Toll-like receptor 4 plays a protective role in pulmonary tuberculosis in mice

Judith Branger1,2, Jaklien C. Leemans1, Sandrine Florquin2, Sebastiaan Weijer1, Peter Speelman1,3 and Tom van der Poll1,3

1 Department of Experimental Internal Medicine, 2 Department of Internal Medicine, Division of Infectious Diseases, Tropical Medicine and AIDS and 3 Department of Pathology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands

Correspondence to: J. Branger, Laboratory of Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, Room G2-105, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: j.branger{at}amc.uva.nl
Transmitting editor: K. Yamamoto


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Toll-like receptors (TLR) play an essential role in the innate recognition of microorganisms by the host. To determine the role of TLR4 in host defense against lung tuberculosis, TLR4 mutant (C3H/HeJ) and wild-type (C3H/HeN) mice were intranasally infected with live Mycobacterium tuberculosis. TLR4 mutant mice were more susceptible to pulmonary tuberculosis, as indicated by a reduced survival and an enhanced mycobacterial outgrowth. Lung infiltrates were more profound in TLR4 mutant mice and contained more activated T cells. Splenocytes of infected TLR4 mutant mice demonstrated a reduced capacity to produce the protective type 1 cytokine IFN-{gamma} upon antigen-specific stimulation, indicating that TLR4 may be involved in the generation of acquired T cell-mediated immunity. These data suggest that TLR4 plays a protective role in host defense against lung infection by M. tuberculosis.

Keywords: inflammation, innate host defense, mice, Mycobacterium tuberculosis, TLR4


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Tuberculosis represents a serious threat to global health. An estimated one-third of the world’s population is infected with Mycobacterium tuberculosis, resulting in ~8 million new cases of tuberculosis and 2 million deaths each year (1). An adequate cell-mediated immune response plays a key role in resistance against tuberculosis (2). In murine models of mycobacterial infection, type 1 cytokines, in particular IFN-{gamma}, are essential for protective immunity (3,4). The development of a protective Th1 response during tuberculosis is driven by IL-12, which is produced by macrophages upon phagocytosis of M. tuberculosis (5) and, like IFN-{gamma}, plays a pivotal role in the control of mycobacterial infection (6).

Innate recognition of mycobacterial products is the first step in a chain of events that results in an effective host defense against infection with M. tuberculosis. The innate immune system serves to mount a rapid protective response in the early phase of an infection and also instructs the (later) adaptive T cell-mediated immune response (7). Until recently it was unclear how M. tuberculosis activates macrophages to initiate an innate immune response. The discovery of the Toll-like receptor (TLR) family has provided important insight into the recognition of various microbial pathogens by the host (8,9). Two members of the TLR family have been demonstrated to mediate M. tuberculosis-induced intracellular signaling in vitro, i.e. TLR2 and TLR4. Indeed, experiments with Chinese hamster ovary cells and murine macrophages transfected with human TLR2 and/or TLR4 have established that viable M. tuberculosis bacilli activate these cells via both TLR2 and TLR4 (10,11). In addition, thioglycollate-elicited peritoneal macrophages from TLR4-deficient mice produced less tumor necrosis factor-{alpha} upon stimulation with whole-cell lysates from M. tuberculosis than normal wild-type macrophages, also indicating that this microorganism activates cells via TLR4 and another receptor, presumably TLR2 (12). TLR2 has emerged as the signaling receptor for purified mycobacterial products, including arabinose-capped lipoarabinomannan (AraLAM), culture supernatant soluble tuberculosis factor, mycolylarabinogalactan–peptidoglycan complex, the 19-kDa antigen and total lipids (10,11,1318).

