Poster: 89The role of macrophage Piezo1 on bacterial clearance in cystic fibrosis

Abstract

Background

Cystic Fibrosis (CF) macrophage dysfunction is an important driver of CF chronic lung infection and infection-induced lung stiffness. In CF, macrophages fail to clear pathogens, such as Pseudomonas aeruginosa, due to failure of phagolysosome acidification and fusion, known as phagolysosome maturation. Our lab has shown that the macrophage mechanosensitive ion channel, Piezo1, augments phagolysosome maturation on infected lung matrix stiffness (25 kPa) to increase bacterial clearance of CF pathogen, P. aeruginosa. Although the lung matrix stiffness is significantly altered in CF, the role of Piezo1 on macrophage function remains poorly understood. Therefore, we seek to determine if changes to Piezo1 expression or Ca²⁺ channel activity drive impaired phagolysosome maturation in CF.

Cystic Fibrosis (CF) macrophage dysfunction is an important driver of CF chronic lung infection and infection-induced lung stiffness. In CF, macrophages fail to clear pathogens, such as Pseudomonas aeruginosa, due to failure of phagolysosome acidification and fusion, known as phagolysosome maturation. Our lab has shown that the macrophage mechanosensitive ion channel, Piezo1, augments phagolysosome maturation on infected lung matrix stiffness (25 kPa) to increase bacterial clearance of CF pathogen, P. aeruginosa. Although the lung matrix stiffness is significantly altered in CF, the role of Piezo1 on macrophage function remains poorly understood. Therefore, we seek to determine if changes to Piezo1 expression or Ca²⁺ channel activity drive impaired phagolysosome maturation in CF.

Methods

To determine the role of macrophage Piezo1 on phagolysosome maturation in CF, we measured phagolysosome maturation ± P. aeruginosa lipopolysaccharide (LPS) (100 ng/mL for 6h, Invivogen) using pHrodo Zymosan particles (Invitrogen) in wild type (WT) and Cftr G542X (Cftr knock-out) bone marrow-derived macrophages (BMDMs). Piezo1 surface expression and Ca²⁺ channel activity were measured by flow cytometry and the FLIPR 5 Ca²⁺ Assay (Molecular Devices), respectively. To determine the effect of Piezo1 on P. aeruginosa lung infection in vivo, we administered a CF clinical strain of P. aeruginosa (PAM 57-15) to Piezo1^fl/fl (Piezo1 sufficient) and Piezo1^LysMCre (Piezo1 myeloid depleted) mice and measured bacterial clearance and bronchoalveolar lavage cell count and differential. To translate our findings to human CF, we performed an Ingenuity pathway analysis on published 10X genomic RNA sequencing data using airway macrophages from healthy and CF expectorated sputum [1]. Finally, Piezo1 mRNA was measured from healthy control (HC) and CF peripheral blood mononuclear cells (PBMCs).

To determine the role of macrophage Piezo1 on phagolysosome maturation in CF, we measured phagolysosome maturation ± P. aeruginosa lipopolysaccharide (LPS) (100 ng/mL for 6h, Invivogen) using pHrodo Zymosan particles (Invitrogen) in wild type (WT) and Cftr G542X (Cftr knock-out) bone marrow-derived macrophages (BMDMs). Piezo1 surface expression and Ca²⁺ channel activity were measured by flow cytometry and the FLIPR 5 Ca²⁺ Assay (Molecular Devices), respectively. To determine the effect of Piezo1 on P. aeruginosa lung infection in vivo, we administered a CF clinical strain of P. aeruginosa (PAM 57-15) to Piezo1^fl/fl (Piezo1 sufficient) and Piezo1^LysMCre (Piezo1 myeloid depleted) mice and measured bacterial clearance and bronchoalveolar lavage cell count and differential. To translate our findings to human CF, we performed an Ingenuity pathway analysis on published 10X genomic RNA sequencing data using airway macrophages from healthy and CF expectorated sputum [1]. Finally, Piezo1 mRNA was measured from healthy control (HC) and CF peripheral blood mononuclear cells (PBMCs).

Results

We found that Cftr G542X BMDMs failed to augment phagolysosome maturation in response to P. aeruginosa LPS. However, treatment with the Piezo1 agonist, Yoda1, restored LPS-induced phagolysosome maturation in Cftr G542X BMDMs. In addition, Piezo1 surface expression and Ca²⁺ channel activity were reduced in Cftr G542X BMDMs compared to WT. In vivo, loss of myeloid Piezo1 decreased bacterial clearance of P. aeruginosa by 87% and increased bronchoalveolar neutrophils, a key mediator of CF lung injury and stiffness, by 55.7%. To determine translational relevance of macrophage dysfunction in CF, we found reduced mRNA transcripts of critical phagolysosome maturation genes in CF airway macrophages compared to HC. As we have shown that Piezo1 regulates phagolysosome maturation on infected lung stiffness (25 kPa), we next found that Piezo1 mRNA transcripts are reduced in PBMCs from pwCF compared to HC, which may contribute to CF macrophage dysfunction.

We found that Cftr G542X BMDMs failed to augment phagolysosome maturation in response to P. aeruginosa LPS. However, treatment with the Piezo1 agonist, Yoda1, restored LPS-induced phagolysosome maturation in Cftr G542X BMDMs. In addition, Piezo1 surface expression and Ca²⁺ channel activity were reduced in Cftr G542X BMDMs compared to WT. In vivo, loss of myeloid Piezo1 decreased bacterial clearance of P. aeruginosa by 87% and increased bronchoalveolar neutrophils, a key mediator of CF lung injury and stiffness, by 55.7%. To determine translational relevance of macrophage dysfunction in CF, we found reduced mRNA transcripts of critical phagolysosome maturation genes in CF airway macrophages compared to HC. As we have shown that Piezo1 regulates phagolysosome maturation on infected lung stiffness (25 kPa), we next found that Piezo1 mRNA transcripts are reduced in PBMCs from pwCF compared to HC, which may contribute to CF macrophage dysfunction.

Conclusions

Herein, we show that macrophage mechanosensitive ion channel Piezo1 restores LPS-induced phagolysosome maturation in Cftr G542X BMDMs. In vivo, loss of myeloid Piezo1 decreased bacterial clearance of P. aeruginosa and increased bronchoalveolar neutrophils. As Piezo1 mRNA expression is reduced in human CF PBMCs, our data suggest that Piezo1 is a potential therapeutic target for CF macrophage dysfunction and CF chronic lung infection.

Herein, we show that macrophage mechanosensitive ion channel Piezo1 restores LPS-induced phagolysosome maturation in Cftr G542X BMDMs. In vivo, loss of myeloid Piezo1 decreased bacterial clearance of P. aeruginosa and increased bronchoalveolar neutrophils. As Piezo1 mRNA expression is reduced in human CF PBMCs, our data suggest that Piezo1 is a potential therapeutic target for CF macrophage dysfunction and CF chronic lung infection.