doi:10.1093/brain/aws058
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BRAIN
A JOURNAL OF NEUROLOGY
Licensing of myeloid cells promotes central
nervous system autoimmunity and is controlled
by peroxisome proliferator-activated receptor c
Stephanie Hucke,1,* Juliane Floßdorf,1,* Berit Grützke,1 Ildiko R. Dunay,2 Kathrin Frenzel,2
Johannes Jungverdorben,3 Bettina Linnartz,3 Matthias Mack,4 Michael Peitz,3 Oliver Brüstle,3
Christian Kurts,1 Thomas Klockgether,5 Harald Neumann,3 Marco Prinz,2 Heinz Wiendl,6
Percy Knolle1,† and Luisa Klotz5,6,†
1
2
3
4
5
6
Institutes of Molecular Medicine and Experimental Immunology, University of Bonn, Bonn, Germany
Department of Neuropathology, University Hospital & BIOSS Center for Biological Signalling Studies, University of Freiburg, Freiburg, Germany
Institute of Reconstructive Neurobiology, LIFE and BRAIN Centre, University of Bonn and Hertie Foundation, Bonn, Germany
Department of Internal Medicine, University of Regensburg, Regensburg, Germany
Clinic for Neurology, University of Bonn, Bonn, Germany
Clinic for Neurology, University of Münster, Münster, Germany
*These authors contributed equally to this work.
†These authors contributed equally to this work.
Correspondence to: Luisa Klotz, MD,
Clinic for Neurology,
University of Münster,
Albert Schweitzer Campus 1; building A10, 48149 Münster, Germany
E-mail: [email protected]
Correspondence may also be addressed to: Percy A. Knolle, MD, Institutes of Molecular Medicine and Experimental Immunology, University of Bonn,
Sigmund-Freud-Str. 25, 53105 Bonn, Germany. E-mail: [email protected]
During central nervous system autoimmunity, interactions between infiltrating immune cells and brain-resident cells are critical
for disease progression and ultimately organ damage. Here, we demonstrate that local cross-talk between invading autoreactive
T cells and auto-antigen-presenting myeloid cells within the central nervous system results in myeloid cell activation, which is
crucial for disease progression during experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. This
T cell-mediated licensing of central nervous system myeloid cells triggered astrocytic CCL2-release and promoted recruitment of
inflammatory CCR2 + -monocytes, which are the main effectors of disease progression. By employing a cell-specific knockout
model, we identify the nuclear receptor peroxisome proliferator-activated receptor c (PPARc) in myeloid cells as key regulator of
their disease-determining interactions with autoreactive T cells and brain-resident cells, respectively. LysM-PPARcKO mice exhibited disease exacerbation during the effector phase of experimental autoimmune encephalomyelitis characterized by
enhanced activation of central nervous system myeloid cells accompanied by pronounced local CCL2 production and inflammatory monocyte invasion, which finally resulted in increased demyelination and neuronal damage. Pharmacological PPARc
activation decreased antigen-specific T cell-mediated licensing of central nervous system myeloid cells, reduced myeloid
cell-mediated neurotoxicity and hence dampened central nervous system autoimmunity. Importantly, human monocytes derived
from patients with multiple sclerosis clearly responded to PPARc-mediated control of proinflammatory activation and production
of neurotoxic mediators. Furthermore, PPARc in human monocytes restricted their capacity to activate human astrocytes leading
Received May 4, 2011. Revised December 20, 2011. Accepted December 30, 2011. Advance Access publication March 24, 2012
ß The Author (2012). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: [email protected]
T cell-licensed myeloid cells drive EAE
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to dampened astrocytic CCL2 production. Together, interference with the disease-promoting cross-talk between central nervous
system myeloid cells, autoreactive T cells and brain-resident cells represents a novel therapeutic approach that limits disease
progression and lesion development during ongoing central nervous system autoimmunity.
Keywords: EAE; multiple sclerosis; inflammatory monocytes; PPAR
Abbreviations: EAE = experimental autoimmune encephalomyelitis; ELISA = enzyme-linked immunosorbent assay; KO = knockout;
LPS = lipopolysaccharide; MOG = myelin oligodendrocyte glycoprotein; PPAR = peroxisome proliferator-activated receptor ;
TNF = tumour necrosis factor ; WT = wild-type
Introduction
The central role of autoreactive encephalitogenic CD4 + T cells has
clearly been demonstrated in the animal model of multiple sclerosis experimental autoimmune encephalomyelitis (EAE) as CNS
autoimmunity can be elicited by adoptive transfer of autoreactive
myelin oligodendrocyte glycoprotein (MOG)-specific CD4 + T cells
(Stinissen et al., 1998). These autoreactive CD4 + T cells are
primed in the periphery within lymphatic tissues, migrate across
the blood–brain barrier, and initiate autoimmune inflammation
upon local reactivation by antigen-presenting cells (Reboldi
et al., 2009). Besides encephalitogenic T cells, myeloid cells are
recruited into the CNS and can outnumber lymphocytes in acute
multiple sclerosis lesions by at least 10–20 times (Prineas and
Graham, 1981; Barnett et al., 2006). During the last years, it
became evident that CNS-infiltrating myeloid cells substantially
contribute to local inflammatory processes as well as demyelination and axonal damage in multiple sclerosis and EAE
(Geissmann et al., 2003; Lassmann, 2004; Mildner et al., 2009).
Recent studies further revealed that the subpopulation of inflammatory CCR2 + Ly-6Chi monocytes (Geissmann et al., 2003) amplifies the effector phase of CNS autoimmunity (Mildner et al., 2009)
and that in vivo depletion of this subpopulation reduces disease
severity (Tran et al., 1998). Moreover, CNS-resident microglial
cells can also contribute to CNS inflammation during EAE
(Heppner et al., 2005). However, the mechanisms driving myeloid
cell activation during sterile autoimmunity induced by autoreactive
T cells have not been resolved. Similarly, the cell-intrinsic factors
that regulate responsiveness of myeloid cells towards such activation remained elusive.
A potent regulator of macrophage activation is the nuclear
receptor peroxisome proliferator-activated receptor (PPAR),
a ligand-activated transcription factor that mediates a broad
range of anti-inflammatory effects (Ricote et al., 1998; Clark
et al., 2000; Storer et al., 2005; Klotz et al., 2007). PPAR
in myeloid cells acts by interference with NFB-target genes
through a transrepression mechanism mediated by ligand-induced
stabilization of the NCoR co-repressor complex at the promoter
sites of pro-inflammatory genes such as inducible nitric
oxide synthase or tumour necrosis factor (TNF) (Glass and
Ogawa, 2006; Straus and Glass, 2007).
Interestingly, peripheral blood mononuclear cells from patients
with multiple sclerosis exhibited strongly decreased PPAR expression levels when compared with healthy controls (Klotz et al.,
2005). As we have previously shown that ablation of PPAR
expression in dendritic cells increases their capacity to prime naı̈ve
CD4 + T cells (Klotz et al., 2007), we reasoned that PPAR may
serve as a cell-intrinsic regulator of myeloid cell activity, and hence,
that diminished PPAR activity in myeloid cells may critically contribute to CNS autoimmunity. In this study, we therefore addressed the
question whether PPAR limits myeloid cell activation during CNS
autoimmunity and thus restricts local innate immune responses
during autoimmune inflammation in the CNS.
Materials and methods
Mice
Mice were bred and maintained under specific pathogen-free conditions at the Haus für Experimentelle Therapie, University of Bonn and
used for experiments at 7–12 weeks of age. C57BL/6 mice and
congenic C57BL/6 (CD45.1) mice were purchased from Charles
River Laboratories. PPAR fl/fl mice were obtained from Jackson
Laboratories. Mice with a conditional PPAR knockout in myeloid
cells (i.e. LysM-PPAR KO mice) were generated by crossing
PPAR fl/fl mice with LysM-Cre transgenic mice, which express the
Cre recombinase under control of the murine M lysozyme gene in
monocytes/macrophages, microglia and neutrophils (Clausen et al.,
1999) (Supplementary Fig. 1). To analyse recombination efficiency,
LysM-Cre mice were crossed to R26-eYFPfl/wt mice (Fig. 1A and
Supplementary Fig. 1A). Mice with a conditional knockout of PPAR
in CNS-resident cells of neuroectodermal origin, i.e. neurons, astrocytes and oligodendrocytes, were generated by crossing PPAR fl/fl
mice with Nestin-Cre transgenic mice (Tronche et al., 1999)
(Supplementary Fig. 3). Lines were backcrossed onto the C57BL/6
background for at least 10 generations. Genotyping of mice was performed by amplifying a portion of the particular gene from tail DNA.
