/Immunology MATURATION OF MURINE MACROPHAGE LIPID RAFTS UPON STIMULATION WITH Mycobacterium tuberculosis SECRETED MOLECULES M. Guadalupe Morales-García1, Gabriela Rodríguez-Luna1, M. Maximina Bertha Moreno-Altamirano1, Diana Aguilar-León2, Rogelio Hernández-Pando2, and F. Javier Sánchez-García1

1Laboratorio de Inmunorregulación, Departamento de Inmunología, Escuela

Nacional de Ciencias Biológicas-Instituto Politécnico Nacional; 2Instituto

Nacional de Ciencias Médicas y de la Nutrición “Salvador Zubirán”, México

D.F., Mèxico.

Corresponding author: F. Javier Sánchez-García, Ph.D.

Departamento de Inmunología

Escuela Nacional de Ciencias Biológicas

Instituto Politécnico Nacional

Carpio y Plan de Ayala, Col. Santo Tomás C.P.

11340

México D.F., México.

Telephone: (5255) 557296300 ext. 62370

Fax: (5255) 55396-3503

e.mail: fsanchez encb.ipn.mx

Running title: maturation of macrophage lipid rafts

1

SUMMARY Lipid rafts, recently termed “membrane rafts” are defined as small (10-200nm),

heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains in

which several cellular processes are compertamentalized.

M. tuberculosis, the causative agent of tuberculosis, and other mycobacteria

enter into their host cells, at least in part, by a membrane rafts-mediated

process. However, the relationship between membrane rafts and secreted

components of mycobacteria is a less studied issue.

Rafts are known to contain hundreds of different proteins with a role in protein

and lipid trafficking, endocytosis and cell signaling and lthough considerable

progress in defining the lipid rafts proteome has been achieved over the last few

years, the protein composition of lipid rafts in different biological settings, such

as in the course of an infection, remains to be analyzed.

This work analyzes the cell membrane compartmentalization of several well

known receptors for mycobacterial ligands, upon stimulation of murine

macrophages with M. tuberculosis-secreted molecules. It is shown that M.

tuberculosis secreted molecules induce the rapid mobilization of rafts, as

assessed by real time confocal microscopy, as well as a differential segregation

of CD14, CD35, CD206, and TLR-4. Moreover, stimulation of murine

macrophages with M. tuberculosis-secreted molecules increases the up-take of

the bacteria. Thus suggesting that previous to macrophage-mycobacteria

contact, their secreted molecules may prepare the host cell, at the level of

membrane rafts, for infection, a process we refer to as “lipid rafts maturation”.

2

INTRODUCTION (2-3 pages)

In spite of the huge efforts to overcome the burden of tuberculosis,

Mycobacterium tuberculosis still kills three million human beings every year

(ref). The selection and spread of multidrug-resistant M. tuberculosis strains

worsen the world scenario for the years to come (refs). Clearly, in addition to

the development of new vaccines, early diagnosis test and pharmacological

treatment, a precise knowledge on mycobacteria-host cell interactions is also

mandatory.

Phagocytosis of M. tuberculosis involves several macrophage receptors such

as the C1R complement receptor (CD35) which recognizes xxx, scavenger

receptors such as CD14 and the mannose receptor (CD206), Toll-like receptors

such as TLR2 and TLR4 which …..(refs)…

On the other hand, cell membrane microdomains termed “lipid rafts” (ref) or

more recently “membrane rafts” (Pike, 2006) have been shown to participate in

both mycobacteria-host cell recognition and internalization processes (Gatfield,

2000; Peyron, 2000 and Maldonado-García, 2004), as has been shown for

other pathogens (Carlos Martínez, 2003-4).

