The β-glucan receptor Dectin-1 functions together with TLR2 to mediated macrophage activation by mycobacteria

doi:10.1182/blood-2006-05-024406Prepublished online July 6, 2006;

Mahesh Yadav and Jeffrey S Schorey macrophage activation by mycobacteria

-glucan receptor Dectin-1 functions together with TLR2 to mediatedβThe

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1

The β-glucan receptor Dectin-1 functions together with TLR2 to mediated macrophage activation by mycobacteria

Mahesh Yadav and Jeffrey S. Schorey

Department of Biological Sciences, Center for Tropical Disease Research and Training

University of Notre Dame, Notre Dame, Indiana, 46556

Corresponding author. Mailing address: Department of Biology, University of Notre Dame, 130 Galvin Life Science Center, Notre Dame, Indiana, 46556.

Phone: (574) 631-3734. Fax: (574) 631-7413.

E-mails: [email protected]

Running title: Dectin-1 and mycobacterial infection

Note: This work was supported through grants AI056979 and AI052439 from the

National Institute of Allergy and Infectious Diseases.

Scientific Heading: Phagocytes

Word count. Abstract (200 words) Total (4,862)

Blood First Edition Paper, prepublished online July 6, 2006; DOI 10.1182/blood-2006-05-024406

Copyright © 2006 American Society of Hematology

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2

ABSTRACT

Pattern recognition receptors (PRRs) play an essential role in a macrophage’s response to

mycobacterial infections. However, how these receptors work in concert to promote this

macrophage response remains unclear. In this study, we used bone marrow-derived

macrophages isolated from Mannose Receptor (MR), Complement Receptor 3 (CR3),

MyD88, Toll-like receptor 4 (TLR4) and TLR2 knockout mice to examine the

significance of these receptors in mediating a macrophage’s response to a mycobacterial

infection. We determined that MAPK activation and TNF-α production in macrophage

infected with M. avium or M. smegmatis is dependent on MyD88 and TLR2 but not

TLR4, MR or CR3. Interestingly, the TLR2 mediated production of TNF-α by

macrophages infected with M. smegmatis required the β-glucan receptor Dectin-1. A

similar requirement for Dectin-1 in TNF-α production was observed for macrophages

infected with M. bovis BCG, M. phlei, M. avium 2151-rough and M. tuberculosis H37Ra.

The limited production of TNF-α by virulent M. avium 724 and M. tuberculosis H37Rv

was not dependent on Dectin-1. Furthermore, Dectin-1 facilitated IL-6, RANTES and G-

CSF production by mycobacteria-infected macrophages. These are the first results to

establish a significant role for Dectin-1, in cooperation with TLR2, to activate a

macrophage’s pro-inflammatory response to a mycobacterial infection.






3

INTRODUCTION

The immune system has the complex task of separating “friend” from “foe”. To

accomplish this mission the immune system has evolved receptors which recognize

molecules present on pathogenic organisms but which show limited interaction with host

components. These receptors referred to as pattern recognition receptors (PRRs),

function to promote an innate immune response and include such members as the

mannose receptor, scavenger receptors and Toll-like receptors among others. PRRs bind

to conserved microbial structures called pathogen associated molecular patterns

(PAMPs). The expression of these receptors allows the immune system to recognize a

wide variety of pathogens which express one or more of these PAMPs and their

engagement initiates the subsequent immune response. Not surprisingly, the PRRs are

expressed on cells of the innate immune system including macrophages, neutrophils,

dendritic cells and NK cells 1.

Binding of microbial products by PRRs elicits a signaling response within the

leukocyte resulting in the production of specific immune modulators. Which PRRs are

engaged and in what combination, along with the specific ligands involved will dictate

the overall response by the immune cell. This complexity allows the immune system to

tailor its response to a specific pathogen, yet remain flexible enough to recognize a large

number of potential pathogens.

One of the most intensively studied PRR is the mannose receptor (MR) which has

been implicated in pathogen recognition through binding terminal mannose, fucose, and

N-acetylglucosamine residues 2,3. The major ligand for MR on mycobacterial surface is

the mannose-capped lipoarabinomannan (ManLAM) from M. tuberculosis and M. bovis






4

BCG 4-6.Another important group of PRRs are the Toll-like receptors (TLRs) which are

also involved in the innate recognition of mycobacteria by the host 7. After ligand

binding, TLRs activate signal transduction pathways by recruiting adaptor molecules

including myeloid differentiation factor 88 (MyD88) 8,9. Stimulation of TLRs by

microbial products activates NF-κB, MAPK and phosphoinositide 3-kinase signaling

pathways leading to activation of inflammatory target genes 10. In vitro studies have

shown a number of potent agonists on mycobacteria for TLR2 11,12 and TLR4 13,14, and

their role in controlling a mycobacterial infection 15,16. TLRs participate with additional

receptors in the innate recognition of microbes, and there is a strong interaction between

the signaling pathways induced by these receptors and signal transduction stimulated by

TLRs (for review 17,18). Dectin-1 is one of the receptors recently shown to complement

TLR signaling in order to generate a pro-inflammatory response 19. Dectin-1 is a C-type

lectin receptor expressed on monocytes, macrophages, neutrophils, Dendritic cells and

Langerhans cells which recognizes fungal wall-derived β-glucans 20,21. Dectin-1 promotes

the phagocytosis of live yeast and fungal-derived zymosan particles, as well as promotes

zymosan or fungal pathogen-induced pro-inflammatory response by macrophages and at

least in some cases cooperates with TLR2 to mediate this response 19,22. Recent studies

have also shown a role for Dectin-1 in the phagocytosis of Haemophilus influenzae by

eosinophils 23. However, the importance of Dectin-1 as a PRR and its role in promoting a

pro-inflammatory response to a mycobacterial infection or to bacterial infections in

general remains undefined.

