Department of Pathology and Program in Molecular Pathology, Graduate School of Biomedical Sciences, University of Texas Houston Health Science Center, Houston, TX, USA
Correspondence
Jeffrey K. Actor
Jeffrey.K.Actor{at}uth.tmc.edu
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ABSTRACT |
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INTRODUCTION |
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Recent studies implicate the major mycolic-acid-containing molecule, trehalose 6,6'-dimycolate (TDM), as an important immunomodulatory component of the mycobacterial cell wall. In mice, immune responses to purified TDM mimic certain aspects of natural M. tuberculosis infection, including production of pro-inflammatory cytokines (IL-1, IL-6 and TNF-
), development of granulomas, increased procoagulant activity and decreased presence of serum cortisol (Behling et al., 1993
; Perez et al., 1994
, 2000
; Actor et al., 2000
). Cytokine production in response to TDM administered in vivo can be reproduced, in part, by macrophages in vitro; high pro-inflammatory cytokine production was evident in murine bone marrow macrophages incubated with TDM-coated beads (Perez et al., 2000
). Additionally, TDM prevents the fusion of phospholipid vesicles (Spargo et al., 1991
; Crowe et al., 1994
; C. Jagannath & R. L. Hunter, unpublished results), suggesting that TDM may contribute to intracellular survival of M. tuberculosis by inhibiting phagosomelysosome fusion events during infection.
We previously characterized macrophage responses to M. tuberculosis subjected to petroleum ether extraction, which specifically removes surface TDM (Indrigo et al., 2002). Delipidated M. tuberculosis demonstrated decreased viability in C57BL/6-derived bone marrow macrophages, compared to native organisms. Furthermore, production of pro-inflammatory cytokines was significantly reduced in macrophages infected with delipidated M. tuberculosis. Reconstitution of delipidated organisms with pure TDM allowed recovery of normal levels of bacterial survival and cellular cytokine production.
In this study, the murine macrophage cell line J774A.1 was infected with delipidated M. tuberculosis to investigate further the role of TDM in bacterial viability and macrophage response in a homogeneous population. Of note, cytokine responses of this cell line were in accordance with bone marrow macrophage responses. Compared to native M. tuberculosis, infection with delipidated organisms led to significantly decreased bacterial survival and altered cellular cytokine production. Moreover, removal of surface lipids led to enhanced trafficking of organisms to acidic compartments. Reconstitution of delipidated organisms with either pure TDM or the petroleum ether extract containing crude surface lipids restored normal responses. Using TDM-coated polystyrene beads, we show that TDM by itself can mediate intracellular trafficking events, as well as influence macrophage production of pro-inflammatory cytokines. Thus, the presence of TDM may be an important determinant for successful infection and survival of M. tuberculosis within murine macrophages.
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METHODS |
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Infection of macrophages.
J774A.1, a murine monocyte/macrophage cell line, was obtained from ATCC. The use of these cells permitted the examination of intracellular trafficking events in a homogeneous population. Cells were maintained at 37 °C with 5 % CO2 in DMEM containing 3·76 g sodium bicarbonate l-1, 0·05 g HEPES l-1, 0·05 g L-arginine l-1 and 5 % FBS. Infection medium was composed of DMEM containing 2 % FBS. Before use in infections, cells were adjusted to 1x106 cells ml-1 (for cytokine quantification) or 1x1055x105 cells ml-1 (for fluorescence microscopy) in 24-well tissue culture plates. In the latter experiments, cells were plated on glass cover slips.