The objective of the present study was to determine the role of TLR4 in host defense against lung tuberculosis. While our experiments were in progress, two investigations on the role of TLR4 in pulmonary tuberculosis were published (19,20). One study reported an increased susceptibility to M. tuberculosis infection in C3H/HeJ mice, which have a non-functional TLR4, as reflected by an enhanced mycobacterial outgrowth and an increased mortality (19); in contrast, however, in the second study no differences in mycobacterial outgrowth or survival between C3H/HeJ and normal C3H/HeN mice were observed (20). Intraperitoneal administration of M. bovis BCG resulted in an increased mortality of C3H/HeJ mice relative to mice with a normal TLR4 gene, as measured 3 days after the infection (11). After i.v. administration of high-dose M. tuberculosis, a small survival disadvantage of C3H/HeJ mice relative to wild-type mice was found, although the difference between the two mouse strains was statistically not significant (21). In the current investigation, we induced lung tuberculosis in wild-type C3H/HeN and TLR4-deficient C3H/HeJ mice by intranasal inoculation with M. tuberculosis (H37Rv), and monitored the course of the infection and host responses.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Pathogen-free 8- to 9-week-old sex-matched C3H/HeJ (TLR4 mutant) and C3H/HeN (wild-type) mice were purchased from Charles River (Someren, The Netherlands), and were maintained in Biosafety Level 3 facilities. C3H/HeJ mice have been demonstrated to have a missense mutation in the third exon of TLR4 predicted to result in a Pro712 -> His substitution, yielding a non-functional TLR4 (22,23). All experiments were approved by the Animal Care and Use Committee of the University of Amsterdam (Amsterdam, The Netherlands).

Experimental infection
Pulmonary tuberculosis was induced exactly as described previously (2426). In brief, a virulent laboratory strain of M. tuberculosis H37Rv was grown in liquid Dubos medium containing 0.01% Tween 80 for 4 days. A replicate culture was incubated at 37°C, harvested at mid-logarithmic phase and stored in aliquots at –80°C. For each experiment, a vial was thawed and washed 2 times with sterile 0.9% NaCl. Mice were lightly anesthetized by inhalation with isoflurane (Upjohn, Ede, The Netherlands) and intranasally inoculated with 50 µl of mycobacterial suspension. The intranasal route of infection has been described previously by our and other laboratories, and results in a reproducible infection of the lung with subsequent dissemination to liver and spleen (24,25,27,28). Bacterial counts recovered from lungs 1 day post-infection were shown previously to be similar to the number of bacteria in the inoculum (25). Exact inoculum strength was determined by plating 10-fold dilutions of the suspension on 7H11 Middlebrook agar plates immediately after inoculation. Mice were inoculated with 105 c.f.u. M. tuberculosis. After 2 and 5 weeks, groups of 10–13 mice per time point were anesthetized by FFM (fentanyl citrate 0.079 mg/ml, fluanisone 2.5 mg/ml and midazolam 1.25 mg/ml in H2O) and sacrificed by bleeding out the vena cava inferior. Lungs, spleen and one lobus of the liver were removed aseptically. For c.f.u. counts and cytokine measurements, lung and liver tissue were homogenized with a tissue homogenizer (Biospec Products, Bartlesville, OK) in 5 volumes of sterile 0.9% NaCl and 10-fold serial dilutions were plated on Middlebrook 7H11 agar plates to determine bacterial loads. Colonies were counted after 21 days of incubation at 37°C. The c.f.u. are provided as total number in the lungs or as total per gram liver tissue. For cytokine measurements, lung homogenates were diluted 1:2 in lysis buffer (150 mM NaCl, 15 mM Tris, 1 mM MgCl.H2O, 1 mM CaCl2, 1% Triton X-100, and 100 µg/ml pepstatin A, leupeptin and aprotinin, pH 7.4) and incubated at 4°C for 30 min. Homogenates were centrifuged at 1500 g for 15 min, after which the supernatants were sterilized using a 0.22-µm filter (Corning, Corning, NY) and stored at –20°C until further use.

Histologic examination
Lungs for histologic examination were harvested at 2 and 5 weeks after infection, fixed in 4% formaldehyde, and then embedded in paraffin. Hematoxylin & eosin and acid-fast staining was performed on 4-µm thick sections, and analyzed for the total area of inflammation and granuloma formation by a pathologist who was blinded for groups.

