miR-146a is a pivotal regulator of neutrophil extracellular trap formation promoting thrombosis

Abstract

Neutrophil extracellular traps (NETs) induce a procoagulant response linking inflammation and thrombosis. Low levels of miR-146a, a brake of inflammatory response, are involved in higher risk for cardiovascular events, but the mechanisms explaining how miR-146a exerts its function remain largely undefined. The aim of this study was to explore the impact of miR-146a deficiency in NETosis both, in sterile and non-sterile models in vivo, and to inquire into the underlying mechanism. Two models of inflammation were performed: 1) Ldlr-/- mice transplanted with bone marrow from miR-146a-/- or wild type (WT) were fed high-fat diet, generating an atherosclerosis model; and 2) an acute inflammation model was generated by injecting lipopolysaccharide (LPS) (1 mg/Kg) into miR-146a-/- and WT mice. miR-146a deficiency increased NETosis in both models. Accordingly, miR-146a-/- mice showed significant reduced carotid occlusion time and elevated levels of NETs in thrombi following FeCl3-induced thrombosis. Infusion of DNAse I abolished arterial thrombosis in WT and miR-146a-/- mice. Interestingly, miR-146a deficient mice have aged, hyperreactive and pro-inflammatory neutrophils in circulation that are more prone to form NETs independently of the stimulus. Furthermore, we demonstrated that community acquired pneumonia (CAP) patients with reduced miR-146a levels associated with the T variant of the functional rs2431697, presented an increased risk for cardiovascular events due in part to an increased generation of NETs.

Figure 1.

miR-146a deficiency enhances neutrophil extracellular trap formation in atherosclerosis. (A) Ldlr^-/- mice were transplanted with bone marrow (BM) from miR-146a^-/- or wild-type (WT) mice. After 4 weeks of recovery, mice were fed a high-fat diet for 8 weeks. Blood and aortic tissues were extracted for quantification of the formation of neutrophil extracellular traps (NET) (n=10-12/group). (B) Plasma cell-free (cf)DNA was measured using Sytox Green. (C) Plasma neutrophil elastase (NE) activity was quantified by enzyme-linked immunosorbent assay. (D) Representative confocal microscopy images of an aortic valve leaflet, an atherosclerotic area, and a NET detail from Ldlr^-/- BM WT, and Ldlr^-/- BM miR-146a^-/ mice immunostained for DNA (DAPI, blue), NE (green), and H2B (red). The dashed yellow line denotes an atherosclerotic lesion (L) boundary; the lumen (Lu), and valves (V) are also marked. White arrowheads point to NET. (E) NET quantification in aortic valve sections from Ldlr^-/- BM WT, and Ldlr^-/- BM miR-146a^-/- mice performed with the Colocalization Colormap Fiji plugin. The correlation index (ICorr) represents the fraction of positively correlated (co-localized) H2B and NE pixels in one representative section per mouse in six WT and six miR-146a^-/- BM-transplanted Ldlr^-/- mice. (F) Fluorescence intensity plots of H2B and NE in a region of interest (yellow line) of a NET and a neutrophil found in the aortic valve from a Ldlr^-/- BM miR-146a^-/- and a Ldlr^-/- BM WT mouse, respectively. The correlation of H2B and NE fluorescence intensity was determined using the Pearson correlation coefficient and Costes method. In the Pearson correlation test, the R value ranges between -1 and 1, with 1 being a perfect correlation, 0 no linear correlation, and −1 a perfect negative linear correlation. Costes analysis compares pixel correlation for no-randomized with randomized images and calculates significance. The Costes P-value was 1 in both cases, indicating that the probability of random images correlating to real images is 0. P-value calculations were performed using the Mann-Whitney U test. Data represent mean ± standard error of mean, *P<0.05, **P<0.01.

Figure 2.

