Lung infection with P. aeruginosa is the major cause of morbidity and mortality in patients with CF. Patients with CF infected with P. aeruginosa have a dysfunctional immune response, which fails to remove the infected pathogens from the airway, but also causes more sustained inflammatory responses, resulting in airway obstruction and extensive lung damage. Currently, the mechanisms underlying this dysfunctional immune response are unclear. For the first time, the results from this study demonstrate that CF bronchial epithelium exhibits a defective ASMase pathway as revealed by in vitro and in vivo experiments. First, the results show a significant induction of ASMase in response to infection with P. aeruginosa in WT cells and animals. Second, the results reveal a clear defect in induction of ASMase after P. aeruginosa infection in CF bronchial epithelial cells and CFTR KO mice. Functionally, the results provide evidence that this defective ASMase induction plays a key role in the overwhelming IL-8 response to P. aeruginosa infection, in bacterial internalization, and in the apoptotic response to bacterial infection.
In a previous study conducted by Grassme and colleagues (18
) in WI-38 cells (normal human fetal lung fibroblast cells), the activation of ASMase after P. aeruginosa
infection was detected very acutely 5 to 10 minutes after infection. ASMase activity and the generated ceramide reached peak levels 10 to 20 minutes after infection, and gradually decreased to basal levels over the next 30 minutes. The authors suggested that these effects are caused by acute activation and translocation of ASMase from intracellular vesicles to the extracellular leaflet of the membrane. In contrast, our results show a prolonged time frame (5–6 h) and a mechanism involving induction of protein levels. The ASMase activity and produced ceramide levels kept increasing until 5 or 6 hours after bacterial infection ( and ). Whether ASMase activation could last longer than 6 hours and whether ASMase/ceramide pathway plays a role in chronic bacterial infection need further study.
The reasons for the compromised ASMase pathway in CF have not been fully elucidated. It is now known that ASMase exists as a lysosomal form, a secretory form, or a surface form. When ASMase is activated, it is translocated from intracellular vesicles to the outer leaflet of the plasma membrane (17
), where it hydrolyzes spingomyelin and generates ceramide. Studies by Di and coworkers (35
) and by Barasch and colleagues (36
) revealed that CFTR regulates the pH value of endosomes and lysosomes due to the counterion effect of Cl-
in promoting luminal H+
accumulation. Normally acidic compartments (such as the Golgi network, endosomes, and lysosomes) were more alkaline in cells with mutant CFTR compared with cells with WT CFTR (35
). Treatment of WT alveolar macrophages with reagents that inhibited CFTR-dependent chloride transport or the vacuolar proton ATPase led to alkalinization of lysosomes (35
). Since ASMase is primarily an endosomal/lysosomal enzyme, which needs an acidic environment to function normally, the increasing pH value in endosome/lysosomes might affect ASMase activity and processing from the lysosomal from to the surface form.
Studies of alternate chloride channels in endosomes/lysosomes also confirm that defects in chloride channels affect the acidification of these membrane organelles. Specifically, defects of the chloride channels CIC-3 (37
); CIC-4 (39
); and CIC-5 (40
) impair the acidification of endosomes/lysosomes. Loss of the chloride channel CIC-7 leads to lysosomal storage disease (41
). The low luminal pH in these membrane organelles is important for several processes, such as receptor–ligand interactions, trafficking along the endosomal pathway, and luminal enzymatic activity (42
). How CFTR protein interacts with lipid metabolism and ASMase activity needs further investigation. Of note, recent studies suggest that sphingolipids interact with CFTR at multiple stages. Lipid rafts have been reported to play a role in recruiting CFTR (18
), and the SMase reaction also affects CFTR ion channel activity (43
In CF with abnormal pH value in lysosomes, there might be less ASMase translocated from lysosomes to the outer leaflet of the plasma membrane, resulting in less ceramide production. A alternative mechanisms for ceramide generation still exist in CF cells and CFTR KO mice. There was a 20% increase in total ceramide in infected IB3-1 cells 300 minutes after bacterial infection and 17% increase of total ceramide in infected CFTR KO mice after 5 × 107
cfu PAO1 infection. Our results are consistent with the finding of Guilbault and coworkers (45
) that patients with CF have significantly lower plasma ceramide levels compared with healthy control subjects. Guilbault and colleagues (45
) also showed that CFTR KO mice displayed diminished ceramide levels in CF-related organs (lungs, pancreas, and ileum) compared with their littermate controls. These findings support a defective ASMase/ceramide pathway in CF. A recent study by Teichgraber and coworkers (46
) revealed that the defect in acidification of endosomes/lysosomes in CF also affects acid ceramidase activity. They reported a constitutive accumulation of ceramide in old (16- to 32-wk-old) CFTR KO mice, but not in 8-week-old CFTR KO mice without bacterial infection. These results are distinct from those of Guilbault and colleagues (45
), who showed that patients with CF (aged 20–59 yr) had a significantly lower plasma levels of ceramide compared with healthy control groups. No correlation was found between the ceramide levels in the lungs and the age of the mice (45
). In the current study, which was conducted in CF bronchial epithelial cells and 8-week-old CFTR KO mice, there was no significant difference of basal levels of ceramide between CF groups and WT controls. The levels of ceramide only exhibited significant difference between CF groups and WT groups after bacterial infection.