Flow cytometry
Pulmonary cell suspensions were obtained using an automated disaggregation device (Medimachine System; Dako, Glostrup, Denmark) and processed as described previously (24,25). Total leukocytes in left lung cell suspensions were counted by using a hemacytometer and Türk’s solution (Merck, Darmstadt, Germany). The number of macrophages, granulocytes and lymphocytes were calculated from these totals, using cytospin preparations stained with modified Giemsa stain (Diff-Quick; Baxter, McGraw Perk, IL). For FACS analysis, cells were brought to a concentration of 4 x 106 cells/ml FACS buffer (PBS supplement with 0.5% BSA, 0.01% NaN3 and 100 mM EDTA). Immunostaining for cell-surface molecules was performed for 30 min at 4°C using directly labeled antibodies against CD3 (anti-CD3–phycoerythrin), CD4 (anti-CD4–CyChrome), CD8 (anti-CD8–FITC, anti-CD8–PerCP), CD25 (anti-CD25–FITC) and CD69 (anti-CD69–FITC). All antibodies were used in concentrations recommended by the manufacturer (PharMingen, San Diego, CA). To correct for non-specific staining, an appropriate control antibody (rat IgG2; PharMingen) was used. The number of positive cells was obtained by setting a quadrant marker for non-specific staining. FACS analysis was performed using CellQuest (Becton Dickinson Immunocytometry Systems, San Jose, CA). The results are expressed as the percentage of CD4+, CD8+, CD25+ and CD69+ T cells within the CD3+ population in the left lung.

Splenocyte stimulation
Single-cell suspensions were obtained by crushing spleens through a 40-µm cell strainer (Becton Dickinson, Franklin Lakes, NJ). Erythrocytes were lysed with ice-cold isotonic NH4Cl solution (155 mM NH4Cl, 10 mM KHCO3 and 100 mM EDTA, pH 7.4) and the remaining cells were washed twice. Splenocytes were resuspended in RPMI medium (Biowhittaker, Walkersville, MD) containing 10% FCS and 1% antibiotic/antimycotic (Life Technologies, Grand Island, NY), seeded in 96-well round-bottom culture plates at a cell density of 1 x 106 cells in triplicate and stimulated with 10 µg/ml tuberculin-purified protein derivative (PPD; Statens Seruminstitut, Copenhagen, Denmark). In a separate experiment, round-bottom plates were coated overnight with anti-CD3 antibody (clone 145.2c11) and washed twice with sterile PBS. Aliquots of 1 x 106 cells (in triplicate) were added to each well and diluted with RPMI containing anti-CD28 antibody (1 µg/ml; PharMingen). Supernatants of both experiments were harvested after a 48-h incubation period at 37°C in 5% CO2. Cytokine levels were analyzed by ELISA.

Cytokine measurements
IFN-{gamma} and IL-4 were measured in lung homogenates and spleen cell supernatants by specific ELISA according to the manufacturer’s instructions (detection limits for both assays 62.5 pg/ml; R & D Systems, Minneapolis, MN).

Delayed-type hypersensitivity (DTH) response to PPD
To measure DTH responses, the swelling responses of the dorsal side of feet in mice were examined. In brief, TLR4 mutant and wild-type mice were immunized intradermally at the base of the tail with 0.1 mg of heat-killed M. tuberculosis H37Ra (Difco, Detroit, MI) in 0.1 ml of mineral oil (Sigma, St Louis, MO). Fourteen days after immunization, mice were challenged with 40 µg PPD in 50 µl saline into the dorsal side of the left hind foot and in the right with 50 µl saline alone. Measurements of the hind feet were performed before and 24 h after PPD injection with a Mitutoyo model 7326 engineer’s micrometer (Mitutoyo MTI, Aurora, IL). The swelling was calculated as the difference in thickness of the hind feet between the 0- and 24-h measurements. Specific DTH reactivity was calculated as the difference between the swelling of the PPD-injected feet and the swelling of the saline-injected feet.

