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Toll-like receptors participate in macrophage activation and intracellular control of Leishmania 1

(Viannia) panamensis. 2

3

Running title: TLR-dependent macrophage activation by L. panamensis 4

5

Carolina Gallego1, Douglas Golenbock2, Maria Adelaida Gomez1, Nancy Saravia1* 6

7

1Centro Internacional de Entrenamiento e Investigaciones Medicas-CIDEIM, Cali- Colombia. 8

2Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, 9

Worcester, Massachusetts 01605, USA. 1 0

1 1

*Corresponding Author: CIDEIM, Carrera 125 No. 19-225, Cali-Colombia. Phone: (57) 2-5552164. Fax 1 2

(57) 2-5552638. E-mail: [email protected] 1 31 4

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.01388-10 IAI Accepts, published online ahead of print on 25 April 2011

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Abstract 1 5

Toll-like receptors (TLRs) play a central role in macrophage activation and control of parasitic 1 6

infections. Their contribution to the outcome of Leishmania infection is just beginning to be deciphered. 1 7

We examined the interaction of Leishmania panamensis with TLRs in the activation of host 1 8

macrophages. L. panamensis infection resulted in up-regulation of TLR1, TLR2, TLR3 and TLR4 1 9

expression, and induced TNFc"secretion by human primary macrophages at comparable levels and 2 0

kinetics as specific TLR ligands. The TLR-dependence of the host cell response was substantiated by 2 1

the absence of TNFc production in MyD88/TRIF-/- murine bone marrow derived macrophages and 2 2

mouse macrophage cell lines in response to promastigotes and amastigotes. Systematic screening of 2 3

TLR-deficient macrophages revealed that TNFc production was completely abrogated in TLR4-/- 2 4

macrophages, consistent with the increased intracellular parasite survival at early time points of 2 5

infection. TNFc secretion was significantly reduced in macrophages lacking endosomal TLRs, but was 2 6

unaltered by lack of TLR2 or MD-2. Together, these findings support the participation of TLR4 and 2 7

endosomal TLRs in the activation of host macrophages by L. panamensis and in the early control of 2 8

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Introduction 3 1

Protozoan parasites of the genus Leishmania are obligate intracellular pathogens that infect and 3 2

impact the lives of millions of people worldwide. Leishmania species induce a broad spectrum of 3 3

pathologies in humans, ranging from cutaneous lesions to progressive fatal visceral disease (43). 3 4

Although the determinants of pathogenesis remain unclear, the host immune response and the 3 5

infecting Leishmania species contribute to the diversity of clinical manifestations (57). 3 6

3 7

Macrophages act as the principal host cells for Leishmania, playing a central role in the development 3 8

of the immune response to these parasites via antigen presentation, secretion of microbicidal 3 9

products, and by production of inflammatory mediators (cytokines and chemokines). These early 4 0

events during host cell-parasite interaction can ultimately determine the outcome of infection (9). 4 1

Previous studies in naturally infected humans have provided evidence of differences in the innate and 4 2

acquired immune responses to L. Viannia subspecies associated with the clinical outcome of infection. 4 3

Macrophages from naturally exposed individuals presenting recurrent or chronic disease (clinical 4 4

susceptibility) were more permissive to in vitro infection than cells from individuals with subclinical 4 5

infection (clinical resistance) (9, 52). 4 6

4 7

Experimental studies have revealed that a diverse repertoire of receptors is involved in Leishmania-4 8

macrophage interactions (5, 11, 37, 39), nevertheless, the relationship between these receptors, host 4 9

cell activation and outcome of infection remains largely unknown. Toll-like receptors (TLRs) provide a 5 0

link between innate and adaptive immunity allowing recognition of a wide range of microbial products 5 1

including those of protozoan pathogens (22, 24, 35). TLR-ligand interaction initiates signal 5 2

transduction through the adaptor proteins MyD88 and/or TRIF resulting in downstream activation of 5 3

NF-mB, interferon regulatory factors and the subsequent transcription of inflammatory mediators such 5 4


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as TNFc, type I interferons and IL-12, and the co-stimulatory molecules CD80 and CD86 in 5 5

monocytes/macrophages and dendritic cells. 5 6

5 7

Gene knockout studies in mice have revealed a critical role for TLR signaling in the immune response 5 8

against Leishmania parasites (32, 60). MyD88-/- and TLR4-/- C57BL/6 mice were more susceptible to 5 9

disease following L. major infection (18, 19, 30, 31, 42), and depletion of TLR9 impaired NK cell 6 0

activation both in murine models of visceral leishmaniasis caused by L. infantum and cutaneous 6 1

leishmaniasis produced by L. major (33, 55). A link between TLRs and microbicidal capacity was 6 2

evidenced by the requirement of TLR2 and TLR3 for intracellular killing of L. donovani in IFN-け-primed 6 3

macrophages (21). Notably, absence of TLR2 was recently shown to promote IL-12 production by DCs 6 4

during L. braziliensis infection, suggesting that this receptor may also play a negative regulatory role 6 5

(61). 6 6

6 7

Although knowledge of the nature and identity of Leishmania molecules that interact with individual 6 8

TLRs is limited, some parasite ligands and cognate TLR receptors on different cells participating in the 6 9

immune response have been described. L. major lipophosphoglycan (LPG) was shown to activate 7 0

human NK cells and murine monocytes through TLR2 (3, 18) and has been implicated in the induction 7 1

of nitric oxide (NO) production by human peripheral blood mononuclear cells (PBMCs) via TLR2 (27). 7 2

