Proposed origins of CF lung disease include reduced mucociliary clearance due to decreased ASL volume or altered mucus, reduced bacterial killing by ASL antimicrobials, defective bacterial elimination by phagocytes, abnormal inflammatory responses, reduced or increased bacterial binding by airway epithelia, and other defects 2–6
. One or more of these defects could be responsible. Two factors have made it difficult to distinguish between hypotheses and identify initiating insults. First, as with many diseases, clinical manifestations may not reflect original defects, but it is problematic to study CF at its onset in newborn human infants. Second, mice with disrupted CFTR
genes do not develop typical CF 14
To circumvent these obstacles, we generated CFTR−/−
pigs (CF pigs) 7
. Within months of birth, CF pigs spontaneously develop airway inflammation, infection, tissue remodeling, mucus accumulation, and airway obstruction 7,8
, hallmark features of CF lungs. Although at birth they exhibit none of these features, they already manifest a host defense defect against bacteria. Thus, newborn CF pigs provide an unprecedented opportunity to investigate mechanisms impairing host defense and initiating disease because they allow CF:non-CF comparisons without secondary confounds.
In previous work, we instilled Staphylococcus aureus
into airways and four hours later found more bacteria in CF than non-CF pigs 8
. However, that study revealed little about responsible mechanisms; we do not know whether bacteria were removed or killed within the lung, whether bacteria grew following instillation, whether phagocytic cells eliminated bacteria, whether bacteria bound to surfaces, or whether deposition and sampling were identical in all animals.
To investigate initial host defense defects, we developed a simple assay that tested viability of individual bacteria. We chemically linked biotin to S. aureus
, bound streptavidin to gold grids, and combined them to attach S. aureus
to grids (). We chose S. aureus
because we frequently isolate it from porcine CF lungs, and it is the most common organism isolated from young children with CF 8,15
. A fluorescent live/dead stain revealed the state of bacteria. Exposing grids to ethanol killed most S. aureus
(). Importantly, placing grids on the porcine tracheal surface in vivo
also killed bacteria.
Bacterial killing is impaired in CF ASL
In 6–15 hr-old pigs, we made a small tracheal incision and placed bacteria-coated grids on the airway surface. Even 30-sec applications on non-CF airways killed S. aureus
(). Applying grids to littermate CF pigs killed approximately half as many bacteria. We administered methacholine to stimulate secretion of submucosal glands, which produce substantial amounts of antimicrobials 16,17
, and to allow us to collect ASL for other studies. After methacholine, CF:non-CF differences persisted (). We predicted that antimicrobial activity would also be detected if we removed methacholine-stimulated ASL and studied it with conventional colony-forming unit (CFU) assays. Indeed, bacterial killing was reduced in CF secretions ().
We also applied S. aureus
-coated grids to primary cultures of porcine airway epithelia and found reduced killing in CF (). Previous data suggest that the host-defense defect involves many different bacteria 8,15
. Therefore, we tested Pseudomonas aeruginosa
-coated grids and found defective killing by CF epithelia (). We also added S. aureus
directly to cultured epithelia. Most non-CF epithelia eliminated low inocula of bacteria, but bacteria grew on most CF epithelia (). At the highest inocula, S. aureus
infected both CF and non-CF epithelia.
These data indicate that ASL rapidly kills bacteria, and CF impairs killing. The defect was partial, as CF ASL retained some activity. The assays allow several conclusions. a) Defective bacterial killing was not due to dysfunctional mucociliary clearance or abnormal killing by phagocytes; neither would explain results with grids in vivo
or studies of cultured epithelia. b) We cannot attribute CF:non-CF differences to altered bacterial-epithelial binding because we saw the difference with bacteria attached to grids and with ASL studied ex vivo
. c) Our earlier finding that newborn CF airways lack inflammation 8
and the killing defect in cultured epithelia indicate that abnormal inflammation was not responsible. d) Our bacteria-coated grid method also excludes differences in bacterial delivery, sampling, or growth. Therefore, we reasoned that defective killing arose either from reduced amounts of ASL antimicrobial factors or inhibition of their function.
We investigated antimicrobials by measuring mRNA, protein, and aggregate activity under optimal conditions. The abundance of transcripts for secreted antimicrobial proteins (Table S1, Fig. S1
) and proteins with known host defense functions revealed no consistent differences between genotypes (Table S2
). In methacholine-stimulated ASL, concentrations of the two most abundant antimicrobials, lysozyme and lactoferrin, as well as PLUNC and SP-A did not differ by genotype (). To assay aggregate ASL antimicrobial function, we did four experiments in which we maximized activity by reducing ionic strength close to zero 16,18,19
. First, we added isotonic, salt-free buffer to apical surfaces of cultured airway epithelia. Under these controlled conditions, both genotypes showed equivalent killing of bacteria on grids (). Second, ASL removed from cultured CF and non-CF epithelia with water killed bacteria to the same extent (, S2
). Third, ASL removed from pigs and diluted 1:100 with water showed genotype-independent killing (). Fourth, radial diffusion assays with 10 mM Na phosphate in 1% agarose revealed areas of clearance for S. aureus
and E. coli
that were similar for both genotypes (). These data indicate that non-CF and CF ASL had similar amounts of antimicrobials. Thus, they suggested that CF:non-CF bacterial killing disparities derived from other differences in ASL composition.
