Department of Pathology, Program in Molecular Pathology, University of Texas-Houston Medical School, MSB 2.214, 6431 Fannin, Houston, TX 77030, USA1
Author for correspondence: Jeffrey K. Actor. Tel: +1 713 500 5344. Fax: +1 713 500 0730. e-mail: Jeffrey.K.Actor{at}uth.tmc.edu
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ABSTRACT |
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Keywords: Mycobacterium tuberculosis, cytokines, chemokines, cord factor
Abbreviations: BCG, Bacille CalmetteGuérin; BMM, bone-marrow-derived macrophage; FBS, fetal bovine serum; MTB, Mycobacterium tuberculosis; TDM, trehalose 6,6'-dimycolate
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INTRODUCTION |
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Virulent MTB strains subjected to surface lipid extraction by petroleum ether treatment became avirulent and unable to form cords, but they retained their viability (Bloch, 1950 ). The petroleum ether fraction was termed the cord factor and was later identified to be composed primarily of trehalose 6,6'-dimycolate (TDM; Noll et al., 1956
). Silva et al. (1985)
showed that TDM from the vaccine strain M. bovis Bacille CalmetteGuérin (BCG) modulated infection in Swiss mice. Mice infected with delipidated BCG, which lost TDM due to petroleum ether extraction, exhibited fewer granulomatous lesions and less intense delayed-type hypersensitivity than mice infected with native BCG (Silva et al., 1985
). Furthermore, the viability of delipidated BCG was significantly reduced in all organs by 4 days after infection, whereas native BCG remained viable for at least 24 days. When delipidated BCG was reconstituted with purified TDM, the native phenotype was almost entirely restored (Silva et al., 1985
).
Recent studies have demonstrated that TDM is able to reproduce many of the pathophysiological characteristics of MTB infection when injected intravenously into mice and rabbits, including the induction of hypersensitive-type pulmonary granulomas and the concurrent production of inflammatory cytokines, as well as increased procoagulant activity (Hamasaki et al., 2000 ; Perez et al., 1994
, 2000
; Yamagami et al., 2001
). The inflammatory responses parallel, in part, responses identified during acute experimental infection with MTB, most notably those involved in and appearing during the formation of pulmonary granulomas (TNF-
, IL-1ß and IL-6) (Actor et al., 2000
; Watson et al., 2000
). It has been suggested that the responses elicited by TDM during infection are regulated through a combination of T-cell-dependent and T-cell-independent mechanisms (Yamagami et al., 2001
).
The contribution of MTB surface lipids, primarily TDM, to the survival of MTB and to the induction of T-cell-independent inflammatory responses warranted further investigation. We hypothesized that the removal of MTB surface lipids and of TDM would lead to alterations in the innate macrophage response, especially in the production of inflammatory mediators such as chemokines and nitric oxide. Non-covalently bound cell-wall lipids were removed from MTB by petroleum ether extraction methods; these delipidated organisms were used to infect purified populations of C57BL/6 bone-marrow-derived macrophages (BMMs). The survival of organisms was monitored, and the cytokine and chemokine responses were evaluated and compared to native organisms as well as to delipidated organisms reconstituted with purified TDM.
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METHODS |
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Mycobacteria.
MTB (Erdman, ATCC 35801) was cultured to the exponential phase of growth in Dubos broth (Difco) supplemented with 5% BSA and 7·5% dextrose. For delipidated MTB, mycobacterial surface lipids were extracted with petroleum ether, as described by Silva et al. (1985) . After the addition of petroleum ether (Sigma), the bacteria were vortexed vigorously for 2 min, followed by 5 min incubation at room temperature. The culture was centrifuged at 500 g for 10 min. The supernatant, which contained extracted material, was removed and the extraction process was repeated twice more. After the last extraction, the bacteria were washed to remove any residual petroleum ether; they were then resuspended in PBS. Petroleum ether extraction by these methods does not affect the viability nor the acid-fastness of organisms. Delipidated mycobacteria demonstrated no reduction in their viability (Fig. 1
) through 14 days of growth in Dubos broth. Growth was measured by the incorporation of a fluorescent nucleic acid stain into the bacteria, using the BacLight kit (Molecular Probes) according to the manufacturers directions. Relative fluorescence units (485 nm excitation/527 nm emission) were measured on a Fluoroskan Ascent CF (Thermo Labsystems) fluorometer. Delipidated organisms retained their acid-fastness (not shown), as indicated by staining with the fluorescent Bacto TB Auramine M kit (Difco). Organisms treated with petroleum ether reconstituted TDM following delipidation (Fig. 2
). Delipidated organisms were monitored for their recovery of surface glycolipids; the level of surface glycolipids recovered was statistically indistinguishable from native organisms by day 7 post-delipidation.