50 -TGTAATGGAAGG-30 and 50 -TGGCTTCCAGTGCATAAGTT-30 primers were used to detect truncated or wild-type form of PPAR
gene in PPAR fl/fl mice. Genotyping of Cre recombinase in LysMPPAR KO mice was performed using the primers: 50 -CTTG
GGCTGCCAGAATTTCTC-30 , 50 -TTACAGTCGGCCAGGCTGAC-30 and
50 -CCCAGAAATGCCAGATTACG-30 . Nestin-Cre genotyping was performed using the primers 50 -TCCATGAGTGAACGAACCTGGTCG-30
and 50 -TCCCTTCTCTAG-TGCTCCACGTCC-30 . In some experiments,
MHC-II-restricted T cell receptor-transgenic 2D2 mice specific for
the myelin oligodendrocyte glycoprotein35–55 (MOG35–55) peptide
and ovalbumin (OVA)-specific T cell receptor-transgenic OT-II mice
were used (Bettelli et al., 2003). All animal experiments were
performed according to the guidelines of the animal ethics committee and were approved by the government authorities of
Nordrhein-Westfalen, Germany.
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Figure 1 PPAR in myeloid cells modulates the effector phase of EAE. (A) LysM-Cre mice were crossed to R26-eYFPfl/wt reporter mice to
analyse successful recombination i.e. YFP expression, by flow cytometry in different immune cell populations. Graph shows the recombination efficiency in the named immune cell populations of n = 3 cre + mice. See Supplementary Fig. 1A for corresponding histograms.
Numbers indicate mean percentage of YFP + cells. (B) MOG35–55-EAE was induced by active immunization of LysM-PPAR WT (n = 12) or
LysM-PPAR KO (n = 13) mice, and mice were clinically monitored. The mean clinical score SEM of one representative experiment out of
five is shown. (C and D) Histopathological profile (left) and quantification (right) of mice at Day 35 after immunization. (C) Demyelination
[luxol fast blue (LFB)] and axonal damage [amyloid precursor protein (APP)] (n = 5 per group); (D) Quantification of immune cell infiltration (MAC-3 for macrophages/microglia, CD3 for T cells) (n = 5). Scale bars indicate 500 mm in the overview and 50 or 200 mm in the
magnified detail. Graphs depict mean results SEM. (E and F) Expression of (E) inflammatory mediators and (F) chemokines in the CNS of
EAE-diseased LysM-PPAR WT and LysM-PPAR KO mice was determined by quantitative real-time reverse transcriptase polymerase chain
reaction on Day 21 (n = 10 per group). Data were normalized to endogenous GAPDH and are displayed as n-fold induction compared
with healthy LysM-PPAR WT or LysM-PPAR KO mice, respectively. Data are expressed as mean SEM. (G) MOG-EAE was induced in
Nestin-PPAR WT (n = 6) and Nestin-PPAR KO mice (n = 6) and disease course was monitored. The mean clinical score SEM of one
representative experiment out of four is shown. *P 5 0.05; **P 5 0.01; ***P 5 0.001. iNOS = inducible nitric oxide.
T cell-licensed myeloid cells drive EAE
Cell culture
For preparation of mixed glial cultures, the cerebrum from post-natal Days
1–5 LysM-PPAR KO and LysM-PPAR WT or C57BL/6 mice was stripped
of meninges. The cortices were mechanically dissociated as described
previously (Wang and Neumann, 2010), seeded into culture flasks in
Dulbecco’s Modified Eagle’s Medium supplemented with 10% foetal
calf serum, 50 mM b-mercaptoethanol, 2 mM L-glutamine, 1% nonessential amino acids, 105 U penicillin and 0.1 g/l streptomycin. After
2 weeks, microglial cells were harvested by vigorously tapping the flasks,
the remaining astrocytes were used for experiments where indicated.
Primary neuronal cultures were prepared from hippocampi of
C57BL/6 mice embryos (embryonic Day15–16). Hippocampi were isolated, dispersed mechanically and seeded on 4-well chamber culture
dishes, which were pretreated with 0.01 mg/ml poly-L-ornithine and
10 mg/ml laminin. The cells were cultured in basal medium Eagle supplemented with 2% B-27 supplement (Gibco-BRL, Invitrogen), 1%
glucose (Sigma), and 1% foetal calf serum. Cells were cultured for
4 days to obtain morphologically mature neurons. Then, neurons
were co-cultured with microglial cells.
Bone marrow-derived macrophages were generated as described
previously (Racoosin and Swanson, 1989). For PPAR-activation in
microglia or bone marrow-derived macrophages, 10 mM pioglitazone
(Axxora) was added for 7 days prior to stimulation. Bone marrow-derived
macrophages were stimulated with 100 ng/ml lipopolysaccharide (LPS)
(Sigma) and 10 ng/ml IFN (eBioscience); primary microglia and primary
astrocytes were stimulated with 1 mg/ml LPS and 10 ng/ml IFN.
MOG35–55-transgenic (2D2) or OVA323–33-transgenic (OT-II)
CD4 + T cells were isolated from spleen and lymph nodes by immunomagnetic separation using CD4-MACS beads (Miltenyi Biotec)
according to the manufacturer’s protocol. Cells were activated with
plate-bound 4 mg/ml CD3 antibody (Clone: 145-2C11) and 4 mg/ml
CD28 antibody (Clone: 37.51; eBioscience) for 72 h.
Human monocytes were differentiated into macrophages as described
(Schwab et al., 2008). Written consent was obtained from all donors
and blood sampling was approved by the local ethics committee and
was performed according to the Declaration of Helsinki. Briefly, after
peripheral blood mononuclear cells isolation from healthy volunteers
and patients with multiple sclerosis, monocytes were selected by adhesion to plastic flasks for 1 h in X-vivo medium (density 2–10 107 per
75 cm2 flask). Then, cells were washed thoroughly and adherent monocytes were cultured in Roswell Park Memorial Institute medium (RPMI)
supplemented with 10% foetal calf serum in the presence of 100 ng/ml
granulocyte-macrophage
colony-stimulating
factor
(GM-CSF)
(Peprotech) and 10 ng/ml macrophage colony-stimulating factor
(M-CSF) (R&D Systems) in the presence or absence of 10 mM rosiglitazone (Enzo Life Sciences). Every other day, cytokines and rosiglitazone
were freshly added. For analysis of surface marker expression and cytokine production, monocytes were stimulated with 1 mg/ml LPS for 24 h.
THP-1 cells were cultured in RPMI supplemented with 10% foetal
calf serum. The human astrocytoma cell line U251 MG was cultured in
minimum essential medium Eagle supplemented with 2 mM L-glutamine, and Earle’s balanced salt solution adjusted to contain 1.5 g/l
sodium bicarbonate, 0.1 mM non-essential amino acids and 1.0 mM
sodium pyruvate and 10% foetal bovine serum.
Human-induced pluripotent stem cell derivation and validation is
described elsewhere (Koch et al., 2011). Differentiation of induced
pluripotent stem cells into long-term neuroepithelial stem cells was
performed following an established protocol (Koch et al., 2009). For
astroglial differentiation, human-induced pluripotent stem cell-derived
long-term neuroepithelial stem cells were seeded at a density of
4.2 104/cm2 in long-term neuroepithelial stem cell proliferation
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medium [Dulbecco’s Modified Eagle’s Medium/F12 containing 1:100
N2-supplement (Invitrogen), 10 ng/ml basic fibroblast growth factor,
10 ng/ml epidermal growth factor (both R&D systems), 1:1000
B27-supplement (Invitrogen)] on dishes coated with MatrigelTM
(BD Biosciences). Medium was changed the next day to glial differentiation medium [Dulbecco’s Modified Eagle’s Medium/F12 containing
1:100 N2-supplement, 10% foetal calf serum (Gibco), 30 ng/ml CTNF
(R&D systems), 45 nM triiodothyronine (Sigma)]. Medium was changed every other day. Cells were trypsinized at 90% confluency (usually
Days 5–6 of differentiation) and replated on gelatine-coated dishes at
a ratio of 1:2 in glial proliferation medium (Dulbecco’s Modified
Eagle’s Medium/F12 containing 1:100 N2-supplement, 20 ng/ml
basic fibroblast growth factor, 20 ng/ml epidermal growth factor,
45 nM triiodothyronine). After two additional passages glial proliferation medium was supplemented with 10 mM forskolin for further
cultivation.
Astrocyte co-cultures
For all co-culture experiments, murine/human astrocytes were cocultured with murine bone marrow-derived macrophages or human
monocytes at a ratio of 1:2 and CCL2 release into the supernatant
was determined by ELISA after 24 h. Before set-up of co-cultures,
PPAR WT or PPAR KO bone marrow-derived macrophages were
pre-stimulated with LPS and IFN for 24 h, thoroughly washed, and
subsequently cultured with primary astrocytes. Alternatively, pioglitazone-treated PPAR WT, PPAR WT and PPAR KO bone marrow-derived
macrophages were co-cultured with pre-activated OT-II T cells in the
presence of 5 mg/ml OVA323–335 (antigen-specific) or 20 mg/ml
MOG35–55 (unrelated antigen). After 24 h, bone marrow-derived
macrophages were isolated by immunomagnetic bead separation
using CD11b + beads according to the manufacturer’s protocol
(Miltenyi Biotec) and co-cultured with primary astrocytes. For human
co-culture, monocytes were prestimulated with 1 mg/ml LPS and
1000 U/ml IFN for 48 h.
Induction of experimental autoimmune
encephalomyelitis
For active immunization, EAE was induced as described previously
(Klotz et al., 2009). For adoptive transfer EAE, mice were immunized
with 50 mg MOG35–55 peptide (Biotrend) emulsified in complete
Freund’s adjuvant (CFA) subcutaneously in the flank. After 10 days,
splenic cells were isolated and cultured for 72 h in the presence of
20 mg/ml MOG35–55 (Pineda) and 20 ng/ml IL-12 (R&D Systems).
