S110 Targeted deletion of G q/G 11 in surfactant protein C-positive epithelial cells reduces TGFss activation and results in inflammation and alveolar airspace enlargement

Abstract

Activation of latent TGFs by the epithelially-restricted αvs6 integrin is critical in the pathogenesis of lung injury and fibrosis, and disruption of this pathway promotes emphysema development. We have previously shown that Gαq and RhoA signalling pathways are central to avb6 integrin induced TGFs activation in vitro. To assess the role of the Gq/11 signalling pathway in the lungs, we generated mice with deletion of the Gq and G11 α-subunits in Surfactant protein C (SftpC)-positive epithelial cells (Gq/11DKO). SftpC-Cre mice were crossed with constitutive Gα11-deficient animals ( Gna11 −/− ; G11KO) carrying floxed alleles of the Gaq gene ( Gnaq fl/fl ) and then backcrossed onto appropriate null mice. Lungs were perfused, inflated and fixed prior to processing for histological and immunohistochemical analysis at 2, 4, 6 and 8 weeks. Bronchoalveolar lavage (BAL) cells were collected at 6 weeks for mRNA, nuclear protein extraction or histological analysis. Focal inflammatory infiltrates were visible in the Gq/11DKO lungs as early as 2 weeks, but became larger and more widespread at later timepoints. Gq/11DKO mice also exhibited significant age-related airspace enlargement compared with G11KO mice from 4 weeks onwards. From 6 weeks, inflammation was closely associated with localised disruption of the alveolar architecture and the appearance of enlarged and vacuolated macrophages within the airspaces. BAL fluid from Gq/11DKO mice contained significantly higher cells numbers (12.5±2.5×10 5 ) than G11KO mice (0.96±0.2×10 5 ) with increases in the percentage of neutrophils, lymphocytes and enlarged and vacuolated alveolar macrophages. mRNA analysis of Gq/11DKO BAL cells showed significantly increased MMP12, RELMα and Arginase 1 suggesting an increase in the number of alternatively activated macrophages. To assess levels of active TGFs in the lungs, phosphorylated SMAD2 (pSMAD2), a component of the TGFs signalling pathway, was measured by ELISA of nuclear extracts from BAL cells. Gq/11DKO BAL cells contained significantly lower levels of pSMAD2 than those from G11KO mice, suggesting decreased levels of active TGFs in the lungs of Gq/11DKO mice. These data suggest that the Gαq/11 signalling pathway in SftpC-positive epithelial cells regulates TGFs activation in the lungs and that deficiency in this pathway results in pulmonary inflammation and disruption of the alveolar architecture of the lung.