Alterations in ceramide concentration and pH determine the release of reactive oxygen species by Cftr-deficient macrophages on infection
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Updated: Jun 14, 2026
Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcγR Cross-linking
Published on: March 7, 2019
Yang Zhang1, Xiang Li, Heike Grassmé
1Department of Molecular Biology, University of Duisburg-Essen, Hufelandstrasse 55, 45122, Essen, Germany.
Abstract:
We recently demonstrated that the accumulation of ceramide in Cftr-deficient epithelial cells is important for the pathophysiology of CF. However, the role of ceramide in other lung cells, particularly lung macrophages, requires definition. In this study, we report that ceramide is accumulated in Cftr-deficient lung macrophages. Alveolar macrophages contain a vesicle population, which is stained with LysoSensor probes but not by tetramethylrhodamine dextran. These vesicles, presumably secretory lysosomes, exhibit a higher pH in Cftr-deficient macrophages than the corresponding vesicles in lung macrophages isolated from wild-type (WT) mice. Alkalinization of these vesicles in Cftr-deficient macrophages correlates with a failure of the macrophages to respond to infection with various Pseudomonas aeruginosa strains by acutely activating acid sphingomyelinase, releasing ceramide, forming ceramide-enriched membrane platforms that serve to cluster gp91(phox), and, most importantly, releasing reactive oxygen species (ROS). In contrast, these events occur rapidly in WT lung macrophages postinfection. Inhibiting ROS in WT macrophages prevents the killing of P. aeruginosa. These findings provide evidence for a novel pH-controlled pathway from acid sphingomyelinase activation via ceramide and clustering of gp91(phox) to the release of ROS in lung macrophages.
Insights
Ceramide accumulation in cystic fibrosis (CF) lung macrophages impairs their ability to fight Pseudomonas aeruginosa infection by disrupting reactive oxygen species (ROS) production. This study reveals a novel pH-controlled pathway affecting macrophage function in CF.
Area of Science:
- Cell Biology
- Immunology
- Pulmonology
Background:
- Ceramide accumulation is implicated in cystic fibrosis (CF) pathophysiology.
- The role of ceramide in lung macrophages in CF remains unclear.
Purpose of the Study:
- To investigate ceramide accumulation and function in CF lung macrophages.
- To define the role of CFTR deficiency in macrophage response to Pseudomonas aeruginosa infection.
Main Methods:
- Isolation and analysis of alveolar macrophages from wild-type (WT) and Cftr-deficient mice.
- LysoSensor and tetramethylrhodamine dextran staining to characterize vesicle pH.
- Assessment of acid sphingomyelinase activation, ceramide release, gp91(phox) clustering, and reactive oxygen species (ROS) production post-Pseudomonas aeruginosa infection.
Main Results:
- Ceramide accumulates in Cftr-deficient lung macrophages.
- Vesicles in Cftr-deficient macrophages exhibit higher pH, correlating with impaired acid sphingomyelinase activation and reduced ceramide release upon infection.
- This leads to a failure in forming ceramide-enriched platforms, gp91(phox) clustering, and subsequent ROS production, hindering Pseudomonas aeruginosa killing.
Conclusions:
- CFTR deficiency in lung macrophages leads to vesicular alkalinization, disrupting a pH-controlled pathway essential for ROS-mediated bacterial killing.
- This identifies a novel mechanism by which CFTR dysfunction impairs macrophage antimicrobial function, contributing to CF lung pathology.
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