Msh expression of mcr4 expresses PPAR-y an, IL-10, Ag1 and reduced Tlr4 and Il-4rα and prevents TLR4 induced p65 activation, inhibits TLR2 which then inhibit NFkb and its subunits p65/RelA, p50, p52, c-Rel and RelB

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Research Article

[Nle4, D-Phe7]-α-MSH Inhibits Toll-Like Receptor (TLR)2- and TLR4-Induced Microglial Activation and Promotes a M2-Like Phenotype

  • Lila Carniglia,

    Affiliation: Institute of Biomedical Research (INBIOMED -UBA- CONICET), School of Medicine, University of Buenos Aires, Buenos Aires, 1121, Argentina

    ⨯
  • Delia Ramírez,

    Affiliation: Institute of Biomedical Research (INBIOMED -UBA- CONICET), School of Medicine, University of Buenos Aires, Buenos Aires, 1121, Argentina

    ⨯
  • Daniela Durand,

    Affiliation: Institute of Biomedical Research (INBIOMED -UBA- CONICET), School of Medicine, University of Buenos Aires, Buenos Aires, 1121, Argentina

    ⨯
  • Julieta Saba,

    Affiliation: Institute of Biomedical Research (INBIOMED -UBA- CONICET), School of Medicine, University of Buenos Aires, Buenos Aires, 1121, Argentina

    ⨯
  • Carla Caruso,

    Affiliation: Institute of Biomedical Research (INBIOMED -UBA- CONICET), School of Medicine, University of Buenos Aires, Buenos Aires, 1121, Argentina

    ⨯
  • Mercedes Lasaga

    * E-mail: mla...@fmed.uba.ar

    Affiliation: Institute of Biomedical Research (INBIOMED -UBA- CONICET), School of Medicine, University of Buenos Aires, Buenos Aires, 1121, Argentina

    ⨯

[Nle4, D-Phe7]-α-MSH Inhibits Toll-Like Receptor (TLR)2- and TLR4-Induced Microglial Activation and Promotes a M2-Like Phenotype

  • Lila Carniglia, 
  • Delia Ramírez, 
  • Daniela Durand, 
  • Julieta Saba, 
  • Carla Caruso, 
  • Mercedes Lasaga
PLOS
x

Abstract

α-melanocyte stimulating hormone (α-MSH) is an anti-inflammatory peptide, proved to be beneficial in many neuroinflammatory disorders acting through melanocortin receptor 4 (MC4R). We previously determined that rat microglial cells express MC4R and that NDP-MSH, an analog of α-MSH, induces PPAR-γ expression and IL-10 release in these cells. Given the great importance of modulation of glial activation in neuroinflammatory disorders, we tested the ability of NDP-MSH to shape microglial phenotype and to modulate Toll-like receptor (TLR)-mediated inflammatory responses. Primary rat cultured microglia were stimulated with NDP-MSH followed by the TLR2 agonist Pam3CSK4 or the TLR4 agonist LPS. NDP-MSH alone induced expression of the M2a/M2c marker Ag1 and reduced expression of the M2b marker Il-4rα and of the LPS receptor Tlr4. Nuclear translocation of NF-κB subunits p65 and c-Rel was induced by LPS and these effects were partially prevented by NDP-MSH. NDP-MSH reduced LPS- and Pam3CSK4-induced TNF-α release but did not affect TLR-induced IL-10 release. Also, NDP-MSH inhibited TLR2-induced HMGB1 translocation from nucleus to cytoplasm and TLR2-induced phagocytic activity. Our data show that NDP-MSH inhibits TLR2- and TLR4-mediated proinflammatory mechanisms and promotes microglial M2-like polarization, supporting melanocortins as useful tools for shaping microglial activation towards an alternative immunomodulatory phenotype.

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Citation: Carniglia L, Ramírez D, Durand D, Saba J, Caruso C, Lasaga M (2016) [Nle4, D-Phe7]-α-MSH Inhibits Toll-Like Receptor (TLR)2- and TLR4-Induced Microglial Activation and Promotes a M2-Like Phenotype. PLoS ONE 11(6): e0158564. doi:10.1371/journal.pone.0158564

Editor: Karin E. Peterson, National Institute of Allergy and Infectious Diseases - Rocky Mountain Laboratories, UNITED STATES

Received: December 18, 2015; Accepted: June 19, 2016; Published: June 30, 2016

Copyright: © 2016 Carniglia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper.

Funding: This research was supported by grants from ANPCyT (Agencia Nacional de Promoción Científica y Técnica) PICT 0894, CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas) PIP 630 and University of Buenos Aires 20020130100120BA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Microglial cells are key players in the inflammatory processes within the central nervous system (CNS). They are in charge of immune surveillance and respond to injury or noxious stimuli by releasing inflammatory mediators, recruiting other immune cells to the injured zone and orchestrating an effective inflammatory response. Afterwards, they release anti-inflammatory mediators leading to resolution of the inflammatory response and return to homeostasis. Alterations in this delicate process may lead to tissue damage, neuroinflammation and neurodegeneration. Microglia can be found in two main activation states, termed M1 and M2. M1 microglia display a classical pro-inflammatory program which can be induced by TH1 cytokines such as IFN-γ or bacterial moieties such as lipopolysaccharide (LPS), and is characterized by the production of tumour necrosis factor (TNF)-α, Interleukin (IL)-1β, IL-12, reactive oxygen species (ROS) and nitric oxide (NO), among other mediators. M2, in turn, refers to alternatively activated microglia, a phenotype induced by TH2 cytokines such as IL-4 and IL-13, whose main markers include Arginase-1 (AG1), IL-10, transforming growth factor (TGF)-β and peroxisome proliferator-activated receptor (PPAR)-γ, among others [1].

As immunocompetent cells, microglia express a variety of innate immunity receptors, such as Toll-like receptors (TLRs) [2]. TLRs comprise a family of membrane-associated receptors that bind a diversity of pathogen-associated molecular patterns (PAMPs) [3]. Alternatively, TLRs can be activated by the presence of endogenous molecules released after tissue damage, termed damage associated molecular patterns (DAMPs) [4]. In particular, TLR2 and TLR4 can mediate the actions of the DAMP high-mobility group box 1 (HMGB1), which can be released to the extracellular milieu during traumatic cell death or upon stimulation by a number of inflammatory mediators such as TLR agonists, and potentiate the inflammatory response [4, 5]. Plus, activation of microglial TLR2 and TLR4 has been associated with neurotoxicity in various CNS diseases [6], further underscoring the importance of studying the modulation of TLR signaling in these cells. The triacylated lipoprotein Pam3CSK4 and LPS are frequently used as TLR2 and TLR4 agonists, respectively [7]. The binding of Pam3CSK4 or LPS to their receptors leads to the activation of the nuclear transcription factor NF-κB. NF-κB family is composed of five subunits, p65/RelA, p50, p52, c-Rel and RelB, which can form homo or heterodimers. The C-terminal transcription activation domain, necessary for positive regulation of target genes, is present in p65, c-Rel and RelB but not in p50 and p52. Therefore, p50/p50 and p52/p52 homodimers are considered repressors of transcription [8]. The p50/p65 heterodimers are activated in most cell types whereas c-Rel-containing dimers are mainly activated in cells of the hematopoietic lineage [9]. Once activated, NF-κB translocates to the nucleus and promotes the expression of its target genes, such as TNF-α [10]. This cytokine in turn promotes and amplifies the inflammatory response. The release of inflammatory mediators is followed by production of immunomodulatory factors such as IL-10, a key player in resolution of the immune response [11]. When the inflammatory response is not properly resolved this otherwise necessary and beneficial process may turn against the organism and contribute to the development of neuroinflammatory disorders in the CNS. Therefore, identifying modulators of microglial activation is of paramount importance in the context of neuroinflammation and neurodegeneration.

α-melanocyte-stimulating hormone (α-MSH) is a member of the melanocortin family, a group of peptides derived from post-translational cleavage of pro-opiomelanocortin (POMC). Melanocortins exert their action through the activation of five melanocortin receptor subtypes (MC1R to MC5R) coupled to G-protein which upon activation induce production of cyclic AMP (cAMP) [12]. In the CNS melanocortins have proven to be anti-inflammatory and neuroprotective peptides in models of brain ischemia [13, 14] and sepsis [15] and to improve cognitive function, acting through MC4R, in a model of Alzheimer’s disease (AD) [16] or after cytokine treatment [17–19]. In a microglial cell line α-MSH was shown to inhibit the production of TNF-α, IL-6 and nitric oxide induced by LPS+IFN-γ [20]. We previously showed that rat cultured microglial cells express MC4R and that the synthetic peptide [Nle4, D-Phe7]-α-MSH (hereon NDP-MSH), an analog of endogenous α-MSH, induces PPAR-γ expression and IL-10 release from microglia, thereby promoting development of an anti-inflammatory phenotype [21]. Given the importance of modulating glial activation in the context of neuroinflammatory processes, this study tested the ability of NDP-MSH to modulate inflammatory mechanisms triggered by TLR2 and TLR4 agonists in microglial cells.

