| Abstract: | Purpose: Delayed radiation enteritis is a frequent side effect of abdominal radiotherapy. Recent studies reveal bone marrow (BM) cells repopulate damaged tissue and contribute to repair in non-hematopoietic tissues. Fusion between BM and somatic cells is enhanced by chronic inflammation. Furthermore, BM cells (BMC) can trigger fibrotic response in damaged organ. The role of cell fusion in the development of chronic intestine fibrosis after radiotherapy needs further investigation. Materials and Methods: To evaluate cell fusion and fibrosis, gender-mismatched BM transplantation (BMT) were used. Immunoblots of macrophage fusion receptor (MFR), CD47 and fibronectin, connective tissue growth factor (CCN2) were measured. BMT with CD11b (+), CD3 (+) or B220 (+) enriched BMC were applied. The mice were treated with macrophage depleting agents, clodronate liposome, after radiotherapy and BMT. In vitro co-culture study of human intestine stromal and mouse BMC was established. Furthermore, we used Rac 1 inhibitor, MMP9 neutralizing antibody and siRNA of MFR within co-culture system and animal model. Results: Using triple stains of Y chromosome fluorescence in situ hybridization, GFP and hematopoietic cell markers including F4/80, CD3, B220, we found BM derived macrophages contributed majorly to cell fusion (63.4%, 47.5% and 9.6%, respectively, p=0.033) within intestine of mice after radiotherapy and BMT. The fusion within intestine decreased 57% after treatment with clodronate liposome. BMT from CD11b(+), CD3(+) or B220(+) enriched donor BMC also revealed prominent fusion only in mice receiving CD11b(+) one. The level of intestine fibrosis was decreased by clodronate liposome or BMT of CD11b(-) BMC. In vitro study showed the rate of fusion decreased from 12.8% to 6.8% between intestine stromal cells with CD11b (-) compared to that with whole mouse BMCs. Fusion rate could be increased by increasing CD11b(+) BMCs (3.5% to 19.7%). Using Rac1 inhibitor, MMP9 antibody or MFR siRNA, the fusion rate decreased (19.7% to 4.6%; 11.8% to 5.6%; 31% to 19.6%, respectively). The expression of fibronectin, CCN2, MFR and CD47 paralleled the findings of fusion. Using MMP9 neutralizing antibody in mice receiving radiotherapy and BMT, we found decreased cell fusion and fibrosis within intestine. Conclusions:BM derived CD11b(+) myelomonocytic cells or macrophage contribute majorly to cell fusion and fibrosis within intestine of mice after radiotherapy Depleting macrophage or targeting cell fusion machinery of macrophage suppresses intestine fibrosis after radiotherapy. Our study impacts not only for developing novel therapies to prevent radiation enteritis but also to avoid fibrosis after BM cell therapy in organ dysfunction. |
