/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