Fluorescence imaging of ATP in neutrophils from patients with sepsis using organelle-localizable fluorescent chemosensors

Sueyoshi, Koichiro; Sumi, Yasuo; Inoue, Yoshiaki; Kuroda, Yoko; Ishii, Kumiko; Nakayama, Hitoshi; Iwabuchi, Kazuhisa; Kurishita, Yasutaka; Shigemitsu, Hajime; Hamachi, Itaru; Tanaka, Hiroshi

doi:10.1186/s13613-016-0175-z

Cited by 6 publications

(5 citation statements)

References 40 publications

(46 reference statements)

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“…As noted earlier, ATP is present in PMN cells [17][18][19]. ATP is a damage-associated molecular pattern (DAMP) molecule, and is associated with PMN chemoattraction, phagocytosis and apoptosis.…”

Section: Plos Onementioning

confidence: 81%

A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells

et al. 2022

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Nociceptin/Orphanin FQ (N/OFQ) is the endogenous opioid agonist for the N/OFQ receptor or NOP. This receptor system is involved in pain processing but also has a role in immune regulation. Indeed, polymorphonuclear cells (PMNs) express mRNA for N/OFQ precursor and are a potential source for circulating N/OFQ. Current measurements are based on ELISA and RIA techniques. In this study we have designed a bioassay to measure N/OFQ release from single PMNs. Chinese Hamster Ovary (CHO) cells transfected with the human (h) NOP receptor and Gαiq5 chimera force receptor coupling in biosensor cells to increase intracellular Ca2+; this can be measured with FLUO-4 dye. If isolated PMNs from healthy human volunteers are layered next to CHOhNOPGαiq5 biosensor cells then stimulated with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) we hypothesise that released N/OFQ will activate the biosensor. PMNs also release ATP and CHO cells express purinergic receptors coupled to elevated Ca2+. In a system where these receptors (P2Y1, P2Y2 and P2X7) are blocked with high concentrations of PPADS and oATP, PMN stimulation with fMLP increases Ca2+ in PMNs then shortly afterwards the biosensor cells. Our data therfore reports detection of single cell N/OFQ release from immune cells. This was absent when cells were preincubated with the selective NOP antagonist; SB-612111. Collectively this is the first description of single cell N/OFQ release. We will deploy this assay with further purified individual cell types and use this to further study the role of the N/OFQ-NOP system in disease; in particular sepsis where there is strong evidence for increased levels of N/OFQ worsening outcome.

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Section: Plos Onementioning

confidence: 81%

A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells

et al. 2022

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“…A higher plasma level of adenosine 5′-triphosphate (ATP) is reported in sepsis patients and in animal models of sepsis181–183 (Figure 3B). Furthermore, higher intracellular ATP is also observed in neutrophils isolated from sepsis patients 184. Further study in this direction has indicated that higher levels of systemic ATP molecules cause an impairment in neutrophil chemotaxis and host defense 185.…”

Section: Inflammasomes In the Immunopathogenesis Of Sepsismentioning

confidence: 97%

Inflammasomes: Pandora’s box for sepsis

Kumar

2018

JIR

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Sepsis was known to ancient Greeks since the time of great physician Hippocrates (460–377 BC) without exact information regarding its pathogenesis. With time and medical advances, it is now considered as a condition associated with organ dysfunction occurring in the presence of systemic infection as a result of dysregulation of the immune response. Still with this advancement, we are struggling for the development of target-based therapeutic approach for the management of sepsis. The advancement in understanding the immune system and its working has led to novel discoveries in the last 50 years, including different pattern recognition receptors. Inflammasomes are also part of these novel discoveries in the field of immunology which are <20 years old in terms of their first identification. They serve as important cytosolic pattern recognition receptors required for recognizing cytosolic pathogens, and their pathogen-associated molecular patterns play an important role in the pathogenesis of sepsis. The activation of both canonical and non-canonical inflammasome signaling pathways is involved in mounting a proinflammatory immune response via regulating the generation of IL-1β, IL-18, IL-33 cytokines and pyroptosis. In addition to pathogens and their pathogen-associated molecular patterns, death/damage-associated molecular patterns and other proinflammatory molecules involved in the pathogenesis of sepsis affect inflammasomes and vice versa. Thus, the present review is mainly focused on the inflammasomes, their role in the regulation of immune response associated with sepsis, and their targeting as a novel therapeutic approach.

