Marked acceleration of atherosclerosis after Lactobacillus casei-induced coronary arteritis in a mouse model of Kawasaki disease.

OBJECTIVE: The purpose of this study was to investigate whether Lactobacillus casei cell wall extract-induced Kawasaki disease (KD) accelerates atherosclerosis in hypercholesterolemic mice. Method and Results- Apolipoprotein E knockout or low-density lipoprotein receptor knockout mice were injected with Lactobacillus casei cell wall extract (KD mice) or PBS, fed high-fat diet for 8 weeks, and atherosclerotic lesions in aortic sinuses, arch (AC), and whole aorta were assessed. KD mice had larger, more complex aortic lesions with abundant collagen, and both extracellular and intracellular lipid and foam cells, compared with lesions in control mice despite similar cholesterol levels. Both apolipoprotein E knockout KD and low-density lipoprotein receptor knockout KD mice showed dramatic acceleration in atherosclerosis versus controls, with increases in en face aortic atherosclerosis and plaque size in both the aortic sinuses and AC plaques. Accelerated atherosclerosis was associated with increased circulating interleukin-12p40, interferon-γ, tumor necrosis factor-α, and increased macrophage, dendritic cell, and T-cell recruitment in lesions. Furthermore, daily injections of the interleukin-1Ra, which inhibits Lactobacillus casei cell wall extract-induced KD vasculitis, prevented the acceleration of atherosclerosis. CONCLUSIONS: Our results suggest an important pathophysiologic link between coronary arteritis/vasculitis in the KD mouse model and subsequent atherosclerotic acceleration, supporting the concept that a similar relation may also be present in KD patients. These results also suggest that KD in childhood may predispose to accelerated and early atherosclerosis as adults.

Interleukin-1ß is crucial for the induction of coronary artery inflammation in a mouse model of Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is the most common cause of acute vasculitis and acquired cardiac disease in US children. Untreated, children may develop coronary artery aneurysms, myocardial infarction, and sudden death as a result of the illness. Up to a third of KD patients fail to respond to intravenous immunoglobulin, the standard therapy, and alternative treatments are being investigated. Genetic studies have indicated a possible role for interleukin (IL)-1ß in KD. We therefore explored the role of IL-1ß in a murine model of KD. METHODS AND RESULTS: Using an established mouse model of KD that involves injection of Lactobacillus casei cell wall extract (LCWE), we investigated the role of IL-1ß and caspase-1 (activated by the inflammasome and required for IL-1ß maturation) in coronary arteritis and evaluated the efficacy of IL-1 receptor antagonist as a potential treatment. LCWE-induced IL-1ß maturation and secretion were dependent on the NLRP3 inflammasome in macrophages. Both caspase-1-deficient and IL-1 receptor-deficient mice were protected from LCWE-induced coronary lesions. Injection of recombinant IL-1ß into caspase-1-deficient mice restored the ability of LCWE to cause coronary lesions in response to LCWE. Furthermore, daily injections of the IL-1 receptor antagonist prevented LCWE-mediated coronary lesions up to 3 days after LCWE injection. CONCLUSIONS: Our results strongly suggest that caspase-1 and IL-1ß play critical roles in the development of coronary lesions in this KD mouse model, blocked by IL-1 receptor antagonist. Therefore, anti-IL-1ß treatment strategies may constitute an effective, more targeted treatment of KD to prevent coronary lesions.

Involvement of innate and adaptive immunity in a murine model of coronary arteritis mimicking Kawasaki disease.

Kawasaki disease (KD) is the most common cause of acquired cardiac disease and acute vasculitis in children in the developed world. Injection of a cell wall extract isolated from Lactobacillus casei (LCCWE) into mice causes a focal coronary arteritis that histopathologically mimics the coronary lesions observed in KD patients. In this study we used this model to investigate the participation of T cells, B cells, and dendritic cells (DC) in the development of coronary arteritis. RAG1(-/-), B cell(null), and wild-type (WT) mice were injected with a single dose of LCCWE (500 microg/mouse i.p.). None of the RAG1(-/-) mice developed coronary arteritis, whereas 70% of WT and 100% of B cell(null) mice developed coronary lesions, indicating that T cells were required for lesion formation. When splenocytes isolated from LCCWE-treated mice were restimulated with LCCWE, we observed significant IFN-gamma secretion in WT but not in RAG1(-/-) mice. Immunohistochemical staining showed F4/80(+) macrophages, activated MIDC-8(+) myeloid DCs (mDC), plasmacytoid DCs, and colocalization of CD3(+) T cells with mDCs in coronary artery lesions, suggesting an Ag-driven process. T cells but not B cells are required for LCCWE-induced coronary arteritis. Similar to human lesions, the coronary lesions contain macrophages, activated mDCs, and plaslmacytoid DCs all in close proximity to T cells, further strengthening the relevance of this mouse model to the immunopathology of coronary disease in KD. These studies are consistent with the interpretation that macrophages and DCs may collaborate with T cells in the pathological mechanisms of coronary arteritis.

