The pro-apoptotic BAX protein influences cell growth and differentiation from the nucleus in healthy interphasic cells

International audienceIt has become more and more evident that the BCL-2 family proteins mediate a wide range of non-apoptotic functions. The pro-apoptotic BAX protein has been reported in interphasic nuclei. Whether the nuclear form of BAX could be involved in non-apoptotic function is still unknown. Our study showed for the first time that BAX was associated with chromatin in vitro. Next, we used gain and loss of function approaches to decipher the potential role of nuclear BAX in non-apoptotic cells. In vitro, nuclear BAX promoted cell proliferation in lung epithelial cells and primary human lung fibroblasts by modulating CDKN1A expression. Interestingly, BAX occupancy of CDKN1A promoter was specifically enriched close to the transcription-starting site. Nuclear BAX also modulated the basal myofibroblastic differentiation and migration of primary human lung fibroblasts. Finally, BAX nuclear localization was associated in vivo with the remodelling of lung parenchyma during development, tumorigenesis as well as fibrosis compared to control adult human lungs. Hence, our study established for the first time, a strong link between the nuclear localization of the pro-apoptotic BAX protein and key basic cellular functions in the non-apoptotic setting

Macrophage Polarization Favors Epithelial Repair During Acute Respiratory Distress Syndrome

International audienceObjectives: Alveolar macrophage polarization and role on alveolar repair during human acute respiratory distress syndrome remain unclear. This study aimed to determine during human acute respiratory distress syndrome: the alveolar macrophage polarization, the effect of alveolar environment on macrophage polarization, and the role of polarized macrophages on epithelial repair.Design: Experimental ex vivo and in vitro investigations.Setting: Four ICUs in three teaching hospitals.Patients: Thirty-three patients with early moderate-to-severe acute respiratory distress syndrome were enrolled for assessment of the polarization of alveolar macrophages.Interventions: Polarization of acute respiratory distress syndrome macrophages was studied by flow cytometry and quantitative polymerase chain reaction. Modulation of macrophage polarization was studied in vitro using phenotypic and functional readouts. Macrophage effect on repair was studied using alveolar epithelial cells in wound healing models.Measurements and main results: Ex vivo, alveolar macrophages from early acute respiratory distress syndrome patients exhibited anti-inflammatory characteristics with high CD163 expression and interleukin-10 production. Accordingly, early acute respiratory distress syndrome-bronchoalveolar lavage fluid drives an acute respiratory distress syndrome-specific anti-inflammatory macrophage polarization in vitro, close to that induced by recombinant interleukin-10. Culture supernatants from macrophages polarized in vitro with acute respiratory distress syndrome-bronchoalveolar lavage fluid or interleukin-10 and ex vivo acute respiratory distress syndrome alveolar macrophages specifically promoted lung epithelial repair. Inhibition of the hepatocyte growth factor pathway in epithelial cells and hepatocyte growth factor production in macrophages both reversed this effect. Finally, hepatocyte growth factor and soluble form of CD163 concentrations expressed relatively to macrophage count were higher in bronchoalveolar lavage fluid from acute respiratory distress syndrome survivors.Conclusions: Early acute respiratory distress syndrome alveolar environment drives an anti-inflammatory macrophage polarization favoring epithelial repair through activation of the hepatocyte growth factor pathway. These results suggest that macrophage polarization may be an important step for epithelial repair and acute respiratory distress syndrome recover

FGF9 prevents pleural fibrosis induced by intra-pleural adenovirus injection in mice

International audienceFibroblast growth factor 9 (FGF9) is necessary for fetal lung development and is expressed by epithelium and mesothelium. We evaluated the role of FGF9 overexpression on adenoviral-induced pleural injury in vivo and determined the biological effects of FGF9 on mesothelial cells in vitro. We assessed the expression of FGF9 and FGF receptors by mesothelial cells in both human and mouse lungs. Intrapleural injection of an adenovirus expressing human FGF9 (AdFGF9) or a control adenovirus (AdCont) was performed. Mice were euthanized at days 3, 5, and 14 Expression of FGF9 and markers of inflammation and myofibroblastic differentiation was studied by qPCR and immunohistochemistry. In vitro, rat mesothelial cells were stimulated with FGF9 (20 ng/ml), and we assessed its effect on proliferation, survival, migration, and differentiation. FGF9 was expressed by mesothelial cells in human idiopathic pulmonary fibrosis. FGF receptors, mainly FGFR3, were expressed by mesothelial cells in vivo in humans and mice. AdCont instillation induced diffuse pleural thickening appearing at day 5, maximal at day 14 The altered pleura cells strongly expressed α-smooth muscle actin and collagen. AdFGF9 injection induced maximal FGF9 expression at day 5 that lasted until day 14 FGF9 overexpression prevented pleural thickening, collagen and fibronectin accumulation, and myofibroblastic differentiation of mesothelial cells. In vitro, FGF9 decreased mesothelial cell migration and inhibited the differentiating effect of transforming growth factor-β1. We conclude that FGF9 has a potential antifibrotic effect on mesothelial cells

Identification of PRRX1 as a key mesenchymal transcription factor in Idiopathic Pulmonary Fibrosis

Matrix remodeling is a salient feature of idiopathic pulmonary fibrosis (IPF) and cells of mesenchymal origin play an active role in this detrimental process. Targeting cells driving matrix production and remodeling could be a promising avenue for IPF treatment. Analysis of public transcriptomic database identified paired-related homeobox protein 1 (PRRX1) as an upregulated mesenchymal transcription factor (TF) in IPF lung. We confirmed that PRRX1a and b isoforms mRNA were up-regulated in pulmonary lung tissue from IPF patients compared to controls. PRRX1 isoforms were also strongly expressed by primary lung fibroblasts. In vitro, PRRX1 expression was up-regulated by cues associated with proliferative and anti-fibrotic properties in control and IPF lung fibroblasts (FGF2, PGE2 and soft culture substrate), while IPF fibroblast-derived matrix also increased PRRX1 TFs expression in a PDGFR dependent manner. Meanwhile, signals promoting myofibroblastic differentiation, such as TGF-1 and stiff culture substrate, decreased PRRX1 TF expression levels. We also demonstrated that PRRX1 overexpression promoted fibroblast survival in serum deprived growth condition while PRRX1 inhibition decreased fibroblast proliferation by downregulating the expression of S phase cyclins and G1 phase marker MKI67. PRRX1 inhibition also impacted myofibroblastic differentiation in response to TGF-β. We demonstrated that PRRX1 controls TGF-1-induced SMAD2/3 phosphorylation through the upregulation of the serine/ threonine phosphatase PPM1A. Finally, in vivo targeted inhibition of Prrx1 TFs with intra-tracheal antisense oligonucleotides (ASO) attenuated fibrotic remodeling in the bleomycin mice model of lung fibrosis. Altogether, our results identified PRRX1 as a mesenchymal transcription factor driving myofibroblastic phenotype and lung fibrogenesis