The role of Peroxisome Proliferator-Activated Receptors (PPAR) in immune responses

Abstract

Peroxisome proliferator-activated receptors (PPARs) are fatty acid-activated transcription factors of nuclear hormone receptor superfamily that regulate energy metabolism. Currently, three PPAR subtypes have been identified: PPARα, PPARγ, and PPARβ/δ. PPARα and PPARδ are highly expressed in oxidative tissues and regulate genes involved in substrate delivery and oxidative phosphorylation (OXPHOS) and regulation of energy homeostasis. In contrast, PPARγ is more important in lipogenesis and lipid synthesis, with highest expression levels in white adipose tissue (WAT). In addition to tissues regulating whole body energy homeostasis, PPARs are expressed in immune cells and have an emerging critical role in immune cell differentiation and fate commitment. In this review, we discuss the actions of PPARs in the function of the innate and the adaptive immune system and their implications in immune-mediated inflammatory conditions.

Introduction: Structure and Classification of PPARs

The transcription factors Peroxisome proliferator activated receptors (PPARs) were discovered 30 years ago in rodents [1] and belong to the subfamily 1 of the nuclear hormone receptor superfamily of transcription factors [2]. PPARs are the best-studied fatty acid-activated nuclear receptors comprising of the following three subtypes: PPARα, PPARγ, and PPARδ (also designated as PPARβ) [3–8]. Although all PPARs play a major regulatory role in energy homeostasis each of them has distinct functions and sites of expression [4–6,8,9].

All PPARs share the basic structural properties of the most nuclear receptors, consisting of four functional domains [10–12] named A/B, C, D and E/F (Figure 1A): The N-terminal A/B domain contains a ligand-independent activation function 1 (AF-1), which is responsible for PPAR phosphorylation. The conserved central DNA binding domain (DBD), also named C domain, is composed of two zinc fingers and is responsible for PPAR binding to the peroxisome proliferator response element (PPRE) in the promoter of PPAR target genes. The D domain is a docking site for various cofactors. The E domain is also named ligand-binding domain (LBD). This is a 12-helix that forms a large hydrophobic binding pocket in the shape of Y and gives the molecule that ability to bind endogenous or exogenous lipophilic ligands. By doing so, this domain provides ligand specificity. Ligand binding stabilizes LBD and promotes conformational changes that enable interaction with co-activator complexes. Recruitment of PPAR cofactors that participate in the transcription process is mediated by the ligand-dependent activation function-2 (AF-2), located in the E/F domain. The full transcriptional activity of PPARs requires binding of cognate lipid ligands, heterodimerization with another nuclear receptor (retinoid-X receptor, RXR), interaction with a number of transcriptional coactivators, including PPARγ coactivator-1 (PGC-1)α and PGC-1β, and binding of the complex to PPAR response elements (PPREs) in the promoter of target genes (Figure 1B) [7,9,13]. Each PPAR member has a different ligand preference that could be explained by differences in the size or the lipophilicity of their binding pockets [8]. Detailed studies using hydrogen-deuterium exchange mass spectrometry have identified how structurally and functionally distinct states of PPARs are induced by binding of agonists vs. antagonists or reverse agonists [14].

Figure 1. PPAR structure and mechanism of action.

(A) All PPARs share the basic structural of the most nuclear receptors, consisting of four functional domains named A/B, C, D and E/F. The N-terminal A/B domain contains a ligand-independent activation function 1 (AF-1). The central DNA binding domain (DBD) or C domain is a conserved domain, composed of two zing fingers, and is responsible for the binding of PPAR to the peroxisome proliferator response element (PPRE) in the promoter of target genes. The D domain is a docking site for various cofactors. The E domain or ligand-binding domain (LBD) binds a variety of endogenous or exogenous lipophilic ligands and provides ligand specificity. Recruitment of PPAR cofactors that participate in the transcription process is mediated by the ligand-dependent activation function-2 (AF-2), located in the E/F domain. (B) Ligand binding promotes conformational changes that enable the interaction with co-activator complexes. The full transcriptional activity of PPARs requires binding of cognate lipid ligands, heterodimerization with another nuclear receptor (retinoid-X receptor, RXR), interaction with a number of transcriptional coactivators, and binding of the complex to PPAR response elements (PPREs) in the promotor of target genes. (FA, fatty acid)

PPARs are encoded by separate genes with distinct but overlapping interspecies sequences. The human PPARα gene is located on chromosome 22, PPARγ, for which three isoforms have been identified [15,16], is located on chromosome 3, and PPARβ/δ is located on chromosomes 6 [9,17,18]. PPAR natural ligands include lipid-derived metabolites such as fatty acids, acyl-CoAs, glycerol-phospholipids, and eicosanoids (Table 1). Dietary lipids activate PPAR, as evidenced by the increased expression of PPARα target genes in the liver and PPARβ/δ target genes in the skeletal muscle induced by high-fat diet (HFD) in mice [19,20].

Table 1:

PPAR subtypes, chromosome localization, natural ligands and biological effects.

Subtype	Chromosome	Site of expression	Natural Ligands	Action
PPARγ	3	WAT, liver, skeletal muscles, intestine, immune cells	Linoleic acid Arachidonic acid Eicosatetraenoic acid PGJ2, Linoleic acid 9-HODE 13-HODE	• FA transport • Lipid synthesis • Adipogenesis • Energy storage • Thermogenesis (BAT) • Glucose homeostasis
PPARα	22	Liver, heart, skeletal muscles, BAT, intestine, kidneys	Palmitic acid Stearic acid Palmitoleic acid Oleic acid Linoleic acid Arachidonic acid Eicosatetraenoic acid Leukotriene B4 Pristanic acid	• FA transport • FA esterification • FAO • BAT browning • Energy dissipation
PPARβ/δ	6	Ubiquitous	FA Retinoic acid Carbaprostacyclin	• FAO • Glucose homeostasis

WAT, white adipose tissue; BAT, brown adipose tissue; PGJ2, Prostaglandin J2; 9-HODE, 9 hydroxyoctadecadienoic acid; 13-HODE, 9-hydroxyoctadecadienoic acid; 15-HETE, 15-Hydroxyeicosatetraenoic acid; FA, Fatty acids; FAO, Fatty acid oxidation

Function of PPARs on lipid metabolism

PPARα and PPARδ are highly expressed in oxidative tissues and regulate genes involved in substrate delivery, substrate oxidation, and oxidative phosphorylation (OXPHOS) [3,9]. PPARα is expressed mainly in the liver, heart, skeletal muscles, brown adipose tissue (BAT), intestine and kidney and activates energy dissipation. PPARα mediates its functions by influencing fatty acid transport, esterification and oxidation. PPARβ/δ is expressed ubiquitously and participates in fatty acid oxidation, but also has a role in the regulation of blood glucose levels. In contrast, PPARγ contributes mainly to energy storage by promoting adipogenesis and lipid synthesis and displays highest expression levels in white adipose tissue (WAT). PPARγ is also expressed in the liver, skeletal muscles, intestine and immune cells [3,9] (Table 1).

