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* Department of Immunology, University of Cape Town, Cape Town, South Africa;
Pasteur Institute, Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 277, and
Centre National de la Recherche Scientifique-Unité Mixte de Recherche 8603, Université Paris V, Paris, France;
Department of Immunology, Saga Medical School, Saga, Japan; and
¶ Institut Transgénose, Centre National de la Recherche Scientifique, Orléans, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-12p40, and monocyte
chemoattractant protein 1 was significantly lower in C3H/HeJ
mice when compared with the wild-type controls. C3H/HeJ-derived
macrophages infected in vitro with M.tb. produced lower
levels of TNF-. Finally, the purified mycobacterial glycolipid,
phosphatidylinositol mannosides, induced signaling in both a TLR2- and
TLR4-dependent manner, thus suggesting that recognition of
phosphatidylinositol mannosides in vivo may influence the development
of protective immunity. In summary, macrophage recruitment and the
proinflammatory response to M.tb. are impaired in TLR4
mutant mice, resulting in chronic infection with impaired elimination
of mycobacteria. Therefore, TLR4 signaling is required to mount a
protective response during chronic M.tb.
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting CD4 T
cells and full activation of the adaptive cell-mediated immunity
(1). Most of the understanding on the role of T cells and
Th1-type cytokines is derived from studies in mice using genetic models
or Ab neutralization studies (2). IFN- derived from T
and NK cells has been shown to be essential, as mice with a disruption
of the IFN- system are unable to restrict the growth of
M.tb. and succumb to the infection (3, 4, 5, 6, 7).
Additional cytokines, which are critical to control infection and mount
protective immunity, include TNF-, IL-12, and IL-18
(8, 9, 10, 11, 12, 13). The prompt recognition of microorganisms via their signature molecules by APC expressing pattern recognition receptors is an exciting and rapidly developing area (14). Receptors involved in sensing M.tb. patterns include the mannose receptor, complement receptors (CR) CR1, CR3, and CR4 (15), and Toll-like receptor (TLR)2 and TLR4. The mycobacterial envelope contains a wide array of chemically diverse lipids and glycolipids that likely mediate specific interactions with host ligands or membranes and have potent biological activity on eukaryotic cells in in vitro systems (10). Mycobacterial lipoprotein-induced mechanisms including cellular activation, mycobacterial killing, and apoptosis have been demonstrated to be mediated by TLR2 (9, 16, 17, 18, 19, 20). Both TLR2 and TLR4 can mediate cellular activation by M.tb., specifically, soluble heat-stable and protease-resistant factors via the TLR2 pathway and heat-sensitive membrane-associated factors via both TLR2 and TLR4 (21, 22). Mutations of the gene lps selectively impair endotoxin signal transduction in C3H/HeJ mice, rendering them resistant to endotoxin, yet highly susceptible to Gram-negative infection. C3H/HeJ mice were shown to have a missense mutation in the third exon of TLR4 (23, 24). In addition, TLR4-deficient mice generated by homologous recombination confirm a critical role for TLR4 in endotoxin signaling (25). During M.tb. infection, early macrophage activation may occur through various microbial-receptor engagements. We hypothesized that lack of TLR4 triggering might impair macrophage activation and the development of a subsequent protective response. To assess the role of TLR4 in vivo, we infected the TLR4 mutants, C3H/HeJ and their wild-type controls, C3H/HeN mice with M.tb. aerogenically. We report that M.tb.-infected C3H/HeJ mice have significantly reduced recruitment of macrophages to the lungs, lower proinflammatory cytokine production, and succumb to chronic infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C3H/HeJ (TLR4 mutant), and C3H/HeOUJ and C3H/HeN (both TLR4 wild-type strains) mice used for the experiments were 8–12 wk old (Charles River Breeding Laboratories, Kisslegg, Germany). Mice were kept under specific-pathogen-free conditions in the animal unit at the University of Cape Town (Cape Town, South Africa). Aerosol M.tb. infections were performed in a biosafety level 3 area and infected animals were maintained in filter-top cages. The University of Cape Town Animal Ethics Committee approved all protocols used in this study.
