Defective Neutrophil Function in Patients with Sepsis Is Mostly Restored by ex vivo Ascorbate Incubation

Kritsanawan Sae-khow,1 Sasipha Tachaboon,2 Helen L Wright,3 Steven W Edwards,4 Nattachai Srisawat,2 Asada Leelahavanichkul,1 Direkrit Chiewchengchol1 1Translational Research in Inflammation and Immunology Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; 2Excellence Ce...

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Autores principales:	Sae-khow K, Tachaboon S, Wright HL, Edwards SW, Srisawat N, Leelahavanichkul A, Chiewchengchol D
Formato:	article
Lenguaje:	EN
Publicado:	Dove Medical Press 2020
Materias:	ascorbate neutrophils sepsis Pathology RB1-214 Therapeutics. Pharmacology RM1-950
Acceso en línea:	https://doaj.org/article/56665d6db5904254b31bb56678ef5a8f
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spelling	oai:doaj.org-article:56665d6db5904254b31bb56678ef5a8f2021-12-02T10:01:53ZDefective Neutrophil Function in Patients with Sepsis Is Mostly Restored by ex vivo Ascorbate Incubation1178-7031https://doaj.org/article/56665d6db5904254b31bb56678ef5a8f2020-06-01T00:00:00Zhttps://www.dovepress.com/defective-neutrophil-function-in-patients-with-sepsis-is-mostly-restor-peer-reviewed-article-JIRhttps://doaj.org/toc/1178-7031Kritsanawan Sae-khow,1 Sasipha Tachaboon,2 Helen L Wright,3 Steven W Edwards,4 Nattachai Srisawat,2 Asada Leelahavanichkul,1 Direkrit Chiewchengchol1 1Translational Research in Inflammation and Immunology Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; 2Excellence Center for Critical Care Nephrology, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand; 3Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK; 4Institute of Integrative Biology, University of Liverpool, Liverpool, UKCorrespondence: Direkrit Chiewchengchol Email cdirekrit@live.comBackground: Neutrophil function is essential for effective defence against bacterial infections but is defective in patients with sepsis. Ascorbate or vitamin C, which is low in the plasma of patients with sepsis, is stored inside human neutrophils and is essential for their normal function.Objective: This study aimed to determine if ascorbate treatment ex vivo improved neutrophil function in patients with sepsis.Patients and Methods: Human blood neutrophils were isolated from 20 patients with sepsis and 20 healthy age-matched controls. Neutrophils were incubated with or without ascorbate (1, 5, 10, 20 and 40 mM) for periods up to 2h. Chemotaxis was evaluated using a chemotactic chamber in response to the chemoattractant, fMLP. Phagocytosis (uptake of pHrodo red stained S. aureus) and apoptosis (annexin-V/propidium iodide staining) were measured by flow cytometry. Neutrophil extracellular trap (NET) formation was detected and quantified using DAPI, anti-myeloperoxidase and anti-neutrophil elastase immuno-fluorescence staining. Quantifluor detected the amount of dsDNA in NET supernatants, while quantitative PCR identified changes in expression of PADI4 gene.Results: Chemotactic and phagocytic activities were decreased in patients with sepsis but increased after treatment with the high concentrations of ascorbate. Apoptosis was increased in the sepsis patients but not altered by ascorbate treatment. Spontaneous NET formation was observed in patients with sepsis. A quantity of 1mM ascorbate decreased spontaneous NETosis to that of normal, healthy neutrophils, while high concentrations of ascorbate (> 10mM) further promoted NET formation.Conclusion: Dysregulated neutrophil function was observed in patients with sepsis which could contribute to disease pathology and outcomes. Exposure to ascorbate could reverse some of these changes in function. These novel discoveries raise the possibility that ascorbate treatment could be used as an adjunctive therapy that could result in improved neutrophil function during sepsis.Keywords: ascorbate, neutrophils, sepsisSae-khow KTachaboon SWright HLEdwards SWSrisawat NLeelahavanichkul AChiewchengchol DDove Medical PressarticleascorbateneutrophilssepsisPathologyRB1-214Therapeutics. PharmacologyRM1-950ENJournal of Inflammation Research, Vol Volume 13, Pp 263-274 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	ascorbate neutrophils sepsis Pathology RB1-214 Therapeutics. Pharmacology RM1-950
