An improved flow cytometry assay to monitor phagosome acidification

Colas, Chloé; Menezes, Shinelle; Gutiérrez-Martínez, Enric; Péan, Claire B.; Dionne, Marc S.; Guermonprez, Pierre

doi:10.1016/j.jim.2014.06.008

Cited by 22 publications

(19 citation statements)

References 55 publications

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“…Although a variety of pH sensors121631 exist, most of the studies performed use fluorescence microscopy and the accumulation of fluorescence in cells is heterogeneous and difficult for large scale measurements such as flow cytometry. In this contribution we demonstrate how PSFC can be used to detect fluorescence lifetime-dependent changes and that those changes correlate to gradual bacteria-macrophage interactions.…”

Section: Discussionmentioning

confidence: 99%

“…nonuniform fluorescence emission) or when the fluorescence emission is measured at a range of continuous times as pH changes. With pH-indicator dyes the total fluorescence emission is not as quantitative because the total increase in fluorescence might be a result of the gradual phagosomal acidification or the increase in the number of phagosomes formed161819202122. Moreover, bacterial species might escape the phagosome owing to their ability to withstand the acidic microenvironment, as shown with Förster Resonance Energy Transfer (FRET) experiments23.…”

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confidence: 99%

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Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

Houston

2017

Sci Rep

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Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

Houston

2017

Sci Rep

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“…Because thiostrepton, similar to bafilomycin A1, is an antibiotic derived from streptomycetes, we were interested to know whether thiostrepton would also exhibit endosomal acidification inhibitory activity. pHrodo Green dextran emits pH-sensitive fluorescence that increases in intensity with increasing acidity (54). To evaluate the effects of thiostrepton and to compare these with the effects of bortezomib and bafilomycin A1, cells were treated with these compounds and subsequently with pHrodo Green dextran.…”

Section: Thiostrepton Contains Dual Functions Of Preventing Endosomalmentioning

confidence: 99%

Identification of Thiostrepton as a Novel Inhibitor for Psoriasis-like Inflammation Induced by TLR7–9

Lai

Yeh

et al. 2015

The Journal of Immunology

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Activation of TLR7–9 has been linked to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and psoriasis. Thus, therapeutic applications of antagonists of these TLRs for such disorders are being investigated. Bortezomib (Velcade) is a proteasome inhibitor known to suppress activation of these TLRs. To identify novel TLR7–9 inhibitors, we searched the Gene Expression Omnibus database for gene expression profiles of bortezomib-treated cells. These profiles were then used to screen the Connectivity Map database for chemical compounds with similar functions as bortezomib. A natural antibiotic, thiostrepton, was identified for study. Similar to bortezomib, thiostrepton effectively inhibits TLR7–9 activation in cell-based assays and in dendritic cells. In contrast to bortezomib, thiostrepton does not inhibit NF-κB activation induced by TNF-α, IL-1, and other TLRs, and it is less cytotoxic to dendritic cells. Thiostrepton inhibits TLR9 localization in endosomes for activation via two mechanisms, which distinguish it from currently used TLR7–9 inhibitors. One mechanism is similar to the proteasome inhibitory function of bortezomib, whereas the other is through inhibition of endosomal acidification. Accordingly, in different animal models, thiostrepton attenuated LL37- and imiquimod-induced psoriasis-like inflammation. These results indicated that thiostrepton is a novel TLR7–9 inhibitor, and compared with bortezomib, its inhibitory effect is more specific to these TLRs, suggesting the potential therapeutic applications of thiostrepton on immunologic disorders elicited by inappropriate activation of TLR7–9.

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“…1 C and D) and localized in acidic organelles, such as lysosomes. This chemical pH indicator-based phagocytosis assay has been widely used because of its specificity and convenience for measuring the phagocytic activity (20)(21)(22). Compared with the negative control, adding pHrodo-conjugated synaptosomes significantly increased the astrocyte population with strong pHrodo intensity (Fig.…”

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confidence: 98%

Novel allele-dependent role for APOE in controlling the rate of synapse pruning by astrocytes

Chung

Verghese

Chakraborty

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

185

151

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The strongest genetic risk factor influencing susceptibility to lateonset Alzheimer's disease (AD) is apolipoprotein E (APOE) genotype. APOE has three common isoforms in humans, E2, E3, and E4. The presence of two copies of the E4 allele increases risk by ∼12-fold whereas E2 allele is associated with an ∼twofold decreased risk for AD. These data put APOE central to AD pathophysiology, but it is not yet clear how APOE alleles modify AD risk. Recently we found that astrocytes, a major central nervous system cell type that produces APOE, are highly phagocytic and participate in normal synapse pruning and turnover. Here, we report a novel role for APOE in controlling the phagocytic capacity of astrocytes that is highly dependent on APOE isoform. APOE2 enhances the rate of phagocytosis of synapses by astrocytes, whereas APO4 decreases it. We also found that the amount of C1q protein accumulation in hippocampus, which may represent the accumulation of senescent synapses with enhanced vulnerability to complement-mediated degeneration, is highly dependent on APOE alleles: C1q accumulation was significantly reduced in APOE2 knock-in (KI) animals and was significantly increased in APOE4 KI animals compared with APOE3 KI animals. These studies reveal a novel allele-dependent role for APOE in regulating the rate of synapse pruning by astrocytes. They also suggest the hypothesis that AD susceptibility of APOE4 may originate in part from defective phagocytic capacity of astrocytes which accelerates the rate of accumulation of C1q-coated senescent synapses, enhancing synaptic vulnerability to classical-complement-cascade mediated neurodegeneration.A lzheimer's disease (AD), the most common cause of dementia, is characterized clinically by a progressive and irreversible loss of cognitive functions. The neuropathological hallmarks of AD include profound synaptic loss and selective neuronal cell death, as well as the formation of extracellular amyloid beta plaques (Aβ) (1) and intracellular neurofibrillary tangles (2). Most cases of AD are the late-onset form, which develops after age 60. The causes of lateonset AD are not yet completely understood; however, genetic studies have shown that apolipoprotein E (APOE) alleles profoundly affect AD susceptibility, with APOE4 being the major genetic risk factor. APOE, a 299-amino acid lipid transport protein, has three common isoforms in humans-APOE2 (Cys-112 and -158), APOE3 (Cys-112 and Arg-158), and APOE4 (Arg-112 and -158)-that are products of alleles at a single gene locus (3, 4). The APOE3 is the most common and mediates intermediate risk for AD. The APOE4 allele is significantly overrepresented in lateonset AD patients and has a dosage effect on the risk and age of onset of AD (5), whereas APOE2 is protective against AD (6). Moreover, recent whole-genome sequencing of humans that age without developing diseases showed that there is a significant depletion of APOE4 alleles in the healthy aging population (7). How do different APOE isoforms influence the age of onset and course of d...

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An improved flow cytometry assay to monitor phagosome acidification

Cited by 22 publications

References 55 publications

Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

Identification of Thiostrepton as a Novel Inhibitor for Psoriasis-like Inflammation Induced by TLR7–9

Novel allele-dependent role for APOE in controlling the rate of synapse pruning by astrocytes

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