PPARγ in immunity and inflammation: cell types and diseases

Biochimica et Biophysica Acta 1771 (2007) 1014 – 1030 www.elsevier.com/locate/bbalip

Review

PPARγ in immunity and inflammation: cell types and diseases Lajos Széles 1 , Dániel Töröcsik 1 , László Nagy ⁎ Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, University of Debrecen, Medical and Health Science Center, Life Science Building, Egyetem ter 1. Debrecen, H-4010, Hungary Received 4 December 2006; received in revised form 23 January 2007; accepted 13 February 2007 Available online 24 February 2007

Abstract The lipid activated transcription factor, PPARγ appears to have multiple functions in the immune system. There are several cell types expressing the receptor, most prominently antigen presenting cells, such as macrophages and dendritic cells. The receptor's activation leads to primary transcriptional activation of many, mostly lipid metabolism-related genes. However, gene regulation also occurs on immunity and inflammation-related genes. Key questions are: in what way lipid metabolism and immune regulation are connected and how activation and/or repression of gene expression may modulate inflammatory and anti-inflammatory responses and in what way can these be utilized in therapy. Here we provide a cell type and disease centric review on the role of this lipid activated transcription factor in the various cells of the immune system it is expressed in, and in some major inflammatory diseases such as atherosclerosis, inflammatory bowel disease and rheumatoid arthritis. © 2007 Elsevier B.V. All rights reserved. Keywords: Peroxisome Proliferator-Activated Receptor gamma; Inflammation; Macrophage; Dendritic cell; Chronic inflammation; Atherosclerosis; Inflammatory bowel disease

1. Introduction More than 15 years have passed since PPARs have been cloned [1–4]. One of them, PPARγ proved to be a key transcription factor of adipocyte differentiation [5,6] lipid and glucose homeostasis and an important target in type 2 diabetes and metabolic syndrome [7]. Besides its role in metabolic tissues it appears to be expressed in several other cell types, including cells of the immune system such as macrophages, dendritic cells, eosinophils, T cells and B cells. Interestingly, it was documented in one of the early publications that this receptor is expressed at high levels in mouse spleen [4] already pointing to a role in the immune system. Besides investigations on the function of PPARγ in macrophages and its role in lipid metabolism [8–14] a new aspect of PPARγ was revealed and developed in parallel: its potential anti-inflammatory activity [15,16]. A lot has been done in the last several years in trying to

⁎ Corresponding author. Tel.: +36 52 416 432; fax: +36 52 314 989. E-mail address: [email protected] (L. Nagy). 1 These authors contributed equally. 1388-1981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2007.02.005

dissect the role of this lipid-activated transcription factor in immune responses at the gene, the cellular and the organismal level. In this review we take a cell and disease centric view and attempt to summarize the available, sometimes conflicting data on the role of the receptor and/or the effect of the activation of the receptor in the various immune cell types in vitro and also in vivo in different disease models as well as in humans with inflammatory diseases. We also review the available evidence showing that some of the effects of PPARγ in lipid metabolism and processing directly contributes to immunoregulation. Finally, we review some of the emerging new technologies that can help to resolve the many outstanding issues on this complex field. We would like to refer to a more gene centric view on PPARγ's role in gene expression regulation, in particular transrepression, in immune cells reviewed by Mercedes Ricote and Chris Glass also appearing in this volume (Ricote and Glass, this issue). Inflammation is a vital response provoked by pathogens, physical harm, ischemic, toxic or autoimmune injury in order to protect the body. Inflammation is also a very complex process including the concerted and often opposing activities of several cell types and dozens of lipid and protein mediators. The initial

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steps of inflammation include changes in local blood flow and accumulation of various inflammatory cells (neutrophils, dendritic cells, monocytes, mast cells and lymphocytes). Pathogenes, cell debris and the inflammatory cells need to be removed afterwards and normal tissue functions and integrity restored. If the delicate balance between inflammation and restoration is broken it leads to the development of chronic inflammatory or autoimmune diseases such as inflammatory bowel disease (IBD), arthritis or asthma. In order to evaluate and define the role and contribution of a given protein to inflammation and inflammatory diseases one needs to consider all the participating cell types the protein is expressed in, and their roles individually and then put it into the context of the entire process. This is a daunting, technically difficult, challenging and very complex task with no easy answers. Immune response can be classified into two fundamental types: innate and adaptive immunity. Innate immunity has evolved to recognize microbes and it is the first line of defense acting before infection occurs. Innate immunity is made up by cells such as epithelial cells, smooth muscle cells, fibroblasts, platelets and endothelial cells that are important in detecting the various stimuli without direct effects on the elimination of microbes. Phagocytic cells – such as neutrophils or macrophages – and natural killer cells (NK) on the other hand have a major role in the elimination process. In addition some plasma proteins such as the complement system are also vital in the innate immune responses. The adaptive immune system consists of lymphocytes (T and B cells) that carry a diverse array of specific receptors to recognize microbes and also nonmicrobial substances called antigens [17,18]. Once stimulated they produce a variety of cytokines and antibodies. In immune responses innate and adaptive immunity are interlocked and complement each other. In the recognition of microbes and antigens several membrane receptors are involved such as mannose receptor or different toll-like receptors (TLRs) [19]. Members of the TLRs detect different microbial products and signal through the activation of transcriptional pathways such as the NFκB pathway regulating the expression of cytokines and other proteins with antimicrobial activity or signaling properties. Signaling in the immune system can be either a direct interaction of cells – cellular immunity – or be mediated by cytokines and antibodies – humoral immunity – that are carrying signals to all cells with the appropriate receptors. This way other cells could also be involved in inflammation such as neurons or cells with roles in maintaining lipid homeostasis of the body. The cross-talk of these distinct cell types and the mechanisms how inflammation affects other physiological programs of the body is determined in large part by gene expression programs at the cellular level. For mediating such processes and cross-talk PPARγ is a good example. It is likely to orchestrate signals and regulate programs of both inflammation and metabolism at the same time in different cell types. One approach to explore the function of this receptor therefore is to identify its role in each of the cell types and then try to reconstruct its combined contribution to inflammation and metabolism.

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2. Activation of PPARγ We are not providing an overview of the structure and activity of the receptor and the molecular details of its interaction with its heterodimeric partner RXR as well as with co-factors. Rather we would like to refer to papers focusing on these topics and also appearing in this volume. However the nature, the source and specificity of the ligand activating the receptor is an important issue in the context of inflammatory gene expression regulation, because it is often the cause of conflicting interpretations of results, therefore we consider it briefly here. Ligand activation of PPARγ shows characteristic differences when compared to activation of classical nuclear receptors. There are two important characteristics of PPARγ's ligand binding. First, the volume of ligand-binding cavity of PPARγ is also much larger than pockets of others nuclear hormone receptors (1619 Å2 (PPARγ) versus 871 Å2 (VDR), 687 Å2 (RXRα), 557 Å2 (Progesteron Receptor) and 476 Å2 (Estrogen Receptor) [20]). This relatively large ligand-binding pocket of PPARγ can bind a broad range of natural and synthetic ligands. Natural ligands include polyunsaturated fatty acids (PUFAs), prostaglandin derivates (most importantly, 15-deoxy-Δ12–14Prostaglandin-J2 (15d-PGJ2) [4,21]), components of oxLDL, such as linoleic acid metabolites 13-HODE and 15-HODE [10]. Synthetic ligands are the insulin sensitizer Thiazolidinediones (TZDs such as englitazone, ciglitazone, pioglitazone, rosiglitazone, troglitazone [22]), non-steroidal anti-inflammatory drugs (i.e. indomethacin, fenoprofen, flufenamic acid [23] and other PPARγ modulators so-called SPPARMs (Selective PPAR Modulators) (reviewed by Balint and Nagy [24]). PPARγ binds its natural agonists, identified so far, with significantly lower affinity than classical endocrine receptors, and the ligands show a significant structural variety. In vivo, several convergent signals, not only fatty acids and prostaglandins, but also RXRactivating rexinoids contribute to the activation status of PPARγ in a given cell. Secondly, some of the synthetic and especially the low affinity natural ligands such as 15d-PGJ2 appear to have PPARγ-independent effects. Here we do not discuss the possible PPARγ-independent mechanisms of 15d-PGJ2, but we would like to point out the most important arguments, which support the notion that 15d-PGJ2 and TZDs can act receptor independently also. (1) 15d-PGJ2 and synthetic agonists have overlapping but distinct effects [25–27]. (2) Overexpression of a dominant-negative PPARγ mutant or treatment with antagonists fails to abolish all the anti-inflammatory actions of 15dPGJ2 or TZDs [28]. (3) PPARγ-null macrophages (and potentially other cell types) show anti-inflammatory effects upon TZD and 15d-PGJ2 treatment [8]. (4) The antiinflammatory effects of synthetic agonists are detected by using much higher concentration than required for transcriptional regulation (i.e. 50 μM rosiglitazone was required to achieve some anti-inflammatory effects, a concentration at least 100-fold above the KD for this receptor [29]). These arguments underscore the notion that extreme caution needs to be taken when interpreting experimental data based on usage of synthetic or natural ligands only.

