Sally M. Lansley

Mesothelial cell differentiation into osteoblast- and adipocyte-like cells.

Serosal pathologies including malignant mesothelioma (MM) can show features of osseous and/or cartilaginous differentiation although the mechanism for its formation is unknown. Mesothelial cells have the capacity to differentiate into cells with myofibroblast, smooth muscle and endothelial cell characteristics. Whether they can differentiate into other cell types is unclear. This study tests the hypothesis that mesothelial cells can differentiate into cell lineages of the embryonic mesoderm including osteoblasts and adipocytes. To examine this, a functional assay of bone formation and an adipogenic assay were performed in vitro with primary rat and human mesothelial cells maintained in osteogenic or adipogenic medium (AM) for 0–26 days. Mesothelial cells expressed increasing levels of alkaline phosphatase, an early marker of the osteoblast phenotype, and formed mineralized bone‐like nodules. Mesothelial cells also accumulated lipid indicative of a mature adipocyte phenotype when cultured in AM. All cells expressed several key osteoblast and adipocyte markers, including osteoblast‐specific runt‐related transcription factor 2, and demonstrated changes in mRNA expression consistent with epithelial‐to‐mesenchymal transition. In conclusion, these studies confirm that mesothelial cells have the capacity to differentiate into osteoblast‐ and adipocyte‐like cells, providing definitive evidence of their multipotential nature. These data strongly support mesothelial cell differentiation as the potential source of different tissue types in MM tumours and other serosal pathologies, and add support for the use of mesothelial cells in regenerative therapies.

Mesothelial cells in tissue repair and fibrosis.

Mesothelial cells are fundamental to the maintenance of serosal integrity and homeostasis and play a critical role in normal serosal repair following injury. However, when normal repair mechanisms breakdown, mesothelial cells take on a profibrotic role, secreting inflammatory, and profibrotic mediators, differentiating and migrating into the injured tissues where they contribute to fibrogenesis. The development of new molecular and cell tracking techniques has made it possible to examine the origin of fibrotic cells within damaged tissues and to elucidate the roles they play in inflammation and fibrosis. In addition to secreting proinflammatory mediators and contributing to both coagulation and fibrinolysis, mesothelial cells undergo mesothelial-to-mesenchymal transition, a process analogous to epithelial-to-mesenchymal transition, and become fibrogenic cells. Fibrogenic mesothelial cells have now been identified in tissues where they have not previously been thought to occur, such as within the parenchyma of the fibrotic lung. These findings show a direct role for mesothelial cells in fibrogenesis and open therapeutic strategies to prevent or reverse the fibrotic process.

Tissue plasminogen activator potently stimulates pleural effusion via a monocyte chemotactic protein-1-dependent mechanism.

Pleural infection is common. Evacuation of infected pleural fluid is essential for successful treatment, but it is often difficult because of adhesions/loculations within the effusion and the viscosity of the fluid. Intrapleural delivery of tissue plasminogen activator (tPA) (to break the adhesions) and deoxyribonuclease (DNase) (to reduce fluid viscosity) has recently been shown to improve clinical outcomes in a large randomized study of pleural infection. Clinical studies of intrapleural fibrinolytic therapy have consistently shown subsequent production of large effusions, the mechanism(s) of which are unknown. We aimed to determine the mechanism by which tPA induces exudative fluid formation. Intrapleural tPA, with or without DNase, significantly induced pleural fluid accumulation in CD1 mice (tPA alone: median [interquartile range], 53.5 [30-355] μl) compared with DNase alone or vehicle controls (both, 0.0 [0.0-0.0] μl) after 6 hours. Fluid induction was reproduced after intrapleural delivery of streptokinase and urokinase, indicating a class effect. Pleural fluid monocyte chemotactic protein (MCP)-1 levels strongly correlated with effusion volume (r = 0.7302; P = 0.003), and were significantly higher than MCP-1 levels in corresponding sera. Mice treated with anti-MCP-1 antibody (P < 0.0001) or MCP-1 receptor antagonist (P = 0.0049) demonstrated a significant decrease in tPA-induced pleural fluid formation (by up to 85%). Our data implicate MCP-1 as the key molecule governing tPA-induced fluid accumulation. The role of MCP-1 in the development of other exudative effusions warrants examination.

IL-2/CD40-driven NK cells install and maintain potency in the anti-mesothelioma effector/memory phase

Murine and human mesothelioma tumors are susceptible to immunotherapy, particularly when immune adjuvants are delivered locally. We have shown that direct injection of IL-2 plus agonist anti-CD40 antibody induces regression of large mesothelioma tumors. These studies aimed to determine if NK cells contribute to IL-2/CD40 antibody-driven tumor eradication. We show that NK cells infiltrate developing mesothelioma tumors; however, their absence (in beige mice or in asialo GM(1) antibody-depleted C57BL/6J mice) does not alter tumor growth rates suggesting that they cannot function as effector cells in this microenvironment. Anti-CD40 antibody treatment did not alter the percent of NK cells in treated tumors or in draining lymph nodes (dLNs), and tumor resolution occurred in the absence of NK cells. However, a two-tumor model showed that NK cells contributed to CD40-driven systemic immunity leading to resolution of untreated distal tumors. IL-2 treatment led to increased proportions of NK cells in tumors and dLNs, and in the absence of NK cells, IL-2 lost its therapeutic effect. In contrast, the absence of NK cells did not reduce the anti-tumor activity of the IL-2/anti-CD40 antibody combination yet tumors recurred in NK-deficient mice and > 37% of tumor cell re-challenged mice were unable to provide protection, implying insufficient memory. Furthermore, untreated distal tumors in NK-depleted mice were less readily cured than in immunologically intact mice. These data show that NK cells infiltrate mesothelioma tumors, which, after local IL-2 and/or anti-CD40 antibody treatment, provide help for the acquisition and/or maintenance of systemic immunity and long-term effector/memory responses.

