Lack of ZNF365 Drives Senescence and Exacerbates Experimental Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant activation of the alveolar epithelium, the expansion of the fibroblast population, and the accumulation of extracellular matrix. Global gene expression of human lung fibroblasts stimulated with TGFß-1, a strong fibrotic mediator revealed the overexpression of ZNF365, a zinc finger protein implicated in cell cycle control and telomere stabilization. We evaluated the expression and localization of ZNF365 in IPF lungs and in the fibrotic response induced by bleomycin in WT and deficient mice of the orthologous gene Zfp365. In IPF, ZNF365 was overexpressed and localized in fibroblasts/myofibroblasts and alveolar epithelium. Bleomycin-induced lung fibrosis showed an upregulation of Zfp365 localized in lung epithelium and stromal cell populations. Zfp365 KO mice developed a significantly higher fibrotic response compared with WT mice by morphology and hydroxyproline content. Silencing ZNF365 in human lung fibroblasts and alveolar epithelial cells induced a significant reduction of growth rate and increased senescence markers, including Senescence Associated ß Galactosidase activity, p53, p21, and the histone variant γH2AX. Our findings demonstrate that ZNF365 is upregulated in IPF and experimental lung fibrosis and suggest a protective role since its absence increases experimental lung fibrosis mechanistically associated with the induction of cell senescence.

Loss of MT1-MMP in Alveolar Epithelial Cells Exacerbates Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal age-related lung disease whose pathogenesis involves an aberrant response of alveolar epithelial cells (AEC). Activated epithelial cells secrete mediators that participate in the activation of fibroblasts and the excessive deposition of extracellular matrix proteins. Previous studies indicate that matrix metalloproteinase 14 (MMP14) is increased in the lung epithelium in patients with IPF, however, the role of this membrane-type matrix metalloproteinase has not been elucidated. In this study, the role of Mmp14 was explored in experimental lung fibrosis induced with bleomycin in a conditional mouse model of lung epithelial MMP14-specific genetic deletion. Our results show that epithelial Mmp14 deficiency in mice increases the severity and extension of fibrotic injury and affects the resolution of the lesions. Gain-and loss-of-function experiments with human epithelial cell line A549 demonstrated that cells with a deficiency of MMP14 exhibited increased senescence-associated markers. Moreover, conditioned medium from these cells increased fibroblast expression of fibrotic molecules. These findings suggest a new anti-fibrotic mechanism of MMP14 associated with anti-senescent activity, and consequently, its absence results in impaired lung repair. Increased MMP14 in IPF may represent an anti-fibrotic mechanism that is overwhelmed by the strong profibrotic microenvironment that characterizes this disease.

Upregulation and Nuclear Location of MMP28 in Alveolar Epithelium of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive aging-associated disease of unknown etiology. A growing body of evidence indicates that aberrant activated alveolar epithelial cells induce the expansion and activation of the fibroblast population, leading to the destruction of the lung architecture. Some matrix metalloproteinases (MMPs) are upregulated in IPF, indicating that they may be important in the pathogenesis and/or progression of IPF. In the present study, we examined the expression of MMP28 in this disease and evaluated its functional effects in two alveolar epithelial cell lines and in human primary bronchial epithelial cells. We found that the enzyme is expressed in bronchial (apical and cytoplasmic localization) and alveolar (cytoplasmic and nuclear localization) epithelial cells in two different groups of patients with IPF. In vitro MMP28 epithelial silencing decreased the proliferation rate and delayed wound closing, whereas overexpression showed opposite effects, protecting from apoptosis and enhanced epithelial-mesenchymal transition. Our findings demonstrate that MMP28 is upregulated in epithelial cells from IPF lungs, where it may play a role in increasing the proliferative and migratory phenotype in a catalysis-dependent manner.

R-Spondin-2 Is Upregulated in Idiopathic Pulmonary Fibrosis and Affects Fibroblast Behavior.

