Identification of the factors that modulate neutrophil response towards NET formation during normal pregnancy and in gestational diabetes mellitus

Circulatory neutrophils exhibit an increased basal activation state in human pregnancy, which is overt in preeclampsia, a severe pregnancy specific disorder. Neutrophils play a defining role in combating infection either by phagocytosis or by degranulation; moreover, there have recently been shown to form neutrophil extracellular traps (NETs), containing extruded DNA, histones and granular proteins. The main activity of the NETs is the entrapment of bacteria, viruses and eukaryotic microorganisms, but more interestingly they seem to play a central role in the regulation of the overall inflammatory response. Pregnancy presents an extraordinary immunological challenge to the maternal immune system. Successful gestation to term, birth and postpartum survival require tolerance to the semi-allogeneic fetus while maintaining the potential to mount a vigorous response against infection. During normal pregnancy insulin sensitivity also declines with advancing gestation, while a compensatory increase in insulin maintains the normal glucose homeostasis. Gestational diabetes mellitus (GDM) is a condition of glucose intolerance first recognized during pregnancy resulting in hyperglycemia of variable severity. GDM develops when the pancreatic beta cells are unable to regulate the increased insulin demand. During pregnancy there is increased degranulation and phagocytic activity of circulating neutrophils indicates that these immune cells play a key role in pregnancy adaptations. The mechanisms remain unknown, though sex hormones provide likely candidates. We therefore investigated and characterized the neutrophil activity during normal pregnancies and in pregnancies affected with gestational diabetes. For this purpose, first we explored neutrophil activity during each trimester of normal pregnancy and in addition we studied the neutrophil response in pregnancies during and after the diagnosis of GDM. Neutrophils were isolated from EDTA blood, while products of NETosis were examined in serum and plasma. Cell-free circulating nucleosomes, myeloperoxidase (MPO), neutrophil elastase (NE), granulocyte-colony circulating factor (G- CSF) and tumor necrosis factor α (TNFα) were determined by ELISA. NET formation was detected by Sytox Green, a fluorescent non-permeable DNA dye and verified by immunofluorescence staining and morphometric analysis. The activated state of neutrophils was also evaluated by monitoring the reactive oxygen species generation. NET associated protein levels were analyzed by Western blots. Our primary results during normal pregnancy show that neutrophil numbers in the periphery increase during gestation, in parallel with circulatory G-CSF. G-CSF induced NET formation and primed neutrophils toward an intense pro-NETotic response at concentrations prevalent in pregnancy. Moreover, detailed investigation of signaling related to the neutrophil activation revealed that the propensity to form NETs was advanced by both chorionic gonadotropin and estrogen/estriol. In contrast, progesterone acted by retaining cells in a primed pro-NETotic state, but inhibited their progress to formation of NETs. This coincided with the prevention of neutrophil elastase translocation from the cytoplasm to the nucleus, an indispensible step for NET release. Neutrophils isolated from pregnant women with GDM were determined to be highly reactive and formed NETs more vigorously when compared to neutrophils isolated from healthy pregnant women, as observed by fluorimetry, immunocytochemistry and morphometric analysis. Moreover, high glucose and TNFα, which are increased in gestational diabetic individuals, primed neutrophils towards NET formation. The infiltration to the placenta of these primed neutrophils led to excessive NE release and the degradation of insulin receptor substrate 1 (IRS1). Our data provide evidence that the increased neutrophil priming and NET formation observed during normal pregnancy is well modulated by the sex steroid hormones and G-CSF. Moreover we propose that neutrophils participate to the pathobiology of GDM. Our data demonstrate that the TNFα driven inflammation, resulting from high glucose levels in the circulation, enhances neutrophil priming and NET formation. The released NE can locally modify glucose tolerance and metabolism in the placenta, which probably results to the anatomical, physiological and functional changes observed in placenta of pregnant women suffering from GDM