CF and non-CF ASL have similar antimicrobial concentrations and aggregate antimicrobial activity under optimal conditions
An increased ionic strength inhibits activity of many antimicrobials 16,18,19
. Studies of human CF airway epithelia in culture or xenografts reported either higher 20,21
or the same 22,23
ASL NaCl concentrations as non-CF, but an in vivo
reported similar concentrations. Therefore, we measured Na+
concentrations in ASL collected from newborn pigs and found that they did not differ by genotype (). In addition, ASL collected after methacholine stimulation showed ion concentrations similar to those measured under basal conditions and only minor differences in K+
concentration between CF and non-CF (). Thus, different ASL Na+
concentrations do not explain defective bacterial killing in CF.
ASL pH is more acidic in CF than non-CF
Earlier studies indicated that pH can affect antimicrobial activity 19,25
. Human and porcine airway epithelia exhibit CFTR-dependent HCO3−
, and CF reduces ASL pH 26,27
. To assess ASL pH in vivo
, we placed a planar pH-sensitive probe on the tracheal surface. pH was lower in CF than non-CF ASL (). Methacholine-stimulated ASL removed from CF pigs and measured with an optical pH probe was more acidic than non-CF (); pH was measured after removal in ambient CO2
, likely contributing to the higher absolute pH values. We also measured ASL pH in primary airway epithelial cultures using a fluorescent pH indicator and found reduced pH in CF (). Although absolute pH values varied in different preparations, in all three, CF ASL was more acidic.
We tested whether pH affects ASL antimicrobial activity by removing ASL from newborn non-CF pigs, adjusting pH, and applying S. aureus
-coated grids. Killing was pH-dependent, increasing as pH increased (, S3
). We also tested lysozyme and lactoferrin; increasing pH increased S. aureus
and E. coli
Increasing ASL pH enhances antimicrobial activity
If pH is responsible for differences in bacterial killing, we predicted that reducing ASL pH would inhibit bacterial killing in wild-type pigs, and raising pH would enhance killing in CF pigs. In non-CF pigs, elevating airway CO2 reduced ASL pH and inhibited bacterial killing (). In CF pigs, we aerosolized NaHCO3 into the trachea. Compared to NaCl, NaHCO3 increased ASL pH and enhanced killing ().
Our results directly link CFTR
mutations to defective bacterial eradication. CFTR is an anion channel that conducts HCO3−
and works in concert with Cl−
exchange and H+
secretion to regulate ASL pH 2,9,10,13
; its loss prevents airway epithelia from secreting HCO3−
. The resulting decreased ASL pH inhibits antimicrobial function, thereby impairing killing of bacteria that enter the lung. Our findings with bacteria-coated grids in vivo
, ASL removed from pigs, and primary epithelial cultures all point to this defect.
What about other defects that might commence CF lung disease? First, progression from the pristine lung of a newborn to the chronically infected, inflamed, remodeled, and obstructed lung of a person with advanced CF entails many steps. Our findings suggest that reduced antibacterial activity may initiate this downward spiral. However, we cannot exclude additional abnormalities at the onset of disease. These might include abnormal mucociliary clearance, bacterial binding, inflammation, or phagocytic function. As disease progresses, the relative importance of various defects may also change. As one example, reduced ASL pH might alter mucus secretion 2
and thereby impair mucociliary clearance either at the genesis of disease or as mucus secretion increases with inflammation and remodeling. Second, ASL contains a complex mixture of peptides, proteins and lipids with individual 16,28,29
and synergistic antimicrobial effects 29,30
. Our data do not assess relative importance or abundance of each factor or CF:non-CF differences.
Several potential fates await bacteria that land on the airway surface. They might be killed. They might remain metabolically active, but unable to reproduce. They might replicate. And/or they might be removed. The balance between these options and their timing determines whether or not infection ensues. Our grid assay tested one of these alternatives independent of the others, and the data indicate that ASL killed many, but not all bacteria. The speed of killing was remarkable, although killing might have continued after grids were withdrawn from ASL, rinsed and placed in indicator solution. Quick action is consistent with rapid bacterial permeabilization by antimicrobial proteins 19
. Our other assays showing reduced numbers of growing bacteria (CFUs) after being placed on epithelial cultures or in ASL reflect consequences of immediate killing plus potential effects of ASL on replication. Results from our earlier delivery of bacteria into porcine airways reflect all these host defenses plus bacterial removal and phagocytosis 8
. Although multiple processes protect lungs when bacteria enter, ASL antimicrobials may be key to rapidly reducing numbers of viable organisms and thereby decreasing the probability that they will replicate, escape other mechanisms, and colonize the lung.
Aerosolizing HCO3− onto CF airways in vivo increased bacterial killing. Our mechanistic findings and this result suggest that correcting ASL pH might prevent the initial CF infection. That might be accomplished by delivering HCO3− into airways, altering pH regulation by airway epithelia, enhancing activity of ASL antimicrobials, delivering pH-insensitive antimicrobials, or targeting mutant CFTR. These results also suggest that adapting the bacteria-coated grid method to assay bacterial killing in patients could be useful for assessing potential therapies. We also speculate that increasing ASL pH might prevent or treat airway infections in other diseases.