Idiopathic pulmonary fibrosis (IPF) is characterized by the expansion of the myofibroblast population, excessive extracellular matrix accumulation, and destruction of the lung parenchyma. The R-spondin family (RSPO) comprises a group of proteins essential for development. Among them, RSPO2 is expressed primarily in the lungs, and its mutations cause severe defects in the respiratory tract. Interestingly, RSPO2 participates in the canonical Wingless/int1 pathway, a critical route in the pathogenesis of IPF. Thus, the aim of this study was to examine the expression and putative role of RSPO2 in this disease. We found that RSPO2 and its receptor leucine-rich G protein-coupled receptor 6 were upregulated in IPF lungs, where they localized primarily in fibroblasts and epithelial cells. Stimulation of IPF and normal lung fibroblasts with recombinant human RSPO2 resulted in the deregulation of numerous genes, although the transcriptional response was essentially distinct. In IPF fibroblasts, RSPO2 stimulation induced the up- or downregulation of several genes involved in the Wingless/int1 pathway (mainly from noncanonical signaling). In both normal and IPF fibroblasts, RSPO2 modifies the expression of genes implicated in several pathways, including the cell cycle and apoptosis. In accordance with gene expression, the stimulation of normal and IPF fibroblasts with RSPO2 significantly reduced cell proliferation and induced cell death. RSPO2 also inhibited collagen production and increased the expression of matrix metalloproteinase 1. Silencing RSPO2 with shRNA induced the opposite effects. Our findings demonstrate, for the first time to our knowledge, that RSPO2 is upregulated in IPF, where it appears to have an antifibrotic role.

Increased Expression of CC16 in Patients with Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease of unknown etiology. The pathogenic mechanisms are unclear, but evidence indicates that aberrantly activated alveolar epithelial cells secrete a variety of mediators which induce the migration, proliferation and activation of fibroblasts and finally the excessive accumulation of extracellular matrix with the consequent destruction of the lung parenchyma. CC16 (approved symbol SCGB1A1), a putative anti-inflammatory protein produced by "club" cells in the distal airways, has not been evaluated in IPF lungs. In this study, we determined the serum and bronchoalveolar lavage (BAL) levels as well as the lung cell localization of this protein. Also, we explored the usefulness of serum levels of CC16 for the differential diagnosis of IPF (n = 85), compared with non-IPF interstitial lung diseases [chronic hypersensitivity pneumonitis (cHP; n = 85) and connective tissue diseases (CTD-ILD; n = 85)]. CC16 was significantly increased in serum and BAL fluids of IPF patients and was found not only in club cells but also in alveolar epithelial cells. When compared with non-IPF patients and controls, serum levels were significantly increased (p<0.0001). Sensitivity and specificity for CC16 (cut-off 41ng/mL) were 24% and 90%, positive predictive value 56% and negative predictive value 69%. These findings demonstrate that CC16 is upregulated in IPF patients suggesting that may participate in its pathogenesis. Although higher than the serum levels of non-IPF patients it shows modest sensitivity to be useful as a potential biomarker for the differential diagnosis.

mTORC1 activation decreases autophagy in aging and idiopathic pulmonary fibrosis and contributes to apoptosis resistance in IPF fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal disease associated with aging. However, the molecular mechanisms of the aging process that contribute to the pathogenesis of IPF have not been elucidated. IPF is characterized by abundant foci of highly active fibroblasts and myofibroblasts resistant to apoptosis. Remarkably, the role of aging in the autophagy activity of lung fibroblasts and its relationship with apoptosis, as adaptive responses, has not been evaluated previously in this disease. In the present study, we analyzed the dynamics of autophagy in primary lung fibroblasts from IPF compared to young and age-matched normal lung fibroblasts. Our results showed that aging contributes for a lower induction of autophagy on basal conditions and under starvation which is mediated by mTOR pathway activation. Treatment with rapamycin and PP242, that target the PI3K/AKT/mTOR signaling pathway, modified starvation-induced autophagy and apoptosis in IPF fibroblasts. Interestingly, we found a persistent activation of this pathway under starvation that contributes to the apoptosis resistance in IPF fibroblasts. These findings indicate that aging affects adaptive responses to stress decreasing autophagy through activation of mTORC1 in lung fibroblasts. The activation of this pathway also contributes to the resistance to cell death in IPF lung fibroblasts.

Fibrocytes contribute to inflammation and fibrosis in chronic hypersensitivity pneumonitis through paracrine effects.