In the present study we assessed the role of various macrophage PRRs in MAPK

activation and cytokine production upon mycobacterial infection. We found TLR2 and






5

Dectin-1 to work in concert to promote macrophage activation upon mycobacterial

infection. These are the first studies to indicate a role for Dectin-1 in promoting a

macrophage pro-inflammatory response to a mycobacteria infection.






6

MATERIALS AND METHODS

Reagents Unless stated otherwise, all chemical reagents were purchased from Sigma (St.

Louis, MO).

Mice Balb/c and C57BL/6 mice were purchased from Harlan (Mutant Mouse Regional

Resource Center, Indiana). TLR2-/- and TLR4-/- mice were purchased from Jackson

Laboratory (Maine, USA). MyD88-/- (in C57BL/6 background) and CR3-/- (in Balb/c

background) were generously provided by Soon-Cheol Hong, Indiana University Medical

School and Tanya Mayadas-Norton, Harvard Medical College respectively. MR-/- and

C57BL/6 MR+/+ were kindly provided by Michel Nussenzweig, Rockefeller University.

Bacteria Mycobacteria stocks were generated by using a single colony to inoculate

Middlebrooks 7H9 media (Difco, Sparks, MD) supplemented with glucose, oleic acid,

albumin, Tween-20 and NaCl (GOATS). For Mycobacterium avium 724 stocks, the

mycobacteria were passaged through a mouse to ensure virulence. Bacteria were grown

for 3-10 days at 37°C with vigorous shaking, centrifuged, re-suspended in

Middlebrooks/GOATS plus 15% glycerol, aliquoted and stored at -80°C. Frozen stocks

were quantitated by serial dilution onto Middlebrooks 7H10 agar/GOATS. M. smegmatis

strain MC2155, M. bovis BCG, M. phlei, M. tuberculosis H37Rv, M. tuberculosis H37Ra

(ATCC) and other M. avium strains were cultured and frozen stocks prepared as

described above. All reagents used to grow mycobacteria were found negative for

endotoxin contamination using the E-Toxate assay and the QCL-1000 Endotoxin test

(Cambrex Bio Science, Walkersville, MD).






7

Complement opsonization Appropriate concentrations of mycobacteria were suspended

in macrophage culture media containing 10% horse serum as a source of complement

components and incubated for 2 hours at 37°C 29. The same concentration of horse serum

was added to uninfected controls for all experiments.

Bone marrow macrophage isolation and Mycobacteria infection BMMφs, used in all

experiments, were isolated from 6-8 week old mice as previously described 24. Infection

assays evaluated by fluorescence microscopy were performed on each stock of

mycobacteria to determine the infection ratio needed to obtain approximately 80% of the

macrophages infected. The MOI required to obtain 80% infection varied from

approximately 20:1 to 50:1 mycobacteria to macrophage and depended on the

mycobacterial species and frozen stock of mycobacteria. Briefly, BMMφs from WT or

KO mice were plated on glass coverslips and infected with different doses of

mycobacteria in triplicate. Infections were halted at either 1 hour or 4 hour and fixed in

1:1 methanol: acetone, washed with PBS, and stained with TB Auramine M Stain Kit

(Becton Dickinson, Sparks,MD) in the case of M. avium and M. bovis BCG, and with

acridine orange (Sigma) in case of M. smegmatis and M. phlei. Slides were visualized

using fluorescent microscopy and the level of infection was quantitated by counting the

number of cells infected and the approximate number of mycobacteria/cell in at least four

fields per replicate. No fewer than 100 cells per replicate were counted.

For all experiments, mycobacteria were added to macrophages on ice and

incubated for 10 minutes and then incubated at 37°C in 5% CO2 for the specified times.

Culture media without antibiotics or L-cell supernatant was used in place of complete






8

media during the infections. For the 24 hour time-points, the BMMφs were incubated for

4 hours with the mycobacteria, washed with PBS 3 times, then fresh media was added

and incubated for an additional 20 hours. All tissue culture reagents were found negative

for endotoxin contamination using either the E-Toxate assay.

Antibody treatment The anti-Dectin-1 antibody clone 2A11 and rat IgG2b isotype control

were purchased from Serotec (Oxford, UK). BMMφs were pre-incubated with 50µg/ml

2A11 or isotype control 1 hour prior to infection in DMEM and then infected with

mycobacteria. After 4 hour incubation, media was removed and cells were washed with

PBS three times and fresh media was added and incubated for an additional 20 hours.

Western blot analysis Blots were performed as previously described 24. Briefly, at

designated times, culture media was collected and saved for subsequent ELISAs and the

cells were washed 3 times with ice-cold PBS containing 1mM per-vanadate. The cells

were then treated for 5-10 minutes with ice-cold lysis buffer and the cell lysates were

removed from the plates and stored at -20°C. Equal amounts of protein, as defined using

the Micro BCA Protein Assay (Pierce, Rockford, IL), were loaded onto 10% SDS-PAGE

gels, electrophoresed and transferred onto PVDF membrane (Millipore, Bedford, MA).

The membranes were incubated with primary antibodies phospho-p38, total p38,

phospho-ERK1/2 or total ERK1/2 from Cell Signaling (Beverly, MA). The blots were

washed and incubated with a secondary antibody, either horseradish peroxidase

conjugated anti-rabbit or anti-mouse IgG (Pierce) in TBST plus 5% powdered milk. The






9

bound antibodies were detected using SuperSignal West Femto enhanced

chemiluminescence reagents (Pierce).

