Macrophage-Specific NF-κB 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-κB transcription factor is a highly regulated, dynamic event in IBD pathogenesis. Using a lentivirus approach, NF-κB-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-κB-regulated luciferase response. The ulcerative colitis (UC) samples appeared only in the hypo-responsive Cluster 1, and in Cluster 2. Conversely, Crohn's disease (CD) patients appeared in all Clusters with their percentage being higher in the hyper-responsive Cluster 3. A positive correlation was seen between NF-κB-induced luciferase activity and the concentrations of cytokines released into medium from stimulated macrophages, but not with serum or biopsy cytokine levels. Confocal imaging of lentivirally-expressed p65 activation revealed that a higher proportion of macrophages from CD patients responded to endotoxin lipid A compared to controls. In contrast, cells from UC patients exhibited a shorter duration of NF-κB p65 subunit nuclear localization compared to healthy controls, and CD donors. Analysis of macrophage cytokine responses and patient metadata revealed a strong correlation between CD patients who smoked and hyper-activation of p65. These in vitro dynamic assays of NF-κB activation in blood-derived macrophages have the potential to segregate IBD patients into groups with different phenotypes and may therefore help determine response to therapy.

Keywords: Crohn's disease; NF-κB; cytokines; inflammatory bowel disease; macrophages; ulcerative colitis.

Figure 1

Endogenous NF-κB-regulated luciferase activity in lipopolysaccharide-stimulated PBMDMs. Graphs of κB-NLSluc luciferase activity measured over time (0–18 h) from differentiated human PBMDMs of two representative patients that were either (A) responsive or (B) non-responsive to stimulation with increasing doses of lipopolysaccharide (LPS); 2 ng/mL (red line), 20 ng/mL (blue line), and 200 ng/mL (black line); n = 5 vials PBMDM cultures for each patient. Nfkb1^−/− murine BMDMs (N = 3 mice, n = 2 replicates) stimulated with 200 ng/mL LPS were used as a negative luciferase activity control (gray dotted line). (C) Flow chart describing the steps that were followed for the clustering analysis (PAM = partitioning around medoids). (D) LPS-stimulated PBMDMs could assigned to one of three clusters based on log₂ fold change in the luciferase activity profile using K-medoids algorithm. Bold lines show mean values, Dashed lines show ± standard error of the mean (SEM), and faint lines show individual cultures.

Figure 2

Pro-inflammatory cytokine levels can reflect differences between NF-κB-regulated luciferase activity defined clusters. (A) Principal component analysis (PCA) based on concentrations of pro-inflammatory cytokines secreted to the medium of 200 ng/mL lipopolysaccharide (LPS)-stimulated patient PBMDMs from Cluster 1 (red), and Cluster 3 (blue) samples. (B) Quantification of individual cytokines levels (pg/mL) released from patient PBMDMs over 20 h stimulation with LPS. Statistical comparisons between clusters were made using the Mann-Whitney U-test. Individual p-values are reported on each chart, with differences considered significant when p < 0.05.

Figure 3

NF-κB-regulated luciferase activity in healthy control and IBD patient donors. Luciferase activity from all patient PBMDMs screened represented as (A) a dynamic, color-coded graph of activity over time in response to 200 ng/mL LPS (blue, healthy controls; red, Crohn's disease (CD) and green, ulcerative colitis (UC), and as (B) area under the curve (AUC) for control, CD, and UC groups. NF-κB-regulated luciferase activity in (C) CD patients, and (D) healthy control donors based on smoking status. Statistical comparisons between disease types were made using the Kruskal-Wallis test, *p < 0.05. Statistical differences between smoking status were tested using the Mann-Whitney U-test, individual p-values are reported on each chart.