Dmitri V. Pechkovsky

Induction of Epithelial–Mesenchymal Transition in Primary Airway Epithelial Cells from Patients with Asthma by Transforming Growth Factor-β1

RATIONALE Airway remodeling in asthma is associated with the accumulation of fibroblasts, the primary cell responsible for synthesis and secretion of extracellular matrix proteins. The process by which the number of fibroblasts increases in asthma is poorly understood, but epithelial-mesenchymal transition (EMT) may play a significant role. OBJECTIVES To evaluate whether EMT occurs in primary airway epithelial cells (AECs), the mechanisms involved, and if this process is altered in asthmatic AECs. METHODS AECs were obtained from subjects with asthma (n = 8) and normal subjects without asthma (n = 10). Monolayer and air-liquid interface-AEC (ALI-AEC) cultures were treated with transforming growth factor (TGF)-beta1 (10 ng/ml) for 72 hours and assayed for mesenchymal and epithelial markers using quantitative polymerase chain reaction, confocal microscopy, and immunoblot. The involvement of BMP-7, Smad3, and MAPK-mediated signaling were also evaluated. MEASUREMENTS AND MAIN RESULTS TGF-beta1-induced EMT in AEC monolayers derived from subjects with asthma and normal donors. EMT was characterized by changes in cell morphology, increased expression of mesenchymal markers EDA-fibronectin, vimentin, alpha-smooth muscle actin, and collagen-1, and loss of epithelial markers E-cadherin and zonular occludin-1. Inhibition of TGF-beta1-induced signaling with Smad3-inhibiting siRNA or TGF-beta1-neutralizing antibodies prevented and reversed EMT, respectively, whereas BMP-7 had no effect. In ALI-AEC cultures derived from normal subjects, EMT was confined to basally situated cells, whereas in asthmatic ALI-AEC cultures EMT was widespread throughout the epithelium. CONCLUSIONS TGF-beta1 induces EMT in a Smad3-dependent manner in primary AECs. However, in asthmatic-derived ALI-AEC cultures, the number of cells undergoing EMT is greater. These findings support the hypothesis that epithelial repair in asthmatic airways is dysregulated.

Characterization of Side Population Cells from Human Airway Epithelium

The airway epithelium is the first line of contact with the inhaled external environment and is continuously exposed to and injured by pollutants, allergens, and viruses. However, little is known about epithelial repair and in particular the identity and role of tissue resident stem/progenitor cells that may contribute to epithelial regeneration. The aims of the present study were to identify, isolate, and characterize side population (SP) cells in human tracheobronchial epithelium. Epithelial cells were obtained from seven nontransplantable healthy lungs and four asthmatic lungs by pronase digestion. SP cells were identified by verapamil‐sensitive efflux of the DNA‐binding dye Hoechst 33342. Using flow cytometry, CD45− SP, CD45+ SP, and non‐SP cells were isolated and sorted. CD45− SP cells made up 0.12% ± 0.01% of the total epithelial cell population in normal airway but 4.1% ± 0.06% of the epithelium in asthmatic airways. All CD45− SP cells showed positive staining for epithelial‐specific markers cytokeratin‐5, E‐cadherin, ZO‐1, and p63. CD45− SP cells exhibited stable telomere length and increased colony‐forming and proliferative potential, undergoing population expansion for at least 16 consecutive passages. In contrast with non‐SP cells, fewer than 100 CD45− SP cells were able to generate a multilayered and differentiated epithelium in air‐liquid interface culture. SP cells are present in human tracheobronchial epithelium, exhibit both short‐ and long‐term proliferative potential, and are capable of generation of differentiated epithelium in vitro. The number of SP cells is significantly greater in asthmatic airways, providing evidence of dysregulated resident SP cells in the asthmatic epithelium.

Mycobacterium bovis BCG Attenuates Surface Expression of Mature Class II Molecules through IL-10-Dependent Inhibition of Cathepsin S

