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

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.

Remodeling of Bone Marrow Hematopoietic Stem Cell Niches Promotes Myeloid Cell Expansion during Premature or Physiological Aging.

Hematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes ß2-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced ß3-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with ß3-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment.

miR-146a deficiency in hematopoietic cells is not involved in the development of atherosclerosis.

BACKGROUND: Atherosclerosis involves activation of the IRAK1/TRAF6/NF-κB inflammatory cascade, which is negatively regulated by miR146a. Previous studies showed that the TT genotype of rs2431697, located near the miR-146a gene, drives lower miR-146a transcription and predicts adverse cardiovascular events in anticoagulated atrial fibrillation patients. Moreover, systemic miR-146a administration protects mice from atherosclerosis. Here we evaluated the ability of miR-146a expression in the hematopoietic component to regulate atherosclerosis in low-density lipoprotein receptor-null mice (Ldlr-/-). METHODS AND RESULTS: Lethally-irradiated Ldlr-/- mice transplanted with bone marrow from wild-type or miR-146a-null mice were fed an atherogenic diet for 8 and 20 weeks. Irak1, Traf6 and MIR146A expression were quantified in thoracic aorta by qRT-PCR and Western blot. Aortic plaque size and composition were characterized by Oil-Red staining and immunohistochemistry and leukocyte recruitment by intravital microscopy. Blood cell counts were similar in fat-fed Ldlr-/-mice with or without hematopoietic miR-146a expression. However, plasma cholesterol decreased in fat-fed Ldlr-/-mice transplanted with bone marrow deficient for miR-146a. Finally, aortic atherosclerosis burden and recruitment of leukocytes into the vessel wall were undistinguishable between the two groups, despite higher levels of Irak1 and Traf6 mRNA and protein in the aorta of fat-fed mice lacking hematopoietic miR-146a expression. CONCLUSIONS: miR-146a deficiency exclusively in hematopoietic cells modulates cholesterol levels in plasma and the expression of its targets in the artery wall of fat-fed Ldlr-/- mice, but does not accelerate atherosclerosis. Atheroprotection upon systemic miR-146a administration may therefore be caused by specific effects on vascular cells.

Cross-reactivity and inhibition of haemolysis by polyclonal antibodies raised against St II, a cytolysin from the sea anemone Stichodactyla helianthus.

The immunogenicity of sticholysin II (St II), a pore-forming polypeptide from the sea anemone Stichodactyla helianthus, was studied in rabbits using two adjuvants, Freund's and aluminium hydroxide. High titres of antibodies were raised against St II with Freund's adjuvant (FA). The structural homology between sticholysins I and II was also revealed by cross-reactivity assays. Since the oil constituent of FA neutralized the St II haemolytic activity, immunizations with St II-Freund's emulsions were carried out with the inactivated cytolysin. Purified anti-St II IgG also neutralized the St II haemolytic activity.