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3. Role of PPARγ in immune cells In vitro studies with cell lines and isolated primary cells proved to be essential in starting to understand the role of PPARγ in immune responses and in evaluating the therapeutic potential of its agonists. These studies helped us to gain insights into the specific contributions of the different cell types in complex processes such as inflammation and inflammatory diseases. In connection with a certain cell type the basic questions are: whether the cell type expresses PPARγ (and if yes, which isoform) in humans and in model animals and what kind of stimuli can regulate its expression level; how the presence of the receptor and its activation or inhibition alter inflammatory responses and which genes and gene networks are directly or indirectly regulated by the receptor. Estimating the availability and/or production of endogenous ligands under physiological conditions in a given cell type is also highly relevant, but quite a challenging issue and rarely addressed. 3.1. Monocytes/macrophages 3.1.1. General features Monocytes and different types of macrophages belong to the Mononuclear Phagocyte System. Macrophages play a central role in innate and adaptive immunity. Macrophages and neutrophils provide the first line of defense against various microorganisms while antigen presentation by macrophages and dendritic cells associated with co-stimulatory signals and cytokines initiate appropriate adaptive immune responses.

Macrophages are essential not only because they eliminate pathogens, senescent and dying cells and present antigens but they also produce a range of secretory products/cytokines, which affect the migration and activation of other immune cells. 3.1.2. PPARγ in monocytes/macrophages The identification of PPARγ in spleen and myelomonocytic leukemia cells [30,4] led us and others to search for PPARγ expression and function in normal human peripheral blood monocytes and tissue macrophages [11]. Ricote et al. demonstrated that murine activated peritoneal macrophages express high levels of PPARγ [31]. Therefore macrophage was the first cell type, where PPARγ and inflammatory diseases got connected, more precisely PPARγ was implicated in atherosclerosis. Using a murine model, we demonstrated that PPARγ was expressed at high level in the nuclei of foam cells within atherosclerotic lesions [11]. Ricote et al. also reported that PPARγ is highly expressed in macrophage-derived foam cells of human atherosclerotic lesions [31]. Connection of PPARγ and atherosclerosis is discussed in details later in this review. Macrophage was the first cell type, where these two seemingly distant fields (lipid metabolism and regulation of immune responses) have been interlocked. We and others showed that in macrophages PPARγ plays an important role in lipid homeostasis [8–14]. These studies suggest that PPARγ coordinately regulates lipoprotein uptake and cholesterol efflux via activation of the scavenger receptor CD36 and the nuclear hormone receptor LXRα and that the two processes are linked at multiple levels (see below) and Fig. 1. Jiang et al. demonstrated that 15d-

Fig. 1. Mechanisms involved in cross-talks between nuclear receptors. In macrophages CD36-mediated uptake of oxidized LDL (ox-LDL) leads to the accumulation of intracellular cholesterol esters, from which oxidized fatty acids and sterols are derived. Oxidized fatty acids and oxysterols can activate PPAR and LXR receptors, respectively. Activation is resulted in the induction of target genes, among them CD36, initiating a positive feedback. LXRα expression is also induced by PPARγ, while ABCA1 is regulated by LXR. The lipid accumulation is counterbalanced by ABCA1-mediated cholesterol efflux to HDL. Production of endogenous LXR activator 27-hydroxycholesterol is enhanced by induction of CYP27. In dendritic cells activated PPARγ not only regulates genes directly, but it turns on retinoic acid synthesis by inducing the expression of retinol and retinal metabolizing enzymes such as retinol dehydrogenase 10 (RDH) and retinaldehyde dehydrogenase type 2 (RALDH2). PPARγ-regulated expression of these enzymes leads to an increase in the intracellular generation of all-trans retinoic acid (ATRA) from retinol. ATRA regulates gene expression via RARα, and RARα acutely induces the expression of CD1d and other target genes. (In dendritic cells CD36 is also regulated by activated PPARγ.) Red arrows indicate transcriptional regulation by nuclear receptors, black arrows represent influx, efflux and metabolism of lipids and nuclear receptor ligands.

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PGJ2 and TZDs could inhibit monocyte elaboration of inflammatory cytokines (TNFα, IL-6 and IL-1β) induced by phorbol myristyl acetate (PMA) [16]. Independently, Ricote et al. showed that these compounds could inhibit the expression of the inducible nitric oxide synthase (iNOS), gelatinase B (MMP9) and scavenger receptor A (SR-A) mRNAs [15]. These and other similar observations led to a series of studies on antiinflammatory effects of PPARγ ligands. A review on transrepression and the proposed molecular mechanisms involved in this repressive activity can be found in this volume by Ricote and Glass (this issue). Studies on macrophages proved to be instrumental to distinguish PPARγ-dependent and -independent effects of 15d-PGJ2 and TZDs. The fact, that 15dPGJ2 cannot be considered as a highly selective ligand for PPARγ, appeared to be a significant obstacle in interpreting anti-inflammatory effects of PPARγ ligands. The observations that the concentration of TZDs required to exert antiinflammatory effects were significantly higher than that for target gene activation [14] and non-TZD PPARγ agonists failed to induce anti-inflammatory responses [27] suggested the existence of at least partly, PPARγ-independent mechanisms. Chawla et al. [8] and Moore et al. [13] independently generated PPARγ−/− embryonic stem cells that could be differentiated into the monocytic lineage. They found that PPARγ is not essential for myeloid development and both 15d-PGJ2 and TZDs have anti-inflammatory effects (inhibition of iNOS and COX2 expression, TNFα and IL-6 production) that are independent of PPARγ. Studies on macrophages helped in identifying novel natural PPARγ ligands and also pathways might be involved in production of PPARγ ligands. We identified [10] components of oxidized LDL, including 9HODE and 13-HODE as activators of PPARγ. These compounds are present in both chemically oxidized LDL and LDL from atherosclerotic plaques, and their concentration correlate with the stage of lesion. In humans, IL-4 induces 15lypoxygenase in cultured human monocytes [32] an enzyme involved in oxidation of LDL. In mouse, its orthologue, 12/15lipoxygenase [33] can generate 13-HODE and 15-HETE from linoleic and arachidonic acid, respectively. Huang et al. [33] reported that IL-4 induces expression of PPARγ and 12/15lipoxygenase in macrophages, suggesting the potential of coordinated induction of both receptor and activating ligands. Therefore it appears likely that PPARγ is a key factor in regulating, at least some aspects, of macrophage lipid metabolism and primarily as a repressor of inflammatory responses. The ways how these two processes are connected and the contribution of macrophage specific PPARγ-induced genexpression and transrepression to inflammatory responses in vivo remains to be explored. 3.2. Dendritic cells 3.2.1. General features Dendritic cells (DCs) are professional antigen presenting cells of myeloid or lymphoid origin. Several subtypes of DCs have been identified to date (the two main categories are conventional DCs such as interstitial DCs and Langerhans cells,

