Human Bronchial Fibroblasts Research Articles

Invasive bronchial fibroblasts derived from asthmatic patients activate lung cancer A549 cells in vitro.

Epidemiological data suggests that there are functional links between bronchial asthma and lung carcinogenesis. Bronchial fibroblasts serve a prominent role in the asthmatic process; however, their involvement in lung cancer progression remains unaddressed. To estimate the effect of the asthmatic microenvironment on the invasiveness of lung cancer cells, the present study compared the behavior of human non-small cell lung cancer A549 cells exposed to the signals from human bronchial fibroblasts (HBFs) derived from non-asthmatic donors (NA HBFs) and from asthmatic patients (AS HBFs). NA HBFs did not significantly affect A549 motility, whereas AS HBFs and the media conditioned with AS HBF/A549 co-cultures increased Snail-1/connexin43 expression and motility of A549 cells. In contrast to NA HBFs, which formed A549-impenetrable lateral barriers, α-SMA+ AS HBFs actively infiltrated A549 monolayers and secreted chemotactic factors that arrested A549 cells within AS HBF/A549 contact zone. However, small sub-populations of A549 cells could release from this arrest and colonize distant regions of AS HBF monolayers. These data indicated that the interactions between lung cancer cells and HBFs in asthmatic bronchi may facilitate the colonization of lung tumors by fibroblasts. It further stabilizes the tumor microenvironment and potentially facilitates collective colonization of novel bronchial loci by cancer cells. Potential mechanistic links between the asthmatic process and lung cancer progression suggest that bronchial asthma should be included in the list of potential prognostic markers for lung cancer therapy.

Fenofibrate Reduces the Asthma-Related Fibroblast-To-Myofibroblast Transition by TGF-Β/Smad2/3 Signaling Attenuation and Connexin 43-Dependent Phenotype Destabilization

The activation of human bronchial fibroblasts by transforming growth factor-β1 (TGF-β1) leads to the formation of highly contractile myofibroblasts in the process of the fibroblast–myofibroblast transition (FMT). This process is crucial for subepithelial fibrosis and bronchial wall remodeling in asthma. However, this process evades current therapeutic asthma treatment strategies. Since our previous studies showed the attenuation of the TGF-β1-induced FMT in response to lipid-lowering agents (e.g., statins), we were interested to see whether a corresponding effect could be obtained upon administration of hypolipidemic agents. In this study, we investigated the effect of fenofibrate on FMT efficiency in populations of bronchial fibroblasts derived from asthmatic patients. Fenofibrate exerted a dose-dependent inhibitory effect on the FMT, even though it did not efficiently affect the expression of α-smooth muscle actin (α-SMA; marker of myofibroblasts); however, it considerably reduced its incorporation into stress fibers through connexin 43 regulation. This effect was accompanied by disturbances in the actin cytoskeleton architecture, impairments in the maturation of focal adhesions, and the fenofibrate-induced deactivation of TGF-β1/Smad2/3 signaling. These data suggest that fenofibrate interferes with myofibroblastic differentiation during asthma-related subepithelial fibrosis. The data indicate the potential application of fenofibrate in the therapy and prevention of bronchial remodeling during the asthmatic process.

Mutual regulation between miR-21 and the TGFβ/Smad signaling pathway in human bronchial fibroblasts promotes airway remodeling