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“…ATP breakdown by apyrase was verified in HBSS as well as in LB medium using a luciferase‐based ATP bioluminescence assay kit (Thermo Fisher Scientific). The cell‐surface targeting fluorescent ATP probe 2‐2Zn (kind gift from Dr. Itaru Hamachi, Kyoto University, Kyoto, Japan) and flow cytometry were used for real‐time monitoring of ATP release from individual cells . PMNs were suspended in HBSS containing 1% heparinized human plasma and stained with 2‐2Zn (500 nM) for 5 min.…”

Section: Methodsmentioning

confidence: 99%

Frontline Science: Escherichia coli use LPS as decoy to impair neutrophil chemotaxis and defeat antimicrobial host defense

Kondo

Ledderose

Slubowski

et al. 2019

Journal of Leukocyte Biology

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Bacterial infections and sepsis are leading causes of morbidity and mortality in critically ill patients. Currently, there are no effective treatments available to improve clinical outcome in sepsis. Here, we elucidated a mechanism by which Escherichia coli (E. coli) bacteria impair neutrophil (PMN) chemotaxis and we studied whether this mechanism can be therapeutically targeted to improve chemotaxis and antimicrobial host defense. PMNs detect bacteria with formyl peptide receptors (FPR). FPR stimulation triggers mitochondrial ATP production and release. Autocrine stimulation of purinergic receptors exerts excitatory and inhibitory downstream signals that induce cell polarization and cell shape changes needed for chemotaxis. Here we show that the bacterial cell wall product LPS dose-dependently impairs PMN chemotaxis. Exposure of human PMNs to LPS triggered excessive mitochondrial ATP production and disorganized intracellular trafficking of mitochondria, resulting in global ATP release that disrupted purinergic signaling, cell polarization, and chemotaxis. In mice infected i.p. with E. coli, LPS treatment increased the spread of bacteria at the infection site and throughout the systemic circulation. Removal of excessive systemic ATP with apyrase improved chemotaxis of LPS-treated human PMNs in vitro and enhanced the clearance of E. coli in infected and LPS-treated mice. We conclude that systemic ATP accumulation in response to LPS is a potential therapeutic target to restore PMN chemotaxis and to boost the antimicrobial host immune defense in sepsis. K E Y W O R D S apyrase, ATP release, bacterial clearance, endotoxin, purinergic signaling 1 INTRODUCTION ATP is an energy carrier that fuels virtually all cellular processes. Damaged and dying cells release ATP into the extracellular space, where ATP acts as a "danger signal" that promotes inflammation and regulates immune responses. 1,2 Elevated plasma ATP levels in the peripheral circulation of trauma and critical care patients may contribute to the systemic inflammatory response and multiple-organ dysfunction syndrome that are the hallmarks of sepsis. 3-5

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Fluorescence imaging of ATP in neutrophils from patients with sepsis using organelle-localizable fluorescent chemosensors

Cited by 6 publications

References 40 publications

A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells

A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells

Inflammasomes: Pandora’s box for sepsis

Frontline Science: Escherichia coli use LPS as decoy to impair neutrophil chemotaxis and defeat antimicrobial host defense

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Fluorescence imaging of ATP in neutrophils from patients with sepsis using organelle-localizable fluorescent chemosensors

Cited by 6 publications

References 40 publications

A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells

A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells

Inflammasomes: Pandora&rsquo;s box for sepsis

Frontline Science: Escherichia coli use LPS as decoy to impair neutrophil chemotaxis and defeat antimicrobial host defense

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Inflammasomes: Pandora’s box for sepsis