Additionally, the P8 proteoglycolipid complex (P8 PGLC) expressed by L. pifanoi has recently been 7 3

shown to promote macrophage activation through TLR4 (62), and L. major DNA to activate DCs 7 4

through TLR9 and thereby promote IFNi production by CD4+ T cells (1). The implications of these 7 5

findings and the participation of TLR signaling in the onset of the immune response during human 7 6

infections with species of the L. Viannia subgenus have yet to be determined. 7 7

7 8


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Th1/Th2 polarization does not explain the clinical outcome in human infections with L. Viannia species, 7 9

which have a propensity to produce latent, relapsing (2) and chronic disease. Early in vitro responses 8 0

are characterized by a mixed profile of pro (IFNi TNFc) and anti-inflammatory (IL-10, IL-13) cytokine 8 1

production (20). Refractory disease presentations are often accompanied by exuberant Th1 responses 8 2

including increased TNFc production that has been implicated in pathogenesis (54). In this study we 8 3

present evidence that TLRs are involved in both activation and secretion of TNFc by human and 8 4

murine macrophages by L. panamensis and in the early control of infection. 8 58 6


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Materials and Methods: 8 7

Parasites: L. panamensis (MHOM/CO/86/1166) promastigotes ygtg" ewnvwtgf" kp" UejpgkfgtÓu"8 8Drosophila medium (Sigma) supplemented with 20% heat-inactivated FBS (HIFBS) (Gibco) at 25°C.

8 9Luciferase and GFP-expressing promastigotes (L.p-LUC and L.p-GFP) were grown in media

9 0supplemented with 50 mg/ml of G418 (Gibco). Promastigotes were harvested during stationary phase,

9 1and opsonized for 1 h at 34°C in RPMI 1640 (Gibco) supplemented with 10% heat inactivated human

9 2AB serum when indicated. Live intracellular amastigotes were obtained by infection of differentiated

9 3adherent U937 cells with opsonized promastigotes (50:1 parasite-to-cell ratio) for 5 days at 34°C and

9 45% CO2. After infection, cells were lysed by passage through a 27G needle and amastigotes were

9 5recovered by centrifugation over a Percoll density gradient (Amersham Biosciences).

9 6

9 7Macrophages: Human promonocytic U-937 cells were cultured as previously described (52). Cell

9 8differentiation was induced by phorbol 12-myristate 13-acetate (PMA) stimulation (100 ng/ml) (Sigma)

9 94 days prior to infection. Immortalized macrophage cell lines generated from wild type (WT),

1 0 0MyD88/TRIF-/-, MyD88-/-, TRIF-/-, TLR2-/-, TLR4-/- and UNC93b mutant C57BL/6 mice were cultured as

1 0 1previously described (23). Clonal lines were tested for the induction of TNFc and IL-6 in response to a

1 0 2panel of TLR ligands, including synthetic lipopeptide, polyinosinic-polycytidylic acid (Poly I:C) , LPS,

1 0 3resiquimod and CpG DNA. In all cases, the predicted responses, based on the known responses of

1 0 4native bone marrow-derived macrophages, were observed (e.g., the TLR4 knockout responded to all

1 0 5TLR ligands except LPS). Cells were maintained in DMEM (Cellgro) supplemented with 10% HIFBS

1 0 6and 10"og of ciprofloxacin/ml (Bayer). Cells were plated and stimulated with TLR ligands or infected

1 0 7with L. panamensis parasites as described in the text.

1 0 8

1 0 9


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All mice were housed and bred in pathogen-free conditions. Bone marrow was harvested from mice as 1 1 0

previously described (62) in accordance with the guidelines set forth by the University of 1 1 1

Massachusetts Medical School and approved by its Institutional Animal Care and Use Committee. 1 1 2

BMM precursors from WT (C57BL/6), MD-2-/-, MyD88/TRIF-/-, MyD88-/-, TRIF-/-, or TLR4-/- mice were 1 1 3

differentiated in culture for 9-10 days in the presence of conditioned medium (DMEM supplemented 1 1 4

with 20% L929 cell culture supernatant, 10% HIFBS, and 10 µg/ml ciprofloxacin) prior to use. 1 1 5

1 1 6

Isolation and culture of human monocyte-derived macrophages: Sixty ml of peripheral blood was 1 1 7

obtained from healthy adult volunteers in accordance with the protocol and informed consent forms 1 1 8

approved by the Institutional Review Board of CIDEIM. PBMCs were obtained from peripheral blood 1 1 9

using Ficoll-hypaque 1077 (Sigma) density gradient centrifugation. Monocytes were allowed to 1 2 0

differentiate to macrophages by culture for 7 days at 37°C and 5% CO2 in complete RPMI (Sigma) 1 2 1

following adherence to plates coated with 2% sterile endotoxin-free gelatin and autologous human 1 2 2

serum. All plasticware, glassware and water used for cell culture were endotoxin-free. 1 2 3

1 2 4

L. panamensis infection and TLR-ligand stimulation: Primary human macrophages were 1 2 5

stimulated for 8 h with LPS (1"og/ml), zymozan (25 og/ml), Pam2CSK4 (10"og/ml), Poly I:C (20 1 2 6

og/ml), or CpG ODN 2216 (5 oM) (Invivogen), or infected with opsonized L. panamensis 1 2 7

promastigotes at a 5:1 parasites-to-cell ratio. Infection was allowed to proceed for 1 h in FBS-free 1 2 8

medium at 34°C and 5% CO2. Extracellular parasites were removed by washing with D-PBS and cells 1 2 9

were incubated for 8 h at 34°C and 5% CO2. 1 3 0

1 3 1

Mouse macrophage cell lines and BMMs were infected with L. panamensis promastigotes and 1 3 2

amastigotes at 50:1 to 200:1 parasites-to-cell ratio for 1 h, washed with D-PBS and incubated at 34°C 1 3 3


