IL-33 targeting attenuates intestinal mucositis and enhances effective tumor chemotherapy in mice

Abstract

Intestinal damage and severe diarrhea are serious side effects of cancer chemotherapy and constrain the usage of most such therapies. Here we show that interleukin-33 (IL-33) mediates the severe intestinal mucositis in mice treated with irinotecan (CPT-11), a commonly used cancer chemotherapeutic agent. Systemic CPT-11 administration led to severe mucosal damage, diarrhea, and body weight loss concomitant with the induction of IL-33 in the small intestine (SI). This mucositis was markedly reduced in mice deficient in the IL-33R (ST2(-/-)). Moreover, recombinant IL-33 exacerbated the CPT-11-induced mucositis, whereas IL-33 blockade with anti-IL-33 antibody or soluble ST2 markedly attenuated the disease. CPT-11 treatment increased neutrophil accumulation in the SI and adhesion to mesenteric veins. Supernatants from SI explants treated with CPT-11 enhanced transmigration of neutrophils in vitro in an IL-33-, CXCL1/2-, and CXCR2-dependent manner. Importantly, IL-33 blockade reduced mucositis and enabled prolonged CPT-11 treatment of ectopic CT26 colon carcinoma, leading to a beneficial outcome of the chemotherapy. These results suggest that inhibition of the IL-33/ST2 pathway may represent a novel approach to limit mucositis and thus improve the effectiveness of chemotherapy.

Figure 1

IL-33 is produced in the small intestine in chemotherapy-induced mucositis. (a) IL-33 and sST2 concentrations in the small intestine (SI) of BALB/c mice after 4 daily injections of CPT-11 (60 mg/kg) or PBS were determined by ELISA. (b) IL-33 production in vitro by SI explants from BALB/c mice after 24 h incubation with CPT-11 or SN-38 measured by ELISA. (c) Representative IHC staining for IL-33 and Annexin V in the SI of PBS or CPT-11-treated mice at day 4. 200X magnification. Arrows indicate positive staining for IL-33 and Annexin V in the villi (black) and crypts (red). Inserts: ISO: 200X, CPT-11: 400X magnification. (d) The frequency of IL-33⁺CD45⁻cytokeratin⁺ epT cells from mice treated with CPT-11 or PBS (vehicle). (e) Representative FACS analysis showing the gating of IL-33⁺CD45⁻Cytokeratin⁺ epithelial cells (epT) isolated from WT mice treated with CPT-11 (50 μM) for 6 h. (f) IL-33 concentration in the supernatant of Caco-2 cells cultured for 48 h with CPT-11 or SN-38 determined by ELISA. Results are representative of two independent experiments (n=3-5 mice per group). *P<0.05, **P<0.01, ***P<0.001

Figure 2

IL-33 is associated with the pathogenesis of chemotherapy-induced mucositis. WT and ST2^−/− mice were treated with CPT-11 or PBS with or without IL-33. (a) Δ Body weight loss (%) was measured daily and (b) clinical score determined on day 4. (c) Whole blood cell (WBC) counts, (d) small intestine (SI) length, and (e) Evans blue leakage were also determined on day 5. (f) Histopathology score, (g) villus/crypt ratio, and (h) Goblet cell counts in the crypt-villus axis were also shown. (i) Representative histology of SI samples on day 5. (j) Expression of Eubacteria 16s rDNA in the blood was evaluated by qPCR on day 4. Data are pool of 2 experiments (n= 4-8 mice per group) and are representative of three independent experiments. *P<0.05, **P<0.01, ***P<0.001.

