Role of Calprotectin as a Modulator of the IL27-Mediated Inflammatory Response of Endothelial Cells

Doctoral thesis (2015)

Cardiovascular diseases (CVD) are the leading causes of death with a morbidity of around 40% worldwide. Their development occur long before any symptoms are clinically evident. An underlying endothelial ... [more ▼]

Cardiovascular diseases (CVD) are the leading causes of death with a morbidity of around 40% worldwide. Their development occur long before any symptoms are clinically evident. An underlying endothelial dysfunction plays a fundamental role in the pathogenesis of cardiovascular events and is the central feature of atherosclerosis, which often appears prior to other CVD. However, the involvement of the chronic inflammatory state of the endothelium in the early mechanisms leading to the atherosclerotic phenomenon, which includes the protein-based communication between leukocytes and inflamed endothelial cells, remains to be further investigated. In this context of atherosclerosis, several key players have already been identified, but the role of the cytokines IL17D, IL27, and IL32γ is less well understood. In addition, evidence for the impact of calprotectin on promoting atherosclerosis has been reported, but its modulatory role on cytokine communication is still unclear. In this study, we first analysed the inflammatory responses of monocytes stimulated with the potent agonist TNFα to simulate a stress-induced inflammatory state. In order to characterize the monocytes as a potential cellular source of IL17D, IL27, IL32γ, and calprotectin, the regulation of gene expression was studied based on microarray data and qPCR. Highly significant up-regulations of IL27 subunit EBI3 and IL32 gene expressions were confirmed in TNFα-stimulated THP-1 cells. Furthermore, we were able to determine elevated intracellular IL27 and calprotectin protein levels in TNFα- stimulated THP-1 cells. In a second part, Human Umbilical Vein Endothelial Cells (HUVECs) were stimulated with IL17D, IL27, IL32γ, and calprotectin to characterize the induced inflammatory responses regarding gene expression. A gene-array qPCR screening demonstrated higher differential gene expression (DGE) after IL27 compared to IL17D, IL32γ, and calprotectin stimulation. Highest IL27-mediated DGEs were ob- served for CXCL10, CXCL11, IL7, and IL15. In order to highlight potential calprotectin modulatory effects on these genes, time course experiments of HUVECs stimulated with IL27+/-calprotectin were assessed by qPCR. We identified modulatory effects of cal- protectin on IL27-induced CXCL10 and IL15 gene expressions. Furthermore a mass spectrometry-based proteomic analysis of IL27+/-calprotectin-stimulated HUVECs enlightened a modulatory role of calprotectin in the late differential regulation of proteins such as the IL27 downstream effector STAT1. A literature-based pathway analysis of significant regulated proteins emphasized that calprotectin-modulated IL27 activity was able to decrease the probability of the development of atherosclerosis. Altogether, our findings suggest that calprotectin may exert a regulatory role on cytokine-mediated inflammatory responses and highlight the need of further investigations in the understanding of atherosclerosis development. [less ▲]