Macrophage migration inhibitory factor enhances Pseudomonas aeruginosa biofilm formation, potentially contributing to cystic fibrosis pathogenesis.

Macrophage migration inhibitory factor (MIF) is a key proinflammatory mediator that we have previously shown to be associated with an aggressive clinical phenotype in cystic fibrosis. It possesses unique tautomerase enzymatic activity. However, to date, no human-derived substrate has been identified that has the capacity to interact with this cytokine's unique tautomerase activity. This led us to hypothesize that MIF may have the capacity to interact with external substrates. We describe for the first time how Pseudomonas aeruginosa can utilize human recombinant MIF (rMIF) to significantly (P < 0.01) enhance its endogenous biofilm formation. Our in vivo studies demonstrate that utilizing a small-molecular-weight inhibitor targeting MIF's tautomerase activity (SCD-19) significantly reduces the inflammatory response in a murine pulmonary chronic P. aeruginosa model. In addition, we show that in in vitro experiments, pretreatment of P. aeruginosa with rMIF is associated with reduced bacterial killing by tobramycin. Our novel findings support the concept of an anti-MIF strategy that targets this enzymatic activity as a potential future antibacterial therapeutic approach.-Tynan, A., Mawhinney, L., Armstrong, M. E., O'Reilly, C., Kennedy, S., Caraher, E., Jülicher, K., O'Dwyer, D., Maher, L., Schaffer, K., Fabre, A., McKone, E. F., Leng, L., Bucala, R., Bernhagen, J., Cooke, G., Donnelly, S. C. Macrophage migration inhibitory factor enhances Pseudomonas aeruginosa biofilm formation, potentially contributing to cystic fibrosis pathogenesis.

Current and Emerging Therapies for the Treatment of Cystic Fibrosis or Mitigation of Its Symptoms.

Clinical presentation of the chronic, heritable condition cystic fibrosis (CF) is complex, with a diverse range of symptoms often affecting multiple organs with varying severity. The primary source of morbidity and mortality is due to progressive destruction of the airways attributable to chronic inflammation arising from microbial colonisation. Antimicrobial therapy combined with practises to remove obstructive mucopurulent deposits form the cornerstone of current therapy. However, new treatment options are emerging which offer, for the first time, the opportunity to effect remission from the underlying cause of CF. Here, we discuss these therapies, their mechanisms of action, and their successes and failures in order to illustrate the shift in the nature of how CF will likely be managed into the future.

Activity of Tobramycin against Cystic Fibrosis Isolates of Burkholderia cepacia Complex Grown as Biofilms.

Pulmonary infection with Burkholderia cepacia complex in cystic fibrosis (CF) patients is associated with more-rapid lung function decline and earlier death than in CF patients without this infection. In this study, we used confocal microscopy to visualize the effects of various concentrations of tobramycin, achievable with systemic and aerosolized drug administration, on mature B. cepacia complex biofilms, both in the presence and absence of CF sputum. After 24 h of growth, biofilm thickness was significantly reduced by exposure to 2,000 µg/ml of tobramycin for Burkholderia cepacia, Burkholderia multivorans, and Burkholderia vietnamiensis; 200 µg/ml of tobramycin was sufficient to reduce the thickness of Burkholderia dolosa biofilm. With a more mature 48-h biofilm, significant reductions in thickness were seen with tobramycin at concentrations of ≥100 µg/ml for all Burkholderia species. In addition, an increased ratio of dead to live cells was observed in comparison to control with tobramycin concentrations of ≥200 µg/ml for B. cepacia and B. dolosa (24 h) and ≥100 µg/ml for Burkholderia cenocepacia and B. dolosa (48 h). Although sputum significantly increased biofilm thickness, tobramycin concentrations of 1,000 µg/ml were still able to significantly reduce biofilm thickness of all B. cepacia complex species with the exception of B. vietnamiensis. In the presence of sputum, 1,000 µg/ml of tobramycin significantly increased the dead-to-live ratio only for B. multivorans compared to control. In summary, although killing is attenuated, high-dose tobramycin can effectively decrease the thickness of B. cepacia complex biofilms, even in the presence of sputum, suggesting a possible role as a suppressive therapy in CF.

Residence in biofilms allows Burkholderia cepacia complex (Bcc) bacteria to evade the antimicrobial activities of neutrophil-like dHL60 cells.

Bacteria of the Burkholderia cepacia complex (Bcc) persist in the airways of people with cystic fibrosis (CF) despite the continuous recruitment of neutrophils. Most members of Bcc are multidrug resistant and can form biofilms. As such, we sought to investigate whether biofilm formation plays a role in protecting Bcc bacteria from neutrophils. Using the neutrophil-like, differentiated cell line, dHL60, we have shown for the first time that Bcc biofilms are enhanced in the presence of these cells. Biofilm biomass was greater following culture in the presence of dHL60 cells than in their absence, likely the result of incorporating dHL60 cellular debris into the biofilm. Moreover, we have demonstrated that mature biofilms (cultured for up to 72 h) induced necrosis in the cells. Established biofilms also acted as a barrier to the migration of the cells and masked the bacteria from being recognized by the cells; dHL60 cells expressed less IL-8 mRNA and secreted significantly less IL-8 when cultured in the presence of biofilms, with respect to planktonic bacteria. Our findings provide evidence that biofilm formation can, at least partly, enable the persistence of Bcc bacteria in the CF airway and emphasize a requirement for anti-biofilm therapeutics.