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and 5% CO2. These parasites to cell ratios using serum-opsonized as well as non-opsonized parasites 1 3 4

were chosen to optimize infection and read out based on prior dose response experiments. 1 3 5

Supernatants were collected and TNFc was evaluated 24 h post-infection. Stimulations with 1 3 6

Pam2CSK4 (4 oM), Poly I:C (100 og/ml), CpG (5 oM) and LPS (1 og/ml) or with the non-TLR ligand 1 3 7

Sendai virus (500 U/ml) were used as positive and negative controls, as indicated. The TLR4/MD-2 1 3 8

antagonist Eritoran (10 µg/ml) (Eisai Research Institute) was used to discriminate activation 1 3 9

attributable to endotoxin contamination. Infection was evaluated in Giemsa stained cells by 1 4 0

microscopy. 1 4 1

1 4 2

WT and TLR4-/- macrophages were seeded on glass cover slips and infected with L. p-GFP 1 4 3

promastigotes at 20:1 parasite to cell ratio to visualize parasite internalization by confocal microscopy 1 4 4

(Leica SP2 AOBS). Cell membranes were stained with CellMask Deep Red stain (Invitrogen). 1 4 5

1 4 6

FACS analysis: Human macrophages were harvested and incubated with 10"og of non-immune 1 4 7

human IgG (BD Biosciences) to block nonspecific binding to FciR and then incubated with PE-labeled 1 4 8

monoclonal anti-TLR1 (clone GD2.F4), anti-TLR2 (clone TL2.1) or anti-TLR4 (clone HTA125) 1 4 9

antibodies. FITC-labeled anti-CD14 was used as monocyte population marker. For intracellular 1 5 0

staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution, washed with PermWash 1 5 1

solution (BD Biosciences), and incubated with monoclonal anti-TLR3 (clone TLR3.7) or anti-TLR9 1 5 2

(eB72-1665). Cells were fixed with 2% para-formaldehyde. PE- IgG1m, PE-mouse IgG2a and PE-1 5 3

mouse IgG1 were used as isotype controls. All antibodies were purchased from e-Biosciences. Cells 1 5 4

were evaluated using a FACSCalibur cytometer (BD Biosciences) and analyzed with the WinMDI 2.9 1 5 5

software (Scripps Research Institute). 1 5 6

1 5 7


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Quantification of TNF : Human TNFc ELISA was performed using antibodies from BD Pharmingen 1 5 8

following the manufacturer´s instructions. Mouse TNFc was quantified by ELISA with the TNFc 1 5 9

FwqUgv" GNKUC" mkv" *T(F+" hqnnqykpi" vjg" ocpwhcevwtgtÓu" kpuvtwevkqpu0" Cnn" samples were evaluated in 1 6 0

duplicate. 1 6 1

1 6 2

Luciferase assay: L. p-Luc promastigotes were used to infect macrophages for 1 h. Non-internalized 1 6 3

parasites were removed by washing and macrophages were further incubated for 4, 16, 24 and 36 h. 1 6 4

Cells were lysed and analyzed using the dual luciferase reporter assay system (Promega) according 1 6 5

to the ocpwhcevwtgtÓu"kpuvtwevkqpu. 1 6 6

1 6 7

Statistical analysis: Fkhhgtgpegu"kp"rcktgf"itqwru"ygtg"gxcnwcvgf"d{"UvwfgpvÓu" t test or the Wilcoxon 1 6 8

rank test. For group comparisons the Kruskal-Wallis test was used ykvj"FwppÓu"cflwuvogpv0"Cpcn{ugu"1 6 9were performed with the GraphPad Prism 5 software (GraphPad Inc., San Diego, CA) and p values of

1 7 0<0.05 were considered significant.

1 7 1

1 7 2Results

1 7 3L. panamensis promastigotes induce TNF production and increased expression of TLR1,

1 7 4TLR2, TLR3 and TLR4 by human macrophages.

1 7 5TNFc production was used as an early readout of TLR-dependent macrophage activation [reviewed in

1 7 6(59)]. TNFc secretion following exposure to live promastigotes peaked at 8 h decreasing by 24 hours

1 7 7(Fig. 1A). The kinetics and level of production of TNFc in response to L. panamensis and specific TLR

1 7 8ligands by human macrophages were comparable (Fig. 1B), consistent with TLR-dependent activation

1 7 9of this host cell by L. panamensis. Stimulation of human macrophages with TLR ligands induced

1 8 0increased expression of the corresponding TLRs (Figure 2 lower panel and data not shown). Similarly,

1 8 1


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co-culture with live L. panamensis significantly increased the cell surface expression of TLR1, TLR2 1 8 2

and TLR4 and the intracellular expression of TLR3, but not TLR9, compared to unexposed 1 8 3

macrophages (Fig. 2). 1 8 4

1 8 5

L. panamensis promastigotes and amastigotes activate TNF production through TLRs. 1 8 6

Based on the results supporting the involvement of TLRs in the L. panamensis-human macrophage 1 8 7

interaction, we investigated the participation of specific TLRs and adaptor molecules in cell activation. 1 8 8

Human HEK293 cells stably transfected with individual human TLRs and a NF-mB-luciferase reporter 1 8 9

construct were used as a surrogate model of infection and cell activation. However, HEK293 cells 1 9 0

deteriorated during co-culture with L. panamensis promastigotes, leading us to examine specific TLR 1 9 1

activation in C57BL/6-derived macrophage cell lines. 1 9 2

1 9 3

TNFc production was induced in a dose-dependent manner in the WT C57BL/6 macrophage cell line 1 9 4

by both promastigotes and amastigotes of L. panamensis over a wide dose range of parasites (Fig. 1 9 5

