Blockade of CD93 in pleural mesothelial cells fuels anti-lung tumor immune responses.

Background: CD93 reportedly facilitates tumor angiogenesis. However, whether CD93 regulates antitumor immunity remains undeciphered. Methods: Lung tumor tissues, malignant pleural effusions (MPEs) were obtained from lung cancer patients. Blood was obtained from healthy volunteers and lung cancer patients with anti-PD-1 therapy. Furthermore, p53fl/flLSL-KrasG12D, Ccr7-/-, Cd93-/- mice and CD11c-DTR mice were generated. Specifically, EM, NTA and western blotting were utilized to identify Tumor extracellular vesicles (TEVs). EV labeling, detection of EV uptake in vitro and in vivo, degradation of EV proteins and RNAs were performed to detect the role of TEVs in tumor progression. Pleural mesothelial cells (pMCs) were isolated to investigate related signaling pathways. Recombinant proteins and antibodies were generated to test which antibody was the most effective one to increase CCL21a in p-pMCs. RNA-Seq, MiRNA array, luciferase reporter assay, endothelial tube formation assay, protein labeling and detection, transfection of siRNAs and the miRNA mimic and inhibitor, chemotaxis assay, immunohistochemical staining, flow cytometry, Real-time PCR, and ELISA experiments were performed. Results: We show that CD93 of pMCs reduced lung tumor migration of dendritic cells by preventing pMCs from secreting CCL21, thereby suppressing systemic anti-lung tumor T-cell responses. TEV-derived miR-5110 promotes CCL21 secretion by downregulating pMC CD93, whereas C1q, increasing in tumor individuals, suppresses CD93-mediated CCL21 secretion. CD93-blocking antibodies (anti-CD93) inhibit lung tumor growth better than VEGF receptor-blocking antibodies because anti-CD93 inhibit tumor angiogenesis and promote CCL21 secretion from pMCs. Anti-CD93 also overcome lung tumor resistance to anti-PD-1 therapy. Furthermore, lung cancer patients with higher serum EV-derived miR-5193 (human miR-5110 homolog) are more sensitive to anti-PD-1 therapy, while patients with higher serum C1q are less sensitive, consistent with their regulatory functions on CD93. Conclusions: Our study identifies a crucial role of CD93 in controlling anti-lung tumor immunity and suggests a promising approach for lung tumor therapy.

Primary and hTERT-Transduced Mesothelioma-Associated Fibroblasts but Not Primary or hTERT-Transduced Mesothelial Cells Stimulate Growth of Human Mesothelioma Cells.

Pleural mesothelioma (PM) is an aggressive malignancy that develops in a unique tumor microenvironment (TME). However, cell models for studying the TME in PM are still limited. Here, we have generated and characterized novel human telomerase reverse transcriptase (hTERT)-transduced mesothelial cell and mesothelioma-associated fibroblast (Meso-CAF) models and investigated their impact on PM cell growth. Pleural mesothelial cells and Meso-CAFs were isolated from tissue of pneumothorax and PM patients, respectively. Stable expression of hTERT was induced by retroviral transduction. Primary and hTERT-transduced cells were compared with respect to doubling times, hTERT expression and activity levels, telomere lengths, proteomes, and the impact of conditioned media (CM) on PM cell growth. All transduced derivatives exhibited elevated hTERT expression and activity, and increased mean telomere lengths. Cell morphology remained unchanged, and the proteomes were similar to the corresponding primary cells. Of note, the CM of primary and hTERT-transduced Meso-CAFs stimulated PM cell growth to the same extent, while CM derived from mesothelial cells had no stimulating effect, irrespective of hTERT expression. In conclusion, all new hTERT-transduced cell models closely resemble their primary counterparts and, hence, represent valuable tools to investigate cellular interactions within the TME of PM.

Update on Novel Targeted Therapy for Pleural Organization and Fibrosis.