Fatty acids involved in PPAR signaling originate from import of exogenous fatty acids or from endogenous de novo synthesis. Importantly, neither source of fatty acids can generate PPAR ligands directly. Instead, a cycle of fatty acid esterification and re-hydrolysis is required [21]. This process, termed lipolysis, involves the hydrolysis of triacylglycerols that generates free fatty acids and glycerol and involves three identified enzymes: adipose triglyceride lipase (ATGL) catalyzes the initial step of lipolysis, converting triacylglycerols to diacylglycerols; heat sensitive lipase (HSL) is hydrolyzes diacylglycerols to monoglycerols; and monoglycerol lipase (MGL), hydrolyzes monoglycerols to generate free fatty acids and glycerol. The indispensable role of lipolysis in PPAR signaling is evident by the fact that mice deficient for ATGL that controls the first step of lipolysis, exhibit a severe defect in PPARα signaling in oxidative tissues such as liver [22], macrophages [23], and BAT [24]. The most dramatic phenotype is observed in cardiac muscle. The reduced expression of PPARα target genes in ATGL knockout animals causes severe mitochondrial dysfu6nction, decreased substrate oxidation and OXPHOS, massive cardiac lipid accumulation, and lethal cardiomyopathy within few months after birth [21]. In addition to PPARα, this process is also required for PPARγ signaling [25].

PPARγ is essential for the development and function of the adipose tissue [26]. In vitro differentiation of adipose tissue from fibroblasts or embryonic stem cells relies on PPARγ [26,27]. Similarly, mice with PPARγ ablation failed to develop all types of fat [28,29]. The role of PPARγ on the adipose tissue is not restricted to its development but extends to lipid metabolism and energy homeostasis. In WAT, PPARγ controls uptake of fatty acids and lipogenesis. Several genes responsible for lipid uptake by adipocytes such as CD36 and fatty acid transport protein (FATP-1), or lipid metabolism such as adipocyte protein 2 (aP2), phosphoenolpyruvate carboxykinase (PEPCK), lipoprotein lipase (LPL) and acyl-CoA synthase, are directly regulated by PPARγ [9]. In BAT, PPARγ induces expression of target genes that regulate mitochondrial biogenesis, such as PGC-1α, and thermogenesis, such as uncoupling protein-1 (UCP-1, also known as thermogenin) [30]. PPARγ isoform 2 can also act as a transcription factor of the electron transport chain (ETC) genes on the mitochondrial DNA of brown adipose tissue cells, regulating cellular respiration [31].

Due to the PPARγ action on metabolism, PPARγ agonists have been used clinically for insulin sensitization (reviewed extensively elsewhere). Thiazolidinediones (TZD), the main medications for glucose homeostasis in patients with DM2, act as ligands for PPARγ [32], and upregulate the c-Cbl-associated protein (CAP) and the glucose transporter type 4 (GLUT4) [32,33]. The regulation of cytokines or hormones such as TNF-α, resistin and adiponectin also contributes to oxidation of fatty acids and to TZD-mediated insulin sensitization [34–36]. Notably, the insulin-sensitizing effect of PPARγ agonists is abolished in mice knocked out for Fibroblast Growth Factor-21 (FGF-21), suggesting that FGF-21 is a crucial mediator of TZD anti-diabetic activity [37]. Obesity-mediated insulin resistance and DM2 are modulated by posttranslational modification of PPARγ [38]. PPARγ phosphorylation reduces its activity in vitro whereas a genetic mutation that prevented PPARγ phosphorylation, enhanced PPARγ function and preserved insulin sensitivity in the context of diet-induced obesity [38]. These findings suggest that compounds that prevent PPARγ phosphorylation might restore insulin sensitivity by enhancing PPARγ function. Conversely, dominant negative PPARγ mutations result in DM2, hypertension and insulin resistance, demonstrating the relationship between PPARγ function and metabolic syndrome [39]. Besides ligand-dependent signaling, PPARγ expression level can be altered by cues of the microenvironment such as proinflammatory cytokines, thereby leading to altered PPAR-mediated events. For example, in patients with ulcerative colitis, PPARγ expression is significantly decreased in the intestinal mucosa and TZD-mediated PPARγ activation can mediate anti-inflammatory effects and improve the symptoms of patients with ulcerative colitis [40].

Studies have provided evidence that PPARα might exert anti-inflammatory action by mediating a direct effect on adipocytes. The proposed mechanism involves sirtuin1 (SIRT1), a NAD(+)-dependent deacetylase, that suppresses the inflammatory response by inhibiting TNF-α induced CD40 expression via the SIRT1-dependent signaling pathway [41,42]. PPARα also plays a role in the regulation of thermogenesis by promoting adipocyte browning and activating thermogenic beige adipocytes [43,44].