ABSTRACTObjective: Airway remodeling is an important pathological feature of asthma. Excessive deposition of extracellular matrix (e.g., collagen) secreted from fibroblasts is a major factor contributing to airway remodeling. Currently, the mechanism by which collagen continues to be oversynthesized in the airway remains unclear. In this study, we investigated the role of the microRNA-21 (miR-21) and TGFβ/Smad signaling pathway in human bronchial fibroblasts (HBFs), and explored the regulatory mechanism of airway remodeling. Methods: HBFs were cultured in vitro and treated with the transforming growth factor β (TGFβ), receptor inhibitor (SB431542), and TGFβ1. miR-21 and Smad7 overexpressing lentiviruses, as well as an miR-21 interfering lentivirus were constructed and transfected into HBFs. Western blotting was used to determine the expression of airway remodeling-related proteins and proteins in the TGFβ/Smad signaling pathway. miR-21 expression was measured by quantitative real-time PCR. Results: The high expression of miR-21 induced by TGFβ1 was reduced following the treatment with the SB431542 in HBFs. Smad7 overexpression inhibited the elevated expression of the COL I protein induced by miR-21 overexpression in HBFs. Inhibiting miR-21 expression upregulated the level of Smad7 protein, thus reducing the expression of airway remodeling-related proteins induced by TGFβ1 stimulation in HBFs. Conclusions: TGFβ1 can induce miR-21 expression in HBFs through the TGFβ/Smad signaling pathway to promote airway remodeling. miR-21 downregulates Smad7, activates the TGFβ/Smad signaling pathway, and promotes airway remodeling. Mutual regulation between miR-21 and the TGFβ/Smad signaling pathway in HBFs promotes airway remodeling.

Glycopyrronium bromide inhibits lung inflammation and small airway remodeling induced by subchronic cigarette smoke exposure in mice

The effects of long-acting muscarinic receptor antagonists (LAMAs) have not been evaluated in a model with simultaneous lung inflammation and small airway remodeling induced by cigarette smoke (CS). We exposed the mice to CS for four weeks with daily treatment with a LAMA (glycopyrronium bromide, NVA237) or its vehicle. Human bronchial epithelial cells (PBECs) and lung fibroblasts were exposed to CS extract (CSE) or acetylcholine with or without NVA237 treatment. We found that NVA237, but not its vehicle, suppressed elevations in inflammatory score, epithelial thickness, and peribronchial collagen deposition in CS-exposed mice. NVA237 alleviated CS-induced increased levels of chemokines, inflammatory cells, and total protein in the bronchoalveolar lavage fluid. NVA237 suppressed acetylcholine- or CSE-induced elevations in IL-8 production in PBECs and elevations in proliferation and collagen production in lung fibroblasts. These phenomena were also prevented by a p44/42 MAPK inhibitor. In conclusion, NVA237 exerted a potent suppressive effect on lung inflammation and small airway remodeling induced by subchronic CS exposure.

Connective tissue growth factor regulates transition of primary bronchial fibroblasts to myofibroblasts in asthmatic subjects

Fibroblast to myofibroblast transition (FMT) contributes to bronchial wall remodelling in persistent asthma. Among other numerous factors involved, transforming growth factor type β (TGF-β) plays a pivotal role. Recently it has been demonstrated that connective tissue growth factor (CTGF), a matricellular protein, combines with TGF-β in the pathomechanism of many fibrotic disorders. However, it is not clear whether this interaction takes place in asthma as well.Primary cultures of human bronchial fibroblasts from asthmatic and non-asthmatic subjects were used to investigate the impact of CTGF and TGF-β1 on the fibroblast to myofibroblast transition. The combined activity of TGF-β1 and CTGF resulted in an average of 90% of FMT accomplished in cell lines derived from asthmatics. In this group FMT was highly dependent on the presence of CTGF produced by the cells, as shown by gene silencing experiments with the specific siRNA.Results support the important role of CTGF biosynthesis in the asthmatic bronchi amplifying FMT. This is evidenced by inhibition of TGF-β1-induced FMT following CTGF silencing in asthmatic bronchial fibroblasts. CTGF is produced by fibroblasts and contributes to the FMT phenomenon in positive loop-back, inducing and boosting TGF-β1 triggered FMT. Thus, CTGF is a promising target for pharmacological intervention in secondary prevention of bronchial remodelling in asthma.