Pleural injury and subsequent loculation is characterized by acute injury, sustained inflammation and, when severe, pathologic tissue reorganization. While fibrin deposition is a normal part of the injury response, disordered fibrin turnover can promote pleural loculation and, when unresolved, fibrosis of the affected area. Within this review, we present a brief discussion of the current IPFT therapies, including scuPA, for the treatment of pathologic fibrin deposition and empyema. We also discuss endogenously expressed PAI-1 and how it may affect the efficacy of IPFT therapies. We further delineate the role of pleural mesothelial cells in the progression of pleural injury and subsequent pleural remodeling resulting from matrix deposition. We also describe how pleural mesothelial cells promote pleural fibrosis as myofibroblasts via mesomesenchymal transition. Finally, we discuss novel therapeutic targets which focus on blocking and/or reversing the myofibroblast differentiation of pleural mesothelial cells for the treatment of pleural fibrosis.

[Chronic toxicity of Multi-walled carbon nanotubes in human pleural mesothelial cells].

Objective: To explore the chronic toxicity and its potential mechanism of multi-walled carbon nanotube (MWCNT) in human pleural mesothelial cells. Methods: A sustainable exposure of MeT-5A cells to MWCNT at 10 µg/cm(2) for one year was conducted in 2016. During the exposure, the cell images and cell proliferation was recorded every 4 weeks. The cell apoptosis, cell cycle, cell migration and cell invasion were compared between the control cells and the cells after MWCNT exposure. Finally, the gene expression was screened with Affymetrix clariom D assay, and some of the significantly differential expressed genes was verified by RT-PCR. Results: Compared with the control group, the proliferation ability of the cells in the 1-year exposed group was significantly increased, and the rate of proliferation was about 2-3 times as that in the Control Group (F=481.32, P<0.05) . MeT-5A cells all showed cell cycle arrest effect, which showed the increase of G1 phase and the decrease of s phase and G2 phase (F=14.94, P<0.05) . The apoptosis rate of cells in the treated group was significantly higher than that in the control group after 6 months (F=15.12, P<0.05) , but the early apoptosis rate and the total apoptosis rate of cells in the treated group were not significantly different from those in the control group after 1 year (F=3.97, P<0.05) . The cell migration and invasion were both promoted by MWCNT. Furthermore, the differentially expressed genes was screened, to find 2, 878 genes with more than 2 folds changes. To further verified, RT-PCR was conducted with PIK3R3、WNT2B、VANGL2、ANXA1, and their expression changes were consistent with above. Conclusion: MWCNT might have a carcinogenic potential to MeT-5A cells after the long term exposure.

NOX1 Promotes Mesothelial-Mesenchymal Transition through Modulation of Reactive Oxygen Species-mediated Signaling.

Pleural organization may occur after empyema or complicated parapneumonic effusion and can result in restrictive lung disease with pleural fibrosis (PF). Pleural mesothelial cells (PMCs) may contribute to PF through acquisition of a profibrotic phenotype, mesothelial-mesenchymal transition (MesoMT), which is characterized by increased expression of α-SMA (α-smooth muscle actin) and other myofibroblast markers. Although MesoMT has been implicated in the pathogenesis of PF, the role of the reactive oxygen species and the NOX (nicotinamide adenine dinucleotide phosphate oxidase) family in pleural remodeling remains unclear. Here, we show that NOX1 expression is enhanced in nonspecific human pleuritis and is induced in PMCs by THB (thrombin). 4-Hydroxy-2-nonenal, an indicator of reactive oxygen species damage, was likewise increased in our mouse model of pleural injury. NOX1 downregulation blocked THB- and Xa (factor Xa)-mediated MesoMT, as did pharmacologic inhibition of NOX1 with ML-171. NOX1 inhibition also reduced phosphorylation of Akt, p65, and tyrosine 216-GSK-3ß, signaling molecules previously shown to be implicated in MesoMT. Conversely, ML-171 did not reverse established MesoMT. NOX4 downregulation attenuated TGF-ß- and THB-mediated MesoMT. However, NOX1 downregulation did not affect NOX4 expression. NOX1- and NOX4-deficient mice were also protected in our mouse model of Streptococcus pneumoniae-mediated PF. These data show that NOX1 and NOX4 are critical determinants of MesoMT.