Materials and Methods

Reagents

NDP-MSH was purchased from Bachem (Bubendorf, Switzerland). Ultrapure LPS (E. coli, 0111:B4) and Pam3CSK4 were purchased from Invivogen (San Diego, CA, USA). Fluorescent microspheres (Molecular Probes, F8823) were kindly provided by Dr. Candolfi (INBIOMED, UBA-CONICET). Foetal bovine serum (FBS) was obtained from Natocor (Córdoba, Argentina). DMEM/F-12, DMEM, L-Glutamine and antibiotics were purchased from Invitrogen Life technologies (CA, USA). All other media and supplements were obtained from Sigma-Aldrich Corporation, unless specified otherwise.

Primary microglial cultures

Wistar rats were bred in the INBIOMED animal housing facility, University of Buenos Aires, Buenos Aires, Argentina. Rats were kept in a 12 h light-dark cycle at 22°C ± 1°C with access to food and water ad libitum. 1–2 day-old Wistar rat pups were decapitated and their brains removed and freed from meninges. Cells were mechanically dispersed and seeded in previously poly-L-lysine-coated culture flasks, and held in DMEM/F-12 supplemented with 10% FBS, 50 μg/ml streptomycin and 50U penicillin, at 37°C in 5% CO2. Medium was replaced twice a week. After 11–14 days, microglial cells were detached from astrocytes by shaking for 1–2 h at 110 rpm, at 33°C in a Thermo Scientific Orbital Shaker (Thermo Fischer Scientific, Germany). Supernatants were collected and centrifuged, and microglia was seeded in uncoated plates in supplemented DMEM containing 10% FBS and 2 mM L-glutamine and left to stabilize overnight at 37°C in a 5% CO2 atmosphere before adding the drugs in fresh supplemented DMEM containing 2% FBS and 2 mM L-glutamine. Cells were stained with a microglial marker (anti-rat CD11b monoclonal antibody, OX-42, Millipore) to assess their purity, which was routinely close to 98%.

Reverse-transcription real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted using Quickzol reagent (Kalium Technologies), following manufacturer’s instructions. 0.5–1 μg of RNA was treated with 1U DNAse (Promega Corp., Madison, WI) and reverse-transcribed as described before [22]. Products of the RT reaction were amplified using specific primers and SYBR Green Master Mix or SYBR Green Select Master Mix (Invitrogen Life Technologies) on a StepOne™ Real-Time PCR System (Applied Biosystems). Primer sequences are detailed in Table 1. PCR product specificity was verified by a melting curve analysis. No-RT controls were performed by omitting addition of the reverse transcriptase enzyme in the RT reaction, and no-template controls were performed by addition of nuclease-free water instead of cDNA. Gene expression was normalized to the endogenous reference gene HPRT by the ΔΔCt method [23] using Step-One Software (Applied Biosystems), and expressed as fold-changes relative to the control group.

Cell viability

Cells were treated with or without 100 nM NDP-MSH for 24 hours, then incubated with or without LPS or Pam3CSK4 (100 ng/ml) for 4.5 or 24 hours. Cell viability was assessed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) reduction assay. Briefly, cells were washed in Krebs buffer and incubated for 4 hours with 110 μl of a 0.5 μg/ml MTT-Krebs buffer solution. Formazan crystals obtained from MTT reduction were dissolved in 100 μl of a 0.04 N HCl-isopropanol solution. OD was measured in a microplate spectrophotometer (Bio-Rad) at 595 nm.

Cytokine release

TNF-α, IL-10 and IL-4 release were assessed by ELISA using commercial kits (BD Biosciences, San Diego, USA). Assays were performed following the manufacturer’s instructions. Cytokine values (pg/ml) were normalized to the viability values obtained by the MTT reduction assay to account for variations in cell numbers caused by the different treatments.

Immunocytochemistry

Cells were washed in PBS and fixed in 4% PBS–PFA for 10–15 minutes at RT. For p65 and c-Rel immunocytochemistry cells were incubated in ice cold methanol for 10 minutes at -20°C. For HMGB1 staining slides were incubated in Citrate buffer and subjected to microwave heating at 350 W for 5 minutes, then left to reach RT for 20 minutes and washed in PBS. Slides were incubated in blocking solution containing 10% donkey serum or 10% goat serum (depending on the source of the secondary antibody) in PBS for 1 hour at RT. Subsequently, cells were incubated overnight at 4°C with the following primary antibodies: anti-CD11b 1:100 (OX-42, Millipore), anti-p65 1:30 (BD Biosciences), anti-c-Rel 1:50 (sc-70, Santa Cruz), anti-HMGB1 1:100 (ab79823, Abcam). After rinsing, slides were incubated with the appropriate secondary antibodies for 1 hour at RT. Cells were washed in PBS and mounted in mounting medium Vectashield (Vector Laboratories). Negative control slides were incubated with blocking solution instead of the primary antibodies. Staining was visualized in a fluorescence microscope (Axiophot; Carl Zeiss, Jena, Germany). Quantification of fluorescence intensity was done with ImageJ Software (NIH, USA).

Phagocytosis assay

Microglial cells (1 x 105) were plated onto glass coverslips and treated with 100 nM NDP-MSH for 24 hours, then stimulated with either Pam3CSK4 or LPS (100 ng/ml) for 24 additional hours. 2 hours before ending the incubation period, 3 μl of a fluorescent carboxylated-latex beads suspension in complete DMEM were added to the cultures in a 100:1 bead to cell ratio. Cells were fixed in 4% PBS-PFA for 10 minutes at RT and processed for immunocytochemistry for the microglial marker CD11b. The number of beads per CD11b positive cell in each experimental group was counted in a Zeiss Axiophot fluorescence microscope under a 40X lens. Cells containing ten or more microspheres were considered positive for phagocytosis, as described elsewhere [24] and the proportion of phagocytosis-positive cells was calculated for each experimental group. To verify that the presence of beads within the cells was due to active phagocytosis, cells were incubated with the bead solution at 4°C for 2 hours, after which no latex beads were detected inside the cells (internal control not shown).

Statistical analysis

Data were analysed by one sample t test, Student’s t test or one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons test, as required by the experimental design. GraphPad Prism 5 Software was used (GraphPad Software, CA, USA). Differences with a value of p<0.05 were considered statistically significant.

Ethics Statement

Experimental procedures were approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) of the School of Medicine, University of Buenos Aires, Argentina (resolution n° 096/2010) and were carried out in compliance with the guidelines of the NIH Guide for the Care and Use of Laboratory Animals.

Results

Effect of NDP-MSH on the expression of M2 markers and inflammatory mediators on microglial cells

To study the effect of NDP-MSH on microglial polarization we assessed the gene expression of the M2 markers Ag1, Socs3 and Il-4rα and the inflammatory mediators Hmgb1, Tlr2 and Tlr4 by RT-qPCR. Treatment with NDP-MSH for 24 hours increased the expression of Ag1 (Fig 1). Conversely, expression of Tlr4 and Il-4rα was reduced by NDP-MSH (Fig 1). Socs3, Tlr2 and Hmgb1 expression levels were not modified by NDP-MSH (Fig 1). Induction of Ag1 expression plus reduction in Tlr4 expression, added to the previously described increase in PPAR-γ expression and IL-10 release [21], support a role for NDP-MSH in microglial polarization towards an alternative M2-like profile.

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Fig 1. Effect of NDP-MSH on the expression of M2 markers and inflammatory mediators.

Cells were treated with 100 nM NDP-MSH for 24 hours. Gene expression was assessed by RT-qPCR. Values are the mean ± SEM of at least 3 independent experiments and are expressed as the percentage of their respective controls (arbitrarily set at 100% and represented by the dotted line). Data were analysed by one-sample t test. *p<0.05 vs. control group.

http://dx.doi.org/10.1371/journal.pone.0158564.g001

NF-κB activation

We tested whether NDP-MSH could inhibit TLR-induced NF-κB activation by determining the intracellular localization of the p65 and c-Rel NF-κB subunits upon treatment with LPS or Pam3CSK4. Immunostaining for p65 was evenly distributed within the cytoplasm and absent from the nucleus in the untreated microglial cells (control group, Fig 2.b). Treatment with LPS strongly increased the percentage of p65-positive nuclei (Fig 2.a and 2.d) and pre-treatment with NDP-MSH reduced the percentage of p65-positive nuclei compared to LPS alone (Fig 2.a and 2.f). Treatment with Pam3CSK4 induced p65 nuclear translocation—although not as robustly as LPS—(Fig 2.a) and pre-treatment with NDP-MSH did not modify the percentage of p65-positive nuclei upon stimulation with Pam3CSK4 (Fig 2.a). NDP-MSH alone did not modify the percentage of p65-positive nuclei compared to the control group (Fig 2.a). These results indicate that NDP-MSH prevents TLR4-induced p65 activation.

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Fig 2. NDP-MSH prevents LPS-induced p65 nuclear translocation.

