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Published ahead of print on September 24, 2004, doi:10.1164/rccm.200404-525OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 1367-1374, (2004)
© 2004 American Thoracic Society
doi: 10.1164/rccm.200404-525OC

Original Article

Non–Mannose-capped Lipoarabinomannan Induces Lung Inflammation via Toll-like Receptor 2

Catharina W. Wieland, Sylvia Knapp, Sandrine Florquin, Alex F. de Vos, Kiyoshi Takeda, Shizuo Akira, Douglas T. Golenbock, Annelies Verbon and Tom van der Poll

Laboratory of Experimental Internal Medicine, Department of Pathology, Department of Internal Medicine, Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; and Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts

Correspondence and requests for reprints should be addressed to Catharina W. Wieland, M.Sc., Laboratory of Experimental Internal Medicine, G2-132, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: c.wieland{at}amc.uva.nl


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Non–mannose-capped lipoarabinomannan (AraLAM) is part of the cell membrane of atypical mycobacteria. To determine the capacity of AraLAM to induce lung inflammation in vivo and to determine the signaling receptors involved herein, wild-type (WT) mice, lipopolysaccharide binding protein knockout mice, CD14-deficient (CD14 KO) mice, Toll-like receptor (TLR) 4 mutant mice, or TLR2 KO mice were intranasally inoculated with purified AraLAM. AraLAM induced high lung levels of tumor necrosis factor, interleukin-1ß, interleukin-6, and cytokine-induced neutrophil chemoattractant (KC) and an influx of neutrophils into the pulmonary compartment of WT mice. Lipopolysaccharide binding protein knockout, CD14 KO, and TLR4 mutant mice displayed similar inflammatory responses as WT mice, whereas in TLR2 KO mice, AraLAM-induced lung inflammation was strongly diminished. In addition, TLR2 KO mice, but not CD14 KO or TLR4 mutant mice, displayed a delayed clearance of pulmonary infection with the atypical AraLAM expressing Mycobacterium smegmatis. These data indicate that TLR2 is the signaling receptor for purified AraLAM in the lung in vivo and that this receptor contributes to an effective clearance of M. smegmatis from the pulmonary compartment.

Key Words: bacterial antigens • knockout • mycobacterium • pulmonary

Toll-like receptors (TLRs) are of critical importance for the initiation of an efficient innate immune response (1–4). TLRs recognize pathogen-associated molecular patterns, which are conserved motifs expressed by microorganisms but not by higher eukaryotes. One of the most prominent pathogen-associated molecular patterns connected with mycobacteria is the glycolipid lipoarabinomannan (LAM). LAM isolated from slowly growing and virulent strains like Mycobacterium tuberculosis or Mycobacterium leprae are capped with mannose to varying degrees (ManLAM) (5, 6). In contrast, the arabinan termini from LAM of rapidly growing, atypical, and avirulent mycobacterial species such as Mycobacterium smegmatis are uncapped (AraLAM) (7). This difference in biochemical structure of ManLAM and AraLAM accounts for the different inflammatory capacities of the purified components in vitro (8). Indeed, AraLAM but not ManLAM is a potent inducer of tumor necrosis factor (TNF) expression in human and murine macrophages (8, 9).

In vitro experiments with transfected cell lines have established that AraLAM signaling is dependent on a functional TLR2 (9–11). Conversely, inhibition of endogenous TLR2 by overexpression of dominant-negative TLR2 protein rendered macrophages unresponsive to AraLAM (12). CD14 likely functions as the ligand binding component of the AraLAM receptor (13), whereas lipopolysaccharide binding protein (LBP) has been found to facilitate the transfer of AraLAM to CD14, thereby enhancing the responsiveness of cells to AraLAM in vitro (10, 13).

