Dataset Information

Neutrophil accumulation and NET release contribute to thrombosis in HIT.

ABSTRACT: Heparin-induced thrombocytopenia (HIT) is an immune-mediated thrombocytopenic disorder associated with a severe prothrombotic state. We investigated whether neutrophils and neutrophil extracellular traps (NETs) contribute to the development of thrombosis in HIT. Using an endothelialized microfluidic system and a murine passive immunization model, we show that HIT induction leads to increased neutrophil adherence to venous endothelium. In HIT mice, endothelial adherence is enhanced immediately downstream of nascent venous thrombi, after which neutrophils undergo retrograde migration via a CXCR2-dependent mechanism to accumulate into the thrombi. Using a microfluidic system, we found that PF4 binds to NETs, leading them to become compact and DNase resistant. PF4-NET complexes selectively bind HIT antibodies, which further protect them from nuclease digestion. In HIT mice, inhibition of NET formation through Padi4 gene disruption or DNase treatment limited venous thrombus size. PAD4 inactivation did affect arterial thrombi or severity of thrombocytopenia in HIT. Thus, neutrophil activation contributes to the development of venous thrombosis in HIT by enhancing neutrophil-endothelial adhesion and neutrophil clot infiltration, where incorporated PF4-NET-HIT antibody complexes lead to thrombosis propagation. Inhibition of neutrophil endothelial adhesion, prevention of neutrophil chemokine-dependent recruitment of neutrophils to thrombi, or suppression of NET release should be explored as strategies to prevent venous thrombosis in HIT.

SUBMITTER: Gollomp K

PROVIDER: S-EPMC6237233 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Neutrophil accumulation and NET release contribute to thrombosis in HIT.

Gollomp Kandace K Kim Minna M Johnston Ian I Hayes Vincent V Welsh John J Arepally Gowthami M GM Kahn Mark M Lambert Michele P MP Cuker Adam A Cines Douglas B DB Rauova Lubica L Kowalska M Anna MA Poncz Mortimer M

JCI insight 20180920 18

Heparin-induced thrombocytopenia (HIT) is an immune-mediated thrombocytopenic disorder associated with a severe prothrombotic state. We investigated whether neutrophils and neutrophil extracellular traps (NETs) contribute to the development of thrombosis in HIT. Using an endothelialized microfluidic system and a murine passive immunization model, we show that HIT induction leads to increased neutrophil adherence to venous endothelium. In HIT mice, endothelial adherence is enhanced immediately do ...[more]

PMID: 30232279

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Project description:Excessive release of neutrophil extracellular traps (NETs) has been reported in various human pathologies, including COVID-19 patients. Elevated NET levels serve as a biomarker, indicating increased coagulopathy and immunothrombosis risks in these patients. Traditional immunoassays employed to quantify NET release focus on bulk measurements of released chromatin in simplified microenvironments. In this study, we fabricated a novel NET-array device to quantify NET release from primary human neutrophils with single-cell resolution in the presence of the motile bacteria Pseudomonas aeruginosa PAO1 and inflammatory mediators. The device was engineered to have wide chambers and constricted loops to measure NET release in variably confined spaces. Our open NET-array device enabled immunofluorescent labeling of citrullinated histone H3, a NET release marker. We took time-lapse images of primary healthy human neutrophils releasing NETs in clinically relevant infection and inflammation-rich microenvironments. We then developed a computer-vision-based image processing method to automate the quantification of individual NETs. We showed a significant increase in NET release to Pseudomonas aeruginosa PAO1 when challenged with inflammatory mediators tumor necrosis factor-α [20 ng mL-1] and interleukin-6 [50 ng mL-1], but not leukotriene B4 [20 nM], compared to the infection alone. We also quantified the temporal dynamics of NET release and differences in the relative areas of NETs, showing a high percentage of variable size NET release with combined PAO1 - inflammatory mediator treatment, in the device chambers. Importantly, we demonstrated reduced NET release in the confined loops of our combined infection-inflammation microsystem. Ultimately, our NET-array device stands as a valuable tool, facilitating experiments that enhance our comprehension of the spatiotemporal dynamics of NET release in response to infection within a defined microenvironment. In the future, our system can be used for high throughput and cost-effective screening of novel immunotherapies on human neutrophils in view of the importance of fine-tuning NET release in controlling pathological neutrophil-driven inflammation.

Dataset Information

Neutrophil accumulation and NET release contribute to thrombosis in HIT.

Publications

Neutrophil accumulation and NET release contribute to thrombosis in HIT.

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