Cells were treated for 24 hours with 100 nM NDP-MSH and then stimulated for 30 minutes with LPS (100 ng/ml) or Pam3CSK4 (100 ng/ml) and processed for p65 immunocytochemistry. (a) The percentage of p65-positive nuclei was calculated for each experimental group. Data are the mean ± SEM of 4 independent experiments and were analysed by one-way ANOVA followed by Bonferroni’s multiple comparisons test ***p<0.001, **p<0.01 vs. control; ^p<0.05 vs. LPS. Representative images are shown of the following groups: (b) and (c) Control; (d) and (e) LPS; (f) and (g) NDP-MSH + LPS. Green: p65. Blue: nuclei stained with DAPI.

http://dx.doi.org/10.1371/journal.pone.0158564.g002

Nuclear c-Rel immunostaining was evident in untreated microglial cells (Fig 3.b). Therefore, we decided to compare the intensity of nuclear c-Rel immunoreactivity rather than positive versus negative nuclei. Treatment with LPS increased nuclear c-Rel immunoreactivity (Fig 3.a and 3.d) and this effect was inhibited by NDP-MSH pre-treatment (Fig 3.a and 3.f), indicating that NDP-MSH also inhibits TLR4-induced c-Rel activation. Treatment with NDP-MSH alone had no significant effect on c-Rel immunoreactivity compared to the control group (Fig 3.a). On the other hand, treatment with Pam3CSK4 did not increase c-Rel nuclear immunoreactivity compared to untreated cells (data not shown).

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Fig 3. NDP-MSH prevents LPS-induced c-Rel nuclear translocation.

Cells were treated for 24 hours with 100 nM NDP-MSH and then stimulated for 30 minutes with LPS (100 ng/ml) or Pam3CSK4 (100 ng/ml) and processed for c-Rel immunocytochemistry. (a) Nuclear fluorescence intensity was semi-quantified using ImageJ software. Data are the mean ± SEM of 4 independent experiments and were analysed by one-way ANOVA followed by Bonferroni’s multiple comparisons test. ***p<0.001 vs. control. ^p<0.01 vs. LPS. Representative images are shown of the following groups: (b) and (c) Control; (d) and (e) LPS; (f) and (g) NDP-MSH + LPS. Green: c-Rel. Blue: nuclei stained with DAPI.

http://dx.doi.org/10.1371/journal.pone.0158564.g003

Cytokine release

In order to determine the effect of NDP-MSH on TLR-induced cytokine release, we determined the concentration of the proinflammatory cytokine (and M1/M2b marker) TNF-α in the culture supernatant upon TLR-stimulation. We also assessed the release of the anti-inflammatory mediator IL-10 and of the M2-inducer IL-4 as a possible autocrine mechanism of NDP-MSH-induced alternative polarization. Both LPS and Pam3CSK4 induced the release of TNF-α to the culture supernatant compared to the control group, and pre-treatment with NDP-MSH inhibited TLR4- and TLR2-induced TNF-α release (Fig 4.a and 4.b, respectively). On the other hand, NDP-MSH did not modify TLR4 and TLR2-induced IL-10 release (Fig 4.c and 4.d, respectively), suggesting that the melanocortin is selectively inhibiting the release of a proinflammatory mediator rather than globally decreasing microglial activity. IL-10 release in the control groups was below the detection limit of the commercial kit (15.6 pg/ml) and therefore these groups are not shown. On the other hand, we detected no IL-4 release either in the control group, or in the NDP-MSH-, LPS- or Pam3CSK4-treated cells, and NDP-MSH also failed to induce IL-4 release in mixed glial cell cultures (data not shown). This result indicates that cultured rat microglial cells do not release IL-4 in the conditions tested and suggests that IL-4 is not a mediator of NDP-MSH effects in vitro.

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Fig 4. NDP-MSH inhibits TLR4- and TLR2-induced TNF-α release but not IL-10 release.

Cells were pre-treated for 24 hours with 100 nM NDP-MSH and then stimulated with LPS (100 ng/ml) or Pam3CSK4 (100 ng/ml). TNF-α release into the culture supernatant after 4.5 hours (a) and (b) and IL-10 release after 24 hours (c) and (d) were assessed by ELISA and normalised to the viability values obtained by the MTT assay for each experimental group (MTT values not shown). The dotted line in (a) and (b) represents values of TNF-α release for control groups. Data are the mean ± SEM of 6 replicates and were analysed by Student’s t test. Experiments were performed twice. (a) ***p<0.001 vs. LPS. (b) ***p<0.001 vs. Pam3CSK4.

http://dx.doi.org/10.1371/journal.pone.0158564.g004

HMGB1 intracellular localization

HMGB1 is a nuclear protein that can be released to the extracellular milieu upon stimulation by TLR agonists and act as a proinflammatory cytokine [25]. Since HMGB1 must translocate from nucleus to cytoplasm prior to its release, the effect of NDP-MSH on TLR-induced HMGB1 translocation was investigated. Cells were pre-treated with or without NDP-MSH for 24 hours and then stimulated with LPS or Pam3CSK4 for 24 additional hours. HMGB1 immunoreactivity was observed in the nuclei of all cells in the control group, and some cells also displayed a faint punctuate pattern of HMGB1 in the cytoplasm (Fig 5.a and 5.b). Upon stimulation with Pam3CSK4 we observed a decrease in nuclear HMGB1 fluorescence intensity and an increase in the punctuate HMGB1 pattern in the cytoplasmic region (Fig 5.a and 5.d) suggesting an active release of HMGB1 from nucleus to cytoplasm. In the NDP-MSH-pre-treated group there was an increase in HMGB1 nuclear fluorescence intensity to the levels of the control group, suggesting that NDP-MSH prevented Pam3CSK4-induced HMGB1 release from nucleus to cytoplasm (Fig 5.a and 5.e). NDP-MSH alone had no effect on HMGB1 intracellular localization compared to the control group (Fig 5.a and 5.c). These results suggest that NDP-MSH inhibits nuclear release of HMGB1 upon TLR2 stimulation. LPS did not significantly induce HMGB1 translocation in the conditions tested (data not shown).

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Fig 5. NDP-MSH prevents Pam3CSK4-induced HMGB1 nucleus-to cytoplasm translocation.

Cells were treated for 24 hours with 100 nM NDP-MSH and then stimulated for another 24 hours with LPS (100 ng/ml) or Pam3CSK4 (100 ng/ml). Cells were immunostained for HMGB1 and nuclear fluorescence intensity was semi-quantified using ImageJ software. (a) Data are the mean ± SEM of 5 independent experiments and were analysed by one-way ANOVA followed by Bonferroni’s multiple comparisons test. *p<0.05 vs. control, ^p<0.05 vs. Pam3CSK4. Representative images are shown of the following groups: (b) Control; (c) NDP-MSH; (d) Pam3CSK4; (e) NDP-MSH + Pam3CSK4. Green: HMGB1.

http://dx.doi.org/10.1371/journal.pone.0158564.g005

Microglial phagocytosis

Both Pam3CSK4 and LPS are known stimulators of microglial phagocytic activity [26, 27]. The effect of NDP-MSH on TLR-induced phagocytosis was investigated through the uptake of fluorescent microspheres; cells containing 10 or more microspheres were considered positive for phagocytosis, as described in Materials and Methods. As expected, the percentage of cells positive for phagocytosis significantly increased upon stimulation with both LPS (Fig 6.a) and Pam3CSK4 (Fig 6.a and 6.d). The effect of Pam3CSK4 was inhibited by pre-treatment with NDP-MSH (Fig 6.a and 6.e). However, NDP-MSH did not affect LPS-induced phagocytosis (Fig 6.a), suggesting that the pathways by which the two TLR agonists initiate microsphere uptake involve different mediators. Finally, NDP-MSH alone had no effect on microglial phagocytosis (Fig 6.a and 6.c).

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Fig 6. NDP-MSH decreases Pam3CSK4-induced microglial phagocytosis of latex beads.

Cells were treated for 24 hours with 100 nM NDP-MSH and then stimulated for another 24 hours with LPS (100 ng/ml) or Pam3CSK4 (100 ng/ml). The percentage of phagocytosis-positive cells was calculated by counting the number of CD11b+ cells containing ten or more microspheres as described in Materials and Methods. At least 10 fields were counted for each experimental group. (a) Values are the mean ± SEM of 8 independent experiments. Data were analysed by one-way ANOVA followed by Bonferroni’s multiple comparisons test. ***p<0.001 vs. control group, ^p<0.05 vs. Pam3CSK4. Representative images are shown of the following groups: (b) Control; (c) NDP-MSH; (d) Pam3CSK4; (e) NDP-MSH + Pam3CSK4. Red: Cd11b. Green: FITC-conjugated microspheres. Blue: nuclei stained with DAPI.

http://dx.doi.org/10.1371/journal.pone.0158564.g006

Discussion

Modulation of microglial activation is of major relevance in the context of inflammatory and neurodegenerative disorders in the CNS, such as AD, multiple sclerosis (MS) and ischemic damage, amongst others. In the present study we show that the synthetic α-MSH analog, NDP-MSH, inhibits some TLR2- and TLR4-triggered inflammatory mechanisms in cultured rat microglial cells and promotes development of an alternative M2-like phenotype. Targeting central MCRs present in neurons and glial cells constitutes a new approach to treating neuroinflammation. The effectiveness of selective MC4R agonists in modulating inflammatory processes plus their low toxicity strongly positions these molecules as useful instruments for treatment of CNS disorders with an inflammatory component [12].