Statistical analysis
All data are expressed as mean ± SEM. Differences between groups were analyzed by Mann–Whitney U-test. For comparison of survival curves Kaplan–Meier analysis with a log-rank test was used. P < 0.05 was considered to represent a statistically significant difference.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Survival
To investigate the role of TLR4 in the outcome of tuberculosis, mice were intranasally inoculated with mycobacteria and were monitored in two separate, consecutive experiments. In the first study, mice were observed for 15 weeks. As shown in Fig. 1(A), no mortality was seen in the wild-type group. In contrast, seven of 12 (58%) of the TLR4 mutant mice died (P = 0.002). In the second experiment, in retrospect, a higher mycobacterial inoculum was given (5 x 105 c.f.u.) and mice were followed for a longer period of time. This experiment showed a trend toward enhanced mortality of TLR4 mutant mice: mortality after 23 weeks in the wild-type group was 10 of 13 (77%), while all of the TLR4 mutant mice had died (non-significant; Fig. 1B).



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Fig. 1. Survival of TLR4 mutant and wild-type mice in two independent experiments after intranasal infection with (A) 105 and (B) 5 x 105 c.f.u. M. tuberculosis. N = 12–13 mice per group in each experiment. *P value indicates difference between groups. NS = non-significant.

 
Mycobacterial outgrowth
Because of the differences in survival between TLR4 mutant and wild-type mice, we determined whether differences in mycobacterial load existed in earlier phases of the infection. At 2 and 5 weeks post-infection, outgrowth of M. tuberculosis in lungs of both mouse strains was compared. At the 2 weeks time-points, lungs of TLR4 mutant mice contained slightly but significantly more viable mycobacteria compared with lungs of wild-type mice (P = 0.004). At 5 weeks post-infection, the difference between mycobacterial numbers recovered from lungs of TLR4 mutant and wild-type mice had grown showing a 2.8-fold increase of mycobacteria in TLR4 mutant mice in comparison with wild-type mice (P = 0.003; Fig. 2A). Since M. tuberculosis is known to disseminate in mice, mycobacterial numbers were also determined in a distant organ—the liver. The mycobacterial load in liver tissue was similar in TLR4 mutant and wild-type mice 2 weeks after M. tuberculosis inoculation. At 5 weeks post-infection, however, the number of c.f.u. in liver tissue of TLR4 mutant mice was 3.2-fold higher compared to the bacterial load in liver tissue of wild-type mice (P = 0.05; Fig. 2B). In a subsequent separate experiment we tried to determine mycobacterial outgrowth at a time point >5 weeks. However, this experiment was confounded due to the fact that in week 6 post-infection, five of 13 (38%) of the TLR4 mutant mice died, while none of the wild-type mice died (P = 0.02), again suggesting that TLR4 mutant are more susceptible to M. tuberculosis infection.



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Fig. 2. Growth of M. tuberculosis in (A) lungs and (B) liver of TLR4 mutant mice compared to wild-type mice at 2 and 5 weeks post-infection. Data are mean and SEM from 10–13 mice per group. *P < 0.05 versus wild-type mice.

 
Histology
Histopathological analysis of the lungs 2 weeks after infection showed a slight interstitial infiltrate consisting of macrophages, lymphocytes and occasional neutrophils adjacent to small airways and vessels in wild-type mice (Fig. 3A). At the same time, the lungs of TLR4 mutant mice showed larger areas of inflammation (Fig. 3B). At 5 weeks post-infection, the inflammatory infiltrates became more diffuse and intense, and involved between 50 and 60% of the analyzed lung surface in wild-type mice (Fig. 3C). At this time point, the inflammation was also more pronounced in TLR4 mutant mice with an involvement of 70–80% of the lung surface (Fig. 3D). Acid-fast staining of lung sections showed that cellular infiltrates in TLR4 mutant lungs contained more infected cells and more bacteria per infected cell than wild-type mouse lungs. All bacteria were cell-associated in both strains of mice (data not shown).



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Fig. 3. Representative slide of wild-type lung tissue 2 weeks post-infection with M. tuberculosis showing a slight inflammatory infiltrate around small vessels and bronchi (hematoxylin & eosin staining, x25) (A). TLR4 mutant lung tissue 2 weeks post-infection showing more diffuse inflammation with in this case involvement of the pleura (hematoxylin & eosin staining, x25) (B). Five weeks after infection, wild-type mouse lungs displayed more intense and diffuse inflammatory infiltrates with small ‘granulomas’ (hematoxylin & eosin staining, x25) (C). At the same time point, TLR4 mutant lungs showed confluent inflammatory infiltrates (D).