3A). In contrast, neither parasite stage induced TNFc production in the MyD88/TRIF-/- double 1 9 6

knockout macrophage cell line in which all TLRs are non-functional (Fig. 3B), supporting the 1 9 7

involvement of TLR-dependent signaling during macrophage activation by L. panamensis. TNFc 1 9 8

secretion induced by opsonized and non-opsonized parasites was indistinguishable (Fig. 3A and data 1 9 9

not shown), ruling out cell activation attributable to interactions with serum opsonins in this 2 0 0

experimental system. 2 0 1

2 0 2

MD-2, a small protein that together with TLR4 forms the LPS-signaling complex is an absolute 2 0 3

requirement for cells to respond to LPS (56) . Eritoran (previously known as B1287) binds to the lipid A 2 0 4

binding site of MD-2, and is a potent inhibitor of LPS mediated activation. Eritoran did not impair 2 0 5


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induction of TNFc by L. panamensis, whereas induction by LPS was blocked (Fig. 3A), thereby 2 0 6

establishing that the macrophage response to L. panamensis could not be attributable to LPS 2 0 7

contamination. 2 0 8

2 0 9

To dissect the TLR signaling pathway activated by L. panamensis in macrophages, we examined the 2 1 0

response of MyD88-/- and TRIF-/- single knockout macrophages to opsonized and non-opsonized 2 1 1

parasites. In contrast to the complete abrogation of TNFc secretion in MyD88/TRIF-/- double knockout 2 1 2

macrophages co-cultured with L. panamensis, specific ablation of either MyD88 or TRIF alone, did not 2 1 3

significantly alter TNFc"production (Fig. 4A-B). Similar results were obtained with opsonized and non-2 1 4

opsonized parasites (Fig. 4A-B). These results indicate that both MyD88 and TRIF are required to fully 2 1 5

induce TNFc production by L. panamensis in this experimental system. 2 1 6

2 1 7

Endosomal TLRs participate in macrophage activation by L. panamensis infection. 2 1 8

UNC93b is an endoplasmic reticulum (ER) membrane protein required for signaling through 2 1 9

endolysosomal TLRs (10) by controlling the trafficking of TLRs 3, 7, 8 and 9 from the ER to the 2 2 0

endolysosomal compartment. Hence, UNC93b mutant cells do not respond to the corresponding TLR 2 2 1

ligands (28). To investigate the role of the endolysosomal TLRs during L. panamensis infection, we 2 2 2

examined the activation of macrophages expressing a non-functional form of UNC93b (58). Induction 2 2 3

of TNFc production by both L. panamensis promastigotes and amastigotes was significantly reduced, 2 2 4

though not completely abrogated, when compared to WT cells""*Fig. 5), suggesting that signaling 2 2 5

through the endolysosomal TLRs also contributes to macrophage activation during L. panamensis 2 2 6

infection. 2 2 7

2 2 8

TLR4 mediates L. panamensis recognition by macrophages 2 2 9


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We next examined the induction of TNFc production in TLR2-/- and TLR4-/- macrophage cell lines. As 2 3 0

shown in Fig. 6A, no significant change in TNFc secretion was observed in TLR2-/- macrophages 2 3 1

compared to WT cells, suggesting that TLR2 is not critical for L. panamensis-mediated macrophage 2 3 2

activation. In contrast, TNFc production was abrogated in TLR4-/- macrophages following infection 2 3 3

with either opsonized or non-opsonized promastigotes or amastigotes (Fig. 6C). These results indicate 2 3 4

that TLR4, but not TLR2, has an important role in the induction of pro-inflammatory cytokine secretion 2 3 5

by macrophages during infection by L. panamensis. 2 3 6

2 3 7

BMMs from MD-2 deficient C57BL/6 mice were used to confirm the specificity of L. panamensis-2 3 8

mediated macrophage activation via TLR4, and unequivocally rule out activation attributable to 2 3 9

endotoxin contamination. Both promastigotes and amastigotes induced similar and significant levels of 2 4 0

TNFc"production in MD-2-/- and WT BMMs, confirming that macrophage activation through TLR4 was 2 4 1

induced by L. panamensis infection (Fig. 6C), and not LPS contamination. 2 4 2

2 4 3

TLR4 is required for induction of leishmanicidal activity of macrophages. 2 4 4

Evaluation of intracellular killing of L. panamensis over the time course of infection from 4 to 48 h 2 4 5

showed that by 16 h post-infection intracellular parasite survival in WT macrophages was reduced by 2 4 6

up to 70%. In contrast, L. panamensis not only survived but replicated during the first 16 h in TLR4-/- 2 4 7

macrophages (Fig. 7). The parasite burden at the initial time point (4 h) of infection was comparable 2 4 8

between WT and TLR4-/- cells (165380 and 143500 RLU, respectively), confirming similar infection 2 4 9

rates in both cell lines. Confocal microscopy of WT and TLR4-/- cells infected with GFP-transfected 2 5 0

parasites corroborated equivalent parasite infectivity and internalization (Fig.7B). By 24 h post-2 5 1

infection, parasite survival decreased in TLR4-/- macrophages to levels comparable to WT host cells 2 5 2

supporting the participation of TLR4 during the very early stages of parasite-macrophage interaction. 2 5 3


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2 5 4

Other MyD88-dependent receptors may participate in macrophage activation in response to L. 2 5 5

panamensis. 2 5 6

To extend the findings obtained in macrophage cell lines to an infection approximating natural host cell 2 5 7

interaction, we systematically evaluated the response of primary C57BL/6 WT BMMs, and knockout 2 5 8