J774A.1 macrophages were infected 24 h after seeding (m.o.i. 5 : 1 for cytokine quantification, m.o.i. 1 : 1 for fluorescence microscopy). Native M. tuberculosis, delipidated M. tuberculosis, delipidated M. tuberculosis reconstituted with purified TDM, delipidated M. tuberculosis reconstituted with CSLE, heat-killed M. tuberculosis or medium alone was added and phagocytosis was allowed to occur for 4 h at 37 °C. Cells were infected in triplicate wells. Cells were then washed to remove extracellular bacteria and fresh DMEM containing 2 % FBS was added. Macrophages were lysed with 0·05 % SDS at 4, 24 and 72 h after infection to assess bacterial survival. Based on results from preliminary experiments, later time points were not used because detachment of the monolayer was evident after 45 days in all infected macrophage groups. Serial dilutions of lysate were plated on Middlebrook 7H11 agar and incubated at 37 °C for 21 days for enumeration of c.f.u. All infections were repeated at least three times.
Cytokine and chemokine protein quantification.
Production of IL-1, IL-6, IL-10, IL-12, TNF-
, IFN-
, MIP-1
and MCP-1 by J774A.1 macrophages was measured in culture supernatants after infection with mycobacteria. Measurements were performed with specific ELISA DuoSet kits purchased from R&D Systems, following the manufacturer's directions. Absorbance was read at 450 nm on an ELISA plate reader (Molecular Devices). The mean of triplicate wells was calculated based on a standard curve constructed for each assay using recombinant cytokine standards (R&D Systems).
Intracellular localization studies.
Macrophages were pre-labelled with LysoTracker Red (1 : 10 000 in DMEM; Molecular Probes) for 1·5 h. Before infection, mycobacteria were labelled with the green fluorescent nucleic acid stain SYTO 9 (Molecular Probes) as instructed by the manufacturer. Macrophages were washed with PBS before infection with mycobacteria as described above. Following 4 h phagocytosis at 37 °C, macrophages were washed extensively with PBS, and DMEM containing LysoTracker Red was added. The medium was replaced after 3 days of infection with fresh DMEM containing LysoTracker Red. At 1, 3 and 7 days after infection, macrophages were fixed with 3·7 % ultrapure formaldehyde (Tousimis Research) by incubating at room temperature for 20 min. Cells were then washed extensively in PBS. Cover slips were mounted on glass slides with Vectashield (Vector Laboratories).
Slides were examined with a Nikon Eclipse E600 fluorescence microscope fitted with a x100 oil immersion objective. Separate images were obtained for green and red wavelengths. Images were merged with MetaVue software (Universal Imaging). At least 100 mycobacteria-containing macrophages from each cover slip (three cover slips per time point) were examined for colocalization of SYTO 9 and LysoTracker Red.
Preparation of lipid-coated beads.
Green fluorescent (480 nm excitation/520 nm emission) 0·78 µm mean diameter polystyrene beads were obtained from Bangs Laboratories. Beads were washed three times with carbonate-bicarbonate buffer (CB buffer; 8 mM sodium carbonate, 17 mM sodium bicarbonate, pH 9·6) by centrifugation at 2200 g for 15 min. Beads to be coated with lipids were further washed once in 30 % ethanol and once in 1 % ethanol. To coat beads with lipids, the amount of TDM required for deposition as a monolayer was calculated according to Retzinger et al. (1981). TDM (150 µg) from M. tuberculosis (Sigma), CSLE or trehalose dibehenate (TDB; Sigma) in CB buffer was added to the beads. Beads were incubated 4 h at 37 °C with constant gentle mixing. Following coating, beads were washed with PBS. Lipid-coated beads were washed in buffers containing 0·1 % F68 to facilitate dispersal. For storage, beads were diluted in PBS containing 0·05 % sodium azide and 0·1 % F68 and kept at 4 °C. A portion of the beads was extracted with chloroform and subjected to TLC to confirm presence of lipid. To coat beads with BSA, 550 µg BSA in CB buffer was added and the beads were incubated at 4 °C overnight with constant gentle mixing. The amount of protein adsorbed to the beads was determined using the Pierce Coomassie Plus Protein Assay Kit according to the manufacturer's instructions. Approximately 430 µg BSA adsorbed to the beads. Beads were washed thoroughly and sonicated before use in experiments. For cytokine quantification, beads were added to cells at a ratio of 10 beads per cell. For fluorescence microscopy, beads were added to LysoTracker Red-labelled macrophages at a ratio of five beads per cell. Following 6 h incubation at 37 °C, cells were washed with PBS and processed for cytokine quantification and colocalization with LysoTracker Red as described above.
