Signal Integration of IFN-I and IFN-II With TLR4 Involves Sequential Recruitment of STAT1-Complexes and NFκB to Enhance Pro-inflammatory Transcription

Atherosclerosis is a chronic inflammatory disease of the blood vessels, characterized by atherosclerotic lesion formation. Vascular Smooth Muscle Cells (VSMC), macrophages (MΦ) and dendritic cells (DC) play a crucial role in vascular inflammation and atherosclerosis. Interferon (IFN)α, IFNγ 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 GAF, and Nuclear Factor-κB (NFκB). 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 IFNα+LPS- and IFNγ+LPS-dependent SI in vascular cells as compared to immune cells, we performed a comprehensive genome-wide analysis of mouse VSMC, MΦ and DC in response to IFNα, IFNγ and/or LPS. Thus we identified IFNα+LPS or IFNγ+LPS induced genes commonly expressed in these cell-types that bound STAT1 and p65 at comparable GAS, ISRE or NFκB 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 IFNγ responses. In addition, similar STAT1-p65 co-binding modes were detected for IFNα+LPS and IFNγ+LPS up-regulated genes, which involved recruitment of STAT1 complexes preceding p65 to closely located GAS/NFκB or ISRE/NFκB composite sites already upon IFNα or IFNγ 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 NFκB, 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 IFNα or IFNγ priming on TLR4-induced activation in vascular and immune cells, with important implications in atherosclerosis.