Evaluation of cellular alterations and inflammatory profile of mesothelial cells and/or neoplastic cells exposed to talc used for pleurodesis.

Introdution: To determine the role of Pleural Mesothelial Cells (PMC) and/or Neoplasic Cells (NC) in the initiation and regulation of acute inflammatory response after exposure to talc for evaluating inflammatory mediators and cellular alterations. MATERIALS AND METHODS: PMC cultures, human lung (A549) and breast (MCF7) adenocarcinoma cells were divided in 5 groups: 100% PMC, 100% NC, 25% PMC + 75% NC, 50% of each type and 75% PMC + 25% NC. All groups were exposed to talc and measured IL-6, IL-1ß, IL-10, TNF-α, TNFRI, pH, LDH, apoptosis and necrosis. STATISTICAL ANALYSIS: One-way Anova. RESULTS: High IL-6, IL-1ß and TNFRI levels were found in PMC and NC exposed to talc. IL-6 was higher at the points of more confluence of PMC. The highest levels of IL-1ß and TNFRI were found in mixed cultures. In pure cultures TNFRI was higher in A549 followed by PMC and MCF7. LDH was higher in A549 than PMC. The lowest pH was found in 100% NC. All cell line exposed to talc reduced viability and increased necrosis. Apoptotic cells exposed to talc were higher in pure cultures of NC than in PMC. Mixed cultures of PMC and A549 showed lower levels of apoptosis in cultures with more NC. CONCLUSIONS: PMC after talc exposure participates in the inflammatory process contributing to production of molecular mediators, necessary for effective pleurodesis. Talc acted in NC causing higher rates of apoptosis, contributing in a modest way to tumoral decrease. Different types of tumor cells may respond differently to exposure to talc.

Crosstalk between pleural mesothelial cell and lung fibroblast contributes to pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic, progressive and fibrosing interstitial pneumonia of unknown cause. The main feature of IPF is a heterogeneous appearance with areas of sub-pleural fibrosis. However, the mechanism of sub-pleural fibrosis was poorly understood. In this study, our in vivo study revealed that pleural mesothelial cells (PMCs) migrated into lung parenchyma and localized alongside lung fibroblasts in sub-pleural area in mouse pulmonary fibrosis. Our in vitro study displayed that cultured-PMCs-medium induced lung fibroblasts transforming into myofibroblast, cultured-fibroblasts-medium promoted mesothelial-mesenchymal transition of PMCs. Furthermore, these changes in lung fibroblasts and PMCs were prevented by blocking TGF-ß1/Smad2/3 signaling with SB431542. TGF-ß1 neutralized antibody attenuated bleomycin-induced pulmonary fibrosis. Similar to TGF-ß1/Smad2/3 signaling, wnt/ß-catenin signaling was also activated in the process of PMCs crosstalk with lung fibroblasts. Moreover, inhibition of CD147 attenuated cultured-PMCs-medium induced collagen-I synthesis in lung fibroblasts. Blocking CD147 signaling also prevented bleomycin-induced pulmonary fibrosis. Our data indicated that crosstalk between PMC and lung fibroblast contributed to sub-pleural pulmonary fibrosis. TGF-ß1, Wnt/ß-catenin and CD147 signaling was involved in the underling mechanism.

Isolation and culture of pleural mesothelial cells.

Purpose: Although the isolation of rat and mouse mesothelial cells has previously been reported, most mesothelial cells used for experimental studies are obtained from peritoneal cells. Here, we describe an optimized method for the isolation and in vitro propagation of rodent pleural mesothelial cells without the requirement for specialized surgical techniques. Materials and Methods: To harvest pleural mesothelial cells, the pleural space of 8-9-week-old rats or older mice was filled with 0.25% trypsin in ethylenediaminetetraacetic acid (EDTA) buffer for 20 min at 37 °C. Cells were then harvested, and incubated at 37 °C in a humidified atmosphere with 5% CO2. Immunofluorescence analysis of plated pleural mesothelial cells was performed using Alexa 546 (calretinin). To investigate optimal proliferation conditions, medium enriched with various concentrations of fetal calf serum (FCS) was used for pleural mesothelial cell proliferation. Results: By day 10, confluent cell cultures were established, and the cells displayed an obvious cobblestone morphology. Immunofluorescence analysis of the cells demonstrated that all stained positive for Alexa 546 (calretinin) expression. Mesothelial cells grew better in medium containing 20% FCS than with 10% FCS. Conclusions: This is a simple procedure for the efficient collection of primary pleural mesothelial cells, which were obtained in defined culture conditions from the euthanized rodent thoracic cavity using trypsin-EDTA treatment. The ability to easily culture and maintain identifiable pleural mesothelial cells from rodents will be helpful for future experiments using these cells.

