Positional Cloning Reveals Strain-Dependent Expression of Trim16 to Alter Susceptibility to Bleomycin-Induced Pulmonary Fibrosis in Mice

摘要

Pulmonary fibrosis is a disease of significant morbidity, with no effective therapeutics and an as yet incompletely defined genetic basis. The chemotherapeutic agent bleomycin induces pulmonary fibrosis in susceptible C57BL/6J mice but not in mice of the C3H/HeJ strain, and this differential strain response has been used in prior studies to map bleomycin-induced pulmonary fibrosis susceptibility loci named Blmpf1 and Blmpf2 . In this study we isolated the quantitative trait gene underlying Blmpf2 initially by histologically phenotyping the bleomycin-induced lung disease of sublines of congenic mice to reduce the linkage region to 13 genes. Of these genes, Trim16 was identified to have strain-dependent expression in the lung, which we determined was due to sequence variation in the promoter. Over-expression of Trim16 by plasmid injection increased pulmonary fibrosis, and bronchoalveolar lavage levels of both interleukin 12/23-p40 and neutrophils, in bleomycin treated B6.C3H- Blmpf2 subcongenic mice compared to subcongenic mice treated with bleomycin only, which follows the C57BL/6J versus C3H/HeJ strain difference in these traits. In summary we demonstrate that genetic variation in Trim16 leads to its strain-dependent expression, which alters susceptibility to bleomycin-induced pulmonary fibrosis in mice. Author Summary Genetic differences within the population influence an individual's susceptibility to the lung disease pulmonary fibrosis. As environmental factors also have a tremendous effect on the development of this disease, investigations in an animal model can reveal the genetic basis of this trait, under controlled circumstances. Starting from previous work that had identified a genomic region linked to fibrosis susceptibility in mice, we assayed the fibrosis response of lines of mice specifically bred to contain reduced portions of the original genetic interval, and we narrowed our study to 13 genes. Genetic evaluation pointed to the gene Trim16 as a prime candidate for affecting fibrosis, and we identified genetic variations to alter its transcription. Our functional studies showed that Trim16 injected into the specifically bred, and bleomcyin-treated, mice significantly increased their pulmonary fibrosis levels. Further evaluation of the mice showed the increase to be associated with known enhancers of fibrosis, neutrophils and interleukin12/23-p40. This study shows that genetic variation in Trim16 affects both the lung tissue inflammatory response and the development of pulmonary fibrosis in mice and thus provides a novel pathway to fibrosis development for subsequent clinical investigation.