[Show abstract][Hide abstract] ABSTRACT: Influenza A virus (IAV) targets airway epithelial cells and exploits the host cell machinery to replicate, causing respiratory illness in annual epidemics and pandemics of variable severity. The high rate of antigenic drift (viral mutation) and the putative antigenic shift (reassortant strains) have raised the need to find the host cell inducible factors modulating IAV replication and its pathogenesis to develop more effective antiviral treatment. In this study, we found for the first time that transcription factor Runx3, a developmental regulator and tumor suppressor, was induced by IAV H1N1 and H3N2, viral RNA, a synthetic analog of viral double-stranded RNA (dsRNA) polyinosinic-polycytidylic acid, and type-II interferon-γ (IFNγ) in human airway epithelial cells. Whereas Runx3 was essentially not induced by type-I IFNα and type-III IFNλ, we show that Runx3 induction by IAV infection and viral RNA is mediated through the innate immune receptor MDA5 and the IκB kinase-β−NF-κB pathway. Moreover, we provide substantial evidence indicating that Runx3 plays a crucial role in airway epithelial cell apoptosis induced by IAV infection and dsRNA through the activation of extrinsic and intrinsic apoptosis pathways. Thus, we have identified Runx3 as an inducible and important transcription factor modulating IAV-induced host epithelial cell apoptosis.
[Show abstract][Hide abstract] ABSTRACT: Tissue Factor (TF) is a transmembrane glycoprotein that plays an essential role in hemostasis by activating coagulation. TF is also expressed by monocytes/macrophages as part of the innate immune response to infections. In the current study, we determined the role of TF expressed by myeloid cells during Mycobacterium tuberculosis (M. tb) infection by using mice lacking the TF gene in myeloid cells (TF(Δ) ) and human monocyte derived macrophages (MDMs). We found that during M. tb infection, a deficiency of TF in myeloid cells was associated with reduced inducible nitric oxide synthase (iNOS) expression, enhanced arginase 1 (Arg1) expression, enhanced IL-10 production and reduced apoptosis in infected macrophages, which augmented M. tb growth. Our results demonstrate that a deficiency of TF in myeloid cells promotes M2 like phenotype in M .tb infected macrophages. A deficiency in TF expression by myeloid cells was also associated with reduced fibrin deposition and increased matrix metalloproteases (MMP)-2 and MMP-9 mediated inflammation in M. tb infected lungs. Our studies demonstrate that TF expressed by myeloid cells has newly recognized abilities to polarize macrophages and to regulate M. tb growth. This article is protected by copyright. All rights reserved.
Full-text · Article · Oct 2015 · European Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: University of Texas health science center, Tyler, TX, Mayo Clinic College of 1 2 3 4 Medicine, Rochester, MN, Mayo Clinic, Rochester, MN, Mayo Clinic Rochester, Rochester, MN, Univ of Texas Hlth Ctr at Tyler, Tyler, TX 5 6 7 The pathogenesis of idiopathic pulmonary fibrosis (IPF) is complex. The current literature suggests that alveolar type II Rationale: epithelial (ATII) cell injury/apoptosis and expansion of the myofibroblast/FL-fibroblast population in IPF leads to progressive deposition of extracellular matrix (ECM) and progressive pulmonary fibrosis (PF). Expression of plasminogen activator inhibitor-1 (PAI-1) has been implicated in the pathogenesis of IPF but PAI-1 has also been implicated as a senescence factor in fibroblasts and its role in the regulation of fibroblast expansion in IPF remains unclear. We sought to determine how PAI-1 regulates primary hFL-fibroblasts from patients with IPF and mFL-fibroblasts from Objectives: bleomycin (BLM)-induced fibrosis in mice and lung tissues and ATII cells to assess the contribution of PAI-1 to the pulmonary fibrogenic response. : ATII cells, Fibroblasts were isolated from patients with IPF and mice with BLM induced PF were analyzed for changes in PAI-1 Methods and components of the fibrinolytic pathway. Transfections of these cells with PAI-1were used to further assess profibrogenic responses. Basal expression of PAI-1 in human and murine FL-fibroblasts (from IPF and BLM-induced PF respectively), was reduced Results: compared to that of fibroblasts from uninjured lungs whereas collagen-I (Col-I) and alpha-smooth muscle actin (a-SMA) were markedly elevated. Conversely, ATII cells surrounding the fibrotic foci as well as those isolated from IPF lungs showed increased apoptosis in situ, and PAI-1 with a parallel reduction in uPA expression. Transduction of an adenovirus PAI-1 cDNA construct suppressed expression of uPA and col-I, and attenuated proliferation in FL-fibroblasts. On the contrary, inhibition of basal PAI-1 in NL-fibroblasts increased col-I and a-SMA. Fibroblasts isolated from PAI-1-deficient mice without lung injury also showed