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and plasmacytoid DCs). DCs are both initiators and regulators of adaptive immunity and they can also be involved in inducing tolerance. Their immature forms residing in the periphery are well equipped to take up antigens by receptor mediated endocytosis, phagocytosis and macropinocytosis. Antigen uptake associated with other signals initiate dramatic changes, DCs almost completely loose their capacity to take up antigens and undergo maturation, which enables them to fulfill their ultimate functions: antigen presentation and activation of different subsets of lymphocytes. Their antigen-presentation function is not restricted to the presentation of peptides by MHC-II molecules, they also present glycolipids in complexes with CD1 molecules and endo- or exogenous antigens with MHC class I molecules (cross-presentation). Co-stimulatory molecules (CD80, CD86 and CD40) are also involved in activation, while released cytokines and chemokines contribute to orchestrating the most effective immune responses. 3.2.2. PPARγ in dendritic cells PPARγ was first detected in murine immature and mature spleen-derived DCs [34]. In human, microarray analyses revealed that PPARγ is highly upregulated in monocyte-derived DCs during differentiation triggered by cytokines [35]. This result was confirmed by several groups [36–38], and the protein product of the PPARγ gene was also detected. The presence of PPARγ was also demonstrated in other DC subtypes such as blood-derived myeloid DCs, S100-positive antigen presenting cells in human tonsils [38], murine Langerhans cells and bone marrow derived murine DCs [39]. As we have observed in macrophages, PPARγ has an impact on both lipid homeostasis and immunoregulation. In DCs, we and others detected upregulation of lipid metabolism and transport related genes (CD36, FABP4, LXRα, PGAR) upon PPARγ ligand treatment. We found that upregulation of these genes could be blocked by PPARγ-specific antagonist suggesting a PPARγ-dependent regulation [38]. To reveal the role of PPARγ ligands as immunoregulators, in several laboratories different processes and immune functions were tested such as antigen uptake, DC migration, modulation of Th1/Th2 balance, cytokine production and antigen presentation. We found that PPARγ, similarly to glucocorticoids and 1α,25-dihydroxyvitamin D3, [40,41], enhanced endocytosis in immature DCs [38]. Antigen uptake and presentation is under tight developmental control: monocytes and immature DCs have the highest capacity to take up antigens. Nuclear receptor ligands can control differentiation and maturation processes of DCs, so it is possible, that PPARγ ligands similarly to the two other nuclear receptor ligands [42] act developmentally, and interfere with endocytosis indirectly. During maturation, DCs migrate to draining lymph nodes, principally due to changes in their chemokine receptor profile, which now includes CCR7. This allows DCs to follow CCL19 and CCL21 chemokine signals released from the lymphatic vessels [43]. Nencioni et al. reported [37] that activation of PPARγ in DCs inhibited the expression of CCR7. Using an experimental murine DC model of Langerhans cell migration induced by TNFα, Angeli et al. found [44] that PPARγ agonist rosiglitazone specifically impairs the departure of LCs from the

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epidermis. This phenomenon might be explained, at least partly, by reduced levels of CCR7. The first papers on PPARγ in murine and human DCs reported that 15d-PGJ2 and/or rosiglitazone down-regulate the CD40-induced secretion of IL-12, a potent Th1-driving factor [34,36]. These and other papers [37,38] also reported a series of observations suggesting that activation of PPARγ in DCs might have an impact on the orientation of immune responses by favoring type 2 responses. Observations included decreased IL-12 production, decreased CD80 and induced CD86 level, down-regulation of chemokines involved in the recruitment of Th1 lymphocytes, namely CXCL10 and RANTES. DCs can initiate and regulate several forms of immunity. The type and amount of antigen presented by DCs to lymphocytes, associated with released molecules are essential factors in determining the immune response. We and others found [37, 38] that activation of PPARγ changed the expression level of CD1a. CD1 molecules bind and present glycolipids and are important in lipid–antigen presentation of DCs. Unexpectedly, we found [38] that PPARγ ligand treatment induced CD1d in human monocyte-derived DCs. Since CD1d mediated lipid presentation is indispensable for the activation and expansion of invariant Natural Killer T (iNKT) cells [45], we hypothesized that increased CD1d protein levels should translate into increased activation of iNKT cells [38]. Further studies showed that DCs treated with PPARγ ligands, induced iNKT cell proliferation, causing a significant increase in the number of iNKT cells in the lymphocyte population. These findings prove that increased CD1d levels in DCs regulated by PPARγ are functional and can play a central role in the immunomodulatory effects of the activated cells (Fig. 1). Our further studies [46] revealed that PPARγ regulates CD1d indirectly by turning on retinoic acid synthesis via the induction of the expression of retinol and retinal metabolizing enzymes such as retinol dehydrogenase 10 and retinaldehyde dehydrogenase type 2 (RALDH2). PPARγ-regulated expression of these enzymes leads to an increase in the intracellular generation of all-trans retinoic acid from retinol. All-trans retinoic acid regulates gene expression via the activation of the RARα in human DCs, and RARα acutely regulates CD1d and other RARα target genes. Interestingly, we also detected a function, regulated by PPARγ, distinct from lipid metabolism, antigen uptake, DC migration, cytokine production and antigen presentation. We found that PPARγ directly induces the expression of ABCG2, a multidrug transporter in human monocyte-derived dendritic cells [47]. Dendritic cells activated by PPARγ express high levels of functional ABCG2 protein, and gain an enhanced capacity to extrude xenobiotics. These features may have important consequences on the survival and drug resistance of human dendritic cells during immune regulation and also have therapeutic ramifications (Fig. 1). These studies established dendritic cells as a very relevant target in PPARγ mediated immune regulation. It remains to be seen what portion of PPARγ's activation in vivo is mediated by receptors residing in dendritic cells. Dendritic cell specific knock outs may prove to be particularly useful in defining the role of the receptor in innate immune responses and autoimmunity.

3.3. T cells 3.3.1. General features There is no adaptive immunity without antigen specific T and B cells. Although adaptive immunity requires a longer period of time to be activated, its key elements, antigen specificity and memory, can guarantee a pathogen specific and more effective response, which is harmless to the host. T cells are derived from bone marrow stem cells and then further develop in the thymus (in adult mammals). The different types of T cells (cytotoxic T cells, T-helper cells and regulatory T cells) have a range of functions, displaying cytotoxic activity, initiating cellular and humoral immune responses or even immune tolerance. The dysfunction of T cells contribute a great deal to several inflammatory and autoimmune diseases, therefore the factors involved in regulating proliferation or cytokine production of T cells have significant therapeutic potential. 3.3.2. PPARγ in T cells Although Greene et al. [30] detected the expression of PPARγ2 in normal neutrophils and peripheral blood lymphocytes in 1995, the first reports [48–50] about the functional significance of PPARγ in T cells were published only a couple of years later. Clark et al. [48,51] were the first to describe the expression and function of PPARγ in mouse T-lymphocytes. They demonstrated that murine SJL-derived Th1 clones and freshly isolated T cell-enriched splenocytes from SJL mice express PPARγ1 mRNA but not PPARγ2. To test its functional significance, they used two PPARγ ligands, 15d-PGJ2 and a TZD, ciglitazone. Both ligands could inhibit antigen (BMBP)induced and anti-CD3 antibody-induced T cell proliferative responses of T cell clones and the freshly isolated T cell enriched splenocytes. In these studies, it was also demonstrated that the two PPARγ ligands mediated inhibition of IL-2 secretion by the T cell clones, whereas inhibition of IL-2 induced proliferation was not detected. In human, in peripheral blood T cells Yang et al. also detected [49] inhibition of PHAinduced proliferation and IL-2 production by 15d-PGJ2 and TZD troglitazone in a dose-dependent manner. When PPARγ2 wild type expression vector was transfected into Jurkat cells, they found that troglitazone and 15d-PGJ2 inhibited transcription and production of IL-2 in Jurkat cells in a PPARγdependent manner. Co-transfection assays with PPARγ and PPRE-driven/IL-2 promoter luciferase reporter constructs revealed that the inhibitory effects of troglitazone and 15dPGJ2 on IL-2 promoter activity are dependent on the expression and activation of PPARγ. Finally, they demonstrated that activated PPARγ inhibited the DNA-binding and activity of transcription factor NFAT regulating the IL-2 promoter in T cells. In their next paper [52] they reported an interesting interaction between monocyte/macrophages and T cells. IL-4 has been shown to induce 12/15 lipoxygenase in monocytes/ macrophages, which in turn produce potential PPARγ ligands [33]. Based on this finding they tested the relevance of the regulation of soluble mediators (PPARγ ligands) released by IL-4 treated monocytes/macrophages on T cell activation. They added medium of macrophages cultured with or without IL-4 to