RATIONALE: Hypersensitivity pneumonitis (HP) represents a lung inflammation provoked by exposure to a variety of antigens. Chronic HP may evolve to lung fibrosis. Bone marrow-derived fibrocytes migrate to injured tissues and contribute to fibrogenesis, but their role in HP is unknown. OBJECTIVES: To assess the possible participation of fibrocytes in chronic HP. METHODS: CD45(+)/CXCR4(+)/Col-I(+) circulating fibrocytes were evaluated by flow cytometry, and the presence of fibrocytes in HP and normal lungs by confocal microscopy. The concentration of CXCL12 in plasma and bronchoalveolar lavage fluids was quantified by ELISA. The effect of fibrocytes on lung fibroblasts and T lymphocytes was examined in co-cultures. MEASUREMENTS AND MAIN RESULTS: The percentage of circulating fibrocytes was significantly increased in patients with HP compared with healthy individuals (5.3 ± 3.4% vs. 0.8 ± 0.7%; P = 0.00004). Numerous fibrocytes were found infiltrating the HP lungs near fibroblasts and lymphocytes. Plasma CXCL12 concentration was significantly increased in patients with HP (2,303.3 ± 813.7 vs. 1,385.6 ± 318.5 pg/ml; P = 0.00003), and similar results were found in bronchoalveolar lavage fluids. The chemokine was primarily expressed by epithelial cells. In co-cultures, fibrocytes induced on lung fibroblasts a significant increase in the expression of α1 type I collagen, matrix metalloprotease-1, and platelet-derived growth factor-ß. Likewise, fibrocytes induced the up-regulation of CCL2 in HP lymphocytes and fibroblasts. CONCLUSIONS: These findings demonstrate that high levels of fibrocytes are present in the peripheral blood of patients with chronic HP and that these cells infiltrate the HP lungs. Fibrocytes may participate in the pathogenesis of HP, amplifying the inflammatory and fibrotic response by paracrine signaling inducing the secretion of a variety of proinflammatory and profibrotic molecules.

Oleic acid modulates mRNA expression of liver X receptor (LXR) and its target genes ABCA1 and SREBP1c in human neutrophils.

PURPOSE: Regulation of liver X receptors (LXRs) is essential for cholesterol homeostasis and inflammation. The present study was conducted to determine whether oleic acid (OA) could regulate mRNA expression of LXRα and LXRα-regulated genes and to assess the potential promotion of oxidative stress by OA in neutrophils. METHODS: Human neutrophils were treated with OA at different doses and LXR target gene expression, oxidative stress production, lipid efflux and inflammation state were analyzed. RESULTS: We describe that mRNA synthesis of both LXRα and ABCA1 (a reverse cholesterol transporter) was induced by OA in human neutrophils. This fatty acid enhanced the effects of LXR ligands on ABCA1 and LXR expression, but it decreased the mRNA levels of sterol regulatory element-binding protein 1c (a transcription factor that regulates the synthesis of triglycerides). Although OA elicited a slight oxidative stress in the short term (15-30 min) in neutrophils, it is unlikely that this is relevant for the modulation of transcription in our experimental conditions, which involve longer incubation time (i.e., 6 h). Of physiological importance is our finding that OA depresses intracellular lipid levels and that markers of inflammation, such as ERK1/2 and p38 mitogen-activated protein kinase phosphorylation, were decreased by OA treatment. In addition, 200 µM OA reduced the migration of human neutrophils, another marker of the inflammatory state. However, OA did not affect lipid peroxidation induced by pro-oxidant agents. CONCLUSIONS: This work presents for the first time evidence that human neutrophils are highly sensitive to OA and provides novel data in support of a protective role of this monounsaturated acid against the activation of neutrophils during inflammation.

Platelet-activating factor downregulates the expression of liver X receptor-α and its target genes in human neutrophils.