Statistical analysis.
Data are expressed as the mean±SD. Differences between groups were analysed using Student's t-test. Results were considered significant at P<0·05.
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RESULTS |
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By 72 h after infection, viability of delipidated M. tuberculosis in macrophages was significantly reduced by 2 log units compared to native M. tuberculosis (Fig. 1). This occurred well prior to full repopulation of TDM on the cell surface of delipidated organisms (Indrigo et al., 2002
). Survival of the delipidated organisms in macrophages could be restored by addition of pure TDM. Additionally, delipidated organisms were reconstituted with the CSLE that had been removed during the delipidation process. At 72 h after infection, survival of delipidated M. tuberculosis that had been reconstituted with CSLE was statistically indistinguishable from native M. tuberculosis. This extracted material contains primarily TDM, although minor amounts of other cell wall components removed by petroleum ether extraction may be present. To test the direct effect of TDM on viability of organisms within macrophages, delipidated organisms were reconstituted with pure TDM. The survival of these TDM-reconstituted organisms was identical both to native M. tuberculosis and to delipidated M. tuberculosis reconstituted with the CSLE. This suggests that TDM alone, in the absence of secondary cell wall components removed during petroleum ether treatment, such as hydrocarbons and free mycolic acids (Silva et al., 1985
), contributes directly to survival of M. tuberculosis in macrophages.
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Representative C-C chemokines were measured because of their central role in in vivo granuloma development. Macrophages infected with delipidated organisms demonstrated a significant delay in MCP-1 production at 4 h (Fig. 3). The level of MCP-1 protein induced by delipidated M. tuberculosis was lower than that of uninfected control cells at this time point. However, the levels of MCP-1 released into supernatants following infection with delipidated organisms at 24 h were indistinguishable from those induced by native M. tuberculosis or by reconstituted organisms. Delipidation also caused a slight decrease in MIP-1
immediately after infection, but no significant differences in MIP-1
production were evident at any time point after infection. For both chemokines, reconstitution of delipidated M. tuberculosis with either TDM or the CSLE was able to restore chemokine production so that responses were practically identical to those induced by infection with native organisms.
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Localization of TDM-coated beads
To examine the role of TDM in intracellular compartmentalization, J774A.1 macrophages were loaded with LysoTracker Red and cultured with green fluorescent TDM-coated beads. At 1, 3 and 7 days after treatment, cells were analysed by fluorescence microscopy for colocalization of beads with LysoTracker (Fig. 7; Fig. 8
). Early colocalization of BSA-coated beads with LysoTracker was evident, as 40 % of beads colocalized to acidic vesicles by 1 day after addition to macrophages, progressing to 64 % at 3 days. Thereafter, vesicles containing BSA-coated beads rapidly acidified, with nearly 100 % of beads colocalizing with LysoTracker at day 7 after treatment. The colocalization of TDB-coated beads with LysoTracker also occurred in a relatively short time period, as nearly 90 % of TDB-coated beads localized to LysoTracker-stained acidic vesicles by 3 days after incubation. In contrast, vesicles containing beads coated with mycobacterial lipids demonstrated a marked delay in acidification. At day 3 only 34 % of beads coated with TDM and 32 % of beads coated with CSLE colocalized with LysoTracker, both of which were significantly reduced relative to BSA- or TDB-coated beads. The delayed colocalization to acidified compartments was transient; by 7 days after incubation, beads from all groups could be found in compartments containing LysoTracker.