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T cells stimulated with anti-CD3 or PHA/PMA. They found that T cells with the conditioned medium from IL-4-treated macrophages produced significantly less IL-2. The medium of IL-4-treated macrophages contained a sufficient amount of 13HODE and anti-13-HODE antibody could neutralize the inhibitory effects of the IL-4-conditional medium on T cell IL-2 production. Harris et al. [50] detected the expression of PPARγ1 in mouse naïve, PMA- and ionomycin-activated T cells. They found that PPARγ agonists (15d-PGJ2, ciglitazone and troglitazone) significantly inhibited T cell proliferation, accompanied by decreases in cell viability, and induced apoptosis. These early studies on PPARγ-induced apoptosis of T cells raised the possibility of utilization of PPARγ agonists in T cell related diseases. Surprisingly, Wang et al. found [53] that at concentrations that induce optimal transcriptional activation, TZD activation of PPARγ protected T cells from apoptosis. Besides inhibition of IL-2 production and induction of apoptosis, later studies revealed other effects of PPARγ ligands on T cells. In murine and/or human systems [54–57] PPARγ ligands inhibited production of further proinflammatory cytokines such as IFNγ and TNFα. Other, PPARγ-independent, effects of 15d-PGJ2 were also reported: induction of proinflammatory cytokine IL-8 in stimulated human T cells [58] or enhanced expression of heme oxygenase-1, an anti-inflammatory enzyme in human lymphocytes [59]. 3.4. B cells 3.4.1. General features B cells are derived from bone marrow stem cells similarly to T cells, but unlike T cells they stay in the bone marrow to further develop (in adult mammals). Depending on the antigens, B cells can be activated by T-helper dependent and independent manner. Following B cell activation, they multiply and differentiate into plasma cells, which produce large amounts of antibodies. Antigen specific antibodies are central to humoral immunity. They contribute to pathogen elimination by pathogen neutralization, coating the surface to enhance phagocytosis and activating the complement system. Although B cells and their differentiated forms are most known for their capacity to produce antibodies, B cells are also able to present antigens to T cells. 3.4.2. PPARγ in B cells The vast majority of papers discussing the role of PPARγ agonists on B cells focus on the induced apoptosis of normal or B lymphoma cells by natural and synthetic agonists. Padilla et al. [60,61] reported for the first time the expression of PPARγ in mouse and human normal B cells and a variety of B lymphoma cells. They found that both 15d-PGJ2 and TZDs could induce apoptosis of these B cells. Treatment with 15dPGJ2 induced a massive increase in MAPK of mouse B-lineage cells. Since both synthetic and natural PPARγ agonists can exert their effects via PPARγ dependent and independent pathways, Setoguchi et al. [62] used not only different kinds of agonists, but they also tested PPARγ+/− heterozygote mutant mice. They found that haploinsufficiency of PPARγ affects B cells but not

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T cells. B cells from mice showed exaggerated proliferative response to each tested stimulus if compared to B cells from wild type littermates. Since Padilla et al. [60] reported that PPARγ induces apoptosis in normal and malignant B-lineage cells they also tested cell viability. Interestingly, they detected increased viability of B cells from PPARγ+/− versus PPARγ+/+ mice, whereas addition of PPARγ agonists (15d-PGJ2, and TZDs such as troglitazone, rosiglitazone and pioglitazone) did not alter this parameter. They concluded that this inconsistency is presumably due to the different mouse strains used by the two groups. Remarkably, Piva et al. [63] also reported that 15d-PGJ2 induced apoptosis was not mimicked by troglitazone. Other results (NF-κB activity even in the resting B cells without stimulation and a significant increase in the serum Ig level) indicated that B cells from PPARγ+/− mice are spontaneously activated. Since B cell hyperreactivity is proposed to be involved in the pathogenesis of autoimmune diseases, including rheumatoid arthritis, they tested the association of B cell hyperreactivity in PPARγ+/− animals with the disease. They found that the induced arthritis in PPARγ+/− animals was more severe if compared to wild type mice. More recent papers [63– 68] revealed other details about the role of PPARγ and RXRα agonists such as activation of MAPKs, inhibition of NF-κB pathways and the role of CD40 activation in the PPARγ agonists-induced apoptosis of B cells. Clearly there are conflicts between the results obtained with different ligands and heterozygous animals calling for more definitive genetic (tissue specific knock out or siRNA mediated gene inactivation) evidence to clarify the receptor's role. 3.5. NK cells 3.5.1. General features NK cells are cytotoxic lymphocytes derived from bone marrow derived lymphoid progenitors. NK cells are important cellular components of the innate immune defense because they recognize and kill virus infected cells and also certain tumour cells. The biological activity of NK cells includes cytokine production (IFNγ, IL-1, GM-CSF) contributing to regulation of hematopoiesis and immune responses, and cytolytic activity by releasing cytotoxic granules in order to eliminate target cells. NK cells use different strategies to identify their targets. These cells can recognize targets coated with antibodies (by so-called antibody dependent cellular cytotoxicity — ADCC) or via CD16 cell surface receptors. Another important activation pathway is essential to distinguish infected and non-infected cells by integrating signals from activating or inhibitory receptors of NK cells. 3.5.2. PPARγ in NK cells Zhang et al. [69] investigated the expression and role of PPARγ in NK cells, using fresh human NK cells and two human NK cell lines. They analyzed the effect of two ligands, the natural ligand 15d-PGJ2 and the synthetic TZD ciglitazone, in the regulation of IFNγ production and the cytolitic activity of NK cells. Their data indicated that PPARγ1 isoform was expressed by human NK cells, and that IL-4 could up-regulate

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PPARγ in NK cells. INFγ production by NK cells was highly up-regulated in response to IL-2, and IFNγ expression was inhibited by 15d- PGJ2. This natural ligand also reduced the IL2 induced cytolitic activity of NK cells. They found that expression of CD69 of NK cells was reduced by 15d-PGJ2 in a dose-dependent manner, while ciglitazone had no effect. Among the investigated cell lines NKL proved to be PPARγ positive (upon IL-4 stimulation) while NK92 was PPARγ negative. Utilizing PPARγ-positive NKL and PPARγ-negative NK92 cell line (and PPARγ-transduced NK92), comparing the effects of 15d-PGJ2 and ciglitazone, they concluded that 15dPGJ2 inhibits IFNγ expression through PPARγ-dependent and -independent pathways, whereas the inhibition of cytolytic activity is independent of PPARγ. 3.6. Mast cells 3.6.1. General features Mast cells are derived from bone marrow stem cells and differentiate at the peripheral tissues. These cells are critical effector cells of allergic immune response and are also implicated in anti-bacterial responses. In rodents, mast cells are classified into two types, connective tissue mast cells (CTMC) and mucosal mast cells (MMCS). Their final differentiation in the tissues is accompanied by granulum formation. The activating stimulus for mast cells is usually an allergen. FcεRI receptors of mast cells recognize the allergen cross-linked with IgE molecules. Upon activation, mast cells release molecules from the preformed granules (within seconds) and start to newly synthesize other factors. In this way a wide variety of biologically active molecules are released into the surrounding tissues: toxic mediators (i.e. histamine and heparin), enzymes (i.e. chymase and tryptase), cytokines, chemokines and lipid mediators (i.e. leukotrienes, arachidonic acid derivatives such as prostaglandin). The consequences of IgE-mediated mast cell activation affects the surrounding tissues, blood vessels and also the migration and activity of other immune cells. 3.6.2. PPARγ in mast cells Sugiyama et al. [70,71] investigated the expression of PPARs in mouse bone marrow-derived mast cells and human cultured mast cells (HCMC). They found that mouse and human mast cells express PPARγ. In mouse, PPARγ is upregulated via antigen stimulation or by calcium ionophor. In mouse, production of GM-CSF and TNFα induced by antigenic stimulation was inhibited by 15d-PGJ2 and troglitazone. In humans, expression of PPARγ1 and PPARγ2 isoforms was detected, the latter of which increased by activators of HCMCs (anti-IgE after IgE sensitization or calcium ionophor plus phorbol ester) and IL-4. Cytokine production and release of chemical mediators were also tested after stimulation with PPARγ agonists. Anti-IgE induced expression of cytokines such as GM-CSF, TNFα, IL-5, MIP-1α was reduced by various ligands. Testing the effects of agonists on chemical mediator release after anti-IgE stimulation they found that prostaglandins inhibited both histamine and leukotriene C4 release. On the

