Alterations in Ceramide Concentration and pH Determine the Release of Reactive Oxygen Species by Cftr-Deficient Macrophages on Infection

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 gp91phox, 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 gp91phox to the release of ROS in lung macrophages.

Ceramide and sphingosine in pulmonary infections

“…A study using biochemical techniques, fluorescence microscopy, and mass spectrometry demonstrated that CFTR deficiency results in the accumulation of ceramide in bronchial, tracheal, and intestinal epithelial cells, in alveolar macrophages in cystic fibrosis mice, and in nasal epithelial cells from cystic fibrosis patients (Teichgräber et al, 2008;Zhang et al, 2009;Becker et al, 2010a,b) (Figure 1). The accumulation of ceramide in lung tissues and cells from cystic fibrosis patients or mice has been confirmed by several studies using human transplant material, mouse lung tissue, and cultured cells (Brodlie et al, 2010;Ulrich et al, 2010;Bodas et al, 2011a,b;Caretti et al, 2014;Itokazu et al, 2014).…”

“…Several studies have demonstrated the activation of acid sphingomyelinase, the translocation of acid sphingomyelinase to the outer leaflet of the plasma membrane, and the release of ceramide after the infection of epithelial cells or macrophages with P. aeruginosa (Grassmé et al, 2003a;Zhang et al, 2008). Ceramide forms membrane platforms that promote the clustering of several molecules, including NADPH-oxidases, CD95, and the cystic fibrosis transmembrane conductance regulator (CFTR) (Grassmé et al, 2003a, Zhang et al, 2009. Upon infection of phagocytes with P. aeruginosa, NADPH-oxidases release ROS, an event that is necessary for killing and eliminating P. aeruginosa (Zhang et al, 2008).…”

“…In particular, normalization of ceramide concentrations normalizes susceptibility to infection; (aseptic) pulmonary inflammation; the number of peribronchial neutrophils, macrophages, and dead cells; and the deposition of DNA in the airways of cystic fibrosis mice (Teichgräber et al, 2008;Zhang et al, 2009;Becker et al, 2010a,b). These findings demonstrate that ceramide plays a crucial role in the high susceptibility of cystic fibrosis patients to bacterial infection.…”

“…Genetic or pharmacological normalization of ceramide concentrations in the lungs of cystic fibrosis mice, which is achieved either by crossing Cftr-deficient (Cftr or by treating mice with functional acid sphingomyelinase inhibitors (acid sphingomyelinase-depleting drugs), normalizes all of the pathological changes and hallmarks associated with cystic fibrosis (Teichgräber et al, 2008;Zhang et al, 2009;Becker et al, 2010a,b). In particular, normalization of ceramide concentrations normalizes susceptibility to infection; (aseptic) pulmonary inflammation; the number of peribronchial neutrophils, macrophages, and dead cells; and the deposition of DNA in the airways of cystic fibrosis mice (Teichgräber et al, 2008;Zhang et al, 2009;Becker et al, 2010a,b).…”

Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH

“…We measured the actual late endosomal pH values in representative high (1A5) and low (21E5) susceptibility clones using LysoSensor Green DND-189, which accumulates in acidic organelles (e.g. late endosomes and lysosomes) and emits green fluorescence at low pH (19,30,31). Confocal microscopy (Fig.…”