Cells (8 106) were injected intraperitoneally into recipient mice,
which also received 100 ng Bordetella pertussis toxin (Sigma) on
Days 0 and 2 after adoptive transfer.
Clinical assessment of EAE was performed daily using a scale ranging
from 0 to 8: 0, no paralysis; 1, limp tail; 2, ataxia or unilateral hind
limb paresis; 3, severe unilateral or weak bilateral hind limb paresis;
4, severe bilateral hind limb paresis; 5, complete bilateral hind limb
plegia; 6, complete bilateral hind limb plegia and partial forelimb
paresis; 7, severe tetraparesis/plegia; and 8, moribund/dead animals.
Oral treatment with pioglitazone
Mice were treated daily with 30 mg/kg body weight pioglitazone
(Actos; Takeda Pharmaceuticals) suspended in 0.5% carboxymethylcellulose by oral gavage. Control mice received the vehicle only.
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Histology
30:37% and the 37:70% interface, washed extensively with
phosphate-buffered saline, and analysed by flow cytometry.
For immunohistological analysis, the CNS of EAE-diseased mice was
perfused on Day 35 post immunization with Gey’s balanced salt solution and heparin (Ratiopharm) and afterwards fixed in 4% paraformaldehyde. The spinal cord was isolated and immunohistochemistry
was performed as described previously (Prinz et al., 2006, 2008).
Detection of nitric oxide, TNFa, IL-6
and CCL2
Depletion of CCR2 + inflammatory
monocytes
CCR2 + inflammatory monocytes were depleted in LysM-PPAR KO or
LysM-PPAR WT mice during EAE by intraperitoneal injection of 20 mg
CCR2 (MC-21) or the isotype control Ig (RatIgG2b; BD Biosciences)
every 24 h from Days 13 to 17 after induction of EAE as described (Bruhl
et al., 2007; Mildner et al., 2009). Specific depletion of inflammatory
monocytes was monitored in the blood 8 h after the first injection.
In-vivo monocyte differentiation
experiments
For in vivo labelling of circulating Ly-6Chi inflammatory monocytes
during EAE, mice were treated as described before (Getts et al., 2008).
Briefly, on Day 12 after MOG-immunization, mice received 200 ml clodronate liposomes intravenously and 12 h later, 0.5 mm FITC-labelled
FluoresbriteÕ polystyrene beads (Polyscience) were injected intravenously, which resulted in stable labelling of Ly-6Chi monocytes. In
contrast, Ly-6Clo monocytes were preferentially labelled in phosphatebuffered saline liposome-treated mice. Analysis of CNS-invading monocytes was performed 12 h after FITC-bead labelling.
On Day 13 after EAE immunization, 1 107 Ly-6C + CD115 +
monocytes, isolated from the bone marrow of C57BL/6 (CD45.2)
mice by immunomagnetic separation, were adoptively transferred
into C57BL/6 mice carrying the congenic marker CD45.1. Twelve
hours later, flow cytometrical analysis of CNS-invading CD45.2 +
monocytes was performed. Transferred monocytes were discriminated
from endogenous monocytes by surface marker staining with antibodies for CD45.1 and CD45.2, respectively.
Transfer of LysM-PPARcWT and
LysM-PPARcKO monocytes
MOG-EAE was induced in wild-type mice. On Days 12, 14, 16 and 19
of EAE, CD115 + monocytes were isolated by immunomagnetic separation from the bone marrow of 15 healthy LysM-PPAR WT and 15
healthy LysM-PPAR KO mice and 1 107 Ly-6C + CD115 + monocytes
per mouse and per time point were transferred. The clinical score was
monitored daily.
Isolation of central nervous system
mononuclear cells
For mononuclear cell isolation, mice were anaesthetized with Avertin
(Sigma), perfused with Gey’s balanced salt solution and heparin, and
brains and spinal cords were removed carefully. Tissue was mechanically dissected and digested with 1 mg/ml collagenase A (Sigma) in
phosphate-buffered saline for 30 min at 37 C while shaking. The
tissue was homogenized, washed and resuspended in 30% isotonic
PercollÕ , which was underlayered with 37 and 70% PercollÕ . After
gradient centrifugation, mononuclear cells were collected from the
To evaluate nitric oxide production, nitrite concentrations were measured in the supernatants of cells after 48 h using the Griess Reagent
(Invitrogen) according to the manufacturer’s protocol. Release of IL-6,
TNF and CCL2 in supernatants was quantified by ELISA after 24 and
48 h, respectively, according to the manufacturer‘s recommendation.
Determination of microglial
neurotoxicity
Pioglitazone-treated or untreated primary microglia were harvested
and stimulated with 1 mg/ml LPS + 10 ng/ml IFN. After 48 h, microglia were resuspended in neuronal medium without LPS and IFN and
added to primary neurons at a ratio of 1:5 (microglia:neurons). After
24 h, immunohistological staining of neurons and analysis of relative
neuronal neurite density was performed as previously described (Wang
and Neumann, 2010).
Flow cytometry
Surface marker staining was performed as described previously, using
a BDCanto II (BD Biosciences) (Burgdorf et al., 2007). For intracellular
IFN and IL-17A staining, T cells were isolated from the CNS of
EAE mice and restimulated ex vivo for 4 h with 5 ng/ml PMA and
200 ng/ml ionomycin in the presence of monensin and brefeldin A
(eBioscience). Staining was performed using the BD Cytofix/
CytopermTM Fixation/Permeabilization Solution Kit (BD Bioscience)
according to the manufacturer’s protocol. Results were analysed with
FlowJo software (Tree Star).
Antibodies
Fluorochrome-labelled antibodies against murine CD3, CD4, CD8,
CD11b, CD11c, CD40, CD45, CD45.1, CD45.2, F4/80, MHC-II,
Ly-6C, Ly-6G, IL-17A, TNF and IFN for flow-cytometric analysis
were obtained from eBioscience and BD Bioscience. Antibodies against
human CD40, CD54, CD80, CD86 and HLA-DR were purchased from
Biolegend and Miltenyi Biotec. Antibodies for ELISA (murine and
human, respectively, TNF, IL-6 and CCL2) were obtained from
Ebioscience and R&D Systems.
Quantitative real-time reverse
transcriptase polymerase chain reaction
Isolation of messenger RNA from microglia and bone marrow-derived
macrophage was performed with RNeasy Mini Kit (Qiagen) according
to the manufacturer’s protocol. Tissue from the upper cervical myelon
was isolated on Day 21 after immunization and messenger RNA was
isolated with Trizol Reagent (Invitrogen) according to the manufacturer’s protocol. Messenger RNA (1 mg) was transcribed into complementary DNA by reverse transcriptase with Superscript III (Invitrogen)
according to the manufacturer’s protocol. Expression of messenger
RNA was quantified in triplicates by quantitative real-time reverse transcriptase polymerase chain reaction using gene-specific primers (IL-1b,
IL-12 p40, TNF, IFN, FIZZ, Ym1/2; Supplementary Table 1),
T cell-licensed myeloid cells drive EAE
QuantiTect Primer Assays [iNOS, murine Arginase 1 (Arg-1)] (Qiagen),
or FAM-labelled TaqMan probes (human Arginase-1, CCL2, CCL3,
CCL5, CXCL10, GAPDH; Applied Biosystems), or Vic-labelled
TaqManÕ probes (18 s). The polymerase chain reaction mixture was
prepared with Power SYBRÕ Green PCR Master Mix or TaqManÕ
Gene Expression Master Mix (Applied Biosystems) according to the
manufacturer’s protocol. Amplification of complementary DNA was
carried out on an AbiPrism 7900 HT cycler. Data quantification was
performed by the CT method and normalized to GAPDH expression
as described in the User Bulletin 2 of the Abi Prism manufacturer.
PPARc-ablation in cells
For detection of PPAR-ablation in various cells, messenger RNA was
isolated and reverse-transcribed into complementary DNA as described
(see above). Reverse transcriptase polymerase chain reaction was performed using primers: 50 -TATCACTGGAGATCTCCGCCAACAGC-30
and 50 -GTCACGTTCTGACAG-GACTGTGTGAC-30 for PPAR. Alternatively, LysMCre mice were crossed to R26-eYFPfl/wt mice. Successful
recombination, i.e. yellow fluorescent protein (YFP) expression, was
analysed by flow cytometry in granulocytes (SSChi CD1b + ), splenic
macrophages (CD11b + F4/80 + ), blood-derived resident monocytes
(SSClo CD11b + Ly-6Clo), blood-derived inflammatory monocytes
(SSClo CD11b + Ly-6Chi), CNS-derived adult microglia (CD11b +
CD45lo), splenic DCs (F4/80 + CD11c + CD11b + ), blood-derived T
cells (CD3 + ) and B cells (B220 + ).