other hand, troglitazone and ciglitazone did not inhibit the histamine release, but they could attenuate leukotriene C4 release. The facts that PGD2 is among the mediators produced and released by mast cells, and this mediator and its metabolites may suppress the mast cell activation raised the possibility of the existence of an autocrine regulation. Maeyma et al. [72] observed that rosiglitazone increased viability of mouse bone marrow derived mast cells from PPARγ +/+ mice by 30%, whereas no increase were detected in the mast cells from PPARγ+/− heterozygote mice in most cases. They also found that rosiglitazone decreased the histamine content and histamine release induced by antigen stimulation. Stimulated bone marrow derived mast cells secrete IL-6 and generate a biphasic (immediate and delayed) PGD2 production [73]. PGD2 is the major prostanoid produced by mast cells, and its generation is dependent on the induced expression of PG endoperoxide synthase-2 (PGHS-2). Diaz et al. [73] examined the regulation of PGHS-2 by different prostaglandins and found that PGHS-2 and IL-6 expression in mouse mast cells (mBMMC) were induced by exogenous (via a paracrine mechanism) but not endogenous (via an autocrine mechanism) prostanoids. Synthetic PPARγ agonists did not mimic the effects of 15d-PGJ2 suggesting that it acts via a PPARγ independent mechanism. 3.7. Eosinophils 3.7.1. General features Eosinophils are granulocytic leukocytes derived from myeloid precursors. They comprise 2–5% of blood leukocytes in healthy individuals. In the periphery, (in the connective tissue, urogenital epithelium and gut) along with macrophages and neutrophils these cells form the first line of defense against parasitic infections. Certain stimuli can trigger eosinophils to degranule releasing highly toxic granule proteins, free radicals (as side-effects, these molecules can cause tissue damages in allergic reactions). Effector functions of eosinophils include synthesis of chemical mediators such as leukotrienes, prostaglandins and also cytokines, which can amplify the inflammatory responses. 3.7.2. PPARγ in eosinophils PGD2, a major mast cell mediator has been identified as an effective chemoattractant for eosinophils [74–76]. Other prostaglandins (e.g. 15d-PGJ2) can enhance eosinophil chemotaxis at low concentrations. Interestingly, Monneret et al. [75] revealed other proinflammatory effects of 15d-PGJ2 such as calcium mobilization, actin polymerization and induction of CD11b expression in human eosinophils. These proinflammatory effects are in contrast to the large number of antiinflammatory effects assigned to this compound in other immune cell types. It should be emphasized though that the concentrations of 15d-PGJ2 required to activate eosinophils are much lower than those required for its anti-inflammatory effects. These experiments focused on the role of different prostaglandins in eosinophil functions. More recent studies started to investigate the functional relevance of the receptor [77–80]. Interestingly and somewhat contradicting previous

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observations, these studies reported anti-inflammatory effects of PPARγ agonists. Ueki et al. [77] reported that purified human eosinophils and Eol-1 cells express PPARγ, and troglitazone reduced the IL-5 stimulated eosinophil survival. Eotaxindirected eosinophil chemotaxis was also inhibited by troglitazone. Based on these observations they raise the possibility that PPARγ agonists could be used in the treatment of allergic diseases such as asthma. Woerly et al. [78] found that PPARγ is expressed in mouse, rat and human eosinophils. However in human samples no correlation was found between PPARγ expression and the activation state of eosinophils. In their experimental system rosiglitazone and ciglitazione were able to inhibit IL-5 and eotaxin induced human eosinophil migration. Eosinophil incubation with PPARγ antagonist along with rosiglitazone led to a loss of chemotaxis inhibition (confirming a PPARγ dependent effect). Comparing effector function of PPARγ agonist treated and non-treated eosinophils, a dosedependent inhibition of human eosinophil-mediated antibodydependent cellular cytotoxicity (ADCC) was observed with rosiglitazone and ciglitazone. Their in vivo mouse models, administration of ciglitazone during ovalbumin (OVA) antigen challenge periods decreased most of the characteristic parameters of airway inflammation (IL-4, IL-5, IL-6, IL-13 and GATA-3 in lung extracts and OVA specific IgE and IgG1 in serum). Ciglitazone reduced airway hyperresponsiveness and decreased eosonophilia in bronchoalveolar lavage. Matsuwaki also reported troglitazone as inhibitor of activation and degranulation of eosinophils [79]. To explain this discrepancy (pro- and anti-inflammatory effect of PPARγ agonists), Kobayashi et al. [80] suggested to consider PPARγ as a biphasic regulator of immune responses of eosinophils. They found, consistently with previous observations, that 15d-PGJ2 and TZDs amplified the migration of these cells in a physiological concentration range (picomolar to low nanomolar) in a PPARγ dependent fashion. On the other hand at higher concentrations, troglitazone and 15d-PGJ2 reduced eosinophil migration. In their experiments, 15d-PGJ2 enhanced eotaxininduced migration was independent of MAPK and NFκB pathways at low concentration. 15d-PGJ2 and TZDs also primed eotaxin-induced cytoskeletal rearrangement and enhanced eotaxin-induced calcium flux via PPARγ at low concentrations. They concluded that exogenous PPARγ agonists in therapeutic concentrations might act as a negative immunomodulator, whereas endogenous ligands at physiological concentrations might have some proinflammatory effects. 3.8. Neutrophils 3.8.1. General features Neutrophils are the most abundant among circulating granulocytes. They are short-lived cells which develop from the same myeloid precursors as monocytes. Neutrophils are recruited to and migrate into tissues at the site of inflammation and recognize, digest and eliminate many pathogens. A large arsenal of antibiotic proteins (i.e. hydrolyses, myeloperoxidase, muramidase, defensins and bacterial permeability proteins) and reactive oxygen species in phagolysosomes of neutrophils efficiently kill pathogens.