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DISCUSSION |
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It was previously demonstrated that TDM could inhibit vesicle fusion when present on a phagosome-like membrane (Spargo et al., 1991; Crowe et al., 1994
) or when embedded in biodegradable poly-DL-lactide-coglycolide (PLGA) particles (C. Jagannath & R. L. Hunter, unpublished results). The experiments described here provide evidence for a role of TDM in mycobacterial survival by mediating trafficking of organisms in macrophages. Because M. tuberculosis is sensitive to low pH found in lysosomal compartments (Gomes et al., 1999
), its ability to persist in macrophages is dependent on arrest of phagosomal maturation. As expected, native M. tuberculosis prevented maturation of early endosomes to lysosomes (Armstrong & Hart, 1971
; Clemens & Horwitz, 1995
; Via et al., 1998
), as evidenced by the lack of colocalization with LysoTracker. TDM-coated beads exhibited a similar trafficking pattern to live native organisms, although the loss of TDM from beads may have contributed to increased localization of beads to acidic vesicles at late time points. Phagosomes containing delipidated organisms, in contrast, progressively matured to acidic compartments over 7 days of infection; reconstitution with pure TDM rescued delipidated organisms from localization to acidic compartments. Future experiments will analyse the expression of surface markers on M. tuberculosis-containing phagosomes, with expectations that native M. tuberculosis will reside in vesicles that retain expression of early endosomal markers (e.g. Rab5), while phagosomes containing delipidated M. tuberculosis will acquire molecules indicative of maturation to lysosomes [early endosome autoantigen (EEA1), Rab7, cellubrevin] (Via et al., 1997
; Fratti et al., 2001
, 2002
).
The mechanisms underlying fusion events during mycobacterial infection are under active investigation (Russell, 2001; Pieters & Gatfield, 2002
). Interestingly, vesicles containing heat-killed M. tuberculosis rapidly fuse with acidic vesicles; these killed organisms have sufficient quantities of surface TDM present. This suggests that an additional factor may also regulate intracellular trafficking, perhaps one produced directly by live organisms or by macrophages upon infection with live organisms. One such candidate factor is mycobacterial lipoarabinomannan (LAM). LAM modulates immune responses during mycobacterial infection by abrogating lymphocyte- and IFN-
-induced macrophage activation (Moreno et al., 1988
; Sibley et al., 1988
) and altering phagosomal maturation (Fratti et al., 2001
). Our experiments also do not rule out differential use of Toll-like receptors (TLR). Indeed, heat-killed mycobacteria have been shown to stimulate cellular activation through TLR2, while live organisms elicit a distinct set of responses by signalling through TLR2 and TLR4 (Means et al., 1999
). Furthermore, mycobacterial uptake by macrophages is mediated by multiple mechanisms, including complement receptors, scavenger receptors and mannose receptors (Schlesinger et al., 1990
; Schlesinger, 1993
). Preferential receptor usage can alter the subsequent compartmentalization and survival of intracellular pathogens (Wright & Silverstein, 1983
; Mosser & Edelson, 1987
; Astarie-Dequeker et al., 1999
). In our experiments, no quantitative differences in phagocytosis were evident between native, delipidated or reconstituted organisms immediately after infection, although qualitative differences, if any, have yet to be explored. The phagocytosis process must also be examined for TDM-coated beads, as bead size may correlate with uptake and intracellular localization. Indeed, it is well documented that the toxic properties of TDM emulsified in oil are dependent upon droplet size (Yarkoni & Rapp, 1977
, 1978
). The polystyrene beads chosen for our experiments were thus as close as possible to size of the bacteria. Although the size of the beads may influence their localization within macrophages, the cytokine induction by TDM-coated beads as described here is in excellent concert with results obtained using larger (5·1 µm diameter) beads (Perez et al., 2000
).
