AIM2 Stimulation Impairs Reendothelialization and Promotes the Development of Atherosclerosis in Mice

Background: Atherosclerosis has been shown to result from chronic inflammation caused by constitutive activation of the pattern recognition receptors (PRR), which are principle effectors of the innate immune system. PRR are present in the endosome or on the cellular membrane and can sense the aberrant release of nucleic acids, which is often a sign of acute or chronic cellular damage. Absent in melanoma 2 (AIM2) is a PRR that is expressed by vascular cells and specializes in detecting cytoplasmic double-stranded DNA (dsDNA). Activation of AIM2 leads eventually to activation of the inflammasome, but the role of AIM2 in vascular disease and atherosclerosis has not been well-studied. Therefore, in this study we took advantage of acute and chronic models of vascular injury to determine the biological role of AIM2 in atherogenesis. Methods and Results: We were able to induce significant release of proinflammatory cytokines in mice through the intravenous injection of a synthetic ligand for AIM2, double-stranded poly dA:dT. This cytokine release was shown to impair reendothelialization of the carotid artery and increase the number of circulating endothelial microparticles (EMP) after acute denudation, compared to treatment with vehicle. We saw an increase in the production of reactive oxygen species in the aorta, the number of circulating EMP, and, most interestingly, atherosclerotic plaque formation in apolipoprotein E-deficient (ApoE−/−) mice when they received continual subcutaneous poly dA:dT, in contrast to vehicle-treated animals. Finally, treatment with poly dA:dT did not impair vascular reendothelialization in AIM2−/− mice compared to vehicle controls in the carotid artery injury model. Conclusion: Overall, our data suggest that AIM2, as a known regulator of the inflammasome, is an active participant in atherogenesis, and highlight the importance of fully understanding the pathological mechanisms involved. It seems to be worth of further exploration as a therapeutic target, and future studies focusing on the effects of AIM2 activation as well as its pharmacological inhibition may reveal promising new therapeutic concepts for the treatment of atherosclerosis. Highlights - Stimulation of AIM2 works in a pro-atherosclerotic manner to promote the disruption of endothelial function, higher production of ROS in the vessels, and a decrease in reendothelialization, and it also promotes the development of atherosclerotic plaques in mice - AIM2 may have an important role in the development and progression of atherosclerosis - AIM2 seems be a therapeutic target that is worth exploring further, and future studies focusing on the effects of AIM2 activation as well as pharmacological inhibition may reveal promising new therapeutic concepts for the treatment of atherosclerosi

High density lipoprotein mediates anti-inflammatory transcriptional reprogramming of macrophages via the transcriptional repressor ATF3

High Density Lipoprotein (HDL) mediates reverse cholesterol transport and it is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional repressor ATF3, as an HDL-inducible target gene in macrophages that down-regulates the expression of Toll-like receptor (TLR)-induced pro-inflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo. Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of novel HDL-based therapies

Proinflammatory Stimulation of Toll-Like Receptor 9 with High Dose CpG ODN 1826 Impairs Endothelial Regeneration and Promotes Atherosclerosis in Mice.

BACKGROUND:Toll-like receptors (TLR) of the innate immune system have been closely linked with the development of atherosclerotic lesions. TLR9 is activated by unmethylated CpG motifs within ssDNA, but also by CpG motifs in nucleic acids released during vascular apoptosis and necrosis. The role of TLR9 in vascular disease remains controversial and we sought to investigate the effects of a proinflammatory TLR9 stimulation in mice. METHODS AND FINDINGS:TLR9-stimulation with high dose CpG ODN at concentrations between 6.25 nM to 30 nM induced a significant proinflammatory cytokine response in mice. This was associated with impaired reendothelialization upon acute denudation of the carotid and increased numbers of circulating endothelial microparticles, as a marker for amplified endothelial damage. Chronic TLR9 agonism in apolipoprotein E-deficient (ApoE-/-) mice fed a cholesterol-rich diet increased aortic production of reactive oxygen species, the number of circulating endothelial microparticles, circulating sca-1/flk-1 positive cells, and most importantly augmented atherosclerotic plaque formation when compared to vehicle treated animals. Importantly, high concentrations of CpG ODN are required for these proatherogenic effects. CONCLUSIONS:Systemic stimulation of TLR9 with high dose CpG ODN impaired reendothelialization upon acute vascular injury and increased atherosclerotic plaque development in ApoE-/- mice. Further studies are necessary to fully decipher the contradictory finding of TLR9 agonism in vascular biology

Proinflammatory response by concentration dependent TLR9 activation in acute vascular injury.