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3.8.2. PPARγ in neutrophils Greene et al. [30] could detect the presence of PPARγ in human neutrophils and peripheral blood lymphocytes by Northern blot analysis. Vaidya et al. [26] investigated the effects of 15d-PGJ2 on some neutrophil functions. They found that 15d-PGJ2 inhibited β2 integrin-dependent production of reactive oxygen intermediates. Several lines of evidence (i.e. the effects of this natural agonist were not mimicked by AD-5075 a synthetic PPARγ agonist) led them to the conclusion that the effects of 15d-PGJ2 in neutrophils are mediated by a PPARγ independent manner. Remarkably, although several studies confirmed the suppressive effects of PPARγ agonists on the infiltration of neutrophils in animal inflammatory model systems [81–83], there are no comprehensive in vitro studies investigating the role of PPARγ in neutrophils. Standiford [84] reported some preliminary studies, without published data, on induction of PPARγ in neutrophils, effects of agonists on chemotactic responses, cytokine production etc. Imamoto et al. [85] found that neutrophil–endothelial cell binding can be influenced by pioglitazone via inhibiting upregulation of CD11b/CD18 on activated neutrophils. 3.9. Basophils 3.9.1. General features Basophils and eosinophils have a common precursor, and growth factors for basophils are also very similar to those of eosinophils. Basophils also share several common features with mast cells. Both cell types express FcεRI (high affinity receptor for IgE) under resting and activated conditions. Basophils and mast cells are among the main effector cells in allergic reactions. They both contain histamine, a vasoactive amine. Basophils are recruited to the sites of inflammation and upon activation by cytokines or antigenes, they release histamine and IL-4 from basophilic granules. 3.9.2. PPARγ in basophils To our knowledge understanding the role of PPARγ in basophils is limited to a human basophilic cell line KU812 and the data obtained in this cell line are fairly inconsistent and controversial. Abe et al. [86] and Fujimura et al. [87] both tested the expression of PPARγ in human basophilic cell line KU812 by RT-PCR. Fujimura could show its expression, while Abe found it undetectable. The effects of 15d-PGJ2 and TZD agonists on basophil cell line are also controversial. Fujimura et al. detected inhibitory effects of 15d-PGJ2 in form of a dose-dependent decrease of the cell surface expression of FcεRI and in the reduction of FcεRImediated histamine release [87]. Nevertheless, treatment with synthetic ciglitazone resulted in no change of FcεRI cell surface expression or histamine release after the cross-linking of FcεRI with anti- FcεRI antibody. In contrast to this, Abe et al., using another TZD, found that troglitazone could decrease both histamine secretion and intracellular histamine concentration and among other TZDs also suppressed cell proliferation [86].

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Fig. 2. Effects of PPARγ ligands on various immune cell types.

In order to provide a global overview we have summarized the effects of PPARγ activators on the various immune cell types in Fig. 2. 4. Acute and chronic inflammation Although essential for gaining mechanistic insights, studies on isolated cell types are rarely predictive on how a complex system is regulated in vivo. The contribution of PPARγ to complex immunological diseases has been addressed in animal models of chronic inflammatory processes (Table 1) and also in clinical studies. Inflammation can be classified based on the dynamics of the response as acute or chronic. Each phase has a characteristic profile of cell types: in the acute phase neutrophils, dendritic cells and macrophages and in chronic inflammation mononuclears such as macrophages, lymphocytes and mast cells dominate. Inflammation such as sore throat, acute hepatitis,

insect bite or reactions in the skin is characterized by an immediate, early response where after the detection of the antigens and/or the damage neutrophils are immediately recruited to the sites by chemoattractive signals followed by monocyte/macrophage invasion that becomes the major cell type after 48 h. The role of neutrophils is phagocytosis of the antigens, which are degraded inside the cells through enzymatic processes. Activation of neutrophils leads to secretion of cytokines that amplifies the inflammatory response and results in lymphocyte infiltrate and also in vascular changes, oedema and enzymatic destruction of not only the antigens but also the surrounding tissues. The goal, and in most cases, the result of the acute phase is the total elimination of the stimulating antigens during the course of a couple of days. However resolution starts already during the early phases of the inflammatory response. Granulocyte entry to the inflammatory site already promotes the switch from proinflammatory arachidonic acid and leukotriene metabolites to lipixins.

Table 1 Models of human diseases used for the study of PPAPγ's role in disease progression Disease

Animal model

Cell type

Effect

Inflammatory bowel disease Psoriasis Rheumatoid arthritis

induced colitis

colonic epithelial cells

irritant and allergic contact dermatitis adjuvant induced arthritis (mouse and Lewis rat) experimental allergic encephalitis (mouse) LDLR knock out mice

keratinocytes ? macrophages B cells synoviocytes T cells macrophages, dendritic cells ? Vascular smooth muscle cells

preventing development of inflammation, inhibition of already persisting inflammation reduced epidermal hyperplasia reduced pannus formation, ameliorated adjuvant induced arthritis ameliorating experimental autoimmune encephalomyelitis decreased lesion size

Multiple sclerosis Atherosclerosis

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Other lipid mediators such as resolvins and protectins initiate neutrophil apoptosis. Macrophages that eliminate apoptotic neutrophils also stimulate resolution by the release of antiinflammatory cytokines such as TGF-beta [88]. It is not difficult to see that the same cell type, most prominently neutrophils, macrophages and dendritic cells have pro- as well as anti-inflammatory roles depending on the immune context and milieu. If the balance between inflammation and resolution is broken that can lead to sustained or chronic inflammation. It has a prolonged duration of weeks or months and it is characterized by active inflammation, tissue destruction and repair proceeding simultaneously. It may follow active inflammation, but more often it starts as a low-grade response and causes tissue damage over a longer period of time such as atherosclerosis and rheumatoid arthritis. Next we review the data available on PPARγ's involvement in inflammatory diseases, focusing on chronic inflammation. 5. Atherosclerosis and PPARγ Atherosclerosis is believed to be a complex disease resulting from changes in lipid metabolism (hyperlipideamia and/or hypercholesterolaemia) and effecting the walls of arteries. It is characterized by intimal lesions protruding to the lumen of arteries and leading to serious complications such as myocardial infarction or ischemic stroke of the brain. The underlying mechanisms in the formation of atherosclerotic lesions is still not known. Many factors could be responsible from high fat diet to infectious agents causing inflammation in the artery wall with accumulation of immune cells and fatty deposits. The initial step is considered to be the activation of the endothelial cells (EC) resulting in high expression of the vascular cell adhesion molecule-1 (VCAM1) on the surface. VCAM-1 is induced on endothelial cells by pro-inflammatory cytokines like TNFα or IL-1β but high cholesterol levels also lead to an increase in its expression. Its major role is attracting leukocytes such as lymphocytes and monocytes to the site promoting their attachment and penetration through the artery walls [89]. Lymphocytes in plaques are derived from the circulation after migration through the wall following chemoattractive stimuli. The chemoattractants for lymphocytes are secreted by the endothelial cells and involve a trio of IFNγ inducible cytokines including inducible protein 10 (IP10), monokine induced by IFNγ (Mig) and IFN inducible T cell a chemoattractant (I-TAC) as reviewed in [89]. These T cells are responsible for orchestrating further inflammatory responses in plaque formation and progression in a Th1 type manner. The migration of monocytes into the arterial wall results in the activation and in the formation of monocytes into macrophages. Activated macrophages in the intima of the wall express high levels of receptors – scavanger receptor A, CD36 and CD68 – capable of uptaking lipids leading to lipid accumulation and foam cell formation that are the hallmarks of macrophages in lesions. Activated macrophages also have an immunological role in chronic inflammation of the wall by secreting cytokines and interacting with lymphocytes and other cells involved in plaque formation like vascular smooth muscle

