Distinct Loci Influence Radiation-Induced Alveolitis from Fibrosing Alveolitis in the Mouse Christina K. Haston, Michelle Begin, Genevieve Dorion, and Sean M. Cory Meakins-Christie Laboratories and Department of Medicine, McGill University, Montreal, Quebec, Canada Abstract Thoracic radiotherapy may produce the morbidity-associated lung responses of alveolitis or fibrosing alveolitis in treated cancer patients. The genetic factors that influence a patient’s likelihood of developing alveolitis and the relationship of this inflammatory response to the development of fibrosis are largely unknown. Herein we use genetic mapping to identify radiation-induced lung response susceptibility loci in recip- rocal backcross mice bred from C3H/HeJ (alveolitis response) and C57BL/6J (fibrosing alveolitis/fibrosis response) strains. Mice were treated with 18-Gy whole thorax irradiation and their survival, lung histopathology, and bronchoalveolar lavage cell types were recorded. A genome-wide scan was completed using 139 markers. The C3H/HeJ alveolitis response included mast cell infiltration and increased neutrophil numbers in the lavage compared with the level in the C57BL/6J strain, which developed fibrosis. In backcross mice, posttreatment survival was dictated by the development of an alveolitis response with increased mast cell, bronchoalveolar lavage total cell, and neutrophil numbers. Fibrosis was measured only in a subset of mice developing alveolitis and, in these mice, was associated with neutrophil count. Geno- typing revealed coinheritance of C3H alleles (chromosomes 2, 4, 19, and X) and C57BL/6J alleles (chromosomes 1, 7, 9, and 17) to result in higher fibrosis scores in backcross mice. Mice that inherited C57BL/6J alleles at the putative alveolitis susceptibility loci were spared this response and lived to the end of the experiment. In this animal model, independent loci control the development of alveolitis from fibrosis, whereas fibrosing alveolitis occurs with the coinheritance of these factors. [Cancer Res 2007;67(22):10796–803] Introduction Radiation treatment of the thorax, which is frequently used as a therapy in breast, lung, and esophageal cancer (1–4) and in Hodgkin’s disease (5, 6), produces alveolitis and fibrosis in the lungs of up to 30% of treated patients (7). These pathologies of excessive inflammation or deposition of extracellular matrix in the lung interstitium can lead to impaired lung function and, ultimately, respiratory failure. The ability to determine a priori which patients are at risk for the development of these treatment-induced effects would allow for the formulation of patient-specific regimens aimed at maximizing therapy while minimizing the incidence of potentially devastating side effects. Indeed, reports of Anscher and Vujaskovic (8) and Kong et al. (7) both indicate that non– small-cell lung cancer could be more aggressively and effectively treated with radiation if higher doses could be delivered to the lung without increasing the patient’s risk for developing the complica- tions of alveolitis and fibrosis. This risk is dictated by radiation dose, tissue volume treated, and yet unknown inherent factors or genetic predisposition to adverse effects. As the identification of causal genetic variations influencing lung disease susceptibility using clinical data alone can be confounded by the effects of multiple genes and their interactions on the phenotype and by the cancer and other treatment modalities, inbred mouse strains that vary in their propensity to develop alveolitis and fibrosis (9, 10) after radiation exposure can be evaluated to genetically dissect the lung response. Specifically, the three clinical outcomes of thoracic radiotherapy, with respect to adverse effects (fibrosing alveolitis, alveolitis, or a subsymptomatic response) and the times at which they occur (3 months posttherapy for alveolitis, 6 months posttherapy for fibrosis; refs. 2–4), are each represented in the radiation response of an inbred mouse strain. We (11) and others (9, 10) have reported that following high-dose whole thorax irradiation, C3H/HeJ (C3H) mice develop a diffuse lethal alveolitis, the C57BL/6J (B6) response is fibrosing alveolitis, whereas male mice of the congenic major histocompatibility strain B6.AKR-H2k present a minimal lung response at 6 months posttreatment (12). The phenotypic difference between B6 and C3Hf/KAM mice has been used to map three loci of susceptibility to radiation-induced pulmonary fibrosis, named Radpf1, Radpf2 , and Radpf3 (12). Susceptibility to the alveolitis response has not been mapped. Defining the genetic basis for the radiation-induced inflamma- tory response of alveolitis has direct clinical implications as this response may be related to the severity of fibrosing alveolitis, a condition for which treatments are minimally effective (13). The relevance of the inflammatory response to the subsequent deposition of fibrosis is, however, not well understood. Hallahan et al. (14) showed intercellular adhesion molecule-1 knockout mice to have both fewer inflammatory cells and less fibrosis after thoracic radiation than did their wild-type littermates, and Adawi et al. (15) reported the administration of an anti-CD40 ligand antibody treatment to reduce both pulmonary inflammation and fibrosis in radiation-treated mice. Other studies have, however, shown that the lung injury of fibrosis can be separated from the alveolitis response (16, 17) and may be initiated by epithelial cell damage (18). In the present study, a genome-wide scan of B6C3H back- cross mice was undertaken to identify loci influencing suscepti- bility to three radiation response outcomes: alveolitis, fibrosing alveolitis, and a sublethal response. Further, the correlation of inflammatory markers (mast cell influx and bronchoalveolar cell counts) to the development of each phenotype was investigated and the potential link between alveolitis and fibrosis severity was assessed. Requests for reprints: Christina Haston, Meakins-Christie Laboratories, 3626 rue St. Urbain, Montreal, Quebec H2X 2P2, Canada. Phone: 514-398-3864, ext. 089714; Fax: 514-398-7483; E-mail: christina.haston@mcgill.ca. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-2733 Cancer Res 2007; 67: (22). November 15, 2007 10796 www.aacrjournals.org Research Article Research. on April 20, 2016. © 2007 American Association for Cancer cancerres.aacrjournals.org Downloaded from