Liver X receptors (LXRs) are ligand-activated members of the nuclear receptor superfamily that regulate the expression of genes involved in lipid metabolism and inflammation, although their role in inflammation and immunity is less well known. It has been reported that oxysterols/LXRs may act as anti-inflammatory molecules, although opposite actions have also been reported. In this study, we investigated the effect of platelet-activating factor (PAF), a proinflammatory molecule, on LXRα signalling in human neutrophils. We found that PAF exerted an inhibitory effect on mRNA expression of TO901317-induced LXRα, ATP-binding cassette transporter A1, ATP-binding cassette transporter G1, and sterol response element binding protein 1c. This negative action was mediated by the PAF receptor, and was dependent on the release of reactive oxygen species elicited by PAF, as it was enhanced by pro-oxidant treatment and reversed by antioxidants. Current data also support the idea that PAF induces phosphorylation of the LXRα molecule in an extracellular signal-regulated kinase 1/2-mediated fashion. These results suggest that a possible mechanism by which PAF exerts its proinflammatory effect is through the downregulation of LXRα and its related genes, which supports the notion that LXRα ligands exert a modulatory role in the neutrophil-mediated inflammatory response.

Matrix metalloproteinase (MMP)-1 induces lung alveolar epithelial cell migration and proliferation, protects from apoptosis, and represses mitochondrial oxygen consumption.

Idiopathic pulmonary fibrosis is a devastating lung disorder of unknown etiology. Although its pathogenesis is unclear, considerable evidence supports an important role of aberrantly activated alveolar epithelial cells (AECs), which produce a large variety of mediators, including several matrix metalloproteases (MMPs), which participate in fibroblast activation and lung remodeling. MMP-1 has been shown to be highly expressed in AECs from idiopathic pulmonary fibrosis lungs although its role is unknown. In this study, we explored the role of MMP-1 in several AECs functions. Mouse lung epithelial cells (MLE12) transfected with human Mmp-1 showed significantly increased cell growth and proliferation at 36 and 48 h of culture (p < 0.01). Also, MMP-1 promoted MLE12 cell migration through collagen I, accelerated wound closing, and protected cells from staurosporine- and bleomycin-induced apoptosis compared with mock cells (p < 0.01). MLE12 cells expressing human MMP-1 showed a significant repression of oxygen consumption ratio compared with the cells with the empty vector. As under hypoxic conditions hypoxia-inducible factor-1α (HIF-1α) mediates a transition from oxidative to glycolytic metabolism, we analyzed activation of HIF-1α. Ηigher activation of this factor was detected in MMP-1-transfected cells under normoxia and hypoxia. Likewise, a significant decrease of both total and mitochondrial reactive oxygen species was observed in MMP-1-transfected cells. Paralleling these findings, attenuation of MMP-1 expression by shRNA in A549 (human) AECs markedly reduced proliferation and migration (p < 0.01) and increased the oxygen consumption ratio. These findings indicate that epithelial expression of MMP-1 inhibits mitochondrial function, increases HIF-1α expression, decreases reactive oxygen species production, and contributes to a proliferative, migratory, and anti-apoptotic AEC phenotype.

Angiogenic and inflammatory markers in acute respiratory distress syndrome and renal injury associated to A/H1N1 virus infection.

Acute kidney injury (AKI) is often associated to acute respiratory distress syndrome (ARDS) due to influenza A/H1N1 virus infection. The profile of angiogenic and inflammatory factors in ARDS patients may be relevant for AKI. We analyzed the serum levels of several angiogenic factors, cytokines, and chemokines in 32 patients with A/H1N1 virus infection (17 with ARDS/AKI and 15 ARDS patients who did not developed AKI) and in 18 healthy controls. Significantly higher levels of VEGF, MCP-1, IL-6, IL-8 and IP-10 in ARDS/AKI patients were detected. Adjusting by confusing variables, levels of MCP-1 ≥150 pg/mL (OR=12.0, p=0.04) and VEGF ≥225 pg/mL (OR=6.4, p=0.03) were associated with the development of AKI in ARDS patients. Higher levels of MCP-1 and IP-10 were significantly associated with a higher risk of death in patients with ARDS (hazard ratio (HR)=10.0, p=0.02; HR=25.5, p=0.03, respectively) even taking into account AKI. Patients with influenza A/H1N1 infection and ARDS/AKI have an over-production of MCP-1, VEGF and IP-10 possibly contributing to kidney injury and are associated to a higher risk of death.