increased col-I, a-SMA and uPA. This study defines a new role of PAI-1 in the control of fibroblast activation and expansion and its role in the pathogenesis of Conclusions: fibrosing lung disease, in particular including IPF Fibroblasts from fibrotic lungs express relatively low levels of PAI-1 compared to cells. from histologically normal lungs and overexpression of PAI-1 attenuated mesenchymal biomarkers in these cells. Fibroblasts from PAI-1 deficient mice demonstrated mesenchymal features. Our findings challenge the generally accepted current concept that PAI-1 exerts exclusively profibrogenic effects in fibrotic lungs. This abstract is funded by: FAMRI, AHA and The Texas Lung Injury Institute Am J Respir Crit Care Med 191;2015:A2354 Internet address: www.atsjournals.org Online Abstracts Issue
[Show abstract][Hide abstract] ABSTRACT: Parenchymal lung inflammation and airway and alveolar epithelial cell apoptosis are associated with cigarette smoke exposure (CSE), which contributes to chronic obstructive pulmonary disease (COPD). Epidemiological studies indicate that people exposed to chronic cigarette smoke with or without COPD are more susceptible to influenza A virus (IAV) infection. We found increased p53, PAI-1 and apoptosis in AECs, with accumulation of macrophages and neutrophils in the lungs of patients with COPD. In Wild-type (WT) mice with passive CSE (PCSE), p53 and PAI-1 expression and apoptosis were increased in AECs as was lung inflammation, while those lacking p53 or PAI-1 resisted AEC apoptosis and lung inflammation. Further, inhibition of p53-mediated induction of PAI-1 by treatment of WT mice with caveolin-1 scaffolding domain peptide (CSP) reduced PCSE-induced lung inflammation and reversed PCSE-induced suppression of eosinophil-associated RNase1 (EAR1). Competitive inhibition of the p53-PAI-1 mRNA interaction by expressing p53-binding 3’UTR sequences of PAI-1 mRNA likewise suppressed CS-induced PAI-1 and AEC apoptosis and restored EAR1 expression. Consistent with PCSE-induced lung injury, IAV infection increased p53, PAI-1 and apoptosis in AECs in association with pulmonary inflammation. Lung inflammation induced by PCSE was worsened by subsequent exposure to IAV. Mice lacking PAI-1 that were exposed to IAV showed minimal viral burden based on M2 antigen and hemagglutination analyses, whereas transgenic mice that overexpress PAI-1 without PCSE showed increased M2 antigen and inflammation after IAV infection. These observations indicate that increased PAI-1 expression promotes AEC apoptosis and exacerbates lung inflammation induced by IAV following PCSE.
[Show abstract][Hide abstract] ABSTRACT: Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD) are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC), cystic fibrosis transmembrane conductance regulator (CFTR), and aquaporin 5 (AQP5) proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of α and β ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI) and II (ATII)-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3) was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD.
[Show abstract][Hide abstract] ABSTRACT: Tissue factor pathway inhibitor (TFPI) is the primary inhibitor of the extrinsic coagulation cascade and its expression is reported to be relatively stable. Various pathophysiologic agents have been shown to influence TFPI activity either by regulating its expression or modifying the protein. It is not clear how TFPI activity is regulated in normal physiology or in injury. Because thrombin and TFPI are both locally elaborated in pleural injury, we sought to determine if thrombin could regulate TFPI in human pleural mesothelial cells (HPMCs). Thrombin significantly decreased TFPI mRNA and protein levels by greater than 70%. Further, thrombin-mediated down-regulation of TFPI promoted factor X activation by HPMCs. The ability of thrombin to significantly decrease TFPI mRNA and protein levels was maintained at nanomolar concentrations. Protease activated receptor (PAR)-1, a mediator of thrombin signaling, is detectable in the mesothelium in human and murine pleural injury. PAR-1 silencing blocked thrombin-mediated decrements of TFPI in HPMCs. Thrombin activates PI3K/Akt and NFκB signaling in HPMCs. Inhibition of PI3K (by PX-866) and NFκB (by SN50) prevented thrombin-mediated TFPI mRNA and protein down-regulation. These are the first studies to demonstrate that thrombin decreases TFPI expression in HPMCs. Our findings demonstrate a novel mechanism by which thrombin regulates TFPI expression in PMCs and promotes an unrestricted procoagulant response. Further, they suggest that interactions between PI3K and NFκB signaling pathways are linked in HPMCs and control TFPI expression. These findings raise the possibility that targeting of this pathway could limit the ability of the mesothelium to support extravascular fibrin deposition and organization associated with pleural injury.
