Abstract Number: PB2020
Meeting: ISTH 2020 Congress
Background: Neutrophil extracellular traps (NETs) are released by neutrophils under diverse pro-inflammatory stimuli. Our group showed that miR-146a deficiency in a murine model increased the intrinsic ability of neutrophils to form NETs in vitro.
Aims: Since it has been described that neutrophils display a wide phenotypic diversity accompanied by important functional changes, we investigate whether the greater capacity of miR-146a-deficient neutrophils to form NETs is associated with neutrophil phenotype.
Methods: Peripheral blood (PB) was extracted from wild type (WT) and miR-146a-/- mice at baseline and 4 hours after intraperitoneal injection of LPS (1mg/Kg). NET components were measured in plasma from these mice: neutrophil elastase (NE) by ELISA, circulating free DNA (cfDNA) by SYTOX Green, and citrullinated Histone H3 (citH3) by western blotting. Ly6G, CD11b, CD62L, Cxcr4, and Tlr4 were measured by flow cytometry in PB neutrophils.
Results: miR-146a-/- mice showed higher plasma levels of all the NET markers analyzed (NE, cfDNA, and citH3) than WT mice after LPS injection. When we compared baseline levels of neutrophil activation markers, we found that miR-146a-/- neutrophils had higher levels of CD11b (MFI 44809 vs. 26011, p< 0.05), and lower levels of CD62L (MFI 2641 vs. 6022, p< 0.01) than WT neutrophils. However, after LPS stimulation both markers reached similar levels, suggesting that miR-146a deficiency confers a pre-activated state. In addition, miR-146a-/- neutrophils had higher levels of Cxcr4 (MFI 1285 vs. 965, p< 0.01) and Tlr4 (MFI 1015 vs. 852, p< 0.05) at baseline, an event that has been associated with an aged phenotype and an increased sensitivity to form NETs.
Conclusions: miR-146a-/- neutrophils produce higher levels of NETs than WT neutrophils upon in vivo stimulation with LPS. We describe that miR-146a deficiency promotes the acquisition of an aged neutrophil phenotype defined by CD11bhigh CD62Llow Cxcr4high Tlr4high markers and an increased ability to form NETs via Tlr4 stimulation.
[Flow cytometry analysis of CD11b, CD62L, Cxcr4, and Tlr4 in WT and miR-146a-/- neutrophils. ]
To cite this abstract in AMA style:Fernandez Perez MP, Arroyo Rodriguez AB, Aguila S, Hernandez-Antolin R, Garcia-Barbera N, de los Reyes-Garcia AM, Reguilon-Gallego L, Zapata-Martinez L, Vicente V, Gonzalez-Conejero R, Martinez C. mir-146a Deficiency Promotes Neutrophil Aging and Increases their Sensitivity to Form Neutrophil Extracellular Traps (NETs) in a Murine Model [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/mir-146a-deficiency-promotes-neutrophil-aging-and-increases-their-sensitivity-to-form-neutrophil-extracellular-traps-nets-in-a-murine-model/. Accessed November 30, 2021.
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