Pseudomonas aeruginosa: Host defence in lung diseases

Authors

  • Bryan J. WILLIAMS,

    Corresponding author
    1. Pulmonary, Allergy, Critical Care and Sleep Medicine, University of Minnesota, Minneapolis, Minnesota
      Bryan J. Williams, Pulmonary, Allergy, Critical Care and Sleep Medicine, University of Minnesota, 420 Delaware St. SE MMC 276, Minneapolis, MN 55455, USA. Email: bjwillia@umn.edu
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  • Joanne DEHNBOSTEL,

    1. Pulmonary, Allergy, Critical Care and Sleep Medicine, University of Minnesota, Minneapolis, Minnesota
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  • Timothy S. BLACKWELL

    1. Division of Allergy, Pulmonary, Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA
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  • The Authors: Bryan J. Williams MD PhD is an assistant professor of pulmonary and critical care medicine at the University of Minnesota in Minneapolis, Minnesota. Joanne Dehnbostel BS is a candidate for the Masters in Science in Immunology at the University of Minnesota. Timothy S. Blackwell MD is a professor and chair of the Division of Allergy, Pulmonary, and Critical Care Medicine at Vanderbilt University in Nashville, Tennessee.

Bryan J. Williams, Pulmonary, Allergy, Critical Care and Sleep Medicine, University of Minnesota, 420 Delaware St. SE MMC 276, Minneapolis, MN 55455, USA. Email: bjwillia@umn.edu

ABSTRACT

Lung infections caused by the opportunistic pathogen Pseudomonas aeruginosa can present as a spectrum of clinical entities from a rapidly fatal pneumonia in a neutropenic patient to a multi-decade bronchitis in patients with cystic fibrosis. P. aeruginosa is ubiquitous in our environment, and one of the most versatile pathogens studied, capable of infecting a number of diverse life forms and surviving harsh environmental factors. It is also able to quickly adapt to new environments, including the lung, where it orchestrates virulence factors to acquire necessary nutrients, and if necessary, turn them off to prevent immune recognition. Despite these capabilities, P. aeruginosa rarely infects healthy human lungs. This is secondary to a highly evolved host defence mechanism that efficiently removes inhaled or aspirated pseudomonads. Many arms of the respiratory host defence have been elucidated using P. aeruginosa as a model pathogen. Human infections with P. aeruginosa have demonstrated the importance of the mechanical barrier functions including mucus clearance, and the innate immune system, including the critical role of the neutrophilic response. As more models of persistent or biofilm P. aeruginosa infections are developed, the role of the adaptive immune response will likely become more evident. Understanding the pathogenesis of P. aeruginosa, and the respiratory host defence response to it has, and will continue to, lead to novel therapeutic strategies to help patients.

INTRODUCTION

Pseudomonas aeruginosa is a common bacterium found in waterborne environments throughout nature.1 It has a large genome containing a myriad of pathogenic and metabolic capabilities allowing it to infect many organisms, including plants, amoebas, nematodes and vertebrate animals.2 Given its pathogenic potential, it is perhaps surprising that human infections from this organism are not more commonplace. Lung infections caused by P. aeruginosa are limited to patients who are immunocompromised, or who have defective mucociliary clearance, previous epithelial injury or foreign body placement. Given its ubiquitous presence in our environment and pathogenic potential, it is clear that a normally functioning host defence is very well adapted to prevent P. aeruginosa infection. Despite this, P. aeruginosa infections can be devastating in the hospitalized or sick. Understanding the failures of the host defence in these patients will help us understand how P. aeruginosa is converted from a common environmental exposure to a deadly pathogen.

PSEUDOMONAS AERUGINOSA RESPIRATORY INFECTIONS

Pseudomonas aeruginosa rarely infects the human lung without an underlying defect in immunity or mechanical barrier. Clinically, P. aeruginosa infections are frequently classified into ‘acute’ and ‘chronic’ infections although the distinctions between these groups are not always clear. New acquisition of P. aeruginosa results in symptomology that prompts an evaluation and its discovery in the lungs as in ventilator-associated pneumonia (VAP) and community-acquired pneumonia (CAP). However, in some diseases such as cystic fibrosis (CF), P. aeruginosa may colonize the lung with little new clinical evidence of disease.3 In this group of patients, ‘exacerbations’ of these chronic infections may clinically resemble a new, acute infection. In the absence of preceding culture data these chronic infections may be mistakenly classified as acute infections. We prefer to categorize the respiratory infections of P. aeruginosa into ‘transient’ and ‘persistent’ to emphasize the importance of microbiological status over one of symptoms. Patients with a mild lung injury following trauma may experience a low-grade infection with P. aeruginosa that can be eradicated with antibiotics and tissue healing.4 Patients with CF may harbour P. aeruginosa for 20 or more years during which time the bacteria adapts to persist in the airways.5,6 The broad range of clinical manifestations of P. aeruginosa lung infections can be attributed to two broad categories: (i) the high pathogenicity and adaptability of P. aeruginosa; and (ii) the magnitude and chronicity of the underlying host defect. In the following sections we will describe these respiratory infections, the features of P. aeruginosa that make it a powerful opportune, and the host defences that are responsible for the prevention of infection in healthy individuals.

Transient lung infections

Nosocomial pneumonias

Pseudomonas aeruginosa is one of the leading causes of nosocomial infections throughout the world.7,8 These infections have been given names such as hospital-acquired pneumonia (HAP), VAP, health care-associated pneumonia (HCAP) and ventilator-associated tracheobronchitis.9,10 By definition, these diseases all require the development of the infection at least 2 days after an inpatient admission to distinguish them from CAP.11 Nosocomial respiratory infections are typically much more lethal than CAP and are caused by a unique class of microbes that rarely cause CAP. P. aeruginosa is the most common Gram-negative organism isolated in nosocomial respiratory infections,12 although wide variations exist between health-care institutions. P. aeruginosa nosocomial pneumonias are associated with a higher mortality than other organisms13 due to its predilection to infect those with the most comorbidities.14 The outcome of these infections depends on the degree host defence impairment,4 unsuccessful early treatment attempts15 and the virulence of the organism.16 The high rate of P. aeruginosa acquisition in these patients is associated with prior use of broad spectrum antibiotics,17 a high carriage rate of multi-drug-resistant P. aeruginosa in hospital wards,18 and varying degrees of lung injury or immune compromise in hospitalized patients.

Of the nosocomial pneumonia subcategories, VAP carries the highest mortality, which is reported to be as high as 30% in some institutions.19 Even after attempts to adjust for comorbidities, the presence of P. aeruginosa in patients with VAP portends to a poorer prognosis.13 While dysfunctions in respiratory clearance and transient immunologic impairment may be to blame in cases of HAP and HCAP, patients with VAP also suffer endotracheal tube-induced epithelial injury4 and possibly ventilator-induced stretch injury.20 The endotracheal tube can serve as a reservoir for P. aeruginosa, which may grow as a biofilm on the plastic surface.21 Management of respiratory secretions is difficult in intubated patients, who depend on hospital personnel (a major source of P. aeruginosa carriage22) to maintain the ventilator circuits. Given the increasing antibiotic resistance profiles of P. aeruginosa in the hospital setting, guidelines have been established to initiate broad anti-pseudomonal therapy in any case of suspected VAP.11 While this strategy has been effective at improving patient outcomes, fatalities associated with VAP continue to occur in cases where the isolate of P. aeruginosa should have responded to the antibiotics administered based on sensitivity testing.13 The recurrence of P. aeruginosa and failure of appropriate antibiotic therapy both suggest that P. aeruginosa may rapidly evolve to a ‘biofilm’ state associated with persistent infection. Antibiotics may be useful in ‘holding off’P. aeruginosa until the normal host defence mechanisms can recover, including removal of the endotracheal tube, which is the most important therapy for VAP.23

Hospital-acquired pneumonia and HCAP are also caused by P. aeruginosa, at a lower frequency than VAP.24 Patients who develop HAP and HCAP tend to be older with multiple chronic illnesses, requiring frequent hospitalization.25 The oropharyngeal flora of patients has been shown to rapidly change upon hospitalization from mostly viridians streptococci, Haemophilus species and anaerobes to Gram-negative bacilli and Staphylococcus aureus.26 Suggested mechanisms of HAP pathogenesis include microaspiration of oropharyngeal flora, mucosal immune impairment or damage from underlying conditions (diabetes, chemotherapy, COPD), and inability to clear secretions with an effective cough.27 As with VAP, resolution typically requires appropriate antibiotics and improvement in the underlying comorbidities that resulted in hospitalization.

