Interferon regulatory factor-5-dependent CD11c+ macrophages contribute to the formation of rupture-prone atherosclerotic plaques.

AIMS: Inflammation is a key factor in atherosclerosis. The transcription factor interferon regulatory factor-5 (IRF5) drives macrophages towards a pro-inflammatory state. We investigated the role of IRF5 in human atherosclerosis and plaque stability. METHODS AND RESULTS: Bulk RNA sequencing from the Carotid Plaque Imaging Project biobank were used to mine associations between major macrophage associated genes and transcription factors and human symptomatic carotid disease. Immunohistochemistry, proximity extension assays, and Helios cytometry by time of flight (CyTOF) were used for validation. The effect of IRF5 deficiency on carotid plaque phenotype and rupture in ApoE-/- mice was studied in an inducible model of plaque rupture. Interferon regulatory factor-5 and ITGAX/CD11c were identified as the macrophage associated genes with the strongest associations with symptomatic carotid disease. Expression of IRF5 and ITGAX/CD11c correlated with the vulnerability index, pro-inflammatory plaque cytokine levels, necrotic core area, and with each other. Macrophages were the predominant CD11c-expressing immune cells in the plaque by CyTOF and immunohistochemistry. Interferon regulatory factor-5 immunopositive areas were predominantly found within CD11c+ areas with a predilection for the shoulder region, the area of the human plaque most prone to rupture. Accordingly, an inducible plaque rupture model of ApoE-/-Irf5-/- mice had significantly lower frequencies of carotid plaque ruptures, smaller necrotic cores, and less CD11c+ macrophages than their IRF5-competent counterparts. CONCLUSION: Using complementary evidence from data from human carotid endarterectomies and a murine model of inducible rupture of carotid artery plaque in IRF5-deficient mice, we demonstrate a mechanistic link between the pro-inflammatory transcription factor IRF5, macrophage phenotype, plaque inflammation, and its vulnerability to rupture.

Synchrotron- and laboratory-based X-ray phase-contrast imaging for imaging mouse articular cartilage in the absence of radiopaque contrast agents.

The mouse model of osteoarthritis (OA) has been recognized as the most promising research tool for the identification of new OA therapeutic targets. However, this model is currently limited by poor throughput, dependent on the extremely time-consuming histopathology assessment of the articular cartilage (AC). We have recently shown that AC in the rat tibia can be imaged both in air and in saline solution using a laboratory system based on coded-aperture X-ray phase-contrast imaging (CAXPCi). Here, we explore ways to extend the methodology for imaging the much thinner AC of the mouse, by means of gold-standard synchrotron-based phase-contrast methods. Specifically, we have used analyser-based phase-contrast micro-computed tomography (micro-CT) for its high sensitivity to faint phase changes, coupled with a high-resolution (4.5 µm pixel) detector. Healthy, diseased (four weeks post induction of OA) and artificially damaged mouse AC was imaged at the Elettra synchrotron in Trieste, Italy, using the above method. For validation, we used conventional micro-CT combined with radiopaque soft-tissue staining and standard histomorphometry. We show that mouse cartilage can be visualized correctly by means of the synchrotron method. This suggests that: (i) further developments of the laboratory-based CAXPCi system, especially in terms of pushing the resolution limits, might have the potential to resolve mouse AC ex vivo and (ii) additional improvements may lead to a new generation of CAXPCi micro-CT scanners which could be used for in vivo longitudinal pre-clinical imaging of soft tissue at resolutions impossible to achieve by current MRI technology.