Bridging innate and adaptive immunity in cardiovascular disease

Cardiovascular disease is the leading cause of death and morbidity in the world. Myocardial infarction and stroke constitute the main manifestations of atherosclerosis that lead to the majority of cardiovascular events. Calcific aortic valve stenosis is the most common valve pathology. Atherosclerosis and aortic valve stenosis share common risk factors such as hypercholesterolemia. Lipid lowering treatment has ameliorated the incidence of fatal events; however, residual risk remains indicating the need to address the inflammatory component in cardiovascular disease. Many inflammatory mediators, such as cytokines and receptors have been implicated to play important role in the pathogenesis and the endpoints caused by an atherosclerotic plaque rupture. The role of pattern recognition receptors has been highlighted in several experimental studies. Both protective and detrimental effects have been described for the members of Tolllike receptor (TLR) family, a class of pattern recognition receptors. Several studies have focused on the role of the cell surface TLRs. The aim of the current thesis is to investigate the role of the intracellular pattern recognition receptor, TLR7. To gain information of the pathophysiological mechanisms that TLR7 is involved, both human cohorts of atherosclerosis and aortic valve stenosis as well as experimental models of atherosclerosis have been utilized. In Paper I, mRNA expression of TLR7 in human carotid plaques was associated with patients´ outcome. Patients that expressed higher levels of TLR7 in their removed plaque had fewer future adverse cardio- and cerebrovascular events. Macrophages and T cells were co-localized with TLR7 in carotid plaques. Furthermore, carotid plaque tissue responded with increased cytokine secretion upon ex vivo stimulation with a synthetic TLR7 ligand. Paper II showed TLR7 mRNA expression in calcified aortic valves. TLR7 mRNA was increased in calcified areas of the aortic valves compare to intermediate and healthy areas. In addition, TLR7 expression was associated with M2 macrophage markers in all parts of the aortic valve. Stimulation of calcified aortic valves ex vivo with a synthetic TLR7 ligand elicited cytokine response that was possibly derived directly or indirectly by macrophages. In Paper III, we investigated the in vivo effects of a synthetic TLR7 ligand in experimental atherosclerosis. Locally, treatment with the synthetic TLR7 ligand led to decrease in lesion size and changes in plaque composition. The lesions of the treated mice presented lesions with smaller necrotic core and fewer apoptotic cells compare to the control. The treatment had effect in the spleen, leading to marginal zone B and regulatory T cell expansion. In the plasma, we observed decrease in cholesterol levels and increase in IgM antibodies against oxidized lowdensity lipoprotein. The three studies presented in this thesis illustrate the protective role of TLR7 in atherosclerosis and aortic valve stenosis. TLR7 was expressed in both myeloid cells and lymphocytes indicating a role of the receptor in bridging innate and adaptive immune. The current results can encourage the investigation of TLR7 ligands as therapeutic intervention in cardiovascular disease

Low TLR7 gene expression in atherosclerotic plaques is associated with major adverse cardio- and cerebrovascular events

AIMS: Processes in the development of atherosclerotic lesions can lead to plaque rupture or erosion, which can in turn elicit myocardial infarction or ischaemic stroke. The aims of this study were to determine whether Toll-like receptor 7 (TLR7) gene expression levels influence patient outcome and to explore the mechanisms linked to TLR7 expression in atherosclerosis. METHODS AND RESULTS: Atherosclerotic plaques were removed by carotid endarterectomy (CEA) and subjected to gene array expression analysis (n = 123). Increased levels of TLR7 transcript in the plaques were associated with better outcome in a follow-up study over a maximum of 8 years. Patients with higher TLR7 transcript levels had a lower risk of experiencing major cardiovascular and cerebrovascular events (MACCE) during the follow-up period after CEA (hazard ratio: 2.38, P = 0.012, 95% CI 1.21–4.67). TLR7 was expressed in all plaques by T cells, macrophages and endothelial cells in capillaries, as shown by immunohistochemistry. In short-term tissue cultures, ex vivo treatment of plaques with the TLR7 ligand imiquimod elicited dose-dependent secretion of IL-10, TNF-α, GM-CSF, and IL-12/IL-23p40. This secretion was blocked with a TLR7 inhibitor. Immunofluorescent tissue analysis after TLR7 stimulation showed IL-10 expression in T cells, macrophages and vascular smooth muscle cells. TLR7 mRNA levels in the plaques were correlated with IL-10 receptor (r = 0.4031, P < 0.0001) and GM-CSF receptor A (r = 0.4354, P < 0.0001) transcripts. CONCLUSION: These findings demonstrate that TLR7 is abundantly expressed in human atherosclerotic plaques. TLR7 ligation elicits the secretion of pro-inflammatory and anti-inflammatory cytokines, and high TLR7 expression in plaques is associated with better patient outcome, suggesting that TLR7 is a potential therapeutic target for prevention of complications of atherosclerosis