Cytokine measurements The levels of cytokines secreted by infected macrophages were

measured using the commercially available ELISA reagent kits for TNF-α (PharMingen,

San Diego, CA), IL-6, RANTES (eBioscience, San Diego, CA) and G-CSF (R&D

Systems, Minneapolis, MN). Culture media collected from the macrophages was

analyzed for cytokines according to manufacturers, instructions and the cytokine

concentrations were determined against standard curves. A cytokine profile analysis was

performed by using the RayBioTM Mouse Cytokine Array (RayBiotech Inc. Atlanta, GA)

with culture supernates from non-infected or infected macrophages.

Statistical analysis Statistical significance was determined with the paired two-tailed

Student’s t-test at P<0.05 level of significance, using InStat/Prism software.






10

RESULTS

MR is not required for phagocytosis of M. avium or M. smegmatis by murine

macrophages.

Prior studies have indicated a role for the MR on human alveolar macrophages in

the phagocytosis of non-opsonized virulent Mycobacterium tuberculosis strains H37Rv

and Erdman but a minimal role in the phagocytosis of the avirulent strain H37Ra 25. With

the availability of mice deficient in the MR, we compared mouse bone marrow-derived

macrophages (BMMφs) from WT and MR-/- mice in their ingestion of complement-

opsonized and non-opsonized M. avium. We found a direct correlation between the

multiplicity of infection (MOI) and the percentage of cells infected; however, there was

no difference in the number of infected cells between WT and MR-/- macrophages at

either 1 or 4 hour infections (Fig 1). Moreover, we did not observe any detectable

differences in the number of mycobacteria per infected macrophage (data not shown). As

expected, we observed a higher number of infected BMMφs, when M. avium was

complement-opsonized compared to non-opsonized at both time points and at different

MOIs. Similar results were seen with non-pathogenic M. smegmatis, as we observed no

difference in its phagocytosis by WT and MR-/- BMMφs at 1 or 4 hour (data not shown).

BMMφs from TLR4-/-, TLR2-/- and MyD88-/- displayed no difference compared to WT

BMMφs in the uptake of complement-opsonized or non-opsonized mycobacteria (data

not shown).

MR, CR3 and TLR4 are not required for MAPK activation or TNF-α production

by BMMφs in response to M. avium or M. smegmatis infection.






11

In addition to serving as phagocytic receptors, PRRs function to promote

macrophage activation characterized by production of cytokines, chemokines and oxygen

and nitrogen reactive species. This macrophage activation requires stimulation of

signaling pathways resulting in a transcriptional response. Activation of the mitogen

activated protein kinase (MAPK) pathways in macrophages has been associated with

PRR stimulation resulting in the production of numerous pro-inflammatory molecules 26-

28. To start addressing the role of various PRRs in promoting macrophage activation, we

first evaluated the signaling response by WT and MR-/- macrophages to infection with

pathogenic M. avium and non-pathogenic M. smegmatis. Specifically, BMMφs from WT

and MR-/- mice were infected with mycobacteria for 1 hour or 9 hour and MAPK

activation was measured by Western blotting for phospho-p38 and phospho-ERK1/2. We

chose to look at 1 and 9 hours, since our previous studies have shown an initial activation

of macrophage MAPKs by all mycobacteria, but a differential activation of MAPKs 9

hours post-infection in macrophages infected with non-pathogenic mycobacteria

compared to M. avium 24,29,30. As shown previously, MAPKs are activated upon

mycobacterial infection and macrophages infected with M. smegmatis show higher levels

of MAPK activation at 9 hours compared to non-infected BMMφs or M. avium infected

cells (Fig. 2A). However, there was no difference in MAPK activation between WT and

MR-/- macrophages following infection with either M. smegmatis or M. avium 724.

Previously we demonstrated the importance of the MAPKs, as well as other signaling

molecules involved in macrophage’s production of TNF-α following a mycobacterial

infection 24,29. Therefore, we looked at the TNF-α production in WT and MR-/- BMMφs

infected with M. smegmatis or M. avium 724. Similar to what we observed with MAPK






12

activation, there was no difference in TNF-α secretion between WT and the KO

macrophages (Fig 2B). Moreover, infection using non-opsonized mycobacteria also

showed similar levels of TNF-α in WT and MR-/- macrophages (Fig. 2B). As previously

observed, M. smegmatis infected macrophages produce significantly higher levels of

TNF-α relative to M. avium infected cells 24.

BMMφs from WT and TLR4-/- mice also showed no difference in MAPK

activation upon infection with either Mycobacterium (Fig 2C). Moreover, TNF-α

production induced upon infection with either M. smegmatis or M. avium 724 did not

differ between TLR4-/- and WT macrophages (Fig. 2D). To test the role of CR3 in the

macrophage response to a mycobacterial infection, we infected BMMφs from WT and

CR3-/- mice. As expected, BMMφs from CR3-/- mice showed decreased phagocytosis of

complement-opsonized mycobacteria, and required a higher mycobacteria to macrophage

ratio in order to obtain a similar infection level as WT macrophages (data not shown).

However, there was no difference in MAPK activation or TNF-α production upon

infection with complement opsonized or non-opsonized mycobacteria in CR3-/-

macrophages compared to WT (data not shown).

Optimal MAPK activation and TNF-α production by mycobacteria infected

macrophages requires TLR2 and MyD88.