Benign Pleural Mesothelial Cells Have Higher Osmotic Water Permeability than Malignant Pleural Mesothelioma Cells and Differentially Respond to Hyperosmolality.

BACKGROUND/AIMS: Cell volume regulation is a critical mechanism for cell homeostasis and depends on the osmotic water permeability (Pf) of the cell plasma membrane. The Pf of human mesothelial cells is unknown although they contribute to serosal fluid turnover. METHODS: In this study we measured the osmotic water permeability of benign human mesothelial cells (MeT-5A) and of epithelioid (M14K) and sarcomatoid (ZL34) malignant pleural mesothelioma (MPM) cells in response to acute hyperosmotic stress. We also assessed the changes in their Pf after preconditioning with 4% glucose for 24 hours. In both cases we also assessed the role of AQP1 inhibition (0.1 mM HgCl2) on the Pf. Finally, we assessed corresponding changes in the AQP1 plasma membrane availability by immunofluorescence. RESULTS: We report that MeT-5A cells have a significantly higher Pf as compared to M14K and ZL34 MPM cells [4.85E-03±2.37E-03 cm/sec (n=17) versus 2.74E-03±0.74E-03 cm/sec (n=11) and 2.86E-03±0.11E-03 cm/sec (n=11)]. AQP1 inhibition significantly decreased the Pf in all cells lines (p<0.001 in all cases). High glucose preconditioning for 24 hours significantly increased MeT-5A Pf (p<0.001), did not influence M14K Pf (p=0.19) and significantly reduced ZL34 Pf (p=0.02). Comparing cell lines after high glucose preconditioning, MeT-5A Pf was significantly higher than that of M14K and ZL34 MPM cells and the AQP1 inhibition effect was significant in MeT-5A and M14K cells. These results were corroborated by AQP1 immunofluorescence. CONCLUSION: We provide evidence for a differential regulation of Pf in benign and MPM cells that require further mechanistic investigation.

miR-4739 mediates pleural fibrosis by targeting bone morphogenetic protein 7.

BACKGROUND: Pleural fibrosis is defined as excessive depositions of matrix components that result in pleural tissue architecture destruction and dysfunction. In severe cases, the progression of pleural fibrosis leads to lung entrapment, resulting in dyspnea and respiratory failure. However, the mechanism of pleural fibrosis is poorly understood. METHODS: miR-4739 levels were detected by miRNA array and real-time PCR. Real-time PCR, western blotting and immunofluorescence were used to identify the expression profile of indicators related to fibrosis. Target gene of miR-4739 and promoter activity assay was measured by using dual-luciferase reporter assay system. In vivo, pleural fibrosis was evaluated by Masson staining and miR-4739 level was detected by In situ hybridization histochemistry. FINDINGS: We found that bleomycin induced up-regulation of miR-4739 in pleural mesothelial cells (PMCs). Over-regulated miR-4739 mediated mesothelial-mesenchymal transition and increased collagen-I synthesis in PMCs. Investigation on the clinical specimens revealed that high levels of miR-4739 and low levels of bone morphogenetic protein 7 (BMP-7) associated with pleural fibrosis in patients. Then we next identified that miR-4739 targeted and down-regulated BMP-7 which further resulted in unbalance between Smad1/5/9 and Smad2/3 signaling. Lastly, in vivo studies revealed that miR-4739 over-expression induced pleural fibrosis, and exogenous BMP-7 prevented pleural fibrosis in mice. INTERPRETATION: Our data indicated that miR-4739 targets BMP-7 which mediates pleural fibrosis. The miR-4739/BMP-7 axis is a promising therapeutic target for the disease. FUND: The National Natural Science Foundation of China.

