Abstract Rationale Cystic fibrosis (CF) is a multisystem disease whereby mutations in the Cystic Fibrosis Transmembrane Regulator (CFTR) gene cause chronic lung infection and resultant lung tissue stiffness. Piezo1 is a mechanosensitive ion channel whose Ca2+ channel activity in response to matrix stiffness augments macrophage bacterial clearance. Thus, we investigated the possibility that Piezo1 mediated chronic lung infection due to Pseudomonas aeruginosa in people with CF. Methods Bacterial clearance was assessed by measuring bacterial load (CFU) by intratracheal instillation of P. aeruginosa (PAM 57-15)-embedded or control agarose beads in wild-type (WT) mice ± Piezo1 inhibitor, GsMTx4. To examine the regulation of Piezo1 in people with CF, Piezo1 mRNA abundance was measured in healthy controls and CF peripheral blood mononuclear cells. Pathways of bacterial clearance that are differentially regulated in CF were identified using published RNA sequencing data from healthy and CF expectorated sputum phagocytes. To determine the effect of lung matrix stiffness on bacterial clearance, WT, Piezo1fl/fl, and Piezo1LysMCre BMDMs were plated on polyacrylamide gels of pathophysiologic range lung stiffness (1 kPa: normal lung, 8-25 kPa: injured lung) or standard culture conditions (106 kPa). Downregulation of Piezo1 was performed using control or Piezo1-targeted siRNA. Phagolysosome maturation was measured ± Piezo1 agonist Yoda1 and P. aeruginosa flagellin using pHrodo particles in mouse and human macrophages. Results Pharmacologic inhibition of Piezo1 impaired bacterial clearance during chronic P. aeruginosa lung infection in vivo. Prolonged stimulation with flagellin (24h) decreased Piezo1 mRNA by 20-fold in WT BMDMs, suggesting chronic exposure to P. aeruginosa virulence factors, such as seen in CF, suppress Piezo1 transcription. These mouse macrophage findings were replicated in CF phagocytes where Piezo1 mRNA decreased by 4.8-fold compared to healthy controls. Publicly available RNA sequencing from CF sputum phagocytes reveals a reduction in genes required for the key bacterial clearance step of phagolysosome maturation. As such, we tested the role of Piezo1 on phagolysosome maturation directly. Activation of Piezo1 increases phagolysosome maturation 3-fold in healthy human alveolar macrophages. Concordantly, loss of Piezo1 function by Piezo1 knockdown reduces macrophage phagolysosome maturation in a matrix stiffness-dependent manner in WT BMDMs. Conclusion These data support the importance of Piezo1 in chronic P. aeruginosa lung infection. They further suggest that P. aeruginosa virulence factors can downregulate Piezo1 mRNA leading to both impaired phagolysosome maturation and bacterial clearance in CF. Therefore, restoring Piezo1 function in CF phagocytes is a potential therapeutic strategy for CF chronic P. aeruginosa lung infection. This abstract is funded by: Cystic Fibrosis Foundation
Orsini et al. (Fri,) studied this question.