Increased levels of prostaglandin E−major urinary metabolite (PGE-MUM) in chronic fibrosing interstitial pneumonia

BackgroundDysregulation of the prostaglandin E2 (PGE2) signaling pathway has been implicated in interstitial pneumonia (IP) pathogenesis. Due to the unstable nature of PGE2, available detection methods may not precisely reflect PGE2 levels. We explored the clinical usefulness of measuring stable prostaglandin E−major urinary metabolite (PGE-MUM) with respect to pathogenesis and extent of chronic fibrosing IP (CFIP), including idiopathic pulmonary fibrosis (IPF), as PGE-MUM is reflective of systemic PGE2 production. MethodsPGE-MUM was measured by radioimmunoassay in controls (n = 124) and patients with lung diseases (bronchial asthma (BA): n = 78, chronic obstructive pulmonary disease (COPD): n = 33, CFIP: n = 44). Extent of lung fibrosis was assessed by fibrosing score (FS) of computed tomography (CT) (FS1-4). Immunohistochemical evaluation of COX-2 was performed to find PGE2 producing cells in IPF. Human bronchial epithelial cells (HBEC) and lung fibroblasts (LFB) were used in in vitro experiments. ResultsCompared to control, PGE-MUM levels were significantly elevated in CFIP. PGE-MUM levels were positively correlated with FS, and inversely correlated with %DLCO in IP (FS 1–3). COX-2 was highly expressed in metaplastic epithelial cells in IPF, but lower expression of EP2 receptor was demonstrated in LFB derived from IPF. TGF-β induced COX-2 expression in HBEC. ConclusionsPGE-MUM, elevated in CFIP, is a promising biomarker reflecting disease activity. Metaplastic epithelial cells can be a source of elevated PGE-MUM in IPF.

Human airway organoid engineering as a step toward lung regeneration and disease modeling.

Organoids represent both a potentially powerful tool for the study cell-cell interactions within tissue-like environments, and a platform for tissue regenerative approaches. The development of lung tissue-like organoids from human adult-derived cells has not previously been reported. Here we combined human adult primary bronchial epithelial cells, lung fibroblasts, and lung microvascular endothelial cells in supportive 3D culture conditions to generate airway organoids. We demonstrate that randomly-seeded mixed cell populations undergo rapid condensation and self-organization into discrete epithelial and endothelial structures that are mechanically robust and stable during long term culture. After condensation airway organoids generate invasive multicellular tubular structures that recapitulate limited aspects of branching morphogenesis, and require actomyosin-mediated force generation and YAP/TAZ activation. Despite the proximal source of primary epithelium used in the airway organoids, discrete areas of both proximal and distal epithelial markers were observed over time in culture, demonstrating remarkable epithelial plasticity within the context of organoid cultures. Airway organoids also exhibited complex multicellular responses to a prototypical fibrogenic stimulus (TGF-β1) in culture, and limited capacity to undergo continued maturation and engraftment after ectopic implantation under the murine kidney capsule. These results demonstrate that the airway organoid system developed here represents a novel tool for the study of disease-relevant cell-cell interactions, and establishes this platform as a first step toward cell-based therapy for chronic lung diseases based on de novo engineering of implantable airway tissues.

Glutathione peroxidase 3 localizes to the epithelial lining fluid and the extracellular matrix in interstitial lung disease.

Aberrant antioxidant activity and excessive deposition of extracellular matrix (ECM) are hallmarks of interstitial lung diseases (ILD). It is known that oxidative stress alters the ECM, but extracellular antioxidant defence mechanisms in ILD are incompletely understood. Here, we extracted abundance and detergent solubility of extracellular antioxidant enzymes from a proteomic dataset of bleomycin-induced lung fibrosis in mice and assessed regulation and distribution of glutathione peroxidase 3 (GPX3) in murine and human lung fibrosis. Superoxide dismutase 3 (Sod3), Gpx3, and Gpx activity were increased in mouse BALF during bleomycin-induced lung fibrosis. In lung tissue homogenates, Gpx3, but not Sod3, was upregulated and detergent solubility profiling indicated that Gpx3 associated with ECM proteins. Immunofluorescence analysis showed that Gpx3 was expressed by bronchial epithelial cells and interstitial fibroblasts and localized to the basement membrane and interstitial ECM in lung tissue. As to human ILD samples, BALF of some patients contained high levels of GPX3, and GPX3 was upregulated in lung homogenates from IPF patients. GPX3 expression in primary human bronchial epithelial cells and lung fibroblasts was downregulated by TNF-α, but more variably regulated by TGF-β1 and menadione. In conclusion, the antioxidant enzyme GPX3 localizes to lung ECM and is variably upregulated in ILD.

IL-1α mediates cellular cross-talk in the airway epithelial mesenchymal trophic unit

ABSTRACTThe bronchial epithelium and underlying fibroblasts form an epithelial mesenchymal trophic unit (EMTU) which controls the airway microenvironment. We hypothesized that cell-cell communication within the EMTU propagates and amplifies the innate immune response to respiratory viral infections.EMTU co-culture models incorporating polarized (16HBE14o-) or differentiated primary human bronchial epithelial cells (HBECs) and fibroblasts were challenged with double-stranded RNA (dsRNA) or rhinovirus.In the polarized EMTU model, dsRNA affected ionic but not macromolecular permeability or cell viability. Compared with epithelial monocultures, dsRNA-stimulated pro-inflammatory mediator release was synergistically enhanced in the basolateral compartment of the EMTU model, with the exception of IL-1α which was unaffected by the presence of fibroblasts. Blockade of IL-1 signaling with IL-1 receptor antagonist (IL-1Ra) completely abrogated dsRNA-induced basolateral release of mediators except CXCL10. Fibroblasts were the main responders to epithelial-derived IL-1 since exogenous IL-1α induced pro-inflammatory mediator release from fibroblast but not epithelial monocultures. Our findings were confirmed in a differentiated EMTU model where rhinovirus infection of primary HBECs and fibroblasts resulted in synergistic induction of basolateral IL-6 that was significantly abrogated by IL-1Ra. This study provides the first direct evidence of integrated IL-1 signaling within the EMTU to propagate inflammatory responses to viral infection.

Human bronchial and parenchymal fibroblasts display differences in basal inflammatory phenotype and response to IL-17A.

Chronic inflammation, typified by increased expression of IL-17A, together with airway and parenchymal remodelling are features of chronic lung diseases. Emerging evidence suggests that phenotypic heterogeneity of repair and inflammatory capacities of fibroblasts may contribute to the differential structural changes observed in different regions of the lung. To investigate phenotypic differences in parenchymal and bronchial fibroblasts, either in terms of inflammation and remodelling or the ability of these fibroblasts to respond to IL-17A. Four groups of primary fibroblasts were used: normal human bronchial fibroblast (NHBF), normal human parenchymal fibroblast (NHPF), COPD human bronchial fibroblast (CHBF) and COPD human parenchymal fibroblast (CHPF). Cytokine and extracellular matrix (ECM) expression were measured at baseline and after stimulation with IL-17A. Actinomycin D was used to measure cytokine mRNA stability. At baseline, we observed higher protein production of IL-6 in NHPF than NHBF, but higher levels of IL-8 and GRO-α in NHBF. IL-17A induced a higher expression of GRO-α (CXCL1) and IL-6 in NHPF than in NHBF, and a higher level of IL-8 expression in NHBF. IL-17A treatment decreased the mRNA stability of IL-6 in NHBF when compared with NHPF. CHPF expressed higher protein levels of fibronectin, collagen-I and collagen-III than CHBF, NHBF and NHPF. IL-17A increased fibronectin and collagen-III protein only in NHPF and collagen-III protein production in CHBF and CHPF. These findings provide insight into the inflammatory and remodelling processes that may be related to the phenotypic heterogeneity of fibroblasts from airway and parenchymal regions and in their response to IL-17A.

Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice.

