Noncanonical NF-κB Signaling Upregulation in Inflammatory Bowel Disease Patients is Associated With Loss of Response to Anti-TNF Agents

Abstract

Objectives: Targeting tumor necrosis factor (TNF) with biologic agents, such as infliximab and adalimumab, is a widely used and effective therapeutic strategy in inflammatory bowel disease (IBD). Unfortunately, a significant number of patients fail to respond or lose response over time to these agents. Previous studies have defined multiple complex roles for canonical NF-κB signaling in the pathogenesis of IBD. However, preliminary evidence suggests that the lesser defined noncanonical NF-κB signaling pathway also contributes to disease pathogenesis and response to anti-TNF agents. The objective of this study was to evaluate this hypothesis in Crohn's disease (CD) and ulcerative colitis (UC) patients. Design: A total of 27 subjects with IBD (19 with CD and 8 with UC) and 15 control subjects were tested. Clinical criteria, patient history, and endoscopic disease activity were factors used to categorize patients and define therapeutic response. Biopsy specimens were collected during colonoscopy and expression was determined for 88 target genes known to be associated with noncanonical NF-κB signaling and IBD. Results: Noncanonical NF-κB signaling was significantly upregulated in IBD patients and was associated with increased gastrointestinal inflammation, epithelial cell death, lymphocyte migration, and Nod-like receptor signaling. Furthermore, noncanonical NF-κB signaling was further upregulated in patients unresponsive to anti-TNF agents and was suppressed in responsive patients. MAP3K14, NFKB2, CCL19, CXCL12, and CXCL13 were significantly dysregulated, as were genes that encode pathway regulators, such as CYLD, NLRP12, and BIRC2/3. Conclusion: Our study identifies a previously uncharacterized role for the understudied noncanonical NF-κB signaling pathway in the pathogenesis of IBD and anti-TNF therapy responsiveness. The genes and pathways identified may ultimately prove useful in IBD management and could potentially be used as biomarkers of drug response.

Keywords: Crohn’s disease; NIK; alternative pathway; anti-TNF agents; inflammatory bowel disease; noncanonical NF-κB pathway; therapeutic response; ulcerative colitis.

FIGURE 1

Noncanonical NF-κB Signaling is Upregulated in Inflammatory Bowel Disease Patients (A) Left: Compared to gene expression from intestinal tissues of controls (n = 15, 3 pools), 39 genes related to noncanonical NF-κB pathway were upregulated in intestinal tissues obtained from untreated CD patients (n = 2, 1 pool) with yellow indicating upregulation and blue indicating downregulation (lines represent 1, 0, and −1 fold change; genes outside of dashed lines are considered significantly dysregulated). Housekeeping genes used for normalization include ACTB, B2M, and GADPH; Right: list of 39 upregulated genes and three downregulated genes and fold change values. (B). Using real-time PCR of individual patient samples, the main noncanonical NF-kB pathway components TNF, NIK, CXCL12, CXCL13, and CXCR4 were found to be upregulated in inflamed areas compared to non-inflamed areas obtained from the same individual patients (inflamed lesions n = 4; non-inflamed tissue n = 4).

FIGURE 2

Noncanonical NF-κB is Downregulated in IBD Patients Responding To Anti-TNF Therapy. (A): Inflamed tissues from anti-TNF nonresponders (n = 10, two pools, CD patients and UC patients) showed 39 upregulated genes related to the noncanonical NF-κB signaling, compared to only 19 genes in anti-TNF responders (n = 5, two pools) (B), with yellow indicating upregulation and blue indicating downregulation (lines represent 1, 0, and −1 fold change; genes outside of dashed lines are considered significantly dysregulated). A list of the involved genes is provided next to each graph. Housekeeping genes used for normalization include ACTB, B2M, and GADPH (C–F): Ingenuity Pathway Analysis revealed that noncanonical NF-κB was a significant signaling hub that was downregulated in patients responding to anti-TNF therapy. Expression levels of the main noncanonical genes NIK (MAP3K14), CXCL12, CXCL13, and CCL21 were upregulated in both untreated patients (n = 2, one pool) (C) and patients treated with non-anti-TNF medications (n = 10, two pools) (D). However, anti-TNF responders (n = 5, two pools) (E), showed a distinct attenuation of expression of these genes. Meanwhile, anti-TNF non-responders showed upregulation of CXCL12 and CXCL13 and downregulation of CCL21. Note: green represents downregulation; red represents upregulation.

FIGURE 3

CXCL12 and CXCL13 are Downregulated in IBD Patients Responding to Anti-TNFα Therapy. (A) CXCL12, CXCL13, CCL19, and CCL21 were significantly upregulated in both untreated IBD patients (n = 2, one pool) and IBD patients treated with non-anti-TNF medications (n = 10, two pools). CXCL12, CXCL13, and CCL19 displayed opposite expression profiles based on anti-TNF responsiveness, with upregulation in anti-TNF nonresponders (n = 10, two pools) and downregulation in responders (n = 5, two pools) for CXCL12 and CXCL13. CCL21 remained downregulated regardless of response to anti-TNF agents. (B) Predicted effect on the migration of naïve lymphocytes to inflamed tissue based on changes of the noncanonical chemokines which are involved in lymphocyte trafficking. In anti-TNF responders, lymphocyte trafficking is reduced. By contrast anti-TNF nonresponders demonstrate increased lymphocyte trafficking. In general, the increase in noncanonical signaling molecules results in a large increase in predicted lymphocyte migration. Note: Blue and orange dashed lines with arrows indicate indirect inhibition and activation, respectively. Yellow and black dashed lines with arrows depict inconsistent effects and no prediction, respectively.