The biological activities of TDM are dependent on the molecule's physical conformation. Specifically, the precise arrangement of TDM on the surface of M. tuberculosis or hydrophobic beads is critical for TDM-induced toxicity (Retzinger et al., 1981). Furthermore, mycolic acid structure correlates with survival in vivo, as M. tuberculosis mutants unable to cyclopropanate or oxygenate mycolic acids fail to persist within mice (Dubnau et al., 2000
; Glickman et al., 2000
). To investigate further the influence of lipid structure on cellular activation and vesicle fusion, macrophages were incubated with beads coated with TDB, a synthetic analogue of TDM containing shorter fatty acid chains. TDB-coated beads demonstrated rapid colocalization with LysoTracker, similar to BSA-coated beads. In addition, beads coated with TDB generally induced significantly less production of inflammatory mediators throughout the incubation period than beads coated with TDM. Taken together, these data indicate that the unique structure of TDM contributes both to the induction of cytokines and to the relatively non-fusogenic trafficking pattern of phagocytosed TDM-coated beads.
Production of pro-inflammatory cytokines IL-1, IL-6, TNF-
and IL-12 was significantly diminished by infection with delipidated M. tuberculosis compared to infection with native M. tuberculosis. Responses were generally restored upon reconstitution of delipidated M. tuberculosis with pure TDM or with the CSLE. Mycobacteria-induced cellular activation, including production of IL-12 and TNF-
, is dependent on signalling through TLR (Brightbill et al., 1999
; Means et al., 1999
; Underhill et al., 1999
). Thus, it can be speculated that delipidation of M. tuberculosis removes a critical TLR agonist, which directly affects (reduces) cytokine responses. Furthermore, other active compounds or TLR agonists in the mycobacterial cell wall (i.e. LAM) may have been altered during petroleum ether treatment, since reconstitution of delipidated mycobacteria with purified TDM only partially restored the IL-1
and IL-12 responses. It is also possible that signal transduction pathways responsible for production of these two cytokines may be regulated differently relative to IL-6 and TNF-
production.
From a larger perspective, the ability of TDM to affect macrophage responses may have important implications for M. tuberculosis infection in vivo. The granulomatous response, in part ascribed to the cellular response directed against TDM (Bekierkunst, 1968; Behling et al., 1993
; Perez et al., 1994
; Hamasaki et al., 2000
), may be required for survival of the organism and overall protection of the host against disease-related pathology. Granulomas successfully contain the spread of M. tuberculosis infection, but in doing so allow the pathogen to survive and persist within the host. A/J- and complement component C5-deficient mice are unable to mount a pulmonary granulomatous response to M. tuberculosis infection; death of the host occurs within 14 days after infection. On the other hand, C57BL/6 mice capable of forming granulomas survive longer with significantly elevated numbers of organisms in their lungs (Actor et al., 1999
, 2001
; Watson et al., 2000
). Macrophages infected with delipidated M. tuberculosis exhibited significantly less production of TNF-
, IL-1
and IL-12, cytokines essential for granuloma formation and maintenance during mycobacterial infection (Kindler et al., 1989
; Cooper et al., 1995
; Juffermans et al., 2000
), than cells infected with native M. tuberculosis. In addition, TDM-coated beads directly induced significant levels of these cytokines. Macrophages infected with delipidated organisms also led to modest decreases in production of MCP-1 and MIP-1
immediately after infection; the diminished chemokine production may have functional consequences as chemokines mediate cellular infiltration and granuloma formation in association with cytokines such as TNF-
(Smith et al., 1997
; Roach et al., 2002
). These data indicate that the presence of TDM initiates an immune response favourable to the development of granulomas and cellular recruitment events, which may enhance long-term survival and persistence of M. tuberculosis within the host. Overall, these data suggest that mycobacterial cell wall glycolipid components can influence the outcome of mycobacterial infection and subsequent immune-mediated pathology.
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ACKNOWLEDGEMENTS |
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Received 9 January 2003;
revised 4 April 2003;
accepted 16 April 2003.
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