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NETosis is induced by high glucose and associated with type 2 diabetes

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Abstract

Aims

The role of neutrophils in diabetes and its complications is unclear. Upon challenge with microbes and inflammatory triggers, neutrophils release enzymes and nuclear material, forming neutrophils extracellular traps (NETs) and thereby dying by NETosis. We herein tested NET formation and NETosis products in high glucose and in the setting of type 2 diabetes (T2D).

Methods

NETosis was assessed in vitro in cells exposed to 0, 5, 25 mM glucose and 25 mM mannitol, DMSO and PMA using immunofluorescence staining for elastase, DNA and chromatin. Single-cell morphometric analysis was used to detect enter of elastase in the nucleus and extrusion of nuclear material. Release of NETs was quantified by staining with Hoechst 33342. In 38 T2D and 38 age- and sex-matched non-diabetic individuals, we determined plasma elastase, mono- and oligonucleosomes and double-strand (ds) DNA, as circulating NETosis products.

Results

NETosis was accurately reproduced in vitro: high (25 mM) glucose increased NETosis rate and release of NETs compared with 5 mM glucose and 25 mM mannitol. T2D patients showed increased plasma elastase, mono- and oligonucleosomes and dsDNA compared with non-diabetic control individuals. A positive correlation was found between HbA1c and mono- and oligonucleosomes, whereas dsDNA was correlated with the presence of nephropathy and cardiovascular disease. Serum IL-6 concentrations were higher in T2D compared with CTRL and correlated with serum dsDNA levels.

Conclusions

High glucose and hyperglycemia increase release of NETs and circulating markers of NETosis, respectively. This finding provides a link among neutrophils, inflammation and tissue damage in diabetes.

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Abbreviations

ACR:

Albumin creatinine ratio

CAD:

Coronary artery disease

CerVD:

Cerebrovascular disease

CVD:

Cardiovascular disease

ds:

Double strand

ESM:

Electronic supplementary material

HRP:

Horseradish peroxidase

IL-6:

Interleukin-6

LOI:

Line of interest

MACE:

Major adverse cardiovascular events

NET:

Neutrophil extracellular trap

PAD:

Peripheral arterial disease

PFA:

Paraformaldehyde

PMA:

Phorbol 12-myristate 13-acetate

ROI:

Region of interest

T2D:

Type 2 diabetes

TNF-α:

Tumor necrosis factor alpha

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Acknowledgments

We acknowledge the technical assistance of Andrea Armani (Department of Biomedical Science, University of Padova) for dsDNA quantification. We thank Dr. Volker Brinkmann, Max Planck Institute for Infection Biology, Berlin, Germany for providing aliquots of the PL2-3 mAb.

Conflict of interest

Lisa Menegazzo, Stefano Ciciliot, Nicol Poncina, Marta Mazzucato, Mariasara Persano, Benedetta Bonora, Mattia Albiero, Saula Vigili de Kreutzenberg, Angelo Avogaro and Gian Paolo Fadini declare no conflict of interest.

Ethical standard

The study was approved by the local ethical committee and carried out in accordance with the principles of the Declaration of Helsinki as revised in 2008. All subjects provided written informed consent.

Human and animal rights disclosure

All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008.

Informed consent disclosure

Informed consent was obtained from all patients for being included in the study.

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Correspondence to Gian Paolo Fadini.

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Managed by Massimo Federici.

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Menegazzo, L., Ciciliot, S., Poncina, N. et al. NETosis is induced by high glucose and associated with type 2 diabetes. Acta Diabetol 52, 497–503 (2015). https://doi.org/10.1007/s00592-014-0676-x

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  • DOI: https://doi.org/10.1007/s00592-014-0676-x

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