Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Foote, Juliet R.; Levine, Adam P.; Béhé, Philippe; Duchen, Michael R.; Segal, Anthony W.

doi:10.3791/55107

Cited by 9 publications

(14 citation statements)

References 31 publications

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“…An acidic environment in phagosomes directly aids in bacterial killing and is also important for the activation of pH-sensitive antimicrobial enzymes ( Sedlyarov et al, 2018 ; Flannagan et al, 2015 ). To determine if the HBC phagosome becomes acidic during maturation, we profiled the uptake of zymosan particles tagged with a pH-sensitive probe (Carboxy SNARF-1 [S-1]; Foote et al, 2017 , 2019 ). After allowing phagocytosis for 20 min, we find that the HBC phagosome becomes rapidly acidic, reaching a pH of ∼4.5 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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Phenotypic and functional characterization of first-trimester human placental macrophages, Hofbauer cells

Thomas

Appios

Zhao

et al. 2020

Journal of Experimental Medicine

124

166

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Hofbauer cells (HBCs) are a population of macrophages found in high abundance within the stroma of the first-trimester human placenta. HBCs are the only fetal immune cell population within the stroma of healthy placenta. However, the functional properties of these cells are poorly described. Aligning with their predicted origin via primitive hematopoiesis, we find that HBCs are transcriptionally similar to yolk sac macrophages. Phenotypically, HBCs can be identified as HLA-DR−FOLR2+ macrophages. We identify a number of factors that HBCs secrete (including OPN and MMP-9) that could affect placental angiogenesis and remodeling. We determine that HBCs have the capacity to play a defensive role, where they are responsive to Toll-like receptor stimulation and are microbicidal. Finally, we also identify a population of placenta-associated maternal macrophages (PAMM1a) that adhere to the placental surface and express factors, such as fibronectin, that may aid in repair.

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Section: Resultsmentioning

confidence: 99%

“…Placental macrophage phagosomal pH measurements were performed by adapting a method using the fluorescent-sensitive pH dye S-1 with a dual-emission spectrum ( Foote et al, 2017 ). Carboxy S-1-succinimidyl ester (Thermo Fisher Scientific) was coupled to zymosan coated beads (Thermo Fisher Scientific) and opsonized with human serum.…”

Section: Methodsmentioning

confidence: 99%

Phenotypic and functional characterization of first-trimester human placental macrophages, Hofbauer cells

Thomas

Appios

Zhao

et al. 2020

Journal of Experimental Medicine

124

166

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“…We confirmed previous findings (13) that R19-S does not react with H 2 O 2 or with MPO in the absence of chloride, and observed that the fluorescence yield was similar when HOCl was added as a reagent or was generated by MPO, chloride, and an equivalent concentration of H 2 O 2 (not shown). It has recently been reported that a pH of 8.5-9 is achieved within neutrophil phagosomes (12). We therefore tested the reactivity of HOCl with R19-S at a higher pH.…”

Section: Reactivity Of R19-s With Other Hypohalous Acids and Haloaminesmentioning

confidence: 99%

“…Several studies involving analysis of phagosomal contents provide evidence that HOCl is produced (3, 6 -10) but there is debate about how much, whether this is sufficient to kill, and whether it does so directly. Others query whether HOCl is produced at all (11,12). To gain a greater understanding of how MPO functions during phagocytosis, real-time monitoring of HOCl at intracellular sites is needed.…”

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confidence: 99%

Heterogeneity of hypochlorous acid production in individual neutrophil phagosomes revealed by a rhodamine-based probe

Albrett¹,

Ashby²,

Dickerhof

et al. 2018

Journal of Biological Chemistry

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The rhodamine-based probe R19-S has been shown to react with hypochlorous acid (HOCl) to yield fluorescent R19, but not with some other oxidants including hydrogen peroxide. Here, we further examined the specificity of R19-S and used it for real-time monitoring of HOCl production in neutrophil phagosomes. We show that it also reacts rapidly with hypobromous acid, bromamines, and hypoiodous acid, indicating that R19-S responds to these reactive halogen species as well as HOCl. Hypothiocyanous acid and taurine chloramine were unreactive, however, and ammonia chloramine and dichloramine reacted only very slowly. MS analyses revealed additional products from the reaction of HOCl with R19-S, including a chlorinated species as a minor product. Of note, phagocytosis of opsonized zymosan or by neutrophils was accompanied by an increase in R19 fluorescence. This increase depended on NADPH oxidase and myeloperoxidase activities, and detection of chlorinated R19-S confirmed its specificity for HOCl. Using live-cell imaging to track individual phagosomes in single neutrophils, we observed considerable heterogeneity among the phagosomes in the time from ingestion of a zymosan particle to when fluorescence was first detected, ranging from 1 to>30 min. However, once initiated, the subsequent fluorescence increase was uniform, reaching a similar maximum in ∼10 min. Our results confirm the utility of R19-S for detecting HOCl in real-time and provide definitive evidence that isolated neutrophils produce HOCl in phagosomes. The intriguing variability in the onset of HOCl production among phagosomes identified here could influence the way they kill ingested bacteria.

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“…The following protocol is described in more detail elsewhere 27 . The wells were washed twice with BSS buffer to remove non-adherent cells, then buffer containing 1 μg/ μl carboxy SNARF-1, AM ester acetate (ThermoFisher) was added for 25 minutes to label the cytosol, and then washed off with BSS buffer.…”

Section: Phagosomal Ph Measurementsmentioning

confidence: 99%

Variations in the phagosomal environment of human neutrophils and mononuclear phagocyte subsets

Foote

Patel

Yona

et al. 2018

Preprint

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count: 183 Text count: 3076 Figure count: 4 Reference count: 59 AbstractThe phagosomal microenvironment has a major influence on the enzyme activity and biology within these organelles. Here we directly compared the phagosomal pH of human neutrophils, monocytes, dendritic cell (DC) and monocyte-derived cells. An 2 unexpected observation was the striking difference in phagosomal environment between the three monocytes subsets. Classical monocytes and neutrophils had alkaline phagosomes, yet non-classical monocytes had more acid phagosomes.Intermediate monocytes had an intermediate phenotype. We next investigated the differences between primary DCs versus in vitro monocyte-derived DCs and established that both these cells had acidic phagosomal environments. We also confirmed reports of an alkaline phagosome in "M1" macrophages, and an acidic one in "M2" macrophages. Across all phagocytes, alkalinisation was dependent upon the activity of the NADPH oxidase, as when it was absent in cells from a patient with chronic granulomatous disease (CGD) or was abolished by an inhibitor of the oxidase, diphenyleneiodonium (DPI). An increased alkalinity in the phagosome was associated with more oxidase activity. These data highlight the heterogenous nature of phagocytic vacuoles within the family of mononuclear phagocytes that will dictate the function of these compartments. Key points:-Phagosomal function depends upon the action of enzymes released into them from cytoplasmic granules.-The substantial differences in the phagosomal pH in the different phagocytes will affect their compliment of enzymes and their functions.

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Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Cited by 9 publications

References 31 publications

Phenotypic and functional characterization of first-trimester human placental macrophages, Hofbauer cells

Phenotypic and functional characterization of first-trimester human placental macrophages, Hofbauer cells

Heterogeneity of hypochlorous acid production in individual neutrophil phagosomes revealed by a rhodamine-based probe

Variations in the phagosomal environment of human neutrophils and mononuclear phagocyte subsets

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