The initial challenge of the innate immune system during mycobacterial infection is the first interaction with intact bacteria. Whole M. tuberculosis stimulates cells through TLR2 as well as TLR4 (9, 11); possible TLR2 ligands expressed by M. tuberculosis include the 19-kD lipoprotein, soluble tuberculosis factor, and phosphatidylinositolmannan, whereas TLR4 ligands still await to be identified (14–19). Consistent with the fact that ManLAM does not activate TLRs, TLR activation by whole M. tuberculosis occurs independent of LAM (11). In line with these in vitro findings, chronically infected TLR2- or TLR4-deficient mice displayed a reduced clearance of mycobacteria from their lungs and a diminished survival during M. tuberculosis infection in vivo (19–21). As observed for LAM, the TLR response to whole bacilli varies with the mycobacterial species. Indeed, M. avium activates cells via TLR2 but not TLR4 (22).

Knowledge of the inflammatory effects of AraLAM in vivo and the role of TLRs herein is not available. Therefore, in this study, we sought to determine (1) whether AraLAM is capable of inducing an inflammatory response in the mouse lung in vivo (2), the contribution of LBP, CD14, TLR2, and TLR4 in these effects, and (3) the role of CD14, TLR2, and TLR4 in the innate immune response to an AraLAM expressing fast-growing mycobacterium, M. smegmatis. Some of these results were presented in the form of an abstract (23).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
LBP knockout (KO) mice (24) backcrossed 11 times to a C57Bl/6 background and TLR2 KO mice (25) backcrossed 6 times to a C57Bl/6 background were generated as described previously. CD14 KO mice backcrossed six times to C57Bl/6 background were obtained from the Jackson Laboratories (Bar Harbor, ME). Wild-type (WT) C57Bl/6 mice were purchased from Harlan Sprague Dawley Inc. (Horst, The Netherlands). C3H/HeJ and C3H/HeN mice were purchased from Charles Rivers (Someren, The Netherlands). The Animal Care and Use of Committee of the University of Amsterdam approved all experiments. Age- and sex-matched animals were used in each experiment.

Reagents
AraLAM was isolated from rapidly growing M. smegmatis and obtained from Dr. J. T. Belisle from the Colorado State University (Fort Collins, CO; under National Institutes of Health Contract NO1-AI-75320). Endotoxin contamination was 0.373 ng/mg of AraLAM as determined by the Limulus Amebocyte lysate assay.

Experimental Design
AraLAM was administered intranasally according to previously described methods (26, 27). In first experiments in WT mice, AraLAM was administered at 0, 0.1, 1, or 10 µg; after 6 hours, mice were anesthetized by an intraperitoneal injection of Hypnorm (Janssen Pharmaceutica, Beerse, Belgium; active ingredients fentanyl citrate and fluanisone) and midazolam (Roche, Mijdrecht, The Netherlands) and were killed by bleeding from the vena cava inferior. Consecutively, WT mice were inoculated intranasally with AraLAM (10 µg) and killed after 3, 6, 12, or 24 hours. In additional experiments, mice were inoculated intranasally with 10 µg of AraLAM and killed after 6 hours.

Bronchoalveolar Lavage
Bronchoalveolar lavage (BAL) and leukocyte differentiation were performed as described previously (26, 28) (see the online supplement for details).

Myeloperoxidase Activity Assay
Myeloperoxidase activity was measured in homogenates as described previously (28) (see the online supplement for details).

Cytokine Measurements
Lungs were homogenized for cytokine measurements exactly as described previously (26, 28, 29). Cytokines and chemokines were measured using specific enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. The detection limits were 8 pg/ml for interleukin (IL)-1ß, 31 pg/ml for IL-6 and TNF, 37 pg/ml for cytokine-induced neutrophil chemoattractant (KC), and 187 pg/ml for macrophage inflammatory protein-2.

Histologic Examination
Unlavaged lungs were removed and fixed in 10% buffered formalin in phosphate-buffered saline (PBS) for 24 hours and embedded in paraffin. Hematoxylin and eosin–stained slides were coded and semiquantitatively scored for inflammatory parameters by a pathologist who was blinded for experimental groups (30) (for details, see the online supplement).

Model of Infection
A laboratory strain of M. smegmatis (ATCC 14468, Rockville, MD) was grown in liquid Dubos medium containing 0.01% Tween 80. Mice were infected intranasally with 2.5 x 105 cfu of M. smegmatis as determined by viable counts on 7H11 Middlebrook agar plates. At 6 hours and 1, 3, and 10 days after infection, mice were killed, and lungs were removed and homogenized. Tenfold serial dilutions of homogenates were plated on 7H11 Middlebrook agar plates and after 4 days cfu were counted. In a different set of infection experiments, mice were intranasally infected with 104 or 106 cfu of M. smegmatis and killed after 10 days.