Full-text · Article · Oct 2014 · American Journal of Respiratory Cell and Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Epithelial sodium channels (ENaC) govern transepithelial salt and fluid homeostasis. ENaC contributes to polarization, apoptosis, epithelial-mesenchymal transformation etc. Fibrinolytic proteases play a crucial role in virtually all of these processes and are elaborated by the airway epithelium. We hypothesized that urokinase-like plasminogen activator (uPA) regulates ENaC function in airway epithelial cells and tested that possibility in primary murine tracheal epithelial cells (MTE). Both basal and cAMP-activated Na(+) flow through ENaC were significantly reduced in monolayers of uPA-deficient cells. The reduction in ENaC activity was further confirmed in basolateral membrane permeabilized cells. A decrease in the Na(+)/K(+)-ATPase activity in the basolateral membrane could contribute to the attenuation of ENaC function in intact monolayer cells. Dysfunctional fluid resolution was seen in uPA-disrupted cells. Administration of uPA and plasmin partially restores ENaC activity and fluid re-absorption by MTEs. ERK1/2, but not Akt phosphorylation was observed in the cells and lungs of uPA-deficient mice. On the other hand, cleavage of γ ENaC is significantly depressed in the lungs of uPA knockout mice vs those of wild type controls. Expression of caspase 8, however, did not differ between wild type and uPA(-/-) mice. In addition, uPA deficiency did not alter transepithelial resistance. Taken together, the mechanisms for the regulation of ENaC by uPA in MTEs include augmentation of Na(+)/K(+)-ATPase, proteolysis, and restriction of ERK1/2 phosphorylation. We demonstrate for the first time that ENaC may serve as a downstream signaling target by which uPA controls the biophysical profiles of airway fluid and epithelial function.
No preview · Article · Aug 2014 · AJP Lung Cellular and Molecular Physiology
[Show abstract][Hide abstract] ABSTRACT: Introduction: Endogenous active plasminogen activator inhibitor 1 (PAI-1) was targeted in vivo with monoclonal antibodies (mAbs) that redirect its reaction with proteinases to the substrate branch. mAbs were used as an adjunct to prourokinase (scuPA) intrapleural fibrinolytic therapy (IPFT) of tetracycline-induced pleural injury in rabbits. Methods: Outcomes of scuPA IPFT (0.25 or 0.0625 mg/kg) with 0.5 mg/kg of mouse IgG or mAbs (MA-33H1F7 and MA-8H9D4) were assessed at 24h. Pleural fluid (PF) was collected at 0, 10, 20, 40 min and 24h after IPFT and analyzed for plasminogen activating (PA), urokinase (uPA), fibrinolytic activities, levels of total plasmin/plasminogen, α-macroglobulin (αM), mAbs/IgG antigens, free active uPA and αM/uPA complexes. Results:Anti-PAI-1 mAbs, but not mouse IgG, delivered with an 8-fold reduction in the minimal effective dose of scuPA (from 0.5 to 0.0625 mg/kg), improved the outcome of IPFT (p<0.05). mAbs and IgG were detectable in PFs at 24h. Compared to identical doses of scuPA alone or with IgG, treatment with scuPA and anti-PAI-1 mAbs generated higher PF uPA amidolytic and PA activities, faster formation of αM/uPA complexes, and slower uPA inactivation. However, PAI-1 targeting did not significantly affect intrapleural fibrinolytic activity or levels of total plasmin/plasminogen and αM antigens. Conclusions: Targeting PAI-1 did not induce bleeding and rendered otherwise ineffective doses of scuPA able to improve outcome in tetracycline-induced pleural injury. PAI-1 neutralizing mAbs improved IPFT by increasing the durability of intrapleural PA activity. These results suggest a novel, well-tolerated IPFT strategy that is tractable for clinical development.