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cells (VSMC). VSMC, via their migration to the sites, also play a major role in the remodeling and thickening of the intima. Implicating nuclear receptors in several of the cell types contributing to lesion formation provided new concepts for understanding disease progression and opened up new avenues for therapeutic interventions. PPARγ was first linked to atherosclerosis by the findings that if activated the receptor can regulate lipid uptake and foam cell formation and that lipids 9-HODE and 13-HODE from oxidized LDL (oxLDL) are activators of the receptor. Activation of PPARγ by its ligands leads to upregulation of CD36 – a receptor for oxLDL – suggesting the existence of a positive feed back loop and providing a possible explanation of how lipid laden macrophages are formed in atherosclerotic plaques [10] [11] (Fig. 1). Identification of another nuclear receptor (LXRα) as a PPARγ target gene completed the picture [9]. PPARγ regulated LXRα expression leads to the expression of LXR target genes such as ABCA1 and regulated cholesterol efflux (Fig. 1). These were supported by in vivo experiments showing that LDLR−/− mice transplanted with PPARγ null bone marrow had an increase in atherosclerosis formation [9]. Furthermore animals treated with PPARγ ligands showed reduced atherosclerosis [90]. A link between the activation of PPARγ and LXR was further strengthened by the identification of cyp27 a P450 enzyme producing 27-OH cholesterol a ligand for LXR and which is regulated by PPARγ and retinoids [91]. PPARγ and LXRα levels and activity seems to be regulated not just by lipid mediators but by other transcription factors also. Adipocyte enhancer-binding protein 1 (AEBP1) was identified as a repressor of PPARγ and LXRα. Overexpression of AEBP1 in macrophages was accompanied by decreased expression of PPARγ, LXRα and their target genes with a diminished cholesterol efflux from macrophages. Furthermore AEBP1−/− macrophages had higher levels of PPARγ and LXRα with impaired increase in the expression of their target genes [92]. These findings established PPARγ as a key regulator of lipid metabolism (uptake and efflux) in macrophages and a relevant target in drug development. Besides lipid metabolism PPARγ has been implicated in regulation of inflammatory gene expression as well. This issue and the mechanism of transrepression is addressed by Ricote and Glass in this volume. However the role for PPARγ in atherosclerosis seems to be more complex involving more than one cell types. In human saphenous vein isolates PPARγ activators – both 15d-PGJ2 and glitazones – inhibited the IFNγ induction of IP-10, Mig and ITAC [93]. Later reports showed that pretreatment of human umbilical vein endothelial cells with PPARγ activators inhibited TNFα induced VCAM-1 expression and these findings were further verified in apoE-deficient mice where troglitazone significantly inhibited the homing of monocytes/macrophages [94]. VSMCs are also possible targets for PPARγ activators according to reports on its inhibitory effect on human vein derived VSMCs. Marx et al. showed a decreased expression of MMP9 in VSMCs after both troglitazone and 15d-PGJ2 treatment with its possible contribution to a decrease in the migration of VSMCs [95]. Other reports also speculate that part of the anti-atherosclerotic affect of PPARγ activators could be

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due to the down regulation of angiotensin II type 1 receptor in VSMC [96]. Findings that PPARγ activators modulate T cells by shifting towards a Th2 response supports the hypothesis that PPARγ activators have a general effect in atherosclerosis targeting many cell types involved in plaque formation [97]. 6. Other chronic inflammatory diseases and PPARγ 6.1. Inflammatory bowel disease Inflammatory bowel disease is a chronic inflammatory disease of the intestine that can either affect small intestine and colon in Crohn disease or the colon in ulcerative colitis. Epithelial cells of the intestine form the barrier between the lumen rich in bacteria and other stimulatory antigens and the body. PPARγ is highly expressed in both colonic epithelial cells and colon cancer cell lines Caco-2 and HT-29 with a function that is not clearly understood. Su et al. reported that when cell lines were treated with either 15d-PGJ2 or rosiglitazone a change in the cytokine profile of the cells could be detected. This was mostly an anti-inflammatory effect where 15d-PGJ2 inhibited IL-1β induced expression of IL-8 and MCP1 and rosiglitazone the IL-8 expression. Animal experiments confirmed the role of PPARγ in the inflammation of the intestine when symptoms of induced colitis were attenuated in a murin model when treated with rosiglitazone [98]. By carrying out analyses on PPARγ+/− and RXRα+/− mice these observations were further supported by showing that both heterozygous mice were more susceptible to induced colitis. As in these animals both the size of the histological lesions and the mRNA levels of inflammatory cytokines such as TNFα and IL-1β were greater the potential role for physiological ligands acting through the receptors in case of colitis was also put forward. Ligand activation of PPARγ and its heterodimeric partner RXR inhibited inflammation in induced colitis alone and in combination resulting in smaller infiltrates, limited oedema and smaller necrosis of the colon mucosa. Moreover, PPARγ agonists were effective not just in preventing development of inflammation but also in cases with already persisting inflammation [99]. However it should also be noted that rosiglitazone might have receptor independent effects as reduced inflammation was also seen after ligand treatment in a mouse model where the PPARγ gene was disrupted in the intestinal epithelial cells in villin-Cre/LoxP-flanked PPARγ mice. [100]. Based on these findings and on studies where rosiglitazone had beneficial effects in the treatment of ulcerative colitis in an open-label trial [101] PPARγ agonists might be potential candidates in the treatment of ulcerative colitis. 6.2. Psoriasis Psoriasis is a chronic inflammation of the skin affecting 1– 2% of the population with hyperproliferation of keratinocytes in the epidermis and infiltration of the dermis mainly by lymphocytes with microvascular proliferation [102]. The pathogenesis of psoriasis is likely to involve a Th1 dependent mechanism mediated by cytokines related to CD4+ T cells such

as TNFα, IFNγ, IL-12 and growth factors leading to the proliferation of keratinocytes [103]. Although many nuclear receptors are known to be involved in skin homeostasis like thyroid hormone-, oestrogen-, glucocorticoid- [104], retinoic acid- [105], vitamin D- [106], LXR- [107], PPARα- [108], and PPARδ- [109,110] receptors less is known about the role of PPARγ. Some studies report a favorable effect of pioglitazone on psoriasis after per os administration to patients with chronic plaque psoriasis [111]. Friedmann et al. showed that during in vitro differentiation of keratinocytes levels of PPARγ increased and when cultures were treated with rosiglitazone a decrease in the levels of MMP1 and MMP9 was detected [112]. Work on epidermis specific PPARγ deficient mice showed that despite patchy hair loss occurring during aging and a slight epidermal hyperplasia the deficient mice had a normal skin as examined both by light and electron microscopy supporting that PPARγ has little if any effect on keratinocytes in maintaining the barrier function of the skin [113]. Studies in a mouse model of hyperproliferative skin disease have also shown that topical administration of PPARγ ligands reduced epidermal hyperplasia and that the treatment had no effect on normal skin [114]. Similar results were obtained on the anti-inflammatory effects of ciglitazone on keratinocytes in mouse models of both irritant and allergic contact dermatitis where ciglitazone inhibited inflammation although the inhibition was to a similar degree in the wild type as in the keratin 14-Cre/LoxP-flanked PPARγ mice where PPARγ was selectively disrupted in epidermal keratinocytes indicating that the PPARγ agonists have either PPARγ receptor or keratinocyte independent effects when altering inflammation of the skin [113]. 6.3. Rheumatoid arthritis Rheumatoid arthritis (RA) is a chronic systemic inflammatory disorder affecting many organs, but is mostly known as a polyarticular joint disease characterized by synovial proliferation in the joint, infiltration of the synovial stroma by B cells, CD4+ helper T cells, plasma cells and macrophages. Other histological features include hypervascularisation, increased osteoclast activity and pannus formation consisting of a mass of synovium, inflammatory cells and fibroblasts causing destruction and ossification. The underlying mechanisms are still not clear in the formation of the disease but it is likely to be an autoimmune disease. Cells derived from the infected joints of patients with RA were found to express PPARγ. In macrophages the expression was at high levels and at moderate levels in synovial cells, endothelial cells and fibroblasts. In the in vitro cultures of synoviocytes both 15d-PGJ2 and troglitazone was found to inhibit proliferation of synoviocytes and induced apoptosis at higher doses [115]. The role for PPARγ in the control of the inflammatory responses of macrophages such as reducing the production of IL-1β, IL-6 and TNFα that are key mediators in RA suggests that application of PPARγ agonists might have multiple targets in RA. This is supported by studies on female Lewis rats where adjuvant induced arthritis was ameliorated by intraperitoneal administration of 15d-PGJ2 or troglitazone with a reduced number of inflammatory cells and

