Biomarkers of Therapeutic Response in the IL-23 Pathway in Inflammatory Bowel Disease

Abstract

Objectives: Interleukin-23 (IL-23) has emerged as a new therapeutic target for the treatment of inflammatory bowel disease (IBD). As biomarkers of disease state and treatment efficacy are becoming increasingly important in drug development, we sought to identify efficacy biomarkers for anti-IL-23 therapy in Crohn's disease (CD).

Methods: Candidate IL-23 biomarkers, downstream of IL-23 signaling, were identified using shotgun proteomic analysis of feces and colon lavages obtained from a short-term mouse IBD model (anti-CD40 Rag2(-/-)) treated preventively with monoclonal antibodies (mAbs) to the IL-23 receptor (IL-23R). The biomarkers were then measured in an IBD T-cell transfer model treated therapeutically with a mAb to IL-23 (p19), confirming their association with IBD. To assess the clinical relevance of these markers, we assessed their concentrations in clinical serum, colon tissue, and feces from CD patients.

Results: We identified 57 proteins up or downregulated in diseased animals that returned to control values when the mice were treated with mAbs to IL-23R. Among those, S100A8, S100A9, regenerating protein 3β (REG), REG3γ, lipocalin 2 (LCN2), deleted in malignant tumor 1 (DMBT1), and macrophage migration inhibitory factor (MIF) mRNA levels correlated with disease score and dose titration of mAbs to IL-23R or IL-23(p19). All biomarkers, except DMBT1, were also downregulated after therapeutic administration of mAbs to IL-23(p19) in a T-cell transfer IBD mouse model. In sera from CD patients, we confirmed a significant upregulation of S100A8/A9 (43%), MIF (138%), pancreatitis-associated protein (PAP, human homolog of REG3β/γ; 49%), LCN2 (520%), and CCL20 (1280%), compared with control samples, as well as a significant upregulation of S100A8/A9 (887%), PAP (401%), and LCN2 (783%) in human feces from CD patients compared with normal controls.

Conclusions: These studies identify multiple protein biomarkers downstream of IL-23 that could be valuable tools to assess the efficacy of this new therapeutic agent.Clinical and Translational Gastroenterology (2012) 3, e10; doi:10.1038/ctg.2012.2; published online 16 February 2012.

Figure 1

Candidate biomarker gene expression and protein concentration in mouse colon tissue or feces of a T-cell-independent model. One day before disease induction, Rag2^−/− mice were injected subcutaneously with isotype control (at 60 mg/kg) or anti-interleukin(IL)-23R neutralizing antibodies (at 60 mg/kg). On the day of disease induction, mice were injected with 50 μg of anti-CD40 mAbs intravenously to induce immune-mediated colitis (see Methods). Mice were killed at days 1, 3, 5, and 7 (N=4 per group). (a) Hematoxylin and eosin staining of proximal colon. The dashed lines indicate mucosal inflammation. Real-time reverse-transcription PCR analysis of (b) IL-22, IL-17A, and tumor necrosis factor-α (TNF-α), (c) S100A8, S100A9, REG3β, REG3γ, DMBT1, LCN2, and MIF transcripts in mouse proximal colon. Results are presented relative to ubiquitin transcripts (RU, relative units). (d) Western blot analysis of S100A8 and REG3γ in feces, (e) REG3γ and DMBT1 in colonic epithelial cells, and (f) LCN2 in colonic lamina propria. Data are representative of two experiments. Mean value is shown±s.d. ^*P<0.05, ^**P<0.01. DMBT1, deleted in malignant tumor 1; LCN2, lipocalin 2; MIF, migration inhibitory factor; REG, regenerating protein.

Figure 2

Expression profiles of the candidate biomarkers in a T-cell-dependent mouse colitis model: preventive and therapeutic treatment with anti-interleukin(IL)-23(p19). (a) Real-time reverse-transcription PCR (real-time RT-PCR) of transcript levels in colon tissue. Bir14 CD4+ T cells or control anti-CD3T cells were transferred intravenously into C3H.SCID mice, and anti-IL-23p19 mAb or control mAb was administered intraperitoneally (100 μg/mouse) on the same day of cell transfer and weekly thereafter. All mice were killed 8 weeks after cell transfer. (b) Bir14 CD4+ T cells or control anti-CD3T cells were transferred into C3H/HeJ SCID mice intravenously. Group 1 was killed after 4 weeks to assess that a colitis was present. Then, groups 2 and 3 were administered either isotype control or anti-IL-23(p19) mAbs at 100 μg/mouse weekly for 4 weeks. Real-time RT-PCR of the markers was performed twice with groups of 3 and 4 mice each time. Results are presented relative to ubiquitin transcripts (RU, relative units). Bars indicate the median values. ^*P<0.05, ^**P<0.01 and ^***P<0.001.

Figure 3

Regulation of the candidate biomarkers by interleukin (IL)-17A, IL-22 and tumor necrosis factor-α (TNF-α) in HT-29 human colonic epithelial cells. Human HT29 colonic epithelial cells were incubated with IL-23 (50 ng/ml), IL-22, IL-17A, and/or TNF-α at 20 ng/ml for 24–48 h. (a) Expression levels were measured by real-time reverse-transcription PCR (real-time RT-PCR) analysis. Results are shown as expression relative to ubiquitin mRNA levels (RU, relative units). (b) The protein accumulation was measured in cell supernatant by enzyme-linked immunosorbent assay (ELISA). Error bars represent s.d.; data are representative of 3 experiments. DMBT1, deleted in malignant tumor 1; LCN2, lipocalin 2; PAP, pancreatitis-associated protein.

Figure 4

Expression of potential interleukin(IL)-23-associated biomarkers in patients with Crohn's disease. (a) Serum concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in normal control subjects (N=18) and Crohn's disease (CD) patients (N=17). Bars indicate the median values. (b) Real-time reverse-transcription PCR for the expression of the candidate biomarkers in colon tissue biopsies from normal control subjects (N=6–7), CD active (N=6–7), and remission patients (N=6). Results are presented relative to ubiquitin transcripts (RU, relative units). Bars indicate the median values. ^*P<0.05, ^**P<0.01, and ^***P<0.001. (c) Fecal concentrations were measured by ELISA in normal control subjects (N=20) and CD patients (N=11). Bars indicate the median values. ^*P<0.05, ^**P<0.01. DMBT1, deleted in malignant tumor 1; LCN2, lipocalin 2; MIF, migration inhibitory factor; PAP, pancreatitis-associated protein.