The mechanisms via which PPARβ/δ impacts lipid metabolism are less well investigated compared to other PPARs. Nevertheless, it is established that PPARβ/δ promotes lipid catabolism in various tissues and serves as mediator of fatty acid oxidation and fat burning [45]. PPARβ/δ activates heat producing uncoupling enzymes in brown adipose tissues and muscles and protects against obesity and fatty liver [46]. Alternatively, or in parallel, PPAR-β/δ might mediate its effects on fat metabolism by regulating the expression of adipokines [47] and by suppressing IL-6-induced STAT3 activation [48]. This effect might contribute to the prevention of IL-6-induced insulin resistance in adipocytes. Thus, therapeutic modulation of PPARβ/δ might be a promising approach for the treatment of obesity and metabolic syndrome. It should be noted that caution is required when pharmacologic modulators of PPARs are employed as several of these compounds may act on more than one PPAR subtypes albeit at different EC50 [49]

Function of PPARs on innate and adaptive immune cells

In the past few years, the role of PPARs in immune cells has been extensively studied. As key regulators of metabolism, PPARs guide the differentiation, expansion and fate commitment of various immune cell types. These effects have significant implications in organs that become targets of immunometabolic aberrations induced upon dysfunction of PPAR members.

PPARγ

This is, by far, the most extensively studied member of the PPAR family and the most common target for therapeutic intervention. PPARγ can control the homeostasis of the immune system by regulating the fate and function of various cell types. In macrophages, PPARγ regulates polarization, maturation, epigenetics and metabolism [50–54] (Figure 2). Macrophages adapt to distinct microenvironments by polarizing to either pro-inflammatory M1 macrophages or to anti-inflammatory M2 macrophages [55,56]. Under M2 conditions induced by IL-4, arginase activity, a classic marker of M2 polarization, is substantially diminished in bone marrow derived macrophages (BMDM) generated from mice with conditional deletion of PPARγ in macrophages, compared to WT counterparts [51]. Growth differentiation factor 3 (GDF3), a key mediator of muscle regeneration, is secreted by repair macrophages in response to cardiotoxin under the control of PPARγ [57]. On the other hand, under LPS culture conditions that drive M1 polarization, PPARγ KO macrophages have markedly elevated levels of proinflammatory cytokines such as IL-6, IL-12, IL-1β and TNF-α, that serve as markers of M1 polarization [58]. In contrast to macrophage polarization, PPARγ does not affect macrophage differentiation [59].

Figure 2. PPARs regulate macrophage polarization and function.

Signals mediated via PPARs regulate the differentiation and function of macrophages and dendritic cells.

In addition to regulating macrophage polarization, PPARγ has an important role in other macrophage functions, including formation of foam cells (Figure 2), i.e. macrophages that localize at the walls of blood vessels during lipid accumulation [60,61]. The expression of the macrophage scavenger receptor CD36, which is necessary for foam cell formation and subsequently atherogenesis [62], relies on PPARγ [60,61]. Cholesterol crystals can cause production of reactive oxygen species (ROS) through xanthine oxidase and NADPH, and ROS can activate BTK. The activated BTK phosphorylates p300 protein, leading to p300-STAT1-PPARγ interaction. Binding of the complex at −107 nucleotide on CD36 promoter results in enchased CD36 expression, absorption of oxLDL and formation of foam cells [61]. The significance of this mechanism is underlined by the diminished capacity of PPARγ deficient macrophages to uptake and degrade OxLDL [59]. Notably, not only cholesterol uptake but also efflux, mediated by the cholesterol efflux cassettes of the ABCA1 family, is under the transcriptional control of PPARγ [63]. Consistently, proline/serine-rich coiled-coil 1 (PSRC1) inhibits atherogenesis and foam cell formation by upregulating LXRα and PPARγ in vitro and in apoE^−/− mice [64].

In dendritic cells (DC), which have a key role in regulating immunity vs. anergy (in vitro) and tolerance (in vivo), PPARγ is a central regulator of functional maturation, thereby guiding the ability to induce immunogenic T cell responses vs. immune tolerance (Figure 2). Specifically, sustained agonist-mediated activation of PPARγ in mouse DC reduced maturation-induced expression of costimulatory molecules and IL-12, and profoundly inhibited the ability of DC to prime naive CD4⁺ T cells in vitro [65]. These effects were abrogated when murine DCs were deficient for PPARγ gene, providing evidence that the effects of pharmacologic modulation of PPARγ-signaling were specific. Conversely, PPARγ ablation increased the immunogenicity of DC, indicating that PPARγ might function as a constitutive regulator of DC suppression [65]. In human monocyte-derived DC, activation of PPARγ with 15d-PGJ2 or troglitazone suppressed DC stimulation via the TLR ligands 2, 3, 4, and 7, as determined by down-regulation of costimulatory and adhesion molecules and reduced secretion of cytokines and chemokines [66]. These effects were mediated by inhibition of TLR-mediated activation of MAP kinase and NF-kB pathways and resulted in reduced capacity of DC to stimulate T-cell proliferation, emphasizing the inhibitory effect of PPARγ activation on DC maturation. PPARγ expression in DC also regulates immune tolerance in the airways and is indispensable for inhibition of Th17 hallmark cytokines and induction of Tregs [67].

PPARγ is also critical for the regulation of adaptive immune cells (Figure 3). In one study, PPARγ activation in mouse CD4⁺ T cells selectively suppressed Th17 differentiation [68]. Control of Th17 differentiation in T cells by PPARγ involved inhibition of TGF-β/IL-6-induced expression of RORγt. Pharmacologic activation of PPARγ also prevented RORγt transcription. Importantly this study confirmed the physiologic opposing role of PPARγ on Th17 differentiation by activation of endogenous ligand. In addition, human CD4⁺ T cells from healthy individuals and patients with multiple sclerosis were susceptible to PPARγ-mediated suppression of Th17 differentiation [68]. Further investigations also provided evidence for the role of endogenous PPARγ as a regulator of Th17 responses by showing that human CD4⁺ T cells with PPARγ siRNAs increased IL-17A production [69]. Interestingly, this effect was uniquely detected in female T cells. Consistently, the synthetic PPARγ ligand, rosiglitazone, reduced IL-17A production by CD4⁺ T cells of women but not men [69]. Notably, a different study observed that the signaling axis of TCR–mTORC1-PPARγ and TCR–mTORC1–SREBP1 is essential for the activation of the fatty acid metabolic program in activated CD4⁺ T cells [70]. In this context, PPARγ directly binds on DNA and regulates the induction of genes associated with fatty acid uptake and metabolism. Thus, although PPARγ opposes the generation of effector cells with Th17 properties, it may be involved in the generation of memory cells that depend on fatty acid metabolism.