The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases.

Chemokine release from human rhinovirus–infected airway epithelial cells promotes fibroblast migration

Thickening of the lamina reticularis, a feature of remodeling in the asthmatic airways, is now known to be present in young children who wheeze. Human rhinovirus (HRV) infection is a common trigger for childhood wheezing, which is a risk factor for subsequent asthma development. We hypothesized that HRV-infected epithelial cells release chemoattractants to recruit fibroblasts that could potentially contribute to thickening of the lamina reticularis. We sought to investigate whether conditioned medium from HRV-infected epithelial cells can trigger directed migration of fibroblasts. Human bronchial epithelial cells were exposed to medium alone or infected with HRV-16. Conditioned medium from both conditions were tested as chemoattractants for human bronchial fibroblasts in the xCELLigence cell migration apparatus. HRV-conditioned medium was chemotactic for fibroblasts. Treatment of fibroblasts with pertussis toxin, an inhibitor of Gαi-coupled receptors, prevented their migration. Production of epithelial chemoattractants required HRV replication. Multiplex analysis of epithelial supernatants identified CXCL10, CXCL8, and CCL5 as Gαi-coupled receptor agonists of potential interest. Subsequent analysis confirmed that fibroblasts express CXCR3 and CXCR1 receptors and that CXCL10 and, to a lesser extent, CXCL8, but not CCL5, are major contributors to fibroblast migration caused by HRV-conditioned medium. CXCL10 and CXCL8 produced from HRV-infected epithelial cells are chemotactic for fibroblasts. This raises the possibility that repeated HRV infections in childhood could contribute to the initiation and progression of airway remodeling in asthmatic patients by recruiting fibroblasts thatproduce matrix proteins and thicken the lamina reticularis.

Sunitinib, a Small-Molecule Kinase Inhibitor, Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal disease, characterized by excessive accumulation of fibroblasts, extensive deposition of extracellular matrix, and destruction of alveolar architecture. IPF is associated with an epithelial-dependent fibroblast-activated process, termed the epithelial-to-mesenchymal transition (EMT). However, there is still a lack of strategies to target EMT for the treatment of IPF. Sunitinib, a small-molecule multi-targeted tyrosine kinase inhibitor, targets multiple kinases that may play an important role in developing pulmonary fibrosis. Here, we explored the therapeutic potential of sunitinib using a mouse model of pulmonary fibrosis. Mice received intratracheal instillation of bleomycin (BLM). Then, the mice were intragastrically administrated with sunitinib or normal saline until the end of the experiment. Distinguished destruction of pulmonary architecture, conspicuous proliferation of fibroblasts and extensive deposition of collagen fibers were found in BLM mice. Sunitinib attenuated the pulmonary fibrosis and inhibited the accumulation of fibroblasts in the lung of BLM mice. To investigate if the inhibition of fibroblast accumulation in the lung by sunitinib was associated with EMT, we used human bronchial epithelial cells (HBEs) and W138 human lung fibroblasts. Sunitinib suppressed the degree of EMT induced by TGF-β, a profibrotic factor, in HBEs and the proliferation of WI38 fibroblasts. Moreover, sunitinib reduced the degree of phosphorylation of serine residues on Smad2/3 that was induced by TGF-β in HBEs. As EMT and accumulation of fibroblasts are critical for the development of pulmonary fibrosis, targeting multiple pro-fibrosis signaling pathways with sunitinib may be a novel strategy to treat pulmonary fibrosis.

Characteristics of IL-25 and allergen-induced airway fibrosis in a murine model of asthma.

