Tumour Necrosis Factor Signalling Heterogeneity in Inflammatory Bowel Diseases

Abstract

Inflammatory bowel disease (IBD) is chronic, heterogeneous diseases characterised by excess inflammation in the gastrointestinal system and a dysfunctional mucosal immune response. Tumour necrosis factor (TNF) is a pleiotropic, pro-inflammatory cytokine upregulated in the serum and mucosa of IBD patients, compared to healthy controls (HCs). Anti-TNF treatment neutralises TNF, preventing the induction of downstream signalling pathways in immune cells. Cell death, pro-inflammatory and pro-survival responses can be mediated by TNF-induced ERK, p38 and NFĸBp65 proteins. The functions of these proteins are diverse and context dependent. Up to 40% of IBD patients do not initially respond to anti-TNF. It was hypothesised that the heterogeneity of TNF signalling in individuals may determine the anti-TNF response. TNF signalling pathways in peripheral blood mononuclear cells (PBMCs), derived from IBD patients and HCs, were activated using TNF stimulation or pan-stimulation by PMA/ionomycin. A phosphoflow cytometry protocol was optimised to allow visualisation of phosphorylated (activated) proteins in immune cells, in response to stimulation. Changes in phosphorylation were measured by normalised fold changes in mean fluorescence intensity (MFI). There was no difference in phosphorylated (P) p38, P-ERK or P-NFĸBp65 expression in CD3+ CD4+ T cells or CD14+ CD64+ monocytes between healthy (n=8) and IBD (n=5) groups (all p>0.05, Mann-Whitney U test). However, one IBD patient had comparatively higher P-p38 expression in CD3+ CD4+ T cells (MFI fold change = 3.8) and lower expression of P-NFĸBp65 in CD3+ CD4+ T cells (MFI fold change = 2), in response to PMA/ionomycin stimulation. A second patient had comparatively higher NFĸBp65 phosphorylation (MFI fold change = 6.21) in response to PMA/ionomycin stimulation, compared to IBD and HC individuals. Interestingly, the first patient was an anti-TNF non-responder. This study provides preliminary data showing the extent of individual heterogeneity in immune response. This study supports the individual analysis of IBD patients to inform the heterogeneity of IBD pathogenesis and how this may be implicated in treatment non-response.