Figure 3. PPARs regulate T cell differentiation and function.

Signals mediated via PPARs regulate the development, differentiation and function of T cells.

PPARγ has an indispensable role in the differentiation of Treg cells in the visceral adipose tissue (VAT) [71] (Figure 3). Specifically, PPARγ is a major driver of a unique population of Treg that differentiate and accumulate in the VAT, and are implicated in the control of the inflammatory state of adipose tissue and, thereby, insulin sensitivity [71,72]. The transcriptional profile driving the differentiation of VAT Treg is under the control of PPARγ and depends on the exact same molecular regulation mediated by PPARγ Ser²⁷³, similarly to a PPARγ-mediated transcriptional differentiation of adipocytes [73]. Because Treg survival and function depends on lipid metabolism [74], the specific role of PPARs on Treg maintenance and function remains an open and intriguing question.

The beneficial role of PPARγ in suppressing T cell activation and generation of alloreactive T cells was reported in a model of cardiac transplantation. Macrophage and CD4⁺ T cell infiltration was significantly higher in cardiac allografts of recipient mice with T-cell-specific PPARγ deficiency, which displayed aggravated lesions of chronic allograft rejection. In contrast to WT recipients, infiltrating macrophages in mice with T-cell-specific PPARγ deficiency failed to polarize to M2. This was associated with the higher Thl/Th2 and Th17/Treg ratios among the infiltrating CD4⁺ T cells [75].

PPARα

Besides PPARγ, PPARα also appears to potentiate the polarization of macrophages towards an anti-inflammatory phenotype (Figure 2). Specifically, under LPS/IFN-γ culture conditions, peritoneal macrophages from PPARα deficient mice show a significant increase of various inflammatory parameters [76]. Similarly, treatment with the PPARα agonist WY14643 promotes the switch of peritoneal macrophages from Trypanosoma cruzi-infected mice from the M1 to M2 phenotype [77]. Notably, PPARα demonstrates an important role both in the inhibition of the excessive inflammatory response and the enhancement of innate host defense [78]. Bone marrow-derived macrophages infected with Mycobacterium Tuberculosis exhibit reduction of proinflammatory cytokines but also decreased intracellular mycobacterial levels when treated with PPARα agonists. Further in vitro studies revealed that PPARα, indeed, regulates macrophage antimycobacterial response through the transcriptional activation of autophagy, lysosomal biogenesis and enhancement of phagosome maturation mediated by the transcription factor EB (TFEB), a critical regulator of autophagy activation [78].

PPARα plays an important role in T cell responses and in the development of T cell-mediated autoimmune diseases, in a gender-specific manner. PPARα expression is higher in CD4⁺ T cells from male mice than female mice, which was found to be influenced by androgen levels. Additionally, genetic deletion of PPARα in male murine T cells resulted in augmented Th1 at the expense of Th2 responses through enhancement of NF-kB and c-Jun activity [79]. PPARα also affects the function of human T cells in a gender-specific manner [69]. Naive CD4⁺ T cells from peripheral blood of healthy women produce higher levels of IFNγ in response to anti-CD3 and anti-CD28 stimulation. PPARα, which is increased by androgens, represses IFNγ production in male T cells. Thus, PPARα, similarly to PPARγ may act as androgen-sensitive regulator driving the sex difference in Th cytokine production, and possibly the increased susceptibility of women to certain autoimmune diseases [69]. A proposed mechanism through which PPARα might inhibit IFNγ production involves the recruitment of nuclear receptor corepressor 1 to specific cis-regulatory elements in the Ifng locus and the subsequent reduction of histone acetylation at these sites [80]. In this context, a novel antagonist of PPARα, IS001, was found to increase IFNγ secretion by NK cells, CD4⁺, and CD8⁺ T cells and improve survival of male mice infected with Th1-associated pathogen Listeria monocytogenes. Although the role of PPARα on Th17 differentiation remains uncertain, its effect on Treg generation is better understood (Figure 3). Following antigen challenge, PPARα deficient mice produced lower levels of Tregs that also exhibited impaired suppressive capacity compared to WT mice [81]. PPARα agonists, bezafibrate, GW7647, and ETYA, increase Foxp3 expression in human iTreg through epigenetic modification of the Foxp3 promoter and induced functional Tregs with suppressive properties [82].

PPARβ/δ

The effects of PPARβ/δ on macrophages are less well established (Figure 2). Under M1 culture conditions, treatment of murine macrophages with PPARδ agonists could inhibit expression of proinflammatory mediators iNOS and COX2 [83]. However, other studies reported that peritoneal macrophages from PPARβ-deficient mice have reduced expression of proinflammatory genes [84]. PPARδ deletion renders adipose tissue resident murine macrophages incapable of transitioning to M2 phenotype [85]. In human macrophages, PPARδ activation did not influence polarization [86]. These apparently contradictory observations can be explained by the context-dependent effect of PPARβ/δ on macrophage polarization [87]. At least one proposed mechanism involves an inflammatory switch that is mediated through PPARβ/δ association and disassociation with transcriptional repressors such as Bc16. Unliganded PPARβ/δ has pro-inflammatory effects, while Bc16-ligated PPARβ/δ switches to an anti-inflammatory mediator by releasing Bc16 that suppresses transcription of proinflammatory genes [84]. The anti-inflammatory effects of PPARβ/δ are also exhibited in mast cells. Mast cells from PPARβ/δ KO mice express higher levels of inflammatory cytokines and lower levels of high-affinity IgE receptor compared to WT [88].

PPARβ/δ has implications on T cell development and function (Figure 3). By regulating the metabolic program of developing thymocytes, PPARβ/δ might enhance the proliferative burst of TCRβ-selected thymocytes and the growth of peripheral CD4⁺ T cells [89]. On the contrary, other studies support that activation or overexpression of PPARβ/δ decreases proliferation of double negative stage 4 (DN4) thymocytes through a switch of cellular fuel preference from glucose to fatty acid oxidation [90]. Regardless of the unresolved issues about the precise role of PPARδ in thymic development, the role of PPARδ in inducing tolerance and preventing autoimmunity is well established. In a murine EAE model, PPARδ deficiency augmented Th1 and Th17 polarization and inhibited Th2 and Treg differentiation, while the opposite results were induced with PPARδ activation [91–93]. Notably, mice lacking PPARδ expression only in the myeloid compartment also develop more severe EAE due to Th1/Th17 polarization of CD4⁺ T cells, highlighting the role of PPARδ in the crosstalk between innate and adaptive immune cells [94].

