Measurement of phagosomal pH of normal and CGD‐like human neutrophils by dual fluorescence flow cytometry

Dri, Pietro; Presani, E.; Perticarari, Sandra; Albèri, Lavinia; Prodan, Mario; Decleva, Eva

doi:10.1002/cyto.10123

Cited by 35 publications

(29 citation statements)

References 21 publications

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“…In the present study, however, DPI treatment of Trpm2 −/− mBMDMs failed to alter significantly phagosomal pH, indicating that the increased pH seen in Trpm2 −/− mBMDMs was caused by TRPM2 deletion rather than increased ROS production secondary to TRPM2 deletion (Di et al, 2011). Thus, the dysregulated phagosomal pH in Trpm2 −/− BMDMs, as opposed to the overproduction of ROS, was essential for the phagosomal killing of bacteria as was shown to be the case for CDG neutrophils (Dri et al, 2002;Geiszt et al, 2001;Henriet et al, 2013;Segal et al, 1981;Thrasher and Segal, 2011).…”

Section: (N=3) *P<005 (T-test) (Fg) Adpr Antagonism Increases Phasupporting

confidence: 39%

“…The role of the ROS and NOX pathway in intra-phagosomal bacterial killing has been proposed in part based on findings in chronic granulomatous disease (CGD) (Pollock et al, 1995;Segal, 1996;Segal et al, 2000). CDG is characterized by severe, protracted and fatal infection resulting from the failure of the NOX system to produce superoxide (O 2 − ) and other ROS in neutrophils (Pollock et al, 1995;Segal, 1996;Segal et al, 2000); however, it was later discovered that defective bacterial killing in CGD neutrophils was secondary to dysregulated phagosomal acidification mechanism (Dri et al, 2002;Geiszt et al, 2001;Henriet et al, 2013;Segal et al, 1981;Thrasher and Segal, 2011). We previously showed that TRPM2 inhibited NOX-mediated ROS generation, specifically that there was overproduction of ROS in Trpm2 −/− macrophages infected with E. coli (Di et al, 2011).…”

Section: (N=3) *P<005 (T-test) (Fg) Adpr Antagonism Increases Phamentioning

confidence: 99%

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Role of the phagosomal redox-sensitive TRP channel TRPM2 in regulating bactericidal activity of macrophages

Kiya

Gong

et al. 2017

Journal of Cell Science

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Acidification of macrophage phagosomes serves an important bactericidal function. We show here that the redox-sensitive transient receptor potential (TRP) cation channel TRPM2 is expressed in the phagosomal membrane and regulates macrophage bactericidal activity through the activation of phagosomal acidification. Measurement of the TRPM2 current in phagosomes identified TRPM2 as a functional redox-sensitive cation channel localized in the phagosomal membrane. Simultaneous measurements of phagosomal Ca 2+ changes and phagosome acidification in macrophages undergoing phagocytosis demonstrated that TRPM2 was required to mediate the efflux of cations and for phagosomal acidification during the process of phagosome maturation. Acidification in phagosomes was significantly reduced in macrophages isolated from Trpm2 −/− mice as compared to wild type, and acidification was coupled to reduced bacterial clearance in Trpm2 −/− mice. Trpm2 +/+ macrophages treated with the vacuolar H + -ATPase inhibitor bafilomycin showed reduced bacterial clearance, similar to that in Trpm2 −/− macrophages. Direct activation of TRPM2 using adenosine diphosphate ribose (ADPR) induced both phagosomal acidification and bacterial killing. These data collectively demonstrate that TRPM2 regulates phagosomal acidification, and is essential for the bacterial killing function of macrophages.

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Section: (N=3) *P<005 (T-test) (Fg) Adpr Antagonism Increases Phasupporting

confidence: 39%

Section: (N=3) *P<005 (T-test) (Fg) Adpr Antagonism Increases Phamentioning

confidence: 99%

Role of the phagosomal redox-sensitive TRP channel TRPM2 in regulating bactericidal activity of macrophages

Kiya

Gong

et al. 2017

Journal of Cell Science

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“…It has been reported that the phagosomal pH undergoes a biphasic change, that is a quick and short lasting alkalinization (nearly to pH 7.6) followed by a slower acidification (nearly to pH 6.5) (26,28). Although the fluorescein green emission is expected to decrease at acidic pH (see reference 6 and references cited therein), under our experimental conditions such a decrease, if occurs, does not seem to interfere with the 5(6)-FAM-SE green fluorescence of ingested C. albicans particles.…”