Statistical Analysis
Data are expressed as mean ± SEM. Serial data were analyzed by Kruskall-Wallis test with Mann-Whitney U as post-test using GraphPad Prism version 3.00 (GraphPad Software, San Diego, CA). Two sample comparisons were done by Mann-Whitney U test. A p value of less than 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
AraLAM Induces Lung Inflammation after Intranasal Inoculation
To determine whether non–mannose-capped LAM (AraLAM) is capable of inducing an inflammatory response in the lung in vivo and to establish a dose–response relationship, we intranasally inoculated WT mice with three different doses of AraLAM (0.1, 1, 10 µg); control mice received PBS intranasally. Inoculation with AraLAM was associated with a dose-dependent increase in TNF, IL-1ß, and KC levels in lung homogenates (Figure 1) as well as BAL fluid (data not shown) 6 hours after inoculation. Expression of the chemokine macrophage inflammatory protein-2 was not influenced by inoculation of AraLAM (data not shown). Compared with control mice, the total cell count was significantly increased in BAL fluid of mice inoculated with 10 µg of AraLAM (data not shown). The increase in myeloperoxidase activity (Figure 1) in lung homogenate as well as differential cell counts (data not shown) revealed that this increase was mainly due to the influx of neutrophils into the pulmonary compartment. Based on these results, additional experiments were performed with 10 µg of AraLAM. To characterize further the proinflammatory response induced by intranasal inoculation of AraLAM, we evaluated the kinetics of cytokine production and cell influx in the lungs of WT mice (Tables 1 and 2). Increased levels of cytokines TNF, IL-1ß, and IL-6 were observed in lung homogenates already at 3 hours after intranasal administration of AraLAM. Although the expression of chemokine KC increased in a similar way, no major changes in lung-associated macrophage inflammatory protein-2 levels could be detected. Total cell counts in BAL fluid consistently increased during the first 24 hours after intranasal inoculation, peaking after 6 hours. Differential counts demonstrated that the influx was primarily composed of neutrophils (Table 2).



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  		Figure 1. Non–mannose-capped lipoarabinomannan (AraLAM) induces pulmonary inflammation. Groups of four to eight wild-type (WT) mice were inoculated intranasally with three different doses of AraLAM (0.1, 1, and 10 µg). Control mice received sterile phosphate-buffered saline (PBS). At 6 hours after inoculation, mice were killed and lungs were excised. Interleukin (IL)-1ß, tumor necrosis factor (TNF), cytokine-induced neutrophil chemoattractant (KC), and myeloperoxidase (MPO) activity as a measure of neutrophil influx were determined in lung homogenates. Data are means ± SEM. *p < 0.05 versus WT mice. **p < 0.01 versus WT mice.

 

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  		TABLE 1. Cytokine and chemokine levels in lung homogenates of mice inoculated with non–mannose-capped lipoarabinomannan*

 

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  		TABLE 2. Effect of non–mannose-capped lipoarabinomannan on cellular subsets in bronchoalveolar lavage fluid*

 
TLR2 but Not CD14, LBP, or TLR4 Is Crucial for AraLAM-induced Lung Inflammation
After having established that AraLAM induces pulmonary inflammation characterized by expression of proinflammatory cytokines and chemokines as well as the influx of neutrophils into the pulmonary compartment, we were interested in the role of TLR2, TLR4, CD14, and LBP herein. Therefore, TLR2 KO, CD14 KO, TLR4 mutant, and LBP KO mice were intranasally inoculated with 10-µg AraLAM and killed after 6 hours. In comparison with WT mice, TLR2 KO mice showed a strongly reduced inflammatory response after intranasal challenge with AraLAM. Indeed, TLR2 KO mice displayed much lower lung concentrations of IL-1ß, KC, IL-6 (Figures 2A–2C), and TNF (data not shown) and an almost complete absence of neutrophil influx into the BAL fluid (Table 3). Accordingly, WT mice developed a significant lung interstitial inflammatory infiltrate in contrast to TLR2 KO mice, which displayed normal lung histology (Figure 3). In contrast, CD14-, LBP-, and TLR4-deficient mice were indistinguishable from their respective WT mice with respect to the induction of TNF, IL-1ß, IL-6, and KC in lungs (shown for IL-1ß, KC, and IL-6 in Figures 2D–2L) and with respect to neutrophil influx in the BAL fluid (data not shown). These results demonstrate that TLR2 signaling is crucial for the induction of pulmonary inflammation after AraLAM challenge in vivo and that CD14, LBP, or TLR4 do not play a role of importance herein. Furthermore, from these results, it can be concluded that the used AraLAM preparation was not contaminated with biologically relevant concentrations of lipopolysaccharide (LPS) because no differences in response was detected between C3H/HeN and LPS-unresponsive C3H/HeJ mice.



