NETosis is induced by high glucose and associated with type 2 diabetes.
OBJECTIVE
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.
Department of Medicine, University of Padova, Via Giustiniani, 2, 35128, Padua, Italy.
Lisa
L
Menegazzo
Stefano
S
Ciciliot
Nicol
N
Poncina
Marta
M
Mazzucato
Mariasara
M
Persano
Benedetta
B
Bonora
Mattia
M
Albiero
Saula
S
Vigili de Kreutzenberg
Angelo
A
Avogaro
Gian Paolo
GP
Fadini
D000368:Aged; D017209:Apoptosis; D016022:Case-Control Studies; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism* / Q000503:physiopathology*; D065206:Extracellular Traps / Q000378:metabolism*; D005260:Female; D005947:Glucose / Q000378:metabolism* / Q000633:toxicity; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009504:Neutrophils / Q000378:metabolism / Q000473:pathology*
D015850:Interleukin-6; D005947:Glucose