BMMs lacking specific TLRs and adaptor molecules. As observed in murine cell lines, TNFc 2 5 9

production was significantly reduced in TLR4-/- and MyD88/TRIF-/- BMMs exposed to L. panamensis 2 6 0

(Fig. 8A and B). However, in contrast to the results in knockout cell lines in which deficiency of either 2 6 1

MyD88 or TRIF alone did not significantly alter TNFc production, MyD88-/- BMMs co-cultured with L. 2 6 2

panamensis produced significantly lower TNFc upon exposure to L. panamensis compared with WT 2 6 3

cells (Fig. 8C and D). Deficiency of TRIF did not significantly alter TNF production by BMMs. In 2 6 4

addition, a striking and consistently greater production of TNFc production was induced in BMMs by 2 6 5

opsonized versus non-opsonized parasites. This increased TNFc response coincided with the higher 2 6 6

percentage of infection observed in BMMs exposed to opsonized parasites (data not shown). The 2 6 7

enhancement of TNFc secretion by opsonized parasites in this experimental model is consistent with 2 6 8

the participation of receptors other than TLR4 in L. panamensis-mediated BMM activation. 2 6 92 7 0 on A

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Discussion 2 7 1

2 7 2

Macrophages are central to host defense, from pathogen detection and clearance to the induction of 2 7 3

adaptive immune responses (40). The outcome of Leishmania infection is largely determined by the 2 7 4

ability of macrophages to recognize and respond to the parasite (4, 6, 8, 9, 37, 41, 52). Although 2 7 5

several macrophage receptors evidently participate in the Leishmania-host cell interaction (5, 11, 37, 2 7 6

39, 41), their role in the modulation of host immune responses and clinical outcomes of infection is not 2 7 7

yet clear. This study sought to determine the participation of TLRs in L. panamensis-mediated 2 7 8

modulation of host cell responses. 2 7 9

2 8 0

Contrary to the negative regulation of host cell functions reported in L. donovani infection, strong pro 2 8 1

and anti-inflammatory cytokine production is a hallmark of infection by parasites of the L. Viannia 2 8 2

subgenus (43). TNFc is thought to play an important role during L. braziliensis infection, contributing 2 8 3

to disease control in the early stages and exacerbation of disease at later stages of infection (2, 16, 2 8 4

50, 53). Mucosal leishmaniasis is characterized by high levels of TNFc production, and genetic 2 8 5

polymorphisms in the promoter region of TNF genes have been linked to susceptibility to mucosal 2 8 6

disease (4, 13). The early induction of TNFc production in isolated human macrophages by L. 2 8 7

panamensis promastigotes, provided initial evidence of putative receptor-mediated cell activation. 2 8 8

Concurrence of the kinetics of TNFc production induced by L. panamensis and by specific TLR 2 8 9

ligands, and the up-regulation of TLR1, TLR2, TLR3 and TLR4 expression in human macrophages 2 9 0

exposed to L panamensis promastigotes (Fig. 2) further supported the possibility of TLR mediated 2 9 1

activation and TNFc production. 2 9 2

2 9 3


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Deletion of both MyD88/TRIF, essential adaptor molecules in signal transduction initiated by TLR 2 9 4

engagement, resulted in the abrogation of TNFc secretion by mouse macrophages. This finding 2 9 5

demonstrated that host cell activation by both L. panamensis amastigotes and promastigotes is TLR-2 9 6

dependent. Although differences in macrophage responses to promastigotes and amastigotes of other 2 9 7

Leishmania species are well documented (29), including decreased superoxide and increased anti-2 9 8

inflammatory cytokines in response to infection with amastigotes (15, 46, 47), the induction of TNFc 2 9 9

by these life stages was indistinguishable in this experimental infection model. This finding suggests 3 0 0

that L. panamensis TLR ligand(s) are similarly expressed by both amastigotes and promastigotes, or 3 0 1

alternatively, that different stage-specific molecules can redundantly stimulate TLR signaling. 3 0 2

3 0 3

The most compelling evidence to date for the participation of TLRs in Leishmania infection and 3 0 4

pathogenesis is the enhanced susceptibility of MyD88-deficient mice to L. major, accompanied by 3 0 5

impaired pro-inflammatory cytokine synthesis and diminished Th1 T cell activation (19, 42). However, 3 0 6

this enhanced susceptibility to L major has not been attributed to any specific TLR family member. 3 0 7

Using MyD88-/- and TRIF-/- macrophage cell lines we found that absence of either of these adaptor 3 0 8

molecules did not significantly alter TNFc"production induced by L. panamensis promastigotes, or 3 0 9

amastigotes. These findings suggest that signaling in response to L panamensis occurs through both 3 1 0

pathways, and in the absence of either, the other can compensate, sustaining TLR signaling and host 3 1 1

cell activation. 3 1 2

3 1 3

Studies addressing the role of MyD88 in macrophage activation during infection with the closely 3 1 4

related species L. braziliensis have yielded conflicting results. Recently, Carvalho et al. showed that 3 1 5

TNFc secretion by DCs during L. braziliensis infection was MyD88-independent (14). In contrast, 3 1 6

Vargas and collaborators reported that L. braziliensis-infected MyD88-/- DCs exhibited reduced 3 1 7


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activation and decreased production of IL-12p40, which is fundamental to the generation of protective 3 1 8

immunity (61). Notwithstanding differences in experimental models, these results raise the issue of 3 1 9

intra as well as inter-species variation in TLR mediated activation. Despite the similarity of L. 3 2 0

braziliensis and L. panamensis as related members of the Viannia subgenus, parasites of these 3 2 1

species may trigger different mechanisms of cell activation. Therefore, generalization of results 3 2 2

obtained with one species may not be appropriate or accurately portray the interaction with other 3 2 3