 
Cellular composition of lung infiltrates
To obtain more insight into the cellular composition of the pulmonary infiltrates, we determined whole lung leukocyte counts and differentials, and further analyzed lymphocyte subsets and activation status by flow cytometry (Table 1). In accordance with the histopathology, the lungs of TLR4 mutant mice contained more leukocytes than those of wild-type mice, significantly so at 2 weeks post-infection. The proportional contribution of macrophages, lymphocytes and granulocytes was similar in TLR4 mutant and wild-type mice. The increase in lymphocyte numbers in lungs of TLR4 mutant mice was proportionally distributed over the CD3/CD4+ and the CD3/CD8+ populations compared to wild-type mice. In addition, the numbers of activated (CD25+ or CD69+) T cells were higher in lungs of TLR4 mutant mice, especially at 5 weeks post-infection in the CD3/CD8+ population.


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Table 1. Cell subsets in the left lung in TLR4 mutant and wild-type mice during pulmonary tuberculosis
 
Lung IFN-{gamma} and IL-4 levels
Th1 cells are essential for early host defense in M. tuberculosis infection (2). We, therefore, investigated whether the deteriorated outcome of TLR4 mutant mice was associated with a change in the Th1 and Th2 cytokine profile in the pulmonary compartment in early infection. Th1 (IFN-{gamma}) and Th2 (IL-4) cytokines were measured in lung homogenates. As shown in Fig. 4(A), IFN-{gamma} levels were similar at 2 weeks post-infection. After 5 weeks, IFN-{gamma} was elevated in TLR4 mutant mice compared to wild-type mice (P = 0.005). This finding paralleled the mycobacterial load in the lungs. In contrast, the Th2 cytokine IL-4 was produced in lower concentrations in TLR4 mutant mouse lungs in comparison with wild-type mice at 5 weeks (P = 0.017), while at 2 weeks, no significant difference in IL-4 concentrations was observed (Fig. 4B).



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Fig. 4. IFN-{gamma} and IL-4 concentrations in lungs of TLR4 mutant and wild-type mice. N = 12–13 mice per group. Data are expressed as mean and SEM. *P < 0.05 versus wild-type mice.

 
Th1 response of ex vivo stimulated spleen cells
To obtain more insight in mechanisms contributing to the decreased survival of TLR4 mutant mice and the enhanced mycobacterial outgrowth in TLR4 mutant mouse lungs in comparison with wild-type mice, the ability of splenocytes, harvested 5 weeks post-infection from TLR4 mutant and wild-type mice, to produce a Th1 response after M. tuberculosis infection was tested. IFN-{gamma} production by splenocytes upon ex vivo stimulation with either the T cell stimulator anti-CD3/anti-CD28 or PPD was reduced 3.3 and 4.4 times respectively in TLR4 mutant mice compared with wild-type mice (P = 0.025 and P = 0.004 respectively, Fig. 5). The Th2 cytokine IL-4 was not detectable in supernatants of anti-CD3/anti-CD28- or PPD-stimulated splenocytes from either mouse strain (data not shown).



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Fig. 5. Splenocytes from infected TLR4 mutant mice produce less IFN-{gamma} upon ex vivo stimulation by CD3/CD28 and PPD than splenocytes from wild-type mice. Splenocytes were harvested 5 weeks after intranasal inoculation with M. tuberculosis and stimulated for 48 h. Data are mean and SEM of six mice per group. *P < 0.05 versus wild-type mice.

 
DTH response to PPD
The recruitment of T cells and phagocytes into inflamed areas is critical for the development of a DTH response. To study the role of TLR4 in Th1-mediated immune responses more extensively we determined DTH reactions in TLR4 mutant and wild-type mice. Mice were immunized and subsequently challenged with PPD in the hind feet, after which local swelling was measured. Both TLR4 mutant and wild-type mice showed significant swelling following challenge with PPD in comparison with saline controls (data not shown). As shown in Fig. 6, swelling responses in TLR4 mutant mice were significantly reduced compared with wild-type mice (P = 0.001). This finding parallels the results obtained from ex vivo stimulated splenocytes and suggests that TLR4 mutant mice have an impaired T cell-mediated immunity against M. tuberculosis.