miR-146a deficiency mediates NETosis in endotoxemia. miR- 146a^-/- and wild-type (WT) mice were injected intraperitoneally with a sublethal dose of lipopolysaccharide (LPS) (1 mg/kg). Plasma markers of neutrophil extracellular traps (NET) were measured 4 h and 24 h after LPS treatment (n=9/group). (A) Cell-free (cf)DNA levels were quantified by Sytox Green. (B) Neutrophil elastase (NE) levels were measured by enzymelinked immunosorbent assay (ELISA). (C) Citrilluniated histone 3 (CitH3) plasma levels were analyzed by western blot. (D) Reactive oxygen species (ROS) were quantified by fluorometry 24 h after treatment with LPS. (E) Thrombin-antithrombin (TAT) complex levels were detected by ELISA 4 h after LPS treatment. (F) Morphology of lungs from WT and miR-146a^-/- mice after LPS stimulation. Lung sections from WT and miR-146a^-/- mice treated 4 h with LPS (1 mg/kg) were stained for reticulin (n=9/group) to observe tissue structure and global injury. Representative images of reticulin staining from WT and miR-146a^-/- lungs after LPS at 20x and 40x magnification. (G) Semi-quantitative analysis for reticulin and global lung damage according to the pathologist’s criteria. One section/mouse was scored in a blinded fashion into four grades from 0 to 3 (0=normal, 1=mild, 2=moderate, 3=severe). P-value calculations were performed using one-way analysis of variance on ranks with the Bonferroni post-hoc test or the Mann-Whitney U test, where appropriate. Data represent mean ± standard error of mean, *P<0.05, **P<0.01, ***P<0.001.

Figure 3.

miR-146a determines neutrophil phenotype. (A) Flow cytometry analysis of CD11b, CD62L, and Cxcr4 (P<0.05 for all markers) on neutrophils (Ly6G-positive population) from blood of wild-type (WT) and miR-146a^-/- mice (n=6 mice in both groups). Contour plots from flow cytometry analysis of CD62L and CD11b on neutrophils from a representative WT mouse and an miR-146a^-/- mouse (bottom left panel). Comparison of Cxcr4 expression between WT and miR-146a^-/- representative mice (bottom right panel). (B) Flow cytometry analysis of Cxcr1 on neutrophils from blood of WT and miR-146a^-/- mice (left panel; n=5 mice in both groups; P<0.01). Comparison of Cxcr1 expression on a representative mouse with each genotype (right panel). (C) Tlr4 levels were measured in Cxcr4^high and CD62^Llow (aged) neutrophil subpopulation versus the rest (n=6 mice in both groups; P<0.05). Contour plots from flow cytometry analysis of CD62L and Cxcr4 on peripheral blood pool neutrophils from six WT and six miR-146a^-/- mice (right panel). (D) Bone marrow (BM) isolated neutrophils were incubated with 10 M H2DCFDA for 30 min at 37°C and analyzed by flow cytometry (n=3 mice in both groups). (E) BM neutrophils were seeded on the plate and the oxygen consumption rate (OCR) was measured by a Seahorse Analyzer. (mean ± standard deviation, n=3 mice for each group, samples in quadruplicate). Statistical analyses between groups of mice were performed using the Mann- Whitney test (A-C) or t-test (D, E); *P<0.05, **P<0.01.

Figure 4.

miR-146a deficiency accelerates carotid thrombotic occlusion. (A) Mouse model of FeCl3-induced carotid arterial thrombosis. The carotid artery from wildtype (WT) and miR-146a^-/- mice was isolated and exposed to 7.5% FeCl3 for 2 min (WT n=24, miR-146a^-/- n=17). A Doppler ultrasound flow probe registered the blood flow continuously. (B) Basal blood flow. (C) Time to carotid thrombotic occlusion. (D) Alternatively, WT (n=7) and miR-146a^-/- (n=7) mice were treated with DNase I (10 g, i.v.) 15 min before vessel injury and the carotid occlusion time was measured for a maximum of 30 min. (E) Representative images of neutrophil extracellular traps (citrullinated histone 3 [citH3]-positive nuclei) in the carotid thrombi from WT and miR-146a^-/- mice analyzed by immunofluorescence. For each genotype the upper row shows the complete cross-section of the thrombosed carotid artery (bar corresponds to 100 m) and the lower row a higher magnification of the artery wall (bar corresponds to 20 m) and the detail of a neutrophil (bar corresponds to 5 m) (F-H) Quantification of total infiltrated nucleated cells, citH3-positive cells and the ratio of total nucleated to citH3-positive cells in the thrombi and the adventitial layer of carotid sections from WT and miR-146a^-/- mice (n=10/group). P-value calculations were performed using the unpaired Student t-test and Mann-Whitney U-test. Data represent mean ± standard error of mean, *P<0.05.

Figure 5.

miR-146a plays a relevant role in thrombo-inflammation after sterile and non-sterile stresses. Low miR-146a levels promote an aging phenotype in neutrophils (CD62^low CD11b^high Cxcr4^high Tlr4^high) which primes these cells. Upon sterile or non-sterile stimuli, primed neutrophils would be more prone to NETosis leading to a thrombo-inflammatory process. MiR-146a: microRNA- 146a; LPS: lipopolysaccharide; CAP: community-acquired pneumonia.