Statistics
All data are expressed as mean SEM. Statistical analysis was performed using unpaired Student’s t-test for all experiments except for
EAE experiments, where we used the ANOVA plus Bonferroni test;
P 5 0.05 were considered significant; P 5 0.01 were considered
highly significant.
Results
Cell-intrinsic function of PPARc in
myeloid cells during the effector phase
of experimental autoimmune
encephalomyelitis
Previously, we have shown that PPAR in CD4 + T cells controls
development of TH17 differentiation and TH17-dependent induction
of CNS autoimmunity (Klotz et al., 2007). The effect of
PPAR-ablation in T cells caused earlier disease onset but not aggravation of disease during the effector phase, which suggested that
autoreactive CD4 + T cells mainly contributed to disease initiation but
were not the critical mediators of local autoimmune damage.
Therefore, we wondered whether other immune cells were critically
involved in disease progression. To address this question, we generated myeloid cell-specific PPAR knockout mice by crossing
LysM-Cre mice with transgenic mice carrying loxP sites within the
PPAR gene (LysM-PPAR KO). In order to quantify recombination
efficiency, we crossed LysM-Cre mice with R26-eYFPfl/WT reporter
mice and assessed YFP expression in immune cells by flow cytometry.
As shown in Fig. 1A and Supplementary Fig. 1, LysM-Cre mice exhibited high recombination-efficiency in monocytes, macrophages and
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granulocytes, and lower recombination efficiency in CNS-ex vivoderived adult microglia, whereas dendritic cells, T cells, and B cells
were not affected [Supplementary Fig. 1 and Clausen et al. (1999)].
Upon EAE induction by immunization with MOG35–55,
LysM-PPAR KO mice compared with wild-type mice showed a similar development of disease severity during the T cell-mediated induction phase (Fig. 1B). Instead, LysM-PPAR KO mice exhibited a
significantly aggravated disease course during the effector phase beginning at Day 15 (Fig. 1B) and histopathological analysis of the
spinal cords from LysM-PPAR KO mice during the effector phase
at Day 35 after disease induction revealed exacerbated demyelination and axonal damage (Fig. 1C). At this time point, CNS infiltration by macrophages and T cells was significantly increased (Fig. 1D).
Similar to findings in inflammatory brain lesions in patients with multiple sclerosis (Bruck et al., 1995; Barnett et al., 2006), we found a
clear predominance of macrophages over T cells in LysM-PPAR KO
mice (Fig. 1D). Augmented disease severity during the effector phase
in LysM-PPAR KO mice was mirrored by an increased inflammatory
milieu within the CNS characterized by enhanced expression of
pro-inflammatory mediators and chemokines involved in immune
cell recruitment (Fig. 1E and F). Hence, in the absence of the
cell-intrinsic regulatory function of PPAR, myeloid cells facilitate
immune cell recruitment and generation of a pro-inflammatory
milieu, which contributes to enhanced demyelination and neuronal
damage. As expected, pharmacological PPAR activation by the synthetic agonist pioglitazone not only ameliorated the disease course
(Niino et al., 2001; Klotz et al., 2009) but also attenuated local
pro-inflammatory responses and eventually CNS damage
(Supplementary Fig. 3).
To exclude a role of PPAR in other CNS-resident cells during
EAE, i.e. neurons, astrocytes and oligodendrocytes, we generated
mice that lack PPAR in cells derived from neuronal and glial
precursors [Nestin-PPAR KO mice; Supplementary Fig. 3 and
Tronche et al. (1999)]. Importantly, ablation of PPAR in those
CNS-resident cells did not influence the disease course of EAE at all
(Fig. 1G). Taken together, PPAR in myeloid cells but not in other
CNS-resident cells contributes to modulation of local immune
responses within the CNS during the effector phase of EAE.
Systemic and local T cell responses are
not affected in LysM-PPARcKO mice
As myeloid cells function as antigen-presenting cells to prime T
cells and initiate (auto)immunity, we investigated whether myeloid
cell-specific PPAR-ablation increases naı̈ve T cell priming thereby
contributing to aggravated CNS inflammation. Characterization of
MOG-specific T cell responses in LysM-PPAR KO mice at different
time points after immunization revealed no increase in the frequency of IFN- or IL-17A-producing CD4 + T cells in the draining
lymph nodes of LysM-PPAR KO mice at Day 7 and Day 14 during
disease initiation (Fig. 2A) or within the CNS at Day 14 and Day
27 (Fig. 2B). Moreover, CD8 + T cell responses were also neither
altered at Day 27 in the CNS of LysM-PPAR KO mice (Fig. 2C) nor
at earlier time points in the periphery (data not shown). Thus, lack
of PPAR in LysMpos myeloid cells does not trigger augmented
MOG-specific T cell responses. Additionally, we adoptively
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Figure 2 PPAR in myeloid cells does not affect T cell responses during EAE. (A–C) Analysis of T cell responses in LysM-PPAR WT and
LysM-PPAR KO EAE mice. At indicated time-points, cells were isolated from (A) draining lymph nodes (drLN) or (B and C) the CNS,
ex vivo restimulated and subsequently analysed by flow cytometry for intracellular IFN and IL-17A expression in (A and B) CD4 + and
(C) CD8 + T cells (n = 5 per group for each analysis). Graphs depict mean percentage SEM. (D) Splenocytes were isolated from
MOG-immunized wild-type mice at Day 10 after immunization, restimulated in vitro in the presence of MOG35–55 and recombinant IL-12
for 72 h, and adoptively transferred into either LysM-PPAR WT or LysM-PPAR KO mice. Two independent experiments are shown. On the
left, n = 6 LysM-PPAR WT and n = 7 LysM-PPAR KO mice were used. On the right, n = 16 LysM-PPAR WT or n = 20 LysM-PPAR KO
mice were used. The mean clinical score SEM is depicted. *P 5 0.05; **P 5 0.01; ***P 5 0.001.
transferred activated MOG-specific wild-type T cells into LysMPPAR KO mice or wild-type littermates. Again, LysM-PPAR KO
mice developed an aggravated disease course during the effector
phase of EAE when compared with wild-type controls (Fig. 2D),
which indicates that differences in T cell priming do not substantially contribute to the observed aggravation during the effector
phase of EAE in LysM-PPAR KO mice. Albeit the difference was
somewhat smaller than after active MOG immunization
(Fig. 1A)—probably due to increased overall disease severity in
this particular model—it was consistently observed in independent
experiments incorporating a total of 27 LysM-PPAR KO mice
and 22 LysM-PPAR WT mice. Together, these data indicate
that PPAR-ablation in myeloid cells did not augment T cell
responses but rather aggravated local inflammation within the CNS.
PPARc-ablation results in an enhanced
activation status of myeloid cells during
experimental autoimmune
encephalomyelitis
To further characterize the role of PPAR in myeloid cells during the
effector phase of EAE we quantified the numbers of CNS-infiltrating
macrophages (CD45hiLy-6Chi) and resident microglial cells
(CD45lowLy-6Clow) and observed a 2-fold increase in total cell numbers on Day 21 of EAE in LysM-PPAR KO mice compared with
littermate control mice (Fig. 3A and B), consistent with results
shown in Fig. 1C. We next investigated whether PPAR also influenced the activation status of these cells during EAE and determined
expression of CD40 and MHC-II, molecules critical for interaction
with autoreactive T cells and therefore involved in disease deterioration (Becher et al., 2001; Greter et al., 2005). CNS-infiltrating
macrophages and microglia in LysM-PPAR KO mice showed
increased expression of CD40 (Fig. 3C) and MHC-II (Fig. 3D).
Thus, PPAR ablation in myeloid cells increases not only local myeloid cell numbers in the CNS but also enhances their activation
status, which points to a so far unrecognized role of CNS-located
myeloid cells during the effector phase of EAE.
PPARc controls T cell-mediated
licensing of macrophages
We hypothesized that during EAE, myeloid cells in the CNS
were activated locally by autoreactive T cells and that ablation of
PPAR facilitated this activation step. This prompted us to test
whether antigen-specific interaction with MOG-specific CD4 +
T cells results in myeloid cell activation. Similar to the observation
T cell-licensed myeloid cells drive EAE
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Figure 3 PPAR-ablation results in enhanced activation status of CNS myeloid cells during EAE. (A–D) MOG35–55-EAE was induced in
LysM-PPAR WT and LysM-PPAR KO mice. During the effector phase of EAE (Day 21 post induction) macrophages and microglia were
isolated from the CNS and analysed by flow cytometry. (A) Discrimination of CNS macrophages (CD11b + CD45hiLy-6C + ) and microglia
(CD11b + CD45loLy-6Clo). (B) Absolute cell numbers of macrophages and microglia were determined by flow cytometry using beads for
quantification (mean SEM, n = 4 per group). (C and D) Macrophages and microglia were analysed for (C) CD40 (n = 6 per group) and
(D) MHC-II (n = 5 per group) expression. Numbers in histograms express mean MFI SEM; graphs depict the mean percentage SEM.
