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  • br Acknowledgments br Introduction Inflammation is

    2019-09-11


    Acknowledgments
    Introduction Inflammation is the protective reaction of the body to infection, injury, or irritation with the aim to remove harmful stimuli such as pathogens, damaged cells, or allergic irritants and to initiate the healing process. Inflammatory abnormalities play a crucial role in the pathogenesis of many human diseases including autoimmune disorders such as type 1 diabetes, rheumatoid arthritis, or multiple sclerosis as well as cardiovascular diseases such as atherosclerosis or metabolic disease. Monocyte-derived macrophages are key mediators of the innate immune response and are central for the inflammation process (for recent reviews see [1], [2]). Based on functional properties macrophage phenotypes fall within a spectrum of two extremes [3]. First, pro-inflammatory macrophages (M1 phenotype or classically-activated macrophages) secrete pro-inflammatory cytokines and chemokines, thereby initiating and sustaining inflammation in order to fight microbes and infected cells. Second, the anti-inflammatory, pro-resolution macrophages (M2 phenotype or alternatively-activated macrophages) which are induced by anti-inflammatory factors such as IL-4 or IL-13 and promote resolution events such as clearance of apoptotic ML385 and tissue repair. For mounting an effective immune response by secretion of pro- or anti-inflammatory mediators and phagocytosis of invading pathogens or cellular debris during an infection, deployment of specific subsets of macrophages to the site of inflammation must be temporally and spatially strictly regulated. In a healthy environment this goal is achieved by a highly concerted production and release of different chemokines and the expression of their corresponding cognate receptors. Imbalances between the pro- and anti-inflammatory processes have been demonstrated to underlie the pathophysiology of autoimmunity and autoinflammation [4], [5]. In this study we have explored a functional role of the oxysterol/EBI2 system in this process. In 2011 we and others reported the discovery of oxysterols as ligands for EBI2 [6], [7]. Oxysterols are metabolites generated by hydroxylation of cholesterol and have been linked to a variety of fundamental physiological processes including sterol transportation, bile acid biosynthesis as well as immune cell function [8], [9]. For example oxysterols are ligands for several nuclear hormone receptors (e.g. LXR, RORα, RORγ) which play a role in the immune process. While LXR and their ligands are negative regulators of macrophage inflammatory gene expression [10] and constitute a metabolic checkpoint for immune cell proliferation [11], RORγt is the key transcription factor to orchestrate differentiation of pro-inflammatory Th17 cells [12]. For mouse macrophages it was reported that they up-regulate CH25H expression in a time-dependent manner upon immune challenge with LPS. The increase in mRNA and protein levels leads to a subsequent production of 25-OHC which is released into the cellular environment [13], [14]. In contrast, EBI2 mRNA transcripts are down-regulated after LPS treatment [15], [16]. While the oxysterol/EBI2 complex in murine B cells is well studied its role in primary human cells of the innate immune system, in particular macrophages, has not yet been investigated. In order to develop an integrated understanding of the transcriptional regulation and function of the oxysterol/EBI2 pathway here we describe the expression of the oxysterol-metabolizing enzymes CH25H, CYP27A1, CYP7B1, HSD3B7, and the receptor itself following inflammatory stimuli and show a functional role of this ligand/receptor pair in monocyte-derived macrophages.
    Materials and methods Details on materials and methods can be found in the Supplemental material section.
    Results
    Discussion In our experiments we mimicked inflammation by LPS challenge of M0 macrophages. We discovered a strong increase in expression of CH25H and CYP7B1 while CYP27A1 and HSD3B7 are down-regulated over time. CH25H up-regulation is transient with a peak around 2h. This is similar to the dramatic raise in expression of EBI2, even beyond expression levels of β-actin, which occurs after LPS treatment. Both findings suggest a feedback mechanism by which increasing amounts of oxysterols diminish further expression of enzyme and receptor mRNA. Interestingly, this result is in contrast to the murine system where EBI2 transcript levels are strongly down-regulated upon LPS challenge.