Good Cf Article

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Our studies initially focused on the pathogenesis of established CF

airways infection and, taking clues from these studies, explored whether

these variables could uniquely contribute to the early pathogenesis of P.

aeruginosa infection in CF airways. Morphometric analyses of freshly

excised lungs by three techniques demonstrated that P. aeruginosa grows

as macrocolonies in the airway intraluminal rather than the epithelial

surface compartment (Figure 1, a–c). These findings contradict recent

hypotheses emanating from in vitro model systems that focus on

high-salt/defensin inactivation (26) or luminal epithelial cell binding

(4), which predict bacterial infection of CF airway epithelial cells

themselves (5, 6). However, our data are consistent with those from

animal models that have demonstrated the adherence of P. aeruginosa to

respiratory mucus (27-29), and three previous qualitative studies of CF

postmortem lungs that identified P. aeruginosa in airway lumens rather

than on airway epithelial cells (30-32). Furthermore, they are also

consistent with our studies of NSEs that revealed P. aeruginosa

preferentially bound to mucus rather than epithelial cell surfaces

(Figure 1, d–f). A key extension of the in vivo characterization of CF

airways infection is that P. aeruginosa occupies an intraluminal niche

that is markedly hypoxic (Figure 2, a–. If the CF airways disease

reflects infection of mucus, how is this process initiated and

perpetuated? A sequence consistent with several aspects of the " low

volume/reduced mucus clearance " hypothesis (10, 33) for CF pathogenesis

is outlined in Figure 5. First, as compared with NL airway epithelial

function (compare Figure 5a), data have been reported that CF airway

epithelia excessively absorb Na+ and Cl– (and water) from the lumen,

deplete the periciliary liquid layer (PCL), and slow/abolish mucus

clearance (Figure 5b) (10, 34, 35). Accelerated Na+ absorption, which

reflects the absence of CFTR’s normal inhibitory activity on ENaC (36),

is fueled by an increased turnover rate of ATP-consuming Na+-K+-ATPase

pumps (37, 38) leading to two- to threefold increases in CF airway

epithelial O2 consumption (39).

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  Figure 5. Schematic model of the pathogenic events hypothesized to lead

to chronic P. aeruginosa infection in airways of CF patients. (a) On

normal airway epithelia, a thin mucus layer (light green) resides atop

the PCL (clear). The presence of the low-viscosity PCL facilitates

efficient mucociliary clearance (denoted by vector). A normal rate of

epithelial O2 consumption (QO2; left) produces no O2 gradients within

this thin ASL (denoted by red bar). (b–f) CF airway epithelia. (

Excessive CF volume depletion (denoted by vertical arrows) removes the

PCL, mucus becomes adherent to epithelial surfaces, and mucus transport

slows/stops (bidirectional vector). The raised O2 consumption (left)

associated with accelerated CF ion transport does not generate gradients

in thin films of ASL. © Persistent mucus hypersecretion (denoted as

mucus secretory gland/goblet cell units; dark green) with time increases

the height of luminal mucus masses/plugs. The raised CF epithelial QO2

generates steep hypoxic gradients (blue color in bar) in thickened mucus

masses. (d) P. aeruginosa bacteria deposited on mucus surfaces penetrate

actively and/or passively (due to mucus turbulence) into hypoxic zones

within the mucus masses. (e) P. aeruginosa adapts to hypoxic niches

within mucus masses with increased alginate formation and the creation of

macrocolonies. (f) Macrocolonies resist secondary defenses, including

neutrophils, setting the stage for chronic infection. The presence of

increased macrocolony density and, to a lesser extent neutrophils, render

the now mucopurulent mass hypoxic (blue bar).

Second, persistent mucin secretion into stationary mucus generates

plaques/plugs (16) (Figure 5c). The combination of thickened mucus and

raised O2 consumption by CF epithelia generated steep O2 gradients within

adherent mucus (Figure 2c). Importantly, the steep pO2 gradient in

ASL/mucus was specific for CF epithelia because it was not reproduced in

cultures from another genetic airways disease with an infectious

phenotype, PCD (Figure 2f). Third, bacteria deposited on thickened mucus

can penetrate into hypoxic zones (Figure 5d). When the normal rotational

mucus transport ceased due to excessive volume absorption, the vertical

" currents " within transported mucus were abolished, but motile P.

aeruginosa still penetrated thickened mucus (Figure 3, c and d). Note

that environmental P. aeruginosa strains such as those that characterize

early infection are motile and would likely penetrate mucus readily.

Fourth, P. aeruginosa can grow in hypoxic/anaerobic CF mucus (Figure 4a).

In part, growth under anaerobic conditions may be supported by the

terminal electron acceptor, nitrate (20 µM), contained in ASL.

Furthermore, we show that increased alginate production was a

characteristic feature of PAO1 strains in response to hypoxia,

particularly with growth in low concentrations of nitrate that mimic ASL

(Figure 4, b–d), and this characteristic is also a feature of

environmental P. aeruginosa strains. We speculate that the increased

alginate formation may represent a stress response to hypoxia that is

part of the process that forms biofilmlike macrocolonies, the predominant

phenotype of P. aeruginosa in CF airways (3). Interestingly,

Staphylococcus aureus also responds to the hypoxic environment of CF

mucus with a switch from nonmucoid to a mucoid phenotype (40, 41).

Finally, the capacity of P. aeruginosa to proliferate in hypoxic mucus

will generate fully hypoxic (anaerobic) conditions in patients with

persistent CF airways infection (Figure 1, Figure 4, e and f, and Figure

5e). Hassett et al. reported that P. aeruginosa alginate production was

maintained by anaerobic conditions (21). The reduced O2 tension in the

mucopurulent intraluminal contents of CF airways may, therefore, be one

variable contributing to the persistence of P. aeruginosa macrocolonies

in CF airways. The consequences of the macrocolony growth state have been

explored in detail and include resistance to antibiotics (42) and host

phagocyte killing (Figure 5f) and (42, 43), all of which contribute to

the persistence of P. aeruginosa infection and the chronic destructive

airways disease characteristics of CF. In summary, our data demonstrate

that the P. aeruginosa infection of CF airways occurs within the luminal

(mucus) rather than the epithelial cell surface compartment. Thus, we

speculate that mucus clearance is a key feature of innate lung defense

(44), and a fundamental defect leading to chronic CF lung infections is

the failure to effectively clear mucus that contains bound bacteria from

the lung (10). Hypoxic gradients exist within poorly cleared/adherent

mucus, consequent to CF-specific increases in epithelial O2 consumption,

and inhaled P. aeruginosa respond to hypoxic mucus with alginate

production and macrocolony formation, which allows them to evade host

defenses and produce a chronic destructive lung disease. These data lead

us to conclude that therapeutic strategies to treat CF lung disease

should include novel drugs designed to clear the lung of retained mucus

plaques/plugs, which initiate and perpetuate CF lung disease, and

antibiotics that effectively treat P. aeruginosa growing under

hypoxic/anaerobic conditions.

Becki

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