Innate immunity atherosclerosis : signaling pattern recognition receptors and an antimicrobial peptide

Abstract

Atherosclerosis, a disease characterized by fibroinflammatory lipid lesions in the arterial wall, is the leading cause of mortality in the Western world. For decades research has been aimed at dissecting the pathogenesis of this multifactorial disease. Recently vascular inflammation induced by both innate and adaptive immune responses has been shown to be important for the disease development. Innate immunity is the primary host defense to infections, and elicited by the recognition of microbial ligands by pattern recognition receptors (PRRs). Infections have been implicated in the pathogenesis of atherosclerosis and its complications such as myocardial infarction (MI).

The aim of this thesis was to elucidate the function of innate immunity in human atherosclerosis. Specific objectives were to investigate expression and functional relevance of two types of PRRs: Toll-like receptors (TLRs) and nucleotide oligomerization domain protein 1 (NOD1) and NOD2, and to characterize the antimicrobial peptide, LL-37, an innate host defense peptide with effector and regulatory functions in innate immunity, in the disease.

We investigated the expression of 9 TLRs in human atherosclerotic lesions and normal vessels. Our data show that endothelial cells of normal artery express low levels of TLRs. However, TLR1, TLR2 and TLR4, were upregulated both at the mRNA and protein level in atherosclerotic lesions compared to normal arteries. TLR2 and TLR4 were expressed by macrophages and some endothelial cells, and co-localized with activated NF-κB signaling in the lesions. These findings suggest that TLRs on endothelial cells constitute the first line of vascular defense against infection, and may be linked to the inflammatory response in atherosclerosis.

To assess the impact of TLR4-mediated innate immunity on the risk of MI, the Stockholm Heart Epidemiology Program (SHEEP) was screened for two functional polymorphisms in the TLR4 gene. Hyporesponsive TLR4 variants were associated with a higher risk for MI in men. Although the underlying mechanisms remain to be elucidated, this genetic study indicates that alteration in TLR4- mediated innate immunity may affect progression of coronary artery disease.

In addition to TLRs, vascular endothelial cells also express basal levels of the intracellular PRRs, NOD1 and NOD2. Yet, NOD2 was markedly increased in atherosclerotic lesions and expressed predominantly by macrophages and some endothelial cells. NOD2 expression in human endothelial cells could be induced by proinflammatory cytokines and activation of NOD2 induced the expression of intercellular adhesion molecule 1 (ICAM-1) and monocyte chemoattractant protein 1 (MCP-1), two proteins with important roles in the recruitment and activation of leukocytes during vascular inflammation. These findings imply that NOD2 might be a key mechanism for sensing intracellular infection in atherogenesis.

In an effort to identify innate effectors, we analyzed antimicrobial peptides and found that LL-37 was produced in atherosclerotic lesions, predominantly by macrophages. Expression of the murine homologue of LL-37 could be induced in macrophages by Chlamydia pneumoniae, a bacterium associated with cardiovascular disease. Stimulation of endothelial cells with LL-37 led to dose-dependent induction of a limited set of proinflammatory genes, signifying a novel proinflammatory pathway involved in disease development. Although LL-37 exhibits broad microbicidal activity, this peptide had no microbicidal effect on Chlamydia pneumoniae.

In summary, our results reveal that the endothelium is the first line vascular defense of the artery. It is characterized by expression of a spectrum of PRRs, capable of sensing extracellular as well as intracellular pathogens. Whether infections contribute to atherosclerosis remains controversial but the results of this thesis suggest that the machinery for microbial recognition and effector functions is further enhanced in atherosclerosis. By identifying a large array of PRRs in atherosclerosis together with the characterization of the antimicrobial peptide, LL-37, the work of this thesis hopefully adds novel understanding of innate immunity in the disease.