The above data did not support a role for the MR, CR3 or TLR4 in the activation

of macrophages by M. avium or non-pathogenic M. smegmatis. To determine if the

observed BMMφ’s response to M. avium and M. smegmatis was dependent on TLR2, we

infected TLR2-/- and WT BMMφs with the different mycobacteria. BMMφs from TLR2-/-






13

mice showed a lack of MAPK activation above non-infected controls following an M.

avium 724 infection at both 1 and 9 hours post-infection (Fig. 3A). The low amount of

TNF-α produced by M. avium 724 infected BMMφs was also completely lost in TLR2-/-

BMMφs (Fig. 3B). In macrophages infected with M. smegmatis, no difference in ERK1/2

activation was observed between TLR2-/- and WT macrophages at 1 hour post-infection,

but by 9 hours a significant decrease in MAPK activation was discerned in the TLR2-/-

BMMφs (Fig. 3B). Furthermore, we observed a significant drop but not a complete loss

of TNF-α production in M. smegmatis infected TLR2-/- macrophages (Fig. 3B). The

adaptor protein MyD88 has been shown previously to be critical in TLR signaling 31. To

determine if MAPK activation and TNF-α production was dependent on MyD88, we

infected BMMφs from WT and MyD88-/- mice. Upon infection there was a complete loss

of MAPK activation in MyD88-/- macrophages. As shown in Fig. 3C, this effect was

evident at both 1 and 9 hours post-infection. Similarly there was no TNF-α production

detected in MyD88-/- macrophages upon infection with either M. smegmatis or M. avium

724 (Fig. 3D). These findings differ from our results with the TLR2-/- BMMφs, where

there was no loss in ERK1/2 phosphorylation at 1 hour post-infection and some TNF-α

production in M. smegmatis infected BMMφs.

Induction of TNF-α production upon mycobacterial infection is partially dependent

on Dectin-1.

We anticipated that TLR2 and MyD88 were required, at least in part, for the

mycobacteria-initiated macrophage activation, and our data supports this prediction.

However, TLR2 often functions in concert with other receptors including additional






14

TLRs and non-TLRs to promote cellular activation 17,18. Which combination of receptors

are engaged depends on the ligands involved 32. Dectin-1 is a recently described receptor

expressed on BMMφs 33, which can function together with TLR2 to induce macrophage

activation 19,22.

Dectin-1, a C-type lectin receptor, has been implicated in the innate recognition of

yeasts through its binding to surface β-glucan 34. Recently it has been found to cooperate

with TLR2 in eliciting inflammatory responses to Zymosan 19. Therefore, we were

interested in defining whether Dectin-1 plays a role in TNF-α production upon infection

with mycobacteria. For this purpose we utilized a previously characterized neutralizing

antibody against Dectin-1 33 to block the mycobacterial interaction with the macrophage

Dectin-1 receptor. Pre-treating the macrophages with the anti-Dectin-1 mAb 2A11 had no

affect on the phagocytosis of M. avium or M. smegmatis (data not shown). However, pre-

treating the macrophages with the 2A11 mAb resulted in a significant reduction in TNF-

α production upon infection with M. smegmatis (Fig. 4A). M. avium 724 induces little

TNF-α production and thus the effect of 2A11 was difficult to determine. Therefore, we

used the avirulent M. avium 2151 rough strain which is known to induce significant TNF-

α production 35. Like our findings with M. smegmatis, the production of TNF-α by M.

avium 2151 infected BMMφs was significantly blocked in the presences of anti-Dectin-1

mAb 2A11 (Fig. 4A). Similar reduction in TNF-α secretion upon M. smegmatis

infection was seen in macrophages pretreated for 1 hour with 700 µg/ml Laminarin, a

soluble β-glucan used for blocking the β-glucan receptor (Fig. 4B) 36. Pretreatment with

the polysaccharide Galactan at the same concentration did not affect TNF-α production

in M. smegmatis infected macrophages. Blocking Dectin-1 in BMMφs infected with non-






15

opsonized M. smegmatis also resulted in inhibition of TNF-α production (Fig. 4C).

However, there was no difference in TNF-α production in BMMφs treated with LPS in

the presence of 2A11 (Fig. 4C).

Dectin-1 mediated induction of TNF-α is dependent on TLR2 expression.

Recent papers have suggested a possible cooperation between TLR2 and Dectin-1

in zymosan recognition by HEK 293 cells. Gantner et al., showed that NF-κB activation

in macrophages by Zymosan requires both TLR2 and Dectin-1 19. We hypothesized that

there is a similar cooperation between Dectin-1 and TLR2 in macrophages infected with

mycobacteria. As shown earlier, there was a marked diminished TNF-α production in

TLR2-/- macrophages compared to WT upon infection with M. smegmatis (Fig. 5).

However in contrast to WT, pretreatment of TLR2-/- BMMφs with 2A11 did not further

reduce TNF-α production upon infection with M. smegmatis, suggesting that Dectin-1

mediated induction of TNF-α production was dependent on TLR2 (Fig. 5). No TNF-α

was detected in TLR2-/- BMMφs upon M. avium 724 infection (Fig. 5).

Dectin-1 facilitates TNF-α production in macrophages infected with M. phlei, M.

bovis BCG and M. tuberculosis H37Ra but not H37Rv.