Interleukin (IL)-25 has been implicated in the pathogenesis of human asthma by inducing a Th2 cytokine response, but its possible role in the development of airway remodelling is less clear. We developed a murine surrogate of chronic airway inflammation induced by intranasal application of IL-25 alone. Comparison was with the 'classical' surrogate of ovalbumin (OVA) intranasal instillation into previously sensitized animals. Airway fibrotic biomarkers were analysed by immunohistochemistry and enzyme-linked immunosorbent assay. Additionally, proliferation assay and real-time polymerase chain reaction analysis were performed to assess IL-25's effects on primary human bronchial fibroblasts in vitro. In Balb/c mice, intranasal instillation of IL-25 alone induced florid airway fibrosis, including increased lay down of extracellular matrix proteins such as collagen I, III, V and fibronectin, increased numbers of fibroblasts/myofibroblasts, a profibrotic imbalance in matrix metalloproteinase/tissue inhibitor of metalloproteinase production and increased expression of profibrotic mediators including connective tissue growth factor and transforming growth factor-β1. These changes broadly reproduced those seen with classical intranasal OVA challenge in OVA-sensitized animals. Furthermore, IL-25 induced proliferation and expression of collagen I and III and smooth muscle α-actin in primary human lung fibroblasts. We conclude that chronic exposure of the airways to IL-25 alone is sufficient to cause functionally relevant airway remodelling, with the corollary that targeting of IL-25 may attenuate bronchial remodelling and fibrosis in human asthmatics.

Undifferentiated bronchial fibroblasts derived from asthmatic patients display higher elastic modulus than their non-asthmatic counterparts.

During asthma development, differentiation of epithelial cells and fibroblasts towards the contractile phenotype is associated with bronchial wall remodeling and airway constriction. Pathological fibroblast-to-myofibroblast transition (FMT) can be triggered by local inflammation of bronchial walls. Recently, we have demonstrated that human bronchial fibroblasts (HBFs) derived from asthmatic patients display some inherent features which facilitate their FMT in vitro. In spite of intensive research efforts, these properties remain unknown. Importantly, the role of undifferentiated HBFs in the asthmatic process was systematically omitted. Specifically, biomechanical properties of undifferentiated HBFs have not been considered in either FMT or airway remodeling in vivo. Here, we combine atomic force spectroscopy with fluorescence microscopy to compare mechanical properties and actin cytoskeleton architecture of HBFs derived from asthmatic patients and non-asthmatic donors. Our results demonstrate that asthmatic HBFs form thick and aligned ‘ventral’ stress fibers accompanied by enlarged focal adhesions. The differences in cytoskeleton architecture between asthmatic and non-asthmatic cells correlate with higher elastic modulus of asthmatic HBFs and their increased predilection to TGF-β-induced FMT. Due to the obvious links between cytoskeleton architecture and mechanical equilibrium, our observations indicate that HBFs derived from asthmatic bronchi can develop considerably higher static tension than non-asthmatic HBFs. This previously unexplored property of asthmatic HBFs may be potentially important for their myofibroblastic differentiation and bronchial wall remodeling during asthma development.

Suppression of autophagy by extracellular vesicles promotes myofibroblast differentiation in COPD pathogenesis

Extracellular vesicles (EVs), such as exosomes and microvesicles, encapsulate proteins and microRNAs (miRNAs) as new modulators of both intercellular crosstalk and disease pathogenesis. The composition of EVs is modified by various triggers to maintain physiological homeostasis. In response to cigarette smoke exposure, the lungs develop emphysema, myofibroblast accumulation and airway remodelling, which contribute to chronic obstructive pulmonary disease (COPD). However, the lung disease pathogenesis through modified EVs in stress physiology is not understood. Here, we investigated an EV-mediated intercellular communication mechanism between primary human bronchial epithelial cells (HBECs) and lung fibroblasts (LFs) and discovered that cigarette smoke extract (CSE)-induced HBEC-derived EVs promote myofibroblast differentiation in LFs. Thorough evaluations of the modified EVs and COPD lung samples showed that cigarette smoke induced relative upregulation of cellular and EV miR-210 expression of bronchial epithelial cells. Using co-culture assays, we showed that HBEC-derived EV miR-210 promotes myofibroblast differentiation in LFs. Surprisingly, we found that miR-210 directly regulates autophagy processes via targeting ATG7, and expression levels of miR-210 are inversely correlated with ATG7 expression in LFs. Importantly, autophagy induction was significantly decreased in LFs from COPD patients, and silencing ATG7 in LFs led to myofibroblast differentiation. These findings demonstrate that CSE triggers the modification of EV components and identify bronchial epithelial cell-derived miR-210 as a paracrine autophagy mediator of myofibroblast differentiation that has potential as a therapeutic target for COPD. Our findings show that stressor exposure changes EV compositions as emerging factors, potentially controlling pathological disorders such as airway remodelling in COPD.

