Defective IL-23/IL-17 Axis Protects p47phox-/- Mice from Colon Cancer

Abstract

In the colon, a sophisticated balance between immune reaction and tolerance is absolutely required. Dysfunction may lead to pathologic phenotypes ranging from chronic inflammatory processes to cancer development. Two prominent modulators of colon inflammation are represented by the closely related cytokines interleukin (IL)-12 and IL-23, which initiate adaptive Th1 and Th17 immune responses, respectively. In this study, we investigated the impact of the NADPH oxidase protein p47phox, which negatively regulates IL-12 in dendritic cells, on colon cancer development in a colitis-associated colon cancer model. Initially, we found that IL-12-/- mice developed less severe colitis but are highly susceptible to colon cancer. By contrast, p47phox-/- mice showed lower tumor scores and fewer high grade tumors than wild-type (WT) littermates. Treatment with toll-like receptor 9 ligand CpG2216 significantly enhanced colitis in p47phox-/- mice, whereas tumor growth was simultaneously reduced. In tumor tissue of p47phox-/- mice, the IL-23/IL-17 axis was crucially hampered. IL-23p19 protein expression in tumor tissue correlated with tumor stage. Reconstitution of WT mice with IL-23p19-/- bone marrow protected these mice from colon cancer, whereas transplantation of WT hematopoiesis into IL-23p19-/- mice increased the susceptibility to tumor growth. Our study strengthens the divergent role of IL-12 and IL-23 in colon cancer development. With the characterization of p47phox as a novel modulator of both cytokines our investigation introduces a promising new target for antitumor strategies.

Keywords: DSS/AOM; IL-17; IL-23; IL23p19 knockout mouse; chronic colitis; colon cancer; p47phox.

Figure 1

Tumor and inflammation score of interleukin (IL)-12p35−/− and wild-type (WT) mice. IL-12p35−/− and WT mice were investigated in the DSS/AOM model. (A) Tumor score and (B) inflammation score of IL-12p35−/− and WT mice were determined by colonoscopic examination. Representative endoscopic pictures are shown (Tu, tumor; Th, thickening of the colon; Gr, granularity of the mucosal surface; Fi, fibrin; Vasc, vascularization). Results are mean ± SEM of at least six mice per group. Statistical significance is indicated (*P < 0.05; **P < 0.01).

Figure 2

Tumor and inflammation score of p47phox−/− and wild-type (WT) mice treated with or w/o toll-like receptor 9 ligand CpG. (A) Tumor score and (B) inflammation score of p47phox−/− and WT mice, determined by colonoscopic examination. (C) Tumor score and (D) inflammation score of p47phox−/− and WT mice, weekly treated with 25 µg CpG2216. (E,F) Weight development of mice treated w/o (E) and with CpG (F) during and after the third DSS cycle as percentage of starting weight. The DSS cycle is marked in gray. (A–F) Results are mean ± SEM of three independent experiments with at least seven mice per group. Statistical significance is indicated (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 3

Expression of interleukin (IL)-12, IL-23, and IL-17 mRNA in tumor and inflamed tissue of p47phox−/− and wild-type (WT) mice. (A) Expression of IL-23p19, (B) IL-17, (C) IL-12p35, and (D) IL-12/23p40 mRNA in tumor and inflamed tissue from p47phox−/− and WT mice treated with or w/o CpG was determined by quantitative real-time PCR. Results are mean ± SEM and representative of three independent experiments with n = 12–22 samples. General differences between p47phox−/− and WT mice were tested for statistical significance (*P < 0.05; **P < 0.01). Differences between tumor and inflamed tissue from p47phox−/− and WT mice, respectively, were tested for statistical significance (^#P < 0.05; ^##P < 0.01; and ^###P < 0.001).

Figure 4

Histological analyses of tumor tissue. (A) Numbers of tumors per tumor size calculated from seven mice per group. (B) Colon tissue sections of p47phox−/− and wild-type mice treated with or w/o CpG were stained with hematoxylin and eosin. Grades are indicated as aberrant crypt foci, adenoma, and mucosa carcinoma (MC). One representative section of three to five is shown. Statistical analysis of tumors in histological sections was performed. Results are mean ± SEM of three to five histological samples per group (C) Colon tissue sections were stained for interleukin (IL)-23, Ki-67, and counterstained for hematoxylin. For each stage, one representative section of three to five is shown. (D) Flow cytometry analysis of IL-23 and IL-12 in myeloid cells from tumor and tumor-free tissue was performed. Results are mean ± SEM of five to seven mice per group. Statistical significance is indicated (*P < 0.05; **P < 0.01).

Figure 5

Tumor and inflammation score of interleukin (IL)-23p19−/− (p19KO) and wild-type (WT) recipient mice. (A,B) Tumor (A) and inflammation (B) score of p19KO and WT mice, lethally irradiated and transplanted with bone marrow cells from p19KO and WT mice or vice versa. (C,D) Tumor (C) and inflammation (D) score of CpG-treated, transplanted mice. Results are mean ± SEM and n = 3–6. Statistical significance is indicated (*P < 0.05; **P < 0.01).