BBAMCB-57531; No.

of pages: 11; 4C: 3, 9

Biochimica et Biophysica Acta xxx (2013) xxxxxx

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbalip

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Patrcia E. Almeida a,c, Natlia R. Roque a, Kelly G. Magalhes a, Katherine A. Mattos b, Livia Teixeira a,
Clarissa Maya-Monteiro a, Ceclia J. Almeida a, Hugo C. Castro-Faria-Neto a, Bernhard Ryffel d,e,
Valrie F.J. Quesniaux d,e, Patrcia T. Bozza a,

Differential TLR2 downstream signaling regulates lipid metabolism and

cytokine production triggered by Mycobacterium bovis BCG infection

Laboratrio de Imunofarmacologia, Instituto Oswaldo Cruz, Fundao Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
Laboratrio de Microbiologia Celular, Instituto Oswaldo Cruz, Fundao Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
c
Laboratrio de Biologia Celular, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil
d
National Center for Scientic Research (CNRS), UMR7355, Orleans, France
e
Experimental and Molecular Immunology and Neurogenetics, University of Orleans, France
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i n f o

a b s t r a c t

a r t i c l e

The nuclear receptor PPAR acts as a key modulator of lipid metabolism, inammation and pathogenesis in BCGinfected macrophages. However, the molecular mechanisms involved in PPAR expression and functions during
infection are not completely understood. Here, we investigate signaling pathways triggered by TLR2, the involvement of co-receptors and lipid rafts in the mechanism of PPAR expression, lipid body formation and cytokine
synthesis in macrophages during BCG infection. BCG induces NF-B activation and increased PPAR expression
in a TLR2-dependent manner. Furthermore, BCG-triggered increase of lipid body biogenesis was inhibited by
the PPAR antagonist GW9662, but not by the NF-B inhibitor JSH-23. In contrast, KC/CXCL1 production was
largely dependent on NF-B but not on PPAR. BCG infection induced increased expression of CD36 in
macrophages in vitro. Moreover, CD36 co-immunoprecipitates with TLR2 in BCG-infected macrophages, suggesting its interaction with TLR2 in BCG signaling. Pretreatment with CD36 neutralizing antibodies signicantly
inhibited PPAR expression, lipid body formation and PGE2 production induced by BCG. Involvement of CD36 in
lipid body formation was further conrmed by decreased BCG-induced lipid body formation in CD36 decient
macrophages. Similarly, CD14 and CD11b/CD18 blockage also inhibited BCG-induced lipid body formation,
whereas TNF- synthesis was not affected. Disruption of rafts recapitulates the latter result, inhibiting lipid
body formation, but not TNF- synthesis in BCG-infected macrophages. In conclusion, our results suggest that
CD36-TLR2 cooperation and signaling compartmentalization within rafts, divert host response signaling through
PPAR-dependent and NF-B-independent pathways, leading to increased macrophage lipid accumulation and
down-modulation of macrophage response.
2013 Published by Elsevier B.V.

Article history:
Received 29 April 2013
Received in revised form 4 September 2013
Accepted 1 October 2013
Available online xxxx

Keywords:
Nuclear receptor
TLR
Lipid droplet
CD36
PPARgamma

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1. Introduction

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Mycobacterium tuberculosis is a global health threat, infecting about

one third of the human population and alone is responsible for almost

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Abbreviations: ADRP, adipose differentiation related protein; BCG, Bacillus Calmette

Gurin; IL, interleukin; NF-B, nuclear factor-B; M-CD, methyl--cyclodextrin; PAMP,
pathogen-associated molecular patterns; PGE2, prostaglandin E2; PPAR, peroxisome
proliferator-activated receptor gamma; TLR, Toll-like receptor
This work was funded by Conselho Nacional de Desenvolvimento Cientco e
Tecnolgico (CNPq/Brazil), PAPES-FIOCRUZ, PRONEX (MCT, Brazil), Fundao de Amparo
Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brazil), French National Center for
Scientic Research (CNRS), CNRS/FIOCRUZ 2009 project No. 24201, and Guggenheim
Foundation.
Corresponding author at: Laboratorio de Imunofarmacologia, Instituto Oswaldo Cruz,
Fundao Oswaldo Cruz, Av. Brasil 4365, Manguinhos, 21045-900 Rio de Janeiro, RJ, Brazil.
E-mail address: pbozza@ioc.ocruz.br (P.T. Bozza).

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2 million deaths annually worldwide [1]. The ability of pathogenic

mycobacteria to adapt to the hostile environment of macrophage has
been instrumental in its success as a pathogen. Mycobacteria interfere
with host trafcking pathways by modulating events in endosomal/
phagosomal maturation pathway to create a protected niche for itself,
the mycobacterial phagosome [24]. Moreover, pathogenic mycobacteria may subvert host signaling response, through upregulation and
highjack of specic metabolic pathways, thus regulating host immune
response and enabling access to nutrients, which ultimately favor
infection establishment [510]. The foam-like macrophages are a cell
type found within granulomatous structures in both experimental
animal models and human disease [1113]. As observed for other
pathological conditions, the foam aspect of macrophages during mycobacteria infection is a reection of intracellular lipid accumulation.
Accumulated evidence demonstrated that newly formed lipid bodies
are structurally distinct cytoplasmic organelles involved in lipid mediator
synthesis with immunomodulatory functions during Mycobacterium

1388-1981/$ see front matter 2013 Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.bbalip.2013.10.008

Please cite this article as: P.E. Almeida, et al., Differential TLR2 downstream signaling regulates lipid metabolism and cytokine production
triggered by Mycobacterium bovis BCG infection, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbalip.2013.10.008

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2.1. Animals

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Mice were housed with free access to food and water at 22 to 24 C

and a 12 h light/dark cycle until used. Animals weighing 20 to 25 g
from both sexes were used. CD36 [29] and CD14 [39], provided by the
Transgenose Institute, CNRS, Orlans, France, and TLR2 [40], provided
by CECAL, FIOCRUZ, genetically decient mice in a homogeneous
C57BL/6 background were used. All protocols were approved by the
Institutional animal welfare committees.