Leishmania species, even within the same taxonomic grouping. 3 2 4

3 2 5

The partial abrogation of TNFc production in UNC93b deficient mutant macrophages in response to L. 3 2 6

panamensis infection supports the involvement of signaling through endolysosomal TLRs, and 3 2 7

concurs with evidence of endolysosomal TLR participation in the immune response to other 3 2 8

Leishmania species. TLR3 has been implicated in the recognition of L. donovani by IFNi-primed 3 2 9

macrophages (21), and TLR9 in DC-mediated NK cell activation and T cell responses following L. 3 3 0

infantum and L. major infection, respectively (33, 55). L. major DNA has been shown to promote IFNi 3 3 1production by CD4+ T cells through TLR 9 dependent activation of DCs (1). The potential therapeutic

3 3 2applicability of activation through endolysosomal TLRs has been demonstrated by the enhanced

3 3 3resolution of experimental L. donovani infection and human cutaneous leishmaniasis (12, 36) using

3 3 4the TLR 7/8 ligand imiquimod. Although our results support the participation of endolysosomal TLR

3 3 5signaling during L. panamensis infection the specific TLR(s) involved remain to be identified. Since all

3 3 6of the endolysosomal TLRs recognize nucleotide ligands, it is reasonable to hypothesize that

3 3 7Leishmania nucleic acids are involved in the innate immune response to infection.

3 3 8

3 3 9TLR2 has been reported to be involved in the host response to L. major and L donovani through the

3 4 0recognition of LPG, which activates human NK cells and murine macrophages through this receptor

3 4 1


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(3, 18). Inhibition of TLR2 expression impaired NO production and TNFc secretion in IFNi-primed 3 4 2

mouse macrophages (21). Our results showed that, although L. panamensis induced increased TLR2 3 4 3

expression in human macrophages, absence of TLR2 in murine macrophage lines did not affect TNFc 3 4 4

production by promastigotes or amastigotes. This finding and the observation that parasites of the 3 4 5

Viannia subgenus express 10-fold less LPG than L. donovani (44), suggest that TLR2 may not be a 3 4 6

crucial determinant of the host cell response to L. panamensis. 3 4 7

3 4 8

The importance of TLR4 signaling in Leishmania infection is supported by the increased susceptibility 3 4 9

of TLR4-/- C57BL/6 and BALB/c respectively, to L. major and L pifanoi infection (30, 31). Furthermore, 3 5 0

L. pifanoi P8 PGLC, has been shown to induce TLR4-dependent macrophage cytokine and chemokine 3 5 1

production (62). Our data provide evidence that TLR4 is also important during the early L. 3 5 2

panamensis-macrophage interaction since reduced TNFc production and increased parasite survival 3 5 3

occurred in TLR4-/- infected mouse macrophages. The results were further supported by ruling out any 3 5 4

possibility of attributing activation to endotoxin contamination using MD-2-/- BMMs (Fig. 6C), thereby 3 5 5

providing unequivocal evidence that the TLR4 response was induced by L panamensis parasites. The 3 5 6

significant decrease in TNFc production in MyD88-/- BMMs, is consistent with the ability of TLR4 to 3 5 7

signal preferentially through MyD88 (25) as previously reported for other Leishmania species (17, 18, 3 5 8

42). These findings encourage identification of the nature of the ligand(s) involved in the TLR4-3 5 9

dependent response to L. panamensis. 3 6 0

3 6 1

A noteworthy difference between the activation of immortalized macrophage cell lines versus BMMs 3 6 2

was the enhanced TNFc secretion induced by opsonized parasites in BMMs. Under physiological 3 6 3

conditions, opsonins (including complement components and antibodies) rapidly interact with the cell 3 6 4

surface of promastigotes after their inoculation into the dermis of mammals or with amastigotes when 3 6 5


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they leave their host cells (48). Opsonization promotes the uptake of parasites by phagocytic cells (63) 3 6 6

principally mediated by Fc and complement receptors, and also mediates the release of anti-3 6 7

inflammatory cytokines (26, 38, 39). Opsonization could also favor the synergistic recognition of 3 6 8

Leishmania parasites through various different receptors (7), eliciting enhanced or diminished 3 6 9

activating signals and cytokine production (49, 51). Although not evaluated in this study, differences in 3 7 0

expression levels of TLRs or other receptors that interact with Leishmania, the maturation stage (34) 3 7 1

and the functional integrity of permanent cell lines (45) may account for differences observed in the 3 7 2

response elicited in primary macrophages and macrophage cell lines in response to parasite 3 7 3

opsonization. Interpretation and extrapolation of results obtained with cell lines to primary cell 3 7 4

populations or either of these to whole organisms and vice versa merit consideration of the 3 7 5

characteristics and limitations of each model system. Although the generalization of observations in 3 7 6

these models to natural human infection is uncertain, exploitation of these experimental models 3 7 7

allowed the discrimination of TLRs involved in the L. panamensis-macrophage interaction. Findings in 3 7 8

human macrophages also support the involvement of multiple TLRs, including TLR4, in the host cell 3 7 9

interaction with L. panamensis. 3 8 0

3 8 1

In conclusion, our results demonstrate TLR4-mediated macrophage activation and cooperation with 3 8 2

other TLRs during infection by L panamensis. The findings provide insight into the mechanism of 3 8 3

parasite recognition and evidence that early induction of TNFc production mediated by ligation of 3 8 4

TLRs contribute to control of infection. Understanding of the participation of TLRs in the outcome of 3 8 5

