Material Supplementary 0.DC1http://www.jimmunol.org/content/suppl/2010/07/06/jimmunol.090390

SummaryThe molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1β production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli.

ABCG2, a member of the ATP-binding cassette transporters has been identified as a protective pump against endogenous and exogenous toxic agents. ABCG2 was shown to be expressed at high levels in stem cells and variably regulated during cell differentiation. Here we demonstrate that functional ABCG2 is expressed in human monocyte-derived dendritic cells by the activation of a nuclear hormone receptor, PPAR␥. We identified and characterized a 150-base pair long conserved enhancer region, containing three functional PPAR response elements (PPARE), upstream of the human ABCG2 gene. We confirmed the binding of the PPAR␥⅐RXR heterodimer to this enhancer region, suggesting that PPAR␥ directly regulates the transcription of ABCG2. Consistent with these results, elevated expression of ABCG2 mRNA was coupled to enhanced protein production, resulting in increased xenobiotic extrusion capacity via ABCG2 in PPAR␥-activated cells. Furthermore PPAR␥ instructed dendritic cells showed increased Hoechst dye extrusion and resistance to mitoxantrone. Collectively, these results uncovered a mechanism by which up-regulation of functional ABCG2 expression can be achieved via exogenous or endogenous activation of the lipid-activated transcription factor, PPAR␥. The increased expression of the promiscuous ABCG2 transporter can significantly modify the xenobiotic and drug resistance of human myeloid dendritic cells.

Cholesterol uptake and efflux are key metabolic processes associated with macrophage physiology and atherosclerosis. Peroxisome proliferator-activated receptor gamma (PPAR␥) and liver X receptor alpha (LXR␣) have been linked to the regulation of these processes. It remains to be identified how activation of these receptors is connected and regulated by endogenous lipid molecules. We identified CYP27, a p450 enzyme, as a link between retinoid, PPAR␥, and LXR signaling. We show that the human CYP27 gene is under coupled regulation by retinoids and ligands of PPARs via a PPAR-retinoic acid receptor response element in its promoter. Induction of the enzyme's expression results in an increased level of 27-hydroxycholesterol and upregulation of LXR-mediated processes. Upregulated CYP27 activity also leads to LXR-independent elimination of CYP27 metabolites as an alternative means of cholesterol efflux. Moreover, human macrophage-rich atherosclerotic lesions have an increased level of retinoid-, PPAR␥-, and LXR-regulated gene expression and also enhanced CYP27 levels. Our findings suggest that nuclear receptor-regulated CYP27 expression is likely to be a key integrator of retinoic acid receptor-PPAR␥-LXR signaling, relying on natural ligands and contributing to lipid metabolism in macrophages.Handling of lipids by macrophages is an important metabolic process in the context of hypercholesterolemia and the development of atherosclerotic lesions (20,32,44). For this reason it is critical to understand the regulatory processes associated with cholesterol and fatty acid uptake and release (efflux) in this cell type. A regulatory network has been associated with macrophage lipid metabolism in recent years. First, it has been shown that peroxisome proliferator-activated receptor gamma (PPAR␥), a member of the nuclear receptor superfamily, can be linked to macrophage maturation and uptake of modified (oxidized) low-density lipoprotein (LDL) (35,45). Later, the oxysterol receptor liver X receptor (LXR) was linked to macrophage lipid metabolism by showing that LXR␣ is a direct transcriptional target of PPAR␥ and could induce lipid transporters such as ABCA1 (9, 40) and ABCG1 (26). A coordinated lipid transport is likely to be regulated by these receptors. Linking of the two receptor systems (PPAR␥ and LXR␣) provides an attractive but not well understood pathway to explain lipid and cholesterol uptake and efflux from macrophages.

a b s t r a c tAloe vera has been used in traditional herbal medicine as an immunomodulatory agent inducing antiinflammatory effects. However, its role on the IL-1␤ inflammatory cytokine production has not been studied. IL-1␤ production is strictly regulated both at transcriptional and posttranslational levels through the activity of Nlrp3 inflammasome. In this study we aimed to determine the effect of Aloe vera on the molecular mechanisms of Nlrp3 inflammasome-mediated IL-1␤ production in LPS-activated human THP-1 cells and monocyte-derived macrophages. Our results show that Aloe vera significantly reduced IL-8, TNF␣, IL-6 and IL-1␤ cytokine production in a dose dependent manner. The inhibitory effect was substantially more pronounced in the primary cells. We found that Aloe vera inhibited the expression of pro-IL-1␤, Nlrp3, caspase-1 as well as that of the P2X7 receptor in the LPS-induced primary macrophages. Furthermore, LPS-induced activation of signaling pathways like NF-B, p38, JNK and ERK were inhibited by Aloe vera in these cells.Altogether, we show for the first time that Aloe vera-mediated strong reduction of IL-1␤ appears to be the consequence of the reduced expression of both pro-IL-1␤ as well as Nlrp3 inflammasome components via suppressing specific signal transduction pathways. Furthermore, we show that the expression of the ATP sensor P2X7 receptor is also downregulated by Aloe vera that could also contribute to the attenuated IL-1␤ cytokine secretion. These results may provide a new therapeutic approach to regulate inflammasome-mediated responses.

