Research ArticleFibrosis

Eosinophil depletion suppresses radiation-induced small intestinal fibrosis

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Science Translational Medicine  21 Feb 2018:
Vol. 10, Issue 429, eaan0333
DOI: 10.1126/scitranslmed.aan0333
  • Fig. 1 Abdominal irradiation induces fibrosis of small intestinal SM regardless of lymphocyte deficiency.

    (A) Magnetic resonance imaging (MRI) of abdomen of wild-type (WT) mice at 12 weeks after abdominal irradiation (12 Gy). The lower panel shows bowel wall thickness (n = 3). Scale bars, 5 mm. (B) Azan staining of small intestines of WT mice at 12 weeks. Muc, mucosa; SM, submucosa; Mus, muscularis externa. Scale bars, 200 μm. The lower panel shows the thickness of the SM fibrous layer, as indicated by red arrows and insets (n = 6 to 7). (C) Immunohistochemistry of α-smooth muscle actin (α-SMA) and CD31 in normal small intestines of mice (n = 3). Scale bar, 100 μm. (D) Time course changes in the location of α-SMA+ cells and collagen deposition in the SM after irradiation. Each panel shows immunohistochemistry of α-SMA (left), zoomed image of inset in left panel (middle), and azan staining (right) at 0, 8, and 12 weeks. Yellow arrowheads denote α-SMA+ cells. Red arrowheads denote collagen deposited between crypts and α-SMA+ cells. Scale bars, 50 μm. (E) Azan staining of the small intestines from Rag2+/+ and Rag2−/− mice at 12 weeks. Scale bars, 200 μm. The lower panel shows the thickness of the SM fibrous layer (n = 4 to 9). Results are the means ± SEM of a representative of two (A) or three (B to E) independent experiments. *P < 0.05, **P < 0.01. N.S., not significant [unpaired t test for (A) and (B); Tukey-Kramer post hoc test for (E)].

  • Fig. 2 RIF is associated with the accumulation of activated eosinophils in the SM.

    (A) Anti–major basic protein (MBP) staining of small intestines of WT mice at 12 weeks after abdominal irradiation (12 Gy). Hematoxylin and eosin (H&E) staining shows a higher magnification of the SM. Red arrowheads denote eosinophils. Scale bars, 100 μm. The lower panels show the numbers of MBP-positive cells in the SM and villous core (n = 6 to 7). (B) H&E staining of the SM of healthy and fibrotic human small intestines. Fibrotic intestine indicated in the image was surgically resected from a patient who received fractionated radiation therapy for colorectal cancer (50 Gy in 25 fractions) at 215 days after the last irradiation. Small magnification images including the area of (B) is shown in the lowest panels of fig. S1. Red arrowheads denote eosinophils. Scale bars, 20 μm. The lower panel shows the numbers of eosinophils infiltrating the SM. Eosinophil numbers were measured at 10 fields per patient (n = 3). (C) Representative transmission electron microscopy image of eosinophils in the small intestinal SM and villi of irradiated WT mice at 12 weeks (n = 3). Scale bars, 10 μm. Results are the means ± SEM. **P < 0.01 (unpaired t test). A representative of three independent experiments is shown (A and C).

  • Fig. 3 Intestinal eosinophils are critical for RIF.

    (A) Flow cytometry of small intestinal lamina propria (LP) cells from WT and ΔdblGATA mice. Diff-Quik staining of CD11bhiCD11cintSiglec-F+ cells. Scale bar, 10 μm. (B) MRI images of the abdomens of WT and ΔdblGATA mice at 12 weeks after abdominal irradiation (12 Gy). Scale bars, 5 mm. The right-hand panel shows bowel wall thickness (n = 3). (C) Azan staining of the small intestines of WT and ΔdblGATA mice at 12 weeks. Scale bars, 200 μm. The right-hand panel shows the thickness of the SM fibrous layer (n = 4 to 6). (D to F) Flow cytometry of LP cells of the small intestines (D), azan staining of the small intestines (E), and the numbers of eosinophils in the SM of small intestines (F) from WT mice, noninjected ΔdblGATA mice, and BM-derived eosinophils (BMEos)–injected ΔdblGATA mice at 12 weeks. Data in (D) to (F) were obtained from the same mice. The right-hand panel in (E) shows the thickness of the SM fibrous layer (n = 5 to 6). Scale bars, 200 μm. Results are the means ± SEM of a representative of two independent experiments. *P < 0.05, **P < 0.01 (Tukey-Kramer post hoc test).

  • Fig. 4 α-SMA+ cells induce eosinophil recruitment via the CCL11/CCR3 axis in RIF.