Mycobacteria
M.tb. H37Rv and H37Ra (obtained from Prof. G. Kaplan, Rockefeller University, New York, NY, and Prof. G. Marchal, Pasteur, Paris, France, respectively) were grown to mid-log phase in Middlebrook 7H9 medium (Difco, Detroit, MI), supplemented with 10% oleic acid/albumin/dextrose catalase (State Vaccine Institute, Pinelands, South Africa) and 1% glycerol (Merck, Munich, Germany) in 5% CO2 at 37°C. Aliquots were prepared and frozen at -80°C. Before use, an aliquot was thawed, briefly vortexed, diluted in sterile saline containing 0.04% Tween 80 (Merck), and clumping was disrupted by aspirating through a 29-gauge needle (Omnican; Braun, Kronberg, Germany) 20 times.
Infection with H37Rv
Mice were infected aerogenically with 50–100 viable CFU using an inhalation exposure system (Glas-Col, Terre Haute, IN). Animals were exposed for 30 min to an aerosol produced by nebulizing 5 ml of a bacterial suspension in 0.9% NaCl solution at a concentration of 2 x 106 bacilli/ml.
Quantitation of viable mycobacteria in organs
The initial infective dose was verified by sacrificing mice 24 h after aerosol exposure. Lungs were aseptically removed, weighed, and homogenized in 0.9% saline containing 0.04% Tween 80, and 10-fold serial dilutions were plated in duplicate onto 7H10 agar (Difco) supplemented with 10% oleic acid/albumin/dextrose catalase and 0.5% glycerol. Plates were incubated at 37°C and CFU were enumerated 14–18 days later. Thereafter, at specific time points, mice were sacrificed and their lungs, livers, and spleens were aseptically removed and weighed. Two-thirds of each organ was homogenized in 0.9% NaCl solution containing 0.04% Tween 80 for CFU enumeration as described above. Data are presented as log10 CFU per organ (n = 3–4 mice per group).
Histopathology
Lungs, livers, and spleens of mice were fixed for 24 h in 10% buffered Formalin (BDH; Laboratory Supplies, Poole, U.K.) and then embedded in paraffin. Sections were stained with H&E or Ziehl-Neelsen for evaluation of pathologic changes and bacillary load, respectively (26).
Immunohistochemistry
Formalin-fixed, paraffin-embedded sections were deparaffinized and rehydrated through decreasing concentrations of alcohol. Sections were stained with a rabbit anti-mouse Ab specific for inducible NO synthase (iNOS; 1/2000; obtained from J. Pfeilschifter, University of Frankfurt, Frankfurt, Germany). Sections were then washed in PBS and incubated for 30 min at room temperature with rat anti-rabbit serum avidin-biotin complexsystem (Vector Laboratories, Burlingame, CA) followed by diaminobenzidine substrate. Finally, sections were mounted in Immunomount (Thermo Shandon, Pittsburgh, PA) (26).
Bronchoalveolar lavage (BAL)
Control and M.tb.-infected mice were administered a lethal dosage of ketamine. A plastic 20-gauge cannula was inserted into the trachea and the lungs were lavaged with 0.5 ml of sterile PBS. The BAL supernatant was frozen at -80°C for later detection of cytokines and chemokines by ELISA. The cell pellets were resuspended in 1% BSA/PBS and counted. Cytospins were prepared and stained with Giemsa for differential counts (300 cells/cytospin counted).
Purification and characterization of phosphatidyl-myo-inositol tetra- and hexamannosides (PIM4–6)
The preparation of PIM4–6 was a generous gift from Dr. G. Puzo (Institut de Pharmacologie et de Biologie Structurale, Toulouse, France). The PIM-containing lipidic extract was obtained by phenolic purification from M.tb. strain H37Rv as described elsewhere (27). Briefly, PIMs were located in the acetone-insoluble fraction of the chloroform/methanol (1:1, v/v) mycobacterial extracts. The contaminating neutral compounds were eliminated by QMA anion exchange chromatography, irrigated with neutral eluents. Then phospholipids were eluted with ammonium acetate-containing organic solvents. The PIM composition was analyzed by electrospray mass spectrometry (EMI-MS) in negative mode. The tri- and tetra-acylated forms of PIMs appeared to be the most abundant acyl forms. The level of contaminating LPS was determined using a quantitative Limulus lysate assay (BioWhittaker, Walkersville, MD) and were <1 pg of endotoxin/µg of material; this amount of LPS does not elicit the degree of activation induced by PIMs.