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2.2. M. bovis BCG and M. smegmatis

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2. Materials and methods

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M. bovis BCG (Moreau strain) dried vaccine (40 mg) was stored at
4 C and M. smegmatis (mc2155) were stored at 20 C. Both
mycobacteria strains were resuspended to a nal concentration of
1 106 colony-forming units (CFU)/mL in RPMI and sonicated just
before use.

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2.3. Macrophage culture and in vitro infection

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Peritoneal cells from nave C57/BL6 or TLR2/ mice were harvested

with sterile RPMI-1640 cell-culture medium. Peritoneal cells were
allowed to adhere for 2 h at 37 C in a 5% CO2 atmosphere, and washed
twice vigorously with phosphate buffer (PBS) to remove non-adherent
cells. Macrophages (1 106 cells/mL) were adhered in cover slides
within culture plates (24 wells) overnight with RPMI-1640 cellculture medium containing 2% FCS. Macrophages were infected with
BCG (MOI, 1:1) or M. smegmatis (MOI, 1:1) for 24 h at 37 C in 5% CO2
atmosphere. In inhibitory studies, macrophages were treated 30 min
before infection with: neutralizing CD36 monoclonal antibody (Cayman
Chemical Co.) or IgA isotype control (eBiosciences) (2 g/mL), antiCD14 (Cayman Chemical Co.), anti-CD11b/CD18 (eBioscience) or IgG
isotype control (10 g/mL), or the drugs JSH (Sigma) (30 M), lipin or
methyl--cyclodextrin (Sigma) (10 M) at 37 C. Then, macrophages
were infected with BCG for 1 h, and washed with PBS (three times)
to remove non-internalized BCG. Vehicle (DMSO 0.01%) was used
as control. The cell-free supernatants were recovered and stored at
20 C. Cell viability was assessed by trypan blue exclusion at the
end of each experiment and was always greater than 90%.

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2.4. Pleurisy induced by M. bovis BCG

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Mice were intrathoracically injected with M. bovis BCG (5 105

bacilli/cavity or 5 106 bacilli/cavity) in 100 L sterile saline. Control
animals received an equal volume (100 L) of sterile saline only. After
24h the animals were killed by CO2 inhalation and their thoracic cavities
were washed with 1 mL of the heparinized PBS (10 UI/mL).

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2.5. Lipid body staining and counting

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Macrophages were xed in 3.7% formaldehyde in PBS (pH7.4), rinsed

in 0.1 M cacodylate buffer (pH 7.4), stained in 1.5% osmium tetroxide
(30 min), rinsed in water, immersed in 1.0% thiocarbohydrazide
(5 min), rinsed in water, rinsed in 0.1 M cacodylate buffer, reincubated
in 1.5% osmium tetroxide (3 min), rinsed in distilled water, dried, and
mounted for further analysis. The morphology of xed cells was
observed, and lipid bodies were enumerated by light microscopy with
a 100 objective lens in 50 consecutive leukocytes in each slide. To
avoid human interference on this counting, we always ensured that

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infections. We demonstrate here that, indeed, lipid body formation, PGE2

production and PPAR expression during M. bovis BCG infection require
the presence of CD36 together with TLR2, suggesting that cellular
activation is a result of synergic interactions among these receptors.

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bovis BCG infection [5,14,15]. In addition, mycobacteria induction and

