ABSTRACT: Atherosclerosis is a chronic inflammatory disease of the blood vessels, characterized by atherosclerotic lesion formation. Vascular Smooth Muscle Cells (VSMC), macrophages (M?), and dendritic cells (DC) play a crucial role in vascular inflammation and atherosclerosis. Interferon (IFN)?, IFN?, and Toll-like receptor (TLR)4 activate pro-inflammatory gene expression and are pro-atherogenic. Gene expression regulation of many pro-inflammatory genes has shown to rely on Signal Integration (SI) between IFNs and TLR4 through combinatorial actions of the Signal Transducer and Activator of Transcription (STAT)1 complexes ISGF3 and ?-activated factor (GAF), and Nuclear Factor-?B (NF?B). Thus, IFN pre-treatment ("priming") followed by LPS stimulation leads to enhanced transcriptional responses as compared to the individual stimuli. To characterize the mechanism of priming-induced IFN? + LPS- and IFN? + LPS-dependent SI in vascular cells as compared to immune cells, we performed a comprehensive genome-wide analysis of mouse VSMC, M?, and DC in response to IFN?, IFN?, and/or LPS. Thus, we identified IFN? + LPS or IFN? + LPS induced genes commonly expressed in these cell types that bound STAT1 and p65 at comparable ?-activated sequence (GAS), Interferon-stimulated response element (ISRE), or NF?B sites in promoter proximal and distal regions. Comparison of the relatively high number of overlapping ISRE sites in these genes unraveled a novel role of ISGF3 and possibly STAT1/IRF9 in IFN? responses. In addition, similar STAT1-p65 co-binding modes were detected for IFN? + LPS and IFN? + LPS up-regulated genes, which involved recruitment of STAT1 complexes preceding p65 to closely located GAS/NF?B or ISRE/NF?B composite sites already upon IFN? or IFN? treatment. This STAT1-p65 co-binding significantly increased after subsequent LPS exposure and correlated with histone acetylation, PolII recruitment, and amplified target gene transcription in a STAT1-p65 co-bound dependent manner. Thus, co-binding of STAT1-containing transcription factor complexes and NF?B, activated by IFN-I or IFN-II together with LPS, provides a platform for robust transcriptional activation of pro-inflammatory genes. Moreover, our data offer an explanation for the comparable effects of IFN? or IFN? priming on TLR4-induced activation in vascular and immune cells, with important implications in atherosclerosis.