Lipid rafts are lateral assemblies of sphingolipids and cholesterol that form a

separate liquid–ordered phase in the liquid-disordered matrix of the cell

membrane lipid bilayer (Simons K et al, 1997). Lipid rafts are small (10-200

nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains

in which several cellular processes are compertamentalized (Pike, 2006). 40-

70% of total plasma membrane is indeed in the detergent-resistant membrane

3

fraction (i.e., liquid-ordered phase) (Piereini et al, 2001; Fridriksson, 1999;

Mayor S, 1995).

The biological functions attributed to lipid rafts include endocytosis (ref), protein

and lipid trafficking (ref), cell signalling (ref) and as pathogen portals

(Rosenberger, 2000; Gatfield and Pieters, 2000; Wooldridge KG, 1996; Shin JS,

2000; Manie SN, 2000).

Considerable progress has been made in the last few years regarding the

protein composition of lipid rafts. Interestingly, by using unbiased proteomic

approaches, it has been shown that they are enriched in proteins involved in

cell signalling (refs). However, there is a paucity of information regarding the

protein composition of lipid rafts in the course of an infection or the exposure to

pathogen-derived products (Triantafilou).

In the macrophage-mycobacteria interaction there is a set of well characterized

macrophage receptors and the corresponding mycobacterial ligands. However

their relation with membrane rafts remains to be analysed.

Hence, we set up to investigate if the cell membrane location of the complement

receptor CD35, the scavenger receptors CD14, MARCO and CD206, and the

Toll-like receptors TLR-2 and TLR-4 varies in relation to the lipid rafts marker

GM-1 and the low or high density, characteristic of lipid rafts and non-lipid rafts

membrane fractions, respectively, during the stimuli of the J774 murine

macrophage cell line with M. tuberculosis-secreted ligands.

It was found that the lipid rafts composition, in terms of these receptors, does

indeed change over time upon mycobacterial stimulation, thus suggesting a

process of “lipid rafts maturation”, perhaps as a way for the host cell to prepare

for infection.

4

MATERIALS AND METHODS

Cells and antibodies

J774 cells were growth in Dulbecco´s modified Eagle´s medium (DMEM)

(Gibco) in plastic Petri dishes (Corning, Corning, NY) at 37oC in a 5% CO2

atmosphere. Cells in logarithmic phase of growth were used for each

experiment. Anti-CD14, anti-CD35, anti-MARCO, anti-TLR-2, anti-TLR4 and

anti-CD206 were from Becton-Dickinson, anti-rat IgG-Biotin was from xxx,

Streptavidin-PE was from xxxx and Extravidin-PO was from Amersham

Mycobacterium tuberculosis growth and secreted molecules preparation

M. tuberculosis xxxx ( ) was grown in xxx medium for 2-3 weeks

under….etc

Confocal microscopy

For real time confocal microscopy, about 1 x106 J774 cells in 6 well culture

plates were labeled with cholera toxin-FITC (Sigma, St. Louis Mo) at a final

concentration of 5 μg/ml for 30 min. Cells were washed with DMEM, and 2 ml of

fresh DMEM were added to each well. Confocal microscopy was set up to take

1 micrograph per minute. The first image was taken just before adding 20 mg/ml

of a preparation of secreted molecules from M. tuberculosis and followed for up

to 30 minutes.

5

For the analysis of colocalization of GM1 with the Different recepotors for

mycobacterial molecules, 1 x 105 J774 cells/well were seeded into 8 well Lab-

Tek chamber slides (Nunc) and cultured overnight at 37oC in 5% CO2

atmosphere. Cells were then incubated in medium alone or in the presence of

M.tuberculosis-secreted antigens (20 μg/ml) for 15, 30, 45 or 60 minutes, cells

were then quickly washed with PBS and fixed with 4% paraformaldehyde in

PBS for 30 minutes. Cells were washed, blocked with 1% BSA in PBS for 45

minutes and then stained with the indicated rat monoclonal antibody, Biotin-

conjugated anti-rat IgG secondary antibody, Streptavidin-PE, and FITC-

conjugated Cholera toxin (5 μg/ml) (Sigma), with PBS washings in between.