Pleural effusions induced by human herpesviruses in the immunocompetent host.

METHODS: A computer-based search of the English literature for articles relative to Human Herpesviruses (HHVs) infection and pleural effusions (PEs) in the immunocompetent host was performed in PubMed and Scopus. The reference lists of the retrieved articles were also reviewed for relevant articles. RESULTS: A total of 20 articles satisfied the selection criteria and were included in the study. In the majority of the articles, PEs were reported as clinical complications of systemic HHV-induced infection. The frequency of HHVs within the reported cases was five for HHV-1/2, one for HHV-3, six for HHV-4, six for HHV-5 and one for HHV-6. One case involved HHV-4 and HHV-5 co-infection. No case of HHV-7 or HHV-8 related PE in the immunocompetent host was retrieved. CONCLUSIONS: Pleural effusions in the immunocompetent host occur in severe viral infections and can be due to comorbidities (or septic complications) or due to the direct HHV pathogenicity although research relative to the susceptibility of pleural mesothelial cells to HHV infection is lacking. HHV pathogenicity needs to be studied further as it could explain undiagnosed PEs.

Anaphylatoxins Enhance Recruitment of Nonclassical Monocytes via Chemokines Produced by Pleural Mesothelial Cells in Tuberculous Pleural Effusion.

In the present study, we sought to elucidate the mechanisms by which monocytes migrate into the pleural space in the presence of anaphylatoxins in tuberculous pleural effusion (TPE). Monocytes in both pleural effusion and blood were counted, and their phenotypic characteristics were analyzed. Activation of the complement system was detected in TPE. The effects of Mpt64 and anaphylatoxins on the production of chemokines in pleural mesothelial cells (PMCs) were measured. The chemoattractant activity of chemokines produced by PMCs for monocytes was observed. Levels of CD14+CD16+ monocytes were significantly higher in TPE than in blood. Three pathways of the complement system were activated in TPE. C3a-C3aR1, C5a-C5aR1, CCL2-CCR2, CCL7-CCR2, and CX3CL1-CX3CR1 were coexpressed in PMCs and monocytes isolated from TPE. Moreover, we initially found that Mpt64 stimulated the expression of C3a and C5a in PMCs. C3a and C5a not only induced CCL2, CCL7, and CX3CL1 expression in PMCs but also stimulated production of IL-1ß, IL-17, and IL-27 in monocytes. C3a and C5a stimulated PMCs to secrete CCL2, CCL7, and CX3CL1, which recruited CD14+CD16+ monocytes to the pleural cavity. As a result, the infiltration of CD14+CD16+ monocytes engaged in the pathogenesis of TPE by excessive production of inflammatory cytokines.

Cigarette smoking aggravates bleomycin-induced experimental pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease that typically leads to respiratory failure and death. The cause of IPF is poorly understood. Although several environmental and occupational factors are considered as risk factors in IPF, cigarette smoking seems to be the most strongly associated risk factor. Here firstly, we treated mice with cigarette (16 mg tar, 1.0 mg nicotine in each cigarette) smoking and tried to explore the role of cigarette smoking in pulmonary fibrosis. Mice were continuously subjected to smoke for about 1 h each day (12 cigarettes per day, 5 days per week) during 40 days. Bleomycin was administrated by intraperitoneal injection at a dose of 40 mg/kg on days 1, 5, 8, 11 and 15. We found bleomycin induced pulmonary fibrosis in mice, and cigarette smoking augmented bleomycin-induced fibrosis reflected by both in fibrotic area and percentages of collagen in the lungs. Then we prepared and employed cigarette smoke extract (CSE) in cell models and found that CSE could induce the activation of p-Smad2/3 and p-Akt, as well as collagen-I synthesis and cell proliferation in lung fibroblasts and pleural mesothelial cells (PMCs). TGF-ß1 signaling mediated CSE-induced PMCs migration. Moreover, in vitro studies revealed that CSE had superimposed effect on bleomycin-induced activation of TGF-ß-Smad2/3 and -Akt signaling. TGF-ß-Smad2/3 and -Akt signaling were further augmented by cigarette smoking in the lung of bleomycin-treated mice. Taken together, these findings represent the first evidence that cigarette smoking aggravated bleomycin-induced pulmonary fibrosis via TGF-ß1 signaling.

