Migration of regulatory T cells into human atherosclerotic lesions is associated with plaque stability and correlates inversely with infiltrated mature dendritic cells

Abstract

Purpose: Atherosclerotic lesions are characterized by an extensive inflammation of the vessel wall, which can contribute to plaque instability. Dendritic cells (DC) play a crucial role in the regulation of inflammatory processes within the atherosclerotic lesions, by inducing a T cell-specific immune response. However, DCs are also involved in immunosuppressive processes, by the induction of regulatory T cells (Tregs). We investigated a possible correlation between infiltrated DCs and Tregs in atherosclerotic plaques. Methods: Cross-sections of 40 human carotid endarterectomy specimens were classified into groups of stable and vulnerable plaques using Trichrome staining. Immunohistochemical staining of plaques was used to detect infiltrated total (S100) and mature DCs (Fascin, DC-LAMP, CD83), Tregs (CD3, Foxp3), and to quantify the inflammatory state of the plaques (CD3, COX-2, CD68). In addition, RNA was isolated from plaque specimens and quantitative real-time PCR was performed to analyze transcription rates of DC markers (CD11c, CD209, HLA-DR), maturation markers (CD80, CD83, CD86), Treg-associated genes (CTLA-4, Foxp3) and of cytokine (TGFβ-family, IL-10, IFN-γ, IL-17α, IL-6). Migration assays and flow experiments were performed, to investigate the effects of Tregs on mature DCs in-vitro. Results: As compared with stable plaques, vulnerable plaques were characterized by increased numbers of COX-2-expressing cells and T lymphocytes, indicating an enhanced inflammatory process in these atherosclerotic lesions. In stable plaques, numbers of total and mature DCs were significantly lower in the inflammatory plaque shoulder. On the contrary, the numbers of Tregs were higher in the plaque shoulder and the fibrous cap of stable as compared to vulnerable plaques. The inverse correlation of mature DCs and Tregs, and the association of Tregs infiltration with plaque stability, were confirmed by PCR analysis showing a decreased transcription of DC- and maturation markers and an increased mRNA expression of Treg-associated genes and anti-inflammatory cytokines in stable atherosclerotic plaques. In-vitro, co-incubation of mature DCs with Tregs resulted in a decreased migration and a suppression of the adhesion to an endothelial cell layer under conditions of non-uniform shear stress. Conclusions: The results of our study suggest the association of a decreased occurrence of Tregs with a vulnerable plaque phenotype.