U373 and Ba-F3 cell cultures and stimulation
The human astrocyte cell line U373 (American Type Culture Collection, Manassas, VA) was cultured in RPMI 1640 medium supplemented with penicillin (100 IU/ml) and streptomycin (100 µg/ml) supplemented with 10% FCS. Ba-F3-transfected cells were cultured in RPMI 1640 with antibiotics, 10% FCS, and IL-3 (28).
U373 cells were cultured in AIM V medium supplemented with penicillin (100 IU/ml) and streptomycin (100 µg/ml) in Terasaki plates or 96-well round-bottom microtiter plates (Nunc, Roskilde, Denmark) in the presence of increasing concentrations of PIM4–6 complexes. Supernatants were harvested 16 h later and stored at -20°C until IL-6 assays by ELISA were performed.
The Ba-F3 cell line was stably expressing TLR4, MD-2, and
p55Ig
Luc, an NF-B-dependent luciferase reporter construct
(28). For the luciferase assay, transformants were washed
and cultured in fresh medium containing heat-inactivated serum.
Individual wells containing 2 x 105 cells
were left untreated or were stimulated with TNF- or
PIM4–6 as indicated. Cells were then incubated
for an additional 6 h before harvesting and lysis in 50 µl in
lysis buffer (Promega, Madison, WI). Luciferase assays were
performed using the Luciferase Assay System (Promega) according to the
manufacturer’s instructions.
Primary macrophage cultures and infection
Bone marrow cells were isolated from femurs and cultivated
(106/ml) for 7 days in DMEM supplemented with
20% horse serum and 30% L929 cell-conditioned medium (as source of
M-CSF, as described in Ref. 29). Three days after washing
and reculturing in the same medium, the cell preparation contained a
homogenous population of macrophages. The bone marrow-derived
macrophages were stimulated with LPS (Escherichia coli,
serotype O111:B4, 10 ng/ml; Sigma-Aldrich, St. Louis, MO) for 4 h
(29). Alternatively the macrophages were infected with
H37Ra at a ratio of 1:1 for 4 h and the supernatants were analyzed
for TNF- by ELISA.
ELISA for cytokines and chemokines
Mice were sacrificed as described above, entire lungs removed, and a piece of equal mass placed in 1 ml of 4°C PBS solution. The contents were transferred into sterile 10-ml perspex mortars and homogenized, centrifuged at 14,000 rpm, supernatants aliquoted, and stored at -80°C until further use. Before use in ELISA, aliquots were thawed and centrifuged at 14,000 rpm, and supernatants were used for cytokine/chemokine quantification by ELISA.
ELISA for TNF-, IFN-, IL-12p40, and monocyte chemoattractant
protein 1 (MCP-1) were performed on BAL supernatants or lung
homogenates (sensitivity <15 pg/ml; R&D Systems, Abingdon, U.K., and
BD PharMingen, San Diego, CA). Quantification of human IL-6 in culture
supernatants was performed using a sandwich ELISA (BD PharMingen) with
a modified protocol using streptavidin-peroxidase (Amersham, Arlington
Heights, IL). Absorbance was measured on a Cytofluor ELISA reader
(PerSeptive Biosystems). Data are expressed as mean ±
SD.
Statistical analysis
Statistical evaluation of differences between the experimental groups was determined with the Mann-Whitney U test and Student’s t tests (level of significance of p < 0.05).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by C3H/HeJ mice in response to endotoxin
and mycobacteria
As previously established, the C3H/HeJ mice with a mutant allele
of the TLR4 gene are protected from endotoxic shock and do
not manifest any of the symptoms which afflict the C3H/HeN mice.
C3H/HeJ mice injected i.p. with 100 µg LPS produce insignificant
levels of TNF- relative to C3H/HeN mice (Fig. 1
A). This hyporesponsiveness
to LPS is confirmed ex vivo by the diminished production of TNF- by
C3H/HeJ-derived whole blood cells in response to LPS stimulation (Fig. 1B).