Immunodeficiency

A hallmark of pseudomonal lung infections is a vigorous neutrophilic response. In neutropenic patients, typically individuals undergoing chemotherapy, P. aeruginosa is capable of infecting a number of sites including the lungs.28P. aeruginosa infections in neutropenic patients were nearly always fatal prior to the development of anti-pseudomonal therapies including carbenicillins, gentamicin and carbapenems.28–30 As these patients are frequently hospitalized, they are exposed to the same reservoirs and risk factors for P. aeruginosa acquisition as patients with HAP; however, many cases are attributed to a community acquisition.28,31 The use of anti-pseudomonal therapies in patients with febrile neutropenia is typically successful at achieving eradication. Failures are most common in those whose neutrophil counts do not increase during the time of their infection28 or require intubation due to respiratory distress.32

HIV patients are also susceptible to infections from P. aeruginosa. These patients have much higher rates of CAP than the standard population and P. aeruginosa is found more frequently in HIV-related CAP than non-HIV-related CAP.33 HIV patients with P. aeruginosa pneumonia are also more likely to become bacteraemic than the general population.34,35 Some patients with chronically decreased CD4 lymphocyte counts may develop recurrent P. aeruginosa infections that resemble those found in CF.36 Domingo and colleagues have observed complete remissions of these persistent P. aeruginosa infections upon initiation of effective HIV therapy and reconstitution of the immune system.37

Community-acquired pneumonia/COPD

Community-acquired pneumonia is rarely caused by P. aeruginosa;38 however, some regions have higher incidences than others.39,40 When P. aeruginosa is isolated from patients in the community, it is almost always in elderly patients with comorbid illnesses33,39,41 especially COPD.42 This is highlighted by an increased incidence of P. aeruginosa infections in nursing home patients.41 Case reports of CAP in healthy individuals exist; however, almost all of these patients were also smokers.43

Patients with COPD represent a significant portion of the patients hospitalized with CAP.44,45P. aeruginosa is isolated from the sputum of 4%–15%46–48 of adults with COPD and is found more commonly in patients with severe disease, those requiring chronic systemic steroids and frequent antibiotic use.49 Clinical manifestations of P. aeruginosa in COPD range from a mild bronchitis to CAP with sepsis. This range of clinical syndromes is exemplified by the heterogeneous pattern of infection with P. aeruginosa that blurs the lines between transient and persistent infections.50 In an analysis by Murphy et al., the authors found that about half of the P. aeruginosa-positive patients were able to clear this organism from their sputum, but half of those took longer than 2 months to do so.50 In those with persistent infections, the P. aeruginosa isolates transformed to the mucoid phenotype and symptomatic exacerbations were caused by relapses of the same organism.

The transient P. aeruginosa infections listed above all demonstrate important key similarities. Most of these patients have comorbidities and are exposed to health-care institutions. Those without permanent lung compromise or severe immunodeficiency acquire P. aeruginosa in the setting of another acute illness. Eradication of P. aeruginosa requires immune reconstitution or improvement in airway clearance mechanisms. Those unable to restore the underlying defect in P. aeruginosa defence either succumb to their illness develop persistent infections.

Persistent lung infections

Cystic fibrosis

Cystic fibrosis is the most common lethal genetic disease in the Caucasian population with a worldwide prevalence of around 30 000 affected individuals.51 The hallmark of CF is progressive lung deterioration with chronic infections and inflammation, ultimately leading to cystic bronchiectasis, severe airflow obstruction and death. The underlying genetic defect lies in the cystic fibrosis transmembrane regulator (CFTR), a cAMP-dependent chloride channel.52 The absence of functional CFTR results in thickened airway surface liquid (ASL) that hinders the mucociliary elevator of the bronchial lining, which is designed to transport inhaled particles and organisms out of the airway.53 With time, bacteria become permanent residents of the normally sterile lower airways. P. aeruginosa infects most adults with CF and has clearly been linked to a more rapid progression of lung disease.54 This connection between CF and P. aeruginosa is so strong that CF organizations and foundations throughout the world have been responsible for funding and advancing a large portion of the research into P. aeruginosa pathogenesis and host defence.55

Pseudomonas aeruginosa infects infants with CF, and becomes a persistent pathogen found in the sputum of patients in their teens. When more sensitive culturing is performed in children by BAL, evidence of P. aeruginosa infections exists in most children by the age of 3.3 Relatively recent evidence has shown P. aeruginosa can grow as a biofilm56 and anaerobically in the CF lung,57 which has suggested a need for new clinical methods of organism detection as well as antimicrobial resistance testing.58,59P. aeruginosa's persistence in the airways is due to its ability to form a biofilm,56 which is a tightly associated colony of bacteria encased in a polysaccharide or polypeptide mesh of bacterial origin.60 This mode of growth resists the actions of both the inflammatory response and antibiotics. Many authors have speculated that the persistent P. aeruginosa infection drives the intense, but ineffective, neutrophilic response that probably creates most of the damage to the airways in CF lungs.53

Non-cystic fibrosis bronchiectasis

Although much more common than CF, bronchiectasis due to other causes has received far less attention.61,62 Non-CF bronchiectasis has a large array of aetiologies but is most commonly associated with a post-infectious sequelae of childhood pneumonia that permanently damages and dilates the lower airways.63 Bronchiectatic damage is frequently isolated to a particular lobe, in the cases of a prior lobar pneumonia, and does not progress to other lobes. These airways may become persistently infected with organisms typical for CF, including P. aeruginosa. Most of these patients do not have recognized defects in their immune systems and thus the host impairment is mostly one of mechanical clearance of the affected airways.

Rarer forms of bronchiectasis can affect the entire lung. Primary ciliary dyskinesia (PCD) also impairs the mucociliary elevator preventing mucus transport out of the distal airways into the central airways.64 These patients display many similarities to patients with CF, including persistent infection with P. aeruginosa. PCD research has not identified an intrinsic hyperresponsive inflammatory state as described in CF, although sputum studies suggest cytokine profiles are at least as high as they are in CF.65 The ASL in PCD appears to be preserved, and while the mucociliary elevator is impaired, intermittent coughing may provide enough mucus elevation to prevent stagnation and infection with inhaled organisms.66 This may explain why patients with PCD typically progress slower and live longer than patients with CF.