Classically, microglial cells were classified based on morphological features often associated with distinct functional roles [28]. However, in recent years great efforts have been made to identify more specific features to help describe the extremely diverse phenotypes that microglial cells and macrophages can acquire in response to a particular set of stimuli. As a consequence, two categories have been proposed in order to describe the two main states in which these cells can be found, termed M1 and M2. The M1 category describes a population of macrophages or microglial cells that become classically activated in response to TH1 cytokines such as IFN-γ, bacterial moieties such as LPS or other TLR agonists, and whose main feature is production of proinflammatory mediators such as IL-1β, TNF-α and IL-12 [29]. The M2 category comprises a population of cells whose main functions include immunomodulation, immunosuppression and tissue repair [30]. It is in turn divided into four subcategories: M2a, M2b, M2c and M2d each possessing its own set of markers and putative predominant functions (for a comprehensive review see: [1]). The M2a profile is driven mainly by TH2 cytokines such as IL-13 and IL-4. The M2b phenotype is induced by LPS and immune complexes and the M2c by IL-10 and TGF-β, whereas the M2d is applied to tumour associated macrophages which typically display M2-like properties [31]. However, increasing evidence indicates that microglial cells are a highly plastic and dynamic population and that fitting their behaviour into one specific category is a difficult task, a fact highlighted by the great overlapping of markers between the different proposed phenotypes.

We previously showed that microglial cells express the MC4 receptor and that they respond to the melanocortin analog NDP-MSH by releasing the anti-inflammatory cytokine IL-10 and by increasing the expression of the nuclear receptor PPAR-γ [21]. Both IL-10 and PPAR-γ are—together with AG1—markers of the M2a/c alternative profiles in microglia and macrophages [1]. Here we show that upon NDP-MSH stimulation microglial cells increase the expression of Ag1, further supporting a polarizing role of melanocortins towards a M2-like phenotype. Similarly, it has been shown that α-MSH and neuropeptide Y derived from the ocular microenvironment alternatively activate resting macrophages to co-express AG1 and NOS2 [32]. Activity of AG1 leads to generation of L-ornithine and polyamines, important mediators of collagen production for tissue repair and in cell differentiation and proliferation, respectively [33], thus influencing key steps of the inflammatory response. On the other hand, NDP-MSH reduced expression of Il-4rα and did not modify that of Socs3, both markers that have been associated with the M2b/c phenotypes [29]. NDP-MSH also reduced expression of Tlr4 in microglial cells, although it did not significantly affect expression of Tlr2. TLR4 has been reported to mediate microglial activation and associated neuroinflammation [34]. Also, microglial TLR4 is required for efficient lymphocyte recruitment to the CNS in response to LPS [35]. Thus, the finding that the MC4R agonist can decrease microglial TLR4 expression suggests that melanocortins could be useful for reducing TLR4-mediated neurotoxicity in neurodegenerative diseases. Previous studies revealed an important role for HMGB1 in promoting M1 macrophage polarization [36]. In our system, no changes in Hmgb1 expression levels were found after treatment with NDP-MSH. Collectively, our data indicate that NDP-MSH polarizes microglial cells towards an alternative profile which shares features with the M2a and M2c subcategories. However, we must note that changes in mRNA expression do not necessarily reflect changes in protein expression; hence further investigation is required for better understanding the effect of NDP-MSH on protein expression of the different microglial phenotype markers.

Stimulation of TLR2 and TLR4 leads to activation of the transcription factor NF-κB. NF-κB family is composed of five subunits, p65/RelA, p50, p52, c-Rel and RelB. Upon activation, these subunits can form homo or heterodimers and translocate to the nucleus where they modulate expression of several genes involved in cell survival and in the inflammatory response [37]. In the present work we found that NDP-MSH prevented LPS-induced p65 nuclear translocation. It has been reported that α-MSH inhibits NF-κB activation in several culture systems, including monocytes [38], macrophages, neutrophils and Jurkat cells [39]. However, α-MSH was also reported to fail to prevent NF-κB activation in a glioma cell line, where its inhibitory effects were instead explained by a reduction in the LPS co-receptor CD14 [39]. In addition, previous results from our group demonstrated that α-MSH did not prevent the increase in nuclear p65 protein levels induced by LPS+IFN-γ in primary cultured rat astrocytes [22], further suggesting that this protective mechanism might be cell-specific.

Our results also show that LPS stimulation increased c-Rel immunoreactivity within the nucleus and that pre-treatment with NDP-MSH prevented this effect. c-Rel is essential for production of the IL-12p40 subunit in LPS-treated microglial cells [40] and in macrophages [41]. This subunit is shared by proinflammatory cytokines IL-12 and IL-23. IL-12 stimulates TH1 T-cell development and thereby helps to establish a nexus between innate and adaptive immunity [42]. IL-23 promotes expansion of the TH17 T-cell population, which is linked to autoimmune inflammatory disease such as MS [43]. In experimental autoimmune encephalomyelitis (EAE), an animal model of MS, treatment with α-MSH diminishes the severity of the disease [44]. Thus, specific inhibition of microglial c-Rel by NDP-MSH in an inflammatory context may prevent amplification of the immune response and could be linked to beneficial effects of melanocortins in CNS autoimmune inflammatory disease.

In our system, the effects of NDP-MSH on LPS-induced p65 and c-Rel translocation may be partially explained by the reduction in Tlr4 expression. In addition, IL-10 has been shown to inhibit LPS-induced p65 (and p50) activation in microglial cells [45]. Since we previously showed that NDP-MSH induces IL-10 release from primary cultured microglial cells [21], it is possible that this cytokine may be partially responsible for the effect of NDP-MSH on TLR4-induced p65 translocation. Our findings raise the possibility that a reduction in microglial TLR4 expression and of p65 and c-Rel activation may be linked to the neuroprotective effects of melanocortins in acute and chronic inflammation in the brain.

Nuclear translocation of p65 in response to stimulation with Pam3CSK4 was significantly increased, although not as robustly as with LPS stimulation. However, the effect of Pam3CSK4 was not prevented by NDP-MSH. This lack of effect of NDP-MSH is consistent with the absence of an inhibitory action on Tlr2 expression and may also indicate that different mechanisms are involved in the induction of p65 translocation upon TLR2 and TLR4 ligation.

Pam3CSK4 has been demonstrated to strongly induce c-Rel nuclear translocation in macrophages after 2 hours of incubation [46]. However, treatment with Pam3CSK4 did not significantly increase c-Rel immunoreactivity within the nucleus in our experimental conditions. The discrepancies between previous results and ours could be due to the different cell type involved.

TLR-induced activation of NF-κB typically results in a rapid release of the proinflammatory cytokine TNF-α, followed by a delayed production of the immunomodulatory factor IL-10, which acts as a negative modulator of the inflammatory response [47]. Our results show that pre-treatment with NDP-MSH inhibits early TLR2- and TLR4-induced TNF-α release, whereas it does not affect late IL-10 release. NF-κB subunit p65 has been proposed to mediate Pam3CSK4- and LPS-induced TNF-α production in microglial cells [48] and macrophages [10], respectively. In contrast, IL-10 production upon Pam3CSK4 stimulation has been linked to activation of the non canonical (p100/p52) pathway in monocytes [49] and of the p50/p50 homodimers in macrophages [50]. The inhibitory effect of NDP-MSH on LPS-induced TNF-α release could therefore be explained by its effect on p65 activation. However, even though NDP-MSH did not prevent Pam3CSK4-induced p65 nuclear translocation, it still decreased Pam3CSK4-induced TNF-α release. One possible explanation for this effect is that NDP-MSH may be inhibiting NF-κB transcriptional activity without inhibiting its translocation to the nucleus, a mechanism previously described for IL-10 in macrophages [51]. Another possibility is that NDP-MSH may also be affecting translocation or activation of the NF-κB p50 subunit, a molecule suggested to partner with p65 in the induction of TNF-α transcription in monocytes [52, 53].

CNS-derived IL-4 is a known inducer of the alternative M2 phenotype in microglial cells [54]. Working with the hypothesis that NDP-MSH drives microglial cells towards an alternative activation profile and knowing that microglial cells express the IL-4 receptor [55], we set out to determine whether IL-4 could be mediating the effects of NDP-MSH on microglial cells in an autocrine manner. However, we detected no IL-4 release to the culture supernatant either in the control group or in the NDP-MSH-, LPS- or Pam3CSK4-treated cells. Furthermore, NDP-MSH also failed to induce IL-4 release in mixed glial cell cultures (data not shown). Thus, we conclude that cultured rat microglial cells do not release IL-4 in the conditions tested and that the effects of NDP-MSH observed are not mediated by this cytokine. In animal models of inflammatory brain disease such as EAE, microglial cells have been proved to be an important source of IL-4 [54], suggesting that factors present in the CNS microenvironment (absent in the culture system) are essential for production of this cytokine.