To determine whether the importance of Dectin-1 in mediating macrophage

activation is limited to the avirulent M. avium 2151 rough strain and M. smegmatis, we

infected BMMφs with non-pathogenic M. phlei, attenuated M. bovis BCG, avirulent M.

tuberculosis H37Ra and virulent H37Rv. We measured infected BMMφs for TNF-α






16

production in the presence of 2A11 or isotype control antibody. As observed previously,

macrophages infected with M. phlei or M. bovis BCG induce high levels of TNF-α

production 24. However, this TNF-α production was reduced significantly in the presence

of the 2A11 antibody while no block was observed with the isotype control antibody

(Fig. 6A). Moreover, we observed significantly higher levels of TNF-α secreted by M.

tuberculosis H37Ra compared to H37Rv infected BMMφs, as observed previously 37.

The TNF-α produced by H37Ra but not H37Rv infected BMMφs was also significantly

decreased in cells treated with the 2A11 blocking antibody compared to untreated or

isotype control (Fig. 6B).

Dectin-1 promotes production of inflammatory mediators in addition to TNF-α in

mycobacteria infected macrophages.

To determine if the importance of Dectin-1 is limited to TNF-α production, we

measured the levels of other pro-inflammatory mediators in the presence of the Dectin-1

blocking antibody. We found that in addition to TNF-α, 2A11 significantly blocked the

BMMφ production of IL-6, G-CSF and RANTES following an M. smegmatis or M. bovis

BCG infection (Fig. 7A). A similar 2A11 antibody-mediated block in IL-6, RANTES

and G-CSF production was observed for BMMφs infected with M. avium 2151 rough or

M. tuberculosis H37Ra (Fig. 7B and data not shown). Infection of BMMφs with M.

avium 724 or H37Rv did not lead to significant levels of IL-6 or G-CSF in culture

supernatants (Fig. 7B and data not shown). However, RANTES production was

increased in M. avium 724 infected BMMφs and this was significantly reduced in 2A11

treated cells (Fig. 7B). Production of the different mediators was not affected by the






17

isotype control antibody. Together, our data indicates that Dectin-1 promotes the

macrophage’s ability to produce a broad spectrum of pro-inflammatory mediators upon

mycobacterial infection.






18

DISCUSSION

The tailored reaction by macrophages to pathogenic organisms as well as their

response to host cell debris and apoptotic cells is dependent on the combination of

macrophage receptors engaged which together initiate a specific signaling response. Key

to understanding macrophage biology requires a definition of the macrophage receptors,

their ligands, the signaling reactions induced upon receptor engagement and the

consequence of their activation (e.g. production of pro-inflammatory mediators). In this

report we focused on a number of macrophage receptors including CR3, MR, TLR2,

TLR4 and Dectin-1 which have been characterized as phagocytic and/or signaling

receptors in the context of various infection models. The purpose of the present study

was to better understand how these receptors are involved in eliciting a macrophage

response following an infection with pathogenic and non-pathogenic mycobacteria.

The mannose receptor has been well studied and reported to bind a wide variety

of pathogens including Candida albicans 38, Leishmania donovani 39, Pneuomcystis

carinii 40 and M. tuberculosis 25. It has been shown to bind Mannose capped LAM (Man-

LAM) from pathogenic but not the phosphoinositol capped LAM from non-pathogenic

mycobacteria 6 and was revealed to mediate phagocytosis of pathogenic M. tuberculosis.

However, we observed that the MR was not necessary for the phagocytosis of

complement opsonized or non-opsonized M. avium 724 or M. smegmatis. The observed

differences in mannose receptor’s role in the present study (using murine BMMφs) and

prior studies (using primary human alveolar macrophages) may in part reflect differences

in the expression and function of the MR, or the redundancy of other phagocytic

receptors in murine macrophages relative to human macrophages. In addition, the MR on






19

human macrophages was shown to bind terminal mannnosyl residues of LAM 5,6 and

studies by Khoo et al., indicate a terminal mono-mannosylation of M. avium Man-LAMs

compared to a di-mannosylation of Man-LAM in M. tuberculosis 41. MR on human

macrophages was also shown to be involved in the phagocytosis of M. smegmatis 42.

However, we observed no difference in the uptake of M. smegmatis between murine WT

and MR-/- BMMφ (data not shown). Similarly, there was no difference in TNF-α

production between WT and MR-/- BMMφs upon infection with mycobacteria. Therefore

our data does not support a role for the MR in MAPK activation or TNF-α production

upon mycobacterial infection of mouse BMMφs. However, we cannot rule out the

contribution of the MR in the activation of other macrophage signaling pathways upon

mycobacterial infection or in MAPK activation upon stimulation with other MR ligands.

The Toll-like receptors have also been extensively characterized in the context of

mycobacterial infections through both in vitro and in vivo studies 7,43. Studies by Feng et

al., indicated that MyD88, and to a lesser extent TLR2, deficient mice were highly

susceptible to an M. avium infection. However, TLR4-/- mice were comparable to WT in

controlling an M. avium infection 16. This is in agreement with in vitro studies which

indicate that M. avium can stimulate a response through TLR2 but not TLR4 44. The M.

avium ligand for TLR2 has recently been defined as glycopeptidolipids (Sweet, L and

Schorey, J.S. J. Leukocyte Biology, In press). This finding contrasts with M.

tuberculosis, which can engage both TLR2 through the 19 KDa lipoprotein and PIM 45,46

and TLR4 through heat shock protein 65 47. Non-pathogenic mycobacteria like M.

smegmatis also express ligands for both TLR2 and TLR4 but whether whole bacteria can

activate both receptors has not been defined. At least in the context of MAPK activation






20

and TNF-α production, we failed to observe a role for TLR4 in a macrophage response to

a M. smegmatis infection.

In contrast, we have observed that both M. smegmatis and M. avium infected

macrophages require TLR2 and MyD88 for macrophage activation. The adaptor

molecule MyD88 is critical for TLR2 signaling and for signaling by other Toll-like

receptors, although TLR4 can signal through a MyD88 independent manner as well 9,31.

