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*
Pulmonary Center, Boston University School of Medicine, and
Laboratory for Infectious Diseases, Boston Medical Center, Boston, MA 02118; and
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation. Coexpression of both
dominant negative proteins blocked virtually all Mtb-induced NF-B
activation. The role of the TLR4 coreceptor MD-2 was also examined.
Unlike LPS, Mtb-induced macrophage activation was not augmented by
overexpression of ectopic MD-2. Moreover, cells expressing an
LPS-unresponsive MD-2 mutant responded normally to Mtb. We also
observed that the lipid A-like antagonist E5531 specifically inhibited
TLR4-dependent Mtb-induced cellular responses. E5531 could
substantially block LPS- and Mtb-induced TNF-
production in both RAW
264.7 cells and primary human alveolar macrophages (AM
). E5531
inhibited Mtb-induced AM apoptosis in vitro, an effect that was a
consequence of the inhibition of TNF- production by E5531. In
contrast, E5531 did not inhibit Mtb-induced NO production in RAW 264.7
cells and AM. Mtb-stimulated peritoneal macrophages from TLR2- and
TLR4-deficient animals produced similar amounts of NO compared with
control animals, demonstrating that these TLR proteins are not required
for Mtb-induced NO production. Lastly, we demonstrated that a dominant
negative MyD88 mutant could block Mtb-induced activation of the TNF-
promoter, but not the inducible NO synthase promoter, in murine
macrophages. Together, these data suggest that Mtb-induced TNF-
production is largely dependent on TLR signaling. In contrast,
Mtb-induced NO production may be either TLR independent or mediated by
TLR proteins in a MyD88-independent manner. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, secretion of
inflammatory mediators (e.g., TNF-), release of the reactive
nitrogen intermediate NO, and secretion of several chemokines (reviewed
in Ref. 3). Until recently, the signaling pathways that
elicit the production of these mediators have remained unknown. Members
of the mammalian Toll-like receptor (TLR) family have been implicated
in the activation of macrophages by a variety of chemically diverse
bacterial products (reviewed in Ref. 4, 5, 6). Both TLR2 and
TLR4 have been shown to mediate Mtb-induced intracellular signaling in
vitro. Means et al. demonstrated that viable Mtb bacilli contain
distinct ligands that activate cells via TLR2 and TLR4
(7), whereas heat-killed Mtb failed to activate cells via
TLR4. Several purified mycobacterial ligands have now been identified
as TLR2 agonists (4), but the identity of the Mtb TLR4
agonist remains unknown. Mtb binding to macrophages induces both
phagocytosis of the bacilli and the activation of innate immune
response genes. The potential roles of TLR proteins in both processes
has been examined. Underhill et al. demonstrated that TLR2 is
selectively associated with macrophage phagosomes that contain yeast
particles or zymosan (8). Abs against TLR2 failed to block
zymosan uptake, although they did block zymosan-induced TNF-
production. These findings suggest that TLR proteins mediate cellular
responses to micro-organisms, but are not required for
phagocytosis. Mtb is an intracellular pathogen that resides mainly within macrophages. Paradoxically, the normal function of macrophages is to engulf and destroy micro-organisms. Mtb possess specific mechanisms to evade destruction by the host macrophage. Phagosomes that contain live Mtb bacilli resist fusion with acidified lysosomes, whereas phagosomes containing heat-killed Mtb fuse freely with lysosomes (9). The ability of live Mtb to inhibit acidification of the phagosome is thought to be a major mechanism for intracellular Mtb survival. However, the exact molecular mechanisms by which the mycobacterial phagosome resists lysosomal fusion remains unknown. Several studies have demonstrated that phagosomes containing live Mtb express early endosomal markers, but lack markers that are specific for later stages of the endocytic pathway (9, 10). An intracellular protein termed TACO (for tryptophan aspartate-containing coat protein) has recently been shown to normally dissociate from phagosomes during the course of phagosome maturation, and this dissociation is required for subsequent lysosomal fusion. Ferrari et al. recently demonstrated that TACO is retained by phagosomes that contain live Mtb, but not by phagosomes containing heat-killed bacteria (11). These data suggest that Mtb trigger the retention of TACO on the phagosome as a means to prevent phagosome-lysosome fusion and bacterial destruction.
Given the spread of drug-resistant Mtb strains, there is a pressing
need to develop treatments that do not rely on new antibiotics. One
novel approach would be to develop therapeutics that antagonize TLR
proteins. This has been accomplished for one TLR4 agonist,
Gram-negative bacterial LPS, and its pharmacophore lipid A. Three lipid
A analogs, lipid IVA, Rhodobacter
sphaeroides lipid A (RSLA), and E5531 have all been reported to
function as LPS antagonists when tested both in vitro and in vivo
(12, 13, 14). These antagonists were all shown to be
selective, because they were all capable of blocking NF-B activation
by LPS, but not NF-B activation induced by the TLR-independent
agonist IL-1
(15, 16, 17). Interestingly, we previously
demonstrated that RSLA could also block signaling by a TLR2 agonist,
the mycobacterial glycolipid lipoarabinomannan (18).
Together, these data suggest that certain lipid A structural
antagonists are capable of blocking TLR-dependent activation by
molecules that are chemically dissimilar to LPS.