Transcription of liver X receptor is down-regulated by 15-deoxy-Δ(12,14)-prostaglandin J(2) through oxidative stress in human neutrophils.

Liver X receptors (LXRs) are ligand-activated transcription factors of the nuclear receptor superfamily. They play important roles in controlling cholesterol homeostasis and as regulators of inflammatory gene expression and innate immunity, by blunting the induction of classical pro-inflammatory genes. However, opposite data have also been reported on the consequences of LXR activation by oxysterols, resulting in the specific production of potent pro-inflammatory cytokines and reactive oxygen species (ROS). The effect of the inflammatory state on the expression of LXRs has not been studied in human cells, and constitutes the main aim of the present work. Our data show that when human neutrophils are triggered with synthetic ligands, the synthesis of LXRα mRNA became activated together with transcription of the LXR target genes ABCA1, ABCG1 and SREBP1c. An inflammatory mediator, 15-deoxy-Δ(12,14)-prostaglandin J(2) (15dPGJ(2)), hindered T0901317-promoted induction of LXRα mRNA expression together with transcription of its target genes in both neutrophils and human macrophages. This down-regulatory effect was dependent on the release of reactive oxygen species elicited by 15dPGJ(2), since it was enhanced by pro-oxidant treatment and reversed by antioxidants, and was also mediated by ERK1/2 activation. Present data also support that the 15dPGJ(2)-induced serine phosphorylation of the LXRα molecule is mediated by ERK1/2. These results allow to postulate that down-regulation of LXR cellular levels by pro-inflammatory stimuli might be involved in the development of different vascular diseases, such as atherosclerosis.

Role of Sonic Hedgehog in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease of unknown etiology and uncertain pathogenic mechanisms. Recent studies indicate that the pathogenesis of the disease may involve the abnormal expression of certain developmental pathways. Here we evaluated the expression of Sonic Hedgehog (SHH), Patched-1, Smoothened, and transcription factors glioma-associated oncogene homolog (GLI)1 and GLI2 by RT-PCR, as well as their localization in IPF and normal lungs by immunohistochemistry. The effects of SHH on fibroblast proliferation, migration, collagen and fibronectin production, and apoptosis were analyzed by WST-1, Boyden chamber chemotaxis, RT-PCR, Sircol, and annexin V-propidium iodide binding assays, respectively. Our results showed that all the main components of the Sonic signaling pathway were overexpressed in IPF lungs. With the exception of Smoothened, they were also upregulated in IPF fibroblasts. SHH and GLI2 localized to epithelial cells, whereas Patched-1, Smoothened, and GLI1 were observed mainly in fibroblasts and inflammatory cells. No staining was detected in normal lungs. Recombinant SHH increased fibroblast proliferation (P < 0.05), collagen synthesis, (2.5 ± 0.2 vs. 4.5 ± 1.0 µg of collagen/ml; P < 0.05), fibronectin expression (2-3-fold over control), and migration (190.3 ± 12.4% over control, P < 0.05). No effect was observed on α-smooth muscle actin expression. SHH protected lung fibroblasts from TNF-α/IFN-γ/Fas-induced apoptosis (14.5 ± 3.2% vs. 37.3 ± 7.2%, P < 0.0001). This protection was accompanied by modifications in several apoptosis-related proteins, including increased expression of X-linked inhibitor of apoptosis. These findings indicate that the SHH pathway is activated in IPF lungs and that SHH may contribute to IPF pathogenesis by increasing the proliferation, migration, extracellular matrix production, and survival of fibroblasts.