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levels were
quantified in the supernatant 4 h later. The TNF- production in
macrophages from C3H/HeJ mice was significantly reduced when compared
with that from C3H/HeOUJ control mice (Fig. 1
C). The
residual TNF- from C3H/HeJ macrophages is likely mediated via TLR2
triggering, as macrophages deficient in TLR2 released significantly
reduced TNF- after infection with M.tb. H37Ra (data not
shown). Reduced survival of C3H/HeJ mice infected with mycobacteria
To assess the susceptibility of the TLR4 mutants to an
M.tb. infection, C3H/HeN and C3H/HeJ mice were infected by
aerosol (100 CFU/lung). The mice were weighed routinely and mortality
was recorded. Although one of five C3H/HeN mice succumbed to the
infection during the 250 days, all C3H/HeJ mice displayed a decrease in
body weight from 4 mo onward (data not shown) and died between 160 and
200 days postinfection (Fig. 2). The
experiment was repeated twice, confirming the reduced resistance of the
TLR4 mutant mice. The present data suggest that TLR4 signaling confers
protection against M.tb. infection, which occurs in the late
chronic stage.
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In view of the increased susceptibility of C3H/HeJ mice with a
late lethal outcome, the distribution, growth and elimination of
mycobacteria was investigated. The infective dose in the lungs was
determined to be 
100 CFU/lung at day 1. CFU were not detectable in
the liver and spleen at this time point. C3H/HeN mice appear to control
the M.tb. infection as noted by a slight, but not
significant decline of viable bacilli in the lungs 2 mo postinfection
(Fig. 3A) and insignificant
growth at extrapulmonary sites (Fig. 3, B and C).
In contrast, C3H/HeJ mice have a reduced ability to control
M.tb. infection as noted by a significant increase in lung
CFU 2 mo postinfection, increasing further at 4 mo to 100-fold higher
levels when compared with C3H/HeN mice (Fig. 3A).
Dissemination from the lungs occurred at the same rate initially (at 1
mo) as in the C3H/HeN mice, but the infection progressed more rapidly
in C3H/HeJ mice, reaching 100-fold higher CFU levels in liver and
spleen, respectively (Fig. 3, B and C).
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Chronic inflammatory responses in C3H/HeJ mice infected with mycobacteria
Postmortem analysis revealed nodular changes in the lung, which
were more prominent in C3H/HeJ mice (data not shown), and the lung
weights showed a slight increase in relative weights, which did not
reach significance (data not shown). The relative organ weights are an
indirect indication of generalized inflammatory cell recruitment and
were significantly increased in the spleen of C3H/HeJ mice (data not
shown). The microscopy of the lungs revealed an early inflammatory
response with small granulomas at 28 days postinfection in the lungs of
C3H/HeN mice (Fig. 4A) and
C3H/HeJ mice (Fig. 4B); the C3H/HeJ mice, however, had more
prominent neutrophil infiltration in the lung that can also be seen in
the spleen. At 140 days postinfection, massive chronic inflammation
with abundant mononuclear cell infiltrates and neutrophils prevailed in
the lungs of the C3H/HeJ mice (Fig. 4D), resulting in the
obliteration of most of the airspace, which was less apparent in
C3H/HeN lungs (Fig. 4C). In contrast, the extent of the
granulomatous response was slightly more prevalent in the lung of
control C3H/HeN mice.
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, A and
B), but at 140 days a significant difference was observed.
Although bacilli were noticed primarily in foamy macrophages of the
C3H/HeJ lungs (Fig. 5D), they were virtually undetectable in
the C3H/HeN lungs (Fig. 5C). The expression of iNOS as
assessed by immunostaining was comparable in the lung of C3H/HeJ mice
when compared with C3H/HeN mice (Fig. 5, F and
E).
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A) and C3H/HeJ mice (Fig. 6B). At 140 days postinfection, the hepatic granulomas were
increased in size and numbers in C3H/HeJ mice (Fig. 6D) when
compared with C3H/HeN mice (Fig. 6C). Therefore, the
inflammatory response in the liver was more prominent in C3H/HeJ mice
coincident with the increased bacterial load and extrapulmonary
dissemination.
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To quantify the inflammatory cell recruitment, BAL was performed
on C3H/HeN and C3H/HeJ mice 4 wk after aerogenic M.tb.
infection, and BAL cells retrieved from the lavages were counted and
differentiated to determine the type of cells recruited into the
bronchoalveolar space. Although the total cell counts were comparable
for the two groups, the C3H/HeN mice had a significantly larger
proportion of macrophages than the C3H/HeJ mice 4 wk postinfection
(Fig. 7A), but the lymphocyte
population appeared to be comparable (Fig. 7B).