We have now extended these findings using the
well-characterized synthetic lipid A-like antagonist, E5531, to block
TLR4-dependent signaling. Here we show that E5531 is capable of
inhibiting some Mtb-induced macrophage responses, namely apoptosis and
TNF- secretion. In contrast, E5531 did not block Mtb-induced NO
production. Additional data demonstrate that Mtb-induced NO production
by macrophages is a TLR-independent response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The RAW 264.7 murine macrophage (TIB-71) and CHO-K1 fibroblast
(CCL-61) cell lines were obtained from American Type Culture Collection
(ATCC, Manassas, VA). RAW 264.7 macrophages, CHO fibroblasts, and
CHO-derived cell lines were cultured as we have previously described
(7). The CHO-derived cell lines 3E10 (CHO/CD14/ELAM-CD25),
3E10/TLR2, and 3E10/TLR4 were generated and cultured as previously
described (16, 19, 20). These 3E10/TLR cells expressed
FLAG-tagged human TLR proteins. Clones that expressed similar levels of
TLR proteins, measured by flow cytometry using an anti-FLAG Ab,
were selected for further study. Furthermore, the TLR2- and
TLR4-expressing cell lines were generated from the same CHO/CD14
parental clones; therefore, each line also expresses the same levels of
CD14. A mutant 3E10 line that lacks functional MD-2 was generated by
chemical mutagenesis as previously described (19). This
line (designated 7.19) was found to be LPS unresponsive, yet fully
responsive to exogenous
IL-1.4 Sequence
analysis of the MD-2 cDNA from these cells revealed a single point
mutation in a highly conserved region resulting in a C95Y amino acid
exchange. All medium components were <10 pg/ml final concentration as
measured by Limulus amebocyte lysate kit (BioWhittaker,
Walkersville, MD).
In experiments using 3E10 cells, which contain a stably transfected
CD25 reporter gene under the control of the NF-B-dependent ELAM-1
promoter, CD25 expression was measured by flow cytometry as previously
described (19). Data were collected using CellQuest
software (Becton Dickinson, Mountain View, CA) and expressed as either
mean channel fluorescence or the ratio (fold activation) of the
percentage of CD25+ cells in unstimulated and
stimulated cell populations (gated to exclude the lowest 5% of cells
based on mean fluorescence). The 95% confidence limit for nonspecific
fluorescence was established using isotype control Abs. Each experiment
was repeated at least three times.
Reagents
Mtb (strain H37Ra, ATCC 25177) and Mycobacterium
bovis bacillus Calmette-Guérin (BCG; ATCC 35734) were
purchased from the ATCC. Bacterial cultures were grown in LPS-free
Middlebrook 7H9 medium supplemented with Tween 80 and ADC (Difco,
Detroit, MI) at 37°C under biosafety level 3 conditions.
Protein-free, phenol/water-extracted LPS (purified from
Escherichia coli K235) was prepared as described previously
(21, 22). E5531 was obtained from Eisai Research Institute
(Eisai Research Institute, Andover, MA). Arabinose-capped mycobacterial
lipoarabinomannan, purified from a rapidly growing avirulent
Mycobacterium species (AraLAM), was provided by Dr. John
Belisle (Colorado State University, Fort Collins, CO). Preparation of a
soluble H37Ra-conditioned culture filtrate, termed soluble tuberculosis
factor (STF), was previously described (7). Levels of
contaminating LPS in the AraLAM and STF preparations were measured
using a quantitative Limulus lysate assay (BioWhittaker),
and were <10 pg/ml final concentration in all experiments Recombinant
human TNF- was purchased from BioSource (Camarillo, CA).
Staurosporine was purchased from Sigma (St. Louis, MO).
Animals
C3H/OuJ and C3H/HeJ mice (female, 5–7 wk old) were obtained from The Jackson Laboratory (Bar Harbor, ME). Chinese and Syrian hamsters (female, 8–10 wk old) were obtained from Cytogen Research and Development (Boston, MA). All mice and hamsters were used within 2 wk of receipt. Mice and hamsters were maintained in a laminar flow hood in cages fitted with polyester filter hoods and fed standard lab food and acid water ad libitum. Peritoneal macrophages were elicited by i.p. injection of 2 ml of sterile fluid thioglycolate medium (3%). Four days after injection peritoneal macrophages were recovered from mice or hamsters by instilling and withdrawing 10 ml of sterile serum-free DMEM medium from the peritoneal cavity. Peritoneal macrophages were allowed to adhere for 2 h to the culture dishes, the nonadherent cells were removed, and the adherent macrophages were cultured for 3–4 days before use.