Using a combination of approaches, the current study reveals an important role for ASMase in attenuating IL-8 responses to P. aeruginosa
infection in CF, and it suggests a role for ceramide in a feedback mechanism that regulates the IL-8 response. These results are consistent with those of Vilela and coworkers (47
), who revealed that generation of ceramide by fenretinide inhibited TNF-α–induced IL-8 production in CF tracheal epithelial cells. Since induction of IL-8 appears to be a major detriment in patients with CF, a better understanding of the mechanisms behind the IL-8 response, including the failure to up-regulate ASMase in infected CF cells, is critical to the development of new therapies for airway inflammation. Our results suggest that the ASMase/ceramide pathway is one of multiple pathways that contribute to cytokine regulation. CF cells have been reported to have higher levels of nuclear factor (NF)-κB, higher level of activator protein-1 (AP-1) activity, and higher extracellular signal–regulated kinase (ERK) phosphorylation (48
). How the ASMase/ceramide pathway interacts with those transcriptional factors and protein kinases needs further study.
In addition to the effects of ASMase on airway inflammation in CF, our results support a critical role for ASMase in mediating/enhancing bacterial uptake in infected cells. CF bronchial epithelial cells (IB3-1 cells) had decreased bacterial uptake with defective ASMase activity as compared with control S9 cells (). This observation extends earlier findings by Pier and colleagues (6
) that cells with wild-type CFTR ingested more infected P. aeruginosa
than cells with mutant CFTR gene. Although there is only an approximate 2-fold difference of bacterial uptake between IB3-1 cells and S9 cells after P. aeruginosa
infection, when we take into consideration the fact that twice as many S9 cells underwent apoptosis as IB3-1 cells, the difference of ingested bacteria between cell lines is approximately 4-fold. This is consistent with the finding of Schroeder and coworkers (49
) that WT C57BL/6 mice ingested 3.9 times more bacteria than did the CF mice. This study demonstrates that the decreased bacterial ingestion can be fully attributed to the defective ASMase activity in CF. Silencing ASMase activity by ASMase RNAi in S9 cells (with normal ASMase activity) significantly decreased the amount of internalized P. aeruginosa
, down to the levels seen in IB3-1 cells ().
Once the bacteria are ingested, the infected cells normally undergo apoptosis as a means of clearing the bacteria. In addition to its role in bacterial uptake, ASMase is involved in regulating the apoptotic response to infection with P. aeruginosa
. We demonstrated a reduced apoptotic response to P. aeruginosa
infection in IB3-1 cells compared with the S9 cells (). This observation is in line with the findings of Cannon and colleagues (7
) that cells expressing mutant CFTR had an approximately 2-fold less apoptotic rate with P. aeruginosa
infection than cells with the WT CFTR. Furthermore, the current study also shows a decreased apoptotic response after P. aeruginosa
infection in CF mice in vivo
(). The addition of exogenous bacterial SMase to IB3-1 cells greatly increased the necrotic/apoptotic response after P. aeruginosa
infection (). In CF, with decreased bacterial uptake and decreased bacterial killing, there is increasing bacterial burden. Schroeder and coworkers (49
) revealed that mice with at least one WT CFTR allele had a 1.1- to 3.4-fold increase in cfu in the lungs over the infecting inoculums after 4.5 hours of P. aeruginosa
infection, whereas homozygous ΔF508 CFTR mice had a 7.5-fold increase in the cfu of P. aeruginosa
in the lungs. The increased bacterial burden in CF in turn could stimulate immune response and finally result in an overwhelming chronic infection.
In conclusion, the current study demonstrates a defective ASMase/ceramide pathway with P. aeruginosa infection in CF bronchial epithelial cells as well as in CFTR KO mice. This defective ASMase pathway is associated with the overwhelming IL-8 cytokine response, decreased bacterial uptake and reduced apoptotic response in CF with P. aeruginosa infection. This compromised ASMase response might permit P. aeruginosa to escape from killing and in turn cause unabated proinflammatory cytokine response in CF. Understanding the mechanisms involved in the predilection for P. aeruginosa infection in CF and the emergence of a chronic infection/inflammation is critical to the development of novel therapeutics aimed at preventing or clearing infection and decreasing inflammation in CF airways.