Altered Degranulation and pH of Neutrophil Phagosomes Impacts Antimicrobial Efficiency in Cystic Fibrosis

Abstract

Studies have endeavored to understand the cause for impaired antimicrobial killing by neutrophils of people with cystic fibrosis (PWCF). The aim of this study was to focus on the bacterial phagosome. Possible alterations in degranulation of cytoplasmic granules and changes in pH were assessed. Circulating neutrophils were purified from PWCF (n = 28), PWCF receiving ivacaftor therapy (n = 10), and healthy controls (n = 28). Degranulation was assessed by Western blot analysis and flow cytometry. The pH of phagosomes was determined by use of BCECF-AM-labelled Staphylococcus aureus or SNARF labelled Candida albicans. The antibacterial effect of all treatments tested was determined by colony forming units enumeration. Bacterial killing by CF and healthy control neutrophils were found to differ (p = 0.0006). By use of flow cytometry and subcellular fractionation the kinetics of intraphagosomal degranulation were found to be significantly altered in CF phagosomes, as demonstrated by increased primary granule CD63 (p = 0.0001) and myeloperoxidase (MPO) content (p = 0.03). In contrast, decreased secondary and tertiary granule CD66b (p = 0.002) and decreased hCAP-18 and MMP-9 (p = 0.02), were observed. After 8 min phagocytosis the pH in phagosomes of neutrophils of PWCF was significantly elevated (p = 0.0001), and the percentage of viable bacteria was significantly increased compared to HC (p = 0.002). Results demonstrate that the recorded alterations in phagosomal pH generate suboptimal conditions for MPO related peroxidase, and α-defensin and azurocidine enzymatic killing of Staphylococcus aureus and Pseudomonas aeruginosa. The pattern of dysregulated MPO degranulation (p = 0.02) and prolonged phagosomal alkalinization in CF neutrophils were normalized in vivo following treatment with the ion channel potentiator ivacaftor (p = 0.04). Our results confirm that alterations of circulating neutrophils from PWCF are corrected by CFTR modulator therapy, and raise a question related to possible delayed proton channel activity in CF.

The Distribution and Role of the CFTR Protein in the Intracellular Compartments

“…CFTR localization in lysosomes of macrophages was demonstrated. Recent studies also support the concept of defective acidification in CFTR-deficient cell phagosomes, as CFTR inhibition was shown to increase the vesicle pH in human macrophages 07] and neutrophils [108].…”

Alpha-1 Antitrypsin Augmentation Inhibits Proteolysis of Neutrophil Membrane Voltage-Gated Proton Channel-1 in Alpha-1 Deficient Individuals

“…Cell activation induced a significant increase in HVCN1 expression on plasma membranes after 10 min ( p = 0.002), but after 30 min a reduction in HVCN1 levels had commenced ( p = 0.002) ( Figure 2 a). As TNF-α/fMLP stimulation can cause degranulation and the release of proteolytic enzymes to the outside of the cell [ 29 , 41 , 42 ], a systematic approach was adopted to investigate the possible cleavage of HVCN1 from plasma membranes and to categorize the proteases involved. Nonspecific protease inhibitors targeting the main protease families were employed including E64, an inhibitor of cysteine proteases, EDTA a general metalloprotease inhibitor, and pefabloc a potent serine protease inhibitor [ 43 ].…”

“…Phagocytic protease and peroxidase activity is supported by changes in phagosomal pH, governed by HVCN1 activity [ 51 ]. Alkalinization of phagocytic vacuoles due to reduced levels of HVCN1 would support serine proteases activity [ 55 ], but would be less supportive of enzymes with low pH optima, including myeloperoxidase, azurocidin or α-defensin [ 41 , 56 , 57 ]. In line with this, killing of S. aureus by Hvcn1 -/- bone marrow cells is impaired [ 51 ].…”

In vitroandin vivomodulation of NADPH oxidase activity and reactive oxygen species production in human neutrophils by α1-antitrypsin

“…Blood neutrophils were isolated as previously described [ 31 ] and determined 98% pure by flow cytometric analysis employing a monoclonal antibody to CD16b [ 11 , 32 ]. Neutrophil viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and found to be >98%.…”