    (A) C-C chemokine receptor 3 (CCR3) and CCR5 expression by intestinal eosinophils of unirradiated mice (n = 3). (B) C-C motif chemokine 11 (CCL11)–mediated eosinophil migration in the presence of CCR3 antagonist SB-328437 (10 μM). Migration index is calculated as the ratio of the numbers of cells migrating in response to CCL11 relative to the numbers of cells migrating in response to media alone (n = 3). (C) Anti-MBP staining of the small intestines of Ccr3+/+ and Ccr3−/− mice at 12 weeks after abdominal irradiation (12 Gy). Scale bars, 100 μm. Red arrowheads denote eosinophils. The lower panel shows eosinophil numbers in the SM (n = 4 to 6). (D) Azan staining of the small intestines of Ccr3+/+ and Ccr3−/− mice at 12 weeks. Scale bars, 200 μm. The lower panel shows the thickness of the SM fibrous layer (n = 4 to 6). (E) Thickness of fibrous layer (left) and eosinophil numbers (right) in the small intestinal SM of 83103-treated WT mice were histologically measured at 12 weeks. Histological images are shown in fig. S7 (n = 5 to 6). (F) In situ hybridization (ISH) of Ccl11 mRNA with immunohistochemistry for α-SMA in the small intestines of WT mice at 4 weeks. Each panel shows ISH only (left), immunohistochemistry only (middle), and their combination (right). Arrows indicate Ccl11 mRNA-expressing α-SMA+ cells. Scale bars, 50 μm. Results are the means ± SEM of a representative of three (A and B) or two (C to F) independent experiments. *P < 0.05, **P < 0.01 [unpaired t test for (B) and (C); Tukey-Kramer post hoc test for (D) and (E)].

  • Fig. 5 Abdominal irradiation causes crypt necrosis and chronic ATP release.

    (A) Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) and anti-cleaved caspase-3 staining of small intestines after abdominal irradiation (12 Gy). Yellow arrowheads indicate TUNEL-positive crypt cells. Scale bars, 50 μm. (B) The numbers of TUNEL-positive crypt cells after abdominal irradiation (n = 4). HPF, high-power field. (C) Azan and anti-MBP staining of the small intestines of PPADS (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid)–treated WT mice at 12 weeks. PPADS was intraperitoneally administered three times per week after irradiation. Scale bars, 100 μm. Red arrowheads denote eosinophils. The right-hand panels show thickness of fibrous layer and eosinophil numbers in the SM (n = 6). (D) Adenosine triphosphate (ATP) concentration in culture supernatant of ex vivo–cultured small intestines of WT mice collected after abdominal irradiation (n = 4). Results are the means ± SEM of a representative of three (A and B) or two (C and D) independent experiments. *P < 0.05, **P < 0.01 [Tukey-Kramer post hoc test for (B) and (C); unpaired t test for (D)].

  • Fig. 6 CCL11 and GM-CSF from α-SMA+ cells induce eosinophil recruitment and activation.

    (A) Chemokine array analysis of cell lysates from intestinal α-SMA+ cells of WT mice treated with ATP (0 or 2.5 mM) for 2 days. The array images are shown in fig. S11. The signal intensity of the dot blot was measured in duplicate. (B) Quantitative real-time polymerase chain reaction (PCR) of Ccl11 mRNA expression in α-SMA+ cells stimulated with ATP for 1 day (n = 3). (C) Quantitative real-time PCR of mRNA expression of eosinophil activation-associated cytokines of intestinal α-SMA+ cells at 1 day after stimulation with ATP (0 or 2.5 mM; n = 3). (D) Flow cytometry of granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) expression by intestinal eosinophils of unirradiated WT mice (n = 3). (E) Immunohistochemistry for GM-CSF in the small intestine of WT mice at 4 weeks after irradiation. Red arrowheads denote pericryptal α-SMA+ cells. Scale bars, 50 μm. (F) Quantitative real-time PCR of Tgfb1 mRNA in intestinal eosinophils stimulated with GM-CSF for 4 hours (n = 4). (G) Col1a1 and Col1a2 mRNA in intestinal α-SMA+ cells at 1 day after incubation with supernatant from intestinal eosinophils, which were cultured with or without GM-CSF (10 ng/ml). For neutralizing the effects of transforming growth factor–β1 (TGF-β1), the supernatant was pretreated with anti-mouse TGF-β1 Ab (10 ng/ml) for 2 hours (n = 3). (H) Eosinophil peroxidase (EPX)–specific enzyme-linked immunosorbent assay analysis of the culture supernatants of intestinal eosinophils stimulated with GM-CSF (10 ng/ml) and CCL11 (100 ng/ml) for 2 hours (n = 3). Results are the means ± SEM of a representative of two independent experiments. **P < 0.01. [Tukey-Kramer post hoc test for (B), (F), (G), and (H); unpaired t test for (A) and (C)].

  • Fig. 7 cmIL5Ra1b12 effectively ameliorates RIF.