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Infection of BMMs.
Matured BMMs were infected 24 h after seeding (m.o.i. 5:1). Immediately before their use, the monolayers were washed extensively with PBS. One millilitre of native MTB, delipidated MTB, delipidated MTB reconstituted with purified TDM or medium alone was added to the macrophages and the infection was allowed to proceed for 4 h at 37 °C, with gentle rotation. Cells were infected in triplicate wells. Upon completion of the infection period, cells were washed to remove extracellular bacteria. Fresh DMEM containing 2% FBS was then added to the wells.
The BMMs were lysed with 0·05% SDS at 4, 24 and 96 h after infection to assess bacterial survival. Serial dilutions of the lysates were plated onto Middlebrook 7H11 agar and incubated at 37 °C for 21 days for enumeration of the c.f.u. values.
Measurement of inflammatory mediators.
After infection with mycobacteria, the production of IL-1ß, IL-6, IL-10, IL-12, TNF- and MIP-1
by the BMMs was measured in culture supernatants by ELISA. Measurements were performed with specific ELISA DuoSet kits (R&D Systems), following the manufacturers directions. Costar 96-well high-binding plates were coated with capture antibody overnight. Plates were washed three times with wash buffer (0·05% Tween 20 in PBS). Blocking buffer (1% BSA, 5% sucrose and 0·05% NaN3 in PBS) was added to the wells for 2 h. After three washes, 100 µl of the cell-culture supernatant was added and the plates were incubated for a further 2 h. After this time, the plates were washed extensively and biotin-conjugated secondary antibodies were added to the wells. Finally, the plates were incubated for 2 h, washed and then developed using streptavidinhorseradish peroxidase and the TMB Microwell Peroxidase Substrate (Kirkegaard and Perry Laboratories). Absorbance was read at 450 nm, with a background correction set at 570 nm, on an ELISA plate reader (Molecular Devices). The mean amount of each inflammatory mediator present (from triplicate wells) was calculated based on a standard curve constructed for each assay using recombinant murine controls (R & D Systems). The limit of sensitivity was 510 pg ml-1 for each assay.
Nitric oxide production was measured using the colorimetric Griess assay, which detects nitrite in culture supernatants. Griess reagent (100 µl; 0·1% N-1-napthylenediamine HCl in water and 1% sulfanilamide in 2·5% phosphoric acid) was added to 100 µl of the BMM culture supernatants collected at various time points after infection. The plates were incubated at room temperature for 15 min and were protected from light. Absorbance values were read at 550 nm on a Molecular Devices plate reader. The absorbance values obtained for the culture supernatants from uninfected cells were subtracted from the absorbance values obtained for the experimental groups.
Statistical analysis.
All experiments were performed in triplicate wells using BMMs obtained from three mice; growth curves and the measurement of molecular mediators are representative of three independent experiments. The differences between groups were analysed for significance using Students t-test. A P value 0·05 was considered significant.
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RESULTS |
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The BMMs produced a high level of IL-6 protein upon infection. The production of this protein began immediately after infection [206 pg (106 macrophages)-1] with native MTB and increased through the 96 h [468 pg (106 macrophages)-1; Fig. 5c]. BMMs infected with delipidated MTB did not produce IL-6 initially; however, by 24 h after infection the level of IL-6 was detectable [102 pg (106 macrophages)-1] and its levels rose through the 96 h. In all cases, the level of protein production in the delipidated-MTB-infected macrophages remained significantly lower than in the native-MTB-infected cells (P<0·01 at all time points). The addition of purified TDM to delipidated MTB restored increases in IL-6 production that closely resembled the pattern seen for native-MTB-infected BMMs; this level was significantly elevated (P<0·001) when compared to the levels of IL-6 produced by delipidated-MTB-infected cells.