Plasmids
The NF-B-dependent ELAM-Luc reporter plasmid and the human
FLAG-tagged TLR2 and TLR4 expression plasmids were previously described
(7). The mutant murine TLR2 dominant negative (DN)
expression plasmid contains a FLAG-tagged TLR2 fusion cDNA, which lacks
the C-terminal 13 aa of the TLR2 coding sequence; this cDNA was
generated by PCR and subcloned into the
5'NheI/3'XhoI site of pcDNA3.1 (Invitrogen,
Carlsbad, CA). The murine TLR4-DN expression plasmid containing a
FLAG-tagged mutant TLR4 cDNA (P712H) was subcloned into the
5'NotI/3'SalI site of the pFLAG-CMV-1 expression
vector and was previously described (17). A murine MyD88
cDNA fragment was generated by RT-PCR using mRNA purified from RAW
264.7 cells and PCR primers based on the N- and C-terminal sequences of
murine MyD88 (GenBank accession no. P22366). A cDNA fragment containing
only the C-terminal half of MyD88 (aa 146–296) plus a new synthetic
N-terminal methionine codon were cloned into the expression plasmid
pcDNA3.1/Hyg. The murine MD-2 expression plasmid was a gift from Dr.
Kensuke Miyake (Saga Medical School, Saga, Japan) and was previously
described (23). The hamster MD-2 expression plasmid was
cloned from CHO-K1 cells (see Footnote 4). The luciferase reporter
plasmid pGL3/mTNF(-1059)Luc contains 1064 bp of the murine TNF-
promoter and was previously described (24). The luciferase
reporter plasmid pGLH/H2, containing 1755 bp of the murine iNOS
promoter, was a gift from Dr. William J. Murphy (University of Kansas
Medical Center, Kansas City, KS) and was previously described
(25). Plasmid DNA was prepared using Qiagen (Valencia, CA)
plasmid DNA purification columns, DNA was eluted from the columns using
LPS-free buffers, and contaminating LPS levels were <10 pg/ml.
Transient transfections
Transient transfections of RAW 264.7 and CHO-K1 cell lines were performed using SuperFect reagent (Qiagen) according to the manufacturer’s instructions and as previously described (26). Each DNA mixture (3 µg of total DNA) was prepared individually, and each condition was performed in triplicate. DNA mixtures were incubated with the cells for 2–3 h, whereupon the cells were washed and cultured in fresh medium containing serum. On the following day, individual wells were left untreated or were stimulated with Mtb, LPS, STF, or AraLAM as indicated in the text and figures. Cells were then incubated for an additional 5 h before harvesting and lysis. Luciferase assays were performed using the Luciferase Assay System (Promega) according to the manufacturer’s instructions. An equal amount of total protein from each lysate was assayed for luciferase activity as measured by light emissions in a Monolight 3010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI) for 20 s. Luciferase activity is expressed as the average total relative light units subtracted from background ± SD. Background luciferase activity was established by assaying lysates from mock-transfected cells.
Measurement of TNF- and NO levels
TNF- protein levels in the culture supernatants were measured
by sandwich ELISA according to the manufacturer’s instructions (R&D
Systems, Minneapolis, MN). Samples were assayed in triplicate, and the
data were expressed as the average ± SD. Recombinant human and
mouse TNF- standards were obtained from R&D Systems. The data were
recorded and analyzed using the SOFTmax version 2.01 software program
(Molecular Devices, Menlo Park, CA). The detection limit was 4 pg/ml
for TNF-. As an indirect indicator of NO production by macrophages,
nitrite levels were measured in culture supernatants using the Greiss
reagent assay (27). Samples were assayed in triplicate,
and the data were expressed as the average ± SEM. The detection
limit was 2 µM for nitrite.
Analysis of macrophage apoptosis
Human alveolar macrophages (AM) were plated at a density of
4 x 104 cells/well in 96-well microtiter
plates. After 2 days portions of the cells were stimulated with Mtb
(five bacilli per macrophage), in the absence or the presence of E5531
(1 or 10 µg/ml). Four hours later the medium was removed and replaced
with fresh medium. After 5 days the supernatant was removed, the
adherent macrophages were resuspended in 200 µl of lysis buffer, and
the lysate was centrifuged at 200 x g for 10 min.
These culture supernatant contain mono- and oligonucleosomes released
by apoptotic cells, and an Ag-capture ELISA was used to measure the
levels of histone-associated DNA fragments (Cell Death Detection ELISA
Plus; Roche, Mannheim, Germany) present in the cell lysates according
to the manufacturer’s instructions. Samples were assayed in
triplicate, and the data are expressed as the average ±
SEM.
Statistical analysis
Statistical analysis was performed using SuperANOVA (version 1.11) software from Abacus Concepts (Berkeley, CA). This computer program applied one-way ANOVA with multiple comparisons on different sample datasets. A 95% confidence level was used for all statistically analyzed data. Statistically significant data are indicated in the figures by an asterisk, and the corresponding p values are listed in the figure legends.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation in
macrophages
We previously reported that live Mtb bacilli can activate CHO
fibroblasts and RAW 264.7 macrophages via TLR2 or TLR4, but not TLR1
(7). These TLR-dependent responses were induced by
distinct secreted and cell-associated bacterial products. Furthermore,
this activation did not require either membrane-bound or soluble CD14.
