Szilvia Benko

Peroxisome Proliferator-activated Receptor γ-regulated ABCG2 Expression Confers Cytoprotection to Human Dendritic Cells

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, PPARγ. 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 PPARγ·RXR heterodimer to this enhancer region, suggesting that PPARγ 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 PPARγ-activated cells. Furthermore PPARγ 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, PPARγ. The increased expression of the promiscuous ABCG2 transporter can significantly modify the xenobiotic and drug resistance of human myeloid dendritic cells.

NLRC5 Limits the Activation of Inflammatory Pathways

Nod-like receptors (NLRs) are intracellular sentinel proteins that are implicated in the detection of microbes and danger signals, thereby controlling several key innate immune pathways. The human genome encodes 22 NLR proteins, the function of many of which remains unknown. In this study, we present the identification and characterization of NLRC5, a NLR protein whose expression is found predominantly in cells of the myeloid and lymphoid lineages. NLRC5 expression was strongly induced by IFN-γ and more modestly by LPS and polyinosinic:polycytidylic acid. Overexpression of NLRC5 in HEK293T cells resulted in a global dampening of NF-κB–, AP-1–, and type I IFN-dependent signaling, most likely through transcriptional repression. Accordingly, NLRC5 was found to shuttle between the cytosol and the nucleus in a CrmA-dependent manner. Knocking down NLRC5 expression in RAW264.7 murine macrophages resulted in a potent upregulation of the proinflammatory responses to IFN-γ and LPS, including increased secretion of TNF, IL-6, and IL-1β, as well as cell surface expression of CD40. Strikingly, NLRC5 expression was also found to be critical for LPS-induced IL-10 production in RAW264.7 macrophages. Collectively, our results identify NLRC5 as a negative modulator of inflammatory pathways.

The microbial and danger signals that activate Nod-like receptors.

Nod-like receptors (NLRs) are a family of intracellular sensors that play key roles in innate immunity and inflammation. While some NLRs, including Nod1, Nod2, NAIP and IPAF, detect conserved bacterial molecular signatures from within the host cytosol, other members of this family seem to have evolved the capacity to sense danger signals perhaps independently of a microbial trigger. This is illustrated by the discovery that Nalp3 and Nalp1 are specifically activated by low concentrations of intracellular potassium. The fact that several stimuli, including bacterial toxins and some viruses, but also sterile crystals made of uric acid, asbestos or aluminium hydroxide, can trigger the Nalp3 inflammasome illustrate the fascinating prospect that microbial infections and certain danger signals may be perceived similarly by host recognition systems. Gaining insight into the function of NLR proteins in general will impact considerably on our understanding of the mechanisms underlying immunity to infection, adjuvanticity and auto-inflammatory disorders. In this review, we summarize the current knowledge on the microbial- and danger-derived signals that activate NLRs.

Nod1 and Nod2 in innate immunity and human inflammatory disorders

Nod (nucleotide-binding oligomerization domain) 1 and Nod2 are intracellular PRMs (pattern-recognition molecules) of the NLR (Nod-like receptor) family. These proteins are implicated in the detection of bacterial peptidoglycan and regulate pro-inflammatory pathways in response to bacteria by inducing signalling pathways such as NF-kappaB (nuclear factor kappaB) and MAPKs (mitogen-activated protein kinases). The Nod proteins act independently of the TLR (Toll-like receptor) cascade, but potently synergize with the latter to trigger innate immune responses to microbes. Most importantly, mutations in Nod2 have been shown to confer susceptibility to several chronic inflammatory disorders, including Crohns disease, Blau syndrome and early-onset sarcoidosis, underscoring the role of Nod2 in inflammatory homoeostasis. This review summarizes the most recent findings in the field of Nod1 and Nod2 research.

Transcriptional Regulation of Human CYP27 Integrates Retinoid, Peroxisome Proliferator-Activated Receptor, and Liver X Receptor Signaling in Macrophages

ABSTRACT Cholesterol uptake and efflux are key metabolic processes associated with macrophage physiology and atherosclerosis. Peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor alpha (LXRα) 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, PPARγ, 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 enzymes 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-, PPARγ-, 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-PPARγ-LXR signaling, relying on natural ligands and contributing to lipid metabolism in macrophages.

The Structural Basis for the Specificity of Retinoid-X Receptor-selective Agonists: New Insights Into the Role of Helix H12

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 hRXRβ 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.