The ability of native-MTB- and delipidated-MTB-infected BMMs to produce the chemokine MIP-1 was also examined. C57BL/6 BMMs infected with native MTB produced a high level of MIP-1
immediately after infection; this level transiently decreased at 24 h before rising again at 96 h (Fig. 5d
). Conversely, BMMs infected with delipidated MTB produced significantly less MIP-1
(P<0·001 at 96 h), with only a slight increase in its level by 96 h after infection. Although this level was reduced when compared to infection by native organisms, responses to the delipidated organisms remained statistically greater than responses to the uninfected controls. At all times examined the infection of BMMs with delipidated MTB reconstituted with purified TDM induced an intermediate level of MIP-1
protein production that was significantly higher (P<0·01) than that seen upon infection of the BMMs with delipidated MTB.
Altered IL-12 and IL-10 production by BMMs following infection with delipidated MTB
Delipidated-MTB-infected BMMs exhibited altered IL-12 and IL-10 production compared to that produced upon infection of BMMs with native MTB. In general, the BMMs produced more IL-12 than IL-10 after infection with native MTB (Table 1). Infection with native MTB yielded IL-12 production that increased throughout the 96 h [1120 pg (106 macrophages)-1]. BMMs infected with delipidated organisms produced markedly less IL-12 (P<0·005 at all time points), although increasing amounts of this protein were evident by 96 h. Reconstitution with TDM restored IL-12 production to levels similar to those seen after infection with native organisms. Infection of the BMMs with native organisms also produced IL-10, with its levels increasing throughout the 96 h. The removal of surface lipids significantly reduced the amount of IL-10 produced (P<0·05 at 96 h), but reconstitution experiments restored IL-10 responses.
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DISCUSSION |
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Multiple methods have been used to remove surface lipids and glycolipids from MTB. Chloroform/methanol extraction is efficient but renders the organisms non-viable (Silva et al., 1985 ). Conversely, petroleum ether extraction allows organisms to survive following delipidation, with the growth of delipidated organisms in broth indistinguishable from that of native organisms (Silva et al., 1985
). Organisms retain their acid-fast nature, presumably due to the removal of exposed glycolipids only. Silva et al. (1985)
found by HPLC analysis that petroleum ether extracts of mycobacteria contain primarily TDM (>95% of the total extract), with only relatively small quantities of free mycolic acid glycerides, menaquinones and hydrocarbons present. TLC analysis performed in this study also demonstrated TDM as the primary extracted component. To specifically examine the influence of TDM, we replenished the mycobacterial surface with the purified molecule. Our central assumption based on the hydrophobic nature of TDM chemistry predicted the interaction of TDM with the mycobacterial surface in a manner consistent with natural TDM associations. Reconstitution of delipidated MTB with purified glycolipid restored macrophage responses. Although postulated, we cannot confirm the repopulation of the organisms surface with TDM in a confirmation mimicking that of native TDM. Indeed, other active compounds or small molecular mass lipids or proteins in the mycobacterial cell wall may have been removed or altered, since reconstitution of delipidated mycobacteria with purified TDM did not completely restore the native phenotype. This is most clearly noted in the partial recovery of the IL-1ß response following the reconstitution experimentation.
TDM inhibits fusion between phospholipid vesicles in vitro (Spargo et al., 1991 ). We hypothesized that delipidated MTB would exhibit reduced viability in macrophages, possibly due to an accelerated maturation of organism-containing phagosomes to lysosomes. Initially, the growth of delipidated MTB in C57BL/6 BMMs was comparable to the growth of native MTB; however, by 96 h after infection the BMMs had significantly reduced the number of intracellular delipidated organisms. Removal of surface lipids can potentially affect the uptake of organisms by macrophages, thus altering the outcome of intracellular compartmentalization and survival once inside the host cell. Mycobacterial uptake by macrophages is mediated by multiple mechanisms including complement receptors, scavenger receptors and mannose receptors. However, no differences were evident in the total c.f.u. values obtained for native, delipidated or reconstituted organisms at 4 h after infection.
