Studying the pathophysiology of CF lung disease is critical for developing novel treatments; however, because there are no adequate controls in the human population, other models must be considered. Animal models of CF lung disease must be used because inflammatory processes are complex and almost surely involve interactions among multiple cell types. However, understanding the strengths and limitations of the animal models available is essential for critically evaluating individual pieces of the disease process and putting those pieces together to make a more complete story.
The data reported here support the hypothesis that CFTR deficiency is sufficient to produce increased inflammatory responses to free-living P. aeruginosa insufflated into the lung. At higher doses, CF mice suffer higher mortality than wild-type mice. At slightly lower doses, they survive, but suffer greater weight loss with slower recovery than their wild-type counterparts. This is, however, not due to excessive or protracted bacterial retention in the airways, for the CF mice cleared the bacteria just as rapidly as wild-type mice. Rather, it is the result of increased and prolonged inflammatory responses.
CF mice had an exaggerated inflammatory response to P. aeruginosa
compared with wild-type control mice both after a single dose and multiple doses, as assessed by the area of the lung that was inflamed and the levels of inflammatory cytokine mediators and neutrophils in BAL fluid. In addition, bacterial clearance was just as rapid in CF mice as in wild-type mice. Control studies showed that these results could not be attributed to the inhalational anesthesia used. The use of the CF mouse strain selected eliminated dietary or nutritional differences as a variable for these experiments (as these mice, although maintaining the CF phenotype in the lung, grow normally, unlike Cftr−/−
mice, which require a liquid diet to survive and remain small compared with wild-type littermates ). In the agarose bead model, the CF mice had pulmonary responses similar to Cftr−/−
). Therefore, we attribute the differences between groups observed in this study to lack of Cftr
activity. Rapid clearance of the bacteria by the CF mice in this study was likely due to the robust, early neutrophil response. Despite the fact that bacteria are cleared within 2 d of administration, CF mice have a prolonged inflammatory response as indicated by excessive levels in the proinflammatory mediator TNF-α and the murine neutrophil chemokine KC compared with wild-type mice.
In another publication using free-living bacteria, mice with Cftr
mutations were reported to have reduced ability to kill P. aeruginosa
compared with wild-type mice (24
). The discrepancy in our results and theirs probably results from differences in experimental conditions, the most important of which is likely to be the mice themselves. In the earlier study, mice from that colony were reportedly spontaneously infected, so the colony was maintained routinely on antibiotics, which were stopped 24 h before challenge. In contrast, our mice were not treated with antibiotics at any time and sentinel animals (both wild-type mice and Cftr
mutants) were not infected with any of the agents reported in the other investigators' colony. Moreover, in our experiments, qualitative bacteriology cultures on BAL fluid in challenged mice showed only the bacteria inoculated; this was not the case in the prior study. In addition, the outcome measures were performed differently. The earlier investigators used a technique designed to detect the difference between bacteria that were ingested by CF and wild-type lung cells, as well as the multiple of infectious inoculum, whereas the method described here simply measured the number of bacteria in the entire lung and these are reported as the number of cfu/lung.
CF mice maintained under specific pathogen–free conditions do not spontaneously acquire lung infections, and they are capable of rapid clearance and killing of even substantial and repeated inocula of pathogenic organisms. In contrast, patients with CF do spontaneously acquire bacterial colonization of the airways early in life and eventually become unable to clear the infecting organisms. Whether bacterial retention is due to (1
) changes in the CF lung and how it responds to the bacterium, (2
) changes in how the bacterium responds to the CF lung, or (3
) a combination of the two is not clear. Recent observations in mouse models, including that presented here, may shed some insight to this conundrum.
mice do not have the same up-regulation of ENaC activity that is observed in the lower airways of patients with CF (25
). Mall and colleagues demonstrated that overexpression of the β subunit of ENaC directed to the airways by a Clara cell–specific promoter in mice led to mucus plugging of the lower airways, mimicking the natural pathophysiology of CF lung disease (6
). In addition, they found that bacterial clearance of a nonmucoid clinical strain of P. aeruginosa
cfu/mouse) was slower than in wild-type control mice, although it did not produce mortality. The differences between Cftr−/−
mice and the ENaC-overexpressing mice probably do not reside entirely in the increase in goblet cells and mucins, however, because induction of increased mucin production and increased goblet cell number in Cftr−/−
mice still did not permit chronic infection with free-living P. aeruginosa
). Second, neutrophil necrosis and the release of DNA and actin likely enhance the conversion of nonmucoid P. aeruginosa
to the mucoid form (27
). Also, mucoid P. aeruginosa
in the presence of alginate is retained more readily in the lungs of CF mice than nonmucoid P. aeruginosa
, and mortality rates were significantly higher in CF mice than in Balb/c wild-type control mice (28
). Last, we report that planktonic mucoid P. aeruginosa
can be cleared readily from the lungs of CF and wild-type mice without mortality.
Therefore, it is likely that trapping and retention of bacteria in the CF lung is a combination of two factors: that of the CF lung and that of the bacterium. First, it requires thick, sticky mucus, which may be attributable to abnormalities in ENaC. The lungs of CF mice appear to be well hydrated, which may be due to normal activity of ENaC and increased activity of a calcium-dependent chloride transporter (25
). Although mucus production is stimulated in both wild-type and CF mice after challenge with P. aeruginosa
, this does not appear to occur to the extent that it does in mice overexpressing the β subunit of ENaC. Second, retention is facilitated by conversion of P. aeruginosa
from a nonmucoid form into a mucoid form, and the alginate coating protects the bacterium from clearance in CF mice (28
). Therefore, it is not surprising that chronic lung infections could not be established in CF mice under these experimental conditions. However, our data clearly show that defective Cftr
, in the absence of both increased function of ENaC in the lower airways and an overabundance of alginate in the challenge bacteria, is sufficient to induce an increased and protracted inflammatory response despite efficient clearance of the bacteria.
There are several possible mechanisms responsible for the inflammatory response after repeated administration of P. aeruginosa, some of them under active investigation in our lab. For instance, if the host already has its defenses recruited to defend against the first inoculum (e.g., neutrophils are already in the airway, macrophages already activated, serum already leaked into the airway), the host may control the second inoculum better, without additional recruitment of cells or increase in cytokines. It is also possible that due to the repeated administration and continual clearance of the bacteria, providing a continual stimulus, a different cell population is now recruited. Indeed, lymphocyte numbers are increasing, as indicated by BAL cell counts and in observing histologic sections over time. The lymphocytic response may be more important later on in the infection. In addition, due to absent Cftr, the CF mice have a more prolonged neutrophilia with the continued production of KC.
In conclusion, the interpretation of our results combined with the work of others suggest that the impaired mucociliary clearance, mucus plugging, and trapping of inhaled bacteria in CF may be more closely associated with the up-regulation of ENaC initially than with deficiency of CFTR activity per se, but that once an infectious or inflammatory stimulus is applied, Cftr deficiency permits the inflammatory response to be exaggerated. This exaggerated response probably allows increased airway damage from neutrophil products and provides a favorable setting for further bacterial invasion. Also, overproduction of alginate by P. aeruginosa protects the organism from clearance by the CF lung, further exacerbating the cycle of chronic infection and exaggerated inflammation. Therefore, the vicious cycle of infection and inflammation in the CF lung probably arises from at least two of the functions of CFTR, one of which has yet to be fully characterized.