It is interesting that the early MAPK activation and some TNF-α production by M.

smegmatis infected BMMφs was independent of TLR2, but required MyD88 suggesting

that M. smegmatis can engage other TLRs to initiate the signaling response. This was not

the case for M. avium whose MAPK activation and TNF-α production was completely

dependent on TLR2. Our results are consistent with previously published in vitro studies

indicating that of the Toll-like receptors; only TLR2 is stimulated by M. avium 16.

Nevertheless our present data demonstrate that TLR2 is vital for an optimal

macrophage response to infection by both pathogenic and non-pathogenic mycobacteria.

A recent study showed that TLR2 associates with Ras to activate ERK1/2 and

TRAF6/TAK1 to activate p38 upon infection with M. avium 48. Further experimentation

is needed to define how the signaling complexes associated with TLR2 differ in

macrophages infected with pathogenic versus non-pathogenic mycobacteria. However,

TLR2 often dimerizes with other Toll-like receptors such as TLR1 and TLR6 following

ligand engagement 49,50. The signaling responses initiated will likely depend on which

combinations of Toll-like receptors are engaged.

Recent studies indicate that TLR2 can also function in concert with receptors

other then TLRs. Of the various receptors which can function in conjunction with TLRs






21

to stimulate a cellular response, we focused our attention on the pattern recognition

receptor Dectin-1. Dectin-1 is a type II C-type lectin receptor which is expressed on

monocytes, macrophages, neutrophils, DCs and Langerhans cells. Dectin-1 binds to β-

glucans and has been implicated in eliciting an inflammatory response to zymosan and

other yeast 21,51. Gantner et al showed that Dectin-1 cooperates with TLR2 to induce NF-

κB activation and TNF-α production upon zymosan treatment 19. Another recent study

showed that Dectin-1 could interact with TLR2 during a fungal infection to stimulate

TNF-α and IL-6 secretion by macrophages 22. However, it is unclear whether Dectin-1

functions in stimulating a macrophage response to organisms other then yeast. Using the

neutralizing monoclonal antibody 2A11 to block Dectin-1, we observed this PRR to

promote macrophage production of TNF-α, RANTES, IL-6 and G-CSF in cells infected

with a number of different mycobacteria including non-pathogenic M. smegmatis and M.

phlei, attenuated M. bovis BCG and avirulent M. avium 2151 rough and M. tuberculosis

H37Ra. However, questions remain as to the importance of Dectin-1 in mediating a

macrophage signaling response to pathogenic mycobacteria. The limited release of TNF-

α, G-CSF and IL-6 by M. avium 724 and M. tuberculosis H37Rv infected BMMφs makes

it difficult to define a role for Dectin-1 in the production of these mediators.

Nevertheless, the release of RANTES, which is markedly increased in M. avium 724

infected BMMφs, is significantly diminished in cells treated with the blocking anti-

Dectin-1 antibody. This suggests that Dectin-1 may play a more restricted role in a

BMMφ’s signaling response to pathogenic mycobacteria. It is intriguing to speculate that

limited engagement/stimulation of Dectin-1 by pathogenic mycobacteria is responsible, at

least in part, for the minimal macrophage pro-inflammatory response induced by these






22

mycobacteria. Our data also indicates that TLR2 is required for the Dectin-1 mediated

pro-inflammatory response since there was no change in TNF-α production in TLR2-/-

BMMφs infected with M. smegmatis in the presence or absence of blocking antibody

2A11.

There have been recent reports that ligation of Dectin-1 results in tyrosine

phosphorylation of the receptor’s ITAM signaling motif leading to recruitment of the

tyrosine kinase Syk and initiation of the downstream signaling response. For example,

Dectin-1 engagement by yeast can stimulate IL-2 and IL-10 production in a Syk kinase

dependent but TLR2 independent mechanism 52. Moreover, in a subset of macrophages,

Dectin-1 mediated production of reactive oxygen species was dependent on Syk kinases

53. The exact mechanism by which Dectin-1 promotes a TLR2 pro-inflammatory

response to mycobacteria is unclear but it might function through an effect on NF-κB.

Future studies will address this important issue and whether Syk kinases are involved.

What ligand(s) on mycobacteria interact with Dectin-1? At present this question

remains unanswered. BCG and M. tuberculosis express α-glucan within the outer

capsule 54,55. In contrast, the presence of β-glucan has not been described in

mycobacteria. Nevertheless, M. tuberculosis binding to the lectin binding site on CR3

was inhibited by the β-glucan Laminarin 56. However, this may simply indicate that the

CR3 lectin site can bind multiple carbohydrates including α and β glucans. Dectin-1 has

also been described to bind an endogenous but undefined ligand on T cells 57, indicating

that Dectin-1 has the potential to bind molecules other then β-glucans. Additional studies

will be needed to define the Dectin-1 ligand on mycobacteria.

To our knowledge a role for the pattern recognition receptor Dectin-1 in






23

stimulating a macrophage response has not been demonstrated for any bacterial pathogen.

Our data suggest that Dectin-1 serves to amplify the TLR2 dependent activation of a

macrophage’s pro-inflammatory response to non-pathogenic and to a lesser extent

pathogenic mycobacteria. Therefore, Dectin-1 may play a significant role in promoting

an immune response against a mycobacterial infection by facilitating macrophage

activation. A test of this hypothesis awaits in vivo studies in Dectin-1 deficient mice.