Figure 3

Neutrophils play a key role in CPT-11/IL-33-induced intestinal mucositis. WT and ST2^−/− mice were treated with CPT-11 with or without IL-33. (a) Representative IHC staining for MPO in the SI on day 2. Arrows indicate positive staining for MPO of the villi. (b) Myeloperoxidase (MPO) activity in the SI on day 4. (c) Gating and representative percentage of blood CD11b⁺Ly6G⁺ neutrophils on day 2. (d) Representative Mean Fluorescence Intensity (MFI) of CXCR2 on blood CD11b⁺Ly6G⁺ neutrophils on day 2. (e) Percentage of CD62L⁺ CD11b⁺ Ly6G⁺ neutrophils in the blood on day 2. (f) Gating and representative percentage of CD11b⁺Ly6G⁺ neutrophils in the SI on day 4. (g) Representative MFI of CXCR2 on CD11b⁺Ly6G⁺ neutrophils in the SI on day 4. (h) Percentage of CD62L⁺CD11b⁺Ly6G⁺ cells in the SI on day 4. SI explants from WT and ST2^−/− mice were cultured for 24 h in the presence of CPT-11 (50 μM), SN-38 (2 μM) or IL-33 (10 ng/ml) and the concentrations of (i) CXCL1/KC and (j) CXCL2/MIP-2 determined by ELISA. (k, l) SI explants from WT or ST2^−/− mice were cultured with CPT-11, SN-38 or IL-33. Supernatants were collected after 24 h and placed in the lower chamber of transwell culture plates. WT LyG6⁺ neutrophils were placed on the upper chamber. (k) The number of neutrophils in the lower chamber was determined 3 h later. (l) The number of trans-migrating neutrophils in the presence of sST2 (10 μg/ml) in the lower chamber was also shown. Results are representative of three independent experiments (n=5-6 mice per group). *P<0.05, **P<0.01, ***P<0.001.

Figure 4

IL-33 blockade ameliorates CPT-11-induced intestinal mucositis. WT mice were treated with PBS or CPT-11 for 4 days and received anti-IL-33 (25 μg/daily/s.c.) or sST2 (100 μg/daily/s.c.). Data were analyzed on day 4. (a) Δ Body weight loss; (b) clinical score; (c) histopathology score; (d) representative histology (H&E staining) of SI; (e) Myeloperoxidase (MPO) activity of SI. (f) CXCL1/KC, (g) CXCL2/MIP-2 and (h) CCL2/JE production in the SI determined by ELISA. Results are representative of two independent experiments (n=5 mice per group). *P<0.05, **P<0.01, ***P<0.001.

Figure 5

Neutrophil depletion attenuates CPT-11-induced intestinal mucositis. BALB/c mice were treated with CPT-11 with or without IL-33. Some mice were injected s.c. with anti-Ly6G (100 μg) or control IgG on days 0 and 2. Data were analyzed on day 4. (a) Δ Body weight loss; (b) Clinical score; (c) Histopathology score; (d) Representative histology of SI (H&E). Intra-vital microscopy was used to assess the rolling (e) and adhesion (f) of leukocytes on the mesenteric microvasculature on day 2 in WT and ST2^−/− mice. Data are pool of 2 experiments (n=8-10 mice per group). (g) Representative pictures of the mesenteric veins. Red dotted lines, blood vessels; white arrows, leukocytes attached to the vessels. Results are representative of two independent experiments (n=5 mice per group). *P<0.05, **P< 0.01, ***P<0.001. Also see Supplementary Videos.

Figure 6

IL-33 targeting attenuates intestinal mucositis and prolongs effective chemotherapy against tumour. WT and ST2^−/− mice were transplanted s.c. with CT26 colon carcinoma cells (1×10⁶ cells). Some mice were treated with CPT-11 at indicated doses. Some WT mice were also injected daily s.c. (day 2-12) with anti-IL-33 (25 μg) or sST2 (100 μg). Tumour growth (mean diameter in cm), clinical score and body weight loss were recorded daily. (a) WT and ST2^−/− mice; (b) WT and anti-IL-33-treated mice; (c) WT and sST2-treated mice. Results are representative of two independent experiments (n=6 mice per group). Dark cross: mice were culled because of severity of disease. *P<0.05, **P<0.01, ***P<0.001. See Supplementary Figure S4 for pictures of tumour-bearing mice.

Figure 7

Schematic representation of the mechanism of IL-33-mediated CPT-11-induced intestinal mucositis and potential therapeutic targets. CPT-11 damages the intestinal mucosa and releases IL-33, which enhances chemokines production from the epithelial cells and up-regulation of CXCR2 on neutrophils, thereby increasing the recruitment of neutrophils to the inflammatory sites, leading to further tissue damage and bacterial translocation. Blocking of IL-33 by anti-IL-33 antibody or sST2 attenuates CXC chemokine production and neutrophil activation/accumulation. Depletion of neutrophils or blocking CXCR2 would also attenuate tissue damage and disease outcome.