Role of PPARs in inflammatory diseases

PPARγ

The function of PPARγ has been associated with several inflammatory and autoimmune conditions. PPARγ null mice develop anti-phospholipid syndrome, an autoimmune disorder associated with glomerular injury and microthrombi [95]. The relationship between PPARγ and anti-phospholipid syndrome is not surprising since hemizygote T cell-specific [96] or myeloid cell-specific deletion [97] of PPARγ is associated with Lupus-like glomerular damage. Mice with PPARγ deletion in the T cell compartment develop splenomegaly and elevated plasma autoantibodies. Reduced expression of sphingosine-1-phosphate receptor 1, a molecule necessary for lymphocyte egress, is proposed to be the reason of splenic accumulation, while enhanced B-cell activation was attributed to increased Th17 polarization and IL-17 signaling [96].

By mediating effects in Kupffer cells, the resident macrophages of the liver, PPARγ can have implications on the development of several liver inflammatory conditions such as nonalcoholic fatty liver disease (NAFLD). High fat containing diet promotes liver steatosis and M1 polarization of the Kupffer cells. Conversely, polyunsaturated fatty acids (n3-PUFA) skew liver macrophages to M2 phenotype via upregulation of PPARγ. In a similar way, a PPARγ agonist converts M1 liver macrophages to M2 and reverses high fat-mediated steatosis [98]. n3-PUFA also promote Treg cell proliferation through upregulation of PPARγ and TGF-β, protecting mice from Con A-induced hepatotoxicity [99]. It is possible that there is an association between PUFA-mediated M2 polarization of Kupffer cells [98] and PUFA-mediated Treg polarization [99]. Tregs induced by PPARγ activation also alleviate Schistosoma mediated immunopathology in the spleen and liver [100]. Similarly to n3-PUFA, administration of 15d-PGJ2, a natural PPARγ ligand, partially prevents the development of hepatitis by attenuating NF-kB activation in macrophages and production of pro-inflammatory cytokines such as TNF-α, IL-2, IL-6 and IL-12 [101].

In the cardiac tissue, in addition to the direct effect of PPARγ deficiency in cardiomyocytes [25], the anti-inflammatory role of PPARγ relies also on its ability to influence macrophage polarization [102], consistent with observations in other tissues [98]. Upregulation of PPARγ, PPARα and Heme oxygenase 1 by epoxyeicosatrienoic acid (EETs) can skew macrophage polarization to M2, and inhibit LPS-induced M1 macrophage myocardial infiltration in vivo [102].

Th2 inflammation, an immune process related to allergic reactions and helminth infections, relies on PPARγ. Mice with conditional deletion of PPARγ in T cells fail to develop type 2 inflammation in response to HDM or nematode infection [103]. Recently, it was determined that Th9 cells, a subpopulation of human Th2 cells that produce IL-9 found in allergic skin conditions, are differentiated under the control of PPARγ [104]. Importantly, the role of PPARγ in the induction of type 2 inflammation is not mediated exclusively by a T cell-intrinsic mechanism. PPARγ controls the migration of CD11b⁺ DCs to the lymph nodes, promoting DC: T cell interaction [105].

PPARγ has a crucial role in regulating CNS immune responses and injury repair [106]. Polarization of microglial cells into anti-inflammatory M2 phenotype relies on PPARγ [107,108]. Adiponectin enhances microglia-mediated scavenging under amyloid β-induced toxicity by inducing M2 polarization through PPARγ, suggesting that PPARγ could have a potential role in the treatment of Alzheimer disease [107]. In an EAE model, PPARγ can protect CNS from autoimmunity by controlling the communication between self-antigen-presenting myeloid cells of the brain and autoreactive CD4⁺ T cells, which invade CNS [109]. In this model, CD4 T cell: myeloid cell interaction in the CNS resulted in CCL2 production by local astrocytes, which contributed to the recruitment of monocytes, that functioned as key mediators of EAE pathology. In mice with myeloid-specific PPARγ deficiency, monocyte invasion, neural demyelination and CCL2 production were higher than their wild type counterparts during EAE. In addition, there was a higher expression of CD40 and MHC-II in microglial cells and infiltrating macrophages, highlight ing the importance of PPARγ in attenuating the inflammatory response in the CNS. In a similar manner, PPARγ agonist-treated monocytes from multiple sclerosis patients demonstrate less prominent upregulation of activation markers after inflammatory stimulation [109].

PPARα

PPARα is involved in the regulation of inflammatory conditions. For example, PPARα attenuates acute liver inflammation. PPARα deficient mice exposed to high-fat diet have higher levels of acute liver inflammation and injury markers, upregulated inflammatory gene expression and increased liver infiltration of macrophages, compared to WT mice. Conversely, PPARα activation with synthetic agonist WY14643 under nonsteatotic conditions, directly downregulates inflammatory gene expression in a PPARα-dependent manner [110]. Similarly, treatment with fenofibrate, a PPARα agonist, in liver-specific PPARα-expressing mice inhibits the hepatic acute-phase response by lowering the levels of pro-inflammatory cytokines TNF, IL-1, and IL-6 [111]. PPARα also attenuates chronic liver inflammation. PPARα deletion in hepatocytes of high-fat fed mice promotes the development of liver inflammation and nonalcoholic fatty liver disease (NAFLD) [112]. Conversely, fenofibrate ameliorated the increased liver inflammatory gene expression and macrophage infiltration in mice with NAFLD [113]. Fenofibrate also improves liver inflammation in patients with NAFLD [114].