spellingShingle	ascorbate neutrophils sepsis Pathology RB1-214 Therapeutics. Pharmacology RM1-950 Sae-khow K Tachaboon S Wright HL Edwards SW Srisawat N Leelahavanichkul A Chiewchengchol D Defective Neutrophil Function in Patients with Sepsis Is Mostly Restored by ex vivo Ascorbate Incubation
description	Kritsanawan Sae-khow,1 Sasipha Tachaboon,2 Helen L Wright,3 Steven W Edwards,4 Nattachai Srisawat,2 Asada Leelahavanichkul,1 Direkrit Chiewchengchol1 1Translational Research in Inflammation and Immunology Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; 2Excellence Center for Critical Care Nephrology, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand; 3Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK; 4Institute of Integrative Biology, University of Liverpool, Liverpool, UKCorrespondence: Direkrit Chiewchengchol Email cdirekrit@live.comBackground: Neutrophil function is essential for effective defence against bacterial infections but is defective in patients with sepsis. Ascorbate or vitamin C, which is low in the plasma of patients with sepsis, is stored inside human neutrophils and is essential for their normal function.Objective: This study aimed to determine if ascorbate treatment ex vivo improved neutrophil function in patients with sepsis.Patients and Methods: Human blood neutrophils were isolated from 20 patients with sepsis and 20 healthy age-matched controls. Neutrophils were incubated with or without ascorbate (1, 5, 10, 20 and 40 mM) for periods up to 2h. Chemotaxis was evaluated using a chemotactic chamber in response to the chemoattractant, fMLP. Phagocytosis (uptake of pHrodo red stained S. aureus) and apoptosis (annexin-V/propidium iodide staining) were measured by flow cytometry. Neutrophil extracellular trap (NET) formation was detected and quantified using DAPI, anti-myeloperoxidase and anti-neutrophil elastase immuno-fluorescence staining. Quantifluor detected the amount of dsDNA in NET supernatants, while quantitative PCR identified changes in expression of PADI4 gene.Results: Chemotactic and phagocytic activities were decreased in patients with sepsis but increased after treatment with the high concentrations of ascorbate. Apoptosis was increased in the sepsis patients but not altered by ascorbate treatment. Spontaneous NET formation was observed in patients with sepsis. A quantity of 1mM ascorbate decreased spontaneous NETosis to that of normal, healthy neutrophils, while high concentrations of ascorbate (> 10mM) further promoted NET formation.Conclusion: Dysregulated neutrophil function was observed in patients with sepsis which could contribute to disease pathology and outcomes. Exposure to ascorbate could reverse some of these changes in function. These novel discoveries raise the possibility that ascorbate treatment could be used as an adjunctive therapy that could result in improved neutrophil function during sepsis.Keywords: ascorbate, neutrophils, sepsis
format	article
author	Sae-khow K Tachaboon S Wright HL Edwards SW Srisawat N Leelahavanichkul A Chiewchengchol D
author_facet	Sae-khow K Tachaboon S Wright HL Edwards SW Srisawat N Leelahavanichkul A Chiewchengchol D
author_sort	Sae-khow K
title	Defective Neutrophil Function in Patients with Sepsis Is Mostly Restored by ex vivo Ascorbate Incubation
title_short	Defective Neutrophil Function in Patients with Sepsis Is Mostly Restored by ex vivo Ascorbate Incubation
title_full	Defective Neutrophil Function in Patients with Sepsis Is Mostly Restored by ex vivo Ascorbate Incubation
title_fullStr	Defective Neutrophil Function in Patients with Sepsis Is Mostly Restored by ex vivo Ascorbate Incubation
title_full_unstemmed	Defective Neutrophil Function in Patients with Sepsis Is Mostly Restored by ex vivo Ascorbate Incubation
title_sort	defective neutrophil function in patients with sepsis is mostly restored by ex vivo ascorbate incubation
publisher	Dove Medical Press
publishDate	2020
url	https://doaj.org/article/56665d6db5904254b31bb56678ef5a8f
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Defective Neutrophil Function in Patients with Sepsis Is Mostly Restored by ex vivo Ascorbate Incubation

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