24

ACKNOWLEDGEMENTS: This work was supported through grants AI056979 and

AI052439 from the National Institute of Allergy and Infectious Diseases. We gratefully

received the M. tuberculosis H37Rv from Colorado State University as part of NIH,

NIAID Contract No. HHSN266200400091C, entitled "Tuberculosis Vaccine Testing and

Research Materials”.






25

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33

FIGURE LEGANDS

FIGURE 1. Mannose receptor is not essential for the phagocytosis of M. avium 724.

BMMφs from WT or MR-/- mice were infected with different ratios of complement

opsonized (A, B) or non-opsonized (C, D) mycobacteria for 1 hour (A, C) or 4 hours (B,

D). After infection, cells were washed, fixed and stained with acridine orange. The

percentage of phagocytic cells having ingested at least one Mycobacterium was measured

by fluorescence microscopy as described in Material and Methods. Values are expressed

as mean + SD. Data are representative of three separate experiments.

FIGURE 2. Mannose Receptor and Toll-like receptor 4 are not essential for MAPK

activation and TNF-α production in BMMφs upon M. smegmatis or M. avium 724

infection. BMMφs from WT and either MR-/- (A and B) or TLR4-/- (C and D) mice were

infected with M. smegmatis or M. avium 724 and screened for MAPK activation at 1 hour

and 9 hour (A and C) and TNF-α production at 24 hour (B and D). (A and C) MAPK

activation was detected by probing cell lysates of infected or non-infected (RC) BMMφs

by Western blot for activated ERK1/2 and p38 using phospho-specific Abs as described

in the Materials and Methods. Total ERK1/2 and total p38 blots were run to show equal

protein loading. (B and D) BMMφs from WT and KO mice were infected with

complement opsonized or non-opsonized mycobacteria and 24 hour later culture

supernates were removed and analyzed by ELISA for TNF-α. Values are expressed as

mean + SD. Data are representative of three separate experiments. Sm and Smeg- M.

smegmatis, Av and Avium- M. avium, RC- non-infected BMMφs.






34

FIGURE 3. MyD88-/- and TLR2-/-

BMMφs show impaired MAPK activation and

TNF-α production upon mycobacterial infection. BMMφs from WT and TLR2-/- mice

(A and B) and MyD88-/- mice (C and D) were infected with M. smegmatis or M. avium

724 and screened for MAPK activation at 1 hour and 9 hour and TNF-α production at 24

h. (A and C) MAPK activation was detected by preparing cell lysates for 1 hour and 9

hour infections and probed for activated ERK1/2 and p38 using phospho-specific Abs as

described in the Materials and Methods. Total ERK1/2 and p38 blots were run to show

equal protein loading. (B and D) BMMφs from WT and KO mice were infected with

complement opsonized or non-opsonized mycobacteria and 24 hour later culture

supernates were removed and analyzed by ELISA for TNF-α. Values are expressed as

mean + SD. Data are representative of three separate experiments. Sm and Smeg- M.

smegmatis, Av and Avium- M. avium, RC- non-infected BMMφs.

FIGURE 4. Dectin-1 functions to promote TNF-α production in M. smegmatis and

M. avium 2151 infected macrophages. BMMφs were infected with (A) M. smegmatis or

M. avium 724 or M. avium 2151 in the presence of PBS or anti-Dectin mAb 2A11 or

isotype control Ab, (B) M. smegmatis in the presence of Laminarin or Galactan and (C)

complement opsonized or non-opsonized M. smegmatis in the presence of PBS, 2A11 or

isotype control Ab. Also shown is LPS treated BMMφs +/- antibodies. Culture supernates

after 24 hour infection were analyzed for TNF-α by ELISA. a, significant to M.

smegmatis plus PBS (p<0.01). b, significant to M. smegmatis alone and M. smegmatis

plus Galactan (p<0.01) Values are expressed as mean + SD. Data are representative of






35

three separate experiments. Iso-Control- Isotype control Ab, Smeg- M. smegmatis, RC-

non-infected BMMφs.

FIGURE 5. Dectin-1 mediated induction of TNF-α by BMMφs infected with M.

smegmatis requires TLR2-/-

. BMMφs from WT or TLR2-/-

mice were infected with M.

smegmatis or M. avium 724 in the presence of anti-Dectin-1 MAb 2A11. After a 4 h

infection, BMMφs were washed and fresh medium was added to the cells and infection

continued for a total of 24 h. Culture supernates were analyzed by ELISA for TNF-α. a,

significant to M. smegmatis plus PBS (p<0.01). Values are expressed as mean + SD. Data

are representative of three separate experiments. Smeg- M. smegmatis, Avium- M.

avium, RC- non-infected BMMφs.

FIGURE 6. Dectin-1 promotes TNF-α production induced in BMMφs upon

infection with non-pathogenic or attenuated mycobacteria but not H37Rv. BMMφs

were infected with M. smegmatis, M. phlei or M. bovis BCG (A) or M. tuberculosis

strains H37Rv and H37Ra (B) in the presence of anti-Dectin-1 MAb 2A11 or isotype

control Ab. After a 4 hour infection, BMMφs were washed and fresh medium was added

to the cells and the infection was continued for a total of 24 h. Culture supernates were

analyzed by ELISA for TNF-α. a, significant to mycobacteria plus PBS (p<0.01). Values

are expressed as mean + SD. Data are representative of three separate experiments.

Smeg- M. smegmatis, RC- non-infected BMMφs, Iso-Con- Isotype control Ab.