Cells were covered with vectashield (Vector) and a glass coverslip and the

edges sealed before observation by confocal microscopy LCM510 (Ziess).

Cytofluorometric analyses J774 cells in 6 well culture plates were left un-stimulated or stimulated with 20

μg/ml of secreted molecules from M. tuberculosis for 15 to 60 minutes, cells

were then washed and fixed with 4% paraformadehyde PBS. Cells were

scraped out of the culture plates and transferred to xxx tubes (Bectin-Dickinson)

after centrifugation, the cell pellets were suspended in 1% BSA-PBS incubated

for 15 min an then stained for GM1 (cholera toxin-FITC), CD14, CD35, CD206,

TLR2, TLR4 as indicated. Cells were analyzed in a FACScan (Becton-

Dickinson) flow cytometer and Cellquest software (Becton-Dickinson).

Mycobacterium tuberculosis uptake

6

Mycobacterium tuberculosis grown in 7H9 medium was stirred for 1h in order to

disrupt bacterial clumps. Bacterial clumps were allowed to sediment for five

minutes and the supernatant containing un-aggregated bacteria were labeled

with PKH-26 (Sigma) as previously described (13). Briefly, 1x108 bacteria were

washed with phosphate-buffered saline (PBS), pH 7.4, and the cell pellet was

dissolved in 300 μl of PKH-26 diluent. PKH-26 fluorochrome was diluted in a

separate eppendorf tube in 300 μl of PKH-26 diluent. The bacterial suspension

was added to the PKH-26-containing eppendorf tube and was gently shaken

and incubated for 15 min at room temperature, after which 600 μl of fetal calf

serum (FCS) (Gibco) was added and further incubated for 5 min at room

temperature. The labeled bacteria were washed twice with PBS, pH 7.4,

suspended in DMEM supplemented with 5% FCS and used for infection, for

which overnight cultured J774 cells in 6-well microplates (Nunc, Naperville, IL,

USA) were exposed to PKH-26-labelled mycobacteria. The infected cultures

were incubated for 30 min at 37oC in 5% CO2 atmosphere, after which cells

were washed, fixed with 4% paraformaldehyde and scraped. Cells were then

transferred to polystyrene tubes. PKH-26 expression (indicative of M.

tuberculosis uptake) was assessed by flow citometry.

RESULTS Mycobacterium tuberculosis-secreted molecules induce rapid

morphological changes and lipid rafts mobilization in murine

macrophages

7

Figure 1 shows representative micrographs at 0, 10, 20 and 30 min of

stimulation with secreted M. tuberculosis molecules, a preparation known to

contain more than 200 proteins (Sonnenberg, 1997). It is observed that lipid

rafts mobilize within the cells, mainly in the membrane extentions or filopodia

(A). Interestlingly, the preparation of mycobacterial molecules also induces

changes in cell shape and even cell motility (B and C). By comparison, in non-

stimulated J774 cells mobilization of lipid rafts is minimal and changes in cell

shape are not observed (D).

CD14, CD35, CD206 and TLR2 co-localize within lipid rafts in M.

tuberculosis-secreted antigens stimulated murine macrophages

Following the observation that lipid rafts mobilize along the cell membrane of

J774 murine macrophages following stimulation with secreted antigens from

Mycobacterium tuberculosis, we set up to analyze the composition of such lipid

rafts in terms of the expression of some well characterized receptors for

mycobacterial ligands, namely the complement receptor CD35, the scavenger

receptors CD14, macrophage xxx (MARCO), mannose receptor (CD206), and

the Toll-like receptors TLR2 and TLR4. Accordingly, J774 cells were stimulated

with the mentioned mycobacterial antigen preparation for different lengths of

time (5-60 minutes) as indicated. Figure 2 shows the location of such

mycobacterial ligand receptors (in red), the location of GM-1 as a lipid rafts

marker (in green), co-localization of both molecules and, a visible light image 30

minutes after stimulation, the time at which the differential location of

mycobacterial receptors was more pronounced.

