Macrophage-specific NF-kappa B activation dynamics can segregate inflammatory bowel disease patients

Abstract The heterogeneous nature of inflammatory bowel disease (IBD) presents challenges, particularly when choosing therapy. Activation of the NF-kappa B transcription factor is a highly-regulated, dynamic event in IBD pathogenesis. We expressed the human NF-kappa B/p65 subunit in blood-derived macrophages, using lentivirus. Confocal imaging of p65 activation revealed that a higher proportion of macrophages from Crohn9s patients responded to lipid-A compared to controls. In contrast, cells from ulcerative colitis (UC) patients exhibited a shorter duration of p65 nuclear localisation compared to healthy controls and Crohn9s donors. Using a similar lentivirus approach, NF-kappa B-regulated luciferase was expressed in patient macrophages, isolated from frozen peripheral blood mononuclear cell samples. Following activation, samples could be segregated into three clusters based on the NF-kappa B-regulated luciferase response. The majority of UC samples appeared in hypo-responsive cluster 1, with Crohn9s patients representing the majority of hyper-responsive cluster 3. A positive correlation was seen between NF-kappa B-induced luciferase activity and cytokine levels released to medium from stimulated macrophages, but not in serum or biopsy. Analysis of macrophage cytokine responses and patient metadata revealed a strong correlation between Crohn9s patients who smoked and hyper-activation of p65. These in vitro dynamic assays of NF-kappa B activation in blood-derived macrophages segregate IBD patients into groups with different phenotypes and therefore may help determine response to therapy.