1/Th17 immune response, UC is characterised by a so-called Th2 immune response. As reviewed in Chapter1 of the thesis, various cytokines, particularly IFN-γ and TNF-α, produced by activated immune cells, play a significant role in the pathogenesis of IBD. TNF-α blocking antibodies, known collectively as anti-TNFs, revolutionised the treatment of IBD in the mid to late 1990s; however, about 60% of patients are classified as inadequate responders, experiencing either no response or losing the response over time to both these anti-TNF drugs and newer targeted therapies. Hence there is a large therapeutic gap in IBD, leading to a clinically unmet need for new potential therapeutic targets that can be targeted in addition to TNF-α to obtain maximum efficacy. In the first study (Chapter 2 of this thesis), we investigated potential mechanisms underpinning synergy between IFN-γ and TNF-α and their ability to induce cell death of intestinal epithelial cells. Through weighted gene co-expression network analysis (WGCNA) of microarray data from HT-29 cells treated with IFN-γ and TNF-α, we identified gene modules that suggested both cytokines can cross regulate components of each other’s receptor-mediated signalling pathways. We confirmed these results through wet-lab experiments and propose the activation of a positive feedback loop when cells are treated with both IFN-γ and TNF-α, leading to enhanced activation of the signalling pathway and cell death. Though genes associated with the TNFR-related apoptotic and necroptotic pathways are increased when cells are treated with IFN-γ+TNF-α, perturbation of these canonical cell death pathways using commercially available inhibitors could not block the cytokine-induced cell death. STAT1 was identified as the main component required for IFN-γ+TNF-α-induced cell death, and we propose that cytokine-mediated pathway cross-regulation drives a positive feedback signalling loop culminating in enhanced activation of STAT1 and STAT1-induced cell death. In the second study (Chapter 3 of this thesis), we identified TNF-α-like cytokines that can exhibit synergy with IFN-γ as an approach to finding new targets to overcome resistance to anti-TNFs. For this, we screened ligands of the TNF superfamily of which TNF-α is a member. We utilised IFN-γ+TNF-α induced cell death and chemokine production as phenotypic readouts to screen a library of recombinant TNFSF ligands. We identified three major hits - TRAIL, TWEAK and LIGHT that could synergise with IFN-γ to induce pro-inflammatory chemokines and cell death in colon cancer cell lines and primary human colonic organoids. In the last chapter (Chapter 4 of this thesis), we selected one of these hits- TRAIL and investigated its synergistic effects with IFN-γ. IFN-γ and TRAIL synergised to induce cell death and production of chemokines, particularly the Th1 chemokine CXCL10. Similar to IFN-γ+TNF-α synergism, the IFN-γ+TRAIL-induced synergistic cell death was independent of canonical TRAIL-R associated apoptosis and necroptosis and was regulated via a caspase-8-JAK1/2-STAT1 pathway with STAT1 activation playing a significant role. Our results show that TRAIL is a TNF-α like molecule from the TNF superfamily and is a potential target for the treatment of IBD. The Casp-8/JAK1/2-STAT1 pathway plays a major role in modulating synergistic responses by the cytokine combinations. These results also explain, to an extent, the success of JAK inhibitors in the treatment of IBD. With further pre-clinical and clinical studies with antibodies targeting the hits from the screen, namely, TWEAK, LIGHT and TRAIL, either alone or in combination with anti-TNFs, new therapies can be developed for clinical trials in IBD.
TNF superfamily , IFN-g+TNF-a , Targets , Anti-TNF resistance , Cross-regulation , Inflammatory bowel diseases
Rajaram, S. 2022. Investigation of the TNF superfamily of ligands as potential therapeutic targets in inflammatory bowel diseases. PhD Thesis, University College Cork.