Interestingly, there were a significant number of neutrophils being
recruited into the alveoli of the C3H/HeJ mice at this time point,
which was not apparent in the C3H/HeN lungs (Fig. 7C). These
data concur with the histological finding of increased lung parenchymal
infiltration by neutrophils in the C3H/HeJ mice (data not shown).
Therefore, pulmonary recruitment of macrophages was reduced, while
recruitment of neutrophils was significantly increased in the TLR4
mutants relative to the C3H/HeN control mice.
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To compare the level of proinflammatory cytokines and chemokines
produced in response to aerosol M.tb. infection, BAL fluid
and lung homogenates were taken at 4 wk postinfection and assessed by
ELISA. The production of IL-12p40 was significantly reduced in the BAL
fluid of the C3H/HeJ mice (Fig. 8A), while no significant
differences were observed for TNF- and IFN- (Fig. 8, B
and C). To further assess the cytokine and chemokine
production in the lung environment, infected lungs were homogenized and
supernatants were quantified. The cytokines IL-12p40 and TNF- were
significantly reduced in the lung homogenates of C3H/HeJ mice when
compared with the C3H/HeN mice (Fig. 9, A and C). The level of IFN- in the C3H/HeJ
lung homogenates was slightly lower than in C3H/HeN homogenates (Fig. 9B). The monocyte and T lymphocyte chemokine MCP-1 was
significantly lower in C3H/HeJ mice than in C3H/HeN mice (Fig. 9D). Therefore, increased susceptibility to M.tb
infection appears to be associated with a reduced induction of
proinflammatory cytokines.
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To define structures that may be associated with the
TLR4-dependent immunity, we have tested the biological activities of a
purified mycobacterial glycolipidic anchor. The main cell wall
glycolipid from Mycobacterium is a lipoarabinomannan (LAM).
Heterogeneity in biological responses to LAM has been linked to the
terminal mannose residues and to mycobacteria strains (30, 31). In contrast, reversed-phase purified PIM is a biosynthetic
precursor of the larger glycolipid LAM and represents a homogeneous
preparation with a phosphatidylinositol moiety with acyl groups
consisting of palmitic and tuberculostearic acids which has been
characterized by mass spectrometry (31). Although LAM and
PIM have been shown to activate via a TLR2-dependent pathway, we
examined the biological activities related to the CD14- and
TLR4-dependent pathways induced by a defined preparation of
PIM4–6 (27). Using the astrocytic U373 cell line, no IL-6
was produced after stimulation with PIM in the absence of serum,
whereas IL-6 production was increased in a dose-dependent manner on the
presence of recombinant CD14 (Fig. 10A). Since U373 are
TLR2-negative and TLR4-positive cells, the phospholipidic anchor PIM
may induce inflammatory cytokine in a CD14-dependent manner through
TLR4. The level of LPS in the PIM4–6 preparation
was found be <1 pg of endotoxin/µg of material by the
Limulus assay, thus excluding LPS contamination. To further
characterize the induction of TLR4 by this lipidic structure, stable
TLR4 and MD-2 Ba-F3 transformants were stimulated with increasing
concentrations of PIM4–6. Whereas no induction
was observed in control cells, PIM4–6 induced
NF-B transactivation in a dose-dependent manner in MD-2- and
TLR4-positive cells (Fig. 10B). As demonstrated previously,
TLR2 transfectants responded to our PIM preparation (data not shown).