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pannus formation in the affected joints [115]. Setoguchi et al. also reported that B cells might also be important in translating the effect of PPARγ in RA. In a model of PPARγ heterozygote (PPARγ+/−) mice B cells were found to have greater proliferative responses to stimulatory agents if compared to wild type and the hyperreactivity of B cells was suppressed when mice were treated with PPARγ agonists. In parallel with increased B cell reactivity PPARγ+/− mice showed an increased joint thickness shortly after antigen challenge in an antigen induced arthritis model compared to wild type mice but without direct evidence that the phenomenon is caused by the change in B cell physiology [62]. 6.4. Multiple sclerosis Multiple sclerosis (MS) is an autoimmune disease of the central nervous system mediated by myelin reactive T cells. Chronic inflammation results in destruction of oligodendrocyte sheet with axonal degeneration contributing to irreversible and so far untreatable disability. The histological examination of the inflammation involved regions shows abundant macrophages and lymphocytes especially in perivascular localization where the majority of lymphocytes are Th1 type. The possible role of PPARγ in MS was tested in a PPARγ+/− mouse model in which experimental allergic encephalitis (EAE) was induced with a protocol causing a phenotype that is very much like the human disease MS. PPARγ+/− mice developed an exacerbated and prolonged EAE that was associated with a more severe demyelinization and inflammation than in wild type. Bright et al. showed that the underlying pathomechanism is the increased T cell activation and proliferation that is seen in heterozygous mice when exposed to neural antigens. As proved by levels of IL-12 and IFNγ productions the applied antigens induced a Th1 response that was increased more in PPARγ+/− mice. The activation also resulted in an increased expression of MHC-II and CD40 in the spleen cells of PPARγ+/− mice [116]. This report confirmed studies of Diab et al. that showed 15dPGJ2 treatment ameliorating experimental autoimmune encephalomyelitis by inhibiting the proliferation and activation of the antigen specific T cells in the used mouse model [117]. 7. New approaches allowing to define the tissue specific function of PPARγ: microarrays, ChIP-on-chip and LCM We tried to illustrate above that identification and validation of the role of a transcription factor in complex physiological and pathological conditions is an inherently difficult task. Fortunately, there are new technologies allowing ablation of the gene or gene product in a temporal and/or cell type specific manner using somatic cell recombination. Also there are technologies to determine cell type specific gene expression profiles in vitro and in vivo and genomic binding sites allowing to develop a more complete picture on the processes regulated by a given transcription factor. In this last part, we review some of the available evidence on PPARγ obtained by using these approaches.

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Commercially available microarrays and genome projects revolutionized the investigations on transcriptional regulation. Microarrays are technically improved from “home-made” spotted cDNA microarrays containing few hundred genes to “one chip one genome“ microarrays. Not only density has increased from low to ultra-dense, sensitivity and reproducibility were also multiplied. Investigators of the nuclear receptor and, in particular the PPAR field, started to utilize these techniques. The combination of highly selective ligands and overexpression of a given PPAR subtype and/or mouse knockouts allowed the identification of regulated genes and gene networks. Obviously, microarray experiments often only provide the starting point for identification of genes as target genes, validation of results by qPCR technique, detecting the changes at the protein level, proving the direct or indirect regulation and revealing functional consequences are also required. These latter parts of the discovery process still prove to be rate limiting. The complexity of microarray experiment designs changed almost parallel with the technique, researchers utilized these to address more and more complex questions. Not surprisingly, microarrays were first used for identification of genes regulated by PPARγ ligands during adipocyte differentiation (i.e. [118–120]). Recently, microarrays were utilized in PPAR related experiments such as comparing PPARγ target genes in different insulin-sensitive tissues [121], detection of isotype selective target genes and identification of the Nterminus of PPARs as a key determinant of selective gene expression [122], crosstalk of PPAR and other nuclear receptors [46], exploring mechanisms by which different members of the nuclear receptor superfamily repress proinflammatory programs of gene expression [123]. Another technique suitable for the identification of nuclear receptor binding sites: chromatin immunoprecipitation was successfully introduced in the studies on nuclear receptors, among them PPARs [124–126]. The obvious advantage of this technique is its ability to reveal molecular mechanisms of the regulation. If the immunoprecipitated DNA is hybridized to special microarrays (Chromatin immunoprecipitation on chip (ChIP on chip) technique [127– 130]), one can get a global view of transcription factor binding sites (among them nuclear receptors [131]). Microarrays used for response element identification, similarly to expression microarrays, were significantly improved. Initially, these chips contained only fractions of non-genomic regions, while today tiling arrays can practically cover complete genomes [131,132]. Studies of Carroll et al. on the estrogen receptor [131] have shown that reconsideration might be required in some aspects of nuclear receptor regulated enhancers (i.e. only 49% of the estrogen receptor binding sites contained a consensus element and the distance between these enhancer elements and the transcriptional start site can be in the order of hundreds of kilobases). The combination of microarray and ChIP on chip data is going to be particularly powerful in exploring regulatory networks. Finally, the availability of Laser Capture Microscopy (LCM) for the dissection of tissues and obtaining protein and RNA from selected cell types will enable us to identify the consequences of receptor activation from in vivo experiments.

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This has been already successfully applied to the analysis of gene expression from foam cells in atherosclerotic models [133]. There is no doubt that using these techniques systematically to dissect the role of nuclear receptors will provide very large datasets and significant insights into the receptor's functions. We attempted to highlight the complexity of the issues concerning the role of PPARγ in immunity and inflammation by showing the proven and in some cases presumed functions of this receptor in the various cell types and in diseases. It appears that we have only took the first few steps on the road to identify and understand the role of PPARγ in immunity and inflammatory diseases. It is, without doubt, still a long ways before we see how this receptor regulates genes and complex processes in the different cell types and how these can contribute to disease progression and most importantly to the basis of future therapies. Acknowledgments The authors thank Drs. Arpad Lanyi, Balint L. Balint for discussions and comments on the manuscript. L.N. is an International Scholar of the Howard Hughes Medical Institute and holds a Wellcome Trust Senior Research Fellowship in Biomedical Sciences in Central Europe. The work in the authors' laboratories is supported by grants from the National Office for Research and Technology (NKFP 2004 OM-00427 and RET-06/2004 to LN). References [1] I. Issemann, S. Green, Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators, Nature 347 (1990) 645–650. [2] C. Dreyer, G. Krey, H. Keller, F. Givel, G. Helftenbein, W. Wahli, Control of the peroxisomal beta-oxidation pathway by a novel family of nuclear hormone receptors, Cell 68 (1992) 879–887. [3] A. Schmidt, N. Endo, S.J. Rutledge, R. Vogel, D. Shinar, G.A. Rodan, Identification of a new member of the steroid hormone receptor superfamily that is activated by a peroxisome proliferator and fatty acids, Mol. Endocrinol. 6 (1992) 1634–1641. [4] S.A. Kliewer, B.M. Forman, B. Blumberg, E.S. Ong, U. Borgmeyer, D.J. Mangelsdorf, K. Umesono, R.M. Evans, Differential expression and activation of a family of murine peroxisome proliferator-activated receptors, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 7355–7359. [5] P. Tontonoz, E. Hu, B.M. Spiegelman, Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor, Cell 79 (1994) 1147–1156. [6] P. Tontonoz, E. Hu, B.M. Spiegelman, Regulation of adipocyte gene expression and differentiation by peroxisome proliferator activated receptor gamma, Curr. Opin. Genet. Dev. 5 (1995) 571–576. [7] R.M. Evans, G.D. Barish, Y.X. Wang, PPARs and the complex journey to obesity, Nat. Med. 10 (2004) 355–361. [8] A. Chawla, Y. Barak, L. Nagy, D. Liao, P. Tontonoz, R.M. Evans, PPARgamma dependent and independent effects on macrophage—gene expression in lipid metabolism and inflammation, Nat. Med. 7 (2001) 48–52. [9] A. Chawla, W.A. Boisvert, C.H. Lee, B.A. Laffitte, Y. Barak, S.B. Joseph, D. Liao, L. Nagy, P.A. Edwards, L.K. Curtiss, R.M. Evans, P. Tontonoz, A PPAR gamma-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis, Mol. Cell 7 (2001) 161–171.

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