Differential expression of Toll-like receptor 2 (TLR2) and responses to TLR2 ligands between human and murine vascular endothelial cells.

Toll-like receptors (TLRs) initiate and maintain host defenses and inflammation, and directly contribute to diseases such as atherosclerosis. It is not completely understood in what cell types proatherogenic TLR-induced signaling arises and, particularly, there is uncertainty regarding the potential functional role of TLR2 in endothelial cells (ECs). We determined TLR2 and TLR4 gene expression in four different human and two different murine primary ECs using gene array analysis, RT-PCR, and flow cytometry and confirmed these data by functional studies by stimulating ECs with the corresponding TLR ligands. TLR4 was expressed in all human and murine ECs and these cells responded to stimulation with LPS. Faint expression of TLR2 was observed in human ECs, whereas murine ECs express considerable amounts of TLR2 mRNA. Human ECs failed to respond to TLR2 ligands while murine ECs responded to TLR2 ligands. Furthermore, in murine ECs, TLR2 was located on the cell surface while in human ECs, TLR2 was sequestered in intracellular compartments. After IFN-gamma or IL-1beta stimulation, TLR2 translocated to the cell surface of human ECs. In conclusion, TLR2 is expressed intracellularly in human ECs and, therefore, TLR2 ligands are inaccessible to the receptor. Murine ECs express membrane TLR2 and respond to TLR2 ligands, but human ECs normally will not respond unless they are first primed with inflammatory stimulation, which appears to trigger translocation of TLR2 to the cell surface.

Activated myeloid dendritic cells accumulate and co-localize with CD3+ T cells in coronary artery lesions in patients with Kawasaki disease.

Emerging evidence implicating the participation of dendritic cells (DCs) and T cells in various vascular inflammatory diseases such as giant cell arteritis, Takayasu's arteritis, and atherosclerosis led us to hypothesize that they might also participate in the pathogenesis of coronary arteritis in Kawasaki disease (KD). Coronary artery specimens from 4 patients with KD and 6 control patients were obtained. Immunohistochemical and computer-assisted histomorphometric analyses were performed to detect all myeloid DCs (S-100(+), fascin(+)), all plasmacytoid DCs (CD123(+)) as well as specific DC subsets (mature myeloid DCs [CD83(+)], myeloid [BDCA-1(+)] and plasmacytoid DC precursors [BDCA-2(+)]), T cells (CD3(+)), and all antigen-presenting cells (HLA-DR(+)). Co-localization of DCs with T cells was assessed using double immunostaining. Significantly more myeloid DCs at a precursor, immature or mature stage were found in coronary lesions of KD patients than in controls. Myeloid DC precursors were distributed equally in the intima and adventitia. Mature myeloid DCs were particularly abundant in the adventitia. There was a significant correlation between mature DCs and HLA-DR expression. Double immunostaining demonstrated frequent contacts between myeloid DCs and T cells in the outer media and adventitia. Plasmacytoid DC precursors were rarely found in the adventitia. In conclusion, coronary artery lesions of KD patients contain increased numbers of mature myeloid DCs with high HLA-DR expression and frequent T cell contacts detected immunohistochemically. This suggests that mature arterial myeloid DCs might be activating T cells in situ and may be a significant factor in the pathogenesis of coronary arteritis in KD.

TLR2 and MyD88 contribute to Lactobacillus casei extract-induced focal coronary arteritis in a mouse model of Kawasaki disease.

BACKGROUND: Kawasaki disease is the most common cause of acquired cardiac disease and acute vasculitis in children, targets the coronary arteries, and can occasionally be fatal. The pathogenesis and the molecular mechanisms remain unknown. After injection of Lactobacillus casei cell-wall extract (LCCWE), mice develop a focal coronary arteritis that histopathologically resembles Kawasaki disease, but the mechanism remains unclear. Here, we tested the hypothesis that signaling by Toll-like receptors (TLRs) through their key downstream adaptor molecule myeloid differentiation factor 88 (MyD88) is required for the cellular activation and coronary arteritis produced by LCCWE. METHODS AND RESULTS: Bone marrow-derived macrophages from TLR2- or MyD88-deficient mice were unresponsive to LCCWE-induced stimulation. In contrast, macrophages obtained from TLR4-deficient mice produced the same amount of interleukin-6 as macrophages from wild-type mice after stimulation with LCCWE. Intraperitoneal injection of LCCWE produced severe focal coronary arteritis in TLR4(-/-) and C57BL/6 control mice but not in TLR2(-/-) or MyD88(-/-) mice. Collectively, these results indicate that LCCWE is a potent inducer of nuclear factor-kappaB via TLR2 but not TLR4 and that this activation proceeds via the MyD88-dependent signaling pathway. In vivo studies suggest that TLR2(-/-) mice are protected from LCCWE-induced coronary arteritis and that this protection is mediated through the adaptor molecule MyD88. CONCLUSIONS: Our results provide important insights into the molecular signaling in this mouse model of coronary arteritis. We show here that LCCWE-induced coronary arteritis is dependent on intact TLR2 and MyD88 signaling.