    (A) Flow cytometry of blood leukocytes of WT mice at 1 week after injection of cmIL5Ra1b12 (25 mg/kg). CD11b+Gr-1int cells among SSChiCD11c leukocytes are eosinophils expressing Siglec-F. (B) Time course changes in blood eosinophil frequency in WT mice injected with cmIL5Ra1b12 (n = 3 to 4). (C) Flow cytometry of small intestinal LP cells of WT mice at 4 weeks after cmIL5Ra1b12 injection (25 mg/kg). Right panels show CD11bhiCD11cintSiglec-F+ eosinophils. (D) Small intestinal eosinophil levels in WT mice at 4 weeks after cmIL5Ra1b12 injection (n = 3). (E) Azan and anti-MBP staining of the small intestines of cmIL5Ra1b12-treated WT mice at 13 weeks after abdominal irradiation. Scale bars, 100 μm. Red arrowheads denote eosinophils. The right-hand panels show thickness of the SM fibrous layer and eosinophil numbers in the SM (n = 5 to 6). Results are the means ± SEM of a representative of two independent experiments. **P < 0.01 [Tukey-Kramer post hoc test for (E)].

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/429/eaan0333/DC1

    Materials and Methods

    Fig. S1. Histology of human RIF.

    Fig. S2. Detection of collagen fibers in mouse small intestine after abdominal irradiation.

    Fig. S3. Migration of α-SMA+ cells from the pericryptal area after abdominal irradiation.

    Fig. S4. Detection of T cells in mouse small intestine after abdominal irradiation.

    Fig. S5. Eosinophil recruitment to the SM in Rag2−/− mice after abdominal irradiation.

    Fig. S6. Mouse BM-derived eosinophils (BMEos).

    Fig. S7. Anti-CCR3 Ab treatment ameliorates RIF.

    Fig. S8. RIF in P2x7r−/− mice after abdominal irradiation.

    Fig. S9. ATP receptor expression of intestinal α-SMA+ stromal cells.

    Fig. S10. Suppression of RIF by blocking ATP-mediated activation of P2X receptors.

    Fig. S11. Chemokine protein array analysis of intestinal α-SMA+ cells.

    Fig. S12. RIF in Il33−/− mice after abdominal irradiation.

    Fig. S13. RIF in Rag2−/−γc−/− mice after abdominal irradiation.

    Fig. S14. In vitro analyses of anti–IL-5Rα activities of cmIL5Ra1b12.

    Fig. S15. Eosinophil depletion by repeated treatment with cmIL5Ra1b12.

    Fig. S16. Hydroxyproline levels of the small intestines of cmIL5Ra1b12-treated WT mice.

    Table S1. Gene expression of intestinal eosinophils after GM-CSF stimulation.

    Table S2. Primary data.

    References (4450)

  • Supplementary Material for:

    Eosinophil depletion suppresses radiation-induced small intestinal fibrosis

    Naoki Takemura, Yosuke Kurashima, Yuki Mori, Kazuki Okada, Takayuki Ogino, Hideki Osawa, Hirosih Matsuno, Lamichhane Aayam, Satoshi Kaneto, Eun Jeong Park, Shintaro Sato, Kouta Matsunaga, Yusuke Tamura, Yasuo Ouchi, Yutaro Kumagai, Daichi Kobayashi, Yutaka Suzuki, Yoshichika Yoshioka, Junichi Nishimura, Masaki Mori, Ken J. Ishii, Mark E. Rothenberg, Hiroshi Kiyono, Shizuo Akira, Satoshi Uematsu*

    *Corresponding author. Email: suematsu{at}chiba-u.jp

    Published 21 February 2018, Sci. Transl. Med. 10, eaan0333 (2018)
    DOI: 10.1126/scitranslmed.aan0333

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Histology of human RIF.
    • Fig. S2. Detection of collagen fibers in mouse small intestine after abdominal irradiation.
    • Fig. S3. Migration of α-SMA+ cells from the pericryptal area after abdominal irradiation.
    • Fig. S4. Detection of T cells in mouse small intestine after abdominal irradiation.
    • Fig. S5. Eosinophil recruitment to the SM in Rag2−/− mice after abdominal irradiation.
    • Fig. S6. Mouse BM-derived eosinophils (BMEos).
    • Fig. S7. Anti-CCR3 Ab treatment ameliorates RIF.
    • Fig. S8. RIF in P2x7r−/− mice after abdominal irradiation.
    • Fig. S9. ATP receptor expression of intestinal α-SMA+ stromal cells.
    • Fig. S10. Suppression of RIF by blocking ATP-mediated activation of P2X receptors.
    • Fig. S11. Chemokine protein array analysis of intestinal α-SMA+ cells.
    • Fig. S12. RIF in Il33−/− mice after abdominal irradiation.
    • Fig. S13. RIF in Rag2−/−γc−/− mice after abdominal irradiation.
    • Fig. S14. In vitro analyses of anti–IL-5Rα activities of cmIL5Ra1b12.
    • Fig. S15. Eosinophil depletion by repeated treatment with cmIL5Ra1b12.
    • Fig. S16. Hydroxyproline levels of the small intestines of cmIL5Ra1b12-treated WT mice.
    • Table S1. Gene expression of intestinal eosinophils after GM-CSF stimulation.
    • References (4450)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Primary data.

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