Prior to the widespread use of macrolides, 22% of patients with diffuse panbronchiolitis presented with P. aeruginosa infections of their lower airways and within 4 years of diagnosis this increased to 60%.67,68 Left untreated, most patients developed bronchiectasis, resulting in a 10-year survival rate of 33.2%.69 In these patients, survival was markedly worse for those with P. aeruginosa than those without.70 The use of macrolide antibiotics has drastically changed the outcome of this disease with >90% of patients surviving 10 years from diagnosis.69 On macrolide therapy some patients with P. aeruginosa at diagnosis become culture negative,71 although the mortality benefit is seen whether or not P. aeruginosa is eradicated.72 The mechanism of action of the macrolides remains a mystery but it is clear that its effects are not simply antibacterial as macrolides are typically not bactericidal to P. aeruginosa73 and appear to work regardless of bacterial clearance. Macrolides restore host defence to a point where maintenance or even removal of persistent infection with P. aeruginosa is possible.74

Bronchiolitis obliterans syndrome

Three separate studies have documented that the airways of lung transplant recipients are colonized with P. aeruginosa up to 43% of the time, and that 75%–88% of those patients were transplanted for CF.75–77 A study by Botha et al. also showed that patients with newly acquired P. aeruginosa infections were much more likely to develop bronchiolitis obliterans syndrome, also referred to as chronic lung rejection.76 Another study by Vos et al. came to a similar conclusion but also determined persistent P. aeruginosa infection was associated with more bronchiolitis obliterans syndrome development.75,78P. aeruginosa allograft infections in CF patients likely arise from their upper airways as the same isolates have been found in the lower airways before and after transplant.79 A unique aspect of this disease is the drastically different impairments in host defence that occur before and after transplant. To maintain a transplanted lung, a number of strong immunosuppresants targeted at cell-mediated immunity are used to prevent rejection. While cell-mediated immunity has been shown to be important in P. aeruginosa lung defence,80,81 the vast majority of the research field has focused on innate and neutrophil-mediated immunity. Furthermore, the host defence in transplanted lungs is a hybrid system with the epithelial and mucous secreting cells from the donor (below the airway anastomoses) and the leukocytes from the recipient. The CFTR channel has been directly implicated as a pseudomonal receptor82,83 and is responsible for phagolysosome acidification in macrophages.84 The persistence of P. aeruginosa in transplanted lungs suggests that the altered inflammatory response in CFTR bearing leukocytes may be an important mediator in the pathophysiology of CF before and after transplant.

PATHOGENESIS OF PSEUDOMONAS AERUGINOSA

Pseudomonas aeruginosa may be the most versatile bacterial pathogen studied to date. It is a natural pathogen to plants and protozoa, and an opportunistic pathogen of mammals. To achieve such a broad host range it must survive extremes of environmental exposure such as temperature and pH fluctuations, nutrient limitations and antimicrobial assaults from soil microorganisms or patient prescriptions. P. aeruginosa must harbour an extraordinary complement of genes to encode for virulence traits, broad ranging nutrient acquisition pathways and an arsenal of defence mechanisms. It contains one of the largest genomes of any bacterium yet sequenced.85,86 A key feature to its environmental sustainability is its ability to form a biofilm. While this mode of growth may have evolved to attach pseudomonads to environmental substrates, such as rocks in a stream, it is clear that it affords P. aeruginosa a distinct survival advantage in the diseased or damaged lung. This same organism is also capable of epithelial invasion and dissemination causing bacteraemia, sepsis and rapid fatality in some patients. Unravelling the diverse pathogenic potential of P. aeruginosa underscores the immense task of respiratory host defence to keep common environmental pathogens at bay in healthy humans.

Airways adaptation/biofilm formation

Pseudomonas aeruginosa expresses a wide array of virulence traits that are powerful manipulators and destroyers of host cells; however, it is clear that P. aeruginosa does not express all of these at all times. Probably the most important aspect of P. aeruginosa's versatility is its ability to quickly adapt to a new environment, and then accumulate traits/mutations that allow it to persist.87 The waterborne environment on a sink faucet and the airway epithelial lining represent very different challenges to a microorganism, and the ability to change its transcriptional profile rapidly to avoid being killed by host defences requires a complex network of environmental sensors. Cues such as temperature, nutrients, pH and iron all trigger a cascade of events to equip P. aeruginosa to survive in the airway. It is also clear that once in the airway, the adaptations continue as P. aeruginosa infection transitions to one of persistence.6

Two models of infection establishment are used to account for the different clinical manifestations of initial infection and the bacterial physiology observed in P. aeruginosa respiratory infections (Fig. 1). Lau et al. have written an excellent review on these models and the host modulatory properties of P. aeruginosa upon initial infection.88 The first model describes the classic ‘acute’ infections seen in the ICU where patients may become rapidly ill from pseudomonal pneumonia and sepsis. In this model planktonic organisms expressing surface-bound fimbriae and pili can attach to a cellular surface. Upon binding, toxin secretion damages the host cell lining, and modulates the mucosal immunity. Some P. aeruginosa may transverse the epithelial barrier and enter the blood stream. Others begin to secrete an extracellular matrix and begin formation of a biofilm containing a macrocolony of organisms. In the biofilm, the bacteria are resistant to the effects of antibiotics and phagocytosis from neutrophils. Individual cells may break free from the macrocolony in an attempt to spread to other areas of the lung. As planktonic (free living, non-biofilm) cells typically express many more immunogenic traits, these cells are vulnerable to host defences. Those bacteria remaining in the biofilm continue to propagate and are metabolically active. This biofilm colony serves as a reservoir that may never be eradicated if the normal host defences are not restored. It is not clear how long it takes P. aeruginosa to establish a biofilm in the airway; however, in vitro models suggest formation occurs immediately upon successful binding to a surface.89 In VAP, a biofilm can clearly be established on the endotracheal tube21 for which there is no real immunologic defence; thus the best therapy for VAP remains removal of the endotracheal tube.23

Figure 1.

Models of Pseudomonas aeruginosa infection establishment. In (a) P. aeruginosa is equipped with a full arsenal of virulence traits including pili, flagella, type 3 secretion systems (T3SS) and secreted virulence factors. Epithelial cell binding occurs via flagella and pili to various structures including asialoGM1. Toxin elaboration injures the surrounding host tissue. The significance of cytosolic invasion during human infections is not clear, but breach of the epithelial surface is thought to occur after cell death from toxin injury. Epithelial injury also results in loss of mechanical clearance mechanisms and establishment of the pseudomonal biofilm leading to a persistent infection. In (b) P. aeruginosa infects an already inflamed surface with a defective mucociliary elevator. The infecting organisms may or may not be piliated, and may already exist in a ‘biofilm’ state if acquired from another patient. The infection occurs strictly in the mucous layer where ‘nests’ of pseudomonads bind to cell debris and extracellular DNA rather than the epithelial surface. The pseudomonal microcolonies are a strong inflammatory stimulus, but are resistant to the actions of neutrophils, which may injure surrounding tissue in their efforts to remove the stimulus. Neutrophil death may provide more substrate for pseudomonal growth and more injurious toxins effecting surrounding tissue.

The second model of infection describes the more subtle acquisition of strains into an already diseased lung that may or may not result in much symptomology.88 In the CF lung, P. aeruginosa are almost exclusively found in the thick, airway mucus, and not in contact with cell surfaces, and not within the alveolar spaces (Fig. 2).90,91 In this model the particulates of purulent sputum acts as a surface for biofilm formation, either DNA strands or cell walls from lysed neutrophils, or thickened, dehydrated mucus.92P. aeruginosa can survive in this state for years and undergoes a number of adaptations for this environment including downregulation of most of the surface and secreted virulence traits described below and conversion to a mucoid phenotype dominated by the expression of alginate. Alginate is a copolymeric exopolysaccharide that is readily observed on plate grown P. aeruginosa when cultured from most CF patients.93 While not required for biofilm formation, alginate expression is rarely seen outside a persistently infected lung.94 Alginate has been shown to dampen the local immune response,93,95 supporting the lifestyle mode of persistence rather than destruction.

Figure 2.

Pseudomonas aeruginosa persistent infection of the lung. P. aeruginosa forms microcolonies enmeshed in biofilm within the sputum of patients with persistent infection. These microcolonies are surrounded by a dense neutrophilic infiltrate that does not penetrate into the protective biofilm layer. In patients with ‘stable infections’ the majority of the pseudomonal load is in the conducting airways and not in the alveolar spaces. (a–f) Intrabronchial specimens of a 41-year-old cystic fibrosis patient with a 28-year history of P. aeruginosa lung infection. (a) Gram. (b–c) HE. (d–f) Fluorescence in situ hybridization to P. aeruginosa (red) and DAPI (blue staining eukaryotic nuclei). (a–e) P. aeruginosa microcolonies surrounded by a dense neutrophilic infiltrate. (f) An occasional phagocytosed pseudomonad within a neutrophil. (g–i) FISH and DAPI stains of the alveolar contents of this same patient showing very little infection, but evidence of prior alveolar injury (h) and opsonization of planktonic P. aeruginosa (i). (Figure courtesy of T. Bjarnsholt and copyright permission obtained from Wiley and sons. This figure can be found in reference91 or at http://www3.interscience.wiley.com/journal/122373991/abstract).

