α7 Nicotinic Acetylcholine Receptor Is Expressed in Human Atherosclerosis and Inhibits Disease in Mice—Brief ReportSignificance
Objective—Cholinergic pathways of the autonomic nervous system are known to modulate inflammation. Because atherosclerosis is a chronic inflammatory condition, we tested whether cholinergic signaling operates in this disease. We have analyzed the expression of the α7 nicotinic acetylcholine receptor (α7nAChR) in human atherosclerotic plaques and studied its effects on the development of atherosclerosis in the hypercholesterolemic Ldlr–/– mouse model.
Approach and Results—α7nAChR protein was detected on T cells and macrophages in surgical specimens of human atherosclerotic plaques. To study the role of α7nAChR signaling in atherosclerosis, male Ldlr–/– mice were lethally irradiated and reconstituted with bone marrow from wild-type or α7nAChR-deficient animals. Ablation of hematopoietic cell α7nAChR increased aortic atherosclerosis by 72%. This was accompanied by increased aortic interferon-γ mRNA, implying increased Th1 activity in the absence of α7nAChR signaling.
Conclusions—The present study shows that signaling through hematopoietic α7nAChR inhibits atherosclerosis and suggests that it operates by modulating immune inflammation. Given the observation that α7nAChR is expressed by T cells and macrophages in human plaques, our findings support the notion that cholinergic regulation may act to inhibit disease development also in man.
The autonomic nervous system is a major regulator of homeostasis. Besides its classical role in regulating heart rate, blood pressure and respiration, recent data show that the vagal nerve, which constitutes the major efferent parasympathetic tract, modulates immune activity and inflammation. Several studies demonstrate that vagal stimulation decreases proinflammatory responses in acute experimental models.1–3 The ability of vagal nerve stimulation to control and dampen inflammation has been referred to as the cholinergic anti-inflammatory pathway.4
The effect of vagal anti-inflammatory signaling is mediated by nicotinic receptors containing the α7 subunits (α7nAChR).5 α7nAChR+ macrophages have been identified as direct targets of acetylcholine action and T cells as acetylcholine-synthesizing cells that relay the vagal signal.6 Several acute experimental models provide solid evidence for the interplay between the nervous and the immune system; however, it remains to be explored whether the cholinergic anti-inflammatory system operates to maintain immunologic homeostasis in chronic inflammatory diseases, such as atherosclerosis. In the current study, we set out to determine whether α7nAChR is present in human atherosclerotic lesions and to investigate the effect of α7nAChR-mediated signaling on the development of atherosclerosis in hypercholesterolemic mice. Our data identify an important atheroprotective action of α7nAChR signaling.
Human carotid endarterectomy specimens were examined for α7nAChR mRNA and protein expression. A bone marrow (BM) transfer strategy was used to investigate the role of α7nAChR on hematopoietic cells for progression of atherosclerosis in Ldlr–/– mice. For details, see the online-only Data Supplement.
α7nAChR Is Expressed in Human Atherosclerotic Lesions
Advanced atherosclerotic lesions of the carotid artery were obtained at surgery and analyzed for the expression of α7nAChR protein. Staining for the α7nAChR was detected in 7 of 10 atherosclerotic lesions. Immunoperoxidase staining revealed a significant population of cells present in inflamed regions of plaques (Figure 1). The protein expression pattern of α7nAChR coincided with that of both CD68+ and CD163+ macrophages, as well as T-cell marker CD3, but to a less extent with smooth muscle and endothelial cells (Figure 1A–1F). In inflamed areas of the plaque, where 39±5% of the cells were CD3+ T cells and 19±4% CD68+ macrophages, α7nAChR protein was expressed by 67±5% of all cells. This implies that in inflamed areas, the majority of α7nAChR+ cells were of immune origin. This was confirmed by double-staining, by which α7nAChR protein was identified on CD68+ macrophages (Figure 1G). α7nAChR was also detected on the CD163+ subset of macrophages, suggesting expression in alternatively activated M2 macrophages (Figure 1H). Furthermore, α7nAChR colocalized with a subset of CD3+ T cells in the lesions (Figure 1I). Single-channel micrographs of immunofluorescent staining are shown in Figure I in the online-only Data Supplement.