<p>WT mice were subjected to an electric denudation of the left carotid artery as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146326#pone.0146326.g001" target="_blank">Fig 1</a> and received repetitive s.c. injections of 0.625nM (n = 7), 6.25nM (n = 7), 18nM (n = 6), 30nM CpG ODN (n = 7) or vehicle (n = 7) over 7 days. Splenic CD3 positive lymphocytes from 18nM and 30nM CpG ODN treated mice expressed more IFN-γ and RANTES compared to vehicle controls (a). TLR9 activation by 6.25nM, 18nM and 30nM CpG ODN significantly impaired reendothelialization in WT mice compared to vehicle controls (b). Further, stimulation with 18nM and 30nM CpG ODN elevated levels of circulating CD11b positive cells (c). Data are presented as mean±SEM. Statistical analysis was performed using ANOVA (Holm-Sidak's multiple comparisons test). *p<0.05, **p<0.001, p***<0.0001.</p

Concentration dependent plaque development by TLR9 stimulation.

<p>ApoE^-/- mice were fed a cholesterol rich diet for 8 weeks and treated 3 times a week s.c. with 0.625nM (n = 7) or 18nM CpG ODN (n = 7), or vehicle (n = 8) in the last 7 weeks. Chronic TLR9 stimulation with high concentrations of CpG ODN in ApoE^-/- mice led to a significant increase in aortic atherosclerotic plaque size compared to low dose CpG ODN and vehicle treated mice (a). Immunohistochemical staining demonstrates increased TLR9 expression in aortic plaques of 18nM CpG ODN treated mice (b, representative image). Differential blood analysis shows increased CD3 and CD3/CD8 positive cells, while CD3/CD4 and neutrophil granulocytes (CD11b⁺/Ly6C⁺/CD115^- cells) are reduced (c). TLR9 expression is induced in B-cells, T-helper cells, and non-classical monocytes (CD11b⁺/Ly6C⁺ low/CD115⁺) are by CpG ODN, while it is decreased in neutrophil granulocytes (d). Data are presented as mean±SEM. Statistical analysis was performed using Kruskal-Wallis test (Dunn’s multiple comparison test). *p<0.05, **p<0.001, ***p<0.0001, ˚p<0.05 18nM to 0.625nM CpG ODN.</p

TLR9 stimulation augments atherosclerosis.

<p>ApoE^-/- mice were fed a cholesterol rich diet for 7 weeks and treated 3 times a week with 18nM CpG ODN s.c. for 6 weeks (a). Chronic stimulation of TLR9 in ApoE^-/- mice led to a significant increase in aortic atherosclerotic plaque size compared to vehicle treated mice (b, quantitative and representative Oil-red O image; n = 9). MOMA-2 stained atherosclerotic plaque were similar compared to vehicle treated mice (c, quantification and representative image). Data are presented as mean±SEM. Statistical analysis was performed using 2-tailed, unpaired students t-test. Scale bar 200μm in b and c. **p<0.001.</p

CpG ODN stimulation in acute vascular injury.

<p>WT mice were injected with PBS (n = 4) or 3-30nM CpG ODN (n = 2) i.v., respectively. Activation of TLR9 with 30nM CpG ODN led to the highest plasma IL-6 concentrations in mice compared to vehicle controls (a). For the acute injury, WT mice (n = 12–13) were subjected to an electric denudation of the left carotid artery and received repetitive injections of 30nM CpG ODN or vehicle over 7 days (b). Reendothelialization was greatly impaired in TLR9-stimulated mice compared to vehicle controls (c, representative Evan’s blue stained photomicrograph; and d, quantitative). The number of circulating EMPs (e, n = 5) and the number of circulating sca-1/flk-1 positive cells (f, n = 5) were significantly increased in ODN treated mice. Gating strategy for FACS analysis is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146326#pone.0146326.s003" target="_blank">S3 Fig</a>. Data are presented as mean±SEM. Statistical analysis was performed using 2-tailed, unpaired students t-test. Scale bar 200μm in c. *p<0.05, **p<0.001, ***p<0.0001.</p

High-density lipoprotein mediates anti-inflammatory reprogramming of macrophages via the transcriptional regulator ATF3

High-density lipoprotein (HDL) mediates reverse cholesterol transport and is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional regulator ATF3, as an HDL-inducible target gene in macrophages that downregulates the expression of Toll-like receptor (TLR)-induced proinflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo. Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of new HDL-based therapies