These data describe a novel murine model of PAH, which displays many of the hallmarks of the human disease, thus opening new avenues of investigation to better understand PAH pathophysiology.
Idiopathic pulmonary fibrosis is a generally progressive disorder with highly heterogeneous disease progression. The most common of the idiopathic interstitial pneumonias, idiopathic pulmonary fibrosis is characterized by a steady worsening of lung function and gas exchange cause by diffuse alveolar damage and severe fibrosis. We examined clinical features of patients with idiopathic pulmonary fibrosis to classify them as exhibiting rapid or slowly progressive over the first year of follow-up. We identified differences between the two groups in order to investigate the mechanism of rapid progression. Previous work from our laboratory has demonstrated that Toll-like receptor 9, a pathogen recognition receptor, promotes myofibroblast differentiation in lung fibroblasts cultured from biopsies of patients with idiopathic pulmonary fibrosis. Therefore, we hypothesized that TLR9 functions as both a sensor of pathogenic molecules and a profibrotic signal in rapidly progressive idiopathic pulmonary fibrosis. TLR9 was present at higher concentrations in surgical lung biopsies from rapidly progressive patients than in tissue from normal controls. Fibroblasts from rapid progressors were more responsive to the TLR9 agonist, CpG, than were fibroblasts from control patients. We used a humanized SCID mouse and demonstrated that there was increased fibrosis in murine lungs receiving human lung fibroblasts from rapid progressors than in mice receiving normal fibroblasts. This fibrosis was exacerbated by intranasal CpG challenges. Furthermore, CpG induced the differentiation of blood monocytes into fibrocytes and the epithelial-to-mesenchymal transition of A549 lung epithelial cells. These data suggest that TLR9 may drive the pathogenesis of rapidly progressive idiopathic pulmonary fibrosis and is a potential indicator of this subset of the disease.
BackgroundDefective epithelial repair, excess fibroblasts and myofibroblasts, collagen overproduction and fibrosis occur in a number of respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. Pathological conversion of epithelial cells into fibroblasts (epithelial-mesenchymal transition, EMT) has been proposed as a mechanism for the increased fibroblast numbers and has been demonstrated to occur in lung alveolar epithelial cells. Whether other airway cell types also have the capability to undergo EMT has been less explored so far. A better understanding of the full extent of EMT in airways, and the underlying mechanisms, can provide important insights into airway disease pathology and enable the development of new therapies. The main aim of this study was to test whether primary human bronchial epithelial cells are able to undergo EMT in vitro and to investigate the effect of various profibrotic factors in the process.ResultsOur data demonstrate that primary human bronchial epithelial cells (HBECs) are able to undergo EMT in response to transforming growth factor-beta 1 (TGF-β1), as revealed by typical morphological alterations and EMT marker progression at the RNA level by real-time quantitative polymerase chain reaction and, at the protein level, by western blot. By using pharmacological inhibitors we show that this is a Smad-dependent mechanism and is independent of extracellular signal-related kinase pathway activation. Additional cytokines and growth factors such as tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL1β) and connective tissue growth factor (CTGF) were also tested, alone or in combination with TGF-β1. TNF-α markedly enhances the effect of TGF-β1 on EMT, whereas IL1β shows only a very weak effect and CTGF has no significant effect. We have also found that cell-matrix contact, in particular to fibronectin, an ECM component upregulated in fibrotic lesions, potentiates EMT in both human alveolar epithelial cells and HBECs. Furthermore, we also show that the collagen discoidin domain receptor 1 (DDR1), generally expressed in epithelial cells, is downregulated during the EMT of bronchial epithelium whereas DDR2 is unaffected. Our results also suggest that bone morphogenetic protein-4 is likely to have a context dependent effect during the EMT of HBECs, being able to induce the expression of EMT markers and, at the same time, to inhibit TGF-β induced epithelial transdifferentiation.ConclusionsThe results presented in this study provide additional insights into EMT, a potentially very important mechanism in fibrogenesis. We show that, in addition to alveolar epithelial type II cells, primary HBECs are also able to undergo EMT in vitro upon TGF-β1 stimulation via a primarily Smad 2/3 dependent mechanism. The effect of TGF-β1 is potentiated on fibronectin matrix and in the presence of TNF-α, representing a millieu reminiscent of fibrotic lesions. Our results can contribute to a better understanding of lung fibrosis and to the development of new ...
Macrophages (MΦ) play an essential role in innate immune responses and can either display a pro-inflammatory, classically activated phenotype (M1) or undergo an alternative activation program (M2) promoting immune regulation. M-CSF is used to differentiate monocytes into MΦ and IFN-γ or IL-4+IL-13 to further polarize these cells towards M1 or M2, respectively. Recently, differentiation using only GM-CSF or M-CSF has been described to induce a M1- or M2-like phenotype, respectively. In this study, we combined both approaches by differentiating human MΦ in GM-CSF or M-CSF followed by polarization with either IFN-γ or IL-4+IL-13. We describe the phenotypic differences between CD14hi CD163hi CD206int FOLR2-expressing M-CSF MΦ and CD14lo CD163lo CD206hi GM-CSF MΦ but show that both macrophage populations reacted similarly to further polarization with IFN-γ or IL-4+IL-13 with up- and down-regulation of common M1 and M2 marker genes. We also show that high expression of the mannose receptor (CD206), a marker of alternative activation, is a distinct feature of GM-CSF MΦ. Changes of the chromatin structure carried out by chromatin modification enzymes (CME) have been shown to regulate myeloid differentiation. We analyzed the expression patterns of CME during MΦ polarization and show that M1 up-regulate the histone methyltransferase MLL and demethylase KDM6B, while resting and M2 MΦ were characterized by DNA methyltransferases and histone deacetylases. We demonstrate that MLL regulates CXCL10 expression and that this effect could be abrogated using a MLL-Menin inhibitor. Taken together we describe the distinct phenotypic differences of GM-CSF or M-CSF MΦ and demonstrate that MΦ polarization is regulated by specific epigenetic mechanisms. In addition, we describe a novel role for MLL as marker for classical activation. Our findings provide new insights into MΦ polarization that could be helpful to distinguish MΦ activation states.
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