Lethal (2) giant larvae regulates pleural mesothelial cell polarity in pleural fibrosis.

Pleural fibrosis is barely reversible and the underlying mechanisms are poorly understood. Pleural mesothelial cells (PMCs) which have apical-basal polarity play a key role in pleural fibrosis. Loss of cell polarity is involved in the development of fibrotic diseases. Partition defective protein (PAR) complex is a key regulator of cell polarity. However, changes of PMC polarity and PAR complex in pleural fibrosis are still unknown. In this study, we observed that PMC polarity was lost in fibrotic pleura. Next we found increased Lethal (2) giant larvae (Lgl) bound with aPKC and PAR-6B competing against PAR-3A in PAR complex, which led to cell polarity loss. Then we demonstrated that Lgl1 siRNA prevented cell polarity loss in PMCs, and Lgl1 conditional knockout (ER-Cre+/-Lgl1flox/flox) attenuated pleural fibrosis in a mouse model. Our data indicated that Lgl1 regulates cell polarity of PMCs, inhibition of Lgl1 and maintenance of cell polarity in PMCs could be a potential therapeutic treatment approach for pleural fibrosis.

TLR2 contributes to trigger immune response of pleural mesothelial cells against Mycobacterium bovis BCG and M. tuberculosis infection.

Mycobacterium tuberculosis is a causative agent leading to pleural effusion, characterized by the accumulation of fluid and immune cells in the pleural cavity. Although this phenomenon has been described before, detailed processes or mechanisms associated with the pleural effusion are still not well understood. Pleural mesothelial cells (PMCs) are specialized epithelial cells that cover the body wall and internal organs in pleural cavity playing a central role in pleural inflammation. Toll-like receptors are expressed in various cell types including mesothelial cells and initiate the recognition and defense against mycobacterial infection. In the present study, we investigated direct immune responses of PMCs against two mycobacterial strains, M. bovis vaccine strain Bacille Calmette-Guérin (BCG) and M. tuberculosis virulent strain H37Rv, and the role of TLR2 in such responses. Infection with BCG and H37Rv increased the production of IL-6, CXCL1, and CCL2 in WT PMCs, which was partially impaired in TLR2-deficient cells. In addition, the activation of NF-κB and MAPKs induced by BCG and H37Rv was suppressed in TLR2-deficient PMCs, as compared with the WT cells. TLR2 deficiency led to the decrease of nitric oxide (NO) production through the delayed gene expression of iNOS in PMCs. TLR2 was also shown to be essential for optimal expression of cellular adhesion molecules such as ICAM-1 and VCAM-1 in PMCs in response to BCG and H37Rv. These findings strongly suggest that TLR2 participates in mycobacteria-induced innate immune responses in PMCs and may play a role in pathogenesis of tuberculosis pleural effusion.

miR-18a-5p Inhibits Sub-pleural Pulmonary Fibrosis by Targeting TGF-ß Receptor II.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease that typically leads to respiratory failure and death within 3-5 years of diagnosis. Sub-pleural pulmonary fibrosis is a pathological hallmark of IPF. Bleomycin treatment of mice is a an established pulmonary fibrosis model. We recently showed that bleomycin-induced epithelial-mesenchymal transition (EMT) contributes to pleural mesothelial cell (PMC) migration and sub-pleural pulmonary fibrosis. MicroRNA (miRNA) expression has recently been implicated in the pathogenesis of IPF. However, changes in miRNA expression in PMCs and sub-pleural fibrosis have not been reported. Using cultured PMCs and a pulmonary fibrosis animal model, we found that miR-18a-5p was reduced in PMCs treated with bleomycin and that downregulation of miR-18a-5p contributed to EMT of PMCs. Furthermore, we determined that miR-18a-5p binds to the 3' UTR region of transforming growth factor ß receptor II (TGF-ßRII) mRNA, and this is associated with reduced TGF-ßRII expression and suppression of TGF-ß-Smad2/3 signaling. Overexpression of miR-18a-5p prevented bleomycin-induced EMT of PMC and inhibited bleomycin-induced sub-pleural fibrosis in mice. Taken together, our data indicate that downregulated miR-18a-5p mediates sub-pleural pulmonary fibrosis through upregulation of its target, TGF-ßRII, and that overexpression of miR-18a-5p might therefore provide a novel approach to the treatment of IPF.