Similarly to its anti-inflammatory effects in liver biology, PPARα exhibits a protective role against renal inflammation. Transgenic mice with increased proximal tubule expression of PPARα develop less severe renal fibrosis and inflammation following unilateral urethral obstruction compared to WT mice. Further analyses reveal decreased production of TGF-β, IL-1b, IL-6 and TNF-α, reduced macrophage infiltration, and increased expression of anti-inflammatory cytokine IL-10 and arginase-1 [115]. In contrast, PPARα deficient mice develop increased inflammation, elevated IL-6 and worse sepsis-associated acute kidney injury [116]. An important mechanism by which PPARα protects against sepsis-induced injury and inflammation involves its regulation by the long non-coding RNA CRNDE and the microRNA miR-181-5p [117].

PPARα exhibits an indispensable role in cardiomyocyte metabolism and exhibits a protective role against cardiac inflammation [21]. In a mouse model, administration of the PPARα agonist, GW7647, attenuated cardiac ischemia/reperfusion injury and inflammation and decreased infarct size. This effect was accompanied by reduction in multiple proinflammatory cytokine levels, diminished neutrophil infiltration in the heart and reduced expression of myocardial matrix metalloproteinases-9 and −2. In this model, a tentative mechanism involves inhibition of NF-kB activation and enhanced levels of inhibitor-kBα [118]. In accordance with this, PPARα and PPARβ/δ activation inhibits LPS-induced expression of TNF-α in neonatal rat cardiomyocytes partly by antagonizing NF-kB [119]. Furthermore, fenofibrate treatment to type 1 diabetic mice significantly decreased cardiac dysfunction and inflammation via upregulating FGF21 ameliorating sirtuin1-mediated autophagy [120].

Administration of either PPARα or PPARγ agonists, but not PPARβ/δ, to LDL receptor deficient male mice exposed to high fat/high cholesterol diet results in significant reduction in atherosclerotic lesions in the aorta and inhibits the formation of macrophage foam cells in the peritoneal cavity [121]. PPARα and γ induce cholesterol efflux from foam cells through activation of the LXR-ABCA1 pathway [122], but can also inhibit foam cell formation through ABCA1-indepedent pathways [121]. PPARα and γ may also attenuate foam cell formation by inhibiting platelet-induced differentiation of human CD34⁺ progenitor cells into foam cells [123]. Thus, although the role of PPARγ in mediating foam cell formation is documented [60,61], under certain conditions, the cholesterol efflux mediated by the LXR-ABCA1 pathway might dominate leading to the opposite outcome.

PPARβ/δ

Consistent with its immunosuppressive effects in immune cells, PPARβ/δ attenuates acute liver inflammation. Administration of PPARβ/δ agonist protects mice against chemically induced hepatotoxicity and downregulates expression of proinflammatory mediators, including TNF-α and monocyte chemoattractant protein-1 (MCP-1), through inhibition of NF-kB [124]. Consistently, administration of the PPARβ/δ agonist, GW501516, to hepatoma HepG2 cells and rat primary hepatocytes reduced expression of various IL-6-mediated acute phase proteins by inhibiting the transcriptional activity of STAT [125]. PPARβ/δ also attenuates chronic liver inflammation. PPARβ/δ agonist treatment to high-fat diet-fed mice alleviates liver steatosis and inflammation and suppresses hepatic caspase-1 and IL-1β expression, while in vitro studies reveal that PPARβ/δ agonist administration to HepG2 cells inhibits palmitic acid and LPS-induced inflammasome activation [126]. Similarly, diabetic rats treated with GW0742, a PPARβ/δ agonist, have decreased fatty liver infiltration and significantly decreased hepatic expression of various inflammatory cytokines, TNF-α and MCP-1 compared to the diabetic control group [127].

PPARβ/δ exhibits a renoprotective role as reported in a number of studies. In a mouse model of protein-overload nephropathy, mice receiving the PPARδ agonist GW501516, developed less severe tubulointerstitial lesions, reduced macrophage infiltration, and decreased expression of MCP-1 and TNFα compared to experimental control mice. The anti-inflammatory effect of PPARδ in that model was mediated through direct inhibition of the TGF-β activated kinase 1 (TAK-1)-NF-kB pathway [128]. PPARβ/δ mediates a renoprotective effect on diabetic nephropathy via a similar mechanism [129]. In other studies, in response to IL-4 treatment, PPARδ drives the differentiation of M2 adipose tissue macrophages and liver Kupffer cells [130]. Conversely, PPARδ deficient macrophages treated with LPS have a highly inflammatory profile and adoptive transfer of PPARδ deficient bone marrow caused autoimmune hepatic dysfunction and systemic insulin resistance. In a different model, the importance of PPARδ on self-tolerance was confirmed by the observation that PPARδ-deficient female mice are much more likely to develop autoimmune kidney disease, a lupus-like autoimmunity, compared to their WT counterparts [131]. This was mediated by the effects of PPARβ/δ as a transcriptional sensor of apoptotic cells inducing recognition and phagocytosis by macrophages, thus limiting autoimmune responses. In accordance with these findings, treatment of lupus mice with a PPARβ/δ agonist resulted in improvement of renal inflammation, hypertrophy and injury with concomitant reduction in gene expression of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 in the kidney [132].

PPARβ/δ mediated signals affect the vascular system. PPARβ/δ activation in endothelial cells upregulates antioxidant gene expression and inhibits TNF-α-induced expression of pro-inflammatory adhesion molecules, thus suppressing endothelial-leukocyte adhesion. The proposed mechanism involves the dissociation of Bc1-6 from PPARδ, which relocates to the promoter pro-inflammatory genes mediating transcriptional repression [133]. PPARβ/δ activation inhibits foam cell formation in THP-1 macrophages treated with VLDL by attenuating VLDL-stimulated lipid accumulation and downregulates expression of inflammatory cytokines and adhesion molecules by attenuating VLDL-stimulated ERK1/2 activation and reversing VLDL-mediated inhibition of AKT/FoxO1 phosphorylation [134]. On the contrary, other studies suggest that PPARβ/δ activation promotes foam cell formation and lipid accumulation via altering expression of genes involved in lipid uptake, storage, metabolism and efflux, including scavenger receptor class A and CD36 [135,136]. In accordance with the latter, genetic deletion of PPARβ/δ in hematopoietic cells of LDLR deficient mice results in significant reduction of aortic atherosclerotic lesions, pro-inflammatory gene expression and macrophage infiltration of atherosclerotic lesions [137].