*P 5 0.05; **P 5 0.01.
in vivo, we observed increased production of nitric oxide and TNF
upon antigen-specific interaction of PPAR KO compared with
PPAR WT bone marrow-derived macrophages with autoreactive
MOG-specific effector 2D2 T cells (Fig. 4A). Of note, no production
of these neurotoxic mediators was observed when T cells did not see
their cognate antigen (Fig. 4A). On the other hand, pharmacological
activation of PPAR in macrophages by the synthetic ligand
pioglitazone suppressed production of proinflammatory mediators
elicited by antigen-specific interaction with CD4 + T cells (Fig. 4B).
Importantly, antigen-specific activation of macrophages was not restricted to 2D2 T cells, but was also observed when macrophages
were co-cultured with OVA-specific effector OT-II T cells, which was
similarly found to be modulated by PPAR activity in macrophages
(Fig. 4C). In addition to induction of proinflammatory mediators,
antigen-specific interaction with either MOG- or OVA-specific
CD4 + T cells elicited upregulation of the co-stimulatory molecule
CD40 on macrophages (Fig. 4D). This upregulation was also controlled by cell-intrinsic PPAR-activity, as it was enhanced
in PPAR KO macrophages and reduced in PPAR-activated macrophages (Fig. 4D). Together, these results indicate that encephalitogenic CD4 + T cells license myeloid cells upon antigen-specific
interaction, which results in induced expression of effector molecules that drive CNS inflammation and organ damage (Hendriks
et al., 2005). Importantly, PPAR in macrophages controls this
licensing step.
Based on these findings we next asked whether pharmacological
PPAR-activation in vivo would also influence the functional outcome of myeloid cell cross-talk with disease-triggering T cells
during the effector phase of EAE (Day 21). CNS macrophages
and microglia of pioglitazone-treated mice exhibited reduced
expression of CD40 and MHC-II compared with vehicle-treated
mice (Fig. 4E). Importantly, this reduction in CD40 and MHC-II
expression on CNS-infiltrating macrophages by pioglitazone treatment was mediated by PPAR-activation directly in myeloid cells,
because we did not observe such reduction in pioglitazone-treated
LysM-PPAR KO mice (Fig. 4F). Hence, these data indicate that
in vivo, T cell-mediated licensing of myeloid cells is controlled
by myeloid cell-intrinsic PPAR.
PPARc-mediated control of myeloid cell
activity regulates recruitment of CCR2 +
inflammatory monocytes that drive the
effector phase of experimental
autoimmune encephalomyelitis
Recently, CCR2 + inflammatory monocytes have been identified as
the subpopulation of myeloid cells that crucially contributes to EAE
pathogenesis during the effector phase (Geissmann et al., 2003;
King et al., 2009; Mildner et al., 2009). It has been demonstrated
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Figure 4 PPAR controls T cell-mediated licensing of myeloid cells and regulates their activation status during EAE. (A and B) Bone
marrow-derived macrophages were co-cultured with activated 2D2 T cells at a ratio of 2:1 (BM-M:T) in the presence of their specific
antigen MOG35–55 (5, 10 or 20 mg/ml) or unrelated antigen OVA323–339 (5, 10 or 20 mg/ml). Nitric oxide (NO) or TNF release into the
supernatant was determined by Griess assay after 48 h, and ELISA after 24 h, respectively. (A) Co-culture of PPAR WT or PPAR KO bone
marrow-derived macrophages with activated 2D2 T cells. (B) Bone marrow-derived macrophages were pretreated with 10 mM pioglitazone (PIO) for 7 days or left untreated (w/o PIO), and co-cultured with activated 2D2 T cells. (C) Pioglitazone-treated PPAR WT,
untreated PPAR WT or PPAR KO bone marrow-derived macrophages were co-cultured with pre-activated OT-II T cells in the presence of
the specific antigen OVA (5 mg/ml) or the unrelated antigen MOG (5 mg/ml), and nitric oxide and TNF was determined as in (A). (D)
CD40 expression on CD11b + cells was determined after 24 h by flow cytometry on bone marrow-derived macrophages co-cultured with
activated 2D2 T cells or OT-II T cells in the presence of the cognate antigen or an unrelated antigen as in A–C, respectively. (E) Thirty
milligram per kilogram body weight pioglitazone or vehicle only (carboxymethylcellulose, CMC) was administered daily by oral gavage to
C57BL/6 wild-type mice and on Day 7 EAE was induced. On Day 21 after EAE-immunization, CD40 and MHC-II expression on
CNS-macrophages and microglia was analysed by flow cytometry as described in Fig. 3 (n = 5 per group). (F) LysM-PPAR KO mice were
orally treated with pioglitazone or vehicle only as described in (E), macrophages were isolated from the CNS during the effector phase
(Day 21) and flow cytometrically analysed for CD40 and MHC-II expression (n = 4 per group). Numbers in histograms express mean
MFI SEM; accompanying graphs depict mean percentage SEM.*P 5 0.05; **P 5 0.01; ***P 5 0.001.
(continued)
T cell-licensed myeloid cells drive EAE
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Figure 4 Continued.
that during EAE the chemokine CCL2 is responsible for CCR2 +
immune cell-recruitment (Huang et al., 2001). Interestingly, we
observed that PPAR in myeloid cells regulated local CCL2 expression within the CNS during EAE, as LysM-PPAR KO EAE mice
exhibited significantly enhanced CCL2-messenger RNA levels
within the inflamed CNS, whereas PPAR activation during EAE
resulted in reduced CCL2 expression levels within the CNS
(Fig. 5A). During CNS inflammation in multiple sclerosis and
EAE, astrocytes have been shown to be the main source of
CCL2 both in mice and humans (Van Der Voorn et al., 1999;
Giraud et al., 2010). We therefore wondered whether activated
CNS myeloid cells might modulate astrocytic CCL2 production. In
a first step, we exposed primary murine astrocytes to pre-activated
PPAR WT or PPAR KO bone marrow-derived macrophages, and
subsequently assessed CCL2 production. Activated macrophages
themselves produced only low amounts of CCL2 (Fig. 5B), but
elicited substantial CCL2 production in co-cultured astrocytes
(Fig. 5B), whereas resting macrophages did not induce astrocytic
CCL2 production (Fig. 5B). Importantly, lack of PPAR in myeloid
cells significantly augmented their capacity to elicit astrocytic CCL2
production (Fig. 5B). Following this line further, we asked whether
antigen-specific interaction with activated CD4 + T cells equally
enables macrophages to evoke astrocytic CCL2 production, as
the interaction with autoreactive T cells can be considered as
more disease-relevant stimulus compared with LPS-stimulation.
Macrophages evoked substantial astrocytic CCL2 production
selectively following antigen-specific interaction with CD4 +
T cells (Fig. 5C). With regard to the experimental setup, it is
important to note that following interaction with effector CD4 +
T cells, macrophages were purified prior to astrocyte co-culture.
Moreover, activated CD4 + T cells themselves did not evoke astrocytic CCL2 production upon co-culture (Fig. 5C). As observed
before, lack of PPAR in macrophages augmented, whereas
pharmacological PPAR activation reduced astrocytic CCL2 production (Fig. 5C). Of note, we observed no relevant CCL2 production by either resting or activated TH1 or TH17 cells
(Supplementary Fig. 4A), suggesting that autoreactive T cells
during EAE are not a source of CCL2. To exclude altered astrocyte
function in the CNS of LysM-PPAR KO mice, we determined CCL2
production by astrocytes isolated from LysM-PPAR KO mice or
their wild-type littermates and did not observe any differences
(Supplementary Fig. 4B). Together, these data suggest that CNS
myeloid cells become activated upon antigen-specific interaction
with autoreactive T cells and then engage in further cross-talk
with astrocytes triggering astrocytic CCL2-production; this
cross-talk is modulated by PPAR in myeloid cells.
Based on these results we next addressed the question whether
PPAR also influences the function of CCR2 + inflammatory
monocytes during the effector phase of EAE. To address this experimentally, we depleted CCR2 + Ly-6Chi inflammatory monocytes
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Figure 5 PPAR-mediated control of myeloid cell activity regulates CCR2 + -dependent recruitment of inflammatory monocytes that drive
the effector phase of EAE. (A) EAE was induced in LysM-PPAR WT (n = 10) or LysM-PPAR KO (n = 10) mice (top) or pioglitazone- or
vehicle-treated mice (n = 10; bottom). Mean CCL2 messenger RNA expression SEM in the CNS was determined on Day 21 of EAE by
quantitative real-time reverse transcriptase polymerase chain reaction. Data were normalized to endogenous GAPDH expression and are
displayed as n-fold induction compared with healthy LysM-PPAR WT or LysM-PPAR KO mice (top) and healthy carboxymethylcellulose
(CMC) or pioglitazone (PIO)-treated C57BL/6 mice (bottom), respectively. (B) PPAR WT and PPAR KO bone marrow-derived macrophages were stimulated with LPS and IFN for 24 h (stim) or bone marrow-derived macrophages were prestimulated with LPS and IFN
for 1 h, thoroughly washed, and subsequently co-cultured with unstimulated primary murine astrocytes at a ratio of 1:1 for 24 h. CCL2
secretion was measured by ELISA. ***Statistical significance between wild-type and knockout bone marrow-derived macrophages;
###
Statistical significance between unstimulated astrocytes and co-culture groups (P 5 0.001). (C) Primary murine astrocytes were cultured either alone, with activated OT-II T cells, with unstimulated bone marrow-derived macrophages or with bone marrow-derived
macrophages that had been pre-activated in co-culture with OT-II effector T cells in the presence of OVA323–339 or MOG35–55 for 24 h.