Here we sought to determine the relative contributions of TLR2 and TLR4
to total macrophage activation by Mtb. As shown in Fig. 1
, overexpression of a TLR2-DN protein in
RAW 264.7 macrophages blocked activation by the TLR2 agonist AraLAM,
but not the TLR4 agonist LPS. Similarly, overexpression of a TLR4-DN
protein in these cells specifically blocked LPS-induced activation, but
not AraLAM-induced activation. Overexpression of the TLR2-DN or TLR4-DN
proteins partially blocked Mtb-induced cellular activation. The TLR2-DN
protein alone consistently blocked 70–80% of total Mtb-induced
cellular activation. This is similar to the level of blocking observed
by other investigators using a TLR2-DN protein to block stimulation of
RAW 264.7 cells with heat-killed Mtb (8). In contrast, the
TLR4-DN protein alone blocked only 30–40% of Mtb-induced cellular
activation. Because both DN proteins were maximally overexpressed in
the macrophages, these results suggest that TLR2 mediates a relatively
higher proportion of the cellular response to Mtb compared with TLR4.
Furthermore, coexpression of both DN proteins blocked virtually all
Mtb-induced NF-B activation (Fig. 1). Together, these data suggest
that TLR2 and TLR4 are responsible for virtually all Mtb-induced
NF-B activation.
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The gene responsible for the LPS hyporesponsiveness phenotype of the C3H/HeJ mouse was identified as tlr4 (28, 29, 30). The C3H/HeJ strain of mice possesses a mutant TLR4 protein that contains a missense mutation within the tlr4-coding sequence (P712H), and these mice are functionally TLR4 deficient for LPS signaling. We sought to determine whether these mice were abnormally susceptible to mycobacterial infection compared with normal C3H/OuJ mice. C3H/OuJ and C3H/HeJ mice were injected with 3 x 106 CFU of M. bovis BCG into their peritoneal cavity. Survival was measured 3 days postinfection. M. bovis BCG was selected for this experiment because, like Mtb, these mycobacteria activate cells via both TLR2 and TLR4 (data not shown). We found that 9 of 20 C3H/HeJ mice died, while only 1 of 20 C3H/OuJ mice died. These data suggest that TLR4 serves a protective function in the host response to mycobacterial infection.
Role of MD-2 expression in Mtb-induced NF-B activation
A novel protein called MD-2 was recently demonstrated to associate
with TLR4 and to be necessary for TLR4-dependent cellular activation by
LPS and Taxol (23, 31). We determined whether MD-2 was
required for Mtb-induced cellular activation via TLR4. We transiently
cotransfected RAW 264.7 cells with the NF-B-dependent reporter
plasmid and a murine MD-2 expression plasmid. We then compared the
responses of these cells to different TLR agonists. We found that
overexpression of MD-2 in RAW 264.7 macrophages enhanced cellular
responsiveness to the TLR4 agonist LPS by 2- to 3-fold, whereas it did
not enhance cellular responsiveness to the TLR2 agonist AraLAM (Fig. 2A). Furthermore, MD-2
overexpression did not enhance the capacity of live Mtb to activate RAW
264.7 macrophages. To directly determine whether MD-2 was required for
TLR4-dependent cellular activation by Mtb, we used a mutant 3E10
(CHO/CD14) cell line that expresses a nonfunctional form of MD-2. These
mutant cells were transiently cotransfected with an NF-B-dependent
luciferase reporter plasmid and a hamster TLR4 expression plasmid. A
portion of the cells was also transfected with an expression plasmid
encoding a function MD-2 protein. As shown in Fig. 2B, the
MD-2 mutant 3E10 line was incapable of activation by LPS in the absence
of ectopically expressed functional MD-2. In contrast, this mutant cell
line was fully capable of activation by Mtb, and expression of ectopic
functional MD-2 did not affect the responses of these cells to the
bacilli. Together, these data suggest that MD-2 may not be required for
cellular activation by Mtb.
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B activation
Because both TLR2 and TLR4 mediate cellular activation by Mtb, we
sought to determine the relative contribution of TLR4 to Mtb-induced
macrophage activation. Because blocking anti-murine TLR Abs were
not available, we used the lipid A-like LPS-antagonist E5531 to block
TLR4-dependent signaling (14, 15). We hypothesized that
E5531 could block cellular activation via TLR4 regardless of the
activating ligand based on our earlier finding that another lipid
A-like antagonist, RSLA, blocked macrophage activation by both LPS and
AraLAM (18). RAW 264.7 macrophages were stimulated for
5 h in the absence and the presence of E5531. As shown in Fig. 3A, E5531 blocked 90–100% of
LPS-induced NF-B activity, but only 30–40% of Mtb-induced NF-B
activity in these murine macrophages. A similar level of blocking was
observed using the TLR4-DN mutant, as shown in Fig. 1
, again suggesting
that TLR4 mediates a lower relative proportion of the cellular
responses to Mtb (as judged by NF-B activation) compared with TLR2.
Interestingly, the specificity of E5531 for TLR4-dependent signaling
was demonstrated by the fact that it did not block NF-B activation
by the TLR2 agonist AraLAM (Fig. 3A). Thus, E5531 appears to
block only TLR-4 dependent signaling, whereas RSLA appears to block
both TLR2- and TLR4-dependent signaling (18).