targeting to lipid body may provide an escape mechanism during
infection due to down-modulation of the macrophage response and/or
acquisition of nutrients, leading to enhanced survival and replication in
host cells [5,13,16,17]. However, the signaling pathway and molecular
mechanisms that regulate lipid body biogenesis during mycobacterial
infection and their contribution to the pathophysiology of tuberculosis
are not clear.
Toll-like receptors are germ-lined encoded receptors of the innate
immune system responsible for recognizing a range of pathogenassociated molecular patterns (PAMPs). Mycobacterial components
are recognized by different members of the TLR family including TLR2
in association with TLR1/TLR6, by TLR4 and by TLR9, and have been
implicated to mediate intracellular signaling in the host response to
bacterial infection [18,19]. TLR2 seems to be very promiscuous and
able to recognize the most diverse set of pathogen-associated motifs
[2022]. Its promiscuity has been attributed to its unique ability to
heterodimerize with TLRs 1 and 6, which direct ligand specicity
towards triacylated versus diacylated lipopeptides, respectively [23]. It
was thus suggested that combinatorial signaling among the TLRs
might account for the breadth of their repertoire [24]. However,
identication of co-receptors that enhances TLR functions suggests
that TLRs act together with other cell surface molecules to link PAMP
recognition to the initiation of inammatory responses. Indeed, recognition of TLR2 agonists is inuenced by several accessory receptors,
including CD14, CD36 [25] and CD11b/CD18 integrin [26]. The study
by Hoebe et al. [27] has shed light into TLR2 heterotypic associations
by demonstrating using forward-genetic mutagenesis that TLR2/TLR6
heterodimers also require CD36. In addition, heterotypic associations
of TLR2/6 with CD36 are not pre-formed and are ligand-induced [28].
CD36 is a transmembrane glycoprotein involved in many biological
processes, such as platelet biology, angiogenesis and in the pathophysiology of atherosclerosis and cardiovascular diseases dening it as
a multi-ligand scavenger receptor. CD36 performs homeostatic functions,
including recognition and phagocytosis of apoptotic cells, senescent cells,
cellular debris, and oxidized LDL [29,30]. Monocyte/macrophage CD36
has been shown to play a critical role in the development of atherosclerotic lesions by its capacity to bind and promote endocytosis of
oxidized low density lipoproteins (oxLDL) and to participate in the
formation of foam cells. In atherosclerosis and Alzheimer's disease,
deposition of the altered self-components oxLDL and amyloid-beta
trigger inammatory response through CD36-induced TLR4TLR6 heterodimer activation, suggesting a new model of TLR heterodimerization
triggered by co-receptor signaling events [31].
Recent studies have demonstrated that mycobacterial components
may regulate expression and function of nuclear receptors [8,3234].
PPAR is a member of the lipid activated nuclear receptor family and
has been demonstrated to function as a key transcriptional regulator
of cell differentiation, inammation and lipid metabolism in macrophages and dendritic cells [35]. Indeed, PPAR is highly expressed in
macrophage-derived foam cells within atherosclerotic lesions where it
plays an important role in lipid homeostasis and metabolism [3638].
Our group demonstrated that BCG-induced PPAR expression, lipid
body formation and TNF- generation were drastically inhibited
in TLR2 decient mice, suggesting a requisite role for TLR2 in BCG
mediated macrophage up-regulation of PPAR protein content [8].
Interestingly, activation of macrophages in vitro with the Mycobacterium
smegmatis failed to induce PPAR expression and lipid body formation,
while inducing TLR2-dependent TNF- production. Thus TLR2 activation, although essential for mycobacterial-induced lipid body formation,
is not sufcient to trigger pathways of lipid body formation and other coreceptors might be involved [8,34].
Since TLR activation seems to be associated with lipid-raft-resident
molecules, such as CD14 and CD36, we hypothesized that CD36 and
other co-receptors such as CD14 and CD11b/CD18 might cooperate
with TLRs, including TLR2, to activate macrophages during mycobacterial

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P.E. Almeida et al. / Biochimica et Biophysica Acta xxx (2013) xxxxxx

Please cite this article as: P.E. Almeida, et al., Differential TLR2 downstream signaling regulates lipid metabolism and cytokine production
triggered by Mycobacterium bovis BCG infection, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbalip.2013.10.008

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2.6. NF-B and PPAR immunolocalization

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The slides containing peritoneal macrophages were xed in 3%

formaldehyde at room temperature for 10 min, permeabilized with
0.2% Triton, and then blocked with 1% normal donkey serum. After
washing, slides were incubated overnight at 4 C with anti-NF-Bp65
polyclonal antibody or anti-PPAR polyclonal antibody (Santa Cruz)
diluted in 1% normal donkey serum/PBS solution. Non-immune rabbit
serum was used as control. After 3 washes of 5min, slides were incubated
for 1 h at room temperature with the secondary Alexa 546-labeled goat
anti-rabbit immunoglobulin G antibody (Molecular Probes). Incubation
with DAPI (Sigma) for 5 min was used for nuclei counterstaining.

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after 24 h of infection were collected and stored at 20 C until the 213
day of analysis. Cytokines were analyzed simultaneously using Luminex 214

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2.8. Cytokine analysis

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PGE2 levels were measured directly in the supernatant of cultured

cells obtained 24 h after BCG infection. PGE2 was assayed in the cellfree supernatant by enzyme-linked immunoassay (EIA), according to
the manufacturer's instructions (Cayman Chemical Co.).

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2.7. Prostaglandin E2 measurement

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Immunostained samples were analyzed with a Confocal Laser Scanning 204

Microscope (CLSM 510; Zeiss).
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the person responsible for counting was blinded to the codes for each
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Fig. 1. M. bovis BCG and M. smegmatis induce differential response in macrophages. Mouse peritoneal macrophages obtained from C57BL/6 or TLR2 knockout mice (TLR2/) were infected
in vitro with BCG or M. smegmatis (MOI 1:1). Analysis of lipid body formation, KC synthesis, as well as, NF-B and PPAR expression and localization were performed at 24 h of infection.
(A) Lipid body counting after osmium staining. (B) KC synthesis was analyzed using Luminex technology. Each bar represents the mean SEM from 3 pools of 10 animals. Differences
between control and infected groups are indicated by asterisks (p b 0.05); + represents differences between TLR2+/+ and TLR2/ macrophages infected by BCG. (C) NF-B and
PPAR immunouorescence staining of TLR2+/+ or TLR2/ macrophages unstimulated or stimulated with LPS and non-infected or infected with BCG. DAPI (blue uorescence) was
used in all cell identication of the nuclei. The same magnication was used in all images detection with a 100 objective lens.