After T cell interaction, bone marrow-derived macrophages were purified and subsequently co-cultured with astrocytes for 24 h (ratio 2:1).
CCL2 secretion was determined by ELISA. #Statistical significance between astrocytes alone and indicated co-culture groups; *Statistical
significance within one particular co-culture group. (D–F) CCR2 + inflammatory monocytes were depleted during EAE in LysM-PPAR KO
(continued)
T cell-licensed myeloid cells drive EAE
by repetitive injection of the CCR2 antibody MC-21
(Supplementary Fig. 5) (Bruhl et al., 2007; Mildner et al., 2009)
and determined its effect on disease progression. Depletion of
CCR2 + cells not only ameliorated the EAE disease course, but
also abolished the observed differences in disease severity
between LysM-PPAR KO mice and their wild-type littermates
(Fig. 5D). Concomitantly, depletion resulted in reduced expression
levels of pro-inflammatory mediators within the CNS and furthermore completely abrogated the differences in pro-inflammatory
gene expression between LysM-PPAR KO and LysM-PPAR WT
mice (Fig. 5E). Importantly, depletion of CCR2 + cells during EAE
nearly completely abrogated local CCL2 expression in the CNS
indicating the existence of a self-perpetuating recruitment of
CCL2-responsive cells at later stages of disease (Fig. 5E). At the
single cell level, PPAR KO macrophages from MC-21-treated animals still exhibited increased expression levels of MHC-II and
CD40 (Fig. 5F), suggesting that the antibody treatment did not
alter the activation status of PPAR-ablated CNS-resident myeloid
cells per se.
To more specifically address the relevance of PPAR in
inflammatory monocytes during the effector phase of EAE we
adoptively transferred PPAR KO or PPAR WT monocytes into
MOG-immunized wild-type mice during the effector phase
on Days 12, 14, 16 and 19 after immunization. Transfer of
PPAR WT monocytes slightly enhanced disease severity (Supplementary Fig. 6). Recipients of PPAR KO monocytes, however,
exhibited a more aggravated effector phase, further pointing towards a monocyte-intrinsic effect of PPAR to control disease
activity.
Taken together, these results allow two important conclusions:
first, local activation of CNS myeloid cells evokes astrocytic CCL2
production, which is then maintained by a sustained influx of inflammatory monocytes in a self-perpetuating fashion. Second,
PPAR in myeloid cells controls both recruitment and activity of
CCR2 + inflammatory monocytes as central driving force for progression of CNS autoimmunity during EAE.
Organ-protective role of PPARc in
central nervous system-resident
myeloid cells
Besides inflammatory monocytes, microglia cells also critically
determine the extent of local inflammation and hence disease
severity during the effector phase of EAE (Heppner et al., 2005).
Given the prominent function of inflammatory monocytes in our
experiments we wondered whether these cells might acquire microglial features within the inflammatory CNS environment. Several
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reports demonstrated that under certain conditions, e.g. viral encephalitis, and blood–brain-barrier injury after irradiation (Getts
et al., 2008; Mildner et al., 2008), CNS-invading inflammatory
monocytes can acquire a microglial phenotype. To follow the fate
of inflammatory Ly-6Chi monocytes during EAE in vivo, we labelled
Ly-6Chi monocytes using an in vivo clodronate-liposome-based
bead labelling method as described previously (Getts et al., 2008).
To this end, we injected FITC-labelled polystrene beads 12 h after
depletion of monocytes from the blood by clodronate liposomes.
This resulted in preferential labelling of Ly-6Chi monocytes in clodronate-liposome-treated mice but not in phosphate-buffered saline-liposome-treated mice (Supplementary Fig. 7). Already 12 h after
labelling, we observed a distinct population of FITC-labelled inflammatory monocytes in the CNS of clodronate-liposome-treated EAE
mice, demonstrating rapid recruitment during ongoing autoimmune
inflammation (Fig. 6A and B). This experiment excluded
extravasation of FITC-labelled beads into the inflamed CNS and
subsequent uptake by resident microglial cells, because only very
few labelled cells were found in the CNS of EAE mice that received
phosphate-buffered saline-liposomes (Fig. 6A). We subsequently
analysed CD45 expression levels on FITC + cells, as CD45 is a reliable marker to distinguish macrophages from microglial cells (Getts
et al., 2008), and found that within the short time frame of 12 h
investigated here, nearly 10% of FITC-labelled inflammatory monocytes in the CNS already exhibited a microglial phenotype (Fig. 6A
and B). We corroborated these results by an independent method,
i.e. by transfer of CD45hi CD45.2 + monocytes into EAE-diseased
CD45.1 + hosts (Fig. 6C). Again, we observed that 5% of all
transferred CD45.2 + monocytes now expressed intermediate
levels of CD45 12 h after transfer, thus exhibiting a microglial
phenotype.
Based on our results showing a critical role of PPAR in the
control of myeloid cell-mediated aggravation of EAE during the
effector phase, we next investigated whether PPAR also influences the organ damaging function of myeloid cells. Stimulation of
macrophages and microglia resulted in production of TNF and
nitric oxide (Fig. 6D and E), molecules that mediate neurotoxicity
(Hendriks et al., 2005). Indeed, pharmacological PPAR activation
suppressed release of these mediators and likewise PPAR-ablated
cells showed increased production of these molecules (Fig. 6D and
E). Consistent with a regulatory role of PPAR we found that
in vivo PPAR activation induced an immune-regulatory phenotype in monocytes (Supplementary Fig. 8A) that is also referred
to as alternative or M2 activation (Fairweather and Cihakova,
2009). Of note, a similar induction of an M2 phenotype could
be achieved by PPAR activation in human monocytes
(Supplementary Fig. 8B).
Figure 5 Continued
or LysM-PPAR WT mice by five injections (arrows indicate time points) of CCR2 (MC-21; n = 20 per group) or isotype control (n = 15 per
group). (D) Mean clinical score of two independent experiments SEM is shown. (E) Expression of inflammatory mediators and chemokines in the CNS was determined at Day 21 as in Fig. 1E and F (n = 5 per group, MC-21; n = 10 per group, isotype control Ig). Data are
displayed as mean SEM. (F) MHC-II and CD40 expression on CNS-macrophages at Day 21 after immunization (n = 5 MC-21; n = 3,
isotype control); shown is one representative histogram overlay. *P 5 0.05; **P 5 0.01; ***P 5 0.001. iNOS = inducible nitric oxide.
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Figure 6 Organ-protective role of PPAR in CNS-resident myeloid cells. (A and B) MOG–EAE was induced in C57BL/6 mice. On Day
12 post immunization, Ly-6Chi inflammatory monocytes were FITC-labelled as described in methods. Twelve hours later, percentages of
macrophages and microglia were analysed based on their CD45 expression by flow cytometry. (A) Gating on FITC + CD11b + Ly-6G CNS
resident macrophages and microglia. (B) Mean percentage SEM of CD45hi or CD45int expressing FITC + cells is shown (n = 5). (C)
1 107 CD45.2 + CD115 + monocytes were transferred into EAE-diseased CD45.1 + mice on Day 12 post immunization and after 12 h,
CNS-infiltrating CD45.2 + CD11b + Ly-6G monocytes were analysed for CD45 expression (n = 5). (D and E) Production of neurotoxic
mediators by bone marrow-derived macrophages or microglia after stimulation with LPS and IFN. Nitric oxide was determined by Griess
reaction and TNF- by ELISA (bone marrow-derived macrophages) or by flow cytometry (microglia) (n = 3 per group; one representative
experiment out of at least four is shown). (D) Wild-type bone marrow-derived macrophages (BM-M) or microglia were treated with
10 mM pioglitazone (PIO) or left untreated for 7 days prior to stimulation. (E) Nitric oxide (NO) and TNF- detection in PPAR WT and
(continued)
T cell-licensed myeloid cells drive EAE
Importantly, primary microglial cells also showed PPARmediated control of expression production of neurotoxic mediators
(Fig. 6D and E), which had neuroprotective effects as assessed in
a 24 h co-culture assay with primary neurons. PPAR activation
in microglial cells preserved neurite density under inflammatory
as well as non-inflammatory conditions, indicating a continuous
role for PPAR-mediated control of microglial neurotoxicity
(Fig. 6F).