The global gene expression array database of the BiKE biobank was interrogated for α7nAChR mRNA expression pattern. We found a significant correlation between mRNA for α7nAChR, Chrna7, and M2 markers CD36 and CD163. The expression levels of Chrna7 and CD36 had a Pearson correlation coefficient of 0.346 (P=2.6e–04) and for Chrna7 and CD163 the Pearson correlation coefficient was 0.282 (P=0.0033). Although relatively weak correlations, this further supports that α7nAChR was expressed by alternatively activated M2 macrophages.
Increased Atherosclerosis in α7nAChR–/– BM Chimeric Ldlr–/– Mice
The finding of α7nAChR on immune cells in human atherosclerotic lesions prompted us to explore whether α7nAChR expression by hematopoietic cells affects atherosclerosis in an experimental model. BM was transplanted from Chrna7–/– mice lacking α7nAChR, or wild-type mice to irradiated Ldlr–/– mice that were fed a cholesterol-rich diet for 8 weeks to promote hypercholesterolemia and atherosclerosis. Mice reconstituted with α7nAChR–/– BM displayed a dramatic 72% increase in atherosclerosis in the aortic root (Figure 2A). When compared with lesions of mice transplanted with wild-type BM, those of mice receiving α7nAChR–/– BM were more advanced with large lipid deposits. Body weight was not influenced by the lack of α7nAChR in BM cells or did plasma cholesterol levels change (Table I in the online-only Data Supplement). Immunostaining for the macrophage marker CD68, the adhesion molecule vascular cell adhesion molecule 1 (VCAM-1), or the T-cell marker CD3 did not show any differences between groups (Figure 2B).
Aortic samples from α7nAChR–/–×Ldlr–/– chimeras exhibited increased mRNA expression of the proinflammatory Th1 cytokine interferon-γ (IFNγ; Figure 2C). In line with this, CD4 and CD8 mRNA levels were numerically increased in mice transplanted with α7nAChR–/– BM although this did not reach statistical significance. Aortic mRNA levels of the scavenger receptor CD36 were not altered, nor did we find any differences in expression of tumor necrosis factor-α (TNFα) or the macrophage marker CD68 (Figure 2C). Together, these data suggest that lack of α7nAChR signaling was associated with increased Th1 activity but not with significantly increased inflammatory cell infiltration into lesions.
Analysis of spleen mRNA did not show any difference between α7nAChR–/– and wild-type×Ldlr–/– chimeras with regard to CD68, CD4, CD8, or the mannose receptor, a marker for M2 macrophages (Figure IIA in the online-only Data Supplement). However, mRNA for the transcription factor FoxP3 that is expressed by regulatory T cells was significantly increased in α7nAChR–/–×Ldlr–/– chimeric mice. There were no differences in mRNA levels of the B-cell–specific CD19 marker, nor in antibody titers to oxidized low-density lipoprotein between the groups (Figure IIB in the online-only Data Supplement). Using flow cytometry, we investigated T-cell populations (CD3, CD4, CD8, and FoxP3), macrophages (F4/80 and CD11b), and granulocytes (Gr-1) in the spleen. There were no differences between the groups (data not shown). We further investigated the number of interferon γ positive (IFNγ+) cells in spleen and the coexpression of IFNγ with CD3, CD4, and F4/80. The total number of IFNγ+ cells, Th1 cells (CD3+CD4+IFNγ+ lymphocytes), and IFNγ+ macrophages (F4/80+IFNγ+) were not altered (Figure IIC in the online-only Data Supplement).
To investigate whether α7nAChR deficiency contributed to the increased T-cell activity, a proliferation assay was used to determine proliferative activity after stimulation with the T-cell mitogen, ConA. Splenocyte proliferation was significantly higher in animals lacking α7nAChR (Figure 2D). α7nAChR deficiency caused an increase in IFNγ secretion after ConA stimulation of splenocytes; however, this did not reach statistical significance (P=0.096; Figure 2D).
In the current study, we demonstrate that the absence of α7nAChR from BM cells accelerates atherosclerosis in hypercholesterolemic Ldlr–/– mice. Therefore, a cholinergic signal mediated through this receptor inhibits the disease process. The fact that targeting α7nAChR in BM cells was sufficient to affect disease substantially indicates that hematopoietic cells are major intermediates in this disease-modifying axis. Because the lack of hematopoietic α7nAChR was associated with increased IFNγ expression in atherosclerotic arterial tissue, cholinergic inhibition of atherosclerosis likely involves Th1-type T cells. Furthermore, α7nAChR was identified in human lesions, where it colocalized with a subset of T cells and macrophages. α7nAChR modulation of adaptive and innate immunity likely contributes to the striking effect on atherosclerosis.