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B activity in CHO/CD14/TLR4 cells, a
cell line that does not express TLR2, whereas E5531 did not block the
activation of CHO/CD14/TLR2 cells (Fig. 3B). This implies
that E5531 is highly specific for TLR4 and has no effect of
TLR2-dependent cellular activation by Mtb. E5531 blocks Mtb-induced human alveolar macrophage apoptosis
The data presented above demonstrate that E5531 has the capacity
to specifically block Mtb-induced NF-B activation in macrophages. We
also wanted to determine whether E5531 could block cellular responses
that are known to be part of the innate immune response against Mtb. We
previously proposed that macrophage apoptosis contributes to the host
defense against Mtb infection (32) and hypothesized that
this may be a TLR-dependent response. Both TLR4 and TLR2 have been
previously implicated in macrophage activation and apoptosis.
TLR4-deficient C3H/HeJ macrophages have been shown to be resistant to
LPS-induced cell death (33). In addition, soluble
bacterial lipoproteins have been demonstrated to induce TLR2-dependent
macrophage apoptosis (34, 35). Therefore, we sought to
determine whether TLR4 was required for Mtb-induced apoptosis. As shown
in Fig. 4A, E5531 inhibited
Mtb-induced apoptosis of human AM in vitro in a dose-dependent
manner. Our previous studies demonstrated that TNF- was necessary,
but not sufficient, for Mtb-induced macrophage apoptosis
(32). Thus, our data suggested that E5531 might block
Mtb-induced cell death by blocking Mtb-induced TNF- production. This
possibility was tested by providing the Mtb-infected AM with
exogenous recombinant human TNF- to determine whether E5531 could
still block cell death. As shown in Fig. 4B, addition of
exogenous TNF- abrogated the capacity of E5531 to block Mtb-induced
AM apoptosis. Thus, E5531 may block cell death by inhibiting
TLR4-dependent Mtb-induced TNF- production by AM. In control
experiments we also confirmed that addition of recombinant TNF-
alone was not sufficient to induce AM apoptosis. Staurosporine was
used as a positive control to induce cell death is this assay.
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B activation
and apoptosis by E5531 by its blocking Mtb binding or uptake, and thus
cellular activation. This hypothesis was directly tested by determining
whether E5531 could block the binding of with M. bovis BCG
that constitutively express green fluorescent protein (GFP) to human
AM in vitro. Human AM were incubated with 10 BCG-GFP
bacilli/macrophage in the absence or the presence of E5531 (1–10
µg/ml). The next day, the cells were washed twice with PBS, and
internalization of BCG-GFP by human AM was quantified by manual
counting using a fluorescence microscope. We observed that 28 ±
5% of the human AM contained BCG-GFP after 16 h of infection.
Inclusion of 1–10 µg/ml of E5531 had no effect on the percentage of
BCG-GFP-positive macrophages (data not shown). Thus, E5531 does not
block binding or uptake of Mtb. Together, these data suggest that E5531
blocks Mtb-induced apoptosis indirectly via its ability to block
TNF- production.
E5531 inhibits LPS- and Mtb-induced TNF- production, but not NO
production
We next sought to directly test the possibility that E5531 blocked
Mtb-induced TNF- production by macrophages. We concomitantly
measured the production of NO by these macrophages in the absence and
the presence of E5531. While murine macrophages produce high levels of
NO in response to LPS and IFN-
, human macrophages are not similarly
responsive. However, high levels of NO can be produced by human AM
stimulated in vitro with live Mtb (27, 36). We stimulated
RAW 264.7 cells and primary human AM with Mtb in the presence and
the absence of E5531. One day after stimulation, culture supernatants
were collected and assayed for the production of both TNF- and
nitrite. Nitrite (NO3-) is a
stable catabolite of NO, and the level of nitrite in culture
supernatants is a good indirect indicator of NO production by
macrophages. E5531 substantially blocked Mtb-induced TNF- production
in both RAW 264.7 murine macrophages (Fig. 5, A and C) and
human AM (Fig. 6A). In
contrast, NO production was not affected by E5531 in either
Mtb-stimulated RAW 264.7 murine macrophages (Fig. 5, B and
D) or in human AM (Fig. 6B) regardless of the
concentration of E5531 used. Thus, Mtb-induced TNF- and NO
production were differentially affected by the TLR4-specific antagonist
E5531.
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The data reported above suggest that TLR4 is not necessary for
Mtb-induced NO production, but do not exclude the possibility that
other TLR proteins mediate this response. For example, the purified
19-kDa lipoprotein Ag from Mtb has been shown to activate the iNOS
promoter in transiently transfected murine macrophages in a
TLR2-dependent manner (37). Thus, we used macrophages from
TLR-deficient animals to compare the roles of TLR2 and TLR4 in
Mtb-induced NO production (Fig. 7).
Primary murine peritoneal macrophages from normal C3H/OuJ and
TLR4-deficient C3H/HeJ mice and primary hamster peritoneal macrophages
from normal Syrian and TLR2-deficient Chinese hamsters were stimulated
with Mtb (five bacilli per macrophage) for 16 h. Culture
supernatants were then assayed for nitrite production. We found that
macrophages lacking either TLR2 or TLR4 produced identical levels of
nitrite in response to Mtb compared with control cells. Together, these
data demonstrate that TLR2 and TLR4 are not necessary for Mtb-induced
NO production. Furthermore, these findings suggest that mycobacterial
TLR2 agonists do not significantly contribute to NO production by
macrophages stimulated by live Mtb bacilli in vitro.