HMGB1 is a highly conserved nuclear protein belonging to a group of molecules termed alarmins. Within the nucleus it acts as a regulator of DNA structure and transcription [56]. HMGB1 can be released to the extracellular milieu during traumatic cell death or upon stimulation by a number of inflammatory mediators such as TLR agonists [25]. Once outside the cell, HMGB1 can form complexes with other proinflammatory mediators such as IL-1β or LPS [57] and potentiate TLR-mediated proinflammatory effects, promote tissue damage and immune reactions [5, 25] and, as mentioned before, facilitate M1 macrophage polarization [36]. In view of these effects, reducing the amount of HMGB1 released upon TLR stimulation could be beneficial in the context of neuroinflammation. Our results showed that treatment with Pam3CSK4 induced HMGB1 nucleus-to-cytoplasm translocation (a phenomenon occurring prior to its release) and we observed that NDP-MSH pre-treatment inhibited this effect. This result suggests a protective role of the melanocortin by potentially hampering HMGB1 release, thereby preventing the delayed potentiation of the TLR-triggered inflammatory response, a possibility that requires further investigation.

Microglial activation through TLR2 and TLR4 increases phagocytosis [26, 27]. Here we show that treatment with NDP-MSH prior to stimulation with Pam3CSK4 resulted in reduced phagocytic activity compared to non-pre-treated cells. Phagocytosis in an inflammatory context has generally been considered a beneficial process due to removal of dying cells and cellular debris which ultimately facilitates the return to homeostasis. Furthermore, certain chronic neurodegenerative diseases appear to be associated with dysfunctional microglial phagocytosis [58]. However, recent evidence suggests that microglial phagocytosis in an inflammatory context may not always be desirable and may actually cause neuronal death by a mechanism termed phagoptosis [59]. In this study, the authors demonstrated that microglial activation by TLR2 and TLR4 agonists stimulates neurons to reversibly expose phosphatidyl-serine which in turn acts as an “eat me” signal for activated microglial cells and results in phagocytosis of the otherwise healthy neurons [59]. This finding suggests that prevention of TLR-induced microglial activation could also prevent neuronal death by phagoptosis in an inflammatory context. In view of these results, modulation of microglial activation by NDP-MSH could prove beneficial in neuroinflammatory disorders linked to neuron loss by phagocytosis such as brain ischemia [60]. On the other hand, NDP-MSH did not inhibit LPS-induced phagocytosis, suggesting that it is acting on a TLR2-triggered mechanism most likely not involved in TLR4-mediated phagocytosis.

To summarize, our data show that NDP-MSH prevented several proinflammatory mechanisms triggered by TLR2 and TLR4 activation and promoted development of an alternative M2-like phenotype on rat microglial cells. Our findings provide new insights into mechanisms through which melanocortins modulate microglial activation upon TLR stimulation and support microglial MCRs as potential targets in the treatment of neuroinflammatory disorders.

Acknowledgments

The authors thank Dr. Marianela Candolfi of the Institute of Biomedical Research for kindly providing us with the fluorescent microspheres.

Author Contributions

Conceived and designed the experiments: LC ML. Performed the experiments: LC DR DD JS CC. Analyzed the data: LC DR DD CC ML. Contributed reagents/materials/analysis tools: ML. Wrote the paper: LC ML.

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Recent Insights on the Role of PPAR-β/δ in Neuroinflammation and Neurodegeneration, and Its Potential Target for Therapy

NeuroMolecular Medicine volume 23, pages86–98 (2021)Cite this article

Abstract

Peroxisome proliferator-activated receptor (PPAR) β/δ belongs to the family of hormone and lipid-activated nuclear receptors, which are involved in metabolism of long-chain fatty acids, cholesterol, and sphingolipids. Similar to PPAR-α and PPAR-γ, PPAR-β/δ also acts as a transcription factor activated by dietary lipids and endogenous ligands, such as long-chain saturated and polyunsaturated fatty acids, and selected lipid metabolic products, such as eicosanoids, leukotrienes, lipoxins, and hydroxyeicosatetraenoic acids. Together with other PPARs, PPAR-β/δ displays transcriptional activity through interaction with retinoid X receptor (RXR). In general, PPARs have been shown to regulate cell differentiation, proliferation, and development and significantly modulate glucose, lipid metabolism, mitochondrial function, and biogenesis. PPAR-β/δ appears to play a special role in inflammatory processes and due to its proangiogenic and anti-/pro-carcinogenic properties, this receptor has been considered as a therapeutic target for treating metabolic syndrome, dyslipidemia, carcinogenesis, and diabetes. Until now, most studies were carried out in the peripheral organs, and despite of its presence in brain cells and in different brain regions, its role in neurodegeneration and neuroinflammation remains poorly understood. This review is intended to describe recent insights on the impact of PPAR-β/δ and its novel agonists on neuroinflammation and neurodegenerative disorders, including Alzheimer’s and Parkinson’s, Huntington’s diseases, multiple sclerosis, stroke, and traumatic injury. An important goal is to obtain new insights to better understand the dietary and pharmacological regulations of PPAR-β/δ and to find promising therapeutic strategies that could mitigate these neurological disorders.

Introduction

Peroxisome proliferator-activated receptors (PPAR) belong to the family of hormone and lipid-activated nuclear receptors, which are involved in metabolism of cholesterol, sphingolipids, and fatty acids. The transcriptional activity of PPARs is known to engage in a variety of cellular functions including cell differentiation, proliferation, and development (Hong et al. 2019). These receptors heterodimerize with retinoid X receptor (RXR), and the dimer regulates gene expression in response to dietary-derived fatty acids as well as exogenous agonists. Activation of these receptors by endogenous or exogenous ligands can evoke transduction of signals and induce interaction with lipoproteins, coactivators, or corepressors (Evans and Mangelsdorf 2014; Varga et al. 2011). PPARs not only play a role on regulating lipid metabolism and signaling, but also for maintenance of carbohydrates and glucose homeostasis.

Similar to PPAR-α and PPAR-γ in this family, PPAR-β/δ, which is also known as PPAR-δ, was cloned from the mouse genome and identified as an orphan nuclear receptor in the 90 s (Hong et al. 2019). Subsequently, two existing isoforms of this protein were identified by alternative splicing of gene NR1C2. PPAR-β/δ contains the canonical structure domains common to other nuclear receptor family members, including the amino-terminal AF-1 trans-activation domain, a DNA-binding domain, and a dimerization and ligand-binding domain with a ligand-dependent trans-activation function AF-2 at the carboxy-terminal region (Azhar 2010). The amino-terminal AF-1 trans-activation domain is responsible for transcriptional activation. It provides constitutive activation function independent of ligand binding. The DNA-binding domain (DBD, domain C), which is comprised of two zinc-finger motifs, is involved in DNA recognition and protein–protein interaction. While the hinge domain (domain D) is succeeded by the C-terminal Ligand-binding domain (LBD, domains E/F), which contains not only the ligand-binding pocket, but also regions important for dimerization and the AF-2 domain. Ligand binding is thought to induce structural changes in the AF-2 domain, allowing the recruitment of co-activator proteins important for transcriptional activation, thereby serving as a switch to activate PPARs (Brunmeir and Xu 2018). So far, only one post-translational modification for PPAR-β/δ is known. Koo and colleagues showed that PPAR-β/δ SUMOylation at K104 is removed by SUMO-Specific Protease 2 (SENP2) and this promotes the expression of FAO genes in muscle (Koo et al. 2015).

PPAR-β/δ is comprised of 441 amino acids with a molecular weight of 49.9 kDa. According to Gene Cards, this protein is widely expressed and detected in human tissues, including the brain, pancreas, liver, and heart (Hong et al. 2019). Although PPAR-β/δ is expressed in cells in all brain regions, neurons appear to have the highest expression. Warden et al. (2016) demonstrated localization of PPAR isotypes in the adult mouse and human brain (Fig. 1). Using quantitative PCR and double immunofluorescence microscopy, investigation among brain parts indicated highest level of mRNA and proteins in the prefrontal cortex (Warden et al. 2016). In the brain, although all PPAR isoforms have been detected in neuronal and astrocytes, PPAR-β/δ appeared to have low immunoreactivity in microglia as compared with other PPARs members. Analysis of subcellular localization indicated that PPAR-β/δ in neurons is present both in the cytoplasm and nucleus. Nevertheless, its intracellular localization may change depending on patho-physiological conditions and applied therapy (Gamdzyk et al. 2018).