No abstract

IL-1β is one of the main proinflammatory cytokines that regulates a broad range of immune responses and also participates in several physiological processes. The canonical production of IL-1β requires multiprotein complexes called inflammasomes. One of the most intensively studied inflammasome complexes is the NLRP3 inflammasome. Its activation requires two signals: one signal “primes” the cells and induces the expression of NLRP3 and pro-IL-1β, while the other signal leads to the assembly and activation of the complex. Several stimuli were reported to function as the second signal including reactive oxygen species, lysosomal rupture, or cytosolic ion perturbation. Despite very intensive studies, the precise function and regulation of the NLRP3 inflammasome are still not clear. However, many chronic inflammatory diseases are related to the overproduction of IL-1β that is mediated via the NLRP3 inflammasome. In this review, we aimed to provide an overview of studies that demonstrated the effect of plant-derived natural compounds on NLRP3 inflammasome-mediated IL-1β production. Although many of these studies lack the mechanistic explanation of their action, these compounds may be considered as complementary supplements in the treatment of chronic inflammatory diseases, consumed as preventive agents, and may also be considered as molecular tools to study NLRP3 function.

Ligands that specifically target retinoid-X receptors (RXRs) are emerging as potentially powerful therapies for cancer, diabetes, and the lowering of circulatory cholesterol. To date, RXR has only been crystallized in the absence of ligand or with the promiscuous ligand 9-cis retinoic acid, which also activates retinoic acid receptors. Here we present the structure of hRXR␤ in complex with the RXR-specific agonist LG100268 (LG268). The structure clearly reveals why LG268 is specific for the RXR ligand binding pocket and will not activate retinoic acid receptors. Intriguingly, in the crystals, the C-terminal "activation" helix (AF-2/helix H12) is trapped in a novel position not seen in other nuclear receptor structures such that it does not cap the ligand binding cavity. Mammalian two-hybrid assays indicate that LG268 is unable to release co-repressors from RXR unless co-activators are also present. Together these findings suggest that RXR ligands may be inefficient at repositioning helix H12.The retinoid-X receptors (RXR␣, ␤, and ␥) 1 are nuclear receptors that regulate differentiation, development, and homeostasis in response to ligand (1-5). RXRs can act as either homodimers or heterodimers with other nuclear receptors (1, 6 -8). The ability to serve as the preferred heterodimeric partner of many (Ͼ10) other receptors makes RXRs unique within the family of nuclear receptors and accounts for their role in many diverse signaling pathways. It is generally held that 9-cis retinoic acid (9cRA) is the natural ligand for RXRs (9, 10). This is a promiscuous ligand because it also activates retinoic acid receptors (RARs), which themselves serve as heterodimeric partners for RXR. Chemical approaches have led to the development of synthetic agonists that allow RXR to be activated without influencing the activity of RARs (11,12). It has emerged that RXR-specific agonists such as Targretin and LG268 may serve as beneficial therapies for the treatment of cancer, diabetes, and other metabolic disorders (13-15).LG268 is a substituted stilbene (6-[1-(3,5,5,8 -8-pentamethyl-5,6,7,8-tetrahydronapthalen-2-yl)-cycloproyl]-nicotinic acid) that specifically binds to and activates RXRs, but not RARs, with a slightly higher affinity than 9cRA (9 -11).Structural and biochemical studies of a number of nuclear receptor ligand-binding domains (LBDs) have yielded a relatively simple model for the mechanism by which ligands regulate nuclear receptor activity. The LBDs of nuclear receptors share a common, predominantly helical fold (16,17). In the absence of ligand, co-repressor proteins bind to the surface of the LBD and prevent the C-terminal helix H12 (AF2-helix) from binding to the surface of the receptor (18 -20). Activating ligands bind in a hydrophobic cavity within the core of the LBD and cause the repositioning of helix H12 such that it seals the ligand binding cavity, resulting in the displacement of co-repressor. In this position, helix H12 forms a critical part of the binding site for co-activator proteins (21,22). Significantly...