Using both pharmacologic inhibition and mutation (F508del) of CFTR, we provide evidence that lack of functional CFTR in neutrophils propagates production of NF-κB-dependent proinflammatory cytokines and worsens LPS-induced acute lung inflammation and injury. We also found that CFTR-mutated neutrophils are more inflammatory than wild-type neutrophils and amplify lung inflammation and injury in response to LPS challenge.
It has been reported that human neutrophils express CFTR [10
]. In this study, we also found that mouse neutrophils expressed CFTR as confirmed by immunofluorescence, gene transcript, and protein analysis. More importantly, for the first time, we found that CFTR expression in neutrophils was upregulated at the transcript and protein level after LPS challenge. In a LPS-induced acute lung injury mouse model, CFTR expression in BAL cells (predominated by neutrophils) was also increased by threefold. Inhibition or mutation of CFTR promotes NF-κB p65 translocation from the cytoplasm to the nucleus in LPS-stimulated neutrophils, suggesting that activation of CFTR might counteract NF-κB activation and limit proinflammatory responses in the neutrophils. Lack of functional CFTR could result in excessive NF-κB activation in neutrophils and therefore propagate a hyper-inflammatory response.
Neutrophils can be mobilized from the bone marrow and recruited into the airspaces of the lung after intratracheal LPS challenge [28
]. If CFTR-deficient neutrophils are recruited into the lung, the proinflammatory state in response to acute infection may be amplified [30
]. TNF-α and MIP-2 play important roles in mobilizing neutrophils and promoting LPS-induced acute lung inflammation [32
]. In the present study, inhibition and mutation of CFTR facilitated production of proinflammatory cytokines (TNF-α and MIP-2) in the BAL and promoted neutrophil alveolar translocation and p65 NF-κB translocation in the BAL cells (predominantly neutrophils). Our results suggest that, under inhibition or mutation, the infiltrated neutrophils in the airspaces of the lung can be activated by LPS or bacterial challenge to produce more NF-κB dependent proinflammatory cytokines and worsen pulmonary inflammation. These results provide evidence to support the hypothesis that the lack of functional CFTR in neutrophils promotes proinflammatory responses, which facilitate the development of sepsis-induced acute lung inflammation and injury. In addition, lack of functional CFTR in neutrophils also reduced production of the anti-inflammatory cytokine, IL-10 [34
], suggesting that CFTR might also regulate the anti-inflammatory responses in LPS-challenged neutrophils.
We also conducted experiments with neutrophil or bone marrow reconstitution to confirm that the lack of functional CFTR in neutrophils promotes LPS-induced acute lung inflammation and injury. Under LPS challenge, neutrophil-depleted wild-type mice reconstituted with F508del neutrophils displayed higher BAL neutrophil counts, MIP-2 and TNF-α, and severe pulmonary edema comparing neutrophil-depleted wild-type mice reconstituted with wildtype neutrophils. Wild-type mice reconstituted with F508del bone marrow also demonstrated an elevated alveolar neutrophil transmigration, vascular permeability, and proinflammatory cytokine production (MIP-2) in response to intratracheal endotoxin exposure. Taken together, these findings also indicate that CFTR expressed by neutrophils modulates proinflammatory responses during LPS-induced acute lung inflammation and injury.
There is evidence that CF lung epithelia demonstrate a constitutive upregulation of the pro-inflammatory chemokine IL-8 and can increase IL-8 mRNA expression [35
] with LPS stimulation in a NF-κB dependent mechanism [5
]. We found that F508del mice reconstituted with wild-type bone marrow exhibited higher BAL MPO and protein levels but without an increase in BAL MIP-2 compared to wild-type recipients reconstituted with wild-type bone marrow. The possible explanations include (1) F508del lung epithelial cells are more permeable to wild-type neutrophils; (2) F508del neutrophils are more potent than the wild-type neutrophils to illicit MIP-2 production by interacting with F508del macrophages or lung epithelial cells; and (3) there may be inflammation-limiting factors [37
] in wild-type bone marrow to counteract the proinflammatory responses in F508del mice after LPS challenge.
There has been a long standing debate as to whether CFTR knockout or deltaF508 cells are more prone to inflammatory injury in the presence or absence of infection [38
]; however, in the present experiments, inhibition or mutation of CFTR alone in neutrophils did not induce proinflammatory responses, and this occurred only after challenge with LPS, which also supports the hypothesis that CFTR is a modulator for sepsis-induced acute lung inflammation and injury.
Lung disease is the major cause of death in cystic fibrosis (CF). In a CFTR deficient (cftrm1HGU
) mouse model, capacity to clear Staphylococcus aureus
—two opportunistic lung pathogens closely associated with lung disease in CF subjects—was impaired. In response to repeated microbial exposure, the cftrm1HGU
mice developed severe pulmonary edema, suggesting that lack of CFTR leads CF subjects to be susceptible to lung diseases and hyper-inflammatory responses [40
Recently, Mckeon et al. [41
] found that low levels of CFTR mRNA could be identified in normal human neutrophils, but CFTR protein was not found in human neutrophil lysates or immunoprecipitates. However, Painter et al. [10
] fond that CFTR was present exclusively in the secretory vesicles of neutrophils. The CFTR chloride channel was also detected in phagolysosomes—a special organelle formed after phagocytosis. CFTR-mediated halide transport in the phagosomes of human neutrophils could be inhibited by thiazolidinone and glycine hydrazide [42
]. In our study, we found that mouse neutrophils express CFTR under normal and LPS-stimulated conditions. Thiazolidinone (CFTRinh
-172) and glycine hydrazide (MalH-2) CFTR inhibitors are able to inhibit CFTR expressed both on the cell membrane and in intracellular organelles because they are cell permeable [42
Although our findings may have relevance to CF patients who develop acute Gram negative sepsis from pneumonia or a non-pulmonary source, our findings do not explain the recurrent infection and excessive inflammation in CF airways [44
]. Further, another ion channel, such as NKCC1 [1
], might play an important role in modulating inflammatory responses during sepsis-induced acute lung inflammation and injury.
In summary, we provide new evidence that lack of functional CFTR in neutrophils promotes production of proinflammatory cytokines by activation of NF-κB, and worsens LPS-induced acute lung inflammation and injury.