Leishmania infection may allow the exploitation of these receptors or their ligands in the development 3 8 6

of co-adjuvant immunotherapy. 3 8 7

3 8 8

Acknowledgements 3 8 9


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We thank Liliana Valderrama and Diane McMahon-Pratt for their insights during the development of 3 9 0

this investigation and critical review of this manuscript. The support of Mauricio Perez with statistical 3 9 1

analysis, Adriana Navas with cytometric analysis, Anna Cerny with animal husbandry and the 3 9 2

technical assistance of Kristen Halmen, are gratefully acknowledged. This work was supported by a 3 9 3

NIH/NIAID grant (AI065866), NIH/ John E. Fogarty International Center GID training grant 3 9 4

(TW006589), COLCIENCIAS doctoral training fellowship (041-2005) to C.G. and NIH/NIAID grant 3 9 5

(AI079293) to D.G. 3 9 6

The authors have no financial or other conflict of interest 3 9 7

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5 7 7

Figure legends 5 7 8

5 7 9


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FIGURE 1. L. panamensis infection induces TNF production at comparable level and kinetics 5 8 0

as specific TLR ligands. (A) Kinetics of TNFc"production in human primary macrophages exposed 5 8 1

to different parasite-to-cell ratios demonstrated maximum cytokine secretion 8 h post-infection. Data 5 8 2

are representative of cytokine production by macrophages of 10 individuals and are expressed as the 5 8 3

mean ‒ SEM. (B) Production of TNFc in human primary macrophages from 10 different donors was 5 8 4

measured by ELISA. Cells were stimulated with zymosan, LPS, Poly I:C, infected 8 h with 5 8 5

promastigotes 5:1 parasite-to-macrophage ratio or left untreated as a control. Data are expressed as 5 8 6

mean ± SD. *(p< 0.05), **(p< 0.01) 5 8 7

5 8 8

FIGURE 2. L. panamensis induces increased expression of TLR 1, 2, 3 and 4 in human 5 8 9

macrophages. Human macrophages were cultured without treatment, infected with L. panamensis 5 9 0

promastigotes at 5:1 parasite-to-macrophage ratio or stimulated with zymosan (TLR1), Pam2CSK4 5 9 1

(TLR2), Poly I:C (TLR3), LPS (TLR4) or CpG (TLR9). TLR expression was evaluated after 8 h of 5 9 2

exposure to ligands or promastigotes by flow cytometry. Upper panel: MFI of TLR expression from un-5 9 3

infected (empty triangle) and infected macrophages (filled circle) from 12 different individuals, P values 5 9 4

are indicated. Lower panel: Representative histograms of TLR expression of uninfected macrophages, 5 9 5

and macrophages exposed to specific TLR ligands, or L. panamensis promastigotes. Cells were gated 5 9 6

on the CD14+ population. 5 9 7

5 9 8

FIGURE 3. L. panamensis promastigotes and amastigotes induce TNF production in a dose-5 9 9

dependent manner and activate macrophages through TLR dependent signaling. (A) 6 0 0

TNFc"production by WT macrophages exposed to increasing parasite-to-cell ratios using opsonized 6 0 1

promastigotes or amastigotes, specific TLR ligands (shaded bars), or untreated. (B) MyD88/TRIF-/- 6 0 2


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cells were infected at a 100:1 parasite-to-macrophage ratio. Data are representative of three 6 0 3

independent experiments and are expressed as the mean ‒ SEM. *(p< 0.05). 6 0 4

6 0 5

FIGURE 4. MyD88 or TRIF molecules are required for induction of TNF production during L. 6 0 6

panamensis infection. TNFc production by MyD88-/- (A) and TRIF-/- (B) macrophages co-cultured 6 0 7

with opsonized or non-opsonized L. panamensis promastigotes and amastigotes at 100:1 parasite-to-6 0 8

macrophage ratio, stimulated with specific TLR ligands, Sendai virus (shaded bars) and untreated. 6 0 9

Data are from three independent experiments and are expressed as the mean ‒ SEM. 6 1 0

6 1 1

FIGURE 5. Endolysosomal TLRs play a role in the induction of TNF production during L. 6 1 2

panamensis infection. TNFc production by UNC93b deficient macrophages co-cultured with L. 6 1 3

panamensis promastigotes or amastigotes at 100:1 parasite-to-macrophage ratio, stimulated with 6 1 4

specific intracellular TLR ligands and LPS (shaded bars), and untreated (medium). Data are from three 6 1 5

independent experiments and are expressed as the mean ‒ SEM.*(p< 0.05). 6 1 6

6 1 7

FIGURE 6. L. panamensis induces TNF production by macrophages through TLR4 but not 6 1 8

TLR2. TNFc production by (A) TLR2-/-, (B) TLR4-/- macrophage cell lines and (C) differentiated MD-2-/- 6 1 9

BMM (C57BL/6). BMMs were untreated (medium) or co-cultured with L. panamensis promastigotes 6 2 0

and amastigotes at 100:1 parasite-to-macrophage ratio, or stimulated with specific TLR ligands and 6 2 1

LPS alone or in combination with Eritoran (shaded bars). Figures summarize three independent 6 2 2

experiments and are expressed as the mean ‒ SEM. *(p< 0.05). 6 2 3

6 2 4

FIGURE 7. TLR4 mediates early macrophage leishmanicidal activity. (A) Parasite infection and 6 2 5

survival in WT (C57BL/6) and TLR4-/- macrophages infected with L. p-Luc promastigotes. Figure 6 2 6


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shows the relative light units (RLU) of three independent experiments performed in duplicate. 6 2 7