Senescent bronchial fibroblasts induced to senescence by Cr(VI) promote epithelial-mesenchymal transition when co-cultured with bronchial epithelial cells in the presence of Cr(VI).

Cellular senescence is a physiological process that serves as a powerful barrier for tumorigenesis. However, senescent cells can be deleterious for the tissue microenvironment. Such is the case of senescent fibroblasts that release several pro-tumorigenic factors that promote malignant transformation in the nearby epithelial cells. Occupational exposure to hexavalent chromium [Cr(VI)] compounds is a cause of respiratory cancers. Although Cr(VI) is known to induce senescence in human foreskin fibroblasts, the role of senescent fibroblasts in the Cr(VI)-induced malignant transformation of human bronchial epithelial cells was never assessed. Thus, to study the evolutionary dynamics generated by the interaction between human bronchial epithelial cells and senescent bronchial fibroblasts, the non-tumorigenic human bronchial epithelial BEAS-2B cells were co-cultured with Cr(VI)-induced senescent human bronchial fibroblasts for 4 weeks. Under the pressure of 0.5 µM Cr(VI), senescent fibroblasts promoted the acquisition of mesenchymal features on BEAS-2B cells, e.g. the fusiform shape and increased Vimentin expression, consistent with the occurrence of an epithelial-mesenchymal transition-like process. Features of transformed cells including larger nuclei, as well as nuclei with heterogeneous size, were also observed. Altogether the results obtained demonstrate that besides acting over the epithelium, Cr(VI) also affects bronchial fibroblasts driving them senescent. As a consequence, a paracrine communication loop is established with the above-placed epithelium prompting the epithelial cells for malignant transformation and thus facilitating the initial steps of tumorigenesis.

Early activation of pro-fibrotic WNT5A in sepsis-induced acute lung injury.

IntroductionThe mechanisms of lung repair and fibrosis in the acute respiratory distress syndrome (ARDS) are poorly known. Since the role of WNT/β-catenin signaling appears to be central to lung healing and fibrosis, we hypothesized that this pathway is activated very early in the lungs after sepsis.MethodsWe tested our hypothesis using a three-step experimental design: (1) in vitro lung cell injury model with human bronchial epithelial BEAS-2B and lung fibroblasts (MRC-5) cells exposed to endotoxin for 18 hours; (2) an animal model of sepsis-induced ARDS induced by cecal ligation and perforation, and (3) lung biopsies from patients who died within the first 24 hours of septic ARDS. We examined changes in protein levels of target genes involved in the Wnt pathway, including WNT5A, non-phospho (Ser33/37/Thr41) β-catenin, matrix metalloproteinase-7 (MMP7), cyclin D1, and vascular endothelial growth factor (VEGF) by Western blotting and immunohistochemistry. Finally, we validated the main gene targets of this pathway in experimental animals and human lungs.ResultsProtein levels of WNT5A, non-phospho (Ser33/37/Thr41) β-catenin, total β-catenin, MMP7, cyclin D1, and VEGF increased after endotoxin stimulation in BEAS-2B and MRC-5 cells. Lungs from septic animals and from septic humans demonstrated acute lung inflammation, collagen deposition, and marked increase of WNT5A and MMP7 protein levels.ConclusionsOur findings suggest that the WNT/β-catenin signaling pathway is activated very early in sepsis-induced ARDS and could play an important role in lung repair and fibrosis. Modulation of this pathway might represent a potential target for treatment for septic and ARDS patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-014-0568-z) contains supplementary material, which is available to authorized users.