Section: Discussionmentioning

confidence: 99%

“…albicans cells, obtained from a clinical isolate from the microbiology laboratory of the pediatric hospital IRCCS Burlo Garofolo (Trieste, Italy), were grown in Sabouraud dextrose broth at 30°C for 18 -20 h. Labeling was performed according to the method described by Dri et al (26), with slight modifications. After growing, yeast cells were washed twice at 2,000g for 7 min in physiologic saline and heat killed by boiling for 10 min.…”

Section: Opsonization and Labeling Of C Albicansmentioning

confidence: 99%

A single‐step, sensitive flow cytofluorometric assay for the simultaneous assessment of membrane‐bound and ingested Candida albicans in phagocytosing neutrophils

et al. 2004

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Background: Distinguishing ingested particles from those attached to the cell surface is an essential requirement when performing quantitative studies of phagocytosis. In the present report, we describe a simple, sensitive and reliable flow cytofluorometric method that achieves this goal in a Candida albicans-human neutrophils (PMN) system. Methods: The assay is based on the observation that the vital dye trypan blue (TB), while quenching the green fluorescence of fluorescein-labeled C. albicans, causes them to fluoresce red. PMN were incubated with fluorescein-labeled yeast particles for the required time. Aliquots of the incubation mixtures were then promptly diluted with an equal volume of a TB solution at pH 4.0, and subsequently analyzed by flow cytometry for green and red fluorescence.

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“…Labeling of C. albicans blastospores was performed according to the method described by Dri et al (29), with slight modifications. Serumopsonized yeast particles were diluted in NaHCO 3 buffer (0.1 M, pH 8.3), washed and suspended in the same medium (300 ϫ 10 6 /ml) containing 1.5 g/ml 5(6)-FAM-SE to start labeling.…”

Section: Labeling Of C Albicansmentioning

confidence: 99%

Chloride Movements in Human Neutrophils during Phagocytosis: Characterization and Relationship to Granule Release

Busetto

Trevisan

Decleva

et al. 2007

The Journal of Immunology

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Chloride ion efflux is an early event occurring after exposure of human neutrophils to several soluble agonists. Under these circumstances, a rapid and reversible fall in the high basal intracellular chloride (Cl−i) levels is observed. This event is thought to play a crucial role in the modulation of several critical neutrophil responses including activation and up-regulation of adhesion molecules, cell attachment and spreading, cytoplasmic alkalinization, and activation of the respiratory burst. At present, however, no data are available on chloride ion movements during neutrophil phagocytosis. In this study, we provide evidence that phagocytosis of Candida albicans opsonized with either whole serum, complement-derived opsonins, or purified human IgG elicits an early and long-lasting Cl− efflux accompanied by a marked, irreversible loss of Cl−i. Simultaneous assessment of Cl− efflux and phagocytosis in cytochalasin D-treated neutrophils indicated that Cl− efflux occurs without particle ingestion. These results suggest that engagement of immune receptors is sufficient to promote chloride ion movements. Several structurally unrelated chloride channel blockers inhibited phagocytosis-induced Cl− efflux as well as the release of azurophilic—but not specific—granules. It implicates that different neutrophil secretory compartments display distinct sensitivity to Cl−i modifications. Intriguingly, inhibitors of Cl− exchange inhibited cytosolic Ca2+ elevation, whereas Cl− efflux was not impaired in Ca2+-depleted neutrophils. We also show that FcγR(s)- and CR3/CR1-mediated Cl− efflux appears to be dependent on protein tyrosine phosphorylation but independent of PI3K and phospholipase C activation.

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Measurement of phagosomal pH of normal and CGD‐like human neutrophils by dual fluorescence flow cytometry

Cited by 35 publications

References 21 publications

Role of the phagosomal redox-sensitive TRP channel TRPM2 in regulating bactericidal activity of macrophages

Role of the phagosomal redox-sensitive TRP channel TRPM2 in regulating bactericidal activity of macrophages

A single‐step, sensitive flow cytofluorometric assay for the simultaneous assessment of membrane‐bound and ingested Candida albicans in phagocytosing neutrophils

Chloride Movements in Human Neutrophils during Phagocytosis: Characterization and Relationship to Granule Release

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