Please cite this article as: P.E. Almeida, et al., Differential TLR2 downstream signaling regulates lipid metabolism and cytokine production
triggered by Mycobacterium bovis BCG infection, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbalip.2013.10.008

P.E. Almeida et al. / Biochimica et Biophysica Acta xxx (2013) xxxxxx

2.10. Immunoprecipitation

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Briey, macrophages infected by M. bovis BCG 24 h were washed

twice with ice-cold PBS and scraped into 800 L of lysis buffer (10 mM
Tris, pH 7.5, 50 mM NaCl, 1% Triton X-100, 60 mM octylglucoside),
containing protease inhibitors. For pre-clearing, lysates were incubated
with pre-bound to Protein A Sepharose CL 48 (Amersham Bioscience).
After incubation for 4 h at 4 C, the beads were extensively washed
with TNET buffer (50 mM Tris pH 8.0, 150 mM, NaCl, 5 mM EDTA, 1%
Triton X-100) (six times). After that, pre-cleared lysates were incubated

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The results were expressed as mean SEM and were analyzed 261
statistically by means of variance followed by NewmanKeulsStudent's 262
test or Student's t test with the level of signicance set at p b 0.05.
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Cell lysates were prepared in reducing and denaturing conditions

and subjected to SDS-PAGE. Samples were submitted to electrophoresis
in 10% acrylamide gradient SDS-PAGE gels. After transfer onto nitrocellulose membranes, non-specic binding sites were blocked with 5%
non-fat milk in Tris buffered saline-Tween (TBST; 50 mM TrisHCl,
pH 7.4, 150 mM NaCl, 0.05% Tween 20). Membranes were probed
with the polyclonal antibodies, anti-CD36 and anti-PPAR (Cayman
Chemical Co.), anti-TLR2 (eBioscience Cd282) or anti--actin mAb
(BD Transduction Laboratories) in TBST with 1% non-fat dry milk.
Proteins of interest were then identied by incubating the membrane
with HRP-conjugated secondary antibodies in TBST, followed by detection of antigenantibody complexes by Supersignal Chemiluminescence
(Pierce). For densitometry analysis, images were analyzed in the
software Scion Image (International University Bremen).

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Murine macrophages were detached with ice-cold 6 mM PBS-EDTA

and gentle scraping. Cells were washed in PBS in blocking buffer (2%
FBS in PBS). FITC-conjugated anti-CD36 antibody (Cayman Chemical
Co.) 0.5 mg/mL or with an irrelevant FITC-conjugated isotype (Santa
Cruz) for 1h at 4C. The cells were xed with 4% (w/v) paraformaldehyde,
and then uorescence measured at FL1 channel was detected by a
FACSCalibur ow cytometer (BD Biosciences, Heidelberg, Germany),
and at least 10,000 cells were analyzed/sample. Quantitative data
analysis was performed using WinMDI analysis software and expressed
as percent of CD36 positive cells.

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2.11. Western-blot analysis

2.9. Flow cytometry analysis for CD36 expression

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overnight at 4C with anti-CD36 polyclonal antibody (Cayman Chemical

Co.) or IgG rabbit control (R&DSystems) 2.5 g/mL. Finally, the beads
were resuspended in 3 sample buffer, separated by SDS-PAGE (10%)
and processed for immunoblotting. Each experiment was repeated at
least three times independently, with virtually identical results.

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technology on the Bio-Plex system (Bio-Rad, Hercules, CA). Fifty L of

sample was analyzed using a mouse multiplex cytokine kit obtained
and assayed according to the manufacturer's instructions (Upstate,
Waltham, MA). Data analyses were performed with the Bio-Plex
Manager software.

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Fig. 2. Inhibition of NF-B does not interfere with lipid body formation, although it is able to inhibit cytokine synthesis after infection by M. bovis BCG. Peritoneal macrophages were treated
with vehicle, JSH-23 (10 M or 30 M) or GW9662 (1 M) 30 min before infection with BCG (MOI 1:1). (A and B) Lipid body counting after osmium staining in peritoneal macrophages
treated with vehicle or JSH-23 in non-infected or BCG-infected during 3 h (A) or 24 h (B), (C) parallel enhancement of KC synthesis at 3 h was analyzed using Luminex technology, (D and E)
lipid body counting after osmium staining in peritoneal macrophages treated with vehicle or GW9662 non-infected or infected with BCG during 3 h (D) and 24 h (E); (F) TNF- synthesis
analysis by Luminex technology. Each bar represents the mean SEM from 3 pools of 10 animals. Differences between control and infected macrophages are indicated by asterisks
(p b 0.05). + represents differences between BCG and BCG in the presence of JSH-23 or GW 9662.

Please cite this article as: P.E. Almeida, et al., Differential TLR2 downstream signaling regulates lipid metabolism and cytokine production
triggered by Mycobacterium bovis BCG infection, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbalip.2013.10.008

P.E. Almeida et al. / Biochimica et Biophysica Acta xxx (2013) xxxxxx

Lipid body formation in leukocytes is a highly regulated event that

depends on the interaction of cellular receptors with their ligands, and
newly formed lipid bodies have been associated with intracellular
pathogen survival advantages in HCV [41,42], dengue [43], Trypanosoma
cruzi [44] and mycobacterial infections [5,8,10,15,45]. The TLR-mediated
pathogen recognition and activation in the mechanism of lipid body
formation have been documented [5,46]. We recently demonstrated
that TLR2 activation, although essential, is not sufcient to trigger lipid
body formation during M. bovis BCG infection [8], suggesting the
involvement of cooperative signaling pathways. Here, we rst investigated the requirement of TLR2 and the involvement of NF-B and
PPAR activation in M. bovis BCG or M. smegmatis induced macrophage
activation. As shown in Fig. 1AB, M. bovis BCG infection triggers
both lipid body formation and CXCL1/KC production in macrophages
in a mechanism highly dependent on TLR2 activation. In contrast,
M. smegmatis was unable to induce lipid body biogenesis (Fig. 1A),
while effectively inducing CXCL1/KC in a TLR2-dependent way (Fig. 1B).
As seen in Fig. 1C, M. bovis BCG infection induced both NF-B nuclear
translocation and PPAR expression in wild-type macrophages, a
phenomenon that was drastically inhibited in TLR2/ macrophages.
Thus, differential signaling pathways are triggered by TLR2 in response
to mycobacteria.