Organizing empyema induced in mice by Streptococcus pneumoniae: effects of plasminogen activator inhibitor-1 deficiency.

BACKGROUND: Pleural infection affects about 65,000 patients annually in the US and UK. In this and other forms of pleural injury, mesothelial cells (PMCs) undergo a process called mesothelial (Meso) mesenchymal transition (MT), by which PMCs acquire a profibrogenic phenotype with increased expression of α-smooth muscle actin (α-SMA) and matrix proteins. MesoMT thereby contributes to pleural organization with fibrosis and lung restriction. Current murine empyema models are characterized by early mortality, limiting analysis of the pathogenesis of pleural organization and mechanisms that promote MesoMT after infection. METHODS: A new murine empyema model was generated in C57BL/6 J mice by intrapleural delivery of Streptococcus pneumoniae (D39, 3 × 10(7)-5 × 10(9) cfu) to enable use of genetically manipulated animals. CT-scanning and pulmonary function tests were used to characterize the physiologic consequences of organizing empyema. Histology, immunohistochemistry, and immunofluorescence were used to assess pleural injury. ELISA, cytokine array and western analyses were used to assess pleural fluid mediators and markers of MesoMT in primary PMCs. RESULTS: Induction of empyema was done through intranasal or intrapleural delivery of S. pneumoniae. Intranasal delivery impaired lung compliance (p < 0.05) and reduced lung volume (p < 0.05) by 7 days, but failed to reliably induce empyema and was characterized by unacceptable mortality. Intrapleural delivery of S. pneumoniae induced empyema by 24 h with lung restriction and development of pleural fibrosis which persisted for up to 14 days. Markers of MesoMT were increased in the visceral pleura of S. pneumoniae infected mice. KC, IL-17A, MIP-1ß, MCP-1, PGE2 and plasmin activity were increased in pleural lavage of infected mice at 7 days. PAI-1(-/-) mice died within 4 days, had increased pleural inflammation and higher PGE2 levels than WT mice. PGE2 was induced in primary PMCs by uPA and plasmin and induced markers of MesoMT. CONCLUSION: To our knowledge, this is the first murine model of subacute, organizing empyema. The model can be used to identify factors that, like PAI-1 deficiency, alter outcomes and dissect their contribution to pleural organization, rind formation and lung restriction.

Pleural mesothelial cells in pleural and lung diseases.

During development, the mesoderm maintains a complex relationship with the developing endoderm giving rise to the mature lung. Pleural mesothelial cells (PMCs) derived from the mesoderm play a key role during the development of the lung. The pleural mesothelium differentiates to give rise to the endothelium and smooth muscle cells via epithelial-to-mesenchymal transition (EMT). An aberrant recapitulation of such developmental pathways can play an important role in the pathogenesis of disease processes such as idiopathic pulmonary fibrosis (IPF). The PMC is the central component of the immune responses of the pleura. When exposed to noxious stimuli, it demonstrates innate immune responses such as Toll-like receptor (TLR) recognition of pathogen associated molecular patterns as well as causes the release of several cytokines to activate adaptive immune responses. Development of pleural effusions occurs due to an imbalance in the dynamic interaction between junctional proteins, n-cadherin and ß-catenin, and phosphorylation of adherens junctions between PMCs, which is caused in part by vascular endothelial growth factor (VEGF) released by PMCs. PMCs play an important role in defense mechanisms against bacterial and mycobacterial pleural infections, and in pathogenesis of malignant pleural effusion, asbestos related pleural disease and malignant pleural mesothelioma. PMCs also play a key role in the resolution of inflammation, which can occur with or without fibrosis. Fibrosis occurs as a result of disordered fibrin turnover and due to the effects of cytokines such as transforming growth factor-ß, platelet-derived growth factor (PDGF), and basic fibroblast growth factor; which are released by PMCs. Recent studies have demonstrated a role for PMCs in the pathogenesis of IPF suggesting their potential as a cellular biomarker of disease activity and as a possible therapeutic target. Pleural-based therapies targeting PMCs for treatment of IPF and other lung diseases need further exploration.