Therapeutic exploitation of PPARs

As multifunctional molecules, PPARs are implicated in a variety of a human diseases such as cancer [138–140], metabolic [141] and autoimmune conditions [142]. Therapeutic targeting of PPARs has been attempted in several of these conditions. Several synthetic exogenous ligands of PPAR receptors have been developed and therapeutically exploited (Table 2). Fibrates consist of a large group of synthetic PPARα agonists used for the treatment of hypertriglyceridemia, while TZD is a group of PPARγ agonists used as insulin sensitization in patients with metabolic syndrome and Diabetes Mellitus type 2 (DM2) [32,143]. Furthermore, compounds that activate more than one PPAR members, such as dual agonists, or all PPAR members, such as pan-agonists (Table 3), could be beneficial in both lipid and glucose imbalances, minimizing the needs for dual pharmacological intervention, and potentially diminishing side effects that might arise from the administration of two individual pharmacologic compounds [144]. On the other hand, dual PPAR agonists or pan-agonists might be related with more adverse effects due to lower target selectivity [144,145]

Table 2:

Synthetic PPAR ligands and therapeutic exploitation.

Subtype	Synthetic ligands (agonists)	Therapeutic exploitation
PPARγ	Thiazolidinediones (TZD) (pioglitazone, troglitazone, rosiglitazone) [32] Farglitazar [145] Ibuprofen [182] INT131 (CHS-131) [145] S26948 [183] GW7845 [184] Efatutazone (CS-7017) [145] GED 0507-34-Levo [145] OMS 405 [145]	• DM2 • Metabolic syndrome • Complications of metabolic diseases, such as renal tubulointerstitial fibrosis and hypertension-induced renal fibrosis • Autoimmune diseases (SLE) • Cancer immunotherapy
PPARα	Fibrates (Fenofibrate, clofibrate, gemfibrozil) [143] Pirinixic acid (WY-14643) [185] GW7647 [82,186]	• Dyslipidemia • Autoimmune diseases (EAE) • Cancer immunotherapy
PPARβ/δ	GW501516 [128,145] GW0742 [127,145] Seladelpar (MBX-8025) [145]	• Autoimmune diseases (SLE) • Cancer immunotherapy

DM2, Diabetes mellitus type 2; SLE, Systemic lupus erythematosus; EAE, Experimental autoimmune encephalomyelitis.

Table 3:

Synthetic dual or pan-PPAR ligands and therapeutic exploitation.

Subtype	Dual synthetic agonists	Therapeutic exploitation
PPARα/γ	Saroglitazar [144] Oxeglitazar [145] Lobeglitazone [145]	• Dyslipidemia • DM2
PPARα/β(δ)	Elafibranor (GFT505) [145]	•Hepatic steatosis • NASH • Dyslipidemia • Pre-diabetes • Insulin resistance • Obesity
PPARγ/δ	T3D 959 [187]	• Alzheimer’s Disease
Pan-PPAR	Chiglitazar [144] Lanifibranor (IVA337) [145]	• Dyslipidemia in obese mice • DM2 • Improved skin fibrosis in mice • NASH

DM2, Diabetes mellitus type 2; NASH, Nonalcoholic steatohepatitis

PPARα is involved in lipid and carbohydrate metabolism [146–148]. Synergistic and contrasting actions between PPARα and PPARγ have been reported [149,150]. Although most of the experimental studies have shown that PPARα agonists do not have such a beneficial effect on lipid metabolism and insulin resistance as PPARγ agonists, particularly TZD [151], novel approaches for obesity and metabolic syndrome have explored the effect of PPARα [41,152–155]. This is particularly significant because, despite their greater anti-diabetic potential, clinical application of PPARγ agonists is accompanied by detrimental side effects. Studies have exploited joint targeting of PPARγ and PPARα with a dual PPARα/γ agonists. These dual agonists improve lipid parameters and reduce cardiovascular complications through PPARα in addition to the insulin-sensitizing effects mediated via PPARγ [156]. Combined with a cutting-edge approach for cell-type specific uptake of PPARα/γ dual agonists by peptide-mediated internalization and controlled release into adipocytes, these compounds could potentially reduce the intolerable side effects of PPARγ agonists and provide major benefit for the treatment of DM2 [157].

Another promising approach in reducing the side effects of PPARγ agonists, was the development of selective PPARγ ligands that are structurally and pharmacologically distinct from glitazones. Efforts toward this goal have resulted in the development of non-thiazolidinedione compounds that belong to a distinct family of drugs named selective PPARγ modulators (SPPARMs). Such compounds mediate a distinct pattern of coregulator recruitment to PPARγ that allows improved specificity and diminished toxicity providing an opportunity for the treatment of DM2 and metabolic syndrome with limited side effects on heart and lung, weight gain, hemodilution, and plasma volume [158–161]. Using X-ray crystallography, it was determined that the SPPARM, INT131, forms hydrophobic contacts with the ligand-binding pocket of PPARγ without direct hydrogen-bonding interactions to residues in helix 12, as full agonists [160]. Because of the beneficial effects of classic PPARγ agonists on protection against neurotoxicity from Aβ amyloid oligomer in Alzheimer’s disease (AD), the role of INT131 in the central nervous system was studied in a mouse model of AD [162]. The study determined that INT131 increased dendritic branching, promoted neuronal survival against Aβ amyloid, increased expression of PGC1-1α and modulated neuronal mitochondrial dynamics. Thus SPPARMs, might have applications not only in DM2 and metabolic syndrome but also in other conditions in which PPARγ-mediated signaling might have beneficial effects.

Nuclear receptors have been associated with autoimmune diseases and PPAR agonists could be used for therapeutic purposes in autoimmunity [142]. PPARα agonists improve the clinical manifestation of EAE mice [163]. Similarly, administration of, the PPARβ/δ agonist GW0742 alleviates systemic lupus erythematosus’s (SLE) complications such as albuminuria, splenomegaly, hypertension, renal and cardiac hypertrophy [132]. These complications of SLE are also improved by rosiglitazone through adiponectin induction [164].