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A number of questions remain about how TLR2 and its coreceptors recognize the diverse set of physiological ligands with
TLR2 agonist activity and how they trigger differential intracellular
signaling pathways. We assessed the relative contribution of the
NF-B and the PPAR pathways in cytokine expression versus lipid
body formation by M. bovis BCG activated macrophages.
NF-B is a transcription factor with a central role in TLR-triggered
signaling pathways. It is composed of hetero- or homodimers of members of the Rel family of proteins, such as RelA (p65), RelB, cRel, p50
and p52 [47]. TLR ligands activate downstream signaling molecules
that include MyD88, IL-1 receptor-associated kinase, and tumor necrosis
factor (TNF) receptor-associated factor 6, which activates IB kinase
(IKK) complex [48,49]. IB degradation unmask the nuclear localization
signal of NF-B, allowing the transcription factor to translocate to the
nucleus, and then NF-B binds to the promoter region of immune and
inammatory genes for transcriptional regulation [50]. The aromatic
diamine 4-methyl-N1-(3-phenyl-propyl)-benzene-1,2-diamine (JSH23) compound inhibits nuclear translocation of NF-B p65 without
affecting IB degradation, [51]. It has been demonstrated that JSH-23
causes down-regulation of LPS-inducible cytokines and enzymes, and
inhibits LPS-induced apoptosis of the RAW 264.7 cells [51]. Here, we
used JSH-23 to determine whether inhibition of NF-B activation
would affect cytokine production and lipid body formation in BCGstimulated peritoneal macrophages. As shown in Fig. 2, although the

3.1. M. bovis BCG and M. smegmatis induce differential responses

in macrophages

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3.2. Distinct transcriptionally regulated pathways are required for cytokine 289
and lipid metabolism control during infection by M. bovis BCG
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3. Results

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Fig. 3. M. bovis BCG induces CD36 expression and TLR2 heterodimerization. Murine peritoneal macrophages were cultured in the absence or presence of M. bovis BCG or M. smegmatis
(MOI 1:1) for 24 h. CD36 expression was determined by medium uorescence intensity of FITC (FL1) by ow cytometric analysis, (A) histogram representing macrophage
autouorescence control of non-labeled cells (white) or CD36 labeled cells as follows: non-stimulated cells (black), M. smegmatis-infected cells (light gray), and M. bovis BCG-infected
cells (dark gray). (B) Graph represents percentage of CD36 positivity in macrophages infected or not with M. smegmatis or M. bovis BCG. (C) TLR2 was detected by Western blot with
anti-TLR2 antibody after immunoprecipitation with anti-CD36 antibody as described under Materials and methods section. (D) Murine peritoneal macrophages were pre-treated with
vehicle or anti-CD36 antibody (2 g/mL) for 30 min. After pre-treatment, macrophages were infected or not with M. bovis BCG for 24 h. Total macrophage cell lysates were separated
by SDS-PAGE (10%) and submitted to Western blotting for anti-PPAR. The graph represents the densitometric analysis (arbitrary units (A.U.)) of the Western blotting bands. The
image is representative of at least two different blots.

Please cite this article as: P.E. Almeida, et al., Differential TLR2 downstream signaling regulates lipid metabolism and cytokine production
triggered by Mycobacterium bovis BCG infection, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbalip.2013.10.008

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3.3. CD36 expression interacts with TLR2 and enhances PPAR expression

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Various molecules have been described as co-receptors acting in

conjunction with TLR2, such as CD36, CD14, TLR1 and TLR6. Considering
the role of CD36 in lipid metabolism, macrophage differentiation and
pathogen recognition, we investigated the role of CD36 in lipid body
formation in mouse peritoneal macrophages infected by BCG. As shown
in Fig. 3A and B, M. bovis BCG, but not M. smegmatis infection induces
an increase in CD36 protein expression in macrophages. Increased
CD36 protein content was noted within 90min, and was maximal within
3h, occurring in parallel but preceding the maximal lipid body formation
that is noted within 24 h (data not shown). Several studies in the
literature show that TLR2 and CD36 dimerize upon various stimuli,
such as lipoproteins, lipotechoic acid or diacylglycerides [27,54,55].
Here we demonstrated that CD36 and TLR2 co-immunoprecipitate in

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In addition to CD36, recognition of TLR2 agonists is inuenced by 372

several accessory receptors, including CD14 and CD11b/CD18 integrin, 373

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TNF- expression in response to M. bovis BCG infection
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To investigate the functional role of CD36 activation during M. bovis

BCG infection, we used a neutralizing antibody for this receptor. As
shown in Fig. 4, addition of anti-CD36 antibody signicantly inhibited
lipid body formation during BCG infection (Fig. 4A), while the isotype
control IgA had no effect. The role of CD36 in lipid metabolism regulation during BCG infection was conrmed in CD36 genetically
decient animals. As shown in Fig. 4, BCG-induced lipid body formation
was signicantly decreased when cells from CD36 decient animals
were compared to cells from wild-type animals, both in vitro (Fig. 4B)
and in vivo (Fig. 4C). PGE2 production was also inhibited by pretreatment with the neutralizing antibody anti-CD36 (Fig. 4D), although
TNF- and IL-1 synthesis remained unaltered (Fig. 4E and F), during
BCG infection at 24 h in vitro. These results indicate that CD36 has a
crucial role in signaling lipid body biogenesis and prostanoid production
by M. bovis BCG infection.

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3.4. CD36 is required for lipid body formation and PGE2 production, but not 353
cytokine synthesis during BCG infection
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macrophages 24 h after infection by M. bovis BCG (Fig. 3C lane 4),

which is not observed in non-infected cells (Fig. 3C lane 1).
Next, we evaluated whether CD36 blockade affects PPAR expression.
As shown in Fig. 3D, PPAR expression was partially inhibited after CD36
neutralization (Fig. 3D lane 4). In summary, these results suggest that
CD36 activation favors PPAR expression, while CD36 expression may
be amplied by PPAR in a positive feedback loop during M. bovis BCG
infection.

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pretreatment with JSH-23 (30 M) signicantly inhibited the production of CXCL1/KC (Fig. 2C), it failed to inhibit lipid body formation
(Fig. 2AB) in peritoneal macrophages at 3 and 24 h after infection.
Recent studies have shown that PPAR activation inhibits NF-B
and MAP kinase pathways, two major signaling pathways that regulate
pro-inammatory responses triggered by TLR activation [52,53]. We
next investigated the role of PPAR on M. bovis BCG-induced lipid
body formation and TNF- production in macrophages. As shown in
Fig. 2DF, M. bovis BCG infection signicantly increased lipid body
formation and synthesis of TNF-. Pretreatment with PPAR antagonist
GW9662 was able to inhibit lipid body formation at 3h and 24h (Fig. 2D
and E, respectively), although it failed to modify BCG-induced TNF-
production by macrophages (Fig. 2F). These results suggest that TLR2
activation by M. bovis BCG triggers differential transcriptional regulated
NF-B and the PPAR pathways, leading to cytokine production and
lipid body formation, respectively.

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Fig. 4. Lipid body formation, PGE2 production, but not cytokine synthesis is dependent of CD36 expression during M. bovis BCG infection. Peritoneal macrophages were treated with vehicle
or anti-CD36 antibody (2 g/mL) for 30 min before infection with BCG (MOI 1:1) during 24 h (A, D, E and F). (A, B and C) Lipid body counting was performed after osmium staining (A) in
peritoneal macrophages infected or not with BCG and treated or not with anti-CD36, (B) in peritoneal macrophages from CD36+/+ or CD36/ mice in vitro, and (C) in total cells from the
intrathoracic cavity of CD36+/+ or CD36/ mice in vivo. (D) PGE2 production was measured in medium of macrophage cultures by an enzyme immunoassay. (E) TNF- and (F) IL-1
cytokine in medium of macrophage cultures were analyzed using Luminex technology. Each bar represents the mean SEM from 3 pools of cells from 10 animals each. Differences
between control and infected groups are indicated by asterisks (p b 0.05). + represents differences between BCG and BCG in the presence of anti-CD36. # represents differences between
CD36+/+ and CD36/ infected by BCG.

Please cite this article as: P.E. Almeida, et al., Differential TLR2 downstream signaling regulates lipid metabolism and cytokine production
triggered by Mycobacterium bovis BCG infection, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbalip.2013.10.008

P.E. Almeida et al. / Biochimica et Biophysica Acta xxx (2013) xxxxxx

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3.6. Raft disruption inhibits both lipid body formation and PGE2, but not
TNF- release induced by M. bovis BCG

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4. Discussion

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Mycobacteria-induced manipulation of host cell lipid metabolism is

now recognized as an important event for persistence and successful
establishment of infection in tuberculosis and leprosy. We and others,
have recently established a key role for PPAR in the regulation of
lipid metabolism, lipid body formation and immune response in mycobacterium infection [8,3234]. However, the mechanisms that regulate
PPAR expression and activation are not well understood. Our study
provides novel evidence that CD36-TLR2 cooperation, association with
CD11b and CD14, and signaling compartmentalization within rafts,
divert host response signaling with increased expression and activation
of PPAR through NF-B-independent pathways, leading to increased
macrophage lipid accumulation and down-modulation of macrophage
response.
The signaling cascade initiated by activation of the TLRs results in
translocation of the transcription factor NF-B, which subsequently
induces the expression of cytokines. TLR2 signaling was also demonstrated as an essential step for PPAR expression and macrophage
lipid accumulation following M. bovis BCG infection [8]. To clarify the
downstream mechanism by which TLR2 triggers increased PPARdependent lipid metabolism alterations, we evaluated the role of
NF-B activation in this phenomenon. Notably, JSH-23, a selective
NF-B transcriptional inhibitor, at doses that signicantly inhibited
KC/CXCL-1 production failed to inhibit BCG-induced lipid body formation. Indeed, NF-B inhibition by JSH-23 within 3 h leads to enhanced
lipid body formation induced by BCG, suggesting NF-B as a negative
regulator of lipid body formation. The mechanism and pathways involved in this negative regulation deserve further investigation.
In contrast the PPAR antagonist GW 9662 was able to inhibit BCGinduced lipid body formation, but not TNF- release. Together, these
results suggest that distinct signaling pathways downstream of TLR2
are involved in cytokine expression and lipid metabolism regulation.
Accordingly, macrophage activation in vitro with M. smegmatis, or
Pam3CSK4 (not shown), both potent TLR2 ligands, at doses that signicantly induced TLR2-dependent CXCL1 production, were unable to
induce lipid body biogenesis in macrophages, suggesting that TLR2

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Localization of TLR2 and co-receptors may be important for function. It has been demonstrated in several model systems that CD36,
CD14 and CD11b/CD18 may compartmentalize with TLR2 in rafts.
Thus, we next evaluated the involvement of rafts in BCG infection.
As shown in Fig. 6, drugs that disrupt rafts, including methyl-cyclodextrin and lipin, signicantly inhibited lipid body formation
(Fig. 6A and B). Treatment with methyl--cyclodextrin also inhibited
BCG-induced PGE2 production (Fig. 6C), although TNF- release remained unaltered (Fig. 6D).
In summary, these results indicate that TLR2 cooperates with CD36,
CD14 and CD11b/CD18 to activate lipid body production, prostanoid
and cytokine synthesis, engaging distinct signaling pathways, which
involve NF-B and PPAR.

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alone is not sufcient and that TLR2 associated co-receptors involved 435
in bacterial recognition may account for the recruitment of signaling 436
molecules important for PPAR expression and lipid body formation [8]. 437

which act in concert to modulate TLR activity [18,21]. Thus, we studied

the participation of CD14 and integrins, as putative co-receptors of TLR2
in cell signaling triggered by mycobacterial infection.
As shown in Fig. 5A, lipid body formation in macrophages from CD14
genetically decient animals is signicantly diminished compared to
macrophages from wild type mice. Accordingly, the neutralization of
CD11b/CD18 or CD14 inhibits lipid body formation (Fig. 5B) and PGE2
production (data not shown), but not TNF- synthesis (Fig. 5C).
Therefore, CD14 and CD11b/CD18 are also engaged in BCG-infected
macrophages leading to lipid body formation and PGE2 synthesis.

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Fig. 5. Involvement of CD14 and CD11b/CD18 in lipid body formation, but not TNF-
synthesis during M. bovis BCG infection. (A) Lipid body counting of peritoneal macrophage
obtained from wild type (CD14+/+) or CD14 decient (CD14/) were treated in vitro
with vehicle or BCG for 24 h. (BC) Peritoneal macrophages from wild type mice were treated
with vehicle, anti-CD14 (10 M) or anti-CD11b/CD18 (10 M) for 30 min before infection
with BCG for 24 h. (B) Lipid body counting was performed after osmium staining, (C) TNF in the media of macrophage cultures was analyzed using Luminex technology. Each bar
represents the mean SEM from 3 independent pools of 10 animals each. Differences
between control and infected groups are indicated by asterisks (p b 0.05). + represent
differences between CD14+/+ and CD14/ macrophages infected with BCG. # represents
differences between BCG and BCG in the presence of anti-CD14 or anti-CD11b/CD18.

Please cite this article as: P.E. Almeida, et al., Differential TLR2 downstream signaling regulates lipid metabolism and cytokine production
triggered by Mycobacterium bovis BCG infection, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbalip.2013.10.008

P.E. Almeida et al. / Biochimica et Biophysica Acta xxx (2013) xxxxxx

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It is increasingly recognized that TLR2-dependent signaling is modulated by the concomitant interaction with co-receptors and depending
on the co-receptor usage, distinct downstream signaling pathways
might be recruited, leading to differential signaling compartmentalization and responses [28]. Several co-receptors for TLR2 have been
characterized, which may contribute to the diversity of ligands able to
trigger TLR2-dependent responses. Heterodimerization of TLR2 with
other members of the TLR family forming the TLR2-TLR1 and TLR2TLR6 heterodimers have been well characterized to regulate signaling,
including cytokine production [25]. TLR6 was demonstrated to be
dispensable for regulating macrophage lipid metabolism upon BCG
infection [8]. However, TLR6 participates in the metabolic response
towards M. leprae in Schwann cells [46], suggesting that different coreceptors involved in lipid metabolism signaling may be recruited
according to the pathogen and cell system.
Next, we addressed the involvement of CD36, in cooperation with
TLR2, to modulate the BCG effects on host lipid metabolism. Monocyte/
macrophage CD36 has been shown to play a critical role in the
development of atherosclerotic lesions by its capacity to bind and
promote endocytosis of oxLDL [56]. It has also been implicated in the
formation of foam cells [57]. CD36 was demonstrated to cooperate
with TLR2 in sensing bacteria and bacterial ligands, acting as a coreceptor for the induction of mycobacterial uptake and cytokine
generation [28]. We showed that BCG triggered increased expression
of CD36. BCG-induced PPAR expression, lipid body formation and
PGE2 formation, but not cytokine production, were signicantly inhibited by CD36 neutralizing antibodies. The involvement of CD36 was
further conrmed by the impairment of BCG-induced lipid body
formation in vitro and in vivo in macrophages from CD36 decient
animals, suggesting that host cell lipid metabolism alteration induced
by infection is a result of synergic interactions between CD36 and