**(p<0.01) vs WT cells. (B) Confocal microscopy images of WT and TLR4-/- mouse macrophages 6 2 8

infected with L. p-GFP promastigotes. Cell membranes were stained with CellMask red deep. Images 6 2 9

show parasites internalized after 30 min of infection. Images of entire slides (right side) show a 6 3 0

representative overview of WT and TLR4-/- macrophage infection. 6 3 1

6 3 2

FIGURE 8. Activation of primary bone marrow macrophages by L. panamensis is TLR4 6 3 3

dependent and signaling is preferentially mediated by MyD88. TNFc production by differentiated 6 3 4

(A) TLR4-/-, (B) MyD88-/-, (C) TRIF-/- and (D) MyD88/TRIF-/- (C57BL/6) BMMs untreated (medium), co-6 3 5

cultured with L. panamensis promastigotes at 100:1 parasite-to-macrophage ratio, stimulated with 6 3 6

specific TLR ligands and Sendai virus (shaded bars). Figures summarize data from three independent 6 3 7

experiments expressed as the mean ‒ SEM. *(p< 0.05). 6 3 8


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FIGURE 1

25 min 4 h 8 h 24 h0

500

1000

1500

2000

2500

3000

5 par:cell1 par:cell

10 par:cell

*

*

Time post-exposition

TN

Fc

(p

g/m

l)

Medium Zymosan LPS Poly I:C L. panamensis10

100

1000

10000

100000**

TN

Fc

(p

g/m

l)

A

B


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TLR1(p= 0.018)

0

25

50

75

100

MF

I

TLR2(p= 0.021)

0

50

100

150

200

TLR3(p= 0.007)

0

200

400

600

800

TLR4(p= 0.012)

0

50

100

150

200

TLR9(p= 0.30)

0

100

200

300

FIGURE 2


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FIGURE 3

LPS (TLR

4)

LPS+ Erit

oran

Pam2C

SK4

(TLR

2)

CpG

(TLR

9)

Poly I:

C (T

LR3)

Sendai

Viru

s0

250

500

750

1000

1250

1500

Medium 50 100 200 25 50 100

promastigotes:cell

L. panamensisL. panamensis + Eritoran

amastigotes:cell

TN

Fc

(p

g/m

l)

LPS (TLR

4)

Pam2C

SK4

(TLR

2)

CpG

(TLR

9)

Poly I:

C (T

LR3)

Sendai

Viru

s0

200

400

600

800

1000

1200

1400

1600

1800

promastigotes amastigotes

WT

MediumMyD88/TRIF-/-

*

*

TN

Fc

(p

g/m

l)

A

B


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FIGURE 4

LPS (TLR

4)

Pam2C

SK4

(TLR

2)

Poly I:

C (T

LR3)

Sendai

Viru

s0

200

400

600

800

1000

1200

1400

1600

1800


WT

MyD88-/-Medium

ops non ops ops non ops

TN

Fc

(p

g/m

l)

Poly I:

C (T

LR3)

LPS (TLR

4)

Pam2C

SK4

(TLR

2)

Sendai

Viru

s0

200

400

600

800

1000

1200

1400

1600

1800

WTTRIF-/-Medium

promastigotes amastigotesops non ops ops non ops

TN

Fc

(p

g/m

l)

A

B


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FIGURE 5

CpG

(TLR

9)

Poly I:

C (T

LR3)

LPS (TLR

4)0

200

400

600

800

1000

1200

1400

1600

1800

WTUNC93b mutantMedium

*


* **

TN

Fc

(p

g/m

l)


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FIGURE 6

Pam2C

SK4

(TLR

2)

LPS (TLR

4)0

200

400

600

800

1000

1200

1400

1600

1800

TLR2-/-

WT


Medium

ops non ops ops non ops

TN

Fc

(p

g/m

l)

LPS (TLR

4)

LPS+ Erit

oran

Pam2C

SK4

(TLR

2)0

200

400

600

800

1000

1200

1400

1600

1800

WT

MediumTLR4-/-


**

* *

TN

Fc

(p

g/m

l)

LPS (TLR

4)

LPS+ Erit

oran

Pam2C

SK4

(TLR

2)0

200

400

600

800

1000

1200L. panamensisMedium

L. panamensis + Eritoran


MD-2-/-WT WTMD-2-/-

TN

Fc

(p

g/m

l)

A

B

C


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4 16 24 480

50000

100000

150000

200000

250000

300000

350000WT

TLR4 -/-

**

hours

RL

U

FIGURE 7

A

B

Cell membrane L. panamensis-GFP Overlay

WT

TLR4-/-


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FIGURE 8.

LPS (TLR

4)

Pam2C

SK4

(TLR

2)0

200

400

600

800

1000

1200Medium

opsonized non opsonized

TLR4 -/-

WT

*

*

TN

Fc

(p

g/m

l)

LPS (TLR

4)

Pam2C

SK4

(TLR

2)

Poly I:

C (T

LR3)

Sendai

viru

s0

200

400

600

800

1000

1200


MyD88/TRIF -/-

WT

Medium

*

*

TN

Fc

(p

g/m

l)

LPS (TLR

4)

Pam2C

SK4

(TLR

2)

Sendai

viru

s

Poly I:

C (T

LR3)

0

200

400

600

800

1000

1200


MyD88-/-

WT

Medium

*

*

TN

Fc

(p

g/m

l)

Poly I:

C (T

LR3)

LPS (TLR

4)

Pam2C

SK4

(TLR

2)

Sendai

viru

s0

200

400

600

800

1000

1200

1400

TRIF-/-

WT

Medium


TN

Fc

(p

g/m

l)

A

B

C

D


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Documents

Toll-like receptors participate in macrophage activation ...iai.asm.org/content/early/2011/04/25/IAI.01388-10.full.pdf · < with 20% L929 cell culture ... < following adherence to