8

Figure 3 shows the cell histograms depicting the position and relative amount of

mycobacterial ligand receptors and those of GM-1 at 5, 15, 30, 45 and 60

minutes post-stimulation. It can be seen that by 30 minutes xxx, xxx and xxx is

concentrated in the same position where GM-1 accumulates.

Expression of xxx is up-regulated in the lipid rafts fraction in M.

tuberculosis-secreted antigens- stimulated murine macrophages

Confocal microscopy analyses strongly suggested that lipid rafts composition

varies upon stimulation with mycobacterial antigens. In order to further test this

assumption lipid rafts were obtained from both stimulated and non-stimulated

J774 cells by the gradient centrifugation procedure (ref) and the gradient

fractions used for analysis of the content of GM-1 and the mentioned

mycobacterial ligand receptors both for slot-blotting and for flow-cytometry.

Figure 4 shows the comparative distribution

Pre-stimulation with M. tuberculosis-secreted antigens increases the

extent of M. tuberculosis up-taking by murine macrophages

9

DISCUSSION Earlier studies have shown that mycobacterial infection of macrophages is a

lipid rafts-dependent process in which mycobacteria induce the mobilization of

macrophage lipid rafts towards the cell-bacteria contact site (Gatfiel, 2000;

Peyron, 2000). Moreover, mycobacterial high polarity lipids have been involved

in such a biological process (Maldonado-Garcia, 2004). In this work we aimed

at evaluating whether secreted molecules from M. tuberculosis would act on

lipid rafts. Real time confocal microscopy experiments first revealed that indeed,

M. tuberculosis culture supernatant (10-20 μg/ml), induces lipid rafts

rearrangement on J774 macrophages, along with morphological changes

(figure 1). In an attempt to characterize the composition of lipid rafts in the

course of the stimuli, antibodies directed against well known receptors for

mycobacterial ligands such as CD14, CD35, CD206, TLR2, and TLR4 were

used in conjunction with cholera toxin B (ligand for GM1, a marker of lipid rafts)

to look for the presence of such receptors within or outside lipid rafts, in

colocalization experiments. It was found

Attempts to define the immunologically active components within the M.

tuberculosis-secreted antigens have lead too the identification of the 6 KDa

ESAT6 (Sorensen,1995), 24 KDa MPT64 (Nagai 1991, Roche, 1996), 30 to 31

KDa Ag85 complex (Harth, 1996; Horwitz,1995; Nagai, 1991), 45 KDa MPT32

(Dobos, 1996; Nagai, 1991, Romain, 1993)

10

We propose the concept of “lipid rafts maturation” which implies a different

composition of lipid rafts depending on xxx

It is known that endocytosis, and mycobacterial infections are dependent on

lipid rafts (13, 19, 22, 33 de Moreno Immunology)

11

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Acknowledgments

16

We thank Dr. Alejandra Contreras (Centro Médico Siglo XXI) for expert

assistance with confocal microscopy analyses. This work was financed in part

by a CONACYT (SEP-2003-CO2-44228) and SIP (20060494) grants. MGMG

was the recipient of a CONACYT studentship, MMBMA and FJSG are

COFAA/EDI/SNI fellows, DAL and RHP are SNI fellows.

17

Figure legends

Fig.1. Mycobacterium tuberculosis secreted molecules mobilize lipid rafts and

induce morphological changes in J774 cells. Cholera toxin-FITC labeled J774

cells were exposed to M. tuberculosis-secreted molecules (20 μg/ml) and

followed by real time confocal microscopy for 30 minutes. Representative

images at 0, 10, 20, and 30 minutes are shown (A) arrows indicate lipid rafts

mobilization along a J774 cell membrane extension, (B and C) arrows indicate

changes in shape and lipid rafts distribution. By contrast, (D) un-stimulaed cells

fail to show changes in lipid rafts distribution or cell shape.

