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uncontrolled or aberrant activation of immunothrombosis promotes thrombotic pathologies such as stroke, myocar- dial infarction, deep vein thrombosis, or disseminated intravascular coagulation during sepsis.5,6
A large variety of infectious and sterile inflammatory stimuli have been described as triggers of NETosis. Interestingly, their consequences on neutrophil activation vary depending on the nature and duration of the stimu- lus.7 Two mechanisms resulting in NET formation have been described: lytic and vital NETosis.8 Specific micro- organisms or lipopolysaccharides (LPS) induce vital NETosis directly or indirectly through toll-like receptors (TLR), via a TLR4-activated platelet interaction, by an oxi- dant-independent mechanism.9,10 This strategy aims to limit the spread of bacteria and keeps neutrophils alive for further functions.8 Alternatively, lytic NETosis can be induced by diverse sterile agonists such as phorbol 12- myristate 13-acetate,3,11 cholesterol crystals11 or cytokines (interleukin-6,12 interleukin-8,3,13 interferon-α14). This drives NADPH oxidase activation via protein kinase C and Raf- MEK-ERK, which generates reactive oxygen species (ROS) inducing neutrophil death.15
MicroRNA (miRNA or miR) are a family of small non- coding RNA that regulate gene expression.16 Since their discovery, miRNA have been implicated as key modula- tors of numerous physiological and pathological process- es.17-20 In particular, miR-146a directly mediates thrombo- inflammatory processes as: (i) it inhibits several pro- inflammatory elements of the TLR-NF (nuclear factor)-κB pathway21,22 and (ii) it is predominantly expressed in cells that promote thrombogenesis (monocytes/macrophages, platelets, neutrophils, and endothelial cells).23,24 Our group demonstrated that reduced levels of miR-146a, guided by the T variant of the functional miR-SNP rs2431697, are predictors of adverse cardiovascular events in patients with atrial fibrillation.24 Interestingly, in vitro activation of rs2431697 TT neutrophils exacerbated NET release in accordance with elevated plasma NE levels found in T car- riers, who also had a higher risk of adverse cardiovascular events.25 Overall, our data suggest that miR-146a is crucial- ly involved in linking inflammation, thrombosis, and NETosis. However, the role of miR-146a in immunothrombosis and NETosis is unknown. Here, we investigated the involvement of miR-146a in NET forma- tion leading to arterial thrombosis in both sterile and non- sterile inflammatory murine models. Thus, we character- ized the neutrophil phenotype to explain the exacerbated NETosis. Finally, we evaluated the association between low miR-146a levels, NETosis markers and the occurrence of thrombosis in septic patients.
Methods
Mice models
Atherosclerosis model
Irradiation and bone marrow (BM) transplantation were per- formed as previously described.26 Briefly, Ldlr-/-mice were irradiat- ed and transplanted with BM cells obtained from wild-type (WT) or miR-146a-/- mice. After 4 weeks on a standard diet, transplant- ed mice were fed an atherogenic diet for 8 weeks. Blood was col- lected from the facial vein into 3.2% citrate and plasma samples were stored at -80oC until analysis. Heart and aorta were extract- ed from euthanized mice after in situ perfusion with phosphate- buffered saline and fixed with 4% paraformaldehyde overnight
at 4oC. Tissue was embedded in paraffin and cross-sections from the aortic root were made.
Endotoxemia model
WT and miR-146a-/- mice were injected intraperitoneally with a sublethal dose of LPS (1 mg/kg) (E. coli 0111/B4, Sigma-Aldrich, Madrid, Spain). For morphological analysis, lungs were fixed with 4% paraformaldehyde overnight at 4oC, embedded in paraffin and sectioned. Lung damage was evaluated in one sec- tion/mouse using a semi-quantitative score via a blind method following the pathologist’s criteria.
Ferric chloride-induced arterial thrombosis
Animals were anesthetized by intraperitoneal injection (xylazine hydrochloride 10 mg/kg + ketamine hydrochloride 100 mg/kg). The carotid artery was isolated and the injury was gen- erated by applying a piece of filter paper (5x1 mm) (GE Healthcare Whatman 1003917, Fisher, Madrid, Spain) soaked in
Experiments were stopped after 30 min if no occlusion occurred. For the experiments with DNase I, mice were injected intra- venously (tail vein) with 10 mg of Pulmozyme® (Roche Farma, Madrid, Spain) diluted in saline, 15 min before FeCl3 treatment.
Patients and controls
The study (NEUMONAC study #2011/0219 [05/07/2011]) was approved by the Ethical Committee (ceic@iislafe.es) of our insti- tution and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its subsequent amendments. Consecutive patients admitted with community- acquired pneumonia (CAP) were included from October 2015 to June 2018 at Hospital Universitario y Politécnico La Fe (Valencia, Spain). The patients’ details are provided in the Online SupplementaryMethods.
Healthy subjects were randomly selected among blood donors from our Transfusion Center and gave their informed consent to enrollment into the study.
Statistical analysis
All statistical analyses were conducted using GraphPad Prism 3.8 (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS 22.0 for Windows (SPSS, Inc., Chicago, IL, USA). Differences between two groups were assessed by an unpaired Student t-test or Mann- Whitney U test, where appropriate. For multiple comparisons, one-way analysis of variance on ranks with the Bonferroni post- hoc test was used. Data are shown as mean ± standard error of mean or as median (95% confidence interval), as appropriate. A P-value <0.05 was considered statistically significant.
Further details on the materials and methods are available in the Online Supplementary Methods.
Results
miR-146a deficiency promotes NET formation in an atherosclerosis model
Previous data from our laboratory demonstrated that
haematologica | 2021; 106(6)
a 7.5% ferric chloride (FeCl 3
miR-146a in NETosis and thrombosis
) solution for 2 min (Sigma-Aldrich, Madrid Spain). After washing the arterial surface with saline solution and removing residual FeCl3, a Doppler ultrasound flow probe (Model 0.5 PSB, Transonic Systems, Ithaca, NY, USA) was applied. The probe was connected to a flow meter (Model TS420, Transonic Systems, Ithaca, NY, USA) that continuously registered the blood flow. Occlusion time was defined as the time elapsed from the withdrawal of FeCl to the lack of blood
3
flow (≤ 0.01 mL/min) for at least 3 consecutive minutes.
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