We have previously shown that macrophage infection with Mycobacterium tuberculosis and M. bovis bacillus Calmette-Guérin (BCG) partially inhibits MHC class II surface expression in response to IFN-γ. The present study examined the nature of class II molecules that do in fact reach the surface of infected cells. Immunostaining with specific Abs that discriminate between mature and immature class II populations showed a predominance of invariant chain (Ii)-associated class II molecules at the surface of BCG-infected cells suggesting that mycobacteria specifically block the surface export of peptide-loaded class II molecules. This phenotype was due to inhibition of IFN-γ-induced cathepsin S (Cat S) expression in infected cells and the subsequent intracellular accumulation of αβ class II dimers associated with the Cat S substrate Ii p10 fragment. In contrast, infection with BCG was shown to induce secretion of IL-10, and addition of blocking anti-IL-10 Abs to cell cultures restored both expression of active Cat S and export of mature class II molecules to the surface of infected cells. Consistent with these findings, expression of mature class II molecules was also restored in cells infected with BCG and transfected with active recombinant Cat S. Thus, M. bovis BCG exploits IL-10 induction to inhibit Cat S-dependent processing of Ii in human macrophages. This effect results in inhibition of peptide loading of class II molecules and in reduced presentation of mycobacterial peptides to CD4+ T cells. This ability may represent an effective mycobacterial strategy for eluding immune surveillance and persisting in the host.

A gene expression signature of emphysema-related lung destruction and its reversal by the tripeptide GHK

BackgroundChronic obstructive pulmonary disease (COPD) is a heterogeneous disease consisting of emphysema, small airway obstruction, and/or chronic bronchitis that results in significant loss of lung function over time.MethodsIn order to gain insights into the molecular pathways underlying progression of emphysema and explore computational strategies for identifying COPD therapeutics, we profiled gene expression in lung tissue samples obtained from regions within the same lung with varying amounts of emphysematous destruction from smokers with COPD (8 regions × 8 lungs = 64 samples). Regional emphysema severity was quantified in each tissue sample using the mean linear intercept (Lm) between alveolar walls from micro-CT scans.ResultsWe identified 127 genes whose expression levels were significantly associated with regional emphysema severity while controlling for gene expression differences between individuals. Genes increasing in expression with increasing emphysematous destruction included those involved in inflammation, such as the B-cell receptor signaling pathway, while genes decreasing in expression were enriched in tissue repair processes, including the transforming growth factor beta (TGFβ) pathway, actin organization, and integrin signaling. We found concordant differential expression of these emphysema severity-associated genes in four cross-sectional studies of COPD. Using the Connectivity Map, we identified GHK as a compound that can reverse the gene-expression signature associated with emphysematous destruction and induce expression patterns consistent with TGFβ pathway activation. Treatment of human fibroblasts with GHK recapitulated TGFβ-induced gene-expression patterns, led to the organization of the actin cytoskeleton, and elevated the expression of integrin β1. Furthermore, addition of GHK or TGFβ restored collagen I contraction and remodeling by fibroblasts derived from COPD lungs compared to fibroblasts from former smokers without COPD.ConclusionsThese results demonstrate that gene-expression changes associated with regional emphysema severity within an individuals lung can provide insights into emphysema pathogenesis and identify novel therapeutic opportunities for this deadly disease. They also suggest the need for additional studies to examine the mechanisms by which TGFβ and GHK each reverse the gene-expression signature of emphysematous destruction and the effects of this reversal on disease progression.

Transforming Growth Factor β1 Induces αvβ3 Integrin Expression in Human Lung Fibroblasts via a β3 Integrin-, c-Src-, and p38 MAPK-dependent Pathway

In response to transforming growth factor β1 (TGFβ) stimulation, fibroblasts modify their integrin repertoire and adhesive capabilities to certain extracellular matrix proteins. Although TGFβ has been shown to increase the expression of specific αv integrins, the mechanisms underlying this are unknown. In this study we demonstrate that TGFβ1 increased both β3 integrin subunit mRNA and protein levels as well as surface expression of αvβ3 in human lung fibroblasts. TGFβ1-induced αvβ3 expression was strongly adhesion-dependent and associated with increased focal adhesion kinase and c-Src kinase phosphorylation. Inhibition of β3 integrin activation by the Arg-Gly-Asp tripeptide motif-specific disintegrin echistatin or αvβ3 blocking antibody prevented the increase in β3 but not β5 integrin expression. In addition, echistatin inhibited TGFβ1-induced p38 MAPK but not Smad3 activation. Furthermore, inhibition of the Src family kinases, but not focal adhesion kinase, completely abrogated TGFβ1-induced expression of αvβ3 and p38 MAPK phosphorylation but not β5 integrin expression and Smad3 activation. The TGFβ1-induced αvβ3 expression was blocked by pharmacologic and genetic inhibition of p38 MAPK- but not Smad2/3-, Sp1-, ERK-, phosphatidylinositol 3-kinase, and NF-κB-dependent pathways. Our results demonstrate that TGFβ1 induces αvβ3 integrin expression via a β3 integrin-, c-Src-, and p38 MAPK-dependent pathway. These data identify a novel mechanism for TGFβ1 signaling in human lung fibroblasts by which they may contribute to normal and pathological wound healing.