Mcl-1 is vital for neutrophil survival.

Upon entry to the systemic circulation, neutrophils exhibit a short mean time to cell death. The viability of most cell types in a steady state is preserved by the interplay of the Bcl-2 family of proteins, wherein the anti-apoptotic members inhibit the action of their pro-apoptotic counterparts. Neutrophils, however, display absent or severely reduced expression of several anti-apoptotic Bcl-2 family proteins. Hence, they rely on the expression of Mcl-1, an anti-apoptotic member of the Bcl-2 family, for survival. This protein is uniquely short-lived relative to related proteins and its loss likely precipitates the induction of apoptosis in neutrophils. This review describes the role of Mcl-1 in the neutrophil in the context of apoptosis and highlights the proteins' importance to the cell. We also address neutrophil apoptosis in the broader context of the cells' response to pathogens, focussing particularly on the strategies used by pathogens to manipulate the apoptotic pathway to their own ends.

Virulence of an emerging respiratory pathogen, genus Pandoraea, in vivo and its interactions with lung epithelial cells.

Pandoraea species have emerged as opportunistic pathogens among cystic fibrosis (CF) and non-CF patients. Pandoraea pulmonicola is the predominant Pandoraea species among Irish CF patients. The objective of this study was to investigate the pathogenicity and potential mechanisms of virulence of Irish P. pulmonicola isolates and strains from other Pandoraea species. Three patients from whom the P. pulmonicola isolates were isolated have since died. The in vivo virulence of these and other Pandoraea strains was examined by determining the ability to kill Galleria mellonella larvae. The P. pulmonicola strains generally were the most virulent of the species tested, with three showing a comparable or greater level of virulence in vivo relative to another CF pathogen, Burkholderia cenocepacia, whilst strains from two other species, Pandoraea apista and Pandoraea pnomenusa, were considerably less virulent. For all Pandoraea species, whole cells were required for larval killing, as cell-free supernatants had little effect on larval survival. Overall, invasive Pandoraea strains showed comparable invasion of two independent lung epithelial cell lines, irrespective of whether they had a CF phenotype. Pandoraea strains were also capable of translocation across polarized lung epithelial cell monolayers. Although protease secretion was a common characteristic across the genus, it is unlikely to be involved in pathogenesis. In conclusion, whilst multiple mechanisms of pathogenicity may exist across the genus Pandoraea, it appears that lung cell invasion and translocation contribute to the virulence of P. pulmonicola strains.

Evaluation of in vitro virulence characteristics of the genus Pandoraea in lung epithelial cells.

Pandoraea species are emerging opportunistic pathogens capable of causing chronic lung infections in cystic fibrosis patients. This study examined the interactions of 17 Pandoraea isolates from the five identified species (Pandoraea apista, Pandoraea norimbergensis, Pandoraea pulmonicula, Pandoraea sputorum and Pandoraea pnomenusa) plus two Pandoraea genomospecies isolates with lung epithelial cells and their ability to form biofilms in vitro. Only three isolates showed an ability to invade A549 lung epithelial cells, and only one isolate was able to form biofilms. In contrast, all isolates triggered a pronounced pro-inflammatory response, with elevation of both interleukin (IL)-6 (two- to 19-fold) and IL-8 (10- to 50-fold) above that observed for a control strain of Escherichia coli. This property is likely to be a major factor in the pathogenesis of the genus.

The effect of recombinant human lactoferrin on growth and the antibiotic susceptibility of the cystic fibrosis pathogen Burkholderia cepacia complex when cultured planktonically or as biofilms.

OBJECTIVES: The cystic fibrosis (CF) pathogen Burkholderia cepacia complex (Bcc) is innately resistant to antibiotics and the development of effective therapeutic strategies to treat these infections is a major challenge. The objectives of this study were to investigate the effects of recombinant human lactoferrin (rHL) on planktonic and biofilm cultures of Bcc organisms and to establish whether lactoferrin alters the susceptibility of these cultures to a range of antibiotic therapies. METHODS: Planktonic and biofilm cultures of strains representative of three species of Bcc, Burkholderia multivorans, Burkholderia cenocepacia and Burkholderia dolosa, were exposed to 0-900 mg/L lactoferrin over 0-48 h. Growth was determined using both spectrophotometric and plate counting methods. The ability of these strains to form and develop biofilms in vitro was also examined. Antimicrobial susceptibility in the presence/absence of lactoferrin was assessed using conventional MICs and a modified method was used to determine biofilm susceptibility to various antibiotics. RESULTS: We found that physiological concentrations of lactoferrin inhibited the growth of both planktonic and biofilm cultures of Bcc in vitro. The addition of lactoferrin to rifampicin enhanced the antibiotic susceptibility of the Bcc strains when grown planktonically and as biofilms. CONCLUSIONS: The present study demonstrates the growth inhibitory and antibiofilm activity of rHL against different species of Bcc. Furthermore, the enhanced susceptibility of both planktonic and biofilm cultures to rifampicin in the presence of lactoferrin offers the potential for novel uses of antibiotics in combination with lactoferrin to treat Bcc infections in CF patients.