It has not been possible to identify α7nAChR protein in mouse lesions by immunostaining although mRNA was detected in lesions of ApoE knockout mice, both at 10 and 20 weeks (data not shown). In fact, all antibodies tested by us showed reactivity also in α7nAChR-targeted mice. This is in line with the experience of others.7
α7nAChR signaling has important effects on macrophage function.3,5 Macrophages lacking α7nAChR exhibit increased expression of scavenger receptor CD36 and accumulate cholesterol intracellularly.8 However, CD36 expression was not altered in the α7nAChR–/–×Ldlr–/– chimeric mice of the current study. The cholinergic pathway also modulates adaptive immunity, with significant effects on T- and B-cell function.9,10 Vagotomized mice display increased CD4+ T-cell proliferation and cytokine release,10 suggesting that intact vagal nerve signaling dampens T-cell activation. In line with this, we observed an increased expression of the proatherogenic Th1 cytokine IFNγ in the aorta of α7nAChR–/–×Ldlr–/– chimeric mice and an increased proliferative response in α7nAChR-deficient splenocytes. Surprisingly, splenic FoxP3 mRNA levels were increased in α7nAChR–/–×Ldlr–/– chimeric mice; however, FoxP3 protein was not altered. Thus, α7nAChR signaling does not seem to influence the number of Th1 cells but rather exert an immunomodulating effect on T cells.
Because α7nAChR ablation in hematopoietic cells increased disease, an endogenous mediator operating through α7nAChR inhibits atherosclerosis. An obvious hypothesis is that this mediator is acetylcholine. In line with this notion, pharmacological inhibition of the enzyme responsible for acetylcholine degradation, cholinesterase, diminishes atherosclerosis in Apoe–/– mice.11 α7nAChR ligands include not only acetylcholine but also the antagonist kynurenic acid.12 Interestingly, metabolites of tryptophan degradation through the kynurenine pathway inhibit atherosclerosis.13 Whether such metabolites also could act as agonists and account for α7nAChR-mediated atheroprotection remains to be determined.
It should be mentioned that the nicotinic receptor family is a heterogeneous one with a large variety of nicotinic receptor subtypes. Previous reports show that nicotine, per se, is atherogenic.14,15 To our knowledge, the specific receptor mediating this proatherogenic effect has not been identified and it is possible that several different nicotinic receptors could contribute to this effect.
In conclusion, α7nAChR signaling operating via hematopoietic cells is an important modulator of atherosclerosis. As α7nAChR is expressed in human atherosclerotic lesions, this pathway likely operates also in human disease. Therefore, the α7nAChR pathway may be an interesting target for antiatherosclerotic therapy.
We thank Anneli Olsson, Ingrid Törnberg, and André Strodthoff for technical assistance, and The Göteborg and Umeå Vascular study group for providing human atherosclerotic tissue and Peder Olofsson for helpful comments on the article.
Sources of Funding
Our work was supported by grants from the Swedish Research Council (grants 6816 and 2667) and the CERIC (Center of Excellence for Research on Inflammation and Cardiovascular disease) Linnaeus program (grant 8703), the Swedish Heart-Lung Foundation, the Foundation for Strategic Research, The Osterman, OE and Edla Johansson, Magnus Bergvall, and Nanna Svartz foundations, European Union Seventh Framework Programme HEALTH-F2-2013 to 602222 Athero-Flux and KI foundation for geriatric disease. The FACS Canto II instrument was bought thanks to generous support from the Inga-Britt and Arne Lundberg Foundation. M.E. Johansson was supported by the Swedish Society for Medical Research and KI Joint funding postdoc position.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.303892/-/DC1.
- Nonstandard Abbreviation and Acronym
- α7 nicotinic acetylcholine receptor
- Received October 14, 2013.
- Accepted October 6, 2014.
- © 2014 American Heart Association, Inc.
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Atherosclerosis is an inflammatory disease. Recently, the cholinergic part of the autonomic nervous system was shown to modulate inflammation in acute experimental models. This immune modulation is mediated via the α7 nicotinic acetylcholine receptor (α7nAChR). Here, we demonstrate that the α7nAChR exerts an important atheroprotective effect that is linked to immunomodulatory action. Furthermore, we demonstrate the expression of the α7nAChR in human atherosclerotic plaques. These findings suggest α7nAChR signaling as an important modulator of atherosclerosis and thus an interesting target for antiatherosclerotic therapy.