Fig. 1

PPAR-β/δ expression in different brain parts and cells. PFC prefrontal cortex, NAC nucleus accumbens, AMY amygdala, VTA ventral tegmental area; ***, **, * level of PPAR-β/δ expression; on the basis of data described by Warden et al. (2016), Schnegg and Robbins (2011), Carnigila et al. (2013)

Until recently, studies on the role of PPAR-β/δ were largely carried out with peripheral organs/tissue (Phua et al. 2020). Its expression is detected at early stage of embryogenesis, and disruption of this gene is lethal due to severe placental defects. The knockout animals are characterized by alterations of skin and fat mass, and impairment of brain development. PPAR-β/δ seems to play a key role in embryo development, and its deletion can induce a high rate of mortality around embryonic day 10.5 (E10.5) (Hall et al. 2008; Nadra et al. 2006). At this time of development, the expression of PPAR-β/δ could be detected in all brain regions, including the cerebral cortex, thalamus, cerebellum and brainstem, and reaching peak levels between E 13.5 and E 15.5 (Gofflot et al. 2007; Braissant and Wahli 1998). The expression of PPAR-β/δ was found in neurons, astrocytes, oligodendrocytes, and recently, also in microglia cells (Schnegg and Robbins 2011; Carniglia et al. 2013). In addition, this receptor is also expressed in brain capillary endothelial cells, suggesting an involvement in regulation of blood/brain barrier (Akanuma et al. 2008). Studies using genetically modified PPAR-β/δ null mice indicated changes in brain weight, and concomitantly, the body weight was also smaller as compared to the wide-type control (Peters et al. 2000). Histological study showed disturbances in myelination in the corpus callosum, more frequently in females comparing to males (Markham et al. 2009).

Role of PPAR-δ in Lipid Metabolism and Signaling Pathways

During the past decade, most studies on PPAR-β/δ were carried out with muscle and other peripheral tissue/organs, and relatively few studies were carried out with the brain (Grimaldi 2007; Phua et al. 2020; Wang et al. 2020). The study by Rosenberger et al. (2002) showed significant alterations of phospholipids and esterified fatty acids, together with gender differences in the brain of PPAR-β/δ null mice. Results with PPAR-β/δ null mice also showed defects in brain peroxisomal acyl-CoA utilization and thus projected a role in myelination. PPAR-β/δ also can influence genes engaged in enzymes responsible for fatty acid β oxidation pathway in mitochondria and peroxisome (Grimaldi 2007; Lamichane et al. 2018). Information in Fig. 2 demonstrates the potential roles of PPAR-β/δ in lipid metabolism in the brain. In many instances, these roles are comparable to those demonstrated in hepatocytes and in some tumor cells (Beyaz and Yilmaz 2016).

Fig. 2

The role of PPAR-β/δ in lipid metabolism. FA fatty acids, FAβO fatty acids-β oxidation, FATP fatty acids transport protein, FABP fatty acids binding protein, CPT1A carnitine palmitoyltransferase I, RXR retinoid X receptor, ATP adenosine triphosphate, TCA tricarboxylic acid, acetyl-CoA acetyl coenzyme A, LCFA long-chain fatty acids, CXCL1,2,10 chemokines 1,2,10, iNOS nitric oxide synthases (inducible form), TNF-α tumor necrosis factor α, IFNγ interferon gamma, IL1,6,10 interleukin 1,2,10, NFκB nuclear factor kappa-light-chain-enhancer of activated B cells

PPAR-β/δ can alter brain membrane phospholipids, through post-translational modification via the acylation process. This process may lead to changes in protein functions, such as the myelin proteolipid proteins (PLP) (Campagnoni and Macklin 1988). On the other hand, activation of PPAR-β/δ by long-chain saturated and unsaturated fatty acids (LCSFA, LCUFA) and their metabolites may lead to regulation/modulation of transcription of genes encoding proteins such as fatty acid binding proteins (FABP) and fatty acid translocase (FAT).

PPAR-β/δ is also engaged in regulation of cholesterol release and metabolism. However, despite that adult CNS contains 23% of the total sterol pool in the entire body, little information is available regarding this receptor and cholesterol in the brain (Dietschy and Turtley 2004). Cholesterol is an important constituent of the plasma membrane and is the major component of myelin in adult human brain where it consists 70–80% of the whole brain cholesterol. In human adult brain, cholesterol level reaches 23 mg/g w.bw, however, at birth only 6 mg/g bw and in adult mouse brain about 18 mg/g bw. With participation of apolipoprotein E and A, PPAR-β/δ may alter cholesterol metabolism in the brain, and exert effects on neural and glial cells differentiation. However, the relationship between cholesterol metabolism, PPARs, and neurodegeneration/neuroinflammation is till now not fully elucidated.

In the peripheral system, PPAR-β/δ agonists have been proposed for treatment of metabolic syndrome (MetSD) which is tightly connected with long-chain fatty acid (LCFA) homeostasis (Varga et al. 2011). Nevertheless, molecular sequence of events leading to imbalance of lipid homeostasis is still not well understood. It is suggested that higher levels of circulating LCFA and their availability can induce fat accumulation in adipose tissue, liver, and other tissues, leading to insulin resistance and DMT2. Activation of PPAR-β/δ in animal model leads to improvement of lipid homeostasis and insulin sensitivity (Tanaka et al. 2003). There is evidence that LCFA signaling is mediated by PPAR- β/δ, which also plays a crucial role in lipid absorption and intestinal physiology. Due to the proangiogenic and pro-/anti-carcinogenic properties of PPAR-β/δ ligands, these compounds may serve as therapeutic agents for treating metabolic syndrome, dyslipidemia, and diabetes (Bishop-Bailey and Swales 2008).

PPAR-β/δ plays a crucial role in diseases associated with alterations of lipid and glucose metabolism, including MetSD, DMT2, and atherosclerosis. MetSD is a complex pathological condition together with dyslipidemia, hyperglycemia, central obesity, and hypertension, many are associated with prothrombotic and proinflammatory state. The study by Serrano-Marco et al. (2011) suggested that PPAR-β/δ activation could impede IL6-induced STAT3 activation by inhibition of ERK1/2 and prevention of STAT3 association with Hsp90. This effect may contribute to the suppression of cytokine-induced insulin resistance in adipocytes and possibly may also occur in the brain. Obviously, more studies are needed to better understand the role of this receptor in neurodegenerative and neuroinflammatory diseases.

Role of PPAR-β/δ in Oxidative Stress and Neuroinflammation

PPARs are known to modulate inflammatory processes associated with lipid signaling pathways. Suppressing inflammatory processes in the CNS could lead to reduction of brain damage and improvement of motor and cognitive outcome (Villapol 2018). Resident microglia and infiltrated inflammatory cells were regarded as mediators responsible for this process (Salvi et al. 2017).

PPAR-β/δ is not only a lipid sensor, but also a regulator of mitochondrial function, and may influence oxidative stress and inflammation in brain cells as well as proliferation and angiogenesis in vascular endothelial cells (Bishop-Bailey and Swales 2008). There is evidence that PPAR-β/δ regulates vascular function by enhancing VEGFR expression, phosphorylation of AKT, and subsequently regulating endothelial NO production and reducing ROS and inflammation (Jiang et al. 2019).

Systemic inflammatory responses (SIR) evoked by the endotoxin lipopolysaccharide (LPS) may contribute to neurodegenerative disorders (Brown 2019). PPARs are unique set of fatty acid regulated transcription factors controlling both inflammation and lipid metabolism (Varga et al. 2011; Schnegg and Robbins 2011). It was recently reported that PPAR-β/δ agonists exerted significant anti-inflammatory effects and suppressed the genes encoding iNOS, several chemokines such as CXCL1, CXCL2, CXCL10 interleukins IL1, IL6, and other cytokines TNF-α, IFN-γ, and concomitantly enhanced IL10 (Kuang et al. 2012; Chehaibi et al. 2017; Beyaz and Yilmaz 2016). Agonist of PPAR β/δ such as GWO 742 could decrease neutrophil infiltration into the brain during ischemia and protects against neuroinflammation (Chehaibi et al 2017). However, activation of other members of PPARs evoked also anti-inflammatory effect (Varga et al. 2011; Carniglia et al. 2013; Villapol 2018).

In our previous studies, acute SIR evoked by LPS administered i.p. to mice-induced memory impairment showed alterations of transcription of pro-oxidative, inflammatory genes, and genes engaged in cells death signaling (Czapski et al. 2010; 2016; Jacewicz et al. 2009). During the last decade, there has been increasing interest on the involvement of PPAR-β/δ in inflammatory processes (Bishop-Bailey and Bystrom 2009; Piqueraset al 2009; Schnegg and Robbins 2011).