PPARc in human monocytes dampens
pro-inflammatory responses and
restricts induction of astrocytic CCL2
production
We next questioned whether human monocytes would also be
responsive to PPAR activation. In a first approach we analysed
the inflammatory response of THP-1 cells, a well-characterized
human monocytic cell line, following pharmacological PPAR
activation with rosiglitazone. We observed that upon LPS stimulation, rosiglitazone-treated THP-1 cells expressed significantly
reduced levels of CD40, CD80 and HLA-DR (Fig. 7A) and produced significantly decreased amounts of TNF (Fig. 7B). This
prompted us to investigate whether PPAR activation in primary
human monocytes differentiated from peripheral blood mononuclear cells of healthy donors would also result in dampened inflammatory responses upon stimulation. Indeed, we observed that
PPAR activation resulted in significantly impaired upregulation of
the activation markers CD40, CD54, CD80, CD86 and HLA-DR
(Fig. 7C) as well as decreased production of the proinflammatory
cytokines TNF and IL-6 upon pro-inflammatory stimulation
(Fig 7D). With regard to the development of novel therapies for
patients with multiple sclerosis, we next asked whether monocytes
from patients with multiple sclerosis were also responsive
towards PPAR-mediated control of proinflammatory activation.
Importantly, rosiglitazone-treated monocytes from patients with
multiple sclerosis also exhibited significantly reduced upregulation
of both activation markers (Fig. 7E) and proinflammatory cytokines (Fig. 7F), following inflammatory stimulation. Additionally,
we observed that human monocytes can be stimulated by
pre-activated human Jurkat T cells, which was assessed by production of TNF and IL-6 (Supplementary Fig. 9), and PPAR
activation in human monocytes by rosiglitazone limited such
activation.
Since we observed that activated murine macrophages elicit
CCL2 production in astrocytes (Fig. 5B and C) and that this
CCL2 induction was limited upon PPAR activation in macrophages, we next questioned whether activated human monocytes
were also able to induce CCL2 production by human astrocytes.
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To this end, we set up a co-culture system of primary human
monocytes and human-induced pluripotent stem cell-derived
astrocytes (Supplementary Fig. 10). Also in the human system,
preactivated monocytes both from healthy donors and patients
with multiple sclerosis elicited substantial CCL2 production
by astrocytes (Fig. 7G). Importantly, PPAR activation in human
monocytes significantly restricted this induction of astrocytic CCL2
production (Fig. 7G). These results could be corroborated using a
human astrocytic cell line (U251 MG; Supplementary Fig. 11).
Together, these data indicate that also in humans, PPAR modulates the activation status of myeloid cells from both patients with
multiple sclerosis and controls, and that the interaction between
human astrocytes and activated myeloid cells can elicit substantial
astrocytic CCL2 production.
In summary, our data reveal a crucial role of PPAR in myeloid
cells and in inflammatory monocytes as key players during the
effector phase of EAE. PPAR in myeloid cells controls the outcome of T cell-induced cross-talk among CNS resident cells, which
also dampens their innate effector functions resulting in reduced
neuro-inflammation and amelioration of inflammation-induced
neurotoxicity (Fig. 8).
Discussion
It is generally believed that CNS autoimmunity is a process promoted by autoreactive T cells (Stinissen et al., 1998). However,
within inflammatory multiple sclerosis lesions or in the CNS of EAE
mice, myeloid cells by far outnumber T cells and contribute to
disease progression (Bruck et al., 1995; Barnett et al., 2006;
Mildner et al., 2009). Yet it is not clear why CNS myeloid cells
contribute to an autoimmune disease process that occurs in the
absence of innate inflammatory stimulation. In this study, we
made use of a cell-type specific ablation of the anti-inflammatory
transcription factor PPAR to study the contribution of myeloid
cells to disease progression after encephalitogenic T cell invasion in
CNS autoimmunity. These experiments revealed a hitherto unrecognized role of myeloid cells in driving progression of T cellinitiated CNS autoimmunity characterized by the following
sequence of events: within the CNS, antigen-presenting myeloid
cells were initially licensed by autoreactive T cells upon cognate
antigen-recognition. The local inflammatory response elicited by
this interaction during the early stages of CNS autoimmunity
evokes CCL2 production through local cross-talk with astrocytes
followed by recruitment of CCR2 + inflammatory monocytes into
the CNS, which then function as the main effectors of organ
damage. Furthermore we provide evidence that the nuclear receptor PPAR in myeloid cells serves as a homeostatic control switch
acting at several important steps of myeloid cell-driven
Figure 6 Continued
PPAR KO bone marrow-derived macrophages or microglia after 48 h stimulation. (F) Wild-type microglia were treated with 10 mM
pioglitazone or left untreated, stimulated and co-cultured with primary neurons for 24 h. Cells were fixed and immunohistologically stained
with antibodies against b-tubulin III (neurons, red) and Iba-1 (microglia, green). Scale bars = 100 mm. Graph shows statistical analysis of
the mean relative neuronal neurite density. One representative experiment out of three is shown. All graphs depict mean results SEM.
*P 5 0.05; **P 5 0.01; ***P 5 0.001. w/o = without.
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Figure 7 PPAR activation in human monocytes dampens pro-inflammatory responses and restricts induction of astrocytic CCL2 production. (A and B) Rosiglitazone (RSG)-treated or untreated THP-1 cells were analysed by flow cytometry (A) for expression of the
indicated activation markers or (B) TNF production after 24 h stimulation with LPS and IFN. Graphs depict the mean percentage of
positive THP-1 cells SEM. One representative experiment out of three is shown. (C and D) Rosiglitazone-treated or untreated CD14 +
primary monocytes from healthy donors were stimulated with LPS for 20 h. (C) Expression of the indicated activation markers by CD14 +
cells was analysed by flow-cytometry. Histograms depict expression after LPS stimulation; graphs depict mean percentage (CD40, CD80,
CD86, HLA-DR) or MFI (CD54) SEM. (D) TNF and IL-6 release into the supernatant was determined by ELISA. Data from one
representative healthy donor out of four is shown. (E and F) Analysis of rosiglitazone-treated or untreated monocytes from patients with
multiple sclerosis was performed as in C and D. The results from one representative multiple sclerosis patient out of five are depicted.
(continued)
T cell-licensed myeloid cells drive EAE
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Figure 7 Continued
(G) Rosiglitazone-treated or untreated monocytes from healthy donors or from patients with multiple sclerosis (MS) were
pre-stimulated for 48 h with LPS and IFN thoroughly washed, and co-cultured with human iPS cell-derived astrocytes at a ratio of
2:1 (monocytes:astrocytes) for 24 h. CCL2 production SEM was determined by ELISA. #Statistical significance between astrocytes alone and co-culture group; *Statistical significance within co-culture group. One representative experiment from seven
healthy donors and five patients with multiple sclerosis is depicted. *P 5 0.05; **P 5 0.01; ***P 5 0.001; #P 5 0.05;
###
P 5 0.001. w/o = without.
autoimmune disease progression, i.e. reciprocal interaction between effector T cells and myeloid cells, chemokine-mediated
immune cell recruitment, and direct neuronal protection from inflammatory damage.
In the context of CNS autoimmunity, it was suggested that
innate inflammatory stimuli acting on myeloid cells may participate
in induction and aggravation of disease (Prinz et al., 2006;
Kierdorf et al., 2010). However, this fails to explain how CNS
autoimmunity can be induced by adoptive transfer of autoreactive
effector CD4 + T cells alone in the absence of additional innate
inflammatory activation. Here, we provide experimental evidence
indicating that auto-antigen presentation by local myeloid cells in
the CNS to infiltrating encephalitogenic T cells is crucial for myeloid cell activation and hence, propagation of CNS autoimmunity.
This antigen-specific local cross-talk may represent a so far unrecognized turning point between T cell-driven induction and myeloid
cell-driven effector phase of CNS autoimmunity. While our report
is the first to describe antigen-specific but myeloid cell-mediated
propagation of autoimmunity in the CNS, a similar key role for
antigen-presenting cell licensing was made in autoimmune kidney
disease. In a model of T cell-mediated kidney damage, initial
cross-talk between CD4 + T cells and local antigen-presenting dendritic cells within the target organ was required for recruitment of
autoreactive CD8 + T cells that then initiated progression of organ
damage (Heymann et al., 2009). The importance of the initial
antigen-specific cross-talk was demonstrated as dendritic cell depletion resolved established CD8 + T cell-dependent kidney pathology. Although in EAE it is not possible to selectively ablate
CNS-resident myeloid cells, several arguments support our
model: first, myeloid cell activation upon interaction with autoreactive T cells in vitro was strictly antigen-dependent. Secondly,
adoptive transfer of encephalitogenic wild-type CD4 + T cells still
caused disease aggravation during the effector phase in
LysM-PPAR KO compared with LysM-PPAR WT mice, which reveals a disease-promoting role of myeloid cells besides peripheral
T cell priming. It is important to note that in this system, transferred MOG-specific T cells were the only possible triggers of local
myeloid cell activation. However, it is not possible to dissect
whether encephalitogenic T cells are still required to sustain this
process or alternatively, whether once initiated, myeloid cells function independently to drive immune cell recruitment and organ
damage.