Arginine Methylation Provides Epigenetic Transcription Memory for Retinoid-Induced Differentiation in Myeloid Cells

ABSTRACT Cellular differentiation is governed by changes in gene expression, but at the same time, a cells identity needs to be maintained through multiple cell divisions during maturation. In myeloid cell lines, retinoids induce gene expression and a well-characterized two-step lineage-specific differentiation. To identify mechanisms that contribute to cellular transcriptional memory, we analyzed the epigenetic changes taking place on regulatory regions of tissue transglutaminase, a gene whose expression is tightly linked to retinoid-induced differentiation. Here we report that the induction of an intermediary or “primed” state of myeloid differentiation is associated with increased H4 arginine 3 and decreased H3 lysine 4 methylation. These modifications occur before transcription and appear to prime the chromatin for subsequent hormone-regulated transcription. Moreover, inhibition of methyltransferase activity, preacetylation, or activation of the enzyme PAD4 attenuated retinoid-regulated gene expression, while overexpression of PRMT1, a methyltransferase, enhanced retinoid responsiveness. Taken together, our results suggest that H4 arginine 3 methylation is a bona fide positive epigenetic marker and regulator of transcriptional responsiveness as well as a signal integration mechanism during cell differentiation and, as such, may provide epigenetic memory.

Activation of Liver X Receptor Sensitizes Human Dendritic Cells to Inflammatory Stimuli

Dendritic cells (DCs) respond to changes in their lipid environment by altering gene expression and immunophenotype. Some of these alterations are mediated via the nuclear receptor superfamily. However, little is known about the contribution of liver X receptor (LXR) to DC biology. In this study, we present a systematic analysis of LXR, activated by synthetic ligands or naturally occurring oxysterols in developing human monocyte-derived DCs. We found that LXRs are present and can be activated throughout DC differentiation in monocyte- and blood-derived DCs. Administration of LXR-specific natural or synthetic activators induced target gene expression accompanied by increased expression of DC maturation markers, such as CD80 and CD86. In mature DCs, LXR activation augmented the production of inflammatory cytokines IL-12, TNF-α, IL-6, and IL-8 and resulted in an increased capacity to activate CD4+ T cell proliferation upon ligation with TLR4 or TLR3 ligands. These effects appear to be underpinned by prolonged NF-κB signaling. Supporting such an inflammatory role, we found that LXR positive DCs are present in reactive lymph nodes in vivo. We propose that activation of LXR represents a novel lipid-signaling paradigm that alters the inflammatory response of human DCs.

Dendritic cell subtypes as primary targets of vaccines: the emerging role and cross-talk of pattern recognition receptors

Abstract Preventive vaccination is the most successful approach against infectious diseases and has a great impact on world health. Vaccines operate through the activation of innate immunity that helps to stimulate antigen-specific T- and B-lymphocytes. These events are orchestrated by dendritic cells (DCs) that are able to sample foreign structures and concomitantly sense ‘danger signals’. Thus, DCs provide a functional link between innate and acquired immunity, and due to their regulatory potential are referred to as natural adjuvants. Human conventional and plasmacytoid DCs express different sets of well-characterized Toll-like membrane receptors (TLRs) that recognize a broad range of conserved molecular patterns of pathogens. The recently discovered cytosolic Nod-like receptors (NLRs) and RIG-like helicases (RLHs) also turned out to participate in pathogen recognition and modulation of immune responses through interacting signaling pathways. As a result of their collaboration, the TLR, NLR and RLH recognition systems induce the secretion of different combinations of cytokines that play a fundamental role in T-cell activation and instruction. Ligands of the innate recognition systems emerge as new adjuvants for vaccine design, whereas manipulation of the signaling pathways mediated by these receptors offers new avenues for fine tuning immune responses and optimizing immunotherapies.

Phagocytosis of cells dying through autophagy induces inflammasome activation and IL-1β release in human macrophages.

Phagocytosis of naturally dying cells usually blocks inflammatory reactions in host cells. We have recently observed that clearance of cells dying through autophagy leads to a pro-inflammatory response in human macrophages. Investigating this response further, we found that during engulfment of MCF-7 or 293T cells undergoing autophagic death, but not apoptotic or anoikic ones, caspase-1 was activated and IL-1β was processed, then secreted in a MyD88-independent manner. Autophagic dying cells were capable of preventing some LPS-induced pro-inflammatory responses, such as TNFα, IL-6 and IL-8 induction, but synergized with LPS for IL-1β production. Caspase-1 inhibition prevented macrophage IL-1β release triggered by the dying cells and also other pro-inflammatory cytokines which were not formed in the presence of IL-1 receptor antagonist anakinra either. IL-1β secretion was also observed using calreticulin knock down or necrostatin treated autophagic MCF-7 cells and it required phagocytosis of the dying cells which led to ATP secretion from macrophages. Blocking K+ efflux during phagocytosis, the presence of apyrase, adding an antagonist of the P2X7 receptor or silencing the NOD-like receptor protein NALP3 inhibited IL-1β secretion. These data suggest that during phagocytosis of autophagic dying cells ATP, acting through its receptor, initiates K+ efflux, inflammasome activation and secretion of IL-1β, which initiates further pro-inflammatory events. Thus, autophagic death of malignant cells and their clearance may lead to immunogenic response.