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promoter, but not
the iNOS promoter
The findings presented above suggest that Mtb-induced NO
production is not mediated by either TLR2 or TLR4. This raises the
possibilities that 1) Mtb-induced NO production may by mediated by
other TLR proteins; and 2) Mtb-induced NO production is the consequence
of a TLR-independent mechanism. To discriminate between these
possibilities we determined whether a MyD88-DN protein could block
Mtb-induced NO production. We postulated that such a MyD88-DN mutant
would block cellular activation via any TLR protein, because all known
TLR proteins require MyD88 for intracellular signaling
(38, 39, 40). RAW264.7 macrophages were cotransfected with a
MyD88-DN expression plasmid plus one of three reporter plasmids. The
luciferase reporter plasmids used were under control of the
NF-B-dependent ELAM promoter, the murine TNF- promoter, and the
murine iNOS promoter. The day following transfection, cells were
stimulated with LPS, STF, or Mtb for 5 h. As shown in Fig. 8, overexpression of the MyD88-DN mutant
blocked LPS-, STF-, and Mtb-induced ELAM and TNF- promoter
activities. Similarly, expression of the MyD88-DN mutant blocked LPS-
and STF-induced activation of the iNOS promoter. In contrast,
overexpression of the MyD88-DN mutant failed to block Mtb-induced
activation of the iNOS promoter. These data suggest that Mtb-induced
TNF- activation is a TLR-dependent process, whereas Mtb-induced NO
production is TLR independent (Fig. 9).
Alternatively, Mtb-induced NO production may be mediated by TLR
proteins in an MyD88-independent manner.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B via both TLR2 and TLR4 in macrophages
(7). Here we examined the relative contributions of TLR2
and TLR4 to Mtb-induced cellular responses. The lipid A-like LPS
antagonist E5531 was capable of substantially blocking Mtb-induced
NF-B activation and TNF- production in both RAW 264.7 murine
macrophages and primary human AM. In CHO/CD14/TLR4 cells, E5531
blocked virtually all Mtb-induced NF-B activation. Because these
cells do not express functional TLR2, virtually all Mtb-induced NF-B
activation appears to be mediated by TLR4 when TLR2 is absent or
nonfunctional. The inhibitory activity of E5531 was specific for TLR4
because it did not block NF-B activation induced by TLR2 agonist
AraLAM. Moreover, E5531 did not affect activation of CHO/CD14/TLR2
cells by Mtb, suggesting that additional mycobacterial TLR2 ligands
were not blocked by the antagonist. E5531 also blocked Mtb-induced
human AM apoptosis, an effect that was secondary to the inhibition
of TNF- production. Lastly, we found that the capacity of E5531 to
block Mtb-induced TNF- production was not shared by all
Mtb-inducible macrophage products. Mtb-induced NO production was not
blocked by E5531, did not depend on the presence of TLR2 or TLR4, and
may be mediated by a TLR-independent mechanism.
Together, these data demonstrate E5531 specifically blocks
TLR4-dependent Mtb-induced cellular responses. Because Mtb are not
known to possess glycolipids that are chemically similar to LPS, our
data suggest three potential mechanisms for E5531 antagonism. First,
E5531 may block the assembly of functional TLR4 receptor complexes,
rather than simply competing for binding of an Mtb ligand to TLR4.
Second, E5531 may compete for binding to a common binding site on TLR4
with a mycobacterial TLR4 agonist. Third, E5531 may block a distinct
TLR protein that functions in association with TLR4 to mediate
responsiveness to LPS and mycobacterial ligands. The recent finding
that another lipid A-like antagonist can block Taxol-induced cellular
activation via TLR4 (41) is consistent with our findings,
but does not favor any of these possible mechanisms. Regardless of the
mechanism of E5531 action, this antagonist has proven to be a useful
and selective tool for inhibiting TLR4 signaling by both LPS and
chemically unrelated TLR4 agonists. It is interesting to note that CD14
(7) and MD-2 (this report), which greatly augment LPS and
taxol stimulation via TLR4, do not appear to be required for
Mtb-mediated activation in vitro. Our studies do not address the
possibility that these TLR4 coreceptors modulate macrophage responses
to Mtb TLR4 agonists in vivo. Interestingly, the
2 integrin CD11b/CD18 appears to function as a
TLR4 coreceptor in macrophages stimulated with taxol (42).
The ability of CD11b/CD18 (also known as CR3) to mediate binding of Mtb
to macrophages has been well described (1), but its
potential role in modulating TLR4-dependent cellular activation by Mtb
has not been clearly defined.