No preview · Article · Aug 2014 · American Journal of Respiratory Cell and Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive and usually fatal lung disease of unknown etiology for which no effective treatments currently exist. Hence, there is a profound need for the identification of novel drugable targets to develop more specific and efficacious therapeutic intervention in IPF. In this study, we performed immunohistochemical analyses to assess the cell type-specific expression and activation of protein kinase D (PKD) family kinases in normal and IPF lung tissue sections. We also analyzed PKD activation and function in human lung epithelial cells. We found that PKD family kinases (PKD1, PKD2 and PKD3) were increased and activated in the hyperplastic and regenerative alveolar epithelial cells lining remodeled fibrotic alveolar septa and/or fibroblast foci in IPF lungs compared with normal controls. We also found that PKD family kinases were increased and activated in alveolar macrophages, bronchiolar epithelium, and honeycomb cysts in IPF lungs. Interestingly, PKD1 was highly expressed and activated in the cilia of IPF bronchiolar epithelial cells, while PKD2 and PKD3 were expressed in the cell cytoplasm and nuclei. In contrast, PKD family kinases were not apparently increased and activated in IPF fibroblasts or myofibroblasts. We lastly found that PKD was predominantly activated by poly-L-arginine, lysophosphatidic acid and thrombin in human lung epithelial cells and that PKD promoted epithelial barrier dysfunction. These findings suggest that PKD may participate in the pathogenesis of IPF and may be a novel target for therapeutic intervention in this disease.
[Show abstract][Hide abstract] ABSTRACT: Alveolar type II epithelial (ATII) cell injury precedes development of pulmonary fibrosis. Mice deficient in urokinase-type Rationale: plasminogen activator (uPA) and uPA plasma membrane receptor, (uPAR) are susceptible to lung injury and development of fibrosis, while those deficient in plasminogen activator inhibitor (PAI-1) are resistant. Epithelial mesenchymal transition (EMT) has been considered, at least in part, as a source of myofibroblasts during fibrogenesis. However, the contribution of altered uPA, uPAR and PAI-1 expression on ATII cell EMT during lung injury is not well understood. To investigate whether changes in the expression of uPA, uPAR and PAI-1 by ATII cells contribute to EMT after lung injury. Objectives: : ATII cells were isolated from patients with idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease Methods (COPD), and mice with bleomycin (BLM) or passive cigarette smoke (PCS) induced lung injury and analyzed for changes in EMT markers. We found reduced expression of E-cadherin and Zona occludens-1 while collagen-I and alpha-smooth muscle actin were Results: increased in ATII cells from IPF or COPD lungs or mice with BLM-or PCS-induced lung injuries. These changes were associated with a parallel increase in PAI-1, reduced uPA and uPAR, and activation of Src kinases. Inhibition of Src kinase activity suppressed BLM or PCS-induced EMT and restored uPA and uPAR expression while suppressing PAI-1. Collectively, these studies show that induction of PAI-1 and reciprocal, yet concurrent, inhibition of uPA and uPAR during Conclusions: fibrosing lung injury leads to EMT in ATII cells.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT Receptor tyrosine kinases, including the epidermal growth factor receptors (EGFR), are able to activate the mitogen activated protein kinases (MAPK) via several adaptor proteins and protein kinases such as Raf. EGFR can be activated by a variety of extracellular stimuli including neutrophil elastase, but we are aware of no report as to whether Pseudomonas aeruginosa bacterium produced elastase (PE) could elicit such signaling through EGFR activation. We sought to test the inference that PE modulates inflammatory responses in human lung fibroblasts and that the process occurs by activation of the EGFR/MAPK pathways. We utilized interleukin (IL)-8 cytokine expression as a pathway specific end-point measure of fibroblasts inflammatory response to PE. Western blot analysis was performed to detect phosphorylation of EGFR and signal transduction intermediates. Northern blot, real time polymerase chain reaction (PCR), and ELISA methods were utilized to determine cytokine gene expression levels. We found that PE induces phosphorylation of the EGFR and the extracellular signal-regulated proteins (ERK1/2) of the MAPK pathway, and nuclear translocation of transcription factor kappa B (NF-ĸB). Furthermore, enzymatically active PE enhances IL-8 mRNA and protein secretion. Pretreatment of the cells with specific inhibitors of EGFR, MEK, and NF-ĸB markedly attenuated the PE-induced signal proteins phosphorylation and IL-8 gene expression and protein secretion. Collectively, the data show that PE produced by Pseudomonas aeruginosa can modulate lung inflammation by exploiting the EGFR/ERK signaling cascades and enhancing IL-8 production in the lungs via NF-ĸB activation.
