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A.B. Arroyo et al.
miR-146a deficiency upregulates NETosis in both murine and human in vitro models.25 Thus, we aimed to extrapo- late this finding to different in vivo models. We first uti- lized an atherosclerosis mouse model previously generat- ed by our group.26 Briefly, Ldlr-/- mice were transplanted with BM from miR-146a-/- or WT animals and fed a high- fat diet for 8 weeks (Figure 1A). As reported, transplant efficiency, body weight, circulating blood cell counts, plasma lipid profile, and atherosclerotic burden (athero- ma, lesion, and necrotic core areas) were similar between the experimental groups.26 No differences were found in cell-free (cf)DNA and NE plasma levels between the two groups of mice before they were fed the high-fat diet (data not shown). As expected, after 8 weeks of the athero- genic diet, notable differences in NET components between the two groups were observed. As shown in Figure 1B, plasma cfDNA levels were significantly higher in Ldlr-/- BM miR-146a-/- mice than in Ldlr-/- BM WT litter- mates (348.1 ± 73.0 vs. 177.3 ± 39.4 ng/mL, respectively; P<0.05). Similarly, plasma NE was almost 2-fold higher in BM miR-146a-/- mice than in BM WT mice (228.6 ± 32.6 vs. 113.0± 14.2 ng/mL, respectively; P<0.05) (Figure 1C). In addition, immunofluorescence analysis of aortic valves revealed that, although intact neutrophils were found in both cases (Online Supplementary Figure S1), there were substantial differences in the size and the amount of NET. DNA, and NE, and H2B staining revealed an increase of large NET in the atherosclerotic lesions and adhered to the vascular wall of BM miR-146a-/- mice compared to BM WT mice, in which we only identified scattered NET throughout all the sections (Figure 1D). Quantification of NET in whole sections within aortic roots confirmed that miR-146a-/- mice had more NET than had WT mice (ICorr values 0.78 ± 0.09 vs. 0.53 ± 0.07, respectively; P<0.01) (Figure 1E). In addition, zooming within NETotic areas demonstrated that H2B and NE co-localization was near to 100% (R=0.97; Costes P-value=1), in contrast to the low co-localization found in intact neutrophils (R=0.13; Costes P-value=1) (Figure 1F, Online Supplementary Movies S1 and S2). Collectively, these results demonstrated a role for miR-146a in NET formation during the process of ath- erosclerosis.
miR-146a mediates NETosis and lung damage
in an lipopolysaccharide-induced, sublethal model of inflammation
The molecular mechanisms leading to NETosis differ depending on the triggering stimulus.7 In order to investi- gate whether miR-146a may link different pathways pro- voking NETosis, we next examined the implication of miR-146a on NET formation using a non-sterile inflam- matory mouse model generated by sublethal injection of LPS (1 mg/kg) for 4 h and 24 h. As expected, there was a significant progressive reduction in circulating leukocyte and platelet counts following induction of endotoxemia, although no relevant differences between miR-146a-/- and WT animals (Online Supplementary Figure S2) were observed. As shown in Figure 2, basal plasma NET mark- ers were similar between miR-146a-/- and WT mice. However, following LPS administration at 4 h and 24 h, plasma cfDNA levels were higher in miR-146a-/- mice than in WT mice, with the difference being statistically signif- icant at 4 h (1653.0 ± 216.5 vs. 845.6 ± 294.4 ng/mL, respectively; P<0.01) (Figure 2A). Similarly, LPS injection resulted in significantly higher levels of plasma NE in miR-
146a-/- mice when compared to WT mice, after 4 h (1671.3 ± 95.6 vs. 1206.1 ± 99.2 ng/mL, respectively; P<0.01) and 24 h (2458.0 ± 57.1 vs. 1524.6 ± 61.2 ng/mL, respectively; P<0.001) (Figure 2B). Higher NETosis in miR- 146a-/- mice following LPS challenge was confirmed by western blotting. Plasma citrullinated histone 3 (citH3) levels were higher in miR-146a-/- mice than in WT mice at both 4 h and 24 h (Figure 2C). Finally, additional inflam- matory and coagulation markers were analyzed. LPS- dependent ROS production at 24 h was significantly higher compared with basal levels only in miR-146a-/- mice (178%; P<0.05) (Figure 2D). Additionally, the amounts of thrombin-antithrombin (TAT) complexes were significantly greater 4 h after LPS injection than at baseline only in miR-146a-/- mice (43%; P<0.05) (Figure 2E).
Although all mice indistinctly survived sublethal LPS injection, we explored whether LPS challenge could dif- ferentially damage mice lungs. Staining of lung sections showed a notable increase of reticulin in samples from miR-146a-/- mice compared with their WT littermates after 4 h of LPS (Figure 2F). According to the pathologist’s criteria, the analysis showed that both reticulin score and global lung injury were increased in miR-146a-/- mice com- pared to WT mice (Figure 2G). Therefore, miR-146a-defi- cient mice had higher NETosis, oxidative stress and acti- vation of coagulation and consequently, greater lung damage after LPS injection.
miR-146a deficiency confers an aged, overactive, and pro-inflammatory phenotype to neutrophils
Although neutrophils have been considered to be a rel- atively homogeneous population, evidence demonstrat- ing their heterogeneity is emerging.1 To investigate whether the increased ability of miR-146a-/- neutrophils to form NET in the above-mentioned models was related to a particular phenotype determined by miR-146a defi- ciency, we explored the aged/activated state of neu- trophils by flow cytometry. miR-146a deficiency did not alter the total percentage of neutrophils in the circulation (Online Supplementary Figures S2 and S3). Analysis of neu- trophil surface markers revealed that miR-146a-/- neu- trophils exhibited significantly higher levels of Cxcr4 and CD11b, and lower levels of CD62L than WT neutrophils (Figure 3A). Our results also showed that neutrophils from miR-146a-/- mice had significantly lower Cxcr1 lev- els than neutrophils from their WT littermates (Figure 3B), suggesting an additional pro-inflammatory ability for the miR-146a-/- neutrophil phenotype.27-29 Tlr4 has been implicated in the process of aging30 and is a target of miR-146a.31 Thus, we measured and compared its levels in miR-146a-/- and WT neutrophils from whole blood and found that, as described before, aged neutrophils (defined by Cxcr4high and CD62Llow surface expression) expressed higher levels of Tlr4 than the rest. Interestingly, aged miR-146-/- neutrophils expressed sig- nificantly more Tlr4 than aged WT neutrophils (Figure 3C). Moreover, miR-146a deficiency in isolated neu- trophils produced significantly higher ROS levels (Figure 3D) and elevated oxygen consumption rate (Figure 3E) at basal state compared with those in neutrophils from WT mice. Thus, miR-146a deficiency seems to promote aging and hyperreactivity to neutrophils, a pro-inflam- matory phenotype that could contribute to the exacer- bated NETosis of these cells.
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haematologica | 2021; 106(6)