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

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

Copyright © 2015 by the American Thoracic Society. 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

Streptococcus pneumoniae potently induces cell death in mesothelial cells.

Pleural infection/empyema is common and its incidence continues to rise. Streptococcus pneumoniae is the commonest bacterial cause of empyema in children and among the commonest in adults. The mesothelium represents the first line of defense against invading microorganisms, but mesothelial cell responses to common empyema pathogens, including S. pneumoniae, have seldom been studied. We assessed mesothelial cell viability in vitro following exposure to common empyema pathogens. Clinical isolates of S. pneumoniae from 25 patients with invasive pneumococcal disease and three reference strains were tested. All potently induced death of cultured mesothelial cells (MeT-5A) in a dose- and time-dependent manner (>90% at 107 CFU/mL after 24 hours). No significant mesothelial cell killing was observed when cells were co-cultured with Staphylococcus aureus, Streptococcus sanguinis and Streptococcus milleri group bacteria. S. pneumoniae induced mesothelial cell death via secretory product(s) as cytotoxicity could be: i) reproduced using conditioned media derived from S. pneumoniae and ii) in transwell studies when the bacteria and mesothelial cells were separated. No excess cell death was seen when heat-killed S. pneumoniae were used. Pneumolysin, a cytolytic S. pneumoniae toxin, induced cell death in a time- and dose-dependent manner. S. pneumoniae lacking the pneumolysin gene (D39 ΔPLY strain) failed to kill mesothelial cells compared to wild type (D39) controls, confirming the necessity of pneumolysin in D39-induced mesothelial cell death. However, pneumolysin gene mutation in other S. pneumoniae strains (TIGR4, ST3 and ST23F) only partly abolished their cytotoxic effects, suggesting different strains may induce cell death via different mechanisms

DNase activity after 6 hours incubation with tPA and antimicrobials or tPA and bacteria.

<p>The activity of DNase in the presence of different antimicrobial agents (Panels A & B) or bacteria (Panels C & D) was measured through digestion of 1 μg of pcDNA3 DNA. V+G = Vancomycin and Gentamicin, A = Amoxicillin, C = Cefazolin and F = Fluconazole. The effects of bacteria on DNase activity was assessed at baseline (T = 0) and following 6 hours incubation (T = 6) at 37°C. Samples were incubated with 5 μg/mL tPA and/or 2.5 μg/mL DNase, as applicable.</p

DNase activity in the presence of vancomycin and gentamicin.

<p>The effect of increasing concentrations of gentamicin on DNase activity was measured in the presence of a constant concentration of Vancomycin. Inhibition of DNase activity, visible as smearing (incomplete digestion) of DNA (Lanes 4–8), was evident at Gentamicin concentrations >35 μg/mL. Samples contained 5 μg/mL tPA and 2.5 μg/mL DNase, as appropriate.</p

tPA and DNase do not directly affect the viability of <i>E</i>. <i>coli</i> and <i>S</i>. <i>aureus</i>.

<p>tPA and DNase do not directly affect the viability of <i>E</i>. <i>coli</i> and <i>S</i>. <i>aureus</i>.</p

H&E sections of kidney, liver, peritoneum and spleen.

<p>Microscopic analysis of abdominal organs and peritoneum was performed following intraperitoneal administration of dialysate (A-D) or dialysate with tPA and DNase (E-H) in the presence or absence of LPS (data with LPS not shown). Samples contained 5 μg/mL tPA and 2.5 μg/mL DNase, as appropriate.</p

tPA and DNase do not influence antimicrobial activity.

<p>The effect of tPA and DNase on antimicrobial activity was assessed by measuring the survival of bacteria (with known sensitivity) to the antimicrobial agents over time in the presence or absence of tPA and DNase. All assessments were performed in triplicate and bacterial counts were calculated through spot counting of serial dilutions of bacteria. Data show is the mean number of cfu/mL at 6 and 24 hours as a % of baseline (error bars are the standard deviation). A = Amoxicillin, C = Cefazolin, V&G = Vancomycin and Gentamicin.</p

Baseline patient characteristics.

<p>Data are presented as median (quartile range) or n (%).</p><p>+Significantly higher than the respective values in other groups by <i>post-hoc</i> test.</p>*<p>Significantly lower than the respective values in other groups by <i>post-hoc</i> test.</p