Coagulopathy after cardiac surgery may be influenced by a functional plasminogen activator inhibitor polymorphism.

BACKGROUND: Cytokine-mediated inflammation and coagulopathy may occur after cardiac surgery. In this study we investigated the temporal pattern of plasminogen activator inhibitor-1 (PAI-1) gene expression after cardiac surgery and its relation with PAI genotype, and obtained preliminary data regarding its relation to perioperative morbidity. METHODS: The relative change in PAI-1 mRNA 1, 6, and 24 h after cardiopulmonary bypass (CPB) was measured from mononuclear cells in 82 patients undergoing elective cardiac surgery. DNA was analyzed for carriage of the 4G/5G PAI-1 polymorphism. RESULTS: PAI-1 gene expression decreased after CPB in all patients. A larger reduction in PAI-1 gene expression was observed in homozygous carriers of the 5G allele. Homozygous carriers of the 5G allele were also more likely to receive transfusion of coagulation blood products. There was no relation between change in PAI-1 gene expression and duration of CPB. CONCLUSIONS: PAI-1 gene expression decreased over time after CPB. We found a link between PAI-1 genotype, PAI gene expression, and transfusion of coagulation products after cardiac surgery.

Invasion and biofilm formation of Burkholderia dolosa is comparable with Burkholderia cenocepacia and Burkholderia multivorans.

BACKGROUND: Colonisation with Burkholderia cepacia complex pathogens has been associated with accelerated decline in cystic fibrosis (CF) patients. The two most common species among the CF community are Burkholderia cenocepacia and Burkholderia multivorans. However, Burkholderia dolosa has recently been causing concern due to its transmissibility and virulence in CF patients. METHODS: We have compared the ability of five B. dolosa strains to invade lung epithelial cells in vitro with other members of the Bcc. The bacterial epithelial cell interaction was visualised by transmission electron microscopy. We have also examined the ability of these strains to form biofilms in vitro. RESULTS: We have found that members of this species can invade pulmonary epithelial cells in vitro as readily as those from B. cenocepacia and B. multivorans. Confirmation of intracellular invasion was obtained by transmission electron microscopy. B. dolosa strains were readily observed in membrane bound vesicles inside the lung epithelial cells. In addition, strains from this species were capable of forming strong biofilms at a level comparable to the more clinically relevant species. CONCLUSIONS: B. dolosa shows comparable virulence characteristics in vitro to the two most clinically relevant species indicating precautions should be taken when this species is identified in the CF population.

Tumor necrosis factor-alpha and interleukin-10 gene expression in peripheral blood mononuclear cells after cardiac surgery.

OBJECTIVE: Cytokine response after cardiac surgery may be genetically influenced. A study was carried out to investigate the relation between cytokine gene expression in peripheral blood mononuclear cells, genotype, and clinical events after cardiac surgery. DESIGN: A case-control study was performed. SETTING: Cardiac intensive care unit in a university hospital. SUBJECTS: A total of 82 patients having elective cardiac surgery were divided into those having uncomplicated recovery (n = 48) or recovery complicated by hyperlactatemia or requirement for inotropic support (n = 34). INTERVENTIONS: The relative change in peripheral blood mononuclear cell tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) messenger RNA 1 and 6 hrs after cardiopulmonary bypass was compared with a baseline preoperative level using quantitative reverse transcriptase polymerase chain reaction. DNA was analyzed for carriage of TNF-alpha and IL-10 polymorphic alleles. MEASUREMENTS AND MAIN RESULTS: Cardiopulmonary bypass was longer in duration in the complicated group. TNF-alpha gene expression decreased and IL-10 gene expression increased in peripheral blood mononuclear cells after surgery when compared with preoperative levels. One hour after cardiopulmonary bypass, the complicated group had more TNF-alpha and less IL-10 messenger RNA production than the uncomplicated group. The IL-10/TNF-alpha ratio was greater in uncomplicated than in complicated recovery patients. An IL-10 haplotype was identified that was less frequent in the complicated group. There was no difference between groups in TNF-alpha genotype. On multivariate analysis, cardiopulmonary bypass time and the IL-10/TNF-alpha messenger RNA ratio were independent predictors of outcome. CONCLUSIONS: There is a predominant anti-inflammatory cytokine response after uneventful cardiac surgery. IL-10 may have a protective role after cardiac surgery.