TGFβ1 induces αvβ3 integrin expression in human lung fibroblasts via a β3 integrin, c-Src and p38MAPK dependent pathway

In response to transforming growth factor β1 (TGFβ) stimulation, fibroblasts modify their integrin repertoire and adhesive capabilities to certain extracellular matrix proteins. Although TGFβ has been shown to increase the expression of specific αv integrins, the mechanisms underlying this are unknown. In this study we demonstrate that TGFβ1 increased both β3 integrin subunit mRNA and protein levels as well as surface expression of αvβ3 in human lung fibroblasts. TGFβ1-induced αvβ3 expression was strongly adhesion-dependent and associated with increased focal adhesion kinase and c-Src kinase phosphorylation. Inhibition of β3 integrin activation by the Arg-Gly-Asp tripeptide motif-specific disintegrin echistatin or αvβ3 blocking antibody prevented the increase in β3 but not β5 integrin expression. In addition, echistatin inhibited TGFβ1-induced p38 MAPK but not Smad3 activation. Furthermore, inhibition of the Src family kinases, but not focal adhesion kinase, completely abrogated TGFβ1-induced expression of αvβ3 and p38 MAPK phosphorylation but not β5 integrin expression and Smad3 activation. The TGFβ1-induced αvβ3 expression was blocked by pharmacologic and genetic inhibition of p38 MAPK- but not Smad2/3-, Sp1-, ERK-, phosphatidylinositol 3-kinase, and NF-κB-dependent pathways. Our results demonstrate that TGFβ1 induces αvβ3 integrin expression via a β3 integrin-, c-Src-, and p38 MAPK-dependent pathway. These data identify a novel mechanism for TGFβ1 signaling in human lung fibroblasts by which they may contribute to normal and pathological wound healing.

STAT3-Mediated Signaling Dysregulates Lung Fibroblast-Myofibroblast Activation and Differentiation in UIP/IPF

STAT3 is a latent transcription factor that plays a role in regulating fibroblast function in fibrotic lung diseases. To further understand the role of STAT3 in the phenotypic divergence and function of human lung fibroblasts (LFs), we investigated the effect of basal and cytokine-induced STAT3 activity on indices of LF differentiation and activation, including expression of α-smooth muscle actin (α-SMA), collagen, and adhesion molecules Thy-1/CD90 and α(v) β(3) and β(5) integrins. We identified a population of fibroblasts from usual interstitial pneumonia (UIP)/idiopathic pulmonary fibrosis (IPF) lungs characterized by constitutively phosphorylated STAT3, lower proliferation rates, and diminished expression of α-SMA, Thy-1/CD90, and β(3) integrins compared with control LFs. Staining of UIP lung biopsy specimens demonstrated that phosphorylated STAT3 was not present in α-SMA-positive fibroblastic foci but was observed in the nuclei of cells located in the areas of dense fibrosis. STAT3 activation in LFs did not significantly influence basal or transforming growth factor β(1)-induced collagen I expression but inhibited expression of α-SMA, Thy-1/CD90, and αv β(3) integrins. Suppression of STAT3 signaling diminished resistance of IPF LFs to staurosporine-induced apoptosis and responsiveness to transforming growth factor β(1) but increased basal α-SMA and restored β(3) integrin expression in LFs via an ALK-5-dependent, SMAD3/7-independent mechanism. These data suggest that STAT3 activation regulates several pathways in human LFs associated with normal wound healing, whereas aberrant STAT3 signaling plays a critical role in UIP/IPF pathogenesis.

Human Lung Parenchyma but Not Proximal Bronchi Produces Fibroblasts with Enhanced TGF-β Signaling and α-SMA Expression