Specific innate responses by primary macrophages to TDM during in vivo MTB infection have not been thoroughly investigated. Purified TDM, emulsified in oil or coated onto hydrophobic microspheres, induces pulmonary granulomatous lesions, procoagulant activity and TNF-, IL-1ß and IL-6 mRNA production in mice (Perez et al., 1994
, 2000
; Behling et al., 1993
). These activities have been, in part, ascribed to macrophages (Perez et al., 2000
) but they have not yet been confirmed in vivo. We hypothesized that the extraction of TDM from MTB would result in reduced cytokine production by macrophages. Indeed, the removal of MTB surface lipids severely depressed inflammatory cytokine responses an effect that was most striking in the TNF-
and IL-6 responses. Reconstitution of delipidated MTB with purified TDM restored these responses. Interestingly, IL-6 is evident at 96 h after infection with delipidated MTB, presumably due to natural reconstitution of surface lipids by surviving organisms. Delipidation may also expose lipoarabinomannan (LAM): purified LAM modulates macrophages by down-regulating TNF-
(Chatterjee et al., 1992
; Roach et al., 1993
), IL-6 (Dahl et al., 1996
) and IL-12 (Yoshida & Koide, 1997
) production. Our results are only partially consistent with increased LAM exposure. Hyporesponsiveness due to LAM may be critical for the survival of MTB in macrophages; however, increased killing of delipidated MTB was observed in C57BL/6 BMMs. Furthermore, LAM down-regulates macrophage IL-10 production (Dahl et al., 1996
) the IL-10 response was not affected by delipidation. It is therefore likely that the biological effects of the delipidation of MTB are primarily due to the loss of TDM and not to increased LAM exposure.
The role of IL-6 in mycobacterial infection has been studied, with conflicting results. Ladel et al. (1997) showed that mice genetically deficient in IL-6 succumbed to intravenous infection with MTB more rapidly than wild-type mice. VanHeyningen and colleagues suggested that IL-6 plays an inhibitory role in antigen presentation and subsequent T-cell activation (VanHeyningen et al., 1997
). MTB-infected C57BL/6 mice show a high induction of IL-6 mRNA that coincides with granuloma development and protective immunity (Actor et al., 1999
). In this study, C57BL/6 BMMs infected with native MTB produced copious amounts of IL-6. Furthermore, the level of IL-6 induced by MTB in the BMMs was significantly higher than the levels induced by delipidated MTB, a response that was restored when these organisms were reconstituted with TDM, suggesting that TDM by itself may be sufficient for IL-6 induction in C57BL/6 macrophages.
IL-12 production is a critical innate response for the maintenance and control of tuberculosis disease and the granulomatous response (Cooper et al., 1995 , 1997
; Seder, 1995
). Furthermore, the relative ratios of IL-12 and IL-10 are significant components influencing the outcome and immunopathology of infection (Seah et al., 2000
). IL-12 protein production during infection of the BMMs with delipidated organisms was significantly reduced. Consequently, the relative ratio of IL-12:IL-10 was lowered. Reconstitution of the delipidated organisms with TDM restored the relative IL-12:IL-10 ratios. This suggests a contributory role for TDM and surface lipids in the induction of an environment leading to a Th1 response, a concept that has also been proposed by Yamagami et al. (2001)
in examining TDM-specific hypersensitivity in athymic mice.
Mice demonstrate increased MIP-1 production during acute MTB infection (Actor et al., 1999
, 2001
), presumably for the recruitment of the T cells and monocytes necessary for the development of mature granulomas. In this study delipidated MTB induced less MIP-1
production in the BMMs than when they were infected with native MTB. Normal levels of MIP-1
production were restored when delipidated organisms were reconstituted with TDM. Hence, TDM may play a role in the recruitment of T cells and monocytes during the early stages of infection.
In concert with the TDM-mediated inhibition of fusion of phospholipid vesicles (Spargo et al., 1991 ), we investigated whether TDM could also promote the intracellular survival of MTB by suppressing macrophage nitric oxide production (Garcia et al., 2000
). If true, we expected delipidated MTB to generate more nitric oxide in BMMs than when they were infected with native MTB. However, this was not the case. Infection with native MTB and delipidated MTB generated nearly identical amounts of nitric oxide in the BMMs. The regulation of nitric oxide production is a complex issue. Oswald et al. (1997
, 1999
) demonstrated that inducible nitric oxide synthase mRNA in macrophages is, in part, regulated by TNF-
and IL-12. A reduction in priming by IL-12 should have therefore led to decreased nitric oxide production. We are further investigating nitric-oxide-independent bactericidal mechanisms, specifically by examining the role of TDM in intracellular trafficking events.
In summary, the data presented here describe the responses of C57BL/6 BMMs to infection with delipidated MTB. The BMMs exhibited relatively high production of IL-1ß, TNF-, IL-6, IL-12 and MIP-1
in response to infection with MTB; infection with delipidated organisms abrogated these responses. By using reconstitution experiments, we determined these responses to be directly related to TDM.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 31 December 2001;
revised 20 March 2002;
accepted 21 March 2002.