Long-term adaptations in P. aeruginosa are likely brought about by an increased mutability of its genome while in the biofilm.6,96 These genomic changes include point mutations, gain and loss of genomic islands, genomic rearrangements and recombination events. The mechanisms underlying this hypermutability include oxidant-induced double-strand DNA breaks with aberrant repair and defective mismatch repair as in the hypermutable mutS strains.97,98 It has been suggested that these mutations reduce the overall virulence of these highly adapted strains. Although these strains are less able to cause pneumonia and bacteraemia in mouse models, they are more capable of establishing chronic infections.5 This finding explains the ease with which CF isolates of P. aeruginosa spread between patients with CF compared with environmental isolates.99

Surface-bound virulence determinants

Free living P. aeruginosa contain flagella and pili, which are required for motility and attachment to surfaces. These appendages not only aid in attachment to inanimate surfaces found in nature, but also to respiratory epithelium, specifically to respiratory mucins and asialoGM1, a glycolipid found enriched in repairing epithelium.100 As mentioned below, the binding and recognition of these appendages to certain target molecules such as Toll-like receptor 5 (TLR5) can induce a strong pro-inflammatory response.101 To counter this response, P. aeruginosa quickly downregulates the production of flagella and pili shortly after introduction into the lung. Flagellar downregulation occurs with biofilm formation as the bacterium shifts from a free living organism to a surface-bound organism.102

Most Gram-negative endotoxins (LPS) are potent immune stimulants through their interactions with TLR4.103 LPS isolated from P. aeruginosa clinical strains has been shown to possess immunogenic potential, but is less potent than LPS from other Gram-negative bacteria, including Escherichia coli.104 During the evolution of strains from acute to chronic, the lipid A moiety changes its acylation pattern to one of less immunogenicity and weaker stimulation through TLR4.105,106 Interestingly, work by Pier et al. has shown that the outer core oligosaccharide of P. aeruginosa LPS can be recognized by CFTR and this binding initiates ingestion of the organism into epithelial cells.82 This mechanism may be a factor in explaining the susceptibility of CF patients to P. aeruginosa infection; however, it is unclear that P. aeruginosa interacts with the epithelial surface in established CF infections.107

Secreted virulence determinants

The type 3 secretion system of P. aeruginosa is a well-described mechanism to ‘inject’ cytotoxins into the cytoplasm of a host cell. A complex machinery of proteins forms on the pseudomonal cell surface when attached to a host membrane, forming a small channel through which toxins can be injected. Hauser et al. have recently written an excellent review of this subject.108 There are four proteins injected through this apparatus named ExoS, ExoT, ExoU and ExoY. ExoS and ExoT are both GTPase activating proteins that can target host molecules responsible for cell division and cytoskeleton function. Upon injection of ExoS into host cells, there is an irreversible disruption of the cytoskeletal structure and the cells lose adherence with neighbouring cells, ‘round up’ and die. This type of cell death is similar to apoptosis except that membrane integrity is not as well preserved.109 ExoT has similar effects on host cells but has not been found to be as important in pathogenesis of P. aeruginosa models of infection as ExoS or ExoU.110 ExoU is powerful phospholipase with a broad range of targets including phospholipids, lysophospholipids and neutral lipids.111,112 One to two hours after injection into the host cell, ExoU results in loss of plasma membrane integrity and cell death. ExoY is an adenylyl cyclase that results in an increase in host cell cAMP113 upon injection, disruption of the actin cytoskeleton and inhibition of bacterial uptake by host cells.114 Its role in the pathogenesis of P. aeruginosa has not yet been established.110 Unlike many presumed pathogenic factors, the type 3 secretion system has been shown to be active in human disease and linked to mortality in acutely ill patients.16 As P. aeruginosa forms a persistent infection, the type 3 secretion system is turned off to prevent local injury and inflammation.110,115

Quorum sensing is a mechanism by which bacteria coordinate genetic transcription events among each other using a secreted ‘pheromone’ regulator. There are three overlapping quorum sensing regulators in P. aeruginosa named RhlR, LasR and PQS.116,117 Together these systems may control over 300 genes that presumably work better when multiple bacteria are expressing them at the same time.118,119 Biofilm and toxin production,120–122 pigment production123 and antimicrobial resistance124 have all been linked to quorum sensing control. The pseudomonal quorum sensing pheromones have been detected in the sputum of intubated patients and patients with CF and are thus presumably active in clinical infections.125,126 One proposed benefit of the macrolides in patients with persistent pseudomonal infections is their ability to inhibit the quorum sensing network.127 The LasR quorum sensing pheromone, N-(3-oxododecanoyl)-L-homoserine lactone, can directly interact with host cells inducing apoptosis in neutrophils and stimulating IL-8 production in bronchial epithelial cells.128 This molecule can be inactivated in human airways by host enzymes in the paraoxonase family,129 and pseudomonads from CF patients typically mutate LasR during the course of a persistent infection implying the effects may be minimal in persistent infections.130 However, the RhlR pheromone, N-butyryl-L-homoserine lactone, does not interact with host cells and is apparently not inactivated in human airways, suggesting it may be the dominant quorum sensing molecule in persistent human infections. LasA and LasB are proteases that are regulated by the LasR quorum sensing network. These enzymes combine activities to produce an elastolytic activity that can destroy extracellular matrix.131 LasA has also been implicated in enhancing syndecan-1 shedding, which is normally shed during tissue damage.132 Syndecan-1-deficient mice are resistant to P. aeruginosa infection in the lungs,133 which suggests that syndecan-1 shedding enhances P. aeruginosa virulence possibly by neutralizing cationic antimicrobial peptides (discussed below).

Phenazines (i.e. pyocyanin) are pigmented bacterial metabolites thought to have evolved for microbial competition and virulence.134 The classic blue-green pigmentation of pseudomonal growth can be partially attributed to phenazine production, which is liberally made in the sputum of patients infected with P. aeruginosa. The phenazines exert a number of effects on host cells including direct damage, altered cytokine production, ciliary motion inhibition and interrupted cell signalling pathways.135,136 Mutants of phenazine production are also less virulent in animal models.134

Exotoxin A is a secreted virulence factor that inhibits protein synthesis resulting in apoptosis, superoxide production and mitochondrial dysfunction.137 It enters cells by endocytosis after binding a specific mammalian receptor.138 It is found in CF sputum and its level is higher during periods of ‘exacerbation’ and lower during periods of relative stability.139

Pseudomonas aeruginosa secretes a number of non-specific proteases and phospholipases, which have activity to the cell surfaces and intracellular matrices of a broad range of hosts. A vast array of cytotoxic effects may occur in vitro but the role these compounds play during human infections is unclear. The phospholipases use phosphatidylcholine as a primary substrate, which is abundant in human airway surfactant.140 A class of the proteases, elastases, are capable of cleaving Ig, surfactant proteins A and D, and disrupting epithelial tight junctions.141,142 Rhamnolipids are biosurfactants secreted by P. aeruginosa that induce ciliostasis of airway epithelial cells and may disrupt their barrier function allowing pseudomonal invasion.143 These compounds are also found in the sputum of patients with CF suggesting they play a role in human infection.144

HOST RESPONSE TO PSEUDOMONAS AERUGINOSA

The healthy human respiratory tract is typically devoid of microorganisms below the main carina. Infections of the lower airways and alveolar spaces are associated with an intense, and potentially fatal, inflammatory response generated to remove the offending organism. The evolutionary necessity to keep organisms out of the alveolar space may be one of improved gas exchange, or prevention of easy bacterial or viral dissemination into the blood stream. Bacterial infections of the respiratory tract can be subdivided into two categories: those due to highly virulent bacteria that penetrate a normal host defence and those due to frequently encountered environmental bacteria that are opportunes of an impaired host defence. As described above, P. aeruginosa clearly falls into this second category and understanding these failures of the host defence may provide effective therapeutic targets.

