The antidiabetic drug metformin blunts NETosis in vitro and reduces circulating NETosis biomarkers in vivo
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.
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.
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.
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.
KeywordsType 2 diabetes Pre-diabetes Inflammation Innate immunity
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.
Compliance with ethical standards
Conflict of interest
All the authors declare no conflict of interest.
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 was obtained from all patients for being included in the study.
- 20.Fadini GP, de Kreutzenberg SV, Mariano V et al (2011) Optimized glycaemic control achieved with add-on basal insulin therapy improves indexes of endothelial damage and regeneration in type 2 diabetic patients with macroangiopathy: a randomized crossover trial comparing detemir versus glargine. Diabetes Obes Metab 13:718–725CrossRefPubMedGoogle Scholar
- 26.Tay HM, Dalan R, Li KHH, Boehm BO, Hou HW (2018) A novel microdevice for rapid neutrophil purification and phenotyping in type 2 diabetes mellitus. Small 14(6). https://doi.org/10.1002/smll.201702832
- 28.Wu L, Zhou B, Oshiro-Rapley N et al (2016) An ancient, unified mechanism for metformin growth inhibition in C. elegans and cancer. Cell 167(1705–1718):e1713Google Scholar
- 29.Amulic B, Knackstedt SL, Abu Abed U et al (2017) Cell-cycle proteins control production of neutrophil extracellular traps. Dev Cell 43(449–462):e445Google Scholar