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miR-146a in NETosis and thrombosis
first line of defense in the context of acute inflammation since they do not return to the bone marrow, arrive first at the inflammatory tissues and infiltrate them in a TLR4/CD11b-dependent manner.48 Thus, we investigated neutrophil phenotype in miR-146a-/- mice in order to explain the greater NET formation observed in our differ- ent models. We found that miR-146a-/- resting neutrophils have an aging phenotype CD62Llow CD11bhigh Cxcr4high, and a lower expression of Cxcr1.1
Thus, we speculate that miR-146a deficiency could act as a priming effector on resting neutrophils so that, under any inflammatory stimulus, (sterile or non-sterile) these cells would reach higher activation rates than WT neu- trophils. Our results show that Tlr4, which is a validated target of miR-146a, is overexpressed in aged miR-146a-/- neutrophils in comparison with WT ones. One possible explanation for this finding may be that the deficiency of miR-146a favors a higher deregulation of the Tlr4/Nf-κB axis in aged neutrophils than in non-aged ones. Thus, this hypothesis would also explain in part why circulating aged neutrophils are more prone to make NET, a previous indolent signal being necessary to prime these cells.49,50 Indeed, it has been shown that Tlr4-/- mice or germ-free mice have less aged neutrophils than WT littermates.30
In accord with our hypothesis, we observed higher ROS levels and oxygen consumption rate in neutrophils from miR146-/- mice than WT mice, which may further suggest a primed status in miR-146a-/- neutrophils. Importantly, the aging process is promoted in large part by ROS that produce cellular damage.51 ROS (mainly pro- duced by NADPH oxidase) are also crucial to initiate the process of NET formation.52
There are a few limitations in this study. First, for the in vivo inflammation models, we did not use a complex marker such as a granulocytic enzyme bound to DNA to measure NET. NET were quantified using cfDNA, NE, and citH3 for the LPS model and tissue immunofluores- cence in the atherosclerotic model. Thus, we cannot rule out that part of the cfDNA and NE measured in plasma was due to neutrophil secretion or to apoptotic processes. Another point is that our study was performed in full miR-146a-/- mice or BM transplanted mice. Given the interplay of other cells with neutrophils to promote NETosis, in particular platelets,9,10 it would be of interest in future studies to dissect the mechanisms implicating
miR-146a in the formation of NET using conditional models of miR-146a deficiency.
In summary, our results demonstrate that miR-146a deficiency promotes an aged neutrophil phenotype prim- ing these cells for activation. Upon both sterile and non- sterile stimuli, such sensitized neutrophils are more prone to form NET, leading to thrombosis when physiological control is overwhelmed (Figure 5). Indeed, we demon- strate this hypothesis in a human disease, sepsis, in which patients with low miR-146a levels due to rs2431697 have increased plasma NET markers and a higher risk of cardiovascular events. These data not only strengthen the proposed interconnection between inflam- mation and thrombosis but also open new perspectives for the development of therapeutic tools against NETosis in different thrombo-inflammatory contexts.
Disclosures
No conflicts of interest to disclose.
Contributions
ABA, MPF-P, AdM, SA, RH-A, NG-B, AMR-P and MIA performed research; ABA, MPF-P, AdM, SA and MIA ana- lyzed data; RaM, PG-J, and RoM collected samples/data from patients and analyzed patients’ data; VV critically reviewed the paper; RG-C, and CM conceived and designed research; ABA, RG-C, and CM wrote the paper.
Acknowledgments
The authors thank Dr Juan José Cerón, Alberto Martínez, and Dr. Antonio Moscardó for technical assistance and Clive Drakeford and Javier Corral for reviewing the manuscript.
Funding
This work was supported by research grants from Instituto de Salud Carlos III (ISCIII), Fondo Europeo de Desarrollo Regional “Investing in your future” (PI17/00051 y PI17/01421) (PFIS18/0045 to AMdlR-G) (CD18/00044: to SA), and Fundación Séneca (19873/GERM/15). The CNIC is supported by the ISCIII, the Ministerio de Ciencia, Innovación y Universidades (MCIU), and the Fundación Pro CNIC, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). ABA has a research fellowship from Sociedad Española de Trombosis y Hemostasia (SETH). The MCIU supported AdM. (predoctor- al contract BES-2014-067791).
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