These results identify mycobacterial PIM as one of the simplest defined
structures known to have TLR4 agonist activity. Taken together, PIM
structures induce responses through CD14 and TLR4 and behave in a
fashion similar to the amphiphilic molecule LPS derived from
Gram-negative bacteria.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The recognition of mycobacterial components may include several pattern recognition receptors including the mannose receptors, CRs CR1, CR3, and CR4 (15), and TLR-2 and TLR4. The 19-kDa lipoprotein binds and signals through TLR2, resulting in IL-12 secretion; furthermore, mycobacterial killing and apoptosis are primarily TLR2 dependent (9, 16, 17, 18, 19). Whereas it has been suggested that both TLR2 and TLR4 can mediate cellular activation by M.tb.-derived soluble heat-stable and heat-sensitive cell-associated mycobacterial factors distinct from mycobacterial cell wall glycolipid LAM (22), several reports have described a TLR2-dependent pathway induced by LAM and PIM (21, 32), suggesting that the main signaling pathway appears to be TLR2. These results do not differ significantly from other groups as our preparation of PIM4–6 induced signaling via TLR2 (data not shown) as demonstrated by Jones et al. (32), but additionally signaled via TLR4. We show that PIM samples obtained following a purification protocol for glycolipidic molecules and characterized by mass spectrometry induce, indeed, a TLR4-dependent activation (31). This work demonstrates a potential mechanism for the recognition of M.tb. via PIM in a TLR4-dependent manner, which might be partly responsible for the phenotype observed for the TLR4 mutants.
TLR4 signaling in response to M.tb. appears to be required for long-term resistance as the TLR4 mutant, C3H/HeJ mice, succumbed to chronic M.tb. infection, while the control C3H/HeN survived. These data concur with those obtained recently with the vaccine strain M. bovis bacillus Calmette-Guérin (BCG)-infected C3H/HeJ mice, demonstrating that TLR4 has a protective role, although the i.p. infection route chosen appears to be rather nonphysiologic (33).
These data are in contrast to findings by Chackerian et al.
(34), who demonstrated a high susceptibility in the C3H
strain of mice, which is TLR4 independent. The reasons for the
difference may be related to the route of infection, the high infective
dose, and the mycobacterial strain difference. Several studies have
shown an enhanced sensitivity of TLR4-deficient C3H/HeJ mice when
compared with C3H/HeN mice to a broad range of infectious models
(35), including E. coli-induced pyelonephritis
(36), Salmonella typhimurium, and
Leishmania donovani (37).
Subsequently, several groups demonstrated that the dramatic
susceptibility of the C3H/HeJ mice to Salmonella was due to
a reduced recruitment of macrophages and an inherent activation defect
in these cells (38, 39, 40, 41). The increased
susceptibility of C3H/HeJ mice to M.tb. infection may be
likely due to a decreased TLR4-dependent activation of proinflammatory
cytokines and chemokine production induced by mycobacterial structures
such as PIM. We indeed demonstrate reduced pulmonary TNF-, IL12p40,
and MCP-1 production in C3H/HeJ mice. Tsuji et al. (42)
reported that BCG-derived cell wall skeleton induced maturation of
dendritic cells via TNF- in a TLR4-dependent manner. In the
absence of TLR4, BCG cell wall skeleton induced significantly less
TNF- from macrophages, leading to poor maturation of dendritic cells
concomitant with impaired secretion of IL-12p40, IL-6, and surface
expression of CD40, CD80, CD83, and CD86 (42). The
diminished cytokines/chemokine production might be a consequence of
reduced macrophage recruitment and activation, impairing the control of
mycobacterial growth and leading to a significant increase in the
bacterial load of C3H/HeJ mice. Over time, the clinical pattern of
M.tb.-infected TLR4 mutants show an increasing infiltration
of inflammatory cells and increasing number of granuloma in infected
organs associated with a defective clearance of pathogens leading to a
lethal outcome. The more prominent neutrophil infiltration in organs
from C3H/HeJ mice suggests that neutrophil migration is TLR4
independent. This is in agreement with a study by Haziot et al.
(43), which demonstrated dramatically enhanced neutrophil
recruitment in response to either LPS or E. coli in a
TLR4-independent manner. Consistent with our findings, Means et al.
(33) demonstrate that M.tb.-induced TNF-
production by RAW 264.7 macrophages was blocked by E5531, a TLR4
antagonist. Furthermore, they demonstrate normal production of NO in
the presence of the TLR4 antagonist, which suggests a TLR4-independent
pathway for NO induction, which is in agreement with our findings by
iNOS2 immunostaining. The reduced IL-12p40 production might be a
consequence of reduced recruitment of macrophages and activation
thereof. A reduction in IL-12 levels was observed in C3H/HeJ mice in
endodontic inflammatory lesions in response to a mixed anaerobic
infection (44). This was accompanied by a comparable level
of IFN-, which is analogous to our findings of decreased IL-12p40,
but comparable IFN-. Furthermore, nucleosome remodeling at the
IL-12p40 promoter was reported to be dependent on TLR4 signaling
(45).
