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The antidiabetic drug metformin blunts NETosis in vitro and reduces circulating NETosis biomarkers in vivo

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Abstract

Aims

Diabetes is associated with an excess release of neutrophil extracellular traps (NETs) and an enhanced NETosis, a neutrophil cell death programme instrumental to anti-microbial defences, but also involved in tissue damage. We herein investigated whether the antidiabetic drug metformin protects against NETosis.

Methods

We measured NET components in the plasma of patients with pre-diabetes who were randomized to receive metformin or placebo for 2 months. To control for the effect on glucose, we also measured NET components in the plasma of patients with type 2 diabetes before and after treatment with insulin or dapagliflozin. In vitro, we used static and dynamic imaging with advanced live confocal two-photon microscopy to evaluate the effects of metformin on cellular events during NETosis. We examined putative molecular mechanisms by monitoring chromatin decondensation and DNA release in vitro.

Results

Metformin, as compared to placebo, significantly reduced the concentrations of NET components elastase, proteinase-3, histones and double strand DNA, whereas glucose control with insulin or dapagliflozin exerted no significant effect. In vitro, metformin prevented pathologic changes in nuclear dynamics and DNA release, resulting in a blunted NETosis in response to phorbol myristate acetate and calcium influx. Metformin prevented membrane translocation of PKC-βII and activation of NADPH oxidase in neutrophils, both of which diminished the NETosis response.

Conclusions

Metformin treatment reduced the concentrations of NET components independently from glucose control. This effect was reproducible in vitro and was related to the inhibitory effect exerted by metformin on the PKC-NADPH oxidase pathway.

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Funding

The study was supported by grants from: the Italian Ministry of Health grant to MA (RF-2013-02358024); the University of Padova 2011 Strategic Project DYCENDI Grant to AA; The University of Padova STARS Grant to GPF; the European Foundation for the Study of Diabetes (EFSD)/Lilly Grant 2016 to GPF; an AstraZeneca Grant to AA. The sponsors had not role in study design, data analysis and interpretation and decision to publish.

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Author notes

  1. Lisa Menegazzo and Valentina Scattolini have contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine, University of Padova, Via Giustiniani 2, 35128, Padua, Italy

    Lisa Menegazzo, Valentina Scattolini, Roberta Cappellari, Benedetta Maria Bonora, Mattia Albiero, Giulio Ceolotto, Saula Vigili de Kreutzeberg, Angelo Avogaro & Gian Paolo Fadini

  2. Venetian Institute of Molecular Medicine, 35129, Padua, Italy

    Lisa Menegazzo, Valentina Scattolini, Roberta Cappellari, Benedetta Maria Bonora, Mattia Albiero, Mario Bortolozzi & Gian Paolo Fadini

  3. Department of Physics and Astronomy, University of Padova, 35131, Padua, Italy

    Mario Bortolozzi & Filippo Romanato

  4. IOM-CNR, ss.14 km 163.5, 34149, Basovizza, Trieste, Italy

    Filippo Romanato

Authors
  1. Lisa Menegazzo
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  2. Valentina Scattolini
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  3. Roberta Cappellari
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  4. Benedetta Maria Bonora
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  5. Mattia Albiero
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  6. Mario Bortolozzi
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  7. Filippo Romanato
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  8. Giulio Ceolotto
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  9. Saula Vigili de Kreutzeberg
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  10. Angelo Avogaro
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  11. Gian Paolo Fadini
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Corresponding author

Correspondence to Gian Paolo Fadini.

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Conflict of interest

All the authors 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

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

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

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Menegazzo, L., Scattolini, V., Cappellari, R. et al. The antidiabetic drug metformin blunts NETosis in vitro and reduces circulating NETosis biomarkers in vivo. Acta Diabetol 55, 593–601 (2018). https://doi.org/10.1007/s00592-018-1129-8

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  • DOI: https://doi.org/10.1007/s00592-018-1129-8

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