Matrix metalloproteinase-19 is a key regulator of lung fibrosis in mice and humans.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by epithelial phenotypic changes and fibroblast activation. Based on the temporal heterogeneity of IPF, we hypothesized that hyperplastic alveolar epithelial cells regulate the fibrotic response. OBJECTIVES: To identify novel mediators of fibrosis comparing the transcriptional signature of hyperplastic epithelial cells and conserved epithelial cells in the same lung. METHODS: Laser capture microscope and microarrays analysis were used to identify differentially expressed genes in IPF lungs. Bleomycin-induced lung fibrosis was evaluated in Mmp19-deficient and wild-type (WT) mice. The role of matrix metalloproteinase (MMP)-19 was additionally studied by transfecting the human MMP19 in alveolar epithelial cells. MEASUREMENTS AND MAIN RESULTS: Laser capture microscope followed by microarray analysis revealed a novel mediator, MMP-19, in hyperplastic epithelial cells adjacent to fibrotic regions. Mmp19(-/-) mice showed a significantly increased lung fibrotic response to bleomycin compared with WT mice. A549 epithelial cells transfected with human MMP19 stimulated wound healing and cell migration, whereas silencing MMP19 had the opposite effect. Gene expression microarray of transfected A549 cells showed that PTGS2 (prostaglandin-endoperoxide synthase 2) was one of the highly induced genes. PTGS2 was overexpressed in IPF lungs and colocalized with MMP-19 in hyperplastic epithelial cells. In WT mice, PTGS2 was significantly increased in bronchoalveolar lavage and lung tissues after bleomycin-induced fibrosis, but not in Mmp19(-/-) mice. Inhibition of Mmp-19 by siRNA resulted in inhibition of Ptgs2 at mRNA and protein levels. CONCLUSIONS: Up-regulation of MMP19 induced by lung injury may play a protective role in the development of fibrosis through the induction of PTGS2.

Absence of Thy-1 results in TGF-ß induced MMP-9 expression and confers a profibrotic phenotype to human lung fibroblasts.

Fibroblasts differ in a variety of phenotypic features, including the expression of Thy-1 a glycophosphatidylinositol-linked glycoprotein. Fibroblasts in idiopathic pulmonary fibrosis (IPF) are Thy-1 negative, whereas most fibroblasts from normal lungs are Thy-1 positive. However, the functional consequences of the absence of Thy-1 are not fully understood. We analyzed the expression of Thy-1 in several primary fibroblasts lines derived from IPF, hypersensitivity pneumonitis (HP), and normal human lungs. We found that a high proportion, independently of their origin, expressed Thy-1 in vitro. We identified a primary culture of HP fibroblasts, which did not express Thy-1, and compared several functional activities between Thy-1 (-) and Thy-1 (+) fibroblasts. Thy-1 (-) fibroblasts were smaller (length: 41.3±20.8 µ versus 83.1±40 µ), showed increased proliferative capacity and enhanced PDGF-induced transmigration through collagen I (59.9% versus 42.2% over control under basal conditions, P<0.01). Likewise, Thy-1 (-) fibroblasts either spontaneously or after TGF-ß stimulation demonstrated stronger contraction of collagen matrices (eg, 0.17±0.03 versus 0.6±0.05 cm² after TGF-ß stimulation at 24 h; P<0.01). Thy-1 (-) lung fibroblasts stimulated with TGF-ß1 expressed MMP-9, an enzyme that is usually not produced by lung fibroblasts. TGFß-induced MMP-9 expression was reversible upon re-expression of Thy-1 after transfection with full-length Thy-1. ß-glycan, a TGF-ß receptor antagonist abolished MMP-9 expression. TGF-ß1-induced MMP-9 in Thy-1 (-) fibroblasts depended on the activation of ERK1/2 signaling pathway. Finally, we demonstrated that fibroblasts from IPF fibroblastic foci, which do not express Thy-1 exhibit strong staining for immunoreactive MMP-9 protein in vivo. These findings indicate that loss of Thy-1 in human lung fibroblasts induces a fibrogenic phenotype.

FGF-1 reverts epithelial-mesenchymal transition induced by TGF-{beta}1 through MAPK/ERK kinase pathway.