PPAR-β/δ in AD and Other Neurodegenerative Disorders
Alzheimer’s Disease (AD)

Alzheimer’s disease (AD) is the most prevalent, progressive, and irreversible neurodegenerative disease that leads to dementia. There are many underlying mechanisms towards the pathogenesis of AD, including the widely known amyloid pathogenesis with liberation and oligomerization of amyloid beta peptides (Aβ), and hyperphosphorylation of the microtubule-associated tau protein, and its polymerization into insoluble, neuronal fibrillary tangles (NFTs). These alterations lead to astrocytes and microglia cells activation, and consequently, inflammatory response (Selkoe and Hardy 2016). On the cellular level, alterations of mitochondrial activity/ function and increase of oxidative stress may play a crucial role in AD pathogenesis (Tiwari et al. 2019; Schmitt et al. 2012; Swerdlow 2018). Recent studies further indicated and suggested that abnormal sphingolipids, phospholipids, and fatty acids metabolism could be early and key events in the pathogenesis of AD (Kunkle et al. 2019; Cuyvers and Sleegers 2016; Jęśko et al. 2019a, b; Picard et al. 2018). Among these lipids, fatty acids and their metabolites through specific receptors and PPARs signaling are engaged in regulation of brain function, learning and memory. Recent studies have described the essential role of PPAR-α in regulation of lipid metabolism, neuronal function, synaptic plasticity, and cognition (Wójtowicz et al. 2020; Sáez-Orellana et al. 2020). Due to the complexity of AD pathophysiology, there is advantage for testing agonists that target different isoforms of PPARs (Reich et al. 2019).

Previous studies have shown that downregulation of PPAR-β/δ could be linked to both neuroinflammation and insulin resistance in the brain (de la Monte and Wands 2006). Alzheimer’s disease is often regarded as a brain form of diabetes, and insulin deficiency or resistance to insulin may lead to neurodegeneration (Tong et al. 2016a, b). Insulin plays a fundamental role in regulating Extracellular Signal-Regulated Kinases (ERK), which are essential for learning and memory, and are compromised in early AD (Dineley et al. 2014). Therefore, maintaining the action of insulin in the brain could potentially restore brain function and reduce neurodegeneration (Tong et al. 2016a, b; Jęśko et al. 2019a, b). PPARs are known to modulate insulin-stimulated gene expression, by responding to signals that are transmitted from surface cells membranes (Collino et al. 2008). As compared to other PPARs, PPAR-β/δ seems to be most expressed in the brain (Cimini et al. 2005), and expression of PPAR-β/δ was reduced in the brains of AD patients similar as PPAR-α but the expression of gene for PPAR-γ was selectively upregulated (de la Monte and Wands 2006) (Fig. 3).

Fig. 3

PPAR-β/δ engagement in neurological disorders. AD-Alzheimer’s disease, PD Parkinson’s disease, HD Huntington’s disease, MS multiplex sclerosis, ALS amyotrophic lateral sclerosis

A new PPAR-δ/γ agonist (T3D-959) with 15-fold higher PPAR-β/δ selectivity/potency (comparing to PPAR-γ) is in an exploratory phase II clinical trial on thirty-four mild-to-moderate AD patients. (Chamberlain et al. 2020). Due to PPAR-β/δ, PPAR-γ activation this agonist might have synergistic/ additive effects on glucose metabolism and regulation of glucose homeostasis in the brain (Chamberlain et al. 2020). In a previous study, T3D-959 administration was shown to significantly improve motor functions and normalize structure of white matter in streptozotocin (STZ)-induced animal model of sporadic AD (intra-cerebrally injected STZ). The data also showed good blood–brain barrier penetration, good therapeutic index, and high brain concentration for this compound (Tong et al. 2016a, b). This compound also effectively restored integrity of temporal lobe, hippocampal structure, and IGF-1 sensitivity and inhibited neuroinflammation (de la Monte et al 2017; Malm et al. 2015; Tong et al. 2016a, b). Results from the latest phase of the study showed that T3D-959 is generally safe and well tolerated by AD patients (Tong et al. 2016a, b). Plasma metabolome profile indicated dose-related systemic effects on insulin-related metabolism. Moreover, relative FDG-PET imaging displayed regional, dose-dependent effects of this compound on cerebral metabolic rate of glucose. Studies on cognitive assessments (ADAS-cog11 and DSST) indicated improvements with possible pharmacodynamics related to T3D-959 mechanism of action. Due to the encouraging results of the phase II clinical trial, this drug warrants further investigation in a larger clinical study with a proper placebo-controlled group (Chamberlain et al. 2020).

The insulin sensitizing action of PPAR-β/δ is probably not the only event with possible impact on AD. As mentioned before, PPAR-β/δ has a potent anti-inflammatory effect and it can stabilize myelin sheath, decline Aβ deposits, as well as exert other molecular effects (Collino et al. 2008; Dunn et al. 2010; Sergey et al. 2009) (Fig. 4). Moreover, experimental depletion of PPAR-β/δ indicated not only increases in neuroinflammation, but also oxidative stress, astrogliosis, and Aβ42 deposition (Barroso et al. 2013).

Fig. 4

Potential PPAR-β/δ mechanisms of action, constructive in Alzheimer’s disease

In a transgenic model of AD (5XFAD mice), the PPAR-β/δ agonist GW0742 could decrease parenchymal Aβ deposits, although intraneuronal Aβ was not affected (Malm et al. 2015). The results of this study showed that this agonist not only significantly decreased Aβ load in the cerebral cortex and hippocampus, but also decreased the level of several cytokines (IL1, IL6, CCL2, and TNF-α) and microglial activity surrounding Aβ deposits. The action of GW0742 was also analyzed in hippocampus of mice with Aβ1-42-induced neurotoxicity (An et al. 2016). Administration of aggregated oligomer of Aβ1-42 (410 pmol/mouse) greatly disrupted memory and learning (in Morris Water Maze and Y-maze tests). This perturbation was associated with decreased expression of PPAR-β/δ in the mouse hippocampus (An et al. 2016). Intra-hippocampal infusion of GW0742 could also reverse the decreased expression of hippocampal PPAR-β/δ, repressed neuroinflammation and apoptotic responses triggered by Aβ1-42 oligomers, and enhanced Bcl2/Bax ratio in hippocampus (An et al. 2016).

PPAR-β/δ and other members of these receptor family are involved in neuroinflammation processes in AD as well as other neurodegenerative disorders. The study of Sergey et al. (2009) showed that the PPAR-β/δ agonist, GW0742 could significantly reduce astrocyte activation, thus exerting anti-inflammatory effect on glial cells. These authors also reported that PPAR-β/δ agonist could reduce amyloid burden, an event presumably mediated by its effect on amyloid clearance.

Parkinson’s Disease

There is evidence for protective properties of PPAR-β/δ agonists in Parkinson disease (PD) (Chaturwedi and Beal 2008). The study by Iwashita et al. (2007) demonstrated that PPAR-β/δ agonists GW501516 and L165041 exhibited protective function against striatal dopamine depletion induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). These agonists also inhibited caspase-3 activation, thereby protecting (SHSY-5Y) neuronal cells from action of MPP + (1-methyl-4-phenylpyridinium) and exerted protective effects also in in vivo model of PD. Moreover, the study of Das et al. (2014) demonstrated the effect of PPAR-β/δ agonist—GW0742—in a rat model of PD-associated cognitive impairment. In this study, rats given MPTP resulted in DNA fragmentation and oxidative stress. Subsequent treatment with GW0742 was shown to partially restore cognitive functions impaired by MPTP. Immunochemical (Tunel) assay, and assays of glutathione (GSH) and malondialdehyde (MDA) revealed that GW0742 reduced oxidative stress and DNA fragmentation. In a recent study, intracerebroventricular administration of GW501516, a highly selective agonist for PPAR-β/δ was shown to exert protective effects in PD model induced in mice by MPTP (Chen et al. 2019). In this study, GW501516 not only reduced the movement impairment in the PD mice, it also suppressed dopaminergic neurodegeneration and inhibited activation of the nucleotide-binding domain and leucine-rich-repeat-protein 3 (NLRP3) inflammasome in the astrocytes but not microglia.

Huntington’s Disease

The study of Dickey et al. (2016) documented that PPAR-β/δ-mediated transcriptional alteration could involve mitochondrial abnormalities and bioenergetic defects in Huntington Disease (HD). The study showed that PPAR-β/δ dysregulation is crucial in the pathogenic cascade of HD and it could elicit neuroprotection in neurons from mouse models of HD. Moreover, treatment with its selective agonist evoked a robust positive response. Through testing PPAR (α, γ and β/δ) individually, and with the use of agonist treatment or shRNA knockdown, this study confirmed the important role of PPAR-β/δ in HD. Experiments on transgenic PPAR-β/δ mice revealed the necessity of PPAR-β/δ for neuronal function (Dickey et al. 2016).

Multiple Sclerosis

The role of PPAR-β/δ has been studied in Multiple Sclerosis (MS), a disease involving demyelination of central nervous system and affecting nearly one million people worldwide (Dean et al. 1994; Lucchinetti et al. 2011). MS is known for deficiencies in sensory and motor areas, resulting probably from autoimmune mechanism. MS patients also showed changes in plasma lipid profiles, implicating the role of lipids in MS pathogenesis (Weinstock-Guttman et al. 2011). The most widely used model for studies on MS is Experimental autoimmune encephalomyelitis (EAE) (Constantinescu et al. 2011). Studies showed that PPAR-β/δ agonists, through a negative feedback loop, could reduce inflammation and damage of tissues in EAE models of MS (Polak et al. 2005). The study by Drohomyrecky et al. (2019) also demonstrated that mutant mice hypomorphic for PPAR-β/δ receptor showed a more severe course of inflammatory process in CNS, and this event could be revered by PPAR-β/δ agonists (Drohomyrecky et al. 2019).

