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miR-146a in NETosis and thrombosis
Loss of miR-146a accelerates time to carotid thrombotic occlusion by increasing NET release
Thus, we hypothesized that under a thrombotic stimu- lus, these neutrophils may favor the production of NET, thereby strongly contributing to thrombosis. To test this hypothesis, we induced carotid endothelial injury with FeCl3 in miR-146a-/- and WT mice (Figure 4A) and deter- mined time to arterial occlusion. Although basal blood flow was similar between the two groups of animals (Figure 4B), miR-146a-/- mice showed a significant reduc- tion in time to occlusion compared to the time in WT lit- termates (374.5 ± 21.15 vs. 452.5 ± 26.14 s, respectively; P<0.05) (Figure 4C). The occlusion times of WT and miR- 146a-/- mice were similar upon treatment with DNAse I (Figure 4D). Thus, DNAse I treatment abolished arterial occlusion, supporting the concept that NET are essential for thrombus formation and stability upon FeCl3 damage. To further analyze the association between thrombosis and NETosis, thrombi within carotids were isolated and immunofluorescence for citH3 was performed. FeCl3- induced injury promoted similar infiltration of nucleated cells in both groups (Figure 4E-F); however, the number of DNA-citH3-positive cells within the carotid thrombi was significantly higher in miR-146a-/- mice than in WT ones (19.3 ± 3.3 vs. 10.0 ± 3.6 cells, respectively; P<0.05) (Figure 4G). The ratio of citH3-positive to total nucleated cells found in the thrombi of carotid sections was signifi- cantly higher in miR-146a-/- mice than in WT mice (Figure 4H, Online Supplementary Figure S4).
Of note, no relevant differences in F12 coagulation fac- tor, recently implicated in NETosis,32 was observed between the two groups (data not shown). Thus, miR-146a deficiency may accelerate vessel occlusion due, in part, to increased NETosis associated with the presence of hyper- reactive neutrophils.
Low miR-146a levels in patients with community-acquired pneumonia are associated with an increased risk of cardiovascular events
In order to test the clinical impact of our in vivo results, we recruited 259 patients with CAP with a 30-day fol- low-up period. Online Supplementary Table S1 shows the demographic and clinical characteristics of all 259 patients, as well as the frequencies and distribution of the rs2431697 genotype. We found no significant differences between the main cardiovascular risk factors and geno-
Table 1. Relationship between hospital cardiovascular complications and rs2431697 genotype in patients with community-acquired pneu- monia (n=259).
CC CT+TT P N=58 N=201 RR (95% CI)
CVE,N 1 29 0.008
(%) (1.7) (14.4) 9.61 (1.28-72.15)
TF,N 4 22 0.366
type (Online Supplementary Table S1). In addition, both Sepsis-related Organ Failure Assessment (SOFA) score and neutrophil count at entry were similar in all patients independently of their genotype (Online Supplementary Table S1). One hundred and twelve plasma samples were available for DNA-citH3 measurement. From these, 81 (72.3%) were considered as positive (optical density [OD] values >0.200, the maximum OD value yielded for 30 healthy controls) (Online Supplementary Figure S5, Online Supplementary Table S2). As described for septic patients, plasma levels of DNA-citH3 correlated with clinical out- comes.33,34 Thus, plasma DNA-citH3 levels were signifi- cantly higher in septic patients (n=58) than in non-septic patients (n=23) (P<0.001). Furthermore, DNA-citH3 levels were significantly higher in patients in whom treatment failed or cardiovascular events occurred (n=18) than in the rest of the patients (n=63) (P=0.028) (Online Supplementary Table S2).
Interestingly, we observed significant differences between the occurrence of cardiovascular complications and rs2431697 genotype. As shown in Table 1, 30 of the 259 patients suffered from cardiovascular events when hospitalized and 29 of them were carriers of the T allele (relative risk [RR]=9.61, [95% CI]: 95% confidence inter- val: 1.28-72.15, P=0.008). The risk of cardiovascular events remained significantly higher for CT+TT patients 30 days after hospitalization (RR=2.85, 95% CI: 0.97- 8.37, P=0.049) (Table 1). In addition, 21.4% of the carriers of the T allele developed a cardiovascular event and failed to respond to therapy (RR=2.89, 95% CI: 1.09-7.66, P=0.027) (Table 1). Finally, among patients with the high- est DNA-citH3 plasma levels (≥0.406 OD units) we found a 3-fold higher frequency of T carriers individuals (30%) compared to CC homozygotes (9.5%; P=0.061) (Table 2).
Discussion
In the last years, the functional versatility of neu- trophils has moved towards a fascinating area of research in which the inflammatory capacity of these cells is tight- ly linked to the development of cardiovascular diseases.35 On the other hand, the pathophysiological role of NET in thrombogenesis is now firmly established.36 In this process, known as immunothrombosis, the mechanisms leading to NETosis differ depending on the triggering stimulus.7 Identifying key regulators of NETosis37 is a challenge to further contribute to the development of anti-thrombotic therapeutic tools that would not affect the essential role of NET fighting germs. In this work, we show that miR-146a, a well-known molecular brake to the NF-κB pathway, underlies NETosis-mediated throm- bosis after both sterile and non-sterile stimulation.
First, we demonstrated that sterile inflammation in a
Table 2. rs2431697 genotype frequencies according to DNA-citrulli- nated H3 plasma levels in patients with community-acquired pneumo- nia.
DNA-citH3 < 4Q DNA-citH3 > 4Q P* CC,N 19 2
(%)
CVE+TF, N (%)
CVE-30, N
(%)
(6.9)
5 (8.6)
4
(6.9)
(10.9)
43 (21.4)
35
(17.4)
-
0.027
2.89 (1.09-7.66)
0.049
2.85 (0.97-8.37)
(%)
CT+TT, N
(%)
(90.5)
42
(70.0)
(9.5)
18
(30.0)
RR: relative risk; 95% CI: 95% confidence interval; CVE: cardiovascular events during hospitalization, including pulmonary thromboembolism; TF: treatment failure during hospitalization; CVE+TF: occurrence of cardiovascular event and/or treatment failure; CVE-30: cardiovascular events 30 days after hospitalization.
0.061
4Q indicates 4th quartile (≥0.406 optical density units).Measurements were performed in a total of 81 patients (OD≥0.200). *ƛ2 test.
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