Most of the critical antibacterial properties of respiratory host defence are applicable to multiple species of microorganisms, and are not specific to P. aeruginosa (Fig. 3). However, P. aeruginosa has served as an excellent model pathogen in many investigations as it is an important clinical pathogen, is easy to propagate and isolate, and is readily mutated by standard molecular biology techniques. Much of our knowledge of the innate immune function of the respiratory tract has been generated using P. aeruginosa, or its components, as a stimulant. Compared with other classic respiratory infections, the role of the adaptive immune response to P. aeruginosa is less clearly understood as antibody-mediated ‘immunity’ does not appear to play a large role in prevention of subsequent infection in humans.

Figure 3.

Respiratory defence against Pseudomonas aeruginosa. The respiratory defence against P. aeruginosa requires many mechanical, innate and adaptive defence mechanisms to be in place. The first line of defence upon inhalation of P. aeruginosa is mechanical clearance via binding to mucins in the airway surface liquid (ASL) and clearance by mucociliary clearance. The ASL contains a myriad of anti-pseudomonal molecules that can detect, label or destroy pseudomonads they come in contact with. The epithelium may also apoptose upon ingestion of pseudomonads to prevent further dissemination. The respiratory epithelium is not only a mechanical barrier but a source of many of the microbicidal agents in the ASL. It also contains transmembrane pattern recognition receptors that can signal immune cells through multiple cytokine networks. A number of immune cells have been implicated in the respiratory defence against P. aeruginosa; however, the neutrophil is likely the most critical component in bacterial clearance and resolution of infection. T-helper cells of the three major subtypes have all been implicated in host defence; however, the role of Th17 cells, and their ability to regulate neutrophilic responses, remains relatively underexplored despite their potential usefulness.

A major factor limiting our understanding of the immune system's role in P. aeruginosa defence is the dearth of good animal models of persistent infection.88 Most animal models of P. aeruginosa have lung infections that last only a few days. In particular, in vivo biofilm formation has rarely been shown to occur as most models still study biofilms on plastic dishes or in media flow chambers. As the majority of persistent P. aeruginosa lung infections are probably due to persistence in a biofilm, understanding the defence against organisms within a biofilm will be critical to developing better therapies for these infections.

Mechanical defence mechanisms

Human disease has demonstrated how critical, and perhaps how underappreciated, the mechanical clearance functions of the respiratory tract are. These mechanisms include mucus and water secretion to create the ASL layer, the ciliary forces to provide the elevation of this layer up and out of the lung, and the cellular barrier that prevents entry of organisms into the subcellular space and bloodstream. These functions are responsible for clearing most of the inhaled pathogen load with each breath. In diseases such as CF and PCD, malfunction of the mucociliary elevator makes the lung susceptible to a number of inhaled microorganisms, especially P. aeruginosa.

The epithelial lining of the conducting zone is described as a pseudostratified columnar epithelium that is composed of a number of different morphologic cell types including squamous, ciliated, basal, serous, goblet, neuroepithelial and non-ciliated Clara cells.145 This lining is also studded with tracheal-bronchial glands, which produce mucus and other products of host defence to be discussed below. Most inhaled organisms are not equipped to penetrate the epithelial cell surface or invade between the cells, which are held together with tight junctions.146P. aeruginosa has been shown to enter type I alveolar cells in vitro but it is not clear if this is a mechanism of systemic invasion during acute pneumonia. The cytotoxic effects of P. aeruginosa are likely responsible for disrupting barrier function and allowing access to the bloodstream for dissemination. Despite this potential, systemic dissemination is rare in patients with persistent infection, likely due to a combination of host and pathogen factors. In persistent infections, P. aeruginosa evolve away from cytotoxicity (discussed above) and are probably contained to the conducting zone of the airways where the barrier functions may be more robust. Most of the equipment that P. aeruginosa uses to destroy host cells requires attachment, or at least remaining in one place long enough to secrete a sufficient concentration of toxic products. This reasoning highlights the role of the single greatest defence mechanism of the respiratory tract against P. aeruginosa—the mucociliary elevator.

An excellent review of mucus clearance mechanisms of the mammalian airways has been written by Knowles and Boucher.147 The ASL of the human airway is a complex mixture of water, ions and mucin macromolecules, which are complex glycosylated proteins with long oligosaccharide side chains. To function properly, mucin requires hydration to properly unfold its carbohydrate chains, which can bind to a vast assortment of charged particles including allergens, pollutants and pathogens. This hydrated mucous layer ‘floats’ on the periciliary liquid layer (PCL), which spans the height of the cilia. The PCL is devoid of the thicker, gel forming mucus, thus the viscosity of this layer is lower, allowing for effective ciliary beating at a frequency of 8–15 Hz. The cilia of the airway epithelial beat in a unidirectional manner via a ‘ratcheting’ motion that provides thrust in one direction and recoil and repositioning in the other direction. The effect of this motion is to elevate the floating mucous layer out of the distal airways and into the central airways where secretions are either elevated to the throat and swallowed or expectorated.

The mechanisms that control the hydration, mucus secretion and ciliary beating are starting to be unravelled, mainly through efforts to understand the CFTR chloride channel responsible for the CF phenotype. The epithelial sodium channel (ENaC) and the CFTR channel appear to be the main drivers of ion transport in the human lung responsible for hydration of the ASL and PCL.148 The main chemical mediators of these channels, as well as ciliary beating and mucin secretion, are probably extracellular nucleotides acting through purinergic receptors on the epithelial surface and in goblet cells.149 The environmental sensors that control the secretion of nucleotides are not well understood. As the control of these functions appears to have little input from the autonomous nervous system, intrinsic control sensors have been sought. A recent line of work demonstrated that airway cilia contain chemosensors, in particular, bitter taste receptors.150 When ciliated airway epithelial cells were exposed to bitter substances, their beat frequency increased to speed removal of potentially noxious substances.

Innate immune defence mechanisms

Innate immunity is the first line of defence once the mechanical barriers of the epithelial or endothelial membrane have been breached. The innate immune system can be described to include any effector mechanism that prevents infection without prior immunologic exposure. A critical component to an effective innate immune system is the ability to ‘recognize’ a stimulus as potentially pathogenic and react to it. Innate pattern recognition receptors (PRR) are encoded in the germ line DNA of the host and do not require genetic rearrangement to create a ‘memory’ of prior infection and do not produce a more robust response upon secondary infection by the same organism.

Pathogen recognition receptors

Surveillance by innate immune cells for pathogen-associated molecular patterns is accomplished by the use of PRR. The PRR can be divided into three categories: secreted, transmembrane and cytosolic.151

Secreted pattern recognition receptors.  The ASL contains a number of substances that have antibacterial properties. Some of these work as direct bacterial cytotoxins, some as signalling molecules and some thwart the efforts of the bacteria to acquire metabolites or bind to surfaces. These molecules are usually always present in the ASL as an intrinsic defence; however, some can be upregulated in the setting of immune stimulation.