We found that TLR4-deficient C3H/HeJ mice were significantly more sensitive to lethal mycobacterial infection than normal C3H/OuJ mice. This finding supports the hypothesis that TLR4 is necessary for innate host responses to mycobacteria in vivo. Many studies have shown enhanced sensitivity of C3H/HeJ mice to infection by a broad range of infectious agents (reviewed in Ref. 43 and references therein). One recent study using experimental E. coli pyelonephritis demonstrated that the kidneys of C3H/HeJ and C3H/HeN mice were colonized at a similar rate 24 h after infection; however, only the C3H/HeN mice could clear the infection 5 wk postinoculation (44). In contrast, Hopkins et al. revealed that both C3H/HeJ and C3H/OuJ mice were similarly sensitive to E. coli infection, whereas C3H/HeN mice were more resistant to infection compared with the other C3H strains (45). Furthermore, the degree of inflammation in the bladder and kidneys positively correlated with the extent of infection, except for C3H/HeJ mice, which had minimal inflammation despite high infection levels. These findings are consistent with the earliest reports of LPS hyporesponsiveness in which C3H/HeJ mice failed to respond to i.p. injection of LPS by eliciting a normal inflammatory cell infiltrate into the peritoneal cavity (46). Together, these data suggest that TLR4 is a critical factor in the development of an inflammatory host response to infection, but TLR4 is not the sole determinant of susceptibility to persistent or lethal infection. The comparatively high susceptibility of C3H/HeJ mice to lethal mycobacterial infection may be linked to the TLR4-dependent activation of proinflammatory cytokine and chemokine production in vivo. It has been shown that these potent inflammatory mediators play a protective role in host responses to mycobacterial infection (reviewed in Ref. 47), and it is possible that the infected C3H/HeJ mice fail to elicit a protective early inflammatory response in vivo.
Another innate host response that appears to require TLR proteins is
Mtb-induced AM apoptosis. Macrophage apoptosis has been shown to
correlate inversely with the virulence of the mycobacterial species and
strain used (48). Macrophage apoptosis has also been
proposed to be important in the host defense against Mtb (reviewed in
Ref. 49). Apoptosis may be a means to trap live bacilli
within apoptotic bodies, thereby reducing the dissemination of Mtb from
infected macrophages. Furthermore, Mtb-containing apoptotic cells are
probably rapidly phagocytosed and destroyed by uninfected AM. This
hypothesis is consistent with previous data demonstrating that virulent
Mtb strains induce less macrophage apoptosis, thereby promoting
survival and ability to replicate within the host (32).
Here we demonstrate that Mtb-induced macrophage apoptosis is at least
in part a TLR4-dependent process that can be blocked by E5531. The
simplest explanation for our findings is that E5531 blocks Mtb-induced
macrophage apoptosis as a consequence of its ability to block
Mtb-induced TNF- production. TNF- production is required to
mediate Mtb-induced macrophage apoptosis, and the addition of exogenous
TNF- was sufficient to abrogate the inhibitory activity of E5531.
Alternatively, E5531 may block an Mtb-induced proapoptotic pathway, but
addition of exogenous TNF- may circumvent this blockade, thereby
promoting apoptosis via a parallel pathway. Whether additional TLR or
non-TLR proteins are also involved in other Mtb-induced proapoptotic
responses remains to be determined.
One important and unexpected finding arising from our studies was the
observation that E5531 could block Mtb-induced TNF- production, but
not NO production. This phenomenon was observed using both a murine
macrophage cell line and primary human AM. Furthermore, macrophages
from animals that lack functional TLR2 or TLR4 produced normal levels
of NO when challenged with Mtb in vitro. Subsequently, we demonstrated
that a MyD88-DN mutant could block Mtb-induced activation of the
TNF- promoter, but not the iNOS promoter. The rationale for using a
MyD88-DN mutant comes from previous studies showing that this adapter
protein is required for signaling by all known TLR proteins as well as
other related receptors (IL-1R type I, IL-18R) (38, 39, 40).
Thus, we used the MyD88-DN mutant as a means to block all Mtb-induced
TLR signaling. Together, our findings demonstrate that Mtb-induced
TNF- production is a TLR-dependent process, whereas Mtb-induced NO
production appears to be a TLR-independent process. Alternatively,
Mtb-induced NO production may be mediated by TLR proteins in a
MyD88-independent manner. Both TNF- and NO are known to play
important roles in the host response to Mtb infection.
TNF--deficient and iNOS-deficient mice are both highly sensitive to
lethal Mtb infection compared with normal mice (50, 51, 52).
Thus, it is interesting that these two critical innate host responses
differ in their requirement for TLR proteins. While the significance of
this difference in TLR requirement is not clear, our data suggest that
inhibition of TLR signaling could leave some innate host responses
intact.
Our future studies are aimed at examining the course of Mtb infection
in vivo using TLR2 and TLR4 knockout mice. It will be particularly
important to determine whether iNOS production and NO-dependent
mycobactericidal activities in vivo are affected by the absence of TLR2
or TLR4. TNF- has been shown to augment IFN--induced NO
production by macrophages in vitro, resulting in maximal mycobacterial
growth inhibitory activity (53). Thus, the absence of TLR2
and/or TLR4 signaling may have an indirect effect on NO production and
mycobacterial growth inhibition. Lastly, the study of naturally
occurring TLR mutations in humans may reveal links to disease
susceptibility. Arbour et al. recently reported that TLR4 mutations are
associated with LPS hyporesponsiveness (54), and it will
be interesting to determine whether such mutations are associated with
increased susceptibility to tuberculosis.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Matthew J. Fenton, Pulmonary Center, R-220, Boston University School of Medicine, Boston, MA 02118-2394.