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Fig. 6. DTH response in hind feet of mice. TLR4 mutant and wild-type mice were immunized with heat-killed M. tuberculosis, and challenged with PPD in the dorsal side of the left hind foot and with saline in the right one. Swelling was measured before and 24 h after antigen swelling, and calculated as described in Methods. Data are mean and SEM of eight mice per group. *P < 0.05 versus wild-type mice.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TLR play a pivotal role in the induction of an innate immune response to various infectious agents. In vitro studies have implicated TLR2 and TLR4 in the innate recognition of M. tuberculosis. We here demonstrate that TLR4 plays a protective role in host defense against pulmonary tuberculosis in vivo, as reflected by an increased mortality and mycobacterial load in the lungs of mice with a non-functional TLR4.

While our experiments were in progress, two studies examining the role of TLR4 in pulmonary tuberculosis were published. Abel et al. (19) found an increased susceptibility to M. tuberculosis infection of TLR4 mutant mice in terms of a reduced survival and an impaired mycobacterial clearance. Reiling et al. (20), however, using a similar inoculum, showed no difference in survival or in pulmonary mycobacterial load in TLR4 mutant and wild-type mice. In two other publications, the role of TLR4 in mycobacterial infection in vivo was examined in slightly different models. One study, in which M. bovis BCG was injected i.p. into TLR4 mutant and wild-type mice, reported an enhanced mortality (nine of 20) of TLR4 mutant mice 3 days after the infection when compared with wild-type mice (one of 20) (11). The second study found a modest, but statistically non-significant, increase in mortality of TLR4 mutant mice after i.v. injection of high-dose M. tuberculosis (21). Our present data are largely in line with the earlier findings by Abel et al. (19). Indeed, in two independent experiments, with follow-ups of 15 and 6 weeks respectively, TLR4 mutant mice displayed an enhanced mortality when compared with wild-type mice. Although in a third experiment, with a slightly higher inoculum and a follow-up of 23 weeks, the difference between TLR4 mutant and wild-type mice was not significant, a trend in the same direction was seen. Notably, these experiments were conducted several months apart, and besides the exact inoculum size, the use of different ‘batches’ of mice from different shipments may have contributed to the variation in absolute mortality rates. We would like to emphasize, however, that all comparisons between wild-type and TLR4 mutant mice were done in experiments in which all mice were inoculated at the same time with exactly the same inoculum. Together these data suggest that TLR4 plays a role in protection of the host against lethality due to mycobacterial infection.

TLR4 mutant mice demonstrated an increased outgrowth of M. tuberculosis at 2 and 5 weeks after the infection. Although the difference with wild-type mice was highly statistically significant (P = 0.003), the absolute difference was modest. Indeed, TLR4 mutant mice on average had 3 times more mycobacteria in their lungs than wild-type mice at the latter time point. For comparison, mice that are devoid of IFN-{gamma} or IL-12 activity were reported to have several logs more c.f.u. during mycobacterial infection than their respective wild-type mice (3,4,6). In accordance, IFN-{gamma}- and IL-12-deficient mice display a profoundly reduced survival in models of mycobacterial infection (3,4,6), whereas here we found that the consequence of TLR4 deficiency on survival during lung tuberculosis is more modest.

TLR4 mutant mice tended to have a stronger inflammatory response in their lungs than wild-type mice, which was accompanied by higher IFN-{gamma} concentrations in lung homogenates at 5 weeks post-infection. The higher mycobacterial load in TLR4 mutant may have contributed to these findings. Also, the number of activated CD8+ T cells was higher in TLR4 mutant mice at this time point. Thus, a deficient local production of the protective cytokine IFN-{gamma} or a deficient recruitment of activated CD8+ T cells cannot explain the enhanced mycobacterial outgrowth in TLR4 mutant animals. In any case, the histopathology of the lungs and the evaluation of leukocyte counts and differentials in whole lung cell suspensions clearly establish that TLR4 is not essential for mounting an inflammatory response to pulmonary infection with M. tuberculosis. Furthermore, TLR4 is not indispensable for the recruitment of (activated) CD4+ or CD8+ T cells to the site of infection during lung tuberculosis.