In conclusion, TLR4 is one of several pattern recognition receptors involved in the recognition of M.tb. including TLR-2, CR1, 3, and 4, and the mannose receptor. Early in vitro studies blocking CR3 indicated that CR3 was the major phagocytic receptor for M.tb (46), but subsequent experiments with CR3-deficient mice revealed no differences compared with wild-type mice with respect to survival and bacterial burden (47) The absence of TLR4 can be partly compensated for by the other pattern recognition receptors, but does indeed lead to an impaired activation of APCs as demonstrated by decreased IL-12p40 production. This impaired APC activation might affect the extent of CD4 T cell priming and ultimately antituberculosis immunity. Thus, immune responses to mycobacteria are at least in part TLR4 dependent. C3H/HeJ mice succumbed to chronic M.tb. infection, and diminished proinflammatory response, macrophage recruitment, and activation are prime factors for reduced host immunity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bernhard Ryffel, Centre National de la Recherche Scientifique, GEM2358, Orléans, France. E-mail address: bryffel{at}cnrs-orleans.fr
3 Abbreviations used in this paper: M.tb., Mycobacterium tuberculosis; CR, complement receptor PIM, phosphatidylinositol mannoside; TLR, Toll-like receptor; iNOS, inducible NO synthase; BAL, bronchoalveolar lavage; MCP-1, monocyte chemoattractant protein 1; LAM, lipoarabinomannan.
Received for publication February 11, 2002. Accepted for publication June 28, 2002.
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 G. Ferwerda, B. J. Kullberg, D. J. de Jong, S. E. Girardin, D. M. L. Langenberg, R. van Crevel, T. H. M. Ottenhoff, J. W. M. Van der Meer, and M. G. Netea Mycobacterium paratuberculosis is recognized by Toll-like receptors and NOD2 J. Leukoc. Biol., October 1, 2007; 82(4): 1011 - 1018. [Abstract] [Full Text] [PDF]
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E. Doz, S. Rose, J. Nigou, M. Gilleron, G. Puzo, F. Erard, B. Ryffel, and V. F. J. Quesniaux Acylation Determines the Toll-like receptor (TLR)-dependent Positive Versus TLR2-, Mannose Receptor-, and SIGNR1-independent Negative Regulation of Pro-inflammatory Cytokines by Mycobacterial Lipomannan J. Biol. Chem., September 7, 2007; 282(36): 26014 - 26025. [Abstract] [Full Text] [PDF]
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L. E. Cole, K. A. Shirey, E. Barry, A. Santiago, P. Rallabhandi, K. L. Elkins, A. C. Puche, S. M. Michalek, and S. N. Vogel Toll-Like Receptor 2-Mediated Signaling Requirements for Francisella tularensis Live Vaccine Strain Infection of Murine Macrophages Infect. Immun., August 1, 2007; 75(8): 4127 - 4137. [Abstract] [Full Text] [PDF]
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C. M. Fremond, D. Togbe, E. Doz, S. Rose, V. Vasseur, I. Maillet, M. Jacobs, B. Ryffel, and V. F. J. Quesniaux IL-1 Receptor-Mediated Signal Is an Essential Component of MyD88-Dependent Innate Response to Mycobacterium tuberculosis Infection J. Immunol., July 15, 2007; 179(2): 1178 - 1189. [Abstract] [Full Text] [PDF]
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 M. Yadav and J. S. Schorey The beta-glucan receptor dectin-1 functions together with TLR2 to mediate macrophage activation by mycobacteria Blood, November 1, 2006; 108(9): 3168 - 3175. [Abstract] [Full Text] [PDF]
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I. Fricke, D. Mitchell, J. Mittelstadt, N. Lehan, H. Heine, T. Goldmann, A. Bohle, and S. Brandau Mycobacteria Induce IFN-{gamma} Production in Human Dendritic Cells via Triggering of TLR2 J. Immunol., May 1, 2006; 176(9): 5173 - 5182. [Abstract] [Full Text] [PDF]
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, p <
0.05). B, Reduced TNF-
and C3H/HeJ
mice by •. Data are expressed as mean values ± SD
(n = 4 mice, 