A commercially available preparation of Staphylococcus aureus bio‐products potently inhibits tumour growth in a murine model of mesothelioma

Mesothelioma is an incurable cancer with a rising global incidence. Intrapleural delivery of a commercially available compound made up of proteins produced by Staphylococcus aureus has been used clinically to induce pleurodesis. We investigate if this bacterial compound has anti‐tumoural activities against pleural malignancies, in addition to its pleurodesing effect.

Bacterial Infection Elicits Heat Shock Protein 72 Release from Pleural Mesothelial Cells

Heat shock protein 70 (HSP70) has been implicated in infection-related processes and has been found in body fluids during infection. This study aimed to determine whether pleural mesothelial cells release HSP70 in response to bacterial infection in vitro and in mouse models of serosal infection. In addition, the in vitro cytokine effects of the HSP70 isoform, Hsp72, on mesothelial cells were examined. Further, Hsp72 was measured in human pleural effusions and levels compared between non-infectious and infectious patients to determine the diagnostic accuracy of pleural fluid Hsp72 compared to traditional pleural fluid parameters. We showed that mesothelial release of Hsp72 was significantly raised when cells were treated with live and heat-killed Streptococcus pneumoniae. In mice, intraperitoneal injection of S. pneumoniae stimulated a 2-fold increase in Hsp72 levels in peritoneal lavage (p<0.01). Extracellular Hsp72 did not induce or inhibit mediator release from cultured mesothelial cells. Hsp72 levels were significantly higher in effusions of infectious origin compared to non-infectious effusions (p<0.05). The data establish that pleural mesothelial cells can release Hsp72 in response to bacterial infection and levels are raised in infectious pleural effusions. The biological role of HSP70 in pleural infection warrants exploration.

Malignant pleural fluid from mesothelioma has potent biological activities

Malignant pleural effusion (MPE) affects >90% of mesothelioma patients. Research on MPE has focused on its physical impact on breathlessness; MPE is rich in growth mediators but its contribution to tumour biology has not been investigated. We aimed to examine the potential effects of MPE in promoting growth, migration and chemo‐resistance of mesothelioma.

Preclinical assessment of adjunctive tPA and DNase for peritoneal dialysis associated peritonitis.

A major complication of peritoneal dialysis is the development of peritonitis, which is associated with reduced technique and patient survival. The inflammatory response elicited by infection results in a fibrin and debris-rich environment within the peritoneal cavity, which may reduce the effectiveness of antimicrobial agents and predispose to recurrence or relapse of infection. Strategies to enhance responses to antimicrobial agents therefore have the potential to improve patient outcomes. This study presents pre-clinical data describing the compatibility of tPA and DNase in combination with antimicrobial agents used for the treatment of PD peritonitis. tPA and DNase were stable in standard dialysate solution and in the presence of antimicrobial agents, and were safe when given intraperitoneally in a mouse model with no evidence of local or systemic toxicity. Adjunctive tPA and DNase may have a role in the management of patients presenting with PD peritonitis.

Role of MCP-1 in pleural effusion development in a carrageenan-induced murine model of pleurisy.

Exudative pleural effusions affect over 1500 patients per million population each year. The pathobiology of pleural exudate formation remains unclear. Our recent study revealed monocyte chemotactic protein‐1 (MCP‐1) as a key driver of fibrinolytic‐induced exudate effusion while another study found a role for MCP‐1 in malignant effusion formation. In the present study, we further evaluated the role of MCP‐1 in the development of pleural effusion in a mouse model of acute pleural inflammation.

Human pleural fluid is a potent growth medium for Streptococcus pneumoniae

Empyema is defined by the presence of bacteria and/or pus in pleural effusions. However, the biology of bacteria within human pleural fluid has not been studied. Streptococcus pneumoniae is the most common cause of pediatric and frequent cause of adult empyema. We investigated whether S. pneumoniae can proliferate within human pleural fluid and if growth is affected by the cellular content of the fluid and/or characteristics of pneumococcal surface proteins. Invasive S. pneumoniae isolates (n = 24) and reference strain recovered from human blood or empyema were inoculated (1.5×106CFU/mL) into sterile human malignant pleural fluid samples (n = 11). All S. pneumoniae (n = 25) strains proliferated rapidly, increasing by a median of 3009 (IQR 1063–9846) from baseline at 24hrs in all pleural effusions tested. Proliferation was greater than in commercial pneumococcal culture media and concentrations were maintained for 48hrs without autolysis. A similar magnitude of proliferation was observed in pleural fluid before and after removal of its cellular content, p = 0.728. S. pneumoniae (D39 strain) wild-type, and derivatives (n = 12), each with mutation(s) in a different gene required for full virulence were inoculated into human pleural fluid (n = 8). S. pneumoniae with pneumococcal surface antigen A (ΔpsaA) mutation failed to grow (2207-fold lower than wild-type), p<0.001, however growth was restored with manganese supplementation. Growth of other common respiratory pathogens (n = 14) across pleural fluid samples (n = 7) was variable and inconsistent, with some strains failing to grow. We establish for the first time that pleural fluid is a potent growth medium for S. pneumoniae and proliferation is dependent on the PsaA surface protein and manganese.