In contrast to the modestly elevated lung IFN-{gamma} levels in TLR4 mutant mice, the capacity of splenocytes to produce this prototypic type 1 cytokine upon non-specific stimulation with anti-CD3/CD28 or antigen-specific stimulation with PPD was reduced in these animals. Moreover, the DTH response in TLR4 mutant mice was impaired. The reduced IFN-{gamma}-releasing capacity of splenocytes upon stimulation with a standardized (recall) antigen and the diminished DTH response suggest that TLR4 may be involved in the generation of acquired T cell-mediated immunity to M. tuberculosis. Interestingly, a recent study also suggested a role for TLR signaling in the generation of an antigen-specific type 1 T cell response. Indeed, mice with deficient TLR signaling were found to be incapable of mounting an ovalbumin-specific Th1 response after immunization with ovalbumin in the footpad (29). Additional research is warranted to further explore the role of TLR in acquired T cell-mediated immunity.

In our experiments, TLR4 mutant C3H/HeJ and wild-type C3H/HeN mice were used. C3H/HeJ mice have long been known to be hyporesponsive to lipopolysaccharide, which is related to a point mutation in the TLR4 gene resulting in a Pro712 -> His substitution (22,23), yielding a non-functional TLR4. Several reports compared the lipopolysaccharide responsiveness of C3H/HeJ mice and TLR4 knockout mice in in vitro experiments, showing a similar phenotype (23,30). However, it cannot be excluded that C3H/HeJ mice have other differences in genetic background (in addition to the mutation in TLR4) that have an impact on host defense against infections compared to the wild-type C3H/HeN mice. Studies using TLR4 knockout mice are warranted to definitively establish this. Likely, extensive backcrossing of such mice is required, since the commonly used 129/SvJ background to generate knockout mice is associated with an increased susceptibility for M. tuberculosis infection (31).

If TLR4 only modestly influences the innate host response to M. tuberculosis, how then is this pathogen recognized by the immune system? All TLR known to date share a common signaling component, myeloid differentiation protein 88 (MyD88) (8,9). In the absence of MyD88 M. tuberculosis cannot activate macrophages in vitro, strongly suggesting that signaling via TLR is of major importance in the first step of immune activation by this microorganism (32). Means et al. have documented that viable M. tuberculosis bacilli are recognized by both TLR2 and TLR4 (11,13). In line with these observations, Takeuchi et al. reported that tumor necrosis factor-{alpha} production by TLR4-deficient macrophages upon stimulation with M. tuberculosis lysates was reduced, but not abolished, suggesting that besides TLR4 another receptor is involved in this response (12). Purified components and products of mycobacteria, such as AraLAM, culture supernatant soluble tuberculosis factor, mycolylarabinogalactan–peptidoglycan complex, the 19-kDa antigen and total lipids, invariably utilize TLR2 as signaling receptor (10,11,1318). Using transfected human dermal microvessel endothelial cells, Bulut et al. demonstrated that heterodimerization of TLR2 and TLR6 provides optimal signaling conditions for purified culture supernatant soluble tuberculosis factor (15). Together, these data suggest that M. tuberculosis is recognized by the host by a repertoire of different TLR, among which TLR2, TLR4, TLR6 and possibly others. Indeed, very recently, TLR2-deficient mice were reported to be more susceptible to lung tuberculosis (20). Investigations with mice that are deficient for more TLR are required to resolve the individual and combined roles of different TLR in the innate immune response to M. tuberculosis.


    Acknowledgements
 
This work was supported by grants from The Netherlands Organization of Scientific Research (NWO) to J. C. L. and S. W.


    Abbreviations
 
AraLAM—arabinose-capped lipoarabinomannan

c.f.u.—colony forming units

DTH—delayed-type hypersensitivity

MyD88—myeloid differentiation protein 88

PPD—purified protein derivative

TLR—Toll-like receptor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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