Complement C1q, collectins and surfactant proteins A and D (SP-A, SP-D) form the c-type lectin super family bind to P. aeruginosa cell walls, especially those with ‘rough LPS’, marking them for clearance by alveolar macrophages and granulocytes in the lung.152 Mannose-binding lectin 2 (encoded by MBL2) binds D-mannose and N-acetyl-D-glucosamine on bacterial surfaces and triggers activation of serine proteases, the complement pathway and ultimately phagocytosis through opsonization. Human serum levels of MBL2 vary widely given high genetic variation of MBL2. This variation appears to influence the time to acquisition of P. aeruginosa in children with CF, those with low MBL2 acquire P. aeruginosa earlier than those with high MBL2.153 SP-A opsonizes P. aeruginosa, but also permeabilizes them, resulting in direct cytotoxicity. Pseudomonads with mutations in their flagellar apparatus are more susceptible to SP-A, but this effect is not mediated by motility but possibly an LPS factor augmented by the presence of the flagella.154

Defensins are short cationic peptides with characteristic beta sheets and disulfide bridges that exhibit antimicrobial activity via bacterial membrane permeabilization.155 Neutrophils contain α-defensins, also known as human neutrophil proteins, in their azurophilic granules as well in phagocytic vacuoles. Not only are they bactericidal but have been shown to be effective in the neutralization of bacterial toxins including pseudomonal exotoxin A.156 Four different β-defensins are found in human ASL and their transcription is upregulated via NF-κB-mediated mechanisms.157 CF patients with certain β-defensin-1 polymorphisms are more susceptible to P. aeruginosa colonization.158 The high salt concentration of CF ASL has also been shown to inactivate defensins, potentially explaining another relative immunodeficiency in this disease.159 PLUNC, or palate lung nasal epithelium clone, is a protein secreted by human respiratory epithelial cells that is related to the bactericidal/permeability protein and works as both a surfactant and a bactericidal agent. PLUNC was recently shown to exhibit anti-biofilm activity in an in vitro model.160

The complement system is a complex, modular set of molecules found in the ASL and serum. Three of the molecules along the pathway are the most significant in host defence against P. aeruginosa. C1q is implicated in marking apoptotic cells for ingestion by phagocytic cells in the lung.161 C3a and C5a are anaphylatoxins, which cause the release of granule proteins from mast cells, and granulocytes in response to pathogen stimuli. Elastase produced by P. aeruginosa can inactivate these anaphylatoxins.162

Transmembrane pattern recognition receptors.  The most familiar PRR are TLR.163 In pseudomonal defence, TLR 2, 4 and 5 recognize pilin, LPS and flagella, respectively. TLR are found on inflammatory cells in high concentrations and epithelial cells. Before binding to TLR4, LPS binds to LPS binding protein, which in turn allows it to bind to CD14 and TLR4. TLR function is critical to control P. aeruginosa infection in the mouse lung; however, TLR 2, 4 and 5 have overlapping functions. Removing TLR 2 and 4 from a mouse does not result in a lethal pseudomonal pneumonia unless the pseudomonads are devoid of flagella and the subsequent TLR5 response.164 CFTR defective airway epithelial cells show decreased expression of TLR4 and therefore decreased response to LPS from P. aeruginosa.165

The CFTR protein, defective in CF patients, is part of an epithelial cell PRR for LPS. Pseudomonal binding to the CFTR protein results in lipid raft formation and interaction with caveolin-1 and 4166 and allows internalization of the bacterium. This results in NF-κB activation and translocation via Myd88 and IL-1β/IL-1R dependent means,167 and apoptosis of the desquamated infected epithelial cells. Despite these in vitro findings, it is unclear if this phenomenon is responsible for the predilection of P. aeruginosa to infect CF lungs mostly devoid of CFTR as it does not appear to interact with the epithelial surface in this infection.91

MUC1 (Muc1 in non-primates) is a transmembrane glycoprotein that has been shown to bind P. aeruginosa and signal through the MAP kinase pathway. Overexpression of Muc1 in mice results in a more robust neutrophilic response to P. aeruginosa and a more rapid clearance of the bacterial burden.168

Cytosolic pattern recognition receptors.  In contrast to TLR 2, 4 and 5, TLR9 is located in the cytosol as a lysosomal transmembrane protein.169 Its ability to detect bacterial CpG DNA may also alert the host to the presence of P. aeruginosa. After phagocytosis of the bacterium and subsequent maturation of the lysosome, liberated CpG DNA from the bacterium binds TLR9, which signals through the familiar adapter protein MyD88, which has migrated to the surface of the endosome.170

NOD-1, short for nucleotide oligomerization domain, is a cytosolic PRR that binds gamma glutamyl diaminopimelic acid from Gram-negative bacteria, including P. aeruginosa.171 NOD-2 binds muramyl dipeptide from both Gram-negative and Gram-positive bacteria. These receptors are located in epithelial cells, macrophages and dendritic cells (DC). When activated, they recruit caspases and activate NF-κB in a similar fashion to the TLR. The NOD proteins have also recently been implicated in cellular autophagy of bacterial specific proteins, a mechanism to recycle ageing cellular proteins that may also be used to scour bacterial products from the cytosol.172

Role of specific cell types

Neutrophils.  Unless a patient is neutropenic, a neutrophilic response will be involved in every P. aeruginosa lung infection; and any chance at complete resolution will likely require this response. In both transient and persistent pseudomonal lung infections, a neutrophilic response is usually robust, creating a purulent secretion that is expectorated allowing sputum cultures to correctly identify P. aeruginosa as the causative agent. In persistent infections the neutrophilic response may be unremitting to the point of injuring surrounding tissues, as is proposed in CF.53 Mouse models of acute pseudomonal pneumonia in which neutrophils were depleted have shown excessive mortality.173,174 Neutropenic mice may succumb to as few as 100 colony-forming units, whereas healthy, wild-type mice may require doses of 107–108 colony-forming units for a similar effect.173 As the uninfected lung is nearly devoid of neutrophils in the airways, a robust system of cell recruitment is required. This occurs through the neutrophil chemokines for recruitment and the integrins for adhesion and extravasation through the vascular endothelium into the airspace. Both chemokine signalling (through IL-8 analogue depletion in mice) and the integrin CD11a(b)/18 have been shown to be critical for clearance of pseudomonal lung infections in mice.174,175 Many of the initial signals involved in neutrophil recruitment are generated through interactions with the PRR listed above. Some of the PRR, including the TLR, lead to upregulation of NF-κB, which drives IL-8 production and neutrophil recruitment. Other PRR (C3b, collectins, surfactant proteins) and Ig mark bacterial surfaces for opsonization by neutrophils and lead to their activation. The discovery of Th17 cells and the IL-17/IL-23 cytokine network has implicated a role for the adaptive immune response in neutrophil recruitment.176

Neutrophils generate a number of important microbicidal molecules including reactive oxygen species via production by NADPH oxidase, reactive nitrogen species via inducible nitric oxide synthase,177 elastases and antimicrobial peptides including α-defensin. Lactoferrin and lysozyme are two important neutrophilic products, but they are also secreted by human airway epithelium.178 Lactoferrin is both bactericidal and bacteriostatic to P. aeruginosa and has recently been shown to inhibit bacteria growing as a biofilm.179 Lactoferrin is a non-haem containing glycoprotein that efficiently scavenges iron; an essential element for pseudomonal proliferation. Lysozyme is a cationic polypeptide found in multiple bodily secretions from including tears, saliva and ASL.180 It is secreted by neutrophils and is an effective bacterial membrane disruptant, which hydrolyses peptidoglycan. Its importance in P. aeruginosa defence is highlighted by hypersusceptibility of lysozyme knockout mice in a model of acute pseudomonal pneumonia.181 A novel therapeutic agent, docosahexaenoic acid, has been shown to improve symptoms of CF perhaps due to its cooperative function with lysozyme to break down bacterial cell membranes in Pseudomonas.182