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  		Figure 2. A lack of inflammatory response in lungs of Toll-like receptor knockout (TLR2 KO) mice. Groups of six to eight wild type (WT), TLR2 KO (A–C), TLR4 mutant (D–F), CD14 (G–I), and lipopolysaccharide binding protein (LBP) (J–L) KO mice were inoculated intranasally with 10 µg of AraLAM. After 6 hours, the mice were killed, and lungs were removed. IL-1ß, KC, and IL-6 levels were measured in lung homogenates by ELISA. Data are means ± SEM. **p < 0.01 versus PBS control animals.

 

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  		TABLE 3. Effect of toll-like receptor 2 deficiency cellular subsets in bronchoalveolar lavage fluid in response to non–mannose-capped lipoarabinomannan*

 


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  		Figure 3. Absence of inflammation in lungs of TLR2 KO mice. Representative lung histology of groups of 5 WT (A) and TLR2 KO (B) mice killed 6 hours after intranasal inoculation with 10 µg of AraLAM. Hematoxylin and eosin staining, magnification x20. Bars represent 50 µm.

 
TLR2 Deficiency Delays the Clearance of M. smegmatis Infection
The cell wall LAM from avirulent fast-growing mycobacterium M. smegmatis is predominantly noncapped (6). To determine the role of TLR2, CD14, and TLR4 in the host response to this mycobacterium, we infected TLR2 KO, CD14 KO, and TLR4 mutant mice with 2.5 x 105 cfu of M. smegmatis and killed the mice after 6 hours and 1, 3, and 10 days. Normal WT mice clear this infection during the first 4 weeks of infection (31). TLR2 KO mice displayed a delayed clearance of M. smegmatis when compared with WT mice (Figure 4). Indeed, whereas at 6 hours and 1 and 3 days after infection no differences in bacterial outgrowth were found, at 10 days after infection, the lungs of TLR2 KO mice contained approximately one log more M. smegmatis cfu than the lungs of WT mice. In accordance with the results obtained after AraLAM administration, the early cytokine response to M. smegmatis was reduced in TLR2 KO mice. Despite a similar bacterial burden 6 hours after infection, lung levels of TNF, IL-1ß, IL-6, and KC were significantly reduced at this time, although myeloperoxidase levels were similar in TLR2 KO and WT mice (Table 4). After 1 and 3 days of infection, influx of neutrophils to the interstitium was observed in both groups together with signs of pleuritis. After 10 days of infection, almost no inflammation was found despite the presence of mycobacteria. Regardless of differences in bacterial outgrowth, lung pathology was similar in WT and TLR2 KO mice, although at 3 days after infection, the inflammatory response in the TLR2 KO lung was enhanced (semiquantitative scores of WT 3.9 ± 1.5 vs. 5.2 ± 1.1 of TLR2 KO mice, respectively; p < 0.05; Figure 5). In contrast, the number of M. smegmatis cfu (Figure 4) and pathology (data not shown) did not differ between CD14 KO or TLR4 mutant mice with their respective WT mice at any of these time points. To obtain further insight into the role of TLR2 in host defense against M. smegmatis, we next intranasally infected TLR2 KO and WT mice with a lower (104-cfu) and a higher (106-cfu) mycobacterial inoculum and counted M. smegmatis cfu in lungs 10 days after infection. Only three-eighths of TLR2 KO mice infected with 104-cfu M. smegmatis had completely cleared the mycobacteria from their lungs 10 days after infection versus six-eighths of WT mice; all mice with detectable mycobacteria displayed 100–200 cfu/g lung (the detection limit being 100 cfu/g lung) with the exception of one TLR2 KO mouse that demonstrated 4,500 cfu/g lung. After infection with 106-cfu M. smegmatis, all mice still had detectable mycobacteria in their lungs at 10 days, but TLR2 KO mice harbored significantly more M. smegmatis than WT mice (Figure 6). Like in our studies using 2.5 x 105 cfu, an inflammatory response was barely present in lungs 10 days after infection (data not shown).