TLR7 Expression Is Associated with M2 Macrophage Subset in Calcific Aortic Valve Stenosis

Calcific aortic valve stenosis (CAVS) is a common age-related disease characterized by active calcification of the leaflets of the aortic valve. How innate immune cells are involved in disease pathogenesis is not clear. In this study we investigate the role of the pattern recognition receptor Toll-like receptor 7 (TLR7) in CAVS, especially in relation to macrophage subtype. Human aortic valves were used for mRNA expression analysis, immunofluorescence staining, or ex vivo tissue assays. Response to TLR7 agonist in primary macrophages and valvular interstitial cells (VICs) were investigated in vitro. In the aortic valve, TLR7 correlated with M2 macrophage markers on mRNA levels. Expression was higher in the calcified part compared with the intermediate and healthy parts. TLR7+ cells were co-stained with M2-type macrophage receptors CD163 and CD206. Ex vivo stimulation of valve tissue with the TLR7 ligand imiquimod significantly increased secretion of IL-10, TNF-&alpha;, and GM-CSF. Primary macrophages responded to imiquimod with increased secretion of IL-10 while isolated VICs did not respond. In summary, in human aortic valves TLR7 expression is associated with M2 macrophages markers. Ex vivo tissue challenge with TLR7 ligand led to secretion of immunomodulatory cytokine IL-10. These results connect TLR7 activation in CAVS to reduced inflammation and improved clearance

Transforming growth factor-β2 is associated with atherosclerotic plaque stability and lower risk for cardiovascular events

AIMS: Transforming growth factor-beta (TGF-β) exists in three isoforms TGF-β1, -β2 and -β3. TGF-β1 has been suggested to be important for maintaining plaque stability, yet the role of TGF-β2 and -β3 in atherosclerosis remains to be investigated. OBJECTIVE: This study explores the association of these three isoforms of TGF-β with plaque stability in the human atherosclerotic disease. METHODS AND RESULTS: TGF-β1, -β2 and -β3 proteins were quantified in 223 human carotid plaques by immunoassays. Indications for the endarterectomy were: symptomatic carotid plaque with stenosis &gt;70% or without symptoms and &gt;80% stenosis. Plaque mRNA levels were assessed by RNA sequencing. Plaque components and extracellular matrix were measured histologically and biochemically. Matrix metalloproteinases were measured with ELISA. Monocyte chemoattractant protein-1 (MCP-1) was measured with immunoassays. The effect of TGF-β2 on inflammation and protease activity was investigated in vitro using THP-1 and RAW264.7 macrophages. Patients were followed longitudinally for cardiovascular events.TGF-β2 was the most abundant isoform and was increased at both protein and mRNA levels in asymptomatic plaques. TGF-β2 was the main determinant separating asymptomatic plaques in an Orthogonal Projections to Latent Structures Discriminant Analysis. TGF-β2 correlated positively to features of plaque stability and inversely to markers of plaque vulnerability. TGF-β2 was the only isoform inversely correlated to the matrix-degrading matrix metalloproteinase-9 and inflammation in the plaque tissue. In vitro, TGF-β2 pre-treatment reduced MCP-1 gene and protein levels as well as matrix metalloproteinase-9 gene levels and activity. Patients with plaques with high TGF-β2 levels had a lower risk to suffer from future cardiovascular events. CONCLUSIONS: TGF-β2 is the most abundant TGF-β isoform in human plaques and may maintain plaque stability by decreasing inflammation and matrix degradation