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease characterized by the expansion of the fibroblast/myofibroblast population and aberrant remodeling. However, the origin of mesenchymal cells in this disorder is still under debate. Recent evidence indicates that epithelial-mesenchymal transition (EMT) induced primarily by TGF-beta1 plays an important role; however, studies regarding the opposite process, mesenchymal-epithelial transition, are scanty. We have previously shown that fibroblast growth factor-1 (FGF-1) inhibits several profibrogenic effects of TGF-beta1. In this study, we examined the effects of FGF-1 on TGF-beta1-induced EMT. A549 and RLE-6TN (human and rat) alveolar epithelial-like cell lines were stimulated with TGF-beta1 for 72 h, and then, in the presence of TGF-beta1, were cultured with FGF-1 plus heparin for an additional 48 h. After TGF-beta1 treatment, epithelial cells acquired a spindle-like mesenchymal phenotype with a substantial reduction of E-cadherin and cytokeratins and concurrent induction of alpha-smooth muscle actin measured by real-time PCR, Western blotting, and immunocytochemistry. FGF-1 plus heparin reversed these morphological changes and returned the epithelial and mesenchymal markers to control levels. Signaling pathways analyzed by selective pharmacological inhibitors showed that TGF-beta1 induces EMT through Smad pathway, while reversion by FGF-1 occurs through MAPK/ERK kinase pathway, resulting in ERK-1 phosphorylation and Smad2 dephosphorylation. These findings indicate that TGF-beta1-induced EMT is reversed by FGF-1 and suggest therapeutic approaches to target this process in IPF.

MICA polymorphisms and decreased expression of the MICA receptor NKG2D contribute to idiopathic pulmonary fibrosis susceptibility.

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disorder of unknown etiology. IPF is likely the result of complex interrelationships between environmental and host factors, although the genetic risk factors are presently uncertain. Because we have found that some MHC polymorphisms confer susceptibility to IPF, in the present study we aimed to evaluate the role of the MHC class I chain-related gene A (MICA) in the risk of developing the disease. MICA molecular typing was done by reference strand mediated conformation analysis in a cohort of 80 IPF patients and 201 controls. In addition, the lung cellular source of the protein was examined by immunohistochemistry, the expression of the MICA receptor NKG2D in lung cells by flow cytometry and soluble MICA by ELISA. A significant increase of MICA*001 was observed in the IPF cohort (OR = 2.91, 95% CI = 1.04-8.25; pC = 0.03). Likewise, the frequency of the MICA*001/*00201 genotype was significantly increased in patients with IPF compared with the healthy controls (OR = 4.72, 95% CI = 1.15-22.51; pC = 0.01). Strong immunoreactive MICA staining was localized in alveolar epithelial cells and fibroblasts from IPF lungs while control lungs were negative. Soluble MICA was detected in 35% of IPF patients compared with 12% of control subjects (P = 0.0007). The expression of NKG2D was significantly decreased in gammadelta T cells and natural killer cells obtained from IPF lungs. These findings indicate that MICA polymorphisms and abnormal expression of the MICA receptor NKG2D might contribute to IPF susceptibility.

Fibrocytes are a potential source of lung fibroblasts in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis is characterized by the accumulation of fibroblasts/myofibroblasts and aberrant remodeling of the lung parenchyma. However, the sources of fibroblasts in IPF lungs are unclear. Fibrocytes are circulating progenitors of fibroblasts implicated in wound healing and fibrosis. In this study we evaluated evidence for the presence of fibrocytes in the lung of patients with idiopathic pulmonary fibrosis by immunofluorescence and confocal microscopy. Fibrocytes were identified in tissues in 8 out of 9 fibrotic lungs. Combinations including CXCR4 and a mesenchymal marker stained significantly more fibrocytes/mm(2) of tissue compared with combinations using CD34 or CD45RO with mesenchymal markers: CXCR4/procollagen-I (10.3+/-2.9fibrocytes/mm(2)) and CXCR4/prolyl-4-hydroxylase (4.1+/-3.1), versus CD34/procollagen-I (2.8+/-3.0), CD34/alphaSMA (2.2+/-1.6) and CD45RO/prolyl-4-hydroxylase (1.3+/-1.6); p<0.003. There was a positive correlation between the abundance of fibroblastic foci and the amount of lung fibrocytes (r=0.79; p<0.02). No fibrocytes were identified in normal lungs. The fibrocyte attractant chemokine CXCL12 increased in plasma [median: 2707.5pg/ml (648.1-4884.7) versus 1751.5pg/ml (192.9-2686.0) from healthy controls; p<0.003)] and was detectable in the bronchoalveolar lavage fluid of 40% of the patients but not in controls. In the lung CXCL12 was strongly expressed by alveolar epithelial cells. A negative correlation between plasma levels of CXCL12 with lung diffusing capacity for carbon monoxide (DLCO) (r=-0.56; p<0.03) and oxygen saturation on exercise was found (r=-0.41; p<0.04). These findings indicate that circulating fibrocytes, likely recruited through the CXCR4/CXCL12 axis, may contribute to the expansion of the fibroblast/myofibroblast population in idiopathic pulmonary fibrosis.