PPAR-β/δ in CNS Hypoxia/Ischemia

Neurodegeneration and neuroinflammatory processes play a significant role in brain ischemia/hypoxia pathology. Alteration of lipid metabolism, including polyunsaturated fatty acids and synthesis of several eicosanoids and docosahexanoids were recognized as the early and most important events in ischemia/hypoxia encephalophathy (Bazan 1970; Tang and Sun 1985; Strosznajder and Domanska-Janik 1980; Nalivaeva and Rybnikova 2019). Omega 3 fatty acids supplementation was shown to exert protection via anti-inflammatory action by suppressing microglia response in neonatal hypoxic-ischemic brain injury (Zhang et al. 2010). Study by Saganuma et al. (2013) also demonstrated that docosahexaenoic acid (DHA) supplementation may be beneficial in ischemia hypoxia encephalopathy. In a rodent model of brain ischemia, the data by Song et al. (2019) showed that oleic acid (OA) could mediate neuroprotection through PPAR-γ activation and its anti-inflammatory effect.

Using GW0742, a specific agonist for PPAR-β/δ receptor, the study by Gamdzyk et al. (2018) demonstrated that stimulation of PPAR-β/δ could exert neuroprotective effects in a rat model of neonatal hypoxic–ischemia (HI). In this study, administration of GW0742 reduced brain infarct area, brain atrophy, apoptosis, and improved neurological function at 72 h and 4 weeks post HI. Additionally, GW0742 administration induced several molecular processes, e.g., enhancing the transcription of gene coding PPAR-β/δ, increase in miR-17-5p level, and downregulation of the Thioredoxin Interacting Protein (TXNIP) in the ipsilateral hemisphere. These events also led to inhibition of the Apoptosis Signal-regulating Kinase 1 (ASK1/p38) signaling pathway and reduced apoptotic cell death. In contrary, GSK3787, an antagonist of this receptor, was shown to reverse the protective effects evoked by intranasal administration of GW0742. The study of Hack et al. (2012) and Zaveri et al (2009) demonstrated the effect of PPAR-β/δ antagonists GSK3787, GSK0660, and SR13904, respectively.

In the recent review article, Gamdzyk et al. (2020) compared neuroprotective efficacy of PPAR-β/ δ agonists to PPAR-α and PPAR-γ and conclude that despite of being the most highly expressed in CNS, the available data on the effect of this receptor agonists in stroke as well as other neurological disorders are relatively poor and thus needing further investigations. However, the last results of Chehaibi et al. (2017) demonstrated several ameliorating effects of PPAR-β/δ agonist GW0742 in mice brain ischemia evoked by occlusion of middle cerebral artery (MCA). The significant anti-inflammatory effect was exerted by this agonist, which decreased the neutrophil infiltration, the level of several chemokines and interleukins such as IL-1β, IL6, and other cytokines. Using the same model of brain ischemia. Pialat et al. (2007) previously demonstrated in magnetic resonance imaging (MRI) scan that PPAR-β/δ-null mice comparing to control wild mice indicated significant differences in lesion volume. The effect of GW0742 was also investigated by Tang et al. (2020) in a collagenase-induced intracerebral hemorrhage (ICH) mouse model. In this study, PPAR-β/δ agonist was administered (intraperitoneally in a dose of 3 mg/kg body weight) 30 min before ICH, and its neuroprotective effects included mitigation of behavioral dysfunction and molecular pathways associated with activation of inflammation and apoptosis. A previous study by Paterniti et al. (2010) evaluated the involvement of PPAR-β/δ in spinal cord injury (SCI) in mice evoked by application of vascular clips (force of 24 g) to the dura via a four-level T5 to T8 laminectomy. GW0742 (administered i.p. in a dose of 3 mg/kg body weight) exerted significant neuroprotective effect, and ameliorated the recovery of limb function. The protective effects of this agonist include inhibition of neutrophil infiltration, expression of proinflammatory cytokines and altered molecular processes leading to cell death through changes of transcription of pro- and anti-apoptotic proteins (Fasl, Bax, Bcl2). The protective processes evoked by GW0742 could be eliminated by specific receptor antagonist (GSK0660), which was administered (1 mg/kg bw) at 30 min before GW0742. This high-affinity PPAR-β/δ agonist GW0742 was able to evoke significant neuroprotective effects in secondary damage, during experimental spinal cord injury (SCI) in mice (Paterniti et al. 2010). In this study, GW0742 treatment (0.3 mg/kg−1 i.p) at 1 and 6 h after SCI, significantly reduced inflammation, nitric oxide synthesis, nitrotyrosine formation and activation of apoptotic signaling. Moreover, this agonist protected against edema and showed positive effect on motor recovery score. The study of Esposito et al. (2012) indicated that GW0742, through targeting divergent downstream pathways regulating PPAR-β/δ receptors, could decrease changes on both molecular and cellular levels that take place in spinal cord damage. CNS hypoxia–ischemia hemorrhage and traumatic injury are closely connected with vascular alterations, oxidative stress and hypertension. In a preclinical study, Toral et al. (2016) showed antihypertensive effects of PPAR-β/δ in spontaneously hypertensive rats (SHR) as well as in other animal models. Pharmacological activation of PPAR-β/δ exerted several protective effects, improved the endothelial dysfunction, decreased vascular inflammation and vasoconstriction responses. There is evidence that other isoforms of PPARs can also show protective effects on cerebral ischemia damage. In a study by Wu et al. (2016), cultured neurons were subjected to in vitro oxygen–glucose deprivation (OGD), and treatment with GW9662, an antagonist for PPAR-γ, could ameliorate neuronal apoptosis and inhibit p22phox subunit of NADPH oxidase (Wu et al. 2016). It is possible to suggest that agonist acting simultaneously on PPAR-γ and PPAR-β/δ could be more effective in OGD model and in brain ischemia pathology comparing to PPAR-γ alone. Despite of evidence indicating ability for PPAR-β/δ agonists to exert neuroprotective effects on cerebral ischemia injury, there were also negative results, probably depending on the type of agonists used and method of administration. For example, in a study by Knauss et al. (2018) oral administration with SAR 145, a known lipophilic agonist for PPAR-β/δ, could not improve short or long outcomes after focal cerebral ischemia induced to mice through middle cerebral artery occlusion (Knauss et al. 2018).

PPAR-β/δ in Brain Tumors (Neuroblastomas and Gliomas)

Despite of the recognition of PPAR-β/δ in metabolic and inflammatory diseases, there is increasing interest in developing appropriate ligands/antagonists towards treatment of cancer (Wagner and Wagner 2020; Reil and Lee 2008; Liu et al. 2018). However, the molecular mechanism of PPARs in carcinogenesis is still not fully elucidated, and data from in vitro and in vivo studies are still controversial. Tatenhorst et al. (2008) concluded in their review articles that the agonists of PPARs could be promising for new approaches in human CNS tumor therapy. Subsequently, Youssef and Badr (2011) tried to explain the complexity of these receptor responses and their conformational changes that influence their ability to recruit specific functionally distinct coactivators. For better understanding of the complicated role of PPAR-β/δ in carcinogenesis, these authors recalled the work of Mukherjee et al. (1994), showing that some receptors (such as androgen receptors) exhibited capability to interact with 150 proteins/polypeptides, and thus suggested such a possibility for PPAR in carcinogenesis. It also seems that, in the case of PPAR-β/δ, the complex coactivators and repressors in PPAR-β/δ could be subjected to deeper analysis. Recent studies of Yao et al. (2017) showed that PPAR-β/δ could inhibit human neuroblastoma cell tumorigenesis by inducing protein p-53 and SOX2 mediated cell differentiation. These results suggest that combinatorial activation of retinoic acid receptor, PRAR-α and PPAR-β/δ may be promising therapeutic approach for RA-resistant neuroblastoma patients. Ding et al. (2020) demonstrated the impact of PPAR-β/δ and PPAR-γ polymorphism on glioma risk and prognosis in the Chinese Han population.

Summary and Perspective

Considering their anti-inflammatory, neuroprotective, and anti-tumors properties, PPAR-β/δ agonists are promising treatments of AD and other neurodegenerative disorders. A list of the natural and synthetic agonists for PPAR-β/δ is shown in Table 1. These PPAR-β/δ ligands should be applied in various other pathologies as DMT2, MetSD, atherosclerosis, obesity, hepatosteatosis. The role of PPAR-β/δ in cancer should be better elucidated and understood. Besides PPAR-β/δ, agonists of PPAR-α and PPAR-γ may also be involve in neurodegenerative diseases, in MetSD and dyslipidemia. Therefore, future studies should test PPAR-β/δ ligands in combination with ligands of other PPARs receptor in these neurological disorders and in inflammation.

Table 1 Natural and synthetic agonists of PPAR-β/δ
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