Mesomesenchymal transition of pleural mesothelial cells is PI3K and NF-κB dependent.

Pleural organization follows acute injury and is characterized by pleural fibrosis, which may involve the visceral and parietal pleural surfaces. This process affects patients with complicated parapneumonic pleural effusions, empyema, and other pleural diseases prone to pleural fibrosis and loculation. Pleural mesothelial cells (PMCs) undergo a process called mesothelial mesenchymal transition (MesoMT), by which PMCs acquire a profibrotic phenotype characterized by cellular enlargement and elongation, increased expression of α-smooth muscle actin (α-SMA), and matrix proteins including collagen-1. Although MesoMT contributes to pleural fibrosis and lung restriction in mice with carbon black/bleomycin-induced pleural injury and procoagulants and fibrinolytic proteases strongly induce MesoMT in vitro, the mechanism by which this transition occurs remains unclear. We found that thrombin and plasmin potently induce MesoMT in vitro as does TGF-ß. Furthermore, these mediators of MesoMT activate phosphatidylinositol-3-kinase (PI3K)/Akt and NF-κB signaling pathways. Inhibition of PI3K/Akt signaling prevented TGF-ß-, thrombin-, and plasmin-mediated induction of the MesoMT phenotype exhibited by primary human PMCs. Similar effects were demonstrated through blockade of the NF-κB signaling cascade using two distinctly different NF-κB inhibitors, SN50 and Bay-11 7085. Conversely, expression of constitutively active Akt-induced mesenchymal transition in human PMCs whereas the process was blocked by PX866 and AKT8. Furthermore, thrombin-mediated MesoMT is dependent on PAR-1 expression, which is linked to PI3K/Akt signaling downstream. These are the first studies to demonstrate that PI3K/Akt and/or NF-κB signaling is critical for induction of MesoMT.

Microfluidic gradient device for studying mesothelial cell migration and the effect of chronic carbon nanotube exposure.

Cell migration is one of the crucial steps in many physiological and pathological processes, including cancer development. Our recent studies have shown that carbon nanotubes (CNTs), similarly to asbestos, can induce accelerated cell growth and invasiveness that contribute to their mesothelioma pathogenicity. Malignant mesothelioma is a very aggressive tumor that develops from cells of the mesothelium, and is most commonly caused by exposure to asbestos. CNTs have a similar structure and mode of exposure to asbestos. This has raised a concern regarding the potential carcinogenicity of CNTs, especially in the pleural area which is a key target for asbestos-related diseases. In this paper, a static microfluidic gradient device was applied to study the migration of human pleural mesothelial cells which had been through a long-term exposure (4 months) to subcytotoxic concentration (0.02 µg cm-2) of single-walled CNTs (SWCNTs). Multiple migration signatures of these cells were investigated using the microfluidic gradient device for the first time. During the migration study, we observed that cell morphologies changed from flattened shapes to spindle shapes prior to their migration after their sensing of the chemical gradient. The migration of chronically SWCNT-exposed mesothelial cells was evaluated under different fetal bovine serum (FBS) concentration gradients, and the migration speeds and number of migrating cells were extracted and compared. The results showed that chronically SWCNT-exposed mesothelial cells are more sensitive to the gradient compared to non-SWCNT-exposed cells. The method described here allows simultaneous detection of cell morphology and migration under chemical gradient conditions, and also allows for real-time monitoring of cell motility that resembles in vivo cell migration. This platform would be much needed for supporting the development of more physiologically relevant cell models for better assessment and characterization of the mesothelioma hazard posed by nanomaterials.