Epithelial cells.  As mentioned above, the respiratory epithelium plays a number of important roles in the host defence against P. aeruginosa including generation of a fairly impenetrable cell barrier and production of a number of antibacterial compounds in the ASL. Epithelial cells also contain surface-bound PRR including the TLR and produce a wide array of pro-inflammatory cytokines that alert components of both the innate and adaptive immune response.183 Many of these cytokines belong to the NF-κB family of cytokines including TNF-α, IL-1β, IL-2, IL-6, RANTES and MCP-1.184 NF-κB stimulation is a critical component to the immune response generated by respiratory epithelium. Mice with a doxycycline induced of expression of NF-κB in the respiratory epithelium are protected from P. aeruginosa infection when fed doxycycline. This is in contrast to mice with a doxycycline repressed NF-κB in the respiratory epithelium that are more susceptible to P. aeruginosa when fed doxycycline.185 These mouse models have also demonstrated that the mouse IL-8 equivalent, KC, is a critical downstream mediator of epithelial induced response to P. aeruginosa. Flagellated P. aeruginosa has been shown to interact with TLR5 on human airway epithelial cells to stimulate the NF-κB pathways.186P. aeruginosa stimulation of type II alveolar epithelial cells activates alveolar macrophages through MCP-1 secretion.187 Epithelial cells also interact with components of the adaptive immune response including DC, and T and B cells. IL-15 production by epithelial cells induces differentiation of DC from monocytes.188 Airway epithelial cells can induce migration of Th1 cells via production of the CXCR3 ligands and induce migration of Th2 cells via production of CCL1 and CCR4 ligands.189,190

Apoptosis can be used as a defence mechanism to remove cells that have been invaded by pathogens. Apoptosis induced by interaction with CD95 (Fas) and CD95 ligand was found to be critical to clear P. aeruginosa from mouse lungs.191P. aeruginosa invasion can induce formation of lipid rafts containing CD95 through the activation of acid sphingomyelinase.192 Non-invading P. aeruginosa (i.e. those in biofilms) do not produce such a response.193

Macrophages.  The resident leukocyte of the lung is the alveolar macrophage, which acts as a general scavenger of foreign particles and a sentinel for infectious agents. Activated macrophages are motile with pseudopodia that allow them to follow their ‘prey’. They have transmembrane PRR to recognize potential pathogens, as well as PRR receptors including Fc receptors, complement receptors and macrophage mannose receptor, which have been implicated in the phagocytosis of P. aeruginosa.194 Organisms recognized by these receptors are pulled into an endocytic vacuole or phagosome. Along with the epithelial cell, alveolar macrophages are the major initial source of cytokines and chemokines that result from P. aeruginosa infection including IL-1β, IL-6, IL-8 and TNF-α. There is some controversy as to whether these cells are dispensable in host defence against Pseudomonas. Depletion of alveolar macrophages in mice has resulted in conflicting results ranging from no change in survival or pseudomonal clearance,173,195 to altered neutrophil recruitment and deficient pseudomonal clearance.196 In a similar fashion, the role of alveolar macrophage phagocytic function has also been debated with separate models coming to opposite conclusions.197,198

Macrophages from CFTR-deficient mice display altered phagocytic function and inflammatory signalling properties. While CFTR-deficient macrophages retain the ability to phagocytose pseudomonads, the phagolysosomes are defective in acidification allowing the ingestants to survive.84 The degree of acidification also appears dependent on the severity of the CFTR mutation.199 CFTR macrophages stimulated with LPS produce a more vigorous pro-inflammatory response (increased NOS-2, IL-1β, CCL-2) and a weaker anti-inflammatory response (IL-10) than wild-type macrophages. Treatment with azithromycin blunted some of these pro-inflammatory effects in vitro lending support to its role as an anti-inflammatory in the use of CF and other chronic inflammatory lung conditions.200

Eosinophils.  While eosinophils are classically implicated in allergic diseases such as asthma and parasitic infections, a role for eosinophils in P. aeruginosa has recently been established. Eosinophils have multiple PRR for Gram-negative bacteria including TLR 2, 4, 5 and 9.201 Adoptive transfer of eosinophils was shown to improve clearance of pseudomonads in an eosinophilic mouse model by release of secondary granule proteins. Without eosinophils these mice have a greater bacterial burden following infection with P. aeruginosa.202

Dendritic cells.  Dendritic cells are the bridge between the innate and adaptive phases of the immune response. Antigens from pathogens like P. aeruginosa are collected non-specifically by DC, which are activated and can release cytokines locally or travel to lymph nodes where they present antigens to T and B cells to activate them. A model of secondary pseudomonal pneumonia in mice has shown that DC activated by an initial polymicrobial pneumonia may suppress or ‘overdampen’ the subsequent immune response to P. aeruginosa.203 Intratracheal instillation of bone marrow derived DC from a mouse that recently recovered from a polymicrobial pneumonia induced more lethality in a model of secondary P. aeruginosa pneumonia. The same DC preparation from mice without a prior infectious insult was protective in the same circumstances. This may explain the increased lethality of HAP from P. aeruginosa in patients recovering from sepsis or other infections.

Important molecules in host defence

Cytokines/chemokines.

TNF-α.  TNF-α is a major acute phase reactant that is produced by macrophages and epithelial cells in the lung in response to a number of infectious stimuli including P. aeruginosa. Several studies suggest TNF-α plays an important role in pseudomonal clearance. There is a wide array of pseudomonal susceptibility among the mouse backgrounds used in scientific research, and much of this variability has been attributed to their TNF-α response.204 Transgenic mice deficient in TNF-α production have difficulty clearing P. aeruginosa.205 TNF-α also appears to positively regulate Muc1 (discussed above) as TNF receptor knockout mice do not stimulate Muc1 in response to P. aeruginosa challenge as is seen in WT mice.206

IL-8.  IL-8 is a human CXC (CXCL8) chemokine that is mainly responsible for neutrophil migration. IL-8 (or its mouse analogues KC and MIP2) is rapidly released from epithelial cells and alveolar macrophages upon stimulation with P. aeruginosa namely through the function of TLR. KC and MIP2 signalling are critical for the respiratory defence of P. aeruginosa as mice made deficient in these responses have significant impairments in neutrophilic responses to the lungs, which greatly hinders bacterial clearance.174 CF patients with elevated sputum IL-8 tend to have inferior lung functions, suggesting IL-8 may be a driver of the ‘excessive’ inflammatory response seen in the CF lung.207

IL-1/IL-18.  The IL-1 superfamily of cytokines are produced by macrophages and tend to drive an IFN-γ/Th1-mediated response to infectious stimuli. The roles of IL-1 and IL-18 in P. aeruginosa lung infections appear to be more detrimental than beneficial in acute pneumonia models. Mice deficient in IL-1 receptor,208 or IL-18 production209 are both protected from P. aeruginosa when large inocula are use to create pneumonia. However, recent investigations by Reiniger et al. have shown IL-1 R knockout mice are more susceptible to a chronic pseudomonal pneumonia induced by a sustained low inoculum in drinking water.167 Studies such as this highlight the caution we should take in applying the results of a transient pneumonia model towards persistent human pneumonias that may develop in very different circumstances.

IL-17/IL-23.  The cytokine milieu not only plays a role in upregulation of and modulation of components of the innate immune system, but also modulates numerous components of the adaptive immune response. The T-cell subsets responsible for coordinating the adaptive immune response are often put in place by the cytokine profile elaborated during the initial infection by the first responders of lung infections, epithelial cells, macrophages and DC. The conventional paradigm describing the divergent Th1 and Th2 subsets has been stretched to incorporate new subsets including Th17, Th3 and Tregs. Th17 CD4+ cells are produced in the presence of IL-6 and TGF-β and are maintained by the presence of IL-23, which is produced by alveolar macrophages.176 Th17 cells recruit neutrophils to the site of an extracellular bacterial infection through the actions of IL-17. IL-17 and IL-23 have been shown to be elevated in the sputum of patients with CF.210,211 IL-23 was also found to be important for recruitment of neutrophils to the lung in a pseudomonal pneumonia model in IL-23 knockout mice.212 As the Th17 response is ‘pro-neutrophilic’ future studies will likely continue to show that P. aeruginosa infections, particularly persistent infections, stimulate this arm of the adaptive immune response.

