![Figure 1. IL-33 expression in the murine small intestine increases upon TBI and in the colon of stage IV GVHD patients. (A-B) After TBI of BALB/c mice with 9 Gy, RNA expression levels of multiple genes in the small bowel were analyzed using microarray-based analysis. This experiment was performed once. (A) Shown is the tile display for the most significantly regulated genes encoding for cytokines, expressed by Robust Multichip Average (RMA). Signal values of 4 individual samples in the following groups are shown: untreated, 24 hours, and 48 hours after TBI. Green rectangle: IL-33 is the second most significantly regulated gene. (B) Each RMA value for IL-33 is shown, including the P values. (C-D) Gut tissue from control (day 0), lethally-irradiated (day 2 post-irradiation), and WT or C57BL/6 recipients, that had received either 2.5 × 106 T cells and 1 × 107 BM cells, or BM only from BALB/c donors in GVHD studies (day 7 or day 14) were stained for IL-33, CD45, vimentin, and 4,6 diamidino-2-phenylindole (DAPI). This experiment was performed once. Images are representative of n = 3 mice/group and graphs were generated by averaging 2 to 3 fields (IL-33/DAPI–20×; IL-33/CD45, or IL-33/vimentin–20×) per animal. Fluorescence area/intensity reported as arbitrary units. *P < .05; ***P < .001. This experiment was performed once. (E) Gut tissue from BALB/c mice 7 days after transplantation with 5 × 106 BM cells and 3 × 105 T cells from C57Bl/6 mice. Prior to transplantation on day 0, mice received either TBI (2× 4,5 Gy), busulfuan/cyclophosphamide (Bu/Cy: busulfan day −7 to day −4 [dose: 10 mg/kg]), cyclophosphamide day −3 and day −2 (dose 100 mg/kg), treosulfan/cyclophosphamide (Treo/Cy: treosulfan day −6 to day −4 [dose: 1.5 g/kg]), cyclophosphamide day −3 and day −2 [dose 100 mg/kg]), or thiotepa/cyclophosphamide (Thio/Cy: thiotepa day −6 to day −4 [dose: 10 mg/kg], cyclophosphamide day −3 and day −2 [dose 100 mg/kg]) conditioning regimens. Fluorescence area/intensity reported as in (C-D). (Ei) Bar diagram for the indicated groups. (Eii) Representative tissue sections. Images are representative of n = 2 to 3 mice per group and graphs were generated by averaging 3 to 6 fields (IL-33/DAPI–20×) per animal. This experiment was performed once. (F-G) The amount of IL-33 in human colon biopsies was quantified by immunohistochemistry as shown for one representative section per group ([Fi] no GVHD; [Fii] GVHD IV° of the intestines) and for multiple patients (G). Samples were taken at different time points after allo-HCT in patients without GVHD or with GVHD grade 3/4. Patients’ characteristics, time of biopsy, conditioning, and immunosuppression data are shown in supplemental Table 1.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/125/20/10.1182_blood-2014-10-606830/4/3183f1.jpeg?Expires=1723171046&Signature=SprSDUnW7m5erdhy~8vXzUC5QYhaffqULgU07MSKJHvHE8fRru4JuVSoXM7hqz6XDy86Ny6ebrhLTjwt9R5Nz1v6B4K~XOAhLXeOUr2qyJPvF2zC7~8xUwSrxlTTwHeANC~CVByaG9owgl5jVD2R-1mkHg1nIIta4VkONQc0yCP22ryCO7Oze-hSZo~PFGE68fiHr13LUP1j9ZRezSLD0Gghw0Ngw9CtjoJ0yGWMc~QJ--DvPJxbK~6AbuhYhWP7TxFPguuK8~K9ZvXdh6UVGK1~IOM9mm6rFGBN6E69iyDfCXL-dAdK3MeJeqZy6sj4aK8ZO46Nq3RsfcYuoEUfkA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
IL-33 expression in the murine small intestine increases upon TBI and in the colon of stage IV GVHD patients. (A-B) After TBI of BALB/c mice with 9 Gy, RNA expression levels of multiple genes in the small bowel were analyzed using microarray-based analysis. This experiment was performed once. (A) Shown is the tile display for the most significantly regulated genes encoding for cytokines, expressed by Robust Multichip Average (RMA). Signal values of 4 individual samples in the following groups are shown: untreated, 24 hours, and 48 hours after TBI. Green rectangle: IL-33 is the second most significantly regulated gene. (B) Each RMA value for IL-33 is shown, including the P values. (C-D) Gut tissue from control (day 0), lethally-irradiated (day 2 post-irradiation), and WT or C57BL/6 recipients, that had received either 2.5 × 106 T cells and 1 × 107 BM cells, or BM only from BALB/c donors in GVHD studies (day 7 or day 14) were stained for IL-33, CD45, vimentin, and 4,6 diamidino-2-phenylindole (DAPI). This experiment was performed once. Images are representative of n = 3 mice/group and graphs were generated by averaging 2 to 3 fields (IL-33/DAPI–20×; IL-33/CD45, or IL-33/vimentin–20×) per animal. Fluorescence area/intensity reported as arbitrary units. *P < .05; ***P < .001. This experiment was performed once. (E) Gut tissue from BALB/c mice 7 days after transplantation with 5 × 106 BM cells and 3 × 105 T cells from C57Bl/6 mice. Prior to transplantation on day 0, mice received either TBI (2× 4,5 Gy), busulfuan/cyclophosphamide (Bu/Cy: busulfan day −7 to day −4 [dose: 10 mg/kg]), cyclophosphamide day −3 and day −2 (dose 100 mg/kg), treosulfan/cyclophosphamide (Treo/Cy: treosulfan day −6 to day −4 [dose: 1.5 g/kg]), cyclophosphamide day −3 and day −2 [dose 100 mg/kg]), or thiotepa/cyclophosphamide (Thio/Cy: thiotepa day −6 to day −4 [dose: 10 mg/kg], cyclophosphamide day −3 and day −2 [dose 100 mg/kg]) conditioning regimens. Fluorescence area/intensity reported as in (C-D). (Ei) Bar diagram for the indicated groups. (Eii) Representative tissue sections. Images are representative of n = 2 to 3 mice per group and graphs were generated by averaging 3 to 6 fields (IL-33/DAPI–20×) per animal. This experiment was performed once. (F-G) The amount of IL-33 in human colon biopsies was quantified by immunohistochemistry as shown for one representative section per group ([Fi] no GVHD; [Fii] GVHD IV° of the intestines) and for multiple patients (G). Samples were taken at different time points after allo-HCT in patients without GVHD or with GVHD grade 3/4. Patients’ characteristics, time of biopsy, conditioning, and immunosuppression data are shown in supplemental Table 1.