Fig. 2. M. tubeculosis secreted molecules induce a rapid increase in both GM1

and mannose receptor (MR) (CD206) expression in J774 cells. J774 cells were

leaved un-stimulated or stimulated with secreted molecules from M.

tuberculosis (20 μg/ml) for 30 minutes. Cells were then washed with PBS and

fixed with 4% Paraformaldehyde PBS. After which cells were stained with

cholera toxin-FITC (GM1) and with rat anti-CD206 moAb, followed by anti-Rat

IgG-Texas red (mannose receptor). Confocal microscopy show the relative

amount of GM1 (green) and MR (red) and, GM1 and MR colocalization (white)

for both un-stimulated and M. tuberculosis-stimulated J774 cells.

Fig. 3. M. tubeculosis secreted molecules induce a rapid increase in CD14

expression in J774 cells. J774 cells were leaved un-stimulated or stimulated

with secreted molecules from M. tuberculosis (20 μg/ml) for 30 minutes. Cells

were then washed with PBS and fixed with 4% Paraformaldehyde PBS. After

18

which cells were stained with cholera toxin-FITC (GM1) and with rat anti-CD14

moAb, followed by anti-Rat IgG-Texas red. Confocal microscopy show the

relative amount of GM1 (green) and MR (red) and, GM1 and CD14

colocalization (white) for both un-stimulated and M. tuberculosis-stimulated

J774 cells.

Fig 4. M. tubeculosis secreted molecules induce a rapid increase TLR4

expression in J774 cells. J774 cells were leaved un-stimulated or stimulated

with secreted molecules from M. tuberculosis (20 μg/ml) for 30 minutes. Cells

were then washed with PBS and fixed with 4% Paraformaldehyde PBS. After

which cells were stained with cholera toxin-FITC (GM1) and with rat anti-TLR2

moAb, followed by anti-Rat IgG-Texas red. Confocal microscopy show the

relative amount of GM1 (green) and TLR4 (red) and, GM1 and TLR4

colocalization (white) for both un-stimulated and M. tuberculosis-stimulated

J774 cells.

Fig. 5. Expression of GM1, CD14, CD206 and TLR2 in J774 cells following

stimulation with M. tuberculosis-secreted molecules. J774 cells were stimulated

with M. tuberculosis-secreted molecules for 5, 15, 30, 45, and 60 minutes. Cells

were washed with PBS and fixed with 4% paraformaldehyde-PBS. Cells were

then stained for GM1, CD14, CD206, or TLR2. Expression of (A) GM1, (B)

CD14, (C) CD206, and (D) TLR2 was assessed by flow citometry. Mean

fluorescence intensity is indicated. Results are representative of four

independent experiments.

19

Fig. 6. Uptake of M. tuberculosis by J774 cells is modified by pre-incubation of

cells with M. tuberculosis-secreted molecules. J774 cells were leaved un-

stimulated or stimulated with secreted molecules from M. tuberculosis (20

μg/ml) for 30 minutes. Cells were washed with PBS and exposed for 30

additional minutes to PKH-26 labeled M. tuberculosis H37Rv at a MOI of 20.

Cells were washed, fixed with 4% paraformadehyde- PBS, scraped form the

culture plates. Mycobacterial up-take was assessed by flow cytometry analysis.

Mean Fluoresence intensity is shown for each case. Results are representative

from 5 independent experiments.

20

Fig. 1

0´ 10´ 20´ 30´0´ 10´ 20´ 30´

21

GM1 CD206 MERGE

medium

M. tuberculosis

30 min

GM1 CD206 MERGE

medium

M. tuberculosis

30 min

GM1 CD14 MERGE

medium

M. Tuberculosis

30 min

GM1 CD14 MERGE

medium

M. Tuberculosis

30 min

22

GM1 TLR2 MERGE

medium

M. Tuberculosis

30 min

GM1 TLR2 MERGE

medium

M. Tuberculosis

30 min

23