IL-4.  IL-4 is a product of the Th2 lymphocyte population and augments the differentiation of CD4+ cells to the Th2 profile, which tends to favour B-cell stimulation and antibody responses. Patients with persistent pseudomonal infections tend to have elevated levels of IL-4 in their sputum and Th2 lymphocytes, along with robust antibody responses to P. aeruginosa. Peripheral blood mononuclear cells from patients with chronic P. aeruginosa infections tend to produce Th2 responses (as measured by IL-4/IFN-γ ratios) compared with similar patients without P. aeruginosa infection.213 Mice that overexpress IL-4 in their airways mount a more vigorous neutrophil response and clear P. aeruginosa earlier.214

IFN-γ.  Exogenous administration of IFN-γ enhances bacterial clearance in both acute and chronic models of P. aeruginosa.215 However, another study revealed mice deficient for IFN-γ receptors also had increased bacterial clearance attributed to increased NOS synthesis.216 These data suggest that the endogenous IFN-γ response is detrimental but exogenous IFN-γ may be beneficial. As in the IL-1 response, it is probable that the answers to these discrepancies lay in the timing and duration of the IFN-γ response in each of these models.

Reactive oxygen/nitrogen species.  The NADPH oxidase system within macrophages and neutrophils is responsible for generating reactive oxygen species within phagolysosomes to aid in destroying ingested bacteria. The vigorous inflammatory response to P. aeruginosa also results in spillage of extracellular ROS either through early death of inflammatory cells that spill their contents, or misdirected NADPH oxidase directly to plasma membrane where ROS could be generated extracellularly.217 One of the key components to NADPH oxidase is the p47phox gene product, which has been successfully removed in a mouse line. These mice were more susceptible to P. aeruginosa lung infections than their wild-type counterparts and this was attributed to altered NF-κB signalling.218

The nitric oxide system has a number of roles in inflammatory signalling and generation of reactive nitrogen species that are directly microbicidal to bacteria. Exogenous administration of inhaled nitric oxide to rats with pseudomonal pneumonia resulted in reduced bacterial loads and decreased neutrophil burden.219 In acute pneumonia models NO is elaborated as an acute phase reactant. In contrast, persistent infections with P. aeruginosa are associated with low levels of NOS activity, which can be pro-inflammatory. In an animal model of chronic pseudomonal pneumonia, inhibition of NOS caused greater injury and stimulation of NOS was protective.220 A similar finding is seen in CF patients as patients who are ill with an ‘exacerbation’ of their illness have lower exhaled nitric oxide that increases after treatment with antibiotics. This is shown to be related to activity of sputum arginase, which actively competes for the substrate of NOS.221

Adaptive immune defence mechanisms

The role of the adaptive immune system in defence against P. aeruginosa lung infections has not been clearly deduced. The majority of patients who become susceptible to P. aeruginosa have defects in their mechanical or innate immune defence mechanisms. Those with adaptive immunodeficiencies usually have some component of altered innate immunity, as the immunomodulation, or crosstalk, between the adaptive arm and innate arms is defective. In some cases, infections that result from deficiencies in adaptive immune function, such as pneumonia from encapsulated organisms, may result in enough lung injury (such as bronchiectasis) that mechanical defences are compromised allowing P. aeruginosa to get a foothold. It is clear patients with persistent P. aeruginosa infections generate high antibody titres to this organism; however, it is insufficient to remove them from the lung.222 It is likely that this vigorous antibody response is responsible for preventing systemic spread of P. aeruginosa as patients with persistent infection virtually never develop pseudomonal bacteraemia or sepsis.

Lymphocytes

T lymphocytes have a number of potential important roles in the setting of persistent P. aeruginosa infection; however, few studies have addressed these roles given the difficulty in creating animal models of persistent infection. Some mice strains are more susceptible to P. aeruginosa than others and some of this difference has been attributed to their tendencies to mount a Th1- or Th2-predominant response. Those that form Th1 responses, highlighted by IFN-γ production and cytotoxic T-cell generation, are more likely to clear P. aeruginosa pulmonary infections than those that form Th2 responses.80,223 Relatively recent advances in immunology have determined multiple subtypes of T cells exist including Th17, Tregs and NKT cells. The potential of Th17 cells to be involved in the P. aeruginosa response is discussed above under the IL-17/IL-23 section. Treg cells are responsible for immunosuppression, although their role in bacterial infections has been variable.224,225 The role of Treg cells has been evaluated by Carrigan et al.226 In their model of acute pseudomonal pneumonia depletion of Treg cells did not appear to have an effect on the immunologic response or the bacterial clearance. NKT cells, or CD1d T cells, are also immunoregulatory cells that respond to diverse lipid and glycolipid antigens, but make up a small subset of the total T-cell population. Their role in P. aeruginosa infections has produced conflicting results using two similar models of NKT knockout mice. Nieuwenhuis et al. determined that mice devoid of the CD1d had marked reductions in P. aeruginosa clearance.227 Kinjo et al.228 used mice deficient in a subtype of NKT cells, the Vα14+ lineage, and found no difference between wild type. As CD1d encompasses all NKT cells, it is likely that another subset of NKT cells are more important in the regulation of P. aeruginosa clearance.

Ig

The main Ig in the ASL is the secretory IgA molecule, which has been shown to develop to very high titres against P. aeruginosa in patients with persistent P. aeruginosa infection.229 It is clear that this response is not sufficient to prevent the development of a persistent infection, or acquisition of new strains of P. aeruginosa based on clinical evidence. However, patients with IgA deficiency may be at risk of disseminated pseudomonal infections.230 Secretory IgA has an anti-inflammatory effect during pseudomonal infection. The poly-immunoglobulin receptor in lung epithelial cells functions to transport secretory IgA into the lumen of the lung. Mice with a defective receptor (pIgRKO) are unable to produce secretory IgA in lung secretions. These mice have been shown to have a threefold increase in mortality from intratracheal infection with Pseudomonas.231

The role of Ig in defence against P. aeruginosa has also been addressed by development of vaccine targets. The pseudomonal flagellum is a potent immunogen that has been developed into a vaccine candidate that has recently been tested in phase III trials.232 Earlier trials demonstrated that pseudomonal flagella given as an intramuscular injection induced serum IgG and secretory IgA responses in healthy humans.233 The larger phase III study sought to immunize children without prior P. aeruginosa infection, and reduce the incidence of P. aeruginosa colonization. This study did demonstrate a decrease in P. aeruginosa infections in the vaccine group compared with the placebo arm, particularly in infections associated with the flagellar type used in the vaccine. This trial demonstrates that there may be a role for Ig in prevention of initial infection.

SUMMARY

Lung infections from P. aeruginosa are found throughout the world in hospitals with ICU patients, cancer patients, and those with debilitating lung diseases or immunodeficiences. The spectrum of symptoms varies widely and this is almost exclusively a feature of the underlying deficiency in the patient's host response, and not the virulence of the infecting P. aeruginosa. Understanding the microbiology of P. aeruginosa has also allowed for the development of infectious models that can replicate various aspects of host defence compromise. This scientific research has succeeded in explaining why some clinical therapies do and do not work, and has resulted in new therapeutic interventions to combat both transient and persistent infections. Our use of these therapies has resulted in improved patient outcomes such as diminished mortality in VAP and neutropenic fevers, to the extension of life in CF patients by decades. Despite these advances, large gaps in our knowledge of P. aeruginosa lung infection still exist. How do we prevent dissemination of P. aeruginosa in hospital ICU to our sickest patients? How does P. aeruginosa persist in the bronchiectatic lung despite seemingly effective antibiotic treatment? Can we augment the inflammatory response in persistent infections to become more effective or less injurious to surrounding tissues? Many of these questions will require continued development of complicated models of infection to be developed by researchers, in particular those requiring anaerobic and biofilm modes of P. aeruginosa growth. The development of transgenic animals with targeted immune modifications continues to reveal important data on the host response; however, most of these model systems are designed to study acute infections with planktonic pseudomonads. Combining these transgenic mouse models with mutant pseudomonads, or those grown as a biofilm, may result in new insights into persistent infections and the adaptive immune response that is sorely missing from the current literature.

Ancillary