36

FIGURE 7. Dectin-1 promotes IL-6, RANTES and G-CSF production in

mycobacterial infected ΒΜΜφs. Cells were infected with M. smegmatis and M. bovis

BCG (A) or M. avium 724 and M. avium 2151 (B) in the presence of PBS or anti-Dectin-

1 mAb 2A11 or isotype control Ab as described in figure 6. Culture supernatants were

analyzed for G-CSF, RANTES and IL-6 by ELISA. (B) Values are expressed as mean +

SD. Data are representative of three separate experiments. Smeg- M. smegmatis, 724- M.

avium 724, 2151- M. avium 2151, RC- non-infected BMMφs, Iso-Con- Isotype control

Ab.






37

Figure 1

0

10

20

30

40

50

% in

fect

ied

cells

WTMR-/-

01020304050607080

% in

fect

ed c

ells

WTMR-/-

-5

5

15

25

35

45

% in

fect

ed c

ells

WTMR-/-

01020304050607080

% in

fect

ed c

ells

WTMR-/-

Complement Opsonized 1 h Complement Opsonized 4 h

Non-opsonized 1 h Non-opsonized 4 h

A B

2:1 5:1 10:1 2:1 5:1 10:1

2:1 5:1 10:12:1 5:1 10:1Mycobacteria : Macrophage

Mycobacteria : Macrophage



C D






38

Figure 2

pERK1/2

Total ERK1/2

RC Sm Av Sm Av RC Sm Av Sm Av

WT MR-/-

pp38

Total p38

1 h 9 h 1 h 9 h

0500

10001500200025003000

RC Smeg Avium Smeg Avium

TNF-

α (p

g/m

)l

WTTLR4 -/-

Complement Opsonized

Non-Opsonized

B

RC Sm Av Sm Av Sm Av Sm Av RC

WT TLR4-/- KO

1 h 9 h 1 h 9 h

pERK1/2

Total ERK1/2

pp38

Total p38

A

DC


Non-Opsonized

0

500

1000

1500

2000

RC Smeg Avium Smeg Avium

TNF-

α (p

g/m

l)

WTMR-/- KO






39

Figure 3

0500

1000150020002500

R C Smeg A vium Smeg A vium

TNF-

α (p

g/m

l)

BL/6 WT

MyD88-/-

0500

10001500200025003000

R C Smeg A vium Smeg A vium

TNF-

α (p

g/m

l)

WTTLR2-/-

pp38

pERK1/2

Total ERK1/2

RC Sm A v RC Sm Av RC Sm Av RC Sm Av

9 hours

WT TLR2-/-WT TLR2-/-

1 hour

pp38

pERK1/2

Total ERK1/2

RC Sm Av RC Sm Av RC Sm Av RC Sm Av

9 hours

WT MyD88-/-WT MyD88 -/-

1 hour


Non-Opsonized

A

D

B

C


Non-Opsonized






40

Figure 4

0

500

1000

1500

2000

2500

RC RC Smeg Sm eg Smeg

TNF-

α (p

g/m

l)

Laminarin ─ + ─ + ─ Galactan ─ ─ ─ ─ +

A B

b

0500

1000150020002500300035004000

RC Smeg Smeg LPS

TNF-

α (p

g/m

l)

PBS2A11Iso-Con

Opsonized Non-Opsonized

C

a

a

0200400600800

10001200140016001800

RC Smeg 724 2151

TNF-

α (p

g/m

l)

PBS2A11Iso-Con

a a






41

Figure 5

0200400600800

10001200140016001800

RC Smeg Avium RC Smeg Avium

TNF-

α (p

g/m

l)

PBS2A11

WT TLR2-/-

a






42

Figure 6

0200400600800

10001200140016001800

RC Smeg Phlei BCG

TNF-

α (p

g/m

l)

PBS2A11Iso-Con

aa

a

A

B

a

050

100150200250300350400450500

RC H37Ra H37Rv

TNF-

α (p

g/m

l)

PBS2A11Iso-Control






43

Figure 7

0

5 0 0

1 0 0 0

1 5 0 0

P B S2A11Iso -C o n tro l3 5 0 0

4 0 0 04 5 0 05 0 0 0

pg/m

l

----RC--- ---SMEG--- ----BCG---- ---RC--- ----SMEG--- ----BCG---- ----RC---- ----SMEG--- ----BCG----

---------------IL-6----------------- ------------RANTES------------ -------------G-CSF---------------

0

5 0 0

1 0 0 0

1 5 0 0

P B S2A11Iso -C o n tro l3 5 0 0

4 0 0 04 5 0 05 0 0 0

pg/m

l

----RC--- ---SMEG--- ----BCG---- ---RC--- ----SMEG--- ----BCG---- ----RC---- ----SMEG--- ----BCG----

---------------IL-6----------------- ------------RANTES------------ -------------G-CSF---------------

----RC----- -----724---- -----2151---- ----RC----- -----724----- ----2151---- ----RC---- -----724----- ----2151----0

5 0 0

1 0 0 0

2 0 0 0

2 5 0 0

3 0 0 0

3 5 0 0P B S2 A1 1Is o -C o n tro l

pg/m

l

---------------IL-6----------------- ------------RANTES------------ -------------G-CSF-------------------RC----- -----724---- -----2151---- ----RC----- -----724----- ----2151---- ----RC---- -----724----- ----2151----

0

5 0 0

1 0 0 0

2 0 0 0

2 5 0 0

3 0 0 0

3 5 0 0P B S2 A1 1Is o -C o n tro l

pg/m

l

---------------IL-6----------------- ------------RANTES------------ -------------G-CSF---------------

A

B

a

a

a

a

aa

a

a

a a






The β-glucan receptor Dectin-1 functions together with TLR2 to mediated macrophage activation by mycobacteria

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