(419) The Role of Macrophage CFTR in Lung Inflammation

Background: The clinical outcome of cystic fibrosis (CF) is the progressive loss of lung function, caused by chronic bacterial infections and persistent neutrophilic inflammation. How the loss of the CF Transmembrane conductance Regulator (CFTR) in macrophages contributes to CF lung disease pathogenesis is not fully defined. To address this, we have developed and employed a macrophage-specific CF (Mac-CF) mouse model.

Methods: Mac-CF mice were produced by breeding Cx3cr1-Cre mice with CFTR exon-10 floxed (Cftr^fl/fl) mice. The Mac-CF and the congenic control Cftr^fl/fl mice were intratracheally intubated with 2.0x10⁷ CFU of Pseudomonas aeruginosa (PsA) in 50 µl PBS. At different time points (0-, 2-, 3-, and 5-days post-exposure), the mice were euthanized, and their lungs lavaged. The bronchoalveolar lavage fluid (BALF) was analyzed for bacterial survival by bacterial plating, inflammatory cytokine transcription and translation by RT-PCR and ELISAs, and inflammatory cell recruitment by cytology. Furthermore, bone marrow-derived macrophages (BMDM) from Mac-CF and Cftr^fl/fl were challenged with PsA LPS (20 µg/mL). At various time points (2-, 4- and 8-hours post-exposure), the supernatant was collected and analyzed for cytokine production by ELISAs.

Results: Immediately after the PsA challenge (Day 0), Mac-CF and Cftr^fl/fl mice were exclusively macrophage-dominant in their lungs with no difference in cytokine levels. Two days after PsA exposure, the BALF cells from both mice became neutrophil dominant ( >90%). This neutrophilic inflammation was accompanied by an increase in inflammatory cytokine production (IL-1β, TNF-α, KC, MIP-2, and IL-6). At this time point, the Cftr^fl/fl and Mac-CF lungs exhibited no difference in cellular recruitment, bacterial burden, or cytokine transcription or production. By 3 days post-exposure, the lungs of the Mac-CF mice remained highly neutrophilic, having elevated levels of inflammatory cytokine production (IL-1β, TNF-α, KC, MIP-2, and IL-6). This inflammatory cytokine increase was confirmed by an increase in inflammatory cytokine transcription. In contrast to Mac-CF mice, the control Cftr^fl/fl mouse lungs had significantly more macrophages and displayed a significant decrease in inflammatory cytokine transcription and production. Despite the difference in inflammatory status, both the Cftr^fl/fl and Mac-CF had no difference in bacterial burden, with PsA largely cleared. Five days post-exposure, the BALF cells from both mice were macrophage dominant, and no significant differences in cellular recruitment, or cytokine transcription and production. Consistent with these in vivo results, BMDMs from Mac-CF mice exhibited significantly more production of inflammatory cytokines (TNF-α, KC, MIP-2, and IL-6) than the BMDMs from Cftr^fl/fl mice after LPS exposure.

Conclusions: Loss of CFTR in macrophages does not affect lung bacterial clearance but is correlated to increased production of inflammatory cytokines and decreased ability to resolve inflammation. Therefore, CF macrophages are a critical player in CF lung inflammation.

Acknowledgements: Funding was provided to GW by NIH (AI140088 and HL150370) and the Cystic Fibrosis Foundation (WANG18I0).

References: