of April 13, 2017.This information is current as

Associated with Bacterial PersistenceCells: Immunoinflammatory Platforms

Infected SchwannMycobacterium leprae-TLR6-Driven Lipid Droplets in

N. Sarno, Maria Cristina V. Pessolani and Patricia T. BozzaEuzenirD'Avila, Luciana S. Rodrigues, Roberta O. Pinheiro,

Katherine A. Mattos, Viviane G. C. Oliveira, Heloisa

http://www.jimmunol.org/content/187/5/2548doi: 10.4049/jimmunol.1101344August 2011;

2011; 187:2548-2558; Prepublished online 3J Immunol 

MaterialSupplementary

4.DC1http://www.jimmunol.org/content/suppl/2011/08/04/jimmunol.110134

Referenceshttp://www.jimmunol.org/content/187/5/2548.full#ref-list-1

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The Journal of Immunology

TLR6-Driven Lipid Droplets in Mycobacteriumleprae-Infected Schwann Cells: ImmunoinflammatoryPlatforms Associated with Bacterial Persistence

Katherine A. Mattos,*,1 Viviane G. C. Oliveira,*,1 Heloisa D’Avila,† Luciana S. Rodrigues,*

Roberta O. Pinheiro,‡ Euzenir N. Sarno,‡ Maria Cristina V. Pessolani,*,2 and

Patricia T. Bozza†,2

The mechanisms responsible for nerve injury in leprosy need further elucidation. We recently demonstrated that the foamy

phenotype of Mycobacterium leprae-infected Schwann cells (SCs) observed in nerves of multibacillary patients results from the

capacity of M. leprae to induce and recruit lipid droplets (LDs; also known as lipid bodies) to bacterial-containing phagosomes. In

this study, we analyzed the parameters that govern LD biogenesis by M. leprae in SCs and how this contributes to the innate

immune response elicited by M. leprae. Our observations indicated that LD formation requires the uptake of live bacteria and

depends on host cell cytoskeleton rearrangement and vesicular trafficking. TLR6 deletion, but not TLR2, completely abolished the

induction of LDs by M. leprae, as well as inhibited the bacterial uptake in SCs. M. leprae-induced LD biogenesis correlated with

increased PGE2 and IL-10 secretion, as well as reduced IL-12 and NO production in M. leprae-infected SCs. Analysis of nerves

from lepromatous leprosy patients showed colocalization of M. leprae, LDs, and cyclooxygenase-2 in SCs, indicating that LDs are

sites for PGE2 synthesis in vivo. LD biogenesis Inhibition by the fatty acid synthase inhibitor C-75 abolished the effect ofM. leprae

on SC production of immunoinflammatory mediators and enhanced the mycobacterial-killing ability of SCs. Altogether, our data

indicated a critical role for TLR6-dependent signaling in M. leprae–SC interactions, favoring phagocytosis and subsequent

signaling for induction of LD biogenesis in infected cells. Moreover, our observations reinforced the role of LDs favoring

mycobacterial survival and persistence in the nerve. These findings give further support to a critical role for LDs in M. leprae

pathogenesis in the nerve. The Journal of Immunology, 2011, 187: 2548–2558.

Leprosy is an ancient infectious scourge caused by Myco-bacterium leprae that remains a public health problem inseveral countries (1). Leprosy constitutes an excellent

model for deciphering the mechanisms that regulate innate and

adaptive immunity in humans, because the disease presents asa clinical spectrum that correlates with the level of the immune

response to the pathogen (2–4). At one end of the spectrum,

individuals with polar tuberculoid leprosy (TT) develop a strong

cellular immune response against M. leprae that restrains the

growth of the pathogen. At the opposite end of the spectrum,

failure of the immune system to kill or inhibit M. leprae is a

conspicuous characteristic of polar lepromatous leprosy (LL). In

the absence of antibiotic treatment, a large proportion of LL

patients suffer lifelong debilitating disease in which the myco-

bacteria are able to reproduce to very high numbers, mainly inside

dermal macrophages and Schwann cells (SCs) of the peripheral

nerve system. In these patients, M. leprae can live for years inside

cells and has developed various, but poorly understood, mecha-

nisms for escaping the host immune defenses. Markedly, the

presence of collections of highly infected macrophages/SCs ac-

cumulating large amounts of lipids, described as foamy cells,

are characteristic in LL, but not in TT, lesions (5). The mecha-

nisms that disturb the lipid homeostasis of M. leprae-infected

cells remain nebulous. These lipids were initially thought to be

derived from M. leprae, such as phthiocerol dimycocerosate/

dimycocerosate and phenolic glycolipid-1 (PGL-1) (6, 7). How-

ever, very recently, a more detailed analysis of the lipid metabo-

lism in LL lesions indicated that the foamy aspect is due mostly to

the accumulation of host-derived lipids, such as oxidized phos-

pholipids and cholesterol ester (8, 9).These lipids were shown to

be stored in nonmembrane-bound cytoplasmic organelles known

as lipid bodies or lipid droplets (LDs) (9).LDs are dynamic lipid-storage organelles found in all cell types

that constitute rapidly mobilized lipid sources for many important

*Laboratory of Cellular Microbiology, Oswaldo Cruz Institute, Oswaldo Cruz Foun-dation, Rio de Janeiro, RJ 21045-900, Brazil; †Laboratory of Immunopharmacology,Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, RJ 21045-900,Brazil; and ‡Laboratory of Leprosy, Oswaldo Cruz Institute, Oswaldo Cruz Founda-tion, Rio de Janeiro, RJ 21045-900, Brazil

1K.A.M. and V.G.C.O. contributed equally to this work as co-first authors.

2M.C.V.P. and P.T.B. contributed equally to this work as co-senior authors.

Received for publication May 9, 2011. Accepted for publication July 1, 2011.

This work was supported by the Conselho Nacional de Desenvolvimento Cientıficoe Tecnologico, Programa Estrategico de Apoio a Pesquisa em Saude-FundacaoOswaldo Cruz, the Programa de apoio a Nucleos de Excelencia, and the Fundacaode Amparo a Pesquisa do Estado do Rio de Janeiro. P.T.B. is a Guggenheim Fellow.K.A.M. and V.G.C.O. were recipients of a fellowship from the Fundacao de Amparoa Pesquisa do Estado do Rio de Janeiro.

Address correspondence and reprint requests to Dr. Patricia T. Bozza or Dr. MariaCristina V. Pessolani, Laboratorio de Imunofarmacologia, Instituto Oswaldo Cruz,Fundacao Oswaldo Cruz, Avenue Brasil 4365, Manguinhos, Rio de Janeiro, RJ21045-900, Brazil (P.T.B.) or Laboratorio deMicrobiologia Celular, Instituto OswaldoCruz, Fundacao Oswaldo Cruz, Avenue Brasil 4365, Manguinhos, Rio de Janeiro, RJ21045-900, Brazil (M.C.V.P.). E-mail addresses: pbozza@ioc.fiocruz.br (P.T.B.) orcpessola@ioc.fiocruz.br (M.C.V.P.)

The online version of this article contains supplemental material.

Abbreviations used in this article: ADRP, adipose differentiation-related protein;BCG, bacillus Calmette-Guerin; COX-2, cyclooxygenase-2; CytB, cytochalasin B;CytD, cytochalasin D; EIA, enzyme immunoassay; ER, endoplasmic reticulum; FAS,fatty acid synthase; LAM, lipoarabinomannan; LD, lipid droplet; LL, lepromatousleprosy; MFI, mean fluorescence intensity; MOI, multiplicity of infection; MSM,Mycobacterium smegmatis mc2 155; ORO, oil red O; PGL-1, phenolic glycolipid-1; SC, Schwann cell; TT, tuberculoid leprosy; WT, wild-type.

Copyright� 2011 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/11/$16.00

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biological processes. Contemporary evidence points to LDs asinflammatory organelles involved in the synthesis and secretionof inflammatory mediators (10). Our group recently demonstratedthat the LDs formed in response to bacillus Calmette-Guerin(BCG) and M. leprae constitute sites for eicosanoid synthesis,ultimately leading to increased production of PGE2 by infectedmacrophages (9, 11, 12). PGE2 is a potent immune modulator thatdownregulates Th1 responses and bactericidal activity toward in-tracellular organisms (12–14). Our previous data also indicatedthat Mycobacterium-induced LD formation in infected macro-phages depends on bacterial recognition via TLRs. Accordingly,we showed recently that TLR2 and TLR6 pathways are prefer-entially activated during LD biogenesis triggered by M. bovisBCG and M. leprae infection in macrophages (9, 11, 12, 15).SCs are capable of secreting a vast array of cytokines and in-

flammatory mediators (i.e., IL-1, IL-6, IL-8, TNF-a, IL-10, IL-12,PGs, TGF-b, and NO) and actively participate in the immunoin-flammatory responses in neuritic processes (16–21). We showedrecently that M. leprae induces LD biogenesis and accumulationin bacterial-containing phagosomes in SCs and is responsible, atleast in part, for originating foamy degeneration of M. leprae-infected SCs in LL nerves (22). Accordingly, as was proposed formacrophages in the context of dermal lesions (9, 23), it is rea-sonable to speculate that the lipid-storage phenomenon observedin M. leprae-infected SCs is an important contributor to theimmunoinflammatory function of these cells in LL nerve lesions.In the current study, we investigated the involvement of TLRs inthe induction of LDs by M. leprae in SCs. Moreover, we analyzedthe capacity of M. leprae-infected SCs to secrete immunoin-flammatory mediators, along with the manner in which this se-cretion is related to LD formation. The data indicated that host celllipid metabolism and the innate immune response to M. lepraeinfection are intimately related in SCs, contributing to bacterialsurvival in the nerve and disease progression.

Materials and MethodsPatients and clinical specimens

LL patients were classified according to the criteria of Ridley (24). Nervebiopsy specimens (6-mm diameter) were obtained at the time of diagnosis.The specimens were snap-frozen in liquid nitrogen and stored at 270˚Cuntil sectioned. The procedures described in this work were approved bythe Oswaldo Cruz Foundation Ethics Committee. Written informed con-sent was obtained from each patient.

Human SC cultures

Human primary SCs were isolated from peripheral nerve tissues andprovided by Dr. Patrick Wood (University of Miami, through the OrganProcurement Organization, University of Miami, Miami, FL). The purity ofthese cultures was .95% by labeling with Ab to Ca2+-binding protein(S100; DakoCytomation, Glostrup, Denmark). These cells were cultured aspreviously described (22). Alternatively, the ST88-14 tumor cell line wasused for in vitro assays. ST88-14 was established from malignantschwannomas (neurofibrosarcomas) from patients with neurofibromatosistype 1 and was generously donated by Dr. J.A. Fletcher (Dana FarberCancer Institute, Boston, MA). The cells were grown in RPMI 1640 me-dium (Invitrogen) supplemented with 2% FBS and 20 mM L-glutamate.SCs were suspended in culture medium without antibiotics and cultured at33˚C for 24 h prior stimuli.

Isolation of mouse primary SCs

C57BL/6 (B6) mice were obtained from the Oswaldo Cruz Foundationbreeding unit. TLR2 and TLR6 knockout mice on a homogeneous B6background were donated by Dr. S. Akira (Osaka University, Osaka, Japan).Mouse SCs were prepared from nerve explants from adult mice, as de-scribed previously (25). The purity of SCs was assessed by microscopicexamination after immunostaining with anti-S100 Abs, which revealed.95% S100+ cells. These highly purified SCs were seeded as described

above for the human SCs. The protocols were approved by the AnimalWelfare Committee of the Oswaldo Cruz Foundation.

Mycobacteria strains and staining

M. leprae prepared from footpads of athymic nu/nu mice was kindlyprovided by Dr. J. Krahenbuhl (National Hansen’s Disease Program,Laboratory Research Branch, Louisiana State University, Baton Rouge,LA through American Leprosy Missions, the Order of St. Lazarus, and theNational Institute of Allergy and Infectious Diseases, Bethesda, MD;Contract No. 155262). Part of the M. leprae suspension was killed bygamma irradiation. Mycobacterium smegmatis mc2 155 (MSM) andMycobacterium bovis BCG Pasteur (ATCC 35734) were grown at 37˚Cin Middlebrook 7H9 base ADC enrichment medium (Becton Dickinson,Franklin Lakes, NJ), supplemented with 0.2% glycerol, 0.05% Tween 80(Sigma). Prior to interaction assays with SCs, bacteria were stained witha PKH26 Red Fluorescence Cell Linker Kit (Sigma), according to themanufacturer’s instructions.

Mycobacterium–SC interaction assays

SCs were suspended in culture medium without antibiotics and cultured ata density of 1 3 105 cells/well on 12-well plates precoated with laminin-1for flow cytometric assays and at 7 3 104 cells/well on 24-well platescontaining poly-L-lysin (Sigma)/laminin-coated glass coverslips for mi-croscopy experiments. M. leprae was added to the culture at a multiplicityof infection (MOI) of 50 bacillus/cell (50:1) for 2, 24, and 48 h. In someexperiments, cells were also infected with live BCG (MOI 50:1) or liveMSM (MOI 50:1). Alternatively, SCs were stimulated with LPS (500 ng/ml), mannose-capped lipoarabinomannan (LAM) and non-capped LAM(500 ng/ml), and PGL-1 (500 ng/ml) kindly provided by Dr. Patrick J.Brennan (Colorado State University, Fort Collins, CO; National Institutesof Health/National Institute of Allergy and Infectious Diseases ContractNo. 1Al25469). Cells were also treated with fluorescent latex beads (50:1;Polysciences, Warrington, PA) as phagocytosis control, as well as recom-binant human TNF-a (25 ng/ml) as inflammatory stimulus.

M. leprae–SC interaction assays were also conducted in the presence ofthe following pharmacological agents: taxol and colchicin (1 mM; Sigma),microtubule-binding agents; cytochalasin D (CytD) and cytochalasin B(CytB) (20 mM; Sigma), actin filament-polymerization inhibitors; bre-feldin (2 mg/ml) and monensin (5 mM), known to disrupt the organizationof the endoplasmic reticulum (ER)–Golgi complex; LY294002 (10 mM;BIOMOL Research Laboratories, Plymouth Meeting, PA) and wortmannin(1 mM; Sigma), PI3K inhibitors; NS-398 (N-(2-cyclohexyloxy-4-nitro-phenyl) methane sulfonamide) (1 mM; BIOMOL Research Laboratories),the nonesteroidal drug that inhibits cyclooxygenase-2 (COX-2) and LDformation; and C-75 (1 mM; Sigma), a specific fatty acid synthase (FAS)inhibitor. In these assays, cells were pretreated with the drug or DMSOalone (vehicle control) for 30 min prior to M. leprae addition; after treat-ment, cells were washed and infected for an additional 48 h in mediumwith 2% serum. After incubation, cell viability was .90%, as revealed bythe trypan blue (Sigma) exclusion assay. LD induction was assessed byflow cytometry, and the supernatants were collected, centrifuged for 5 minat 3000 3 g, filtered (0.2 mm), and then frozen until use. All experimentswere performed at 33˚C in 5% CO2 atmosphere.

LD staining and quantification

Cells adhering to coverslips were fixed in 4% paraformaldehyde in Ca2+/Mg2+-free HBSS (pH 7.4) for 10 min, and LDs were analyzed using dif-ferent markers—osmium tetroxide, oil red O (ORO), or BODIPY 493/503dye—as described (22). Coverslips were then mounted in 20% glyceroland 1% n-propyl gallate in PBS (pH 7.8). The morphology of the fixedcells was observed, and LDs were enumerated at 1003 in 50 consecutivelyscanned cells. Alternatively, the area occupied by LDs per cell was esti-mated following LD staining with fluorescent ORO, or LD analysis wasperformed by flow cytometry. Corresponding mean fluorescent intensity(MFI) (green) values were obtained from the histograms. Bacterial associa-tion with cells was measured in the fluorescence channel 2 by PKH26-labeled bacteria. The percentage of eukaryotic cells with bacterial associa-tion was determined from the gated red cells. Index of bacterial associationis expressed as the percentage of cells taking up PKH26–M. leprae.

Immunohistological analysis

Immunohistochemical procedures to detect LD organelles andM. leprae inSCs from LL and healthy nerve were performed, as described (22). Briefly,tissue sections were thawed on sylane-precoated slides and submitted toimmunostaining protocols and to acid fast bacilli with Wade staining,following an standard protocol (26). Immunostaining was performed by

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incubation with primary Abs anti-adipose differentiation-related protein(ADRP; Research Diagnostics, Concord, MA), anti-S100, and anti-M.leprae (anti-whole bacteria or anti-LAM) kindly provided by Dr. Patrick J.Brennan (Colorado State University, Fort Collins, CO; National Institutesof Health/National Institute of Allergy and Infectious Diseases ContractNo. 1Al25469), as well as anti–COX-2 (C terminus; Oxford BiomedicalResearch, Oxford, MI). Two-color immunofluorescence staining was per-formed by serial incubation of sections with combinatorial Abs againstS100 (1:100) and ADRP (1:25), M. leprae (1:25) and ADRP (1:25), COX-2 (1:20) and ADRP (1:25), and M. leprae (1:25) and COX-2 (1:20). Thecorresponding isotype Abs were used as negative controls.

Image acquisition in confocal microscopy

Preparations were examined using a Zeiss Axio Observer microscopeequipped with a Plan Apo 40 or 100 objective (Carl Zeiss, Thornwood, NY)and a CoolSNAP-Pro CF digital camera in conjunction with Axion VisionVersion 4.7.2 software (Carl Zeiss). The images were edited using Axio-Vision software. To determine the colocalization of fluorescent signals,images were acquired with an LSM 510 Zeiss confocal microscope. Imageswere acquired, colored, and merged using LSM 510 Zeiss software. Argonand neon-helium lasers (Mellets Griot) emitting at 488, 543, and 633 nmwere used. Pictures of 20–40 confocal planes through the cell (z-stack),with a step size of 0.2–0.3 mm, were taken with a 633 objective every 30 sfor 5–10 min using LSM image software. For some experiments, z-stacksof images were captured, processed, and rendered using the LSM imagingsystem (Carl Zeiss).

Measurement of PGE2

PGE2 concentration was measured in cell-free supernatants via an enzymeimmunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI), and the as-says were conducted according to the manufacturer’s protocol.

Cytokine measurement

IL-12 p40 and IL-10 concentrations in culture supernatants were deter-mined by ELISA using the specific Duo set kit (R&D Systems, Minne-apolis, MN), according to the manufacturer’s instructions. The sensitivityof these assays was 2 pg/ml.

Nitrite determination

NO synthesis was assessed by measuring the accumulation of nitrite in cellsupernatants, as detected by the Griess reagent (Sigma). Absorption wasmeasured at 550 nm, and nitrite concentrations were determined bycomparison with OD of the NaNO2 standards.

M. leprae viability

A live/dead-staining protocol, based on the LIVE/DEADBaclight BacterialViability Kit (Invitrogen), was applied to determine the percentage of M.leprae viability in SCs treated with NS-398, C-75, or vehicle. In brief, SCs(2 3 106 cells/well) were treated with drugs or vehicle for 30 min at 33˚Cand then infected with M. leprae (50:1) for 72 h in RPMI 1640 cell culturemedium. SC cultures were washed three times with PBS to remove anynoninternalized bacteria and lysed with 0.1% saponin. Bacterial-containingsuspensions were labeled with the LIVE/DEAD kit, and the percentages oflive and dead bacteria were determined by flow cytometry, according tothe manufacturer’s instructions. Flow cytometry measurements were per-formed on a FACSCalibur and analyzed with CellQuest software (bothfrom BD Bioscience).

Statistical analysis

Data analysis was performed using the GraphPad InStat program (GraphPadSoftware, San Diego, CA), and the statistical significance (p , 0.05) wasdetermined by the Student t test.

ResultsIn vitro induction of LDs by M. leprae in SCs depends onbacterial viability

We showed previously that the foam phenotype in infected SCs,described as a characteristic feature seen in nerve lesions of LLpatients, is derived from lipid accumulation in LD organelles, asobserved in in vitro and in vivo studies (22). Double labeling ofADRP/S100 and ADRP/M. leprae showed that there is a pre-dominant association of M. leprae with these lipid-containingvacuoles in foamy SCs, both in the nerves of LL patients (Fig.

FIGURE 1. M. leprae induces LD formation in

SCs. Fluorescent confocal images showing LDs in

nerve biopsy from a LL patient (A) and in in vitro-

infected SCs (B). LDs were labeled with a mAb

specific for ADRP (green) (A) or BODIPY (B); SCs

were labeled with anti-S100 (A); and the bacterial

presence was observed in red usingM. leprae-specific

Ab (A) or PKH26-labeled M. leprae (B). Nuclei

(blue) were labeled with TO-PRO-3. Scale bars, 20

mm. Original magnification363. C–F, The LD levels

were monitored by flow cytometry using BODIPY.

The bar graphs show MFI values of BODIPY. Dose-

response (C) and time-course (D) for LD biogenesis

induced by M. leprae (ML) infection. E, LD bio-

genesis was dependent on bacterial viability. SCs

were exposed to mycobacterial Ags (500 ng/ml) and

LPS (500 ng/ml). F, Cells were stimulated with

a pathogenic mycobacteria, M. bovis BCG, and

a nonpathogenic mycobacteria, M. smegmatis. All

experiments were performed using an MOI of 50;

and LDs were determined after 48 h, except for the

dose-response and temporal-course experiments.

Data are representative of four independent experi-

ments and are expressed as the mean 6 SD of trip-

licates. *p , 0.05, stimulated versus control groups.

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1A) and in vitro cultured SCs (Fig. 1B). M. leprae-induced LDbiogenesis in SCs was shown to be dose and time dependent,reaching significant levels at an MOI of 50 after 24 and 48 h ofincubation (Fig. 1C, 1D). Because M. leprae at an MOI of 50 for48 h turned out to be an optimal condition for LD induction, thisdose and incubation time were adopted in the remaining experi-ments. Given the fact that both live and dead M. leprae are foundin affected nerves of leprosy patients, we next investigatedwhether the capacity to modulate LD formation was dependent onbacterial viability. Primary SCs were stimulated in vitro in eitherlive or irradiated-killed bacteria, and LD formation was monitoredby flow cytometry (Fig. 1E). Live M. leprae-infected SCs showed∼4- and 2-fold higher values of the MFI of BODIPY probe incomparison with dead M. leprae-treated cells and untreated cells,respectively (Fig. 1E). Alternatively, LDs were also enumeratedafter osmium tetroxide staining, and identical results wereobtained (data not shown). Similar results were obtained with theST88-14 schwannoma cell line (data not shown). Accordingly, themycobacterial glycolipids mannose-capped LAM, non-cappedLAM, and PGL-1 were unable to induce LD biogenesis in SCs, aswell as LPS, a potent inductor of LDs in macrophages (Fig. 1E).These data strongly reinforced that different from macrophages,M. leprae viability is necessary to actively upregulate the bio-genesis of LDs in SCs.Next, we investigated the capacity of other viable mycobacterial

species, such as the pathogenic M. bovis BCG and the avirulent

M. smegmatis (MSM), to induce LD in SCs, given the fact thatM. bovis BCG is a good inducer of LD formation in macrophages(12). Interestingly, only M. leprae was able to induce LD forma-tion, whereas BCG and MSM showed LD levels similar to thosefound in unstimulated cells (Fig. 1F). Moreover, SCs incubatedwith fluorescent latex beads showed only basal levels of LDs,despite the fact that they were readily internalized (22), indicatingthat phagocytosis itself is not sufficient to induce LD formationin SCs.

Only SCs bearing bacteria show increased numbers of LDs

In a previous study with macrophages, we showed that, in M.leprae-treated cultures, the LDs were significantly induced both inhuman macrophages bearing bacterium and in cells with no bac-terium. We also showed that soluble factors are secreted upon M.leprae–macrophage interaction and that these factors are capableof inducing LD formation in uninfected cells (9). We then in-vestigated whether the effect of M. leprae on SC LD biogenesisdescribed in this article follows the characteristics previously seenin macrophages. To this end, SCs were exposed to fluorescent-labeled M. leprae. Untreated cells or those exposed to PKH-labeled M. leprae were cultured for 48 h; after staining withBODIPY, the induction of LDs was analyzed by flow cytometryand fluorescence microscopy.Fluorescence images clearly showed increased numbers of LDs

only in cells bearing bacteria (M. leprae+, PKH-labeled M. leprae)

FIGURE 2. LD induction is dependent on M. leprae (ML) phagocytosis by SCs. A, Fluorescent images of SC cultures showing LDs only in cells bearing

M. leprae. PKH26-labeledM. leprae in red; BODIPY-stained LDs in green; and TO-PRO-3–labeled nuclei in blue. Scale bar, 20 mm. B, Dot plots represent

PKH26 fluorescence versus BODIPY fluorescence of human SCs infected or not with PKH26-labeled M. leprae. C, A separate analysis was performed in

cells with and without internalizedM. leprae. The induction of LDs byM. lepraewas compared with control cells without bacterial stimulation. MFI values

of BODIPYare expressed in bar graphs. Results from five representative experiments are shown. *p , 0.05. D and E, Effect of cytoskeleton rearrangement

on LD formation. SCs were infected with M. leprae (MOI = 50) for 48 h after CytD treatment for 30 min. D, The association capacity of the bacteria was

assayed by flow cytometric analysis, quantifying the relative percentages of cells with PKH26-labeledM. leprae internalized by gating the subpopulation of

cells with orange fluorescence. M. leprae-induced LD formation was dependent on bacterial phagocytosis. E, LD formation was determined by MFI of

BODIPY by flow cytometric analysis. The addition of CytD significantly reduced both LD biogenesis and the bacterial-association index. Data are rep-

resentative of five independent experiments. F, Conditioned medium (CM) from M. leprae-infected SCs did not induce LD biogenesis in uninfected cells.

*p , 0.05, control group versus M. leprae-treated cell group; +p , 0.05 between M. leprae-treated SCs pretreated or not with CytD.

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and not in cells without bacterial internalization (M. leprae2),suggesting a close relationship between phagocytosis of M. lepraeand the induction of LD formation (Fig. 2A). Similar results wereobtained when LD biogenesis was analyzed by flow cytometry(Fig. 2B). A representative dot plot showed that M. leprae in-fection induced high levels of BODIPY fluorescence comparedwith control. Fig. 2C shows MFI values of BODIPY observed incells with red fluorescence (cells with internalized M. leprae) andcells without red fluorescence (cells without internalized M. lep-rae). LDs were significantly induced only in SC populationsbearing M. leprae in comparison with the population withoutbacterial association and untreated cells (MFI of 167.4 6 10.84,67.5 6 6.3, and 59.1 6 6.6, respectively). An identical result wasobtained when ST88-14 SCs were stimulated with M. lepraein vitro (MFI of 109.2 6 2.7 in untreated cells, 103.1 6 0.5 incells without internalized M. leprae, and 333.2 6 22.6 in cellswith internalized M. leprae).To investigate whether bacterial phagocytosis is required to elicit

LD formation, SCs were pretreated with the actin polymerizationinhibitor (CytD) before M. leprae infection. The inhibitory effectof CytD on phagocytosis was monitored by flow cytometry usingbacteria prestained with PKH26. Fig. 2D shows that CytD-treatedcells had ∼90% impaired phagocytic ability. Fig. 2E shows thatM. leprae was unable to induce LDs in CytD-treated cells (MFIof 88.10 6 4.47 in M. leprae-treated cells versus 95.6 6 2.8 inuninfected CytD-treated cells). Moreover, conditioned mediumderived from M. leprae-infected SCs was not able to inducethe biogenesis of these organelles in cultures not infected withM. leprae, in contrast to our previous observation of M. leprae-stimulated macrophages (9) (Fig. 2G). These data suggested thatM. leprae internalization is necessary for triggering the signalingpathways that will culminate in LD formation, although othercytoskeleton-dependent mechanisms, in addition to phagocytosisof Mycobacterium, might participate in the mechanisms involvedin LD biogenesis.

ER–Golgi transport and a functional cytoskeleton are essentialfor M. leprae-induced LD biogenesis

Several lines of evidence suggest that LDs are derived from theER–Golgi apparatus pathway and that their formation dependson the microtubule system of the cell (27, 28). To better un-derstand the mechanisms behind the LD formation induced byM. leprae in SCs, a series of experiments was conducted usingpharmacological inhibitors of pathways previously shown toparticipate in LD biogenesis in other cell types (9, 29, 30) (TableI). We inhibited ER–Golgi transport with brefeldin A and mon-esin and observed the significant reduction in formed LDs. Inaddition, we examined the effect of CytB, an inhibitor of actinfilament polymerization at barbed ends. CytB-treated SCsshowed a decrease in LD formation in cells treated with liveM. leprae compared with untreated CytB cells at 48 h post-infection, replicating the inhibitory effects of the actin-disruptingdrug CytD. In addition, the microtubule-binding drugs taxol andcolchicin showed inhibitory effects on LD biogenesis. Finally,the role of PI3K signaling, known to be involved in cytoskeletalrearrangements in Mycobacterium-infected macrophages (31),was investigated. Treatment of SCs with LY294002, an inhibitorof PI3K, blocked LD biogenesis in M. leprae-infected SCs (TableI). This effect was confirmed using wortmannin, an alternativespecific PI3K inhibitor (data not shown). Taken together, theseresults suggested that M. leprae-induced LD biogenesis in SCs isan ER–Golgi and actin- or microtubule-dependent event, con-firming the important role of vesicular traffic in the nascent LDprocess.

TLR6, but not TLR2, is essential for M. leprae-induced LDbiogenesis in SCs

As reported previously, the recognition of M. leprae by the TLR2/TLR6 heterodimer is an essential step leading to LD formation ininfected macrophages (9). We investigated the functional role ofthese receptors on LD formation in the context of SCs. SCs iso-lated from mouse deleted of TLR2 (TLR22/2) or TLR6 (TLR62/2)were infected withM. leprae, and LD biogenesis was evaluated byflow cytometry and microscopic analysis. In marked contrast tomacrophages, TLR22/2 SCs showed M. leprae-induced LD levelscomparable to wild-type (WT) SCs (Fig. 3A–D). In contrast, LDformation was reduced to basal levels in TLR62/2 SCs (Fig. 3A–D). Moreover, in contrast to TLR22/2 SCs, M. leprae inter-nalization was partially impaired in TLR62/2 SCs, suggestinga role for TLR6 as a bacterial phagocytic receptor in these cells(Fig. 3E). These data indicated a critical role for TLR6, in-dependent of its association with TLR2, in M. leprae–SC inter-actions, favoring phagocytosis and subsequent signaling for in-duction of LD biogenesis in infected cells.Interestingly, immunofluorescence images and flow cytometry

analysis of the same cultures indicated the induction of LDs onlyin cells bearingM. leprae (Supplemental Fig. 1A, 1B), as observedbefore in human SCs (Fig. 2A). The profile of LD formation inTLR22/2 SCs, with or without bacteria, was identical to thatobserved in WT cells (Supplemental Fig. 1B). Of note, TLR6deletion caused a significant decrease in LD formation, even incells with internalized M. leprae (Supplemental Fig. 1B), rein-forcing the involvement of TLR6, beyond phagocytosis, as a sig-naling platform necessary for M. leprae-induced LD formation inSCs. Together, this set of results corroborated the idea that themodulation of LD formation in response to M. leprae in SCsrequires bacterial phagocytosis and that TLR6 plays a critical roleas a phagocytic receptor and a trigger of downstream signalsnecessary for LD biogenesis.

LDs are sites of eicosanoid generation in in vivo-infectedfoamy SCs

To investigate whether LDs are involved in PGE2 production inSCs during the natural course of leprosy, cross-sections of nervebiopsies from three LL patients were immune stained with specificAbs that recognize the inducible cyclooxygenase, COX-2, the LDmarker, ADRP, the SC, marker S100, as well as M. leprae, usingan Ab that recognizes LAM, a component of the mycobacterial

Table I. Effect of inhibition of the vesicular traffic onM. leprae-induced LD biogenesis

Stimuli Treatment LD Formationa

Control Vehicle (DMSO) 186.6 6 35.57M. leprae infection Vehicle (DMSO) 625.2 6 17.2**

Disruption of Golgi–ERBrefeldin (2 mM) 307.1 6 23.34*Monesin (5 mM) 174.3 6 3.49*

Actin filament destabilizationCytD (20 mM) 275.6 6 48.83*CytB (20 mM) 90.29 6 3.96*

Microfilament destabilizationTaxol (1 mM) 350.0 6 46.05*Colchicin (1 mM) 376.5 6 40.8*

PI3K inhibitionLY294002 (10 mM) 395.9 6 40.9*

SCs were treated for 30 min before infection with M. leprae (MOI = 50), and LDformation was determined 48 h later by MFI of BODIPY by flow cytometric analysis.

aData are mean 6 SD of five independent experiments.*p , 0.05, M. leprae-stimulated versus control cells; **p , 0.05 between the

different M. leprae-treated cell groups.

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envelope. Fig. 4A shows an infected nerve section stained for acidfast bacilli by the Wade procedure. This image shows SCs witha foamy appearance packed with fuchsin-stained mycobacterialglobi. LL nerve specimens exhibited an intense immune reactivityto COX-2 and M. leprae, confirming the relationship betweenbacterial presence and COX-2 expression (Fig. 4B). Immunoflu-orescent staining for both COX-2 and ADRP demonstrated thatthe COX-2–reactive cells coincide with foamy SCs (Fig. 4C).Worthy of note was the close association between LDs and COX-2in M. leprae-infected SCs (Supplemental Fig. 2). Nerves fromhealthy donors were used as control, showing SCs weakly labeledto ADRP and COX-2 (data not shown). These results reinforcedthe idea that LDs constitute active catalytic organelles of PGE2

synthesis in infected nerves.

M. leprae elicits an innate immune response in infected SCsthat depends on LD formation and TLR6 signaling

Because LDs in leukocytes were shown to be involved in theproduction of inflammatory mediators, we investigated the re-lationship between LDs induced by M. leprae infection and im-

mune response in the context of SCs. As shown in Fig. 5, bothhuman and murine SCs produced significantly higher levels ofPGE2 (Fig. 5A) and IL-10 (Fig. 5B) after 48 h of M. leprae in-fection compared with uninfected cells, coinciding with the timepoint of greatest LD formation. In contrast, M. leprae-infectedSCs produced significantly lower levels of IL-12 (Fig. 5C) and NO(Fig. 5E) compared with the basal levels observed in uninfectedcells. Moreover, M. leprae infection was able to attenuate SCproduction of IL-12 and NO induced by TNF-a, a potent proin-flammatory cytokine (Fig. 5D, 5F). These changes in the pro-duction of immunoinflammatory mediators triggered by liveM. leprae were not observed when cells were treated with deadbacteria (data not shown), coinciding with the inability of deadM. leprae to induce LD formation.Because bacterial phagocytosis was previously shown to be

essential for the induction of LD formation in SCs, we next testedthe effect of CytD on the profile of immunomediators producedby SCs in response to M. leprae. As expected, CytD treatmentcompletely inhibited M. leprae-induced PGE2 (Fig. 5A) and IL-10(Fig. 5B) production. In addition, basal IL-12 and nitrite levelswere not reduced, as observed in untreated cultures (Fig. 5C, 5E).Confirming the results obtained with human SCs, M. leprae

infection induced PGE2 (Fig. 5G) and IL-10 (Fig. 5H) productionand inhibited basal levels of NO generation (Fig. 5I) in murineWT SCs. Moreover, a correlation was observed between theeffects of TLR2 and TLR6 deletion on M. leprae-induced LDformation and inflammatory mediator secretion (Fig. 5G–I). Al-though TLR22/2 SCs showed an identical profile of mediatorsecretion to WT cells in response to M. leprae, it failed to mod-ulate the profile of these mediators in TLR62/2 SCs. These datasuggested a key role for LDs as inflammatory organelles in SCsduring M. leprae infection, with the novel involvement of TLR6signaling independently of TLR2.

FIGURE 4. LD organelles are sites of eicosanoid production in SCs of

LL nerve biopsy. Serial sections of nerve biopsies from LL patients (n = 4)

were analyzed. In situ localization of COX-2 in M. leprae-infected SCs. A,

Wade staining showing foamy SCs with multiple acid-fast bacilli. Scale

bar, 20 mm. B, Double-immunofluorescent labeling with anti-LAM (red)

and anti–COX-2 (green) reveals putative COX-2 production in cells with

a strong bacterial presence. C, COX-2 (red) localization within LD

organelles labeled with ADRP (green) within SCs. Inset: Magnified view

of COX-2/LD colocalization. Nuclei were stained with TO-PRO-3. Scale

bar, 10 mm.

FIGURE 3. TLR6, but not TLR2, is essential for M. leprae-induced LD

biogenesis in SCs. A, Murine WT, TLR22/2, and TLR62/2 SCs were

cultured in the presence of live M. leprae (MOI = 50) for 48 h, and LD

induction was estimated by microscopy after ORO staining. B, The quan-

tification of ORO-stained LDs is plotted as the number of LDs/cell. C,

Measurement of LD area/cell, following LD staining with the fluorescent

ORO dye. D, MFI values for BODIPY. White bars: uninfected cells; gray

bars: M. leprae-infected cells. E, Reduced bacterial association in TLR62/2

SCs compared with TLR22/2 and WT SCs. The phagocytic index was

determined by flow cytometric analysis, quantifying the relative percentages

of cells associated with PKH26-labeled M. leprae. Data are representative

of five independent experiments and represent mean 6 SD of triplicates.

*p, 0.05,M. leprae-stimulated versus control cells; +p, 0.05 between the

different M. leprae-treated cell groups. Scale bar, 10 mm.

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To gain more insight into the role of LDs in the M. leprae-modulated innate immune response in SCs, the effect of the drugsNS-398 and C-75 on PGE2, IL-10, IL-12, and NO production wasinvestigated. NS-398, a nonsteroidal anti-inflammatory drug, andC-75, an inhibitor of FAS, were previously shown to inhibit My-cobacterium-induced LD formation in macrophages (9, 12, 32).As expected, these inhibitors drastically inhibited M. leprae-in-duced LD formation in SCs (Fig. 6A). Next, to confirm the re-lationship between LDs and the innate immune response elicitedby M. leprae in SCs, we pretreated SCs with both drugs, and theprofile of immunoinflammatory mediators was determined. Thesetreatments also completely abolished both M. leprae-inducedPGE2 and IL-10 production in these cells (Fig. 6B, 6C). Addi-tionally, inhibition of the basal levels of NO production was notobserved (Fig. 6E), and in the case of IL-12, instead of inhibition,increased levels of this mediator were produced in the absenceof LD formation (Fig. 6D). These data reinforced the role oflipid metabolism, through LD formation, in modulating the in-nate immune response triggered by M. leprae infection in SCs.

LD biogenesis favors M. leprae intracellular survival in SCs

We showed in a previous study that LDs are promptly recruitedto M. leprae-containing phagosomes in SCs and that impairment

of this recruitment by cytoskeleton drugs decreased the survivalof intracellular bacteria (22). Based on these data, we next in-vestigated whether the inhibition of LD biogenesis by NS-398 orC-75 affected M. leprae intracellular survival. To this end, cellswere treated with the drugs or the vehicle alone, and bacterialviability was monitored by flow cytometry after 72 h of infection,using the LIVE/DEAD Baclight Kit. As shown in Fig. 6F, a sig-nificant decrease in bacterial viability was observed upon in-hibition of LD biogenesis, reinforcing the idea that the modulationof LD formation in SCs constitutes an important pathophysio-logical mechanism triggered by M. leprae to persist in its in-tracellular niche, as proposed in Fig. 7.

DiscussionPathogen-triggered dysregulation of host–cell lipid metabolism areemerging as a key feature in the response to mycobacterial in-fection. Accumulating evidence suggest that modulation of hostlipid metabolism through M. leprae-induced LD formation isimportant in leprosy disease. However, the mechanisms thatgovern M. leprae infection-induced lipid synthesis and lipid ac-cumulation in LDs and the role of LDs in bacterial persistence inthe nerve are poorly understood. Our study offers novel evidencethat M. leprae is able to increase SC lipid accumulation and PGE2

FIGURE 5. The innate immune response of SCs

toward M. leprae (ML) depends on bacterial in-

ternalization and TLR6. Human SCs were pretreated

or not with CytD prior to M. leprae infection.

Supernatants were harvested 48 h later. A, PGE2 pro-

duction was measured by EIA. IL-10 (B) and IL-12

(C) production was measured by ELISA. E, Nitrite

production. Infected SCs were stimulated with TNF-

a, and IL-12 (D) and nitrite (F) were measured.

Murine WT, TLR22/2 and TLR62/2 SCs were

infected withM. leprae, and PGE2 (G), IL-10 (H), and

nitrite (I) were determined in culture supernatants.

Data are presented as the mean 6 SD from six in-

dependent experiments. *p , 0.05, M. leprae-stimu-

lated versus control cells; +p , 0.05 between the

different M. leprae-treated cell groups.

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formation through specific and highly regulated mechanisms in-volving TLR6-signaling activation. Moreover, we demonstratedthat increased LD biogenesis affects SCs’ production of in-flammatory mediators and mycobacterial killing capacity, whichadds support to the growing body of evidence that identifies LDsas a key component in immunity to infections.We showed that the mechanisms of LD induction by M. leprae

in SCs share similarities, but also exhibit some differences, withthose previously observed in macrophages (9). LD biogenesis wasinduced in a time- and dose-dependent–response manner, withbacterial phagocytosis required for the induction. Moreover, byusing selective drugs, we showed that LD biogenesis in M. leprae-infected SCs follows a classical route. LDs are known to originatein the ER–Golgi complex, and the ability of LDs to form and growin size was described to be dependent on motor proteins andmicrotubules (28, 33). Induction of LDs in SCs by M. leprae wasdependent on ER and Golgi apparatus, because it was inhibited bybrefeldin and monesin drugs, known to disorganize the ER–Golgicomplex. In addition, disturbance of microtubule and actin orga-nization by specific drugs demonstrated that M. leprae-inducedLD biogenesis also signals through a pathway dependent oncytoskeleton dynamics. Finally, the ability of M. leprae to in-duce LDs in SCs was abrogated using an inhibitor of PI3K, anenzyme that participates in several signaling pathways, includingcytoskeletal organization.However, the induction of LDs in SCs displayed interesting

unique features compared with macrophages. First, in contrast tomacrophages, live M. leprae, but not dead bacterium or isolatedMycobacterium components, were able to induce LDs in in vitro-cultured SCs, suggesting that, in SCs, the bacteria may play anactive role in LD formation. Second, in M. leprae-infected cul-

tures, M. leprae-induced LD formation was observed inbacterium-associated cells but not in cells with no bacterium. Indeep contrast to macrophages, media conditioned by M. leprae-stimulated SCs were unable to mimic the LD-induction activity ofthe bacteria, confirming the absence of soluble factors secretedby infected SCs able to promote LD biogenesis in neighboring,uninfected cells. A third important difference found between M.leprae-induced LD biogenesis in macrophages and SCs was re-lated to the involvement of TLRs. We demonstrated previouslythat the formation of LDs in response to BCG and M. leprae isdependent on TLR2/TLR6 in infected macrophages (9, 12, 22,32). Although SCs express TLR2 (34), signaling through this re-ceptor was not essential to LD biogenesis, reinforcing our pre-vious data indicating that M. leprae-induced LD biogenesis andrecruitment to bacterial-containing phagosomes in SCs were in-dependent of TLR2 bacterial sensing (22). Moreover, althoughTLR6 was essential in both cell types in sensing M. leprae, a po-tential additional role as a phagocytic receptor was observed in thecase of SCs. Interestingly, a signaling that excludes the TLR2pathway as a heterodimer with TLR6 has not been explored ininflammation in response to microbial infection, but it was re-cently described in sterile inflammation, such as atherosclerosisand Alzheimer’s disease (35). In this study, a new hetero-dimerization model of cellular receptors, consisting of TLR4/6,was identified. Whether TLR4 or other coreceptors participate inconjunction with TLR6 in the recognition and signaling of liveM. leprae in SCs deserves future investigation. Additionally,the specific M. leprae chemical signatures recognized by TLR6need characterization.We investigated the functional role of TLRs in the immune

response triggered byM. leprae in SCs. TLR6 deletion completely

FIGURE 6. M. leprae elicits an innate immune

response in infected SCs that depends on LD for-

mation. Human SCs were pretreated or not with

C-75 or NS-398 prior to M. leprae infection, and

LD formation and inflammatory mediators were

measured. A, LD formation was determined by

MFI of BODIPY by flow cytometric analysis. B,

PGE2 production was determined by EIA. IL-10

(C) and IL-12 (D) production was estimated by

ELISA. E, NO production was estimated by mea-

suring nitrite by the Griess reagent. F, Percentage

of live and dead bacteria was evaluated using

a LIVE/DEAD Baclight Bacterial Viability Kit in

combination with flow cytometry after 72 h of drug

treatment. Data are mean 6 SD from six in-

dependent experiments. *p , 0.05, M. leprae-

stimulated versus control cells; +p , 0.05 between

the different M. leprae-treated cell groups.

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abolished the M. leprae-induced immunomodulatory effect onSCs. In contrast, no involvement of bacterial sensing by TLR2 inthe production of PGE2, IL-10, NO, and IL-12 by SCs in responseto M. leprae was observed. Because TLR6, but not TLR2, wasessential for both M. leprae-induced LD formation and cytokineand PGE2 production in SCs, these results corroborated the ideathat lipid metabolism and immune response are closely related ininfected SCs. TLR expression was found to correlate with theclinical presentation of leprosy (36). The crucial role of TLR6 inleprosy described in this article is in agreement with the reportedexpression profile of TLRs in leprosy lesions. Bleharski et al. (37),while analyzing gene-expression profiles in skin lesions of LL andTT patients, demonstrated that TLR6 was significantly upregu-lated in LL lesions. In contrast, Krutzik et al. (38) showed thatTLR2 was more strongly expressed in TT lesions compared withLL lesions. These data are in agreement with the essential role ofTLR6 in M. leprae-induced LD formation demonstrated in thisarticle, as well as the capacity of LL cells to accumulate LDs.Interestingly, M. leprae-infected SCs were less responsive to

TNF-a, suggesting a deactivating phenotype, similar to the onedescribed for M. leprae-infected macrophages (39). In this study,infection of mouse macrophages with M. leprae was shown torestrict IFN-g–mediated activation, at least in part, by the in-duction of PGE2. TNF-a receptors are constitutively expressed bySCs, and treatment of cells with TNF-a promoted activation ofSCs (40). Furthermore, TNF-a alone or in synergism with TGF-bwas shown to induce apoptosis in SCs (40, 41). Avoidance of hostcell apoptosis is particularly essential for the successful infectionof obligate intracellular pathogens, such as M. leprae, which relyon host cell integrity and metabolic activities to complete theirreplication cycle. Thus, the unresponsive phenotype of infectedSCs to TNF signaling may delay or suppress host cell apoptosisand activation during infection and favor infection persistence inthe nerve during the natural course of leprosy.Because immunocompetence has been recognized as an im-

portant feature of SCs (42), the correlation between lipid metab-olism and the innate immunity generated in response to M. lepraeinfection was investigated. We evaluated whether LDs constitutesites of eicosanoid production in M. leprae-infected SCs, ina similar manner to what was observed in the context of M. leprae

and BGC infection in macrophages (9, 12). The colocalizationbetween ADRP, COX-2, and M. leprae inside SCs of nerve bi-opsies from LL patients was a strong indicator that M. leprae-induced LDs constitute intracellular sites for eicosanoid synthesisin vivo. This was sustained by in vitro assays, in which a signifi-cant correlation between LD formation and PGE2 generation wasobserved in SCs treated with M. leprae. We next evaluated theimpact of the inhibition of LD formation on immunomodulationof host response and killing capacity of SCs infected byM. leprae.First, we tested the effect of NS-398 (COX-2 inhibitor) that, inaddition to its ability to inhibit cyclooxygenase, has COX-independent inhibitory effects on LD biogenesis (43, 44). NS-398 significantly inhibited the LD formation and PGE2 pro-duction in infected SCs. In different cell systems, LD biogenesiswas shown to require new lipid synthesis in a mechanism tightlyregulated by FAS (45–47). Accordingly, the FAS inhibitor C-75significantly inhibited LD formation induced by BCG, Trypano-soma cruzi, and dengue virus infection (32, 46, 48). Although nota cyclooxygenase inhibitor, C-75–induced LD inhibition led toinhibition of PGE2 production. Accordingly, C-75, throughmechanisms dependent on LD inhibition and independent ofinhibition of the COX-2 enzyme, inhibited PGE2 production incancer cells (45) and mycobacterial-infected macrophages (11,32). Interestingly, when other inflammatory mediators were ana-lyzed, infected SCs secreted increasing levels of the anti-inflammatory cytokine IL-10 and reduced levels of IL-12 andNO compared with the basal levels observed in un. Also, in-hibition of either PG production or LD formation resulted indownmodulation of IL-10 production. Furthermore, we observedan increase in IL-12 and nitrite detection in culture supernatants,suggesting that LDs and/or LD-derived PGE2 may negativelymodulate the immune response of SCs toward intracellular in-fection. PGE2 is a potent immune modulator that downregulatesTh1 responses and bactericidal activity toward intracellularorganisms (12–14, 49). Our data are consistent with these obser-vations, because a cytokine profile characterized by predominantIL-10 secretion and low secretion of IL-12 was dependent onPGE2 production by M. leprae-infected SCs. The signaling path-ways that regulate this immune cascade in SCs and whether thehigh IL-10 production involves an autocrine circuit of PGE2, as

FIGURE 7. A model proposing key roles for LDs in M. leprae (ML)-infected SCs for infection persistence in the nerve. M. leprae attachment and

internalization: LDs are promptly recruited to bacterial-containing phagosomes where they accumulate (22). LD recruitment probably constitutes an ef-

fective M. leprae intracellular strategy to acquire host SC lipids as a nutritional source and promote bacterial survival. Sensing of M. leprae by TLR6 leads

to the formation of new LDs that constitute active catalytic sites of PGE2 synthesis.M. leprae-infected SCs also produce IL-10, which, together with PGE2,

favors the inhibition of SCs’ bactericidal activities as downregulation of the cytokine IL-12 and inhibition of NO production. The accumulation of host-

derived lipids dependent on TLR6 signaling in infected SCs favors the generation of an innate immune response that may contribute to a permissive

environment for M. leprae proliferation within LL nerves.

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has been shown in the context of other cell types (50), deservefurther investigation.Strikingly, we demonstrated that treatment with C-75, in parallel

to LD inhibition, favored SCs’ mycobacterial killing. This suggestsa role for newly formed LDs in intracellular M. leprae survivalwithin the nerve. In support of the hypothesis that LDs are in-volved inM. leprae survival, pathogen-induced host LD formationhas been associated with intracellular pathogen survival advan-tages in hepatitis C virus (51, 52), dengue (46), T. cruzi (48), andBCG infections (11, 32). The role of LD inhibition in leading toM. leprae’s improved killing ability of SCs may occur secondaryto the inhibition of PGE2 production and modulation of the cy-tokine milieu. In addition, LDs are being suggested as a richsource of nutrients for mycobacterial intracellular growth (12,53–55). Accordingly, we demonstrated that LDs are promptlyrecruited to the bacterial-containing phagosome during M. lepraeinternalization in SCs and that this event contributes to bacterialsurvival inside the cell (22).In conclusion, a model can be proposed in which the lipid-

storage phenomenon observed in M. leprae-infected SCs playsa key role in leprosy pathogenesis by facilitating bacterial per-sistence in the host through at least two mechanisms (Fig. 7). Asindicated in our previous study, during M. leprae attachment andinternalization, LDs are promptly recruited to bacterial-containingphagosomes, where they accumulate (22). LD recruitment prob-ably constitutes an effective M. leprae intracellular strategy toacquire host SC lipids as a nutritional source and promote bac-terial survival. Second, as shown in the current study, the accu-mulation of host-derived lipids in infected SCs favors the gen-eration of an innate immune response that may contribute to apermissive environment for M. leprae proliferation within LLnerves. Sensing M. leprae by TLR6 leads to the formation of newLDs that constitute active catalytic sites of PGE2 synthesis. M.leprae-infected SCs also produce IL-10, which, together withPGE2, favors the inhibition of SCs’ bactericidal activities and thedownregulation of Th1-type cytokines. Thus, suppression of nor-mal inflammatory function of TLR6 by M. leprae in SCs results inthe expression of an anti-inflammatory phenotype dependent onLD biogenesis.The data presented in this article extend our knowledge of the

underlying disease mechanism in M. leprae-infected nerves.Moreover, they support the idea of SCs as immunocompetentcells that form parts of the local immune circuitry within theperipheral nerve (42). Finally, our data confirm that the lipidmodulation induced by M. leprae has pathophysiological con-sequences for the persistence of infection and open avenuesfor developing novel therapies for controlling mycobacterialinfections, based either on inhibition of PG production or LDformation.

AcknowledgmentsWe thank Dr. J. Krahenbuhl for providing M. leprae and Pedro Paulo

Manso for assistance with the confocal images. We thank the Program

for Technological Development in Tools for Health-Oswald Cruz Foun-

dation for use of its confocal laser scanning microscopy facility.

DisclosuresThe authors have no financial conflicts of interest.

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