[Show abstract][Hide abstract] ABSTRACT: Local derangements of fibrin turnover and plasminogen activator inhibitor-1 (PAI-1) have been implicated in the pathogenesis of pleural injury. However, their role in the control of pleural organization has been unclear. We found that a C57Bl/6j mouse model of carbon black-bleomycin (CBB) injury demonstrates pleural organization resulting in pleural rind formation (14 d). In transgenic (Tg) mice overexpressing human PAI-1, intrapleural fibrin deposition was increased, but visceral pleural thickness, lung volumes and compliance were comparable to WT. CBB-injury in PAI-1-/- mice significantly increased visceral pleural thickness (P<0.001) and lung compliance (P<0.01) while decreasing lung volumes. Collagen, α-SMA, and tissue factor were increased in the thickened visceral pleura of PAI-1-/- mice. Colocalization of α-SMA and calretinin within pleural mesothelial cells (PMCs) was increased in CBB injured PAI-1-/- mice. Thrombin, FXa, plasmin and urokinase induced meso-mesenchymal transition (MesoMT), TF expression and activity in primary human PMCs. In PAI-1-/- mice, D-dimer and thrombin-antithrombin complex concentrations were increased in pleural lavage fluids. The results demonstrate that PAI-1 regulates CBB-induced pleural injury severity via unrestricted fibrinolysis and cross-talk with coagulation proteases. While overexpression of PAI-1 augments intrapleural fibrin deposition, PAI-1 deficiency promotes profibrogenic alterations of the mesothelium that exacerbate pleural organization and lung restriction.
Full-text · Article · Sep 2013 · American Journal of Respiratory Cell and Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Rationale: Intrapleural processing of prourokinase (scuPA) in tetracycline (TCN)-induced pleural injury in rabbits was evaluated to better understand the mechanisms governing successful scuPA-based intrapleural fibrinolytic therapy (IPFT); capable of clearing pleural adhesions in this model. Methods: Pleural fluid (PF) was withdrawn 0-80 min and 24 h after IPFT with scuPA (0-0.5 mg/kg) and activities of free urokinase (uPA), plasminogen activator inhibitor 1 (PAI-1) and uPA complexed with α-macroglobulin (αM) were assessed. Similar analyses were performed using PFs from patients with empyema, parapneumonic, and malignant pleural effusions. Results: The peak of uPA activity (5-40 min) reciprocally correlated with the dose of intrapleural scuPA. Endogenous active PAI-1 (10-20 nM) decreased the rate of intrapleural scuPA activation. The slow step of intrapleural inactivation of free uPA (t1/2(β)=40±10 min) was dose-independent and 6.7-fold slower than in blood. Up to 260±70 nM of αM/uPA formed in vivo (kass=580±60 M(-1)s(-1)). αM/uPA and products of its degradation contributed to durable intrapleural plasminogen activation up to 24 h after IPFT. Active PAI-1, active α2M, and α2M/uPA found in empyema, pneumonia, and malignant PFs demonstrate the capacity to support similar mechanisms in humans. Conclusion: Intrapleural scuPA processing differs from that in the bloodstream and includes (i) dose-dependent control of scuPA activation by endogenous active PAI-1; (ii) two-step inactivation of free uPA with simultaneous formation of αM/uPA; (iii) slow intrapleural degradation of αM/uPA releasing active free uPA. This mechanism offers potential clinically relevant advantages that may enhance the bioavailability of intrapleural scuPA and may mitigate the risk of bleeding complications.
Preview · Article · Aug 2013 · AJP Lung Cellular and Molecular Physiology