In the context of CNS autoimmunity, the identity of signals
provided by T cells during this antigen-specific licensing step
remains elusive, however, several molecules, e.g. CD40L, granulocyte-macrophage colony-stimulating factor and IFN have been
identified as crucial mediators of T cell-mediated activation of
antigen-presenting cells such as dendritic cells, macrophages and
microglial cells (Koya et al., 2003; Koguchi et al., 2007; Min et al.,
2010). Especially, CD40–CD40L interaction represents an interesting candidate in this context, as preformed CD40L exists in secretory lysosomes of effector CD4 + T cells and is quickly expressed in
an antigen-specific fashion (Koguchi et al., 2007). In support of
this hypothesis, we observed that the strength of human monocyte activation by human Jurkat T cells correlated to CD40L
expression-levels on the T cell side (data not shown); however,
it has to be pointed out that this setup did not involve
antigen-specific interaction of monocytes and T cells. The general
importance of CD40–CD40L interaction in CNS autoimmunity has
convincingly been demonstrated (Becher et al., 2001; Chen et al.,
2006), and microglial CD40 expression is essential for their full
activation within the CNS, resulting in further effector T cell recruitment and expansion and hence disease progression
(Ponomarev et al., 2006).
Myeloid cell-dependent propagation of autoimmune CNS disease comprised several key aspects: first, autoantigen-specific
cross-talk with autoreactive T cells caused myeloid cell expression
of molecules known to promote further T cell stimulation, such as
CD40 and MHC class II. These molecules promote T cell-mediated
CNS pathology (Becher et al., 2001) and it is tempting to
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| Brain 2012: 135; 1586–1605
speculate that they may also further accentuate myeloid cell activation in a self-perpetuating circle. Second, activated myeloid cells
induce in a further cross-talk chemokine expression, particularly
CCL2, by astrocytes which is known to trigger recruitment of inflammatory CCR2 + monocytes into the CNS the key cell population that drives disease progression during the effector phase of
EAE (Mildner et al., 2009). Both, during EAE and in acute inflammatory multiple sclerosis lesions, astrocytes seem to be the main
source of CCL2, whereas myeloid cells and T cells do not produce
much CCL2 under these conditions (Van Der Voorn et al., 1999;
Giraud et al., 2010). Although we cannot formally rule out that
myeloid cells or activated T cells contribute to local CCL2 production in vivo, we observed that astrocytic CCL2 production strongly
exceeded CCL2 production by myeloid cells and CD4 + T cells
in vitro. We now provide evidence for the first time that local
interaction between activated myeloid cells and astrocytes is the
key trigger for astrocytic CCL2 production. Importantly, this myeloid cell-induced astrocytic CCL2 production required preactivation of myeloid cells e.g. by antigen-specific interaction
with CD4 + effector T cells, which indicates that this licensing
step facilitates cross-talk of myeloid cells with other cells involved
in CNS autoimmunity. The exact mechanisms governing myeloid
cell-induced expression of CCL2 by astrocytes remain unclear but
it is important to note that astrocytic chemokine production was
modulated by PPAR activity in myeloid cells.
An important question raised by our results is whether endogenous PPAR ligands might be relevant for continuous protection from inappropriate myeloid cell licensing under
physiological conditions. This hypothesis is supported by the
fact that cell-type specific ablation of PPAR augmented disease
progression, indicating that endogenous ligands were involved in
PPAR-mediated control of myeloid cell sensitivity. In other
words, a particular baseline activity of PPAR provided by local
production of endogenous PPAR ligands might serve as endogenous homeostasis signal preventing inappropriate immune
activation and perhaps even contribute to the immune privileged
state of the CNS. In support of this hypothesis, it has been
observed that ablation of the enzyme involved in the production
of several important PPAR ligands, i.e. 12,15-lipoxygenase,
resulted in enhanced CNS autoimmunity (Emerson and LeVine,
2004), whereas oral treatment with its substrate linoleic acid
ameliorated clinical and histopathological signs of EAE (Harbige
et al., 1995). In the CNS, IL-4 stimulation induces production of
endogenous PPAR ligands in microglial cells (Yang et al., 2002;
Ponomarev et al., 2007) and high IL-4 levels are associated with
resolution of CNS inflammation during EAE and in patients with
multiple sclerosis (Falcone et al., 1998; Franciotta et al., 2000).
IL-4 is also known to induce alternative activation of macrophages or M2 macrophages, and these M2 macrophages have
a strong capacity to limit ongoing CNS autoimmunity (Butovsky
et al., 2006; Weber et al., 2007). Interestingly, phenotype and
function of PPAR-activated myeloid cells during CNS autoimmunity are reminiscent of immunoregulatory macrophages.
Following this line, we demonstrate that PPAR activation in
mice also induced alternative activation of circulating and splenic
monocytes (Supplementary Fig. 8A), and likewise of human
monocytes as previously shown (Bouhlel et al., 2007;
S. Hucke et al.
Figure 8 Cell-type specific ablation of PPAR reveals a novel
function of myeloid cells for driving progression of CNS autoimmunity during the effector phase of EAE. (A) Our results
suggest a novel role of local myeloid cell—T cell interactions in
the CNS during the effector phase of EAE where licensing of
antigen-presenting myeloid cells by autoreactive CD4 + T cells
initiates production of inflammatory and neurotoxic mediators
(1). Reciprocal antigen-specific interaction between myeloid cells
and T cells may establish a self-amplifying circle of mutual activation (2). Activated myeloid cells elicit local CCL2 production by
astrocytes (3), which results in recruitment of CCR2 + inflammatory monocytes into the CNS (4). Newly recruited inflammatory monocytes perpetuate local CCL2 production and further
immune cell recruitment and inflammation ultimately leading to
aggravation of neurotoxicity and enhanced demyelination (5).
(B) PPAR in myeloid cells controls T cell-mediated licensing (1)
and decreases the ability of myeloid cells to re-stimulate encephalitogenic T cells (2), thereby attenuating proinflammatory
cytokine and chemokine production (3) and hence inflammatory
monocyte recruitment (4), ultimately resulting in decreased
neurotoxicity and demyelination (5).
T cell-licensed myeloid cells drive EAE
Supplementary Fig. 8B). It is therefore an intriguing concept that
the local microenvironment as a source of endogenous
PPAR-ligands may act as ‘anti-danger’ signal to influence the
outcome of CNS autoimmunity by promoting alternative activation of myeloid cells.
T cell-specific PPAR-ablation revealed a separate function of
PPAR in CNS autoimmunity characterized by enhanced differentiation of PPAR KO T cells into TH17 cells that accelerate disease induction but do not affect the disease activity during the
effector phase. The strategy of cell-specific PPAR ablation
allowed us to dissect the distinct and complementary roles of T
cells and myeloid cells at the different disease stages. An important conclusion from our findings is that therapeutic approaches,
which target myeloid cells, may be more effective for modulation
of ongoing CNS autoimmunity in patients with multiple sclerosis
than those targeting T cell responses. In patients with multiple
sclerosis, we have previously shown that peripheral blood mononuclear cells exhibited decreased PPAR expression levels, and
that PPAR activity inversely correlated with disease activity
(Klotz et al., 2005). It is not possible to determine whether
lower PPAR expression levels are the consequence or cause of
autoimmunity in these patients. However, in light of our findings
presented here, these data suggest that in patients with multiple
sclerosis, reduced PPAR activity in myeloid cells may facilitate
disease exacerbation due to an intrinsically lowered threshold towards T cell-mediated licensing. Importantly, we demonstrate
here that PPAR controls inflammatory activation of human
monocytes and that this also holds true for monocytes from patients with multiple sclerosis. This indicates that myeloid cells
from patients with multiple sclerosis can be targeted by PPAR
activation, which might in turn contribute to correction of
decreased PPAR activity observed in multiple sclerosis (Klotz
et al., 2005). Even more interestingly, we provide evidence that
primary human monocytes elicit CCL2 secretion by human astrocytes, which can be modulated by PPAR in myeloid cells. This
suggests that cross-talk between astrocytes and myeloid cells
within the CNS, which results in CCL2-mediated immune cell
recruitment, is also a feature of human CNS autoimmunity.
Taken together, our data suggest that myeloid cells amplify
the disease-inducing potential of encephalitogenic T cells and perpetuate progression of CNS autoimmunity following initial
antigen-specific interaction. We identify PPAR in myeloid cells
as critical factor in regulating such licensing and hence as a promising target for attenuating myeloid cell-mediated autoimmunity
and ultimately organ damage.
Acknowledgements
We thank Helga Althausen, Corinna Hölscher and Indra Prusseit
for excellent technical assistance and acknowledge the technical
support of the Flow Cytometry Core Facility at the Institutes of
Molecular Medicine and Experimental Immunology as well as of
the central animal facilities of the medical faculty of the University
of Bonn (Haus für Experimentelle Therapie).
Brain 2012: 135; 1586–1605
| 1603
Funding
The German Research Foundation (Grant numbers KFO177 to
L.K., SFB704 to L.K. and KL 2199/3-1 to L.K.).
Supplementary material
Supplementary material is available at Brain online.
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