3 Abbreviations used in this paper: Mtb, Mycobacterium tuberculosis; TLR, Toll-like receptor; TACO, tryptophan aspartate-containing coat protein; AM, alveolar macrophage; BCG, bacillus Calmette-Guérin; AraLAM, arabinose-capped lipoarabinomannan; RSLA, Rhodobacter sphaeroides lipid A; STF, soluble tuberculosis factor; DN, dominant negative; GFP, green fluorescent protein.
4 A. B. Schromm, A. Yoshimura, H. Heine, P. Henneke, B. Monks, K. Miyake, and D. T. Golenbock. Molecular genetic analysis of an endotoxin non-responder mutant cell line: a point mutation in a conserved region of MD-2 abolishes endotoxin-induced signaling. Submitted for publication.
Received for publication September 25, 2000. Accepted for publication January 16, 2001.
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J. Hellman, J. D. Roberts Jr., M. M. Tehan, J. E. Allaire, and H. S. Warren Bacterial Peptidoglycan-associated Lipoprotein Is Released into the Bloodstream in Gram-negative Sepsis and Causes Inflammation and Death in Mice J. Biol. Chem., April 12, 2002; 277(16): 14274 - 14280. [Abstract] [Full Text] [PDF]
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 S. Lehnardt, C. Lachance, S. Patrizi, S. Lefebvre, P. L. Follett, F. E. Jensen, P. A. Rosenberg, J. J. Volpe, and T. Vartanian The Toll-Like Receptor TLR4 Is Necessary for Lipopolysaccharide-Induced Oligodendrocyte Injury in the CNS J. Neurosci., April 1, 2002; 22(7): 2478 - 2486. [Abstract] [Full Text] [PDF]
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R. N. Fichorova, A. O. Cronin, E. Lien, D. J. Anderson, and R. R. Ingalls Response to Neisseria gonorrhoeae by Cervicovaginal Epithelial Cells Occurs in the Absence of Toll-Like Receptor 4-Mediated Signaling J. Immunol., March 1, 2002; 168(5): 2424 - 2432. [Abstract] [Full Text] [PDF]
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K. M. EDWARDS, M. H. CYNAMON, R. K. R. VOLADRI, C. C. HAGER, M. S. DESTEFANO, K. T. THAM, D. L. LAKEY, M. R. BOCHAN, and D. S. KERNODLE Iron-cofactored Superoxide Dismutase Inhibits Host Responses to Mycobacterium tuberculosis Am. J. Respir. Crit. Care Med., December 15, 2001; 164(12): 2213 - 2219. [Abstract] [Full Text] [PDF]
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C. A. Scanga, V. P. Mohan, K. Tanaka, D. Alland, J. L. Flynn, and J. Chan The Inducible Nitric Oxide Synthase Locus Confers Protection against Aerogenic Challenge of Both Clinical and Laboratory Strains of Mycobacterium tuberculosis in Mice Infect. Immun., December 1, 2001; 69(12): 7711 - 7717. [Abstract] [Full Text] [PDF]
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J. Warwick-Davies, A. J. Watson, G. E. Griffin, S. Krishna, and R. J. Shattock Enhancement of Mycobacterium tuberculosis-Induced Tumor Necrosis Factor Alpha Production from Primary Human Monocytes by an Activated T-Cell Membrane-Mediated Mechanism Infect. Immun., November 1, 2001; 69(11): 6580 - 6587. [Abstract] [Full Text] [PDF]
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A. E. Medvedev, P. Henneke, A. Schromm, E. Lien, R. Ingalls, M. J. Fenton, D. T. Golenbock, and S. N. Vogel Induction of Tolerance to Lipopolysaccharide and Mycobacterial Components in Chinese Hamster Ovary/CD14 Cells Is Not Affected by Overexpression of Toll-Like Receptors 2 or 4 J. Immunol., August 15, 2001; 167(4): 2257 - 2267. [Abstract] [Full Text] [PDF]
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E. H. Noss, R. K. Pai, T. J. Sellati, J. D. Radolf, J. Belisle, D. T. Golenbock, W. H. Boom, and C. V. Harding Toll-Like Receptor 2-Dependent Inhibition of Macrophage Class II MHC Expression and Antigen Processing by 19-kDa Lipoprotein of Mycobacterium tuberculosis J. Immunol., July 15, 2001; 167(2): 910 - 918. [Abstract] [Full Text] [PDF]
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B. W. Jones, T. K. Means, K. A. Heldwein, M. A. Keen, P. J. Hill, J. T. Belisle, and M. J. Fenton Different Toll-like receptor agonists induce distinct macrophage responses J. Leukoc. Biol., June 1, 2001; 69(6): 1036 - 1044. [Abstract] [Full Text] [PDF]
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13) and/or a DN mouse TLR4
mutant (muTLR4-P712H). A portion of the cells was stimulated with LPS
(100 ng/ml), AraLAM (1 µg/ml), or Mtb (H37Ra, five bacilli per RAW
264.7 cell) for 5 h. Cells were then harvested, and luciferase
activity was measured as described in the text. All transfection
experiments were performed in triplicate and repeated at least three
times using different plasmid preparations, and a single representative
experiment is shown. Data are expressed as the average luciferase value
from a single experiment (subtracted for background) ± SD.