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Fig. 6. Raft disruption inhibits both lipid body formation and PGE2 production, but not TNF- synthesis induced by M. bovis BCG. Peritoneal macrophages were treated with vehicle, lipin
(10 M) (A) or methyl--cyclodextrin (Sigma) (10 M) (B) for 30 min, and then infected with BCG (MOI 1:1) for 24 h. Lipid body counting in peritoneal macrophages with different
treatments after osmium staining (A and B). (C) PGE2 production and (D) TNF- synthesis in peritoneal macrophages infected or not with BCG and treated with vehicle or methyl-cyclodextrin a. Each bar represents the mean SEM from 3 independent pools of 10 animals each. Differences between control and infected groups are indicated by asterisks
(p b 0.05). + represents differences between BCG and BCG in the presence of lipin, # represents differences between BCG and BCG in the presence of methyl--cyclodextrin.

TLR2. Indeed, impairment of either TLR2 or CD36 inhibited BCGinduced PPAR expression and lipid droplet formation in macrophages.
Accordingly, the co-immunoprecipitation of CD36 and TLR2 in cells
activated with M. bovis BCG infection in vitro suggests that heterodimerization occurred between TLR2 and CD36 and signaling downstream of TLR2/CD36 trigger pathways of increased PPAR expression
and lipid metabolism regulation in macrophages during mycobacterial
infection. Corroborating with the observations that increased PPAR
expression and lipid body biogenesis is associated with detrimental
ability of host cells to control mycobacteria growth acting as an escape
mechanism of intracellular pathogens, Hawkes et al, [58] demonstrated
that CD36 deciency confers resistance to mycobacterial infection.
These authors demonstrated that reduced intracellular survival of mycobacteria in the CD36/ macrophage, as well as, a role of CD36 in the
cellular events involved in granuloma formation that promote early
bacterial expansion and dissemination.
BCG-induced lipid formation was only halved in CD36-decient
cells, indicating that some other co-receptors might be involved and
that compensatory gene expression might occur in genetically decient
mice. Indeed, partial redundancy of the different pattern recognition
receptors was documented and a double deciency in scavenger
receptors CD36 plus SR-A had no marked effect on the control of
M. tuberculosis infection [59].
The mechanisms by which CD14 contributes as a co-receptor to
promote TLR2 signaling are poorly understood. TLR2 is activated by a
variety of bacterial products, including some that depend on CD14 for
sensitive responses. CD14 may participate in trafcking and/or increasing bioavailability of TLR2 ligands [55,60]. Of note, this receptor
was shown to be highly concentrated in lipid raft microdomains of
monocyte lineage [61]. In addition, Nielsen et al. (2008) [54] demonstrated that signaling in response to lipoteichoic acid, preferentially

Please cite this article as: P.E. Almeida, et al., Differential TLR2 downstream signaling regulates lipid metabolism and cytokine production
triggered by Mycobacterium bovis BCG infection, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbalip.2013.10.008

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occurs at the plasma membrane and requires CD36 and CD14 as coreceptors for TLR2. Corroborating with these data, Hajishengallis et al.
have shown in an elegant experiment the induction of a multireceptor
complex in lipid rafts, comprising TLR2, CD14 and CD11b/CD18 upon
stimulation with LPS from Porphyromonas gingivalis and mbriae [62].
Whether similar multireceptor complex in lipid rafts are formed after
mycobacterial infection needs further investigation.
Here, we demonstrate that, beyond TLR2 and CD36, blockade of CD14
and CD11b/CD18 also inhibited M. bovis BCG-induced lipid droplet
formation and PGE2 production, suggesting that in addition to CD36
other TLR2 co-receptors may regulate mycobacterial infection-triggered
alterations in host lipid metabolism. CD36, CD11b/CD18, and CD14 are
all lipid-raft-resident molecules [28], and raft signaling compartmentalization may modulate TLR mechanism of activation and downstream
signaling. Membrane lipid rafts are enriched in cholesterol and play an
important role as signaling platforms. Spatial concentration of receptors
on the membrane can allow rapid and efcient coupling with ligands
and effectors of the intracellular signaling system [63]. Lipid rafts do
not only facilitate receptor interactions by compartmentalization of
molecules on the plasma membrane, but also seem to play a role in the
intracellular activation pathways during mycobacterial infections. Here,
we show that during M. bovis BCG-infection of macrophages, stability
of rafts is important for lipid body formation and PGE2 production,
suggesting that TLR2 and co-receptors interact in these membrane
domains to modulate host cell lipid metabolism.
In conclusion, we uncovered novel connections between TLR2/CD36
activation, in association with CD11b/CD18 and CD14, and PPAR
expression and activation. Moreover, our results suggest that TLR2

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Fig. 7. TLR2/CD36 heterodimers divert intracellular signaling toward increased PPAR expression and lipid accumulation during M. bovis BCG infection. Mycobacterium bovis BCG infection
triggers different TLR2-dependent intracellular signaling cascades with PPAR and NF-B activation. The CD36-TLR2 cooperation and signaling compartmentalization within rafts, with
participation of both CD14 and CD11b/CD18, diverts host response signaling with increased expression and activation of PPAR through NF-B-independent pathways, leading to
increased macrophage lipid accumulation and down-modulation of macrophage response.

signaling compartmentalization within rafts is essential for myco- 528

bacterial induced regulation of host lipid metabolism but not for pro- 529
inammatory cytokine production (Fig. 7).
530

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