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  		Figure 4. Delay in clearance of M. smegmatis in TLR2-deficient mice. Mycobacterial outgrowth in lungs: WT (open symbols) and TLR2 or CD14 KO as well as TLR4 mutant mice (closed symbols) were infected intranasally with 2.5 x 105 cfu of M. smegmatis. At 1, 3, and 10 days after infection, mice were killed, and bacterial loads were determined in lung homogenates. Data are means ± SEM of five to eight mice per group. *p < 0.05.

 

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  		TABLE 4. Effect of toll-like receptor 2 deficiency on the early (6 hours) response to m. smegmatis*

 


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  		Figure 5. Representative histologic sections of lungs of groups of eight WT (A) and TLR2 KO (B) mice killed after infection with 2.5 x 105 cfu of M. smegmatis. Hematoxylin and eosin staining, magnification x20. Bars represent 50 µm.

 


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  		Figure 6. A different dose of M. smegmatis in TLR2-deficient mice. Mycobacterial outgrowth in lungs: WT and TLR2 KO mice were infected intranasally with 106 cfu of M. smegmatis. Ten days after infection, mice were killed, and bacterial loads were determined in lung homogenates. Data are means ± SEM of eight mice per group. *p < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TLRs are the major receptors for the induction of the innate immune response after a microorganism enters the body. They recognize specific bacterial components, among which LAM prominently features in the mycobacterial cell wall. In this study, we investigated the capacity of AraLAM from fast-growing mycobacteria to elicit an inflammatory response in the lung. Thereby, we were particularly interested in signaling receptors involved in AraLAM-induced effects in the respiratory tract in vivo. We here demonstrate that AraLAM is able to induce a profound inflammatory response in the lungs of WT mice. The reaction observed was very similar to the response induced by intranasal LPS administration and characterized by rapid induction of proinflammatory cytokines and chemokines followed by a marked influx of neutrophils into the pulmonary compartment (32). In addition, by using a number of genetically modified or altered mouse strains, we established that TLR2, but not LBP, CD14, or TLR4, is important for triggering lung inflammation by AraLAM.

We chose to investigate the inflammatory response in lungs at 6 hours after intranasal administration of AraLAM for two reasons. First, from our kinetic study during 24 hours after administration, we found 6 hours most suitable for investigating both neutrophil influx and cytokine release. Second, several other studies in our laboratory and elsewhere challenging mice intranasally with different bacterial compounds (LPS, lipoteichoic acid, peptidoglycan, or pneumolysin) have shown that 3–6 hours after challenge is an appropriate time point for studying pulmonary inflammation (26, 32, 33). Potential cells triggered after contact with AraLAM are macrophages, epithelial cells, and most CD11chi dendritic cells (34, 35). All of these cell types express TLR2 on their surface, and all of them potentially contributed to the induction of the inflammation observed in this investigation. Of note, whereas AraLAM induced a rise in lung KC concentrations, it did not influence macrophage inflammatory protein-2 levels. Although a clear explanation for this finding is not available, it should be noted that earlier reports have suggested that the production of these two chemokines in the pulmonary compartment is regulated by at least partially different mechanisms (26, 36).

