Anurag Kumar Gupta

Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated cell death

Neutrophil interaction with activated endothelial cells (EC) is required for transmigration. We examined consequences of this interaction on NETosis. Co‐culture of activated EC with neutrophils induced neutrophil extracellular trap (NET) formation, which was partially dependent on production of IL‐8 by activated EC. Extended neutophil/EC co‐culture resulted in EC damage, which could be abrogated by inclusion of either diphenyleneiodonium to inhibit the NAPDH oxidase pathway required for NETosis, or DNAse to disrupt NETs. These findings offer new insight into mechanisms whereby NETs trigger damage to the endothelium in sepsis, small vessel vasculitis and possibly the villous trophoblast in preeclampsia.

Efficient Neutrophil Extracellular Trap Induction Requires Mobilization of Both Intracellular and Extracellular Calcium Pools and Is Modulated by Cyclosporine A

Excessive or aberrant generation of neutrophil extracellular traps (NETs) has recently become implicated in the underlying aetiology of a number of human pathologies including preeclampsia, systemic lupus erythromatosus, rheumatoid arthritis, auto-antibody induced small vessel vasculitis, coagulopathies such as deep vein thrombosis or pulmonary complications. These results imply that effective pharmacological therapeutic strategies will need to be developed to counter overt NETosis in these and other inflammatory disorders. As calcium flux is implicated in the generation of reactive oxygen species and histone citrullination, two key events in NETosis, we analysed the roles of both extra- and intracellular calcium pools and their modulation by pharmacological agents in the NETotic process in detail. Interleukin-8 (IL-8) was used as a physiological stimulus of NETosis. Our data demonstrate that efficient induction of NETosis requires mobilisation of both extracellular and intracellular calcium pools. Since modulation of the calcineurin pathway by cyclosporine A has been described in neutrophils, we investigated its influence on NETosis. Our data indicate that IL-8 induced NETosis is reduced by ascomycin and cyclosporine A, antagonists of the calcineurin pathway, but not following treatment with rapamycin, which utilizes the mTOR pathway. The action of the G protein coupled receptor phospholipase C pathway appears to be essential for the induction of NETs by IL-8, as NETosis was diminished by treatment with either pertussis toxin, a G-protein inhibitor, the phospholipase C inhibitor, U73122, or staurosporine, an inhibitor of protein kinase C. The data regarding the calcineurin antagonists, ascomycin and cyclosporine A, open the possibility to therapeutically supress or modulate NETosis. They also provide new insight into the mechanism whereby such immune suppressive drugs render transplant patients susceptible to opportunistic fungal infections.

Occurrence of neutrophil extracellular DNA traps (NETs) in pre-eclampsia : A link with elevated levels of cell-free DNA?

Abstract:  Manifest pre‐eclampsia is associated with activation of peripheral neutrophils as well as elevations in maternal cell‐free DNA. For this reason, we were very intrigued by recent reports indicating that activated circulatory neutrophils secrete nuclear DNA to generate extracellular DNA lattices, termed NETs (neutrophil extracellular traps). Our preliminary data indicate that placental syncytiotrophoblast microparticles, which are released in elevated amounts in pre‐eclampsia, can induce NETs in isolated neutrophils. Furthermore, we found evidence for the increased presence of NETs directly in the intervillous space of pre‐eclamptic placentae. Therefore, these newly discovered entities may be implicated in the underlying etiology of this disorder.

Neutrophil NETs: a novel contributor to preeclampsia-associated placental hypoxia?

Recent studies have suggested that the innate immune system is involved in the pathogenesis of preeclampsia. Its pathogenesis involves neutrophil activation and increased levels of cell-free DNA in the maternal plasma. Activation of neutrophils has recently been shown to induce DNA containing neutrophil extracellular traps (NETs) which trap and kill bacteria. Massive NETs induction by the placentally derived factors (IL-8 and placental micro-debris) and their increased presence in preeclamptic placenta suggest that NETs might be involved in the pathogenesis of preeclampsia. Therefore, increased presence of NETs in preeclampsia may play a role in the deficient placental perfusion associated with this disorder.

Disturbances in placental immunology: ready for therapeutic interventions?

Recent studies have provided new insight into aberrations in the immunological interplay between mother and fetus and their potential role in the development of recurrent fetal loss and preeclampsia. The action of anti-phospholipid antibodies in recurrent fetal loss is now proposed to involve the complement system, neutrophil activation and the production of TNFα by immune bystander cells. A clear involvement of the immune system is emerging in preeclampsia, involving mainly the innate arm, especially neutrophils. The activation of peripheral neutrophils by placentally released inflammatory debris triggers the induction of neutrophil extracellular traps (NETs), which may lead to an occlusion of the intervillous space, thereby further promoting a condition of placental hypoxia. It has, hence, been suggested that new therapeutic strategies be developed, including the possible use of TNFα antagonists in cases of recurrent miscarriage. These strategies need to be addressed with caution due to the possible induction of fetal congenital abnormalities.

Decrease in lipid levels of syncytiotrophoblast micro-particles reduced their potential to inhibit endothelial cell proliferation

BackgroundPreeclampsia is characterized by damage to the maternal endothelium that has been suggested to be mediated in part by elevated shedding of inflammatory placental syncytiotrophoblast micro-particles (STBM) into the maternal circulation. Previously, we have shown that STBM, prepared by three different methods: mechanical dissection, in vitro placental explants culture and perfusion of placenta, can inhibit endothelial cell proliferation. Only mechanically prepared STBM induced apoptosis in the endothelial cells. Now, we have examined lipid levels in the three STBM preparations and their differential responses on endothelial cells.MethodsWe examined the lipid levels in the three STBM preparations using thin layer chromatography. Furthermore, the effects of reduced lipid levels in the three STBM preparations using the pharmacological agent methyl-β-cyclodextrin were examined on endothelial cell proliferation and apoptosis.ResultsAmong the three STBM preparations, mechanical STBM contained highest levels of lipids. The reduction in lipid levels in mechanical STBM reduced their potential to inhibit human umbilical vein endothelial cells (HUVEC) proliferation and blocked their potential to induce apoptosis. No similar effect was observed following lipid reduction in the two other STBM preparations.ConclusionsAs it has been suggested that mechanically derived STBM may more closely resemble placental micro-particles generated in preeclampsia, our data suggest that lipid content may play a role in the anti-endothelial defects present in this disease.

Minimal alteration in the ratio of circulatory fetal DNA to fetal corticotropin-releasing hormone mRNA level in preeclampsia

Objectives: We have recently observed that fetal DNA and fetal corticotropin-releasing hormone (CRH) mRNA are associated with in vitro generated syncytiotrophoblast-derived microparticles, and that the ratio of fetal DNA to mRNA (CRH) varied according to whether the particles were derived by predominantly apoptotic, apo-necrotic or necrotic pathways. Hence, we examined whether these ratios varied in maternal plasma samples taken from normotensive and preeclamptic pregnancies in vivo. Methods: Maternal plasma samples were collected from 18 cases with preeclampsia and 29 normotensive term controls. Circulatory fetal CRH mRNA and DNA levels were quantified by real-time PCR and RT-PCR. Results: Circulatory fetal mRNA and fetal DNA levels were significantly elevated in the preeclampsia study group when compared to normotensive controls. Alterations in the fetal mRNA to DNA ratio between the study and control groups were minimal, even when stratified into early (<34 weeks of gestation) and late (>34 weeks of gestation) onset preeclampsia. Conclusions: Our data suggest that although circulatory fetal DNA and mRNA levels are significantly elevated in preeclampsia, the ratios in maternal plasma are not dramatically altered.