Person:

Rosas, Ivan

Background: Autophagy is a basic cellular homeostatic process important to cell fate decisions under conditions of stress. Dysregulation of autophagy impacts numerous human diseases including cancer and chronic obstructive lung disease. This study investigates the role of autophagy in idiopathic pulmonary fibrosis. Methods: Human lung tissues from patients with IPF were analyzed for autophagy markers and modulating proteins using western blotting, confocal microscopy and transmission electron microscopy. To study the effects of TGF-β1 on autophagy, human lung fibroblasts were monitored by fluorescence microscopy and western blotting. In vivo experiments were done using the bleomycin-induced fibrosis mouse model. Results: Lung tissues from IPF patients demonstrate evidence of decreased autophagic activity as assessed by LC3, p62 protein expression and immunofluorescence, and numbers of autophagosomes. TGF-β1 inhibits autophagy in fibroblasts in vitro at least in part via activation of mTORC1; expression of TIGAR is also increased in response to TGF-β1. In the bleomycin model of pulmonary fibrosis, rapamycin treatment is antifibrotic, and rapamycin also decreases expression of á-smooth muscle actin and fibronectin by fibroblasts in vitro. Inhibition of key regulators of autophagy, LC3 and beclin-1, leads to the opposite effect on fibroblast expression of á-smooth muscle actin and fibronectin. Conclusion: Autophagy is not induced in pulmonary fibrosis despite activation of pathways known to promote autophagy. Impairment of autophagy by TGF-β1 may represent a mechanism for the promotion of fibrogenesis in IPF.

Objectives: Matrix metalloproteinase-8 (MMP-8) promotes lung fibrotic responses to bleomycin in mice. Although prior studies reported that MMP-8 levels are increased in plasma and bronchoalveolar lavage fluid (BALF) samples from IPF patients, neither the bioactive forms nor the cellular sources of MMP-8 in idiopathic pulmonary fibrosis (IPF) patients have been identified. It is not known whether MMP-8 expression is dys-regulated in IPF leukocytes or whether MMP-8 plasma levels correlate with IPF outcomes. Our goal was to address these knowledge gaps. Methods: We measured MMP-8 levels and forms in blood and lung samples from IPF patients versus controls using ELISAs, western blotting, and qPCR, and assessed whether MMP-8 plasma levels in 73 IPF patients correlate with rate of lung function decline and mortality. We used immunostaining to localize MMP-8 expression in IPF lungs. We quantified MMP-8 levels and forms in blood leukocytes from IPF patients versus controls. Results: IPF patients have increased BALF, whole lung, and plasma levels of soluble MMP-8 protein. Active MMP-8 is the main form elevated in IPF lungs. MMP-8 mRNA levels are increased in monocytes from IPF patients, but IPF patients and controls have similar levels of MMP-8 in PMNs. Surprisingly, macrophages and airway epithelial cells are the main cells expressing MMP-8 in IPF lungs. Plasma and BALF MMP-8 levels do not correlate with decline in lung function and/or mortality in IPF patients. Conclusion: Blood and lung MMP-8 levels are increased in IPF patients. Active MMP-8 is the main form elevated in IPF lungs. Surprisingly, blood monocytes, lung macrophages, and airway epithelial cells are the main cells in which MMP-8 is upregulated in IPF patients. Plasma and BALF MMP-8 levels are unlikely to serve as a prognostic biomarker for IPF patients. These results provide new information about the expression patterns of MMP-8 in IPF patients.