Given the contribution various fibroblast subsets make to wound healing and tissue remodeling, the concept of lung fibroblast heterogeneity is of great interest. However, the mechanisms contributing to this heterogeneity are unknown. To this aim, we compared molecular and biophysical characteristics of fibroblasts concurrently isolated from normal human proximal bronchi (B-FBR) and distal lung parenchyma (P-FBR). Using quantitative RT-PCR, spontaneous expression of more than 30 genes related to repair and remodeling was analyzed. All P-FBR lines demonstrated significantly increased basal α-smooth muscle actin (α-SMA) mRNA and protein expression levels when compared with donor-matched B-FBR. These differences were not associated with sex, age, or disease history of lung tissue donors. In contrast to B-FBR, P-FBR displayed enhanced transforming growth factor (TGF)-β/Smad signaling at baseline, and inhibition of either ALK-5 or neutralization of endogenously produced and activated TGF-β substantially decreased basal α-SMA protein in P-FBR. Both B-FBR and P-FBR up-regulated α-SMA after stimulation with TGF-β1, and basal expression levels of TGF-β1, TGF-βRI, and TGF-βRII were not significantly different between fibroblast pairs. Blockade of metalloproteinase-dependent activation of endogenous TGF-β did not significantly modify α-SMA expression in P-FBR. However, resistance to mechanical tension of these cells was significantly higher in comparison with B-FBR, and added TGF-β1 significantly increased stiffness of both cell monolayers. Our data suggest that in contrast with human normal bronchial tissue explants, lung parenchyma produces mesenchymal cells with a myofibroblastic phenotype by intrinsic mechanisms of TGF-β activation in feed-forward manner. These results also offer a new insight into mechanisms of human fibroblast heterogeneity and their function in the airway and lung tissue repair and remodeling.

CCR2 and CXCR3 agonistic chemokines are differently expressed and regulated in human alveolar epithelial cells type II

The attraction of leukocytes from circulation to inflamed lungs depends on the activation of both the leukocytes and the resident cells within the lung. In this study we determined gene expression and secretion patterns for monocyte chemoattractant protein-1 (MCP-1/CCL2) and T-cell specific CXCR3 agonistic chemokines (Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11) in TNF-α-, IFN-γ-, and IL-1β-stimulated human alveolar epithelial cells type II (AEC-II). AEC-II constitutively expressed high level of CCL2 mRNA in vitro and in situ , and released CCL2 protein in vitro . Treatment of AEC-II with proinflammatory cytokines up-regulated both CCL2 mRNA expression and release of immunoreactive CCL2, whereas IFN-γ had no effect on CCL2 release. In contrast, CXCR3 agonistic chemokines were not detected in freshly isolated AEC-II or in non-stimulated epithelial like cell line A549. IFN-γ, alone or in combination with IL-1β and TNF-α resulted in an increase in CXCL10, CXCL11, and CXCL9 mRNA expression and generation of CXCL10 protein by AEC-II or A549 cells. CXCL10 gene expression and secretion were induced in dose-dependent manner after cytokine-stimulation of AEC-II with an order of potency IFN-γ>>IL-1β ≥ TNF-α. Additionally, we localized the CCL2 and CXCL10 mRNAs in human lung tissue explants by in situ hybridization, and demonstrated the selective effects of cytokines and dexamethasone on CCL2 and CXCL10 expression. These data suggest that the regulation of the CCL2 and CXCL10 expression exhibit significant differences in their mechanisms, and also demonstrate that the alveolar epithelium contributes to the cytokine milieu of the lung, with the ability to respond to locally generated cytokines and to produce potent mediators of the local inflammatory response.

Human alveolar epithelial cells induce nitric oxide synthase-2 expression in alveolar macrophages

It was hypothesized that celltocell interaction between human alveolar macrophages (AM) and alveolar epithelium, might be an important factor leading to nitric oxide synthase‐2 (NOS2) messenger ribonucleic acid (mRNA) and protein expression by constituent cells of the alveolar wall and/or AM. NOS2 mRNA and the protein expression patterns of human AM and alveolar epithelial cells type II (AECII) isolated from normal parts of lung resections of patients with pulmonary malignancies were determined. In addition, NOS2 mRNA expression in human AM cocultured with autologous AECII in the presence of proinflammatory cytokines interleukin (IL)‐1β, tumour necrosis factor (TNF)‐α, interferon (IFN)‐γ or lipopolysaccharide (LPS) was investigated. The effect of human surfactant protein‐A (SP‐A) on IFN‐γ‐mediated NOS2 mRNA expression in human AM was also studied. Neither NOS2 mRNA nor protein could be detected in freshly isolated, unstimulated or cytokinestimulated AECII. In contrast, freshly isolated AM from bronchoalveolar lavage or lung tissue samples expressed immunoreactivity for NOS2 protein, but no NOS2 mRNA could be detected by reverse transcriptase polymerase chain reaction. All stimuli tested failed to induce NOS2 mRNA expression in human AM in vitro. Only AMAECII coculture in the presence of IFN‐γ led to NOS2 mRNA and protein expression. In situ hybridization of NOS2 mRNA on lung tissue explants and immunohistochemical staining of cytospin preparations of AMAECII cocultures demonstrated that NOS2 is expressed in AM but not in AECII. This coculture effect could not be reproduced by substitution of AECII with SP‐A. These data give evidence of a regulatory network controlling human nitric oxide synthase‐2 expression in the lower respiratory tract.