Density and duration of pneumococcal carriage is maintained by transforming growth factor β1 and T regulatory cells.

Nasopharyngeal carriage of Streptococcus pneumoniae is a prerequisite for invasive disease, but the majority of carriage episodes are asymptomatic and self-resolving. Interactions determining the development of carriage versus invasive disease are poorly understood but will influence the effectiveness of vaccines or therapeutics that disrupt nasal colonization. We sought to elucidate immunological mechanisms underlying noninvasive pneumococcal nasopharyngeal carriage. Pneumococcal interactions with human nasopharyngeal and bronchial fibroblasts and epithelial cells were investigated in vitro. A murine model of nasopharyngeal carriage and an experimental human pneumococcal challenge model were used to characterize immune responses in the airways during carriage. We describe the previously unknown immunological basis of noninvasive carriage and highlight mechanisms whose perturbation may lead to invasive disease. We identify the induction of active transforming growth factor (TGF)-β1 by S. pneumoniae in human host cells and highlight the key role for TGF-β1 and T regulatory cells in the establishment and maintenance of nasopharyngeal carriage in mice and humans. We identify the ability of pneumococci to drive TGF-β1 production from nasopharyngeal cells in vivo and show that an immune tolerance profile, characterized by elevated TGF-β1 and high nasopharyngeal T regulatory cell numbers, is crucial for prolonged carriage of pneumococci. Blockade of TGF-β1 signaling prevents prolonged carriage and leads to clearance of pneumococci from the nasopharynx. These data explain the mechanisms by which S. pneumoniae colonize the human nasopharynx without inducing damaging host inflammation and provide insight into the role of bacterial and host constituents that allow and maintain carriage.

Co-Culture of Human Bronchial Fibroblasts and CD4+ T Cells Increases Th17 Cytokine Signature

BackgroundAirway inflammation is an important characteristic of asthma and has been associated with airway remodelling and bronchial hyperreactivity. The mucosal microenvironment composed of structural cells and highly specialised extracellular matrix is able to amplify and promote inflammation. This microenvironment leads to the development and maintenance of a specific adaptive response characterized by Th2 and Th17. Bronchial fibroblasts produce multiple mediators that may play a role in maintaining and amplifying this response in asthma.ObjectiveTo investigate the role of bronchial fibroblasts obtained from asthmatic subjects and healthy controls in regulating Th17 response by creating a local micro-environment that promotes this response in the airways.MethodsHuman bronchial fibroblasts and CD4+T cells were isolated from atopic asthmatics and non-atopic healthy controls. CD4+T were co-cultured with bronchial fibroblasts of asthmatic subjects and healthy controls. RORc gene expression was detected by qPCR. Phosphorylated STAT-3 and RORγt were evaluated by western blots. Th17 phenotype was measured by flow cytometry. IL-22, IL17, IL-6 TGF-β and IL1-β were assessed by qPCR and ELISA.ResultsCo-culture of CD4+T cells with bronchial fibroblasts significantly stimulated RORc expression and induced a significant increase in Th17 cells as characterized by the percentage of IL-17+/CCR6+ staining in asthmatic conditions. IL-17 and IL-22 were increased in both normal and asthmatic conditions with a significantly higher amount in asthmatics compared to controls. IL-6, IL-1β, TGF-β and IL-23 were significantly elevated in fibroblasts from asthmatic subjects upon co-culture with CD4+T cells. IL-23 stimulates IL-6 and IL-1β expression by bronchial fibroblasts.ConclusionInteraction between bronchial fibroblasts and T cells seems to promote specifically Th17 cells profile in asthma. These results suggest that cellular interaction particularly between T cells and fibroblasts may play a pivotal role in the regulation of the inflammatory response in asthma.