The inflammatory response in the lungs induced by AraLAM was dependent on TLR2 but not on LBP, CD14, or TLR4. With respect to the role of TLR2 in AraLAM signaling, our data are in line with in vitro observations (9–11). By engineering Chinese hamster ovary cell K1 (CHO-K1) fibroblasts, human monocytic leukemia cell line (THP-1), and RAW264.7 murine macrophage cell lines to express CD14, TLR4, or TLR2, Means and colleagues demonstrated that TLR2 but not TLR4 signaling is crucial for activating cells by AraLAM (10). Results obtained with peritoneal macrophages from TLR4 mutant mice also revealed that LPS and AraLAM use different recognition receptors for activation of host cells (37). Subsequent studies confirmed these data and added several other mycobacterial proteins to the list of TLR2 signaling pathogen-associated molecular patterns associated with mycobacteria: the 19-kD protein (16–18), soluble tuberculosis factor and phosphatidylinositol dimannoside from M. tuberculosis (11, 14), and lipomannan from Mycobacterium kansasii (38). Furthermore, heat-killed as well as live whole mycobacteria stimulate macrophages through TLR2 (9, 21) and expression of a dominant-negative TLR2 construct partially inhibited activation of mouse macrophages by heat-killed mycobacteria (12).

Interestingly, TLR2 can cooperate with different TLRs before transmitting a signal into the cytoplasm, in particular TLR1 and TLR6 (3, 39). This cooperation is important for discrimination between different microbial constituents and products. For example, TLR1 and TLR2 cooperate in the recognition of the 19-kD peptide of M. tuberculosis (40). Recently, two studies demonstrated that also AraLAM uses both TLR2 and TLR1 for forming a functional signaling complex in vitro (41, 42). Therefore, it will be interesting to look at the in vivo role of TLR1 and/or TLR6 in pulmonary inflammation induced by AraLAM as well as in M. smegmatis infection. Such studies were not part of the present investigations because mice deficient for TLR1 or TLR6 (in the presence or absence of TLR2 deficiency) are not available to our laboratory at present.

Unlike the important role of TLR2, our data suggest that neither LBP nor cell-associated CD14 nor soluble CD14 are crucial receptors for AraLAM. These findings are in conflict with several in vitro studies (10, 13, 43–47). These studies point out that AraLAM needs binding of the LBP/CD14 complex to activate the cellular receptor. This discrepancy between the in vitro data and our in vivo findings possibly can be explained by the extent of LPS contamination of the AraLAM preparation used. LPS signaling is dependent on binding of LPS to LBP, CD14, or soluble CD14 and subsequent transfer to TLR4. In our study, AraLAM was used that contained 0.373 ng of LPS per mg of AraLAM, which results in intranasal inoculation of 3.73 pg of LPS. This amount of LPS does not induce a detectable inflammatory response in mouse lungs (48). Furthermore, no difference in inflammatory response was found in the lungs of the TLR4 mutant C3H/HeJ mice when compared with their WT C3H/HeN control animals. If LPS contamination was the reason for the inflammatory response elicited by AraLAM administration, a suppressed or absent inflammatory response should have been seen in TLR4 mutant mice because they are LPS unresponsive (49).

Whole microorganisms contain several different pathogen-associated molecular patterns, and the inflammatory response to whole live microorganisms is most likely to be different from the effects of isolated bacterial components. For example, by using antibodies against LAM, it was shown that TLR-dependent cellular activation by M. tuberculosis H37Ra was not purely mediated by LAM (11). M. smegmatis abundantly expresses non–mannose-capped LAM on its surface (6). To facilitate comparisons with results obtained after AraLAM administration, we studied the role of TLR2, CD14, and TLR4 in a model of pulmonary infection with M. smegmatis. In humans, M. smegmatis primarily is associated with soft tissue and wound infections. M. smegmatis rarely causes lung infection in the immunocompetent host (50). However, in severely immunocompromised patients such as patients with inherited interferon {gamma}-R1 deficiency, M. smegmatis infection can disseminate and can be lethal (51). In addition, M. smegmatis can cause pulmonary infections in patients with an underlying condition such as lipoid pneumonia (50, 52–54). Little is known about the pathogenesis of infection by either virulent or avirulent fast-growing mycobacteria (such as M. smegmatis). It should be noted that it has not been established how much AraLAM is contained in M. smegmatis. It may therefore be possible that the amount of AraLAM in the different mycobacterial doses used was below the threshold for stimulating a measurable response by the mycobacterial AraLAM component in vivo. However, our data obtained in TLR2 KO mice early (6 hours) after infection with M. smegmatis point to a possible role for AraLAM in the inflammatory response induced by this microorganism. Indeed, at this time point, TLR2 KO displayed significantly lower cytokine concentrations in their lungs than WT mice, which resembled the findings in TLR2 KO and WT mice administered with AraLAM.