Functional diversity of T-cell subpopulations in subacute and chronic hypersensitivity pneumonitis.

RATIONALE: Hypersensitivity pneumonitis (HP) exhibits a diverse outcome. Patients with acute/subacute HP usually improve, whereas patients with chronic disease often progress to fibrosis. However, the mechanisms underlying this difference are unknown. OBJECTIVES: To examine the T-cell profile from patients with subacute HP and chronic HP. METHODS: T cells were obtained by bronchoalveolar lavage from 25 patients with subacute HP, 30 patients with chronic HP, and 8 control subjects. T-cell phenotype and functional profile were evaluated by flow cytometry, cytometric bead array, and immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: Patients with chronic HP showed higher CD4+:CD8+ ratio (median, 3.05; range, 0.3-15; subacute HP: median, 1.3; range, 0.1-10; control: median, 1.3; range, 0.7-2.0; P < 0.01), and a decrease of gammadeltaT cells (median, 2.0; range, 0.5-3.4; subacute HP: median, 10; range, 4.8-17; control: median, 15; range, 5-19; P < 0.01). Patients with chronic HP exhibited an increase in the terminally differentiated memory CD4+ and CD8+ T-cell subsets compared with patients with subacute HP (P < 0.05). However, memory cells from chronic HP showed lower IFN-gamma production and decreased cytotoxic activity by CD8+ T lymphocytes. Chronic HP displayed a Th2-like phenotype with increased CXCR4 expression (median, 6%; range, 1.7-36, vs. control subjects: median, 0.7%; range, 0.2-1.4; and subacute HP: median, 2.2%; range, 0.1-5.3; P < 0.01), and decreased CXCR3 expression (median, 4.3%; range, 1.4-25%, vs. subacute HP: median, 37%; range, 4.9-78%; P < 0.01). Likewise, supernatants from antigen-specific-stimulated cells from chronic HP produced higher levels of IL-4 (80 +/- 63 pg/ml vs. 25 +/- 7 pg/ml; P < 0.01), and lower levels of IFN-gamma (3,818 +/- 1671 pg/ml vs. 100 +/- 61 pg/ml; P < 0.01) compared with subacute HP. CONCLUSIONS: Our findings indicate that patients with chronic HP lose effector T-cell function and exhibit skewing toward Th2 activity, which may be implicated in the fibrotic response that characterizes this clinical form.

Nitric oxide mediates the survival action of IGF-1 and insulin in pancreatic beta cells.

Generation of low levels of nitric oxide (NO) contributes to beta cell survival in vitro. The purpose of this study was to explore the link between NO and the survival pathway triggered by insulin-like growth factor-1 (IGF-1) and insulin in insulin producing RINm5F cells and in pancreatic islets. Results show that exposure of cells to IGF-1/insulin protects against serum deprivation-induced apoptosis. This action is prevented with inhibitors of NO generation, PI3K and Akt. Moreover, transfection with the negative dominant form of the tyrosine kinase c-Src abrogates the effect of IGF-1 and insulin on DNA fragmentation. An increase in the expression level of NOS3 protein and in the enzyme activity is observed following exposure of serum-deprived RINm5F cells to IGF-1 and insulin. Phosphorylation of IRS-1, IRS-2 and to less extent IRS-3 takes place when serum-deprived RINm5F cells and rat pancreatic islets are exposed to either IGF-1, insulin, or diethylenetriamine nitric oxide adduct (DETA/NO). In human islets, IRS-1 and IRS-2 proteins are present and tyrosine phosphorylated upon exposure to IGF-1, insulin and DETA/NO. Both rat and human pancreatic islets undergo DNA fragmentation when cultured in serum-free medium and IGF-1, insulin and DETA/NO protect efficiently from this damage. We then conclude that generation of NO participates in the activation of survival pathways by IGF-1 and insulin in beta cells.