The role of PPAR on innate and adaptive immune cells in the context of cancer is extensively studied because factors that affect cancer development, evolution and therapeutic response might simultaneously affect the fate of immune cells via interfering with PPAR-mediated signaling. Global or conditional deficiencies of PPAR members affect tumor progression suggesting that PPARs are potential targets for cancer therapy. PPARα or PPARβ deletion in Tregs diminishes anergic properties in the tumor microenvironment [165,166]. Furthermore, combined activation of PPARα and PPARδ improves antitumor efficacy of adoptively transferred CD8⁺ T cells by reprogramming metabolism from aerobic glycolysis toward fatty acid oxidation, thereby increasing their in vivo longevity and enhancing inflammatory signals [167] (Figure 3). Similarly, PPARα-mediated fatty acid catabolism and uptake by CD8⁺ tumor-infiltrating T lymphocytes preserves their effector function and decelerates tumor growth [168]. PPAR activation can also enhance anti-tumor activity when combined with checkpoint inhibitors such as anti-PD-1 [169,170] and anti-CTLA-4 [171].

PPARγ also enhances the anti-tumor capacity of intratumoral iNKT cells. Cholesterol is one of the main precursors for IFNγ synthesis by the iNKT cells. The elevated lactic acid of the tumor microenvironment decreases PPARγ expression in iNKT cells resulting in decreased cholesterol synthesis, diminished IFNγ production, and weaker anti-tumor response. Under these conditions, a PPARγ agonist can restore cholesterol and IFNγ production, improving the antitumor activity of iNKT cells [172]. On the other hand, studies have shown that during obesity, a major risk factor for cancer development, NK cells undergo PPARα/δ-depended accumulation of lipids, resulting in metabolic defects that subsequently diminish their anti-tumor capacity [173]. These metabolic alternations can be restored after PPARα/δ inhibition, suggesting that PPARα/δ blockers could have a role in the treatment of cancer in obese patients, by enhancing the cytotoxic ability of NK cells.

PPAR can also affect the generation and function of myeloid-derived suppressor cells (MDSCs), a population of immature myeloid cells derived from myeloid progenitors during cancer-mediated emergency myelopoiesis, which have immunosuppressive properties and promote tumor progression [174]. Tumor-infiltrating MDSCs preferentially depend on fatty acid uptake and fatty acid oxidation over glycolysis for their survival and function [175]. Detailed studies have shown that upregulation of fatty-acid synthase (FASN) by M-CSF is mandatory for the differentiation of tumor infiltrating myeloid cells into MDSC, expression of MDSC hallmark genes such as IL-10, Arg1 and VEGF, and tumor progression. These events depend on fatty acid-mediated signaling via PPARβ/δ [176]. PPARγ also plays an important role in MDSC differentiation by regulating neutral lipid metabolism that depends on the generation of endogenous fatty acids via lipolysis mediated by lysosomal acid lipase (LAL). In this context, signaling via PPARγ impairs MDSC development and function and suppresses tumor growth [177]. The opposite outcomes were observed when PPARγ signaling in myeloid cells was abrogated by expressing a dominant negative PPARγ construct in a myeloid-specific bitransgenic mouse model [178]. Under these conditions, there was a massive expansion of PPARγ deficient MDSC that mediated potent suppression expansion and function of CD4⁺ and CD8⁺ T cells and resulted in the development of multiple forms of cancers in various organs. Notably, PPARγ expression is elevated in myeloid cells and tumor associated macrophages (TAMs) in PD-1 KO mice or mice with myeloid-specific PD-1 deletion [179]. These results indicate that PPARγ-mediated signals during PD-1 ablation might specifically regulate the effector function of myeloid cells and prevent the generation of MDSC in the context of cancer.

Together these studies indicate that increase of fatty acid availability might promote the generation and function of immunosuppressive and tumor promoting myeloid cells by altering PPAR-mediating signaling. This mechanism might provide a mechanistic explanation for the fact that obesity is a risk factor for carcinogenesis [180,181]. However, as outlined above, it becomes increasingly apparent that PPAR subtypes have distinct and opposing roles in the generation of MDSC [176–178]. Thus, it is unpredictable how metabolic adaptation of immune cells in the context of altered host metabolism, as in diabetes, metabolic syndrome and obesity, might affect distinct PPAR subtypes under patient-specific conditions. Therefore, development of markers related to PPAR signaling in innate and adaptive immune cells might provide critical patient-specific information that might be helpful for guiding selection of treatment strategies exploiting the potential of PPAR signaling in cancer therapy.

Concluding remarks

Members of the PPAR family of nuclear hormone receptors are well-established regulators of lipid metabolism, mitochondrial biogenesis and energy homeostasis. Their activation has central implications in the function of oxidative tissues and organs such as cardiomyocytes, liver and muscle. For many years, PPARs have been attractive therapeutic targets for the treatment of metabolic disorders. The role of various members of this nuclear receptor family is currently emerging in the differentiation and function of immune cells as they guide metabolism-mediated immune cell fate commitment. Well-established as mediators of macrophage polarization, PPARs are also key determinants of T cell activation, expansion and differentiation as well as regulators of MDSC generation. The cell-specific temporal expression and activation of PPARs provide new opportunities for therapeutic intervention to alter the function of innate and adaptive immune cells with the goal to modulate immune cell activation for the treatment of autoimmune diseases, organ transplantation, neurodegenerative disorders, and cancer. Discoveries on the posttranslational regulation of PPARs, will assist the development of new compounds to target for cell-specific activation for therapeutic intervention. New compounds that function as agonists for more than one PPAR subtypes will allow the simultaneous therapeutic exploitation of these nuclear receptors to maximize therapeutic benefit while minimizing toxicity. Cutting edge approaches, which allow the identification of how structurally and functionally distinct states of PPARs are induced by binding of agonists vs. antagonists or reverse agonists, will provide guidance for the design of compounds to promote the desired activation state. Moreover, novel technologies for targeted delivery of PPAR-modulating compounds will provide unprecedented opportunities for targeted therapeutic interventions in the context of immune-mediated diseases.

Highlights.

PPAR in the function on innate immune cells

PPAR function on adaptive immune cells

PPAR role in immune-mediated inflammatory conditions

PPAR therapeutic options in immunology