An earlier study examined the role of TNF during intravenous infection of mice with M. smegmatis (31). In this and in our study, M. smegmatis was cleared from the main target organ, the liver in the intravenous model, and the lung in our model. In the intravenous infection, the lack of TNF resulted in a delayed expression of cytokines and chemokines, reduced cellular recruitment, and an associated delay in clearance of M. smegmatis (31). Different studies using TLR2 KO mice in infectious models with pathogenic mycobacterial strains such as M. tuberculosis, Mycobacterium avium, and Mycobacterium bovis bacillus Calmette-Guérin (BCG) showed divergent results. Despite differences in host response against the pathogens in all studies like an altered inflammatory response, enhanced bacterial outgrowth, and/or reduced survival of the animals, these differences in outcome ranged from subtle to significant (20, 21, 55–58). These differences can be explained by the differences in routes of infection (intravenously vs. aerosol), infection dose (low dose vs. high dose), and/or mouse strains used. To our knowledge, our study is the first to investigate the role of TLR2 in the defense against mycobacteria that are normally cleared by the immunocompetent host. In our model of mycobacterial infection, the host response against the nonpathogenic M. smegmatis was altered, but TLR2 deficiency did not result in a greatly enhanced susceptibility; that is, the clearance of this mycobacterium was delayed but still occurred. Hence, although TLR2 is involved in an effective clearance of M. smegmatis from the lungs, other receptors such as scavenger receptors and other members of the TLR family likely contribute as well. Indeed, although not established for M. smegmatis, heat-sensitive cell-associated mycobacterial factors have been found to activate TLR4, whereas CpG-DNA is recognized by TLR9 (11, 19, 59). Unexpectedly, after 3 days of infection with 2 x 105 cfu, lungs of TLR2 KO mice displayed slightly enhanced inflammation. At this time point, although not significantly different (p = 0.1), bacterial loads in the lungs of TLR2 KO mice were higher and the presence of other pathogen-associated molecular patterns of M. smegmatis bacilli may have triggered other TLRs like TLR4 or TLR9 (discussed previously here), resulting in more stimulation of the inflammatory response than in WT mice. In line, Drennan and colleagues recently reported that although the production of proinflammatory cytokines induced by live M. tuberculosis in resident macrophages in vitro was strictly TLR2 dependent, during M. tuberculosis infection in vivo, TLR2 KO mice demonstrated an exaggerated inflammatory response (21). Of note, in our study, hardly any inflammatory response was observed in lungs of either WT or TLR2 KO mice at 10 days after infection. Conceivably, the low numbers of nonpathogenic M. smegmatis were unable to elicit inflammation during that clearance phase of the infection.

In conclusion, we demonstrate that in vivo administration of the mycobacterial component AraLAM in the lungs triggered acute pulmonary inflammation characterized by induction of proinflammatory cytokines and subsequent neutrophil influx. Furthermore, based on the results from several genetically modified mouse strains, we conclude that TLR2 is indispensable for AraLAM signaling in the lung in vivo and that this receptor contributes to an effective clearance of AraLAM expressing M. smegmatis from the pulmonary compartment.


   Acknowledgments
 
The authors thank Ingvild Kop and Joost Daalhuisen for expert technical assistance.


   FOOTNOTES
 
Supported by grants from Mr. Willem Bakhuys Roozeboom Foundation (C.W.W.), the Fonds zur Foerderung der wissenschaftlichen Forschung in Oesterreich (S.K.), and the Netherlands Organization of Scientific Research (S.F.) and by National Institutes of Health grants GM54060, DK50305, and RR14466 (D.T.G.).

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Conflict of Interest Statement: C.W.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.F.d.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; K.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; D.T.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; T.v.d.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form April 21, 2004; accepted in final form September 17, 2004


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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