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. Author manuscript; available in PMC: 2019 Mar 25.
Published in final edited form as: Pathog Dis. 2013 Dec 16;70(2):132–140. doi: 10.1111/2049-632X.12110

Inhibition of toll-like receptor 2 (TLR-2)-mediated response in human alveolar epithelial cells by mycolic acids and Mycobacterium tuberculosis mce1 operon mutant

Patricia C Sequeira 1,*, Ryan H Senaratne 1, Lee W Riley 1,†
PMCID: PMC6433132  NIHMSID: NIHMS538140  PMID: 24190334

Abstract

In human lungs, the earliest encounter of Mycobacterium tuberculosis, the agent of tuberculosis (TB), involves alveolar epithelial cells. Droplets expectorated by a TB patient are likely to contain a mixed population of M. tuberculosis cells in different physiologic and metabolic states from the lung lesions of the patient. Here, we compared the chemokine expression patterns of human epithelial cell line A549 and RAW 264.7 macrophage cells infected with wild type M. tuberculosis H37Rv against patterns induced by a mutant that accumulates free mycolic acids in its cell wall (Δmce1). We also examined the effect of free mycolic acids on toll-like receptor-2 (TLR-2). Wild type M. tuberculosis induced significantly higher levels of IL-8, MCP-1, RANTES, and IP-10 in both cell types than did Δmce. Free mycolic acids reduced the ability of the mammalian cells to respond to a TLR-2 agonist in a dose-dependent manner. These observations suggest that differences in mycolic acid abundance in the M. tuberculosis cell wall can affect TLR-2-mediated pro-inflammatory response in both epithelial and macrophage cells. The final fate of a new infection may be ultimately determined by the proportion of M. tuberculosis cells expressing free mycolates in the infecting inoculum population.

Keywords: Mycobacterium tuberculosis, tuberculosis, mycolic acid, A549 cells, mce1 operon, alveolar epithelial cells

INTRODUCTION

Mycobacterium tuberculosis is a leading infectious cause of death in adults worldwide [1]. Most often, the initial site of infection in lungs of someone exposed to M. tuberculosis inhaled in droplets released from an index case of TB is the alveolar air space. Most studies that examine host innate immune response to M. tuberculosis have focused on the effector functions of alveolar macrophages and dendritic cells [2–5]. M. tuberculosis has been shown to activate alveolar macrophages in TLR-2- and TLR-4-dependent manner [6], and several lines of evidence suggest the innate immunity-mediated protective role of TLR-2 in mycobacterial infections [7–10]. However, these events that involve alveolar professional phagocytes most likely occur many days or weeks after the initial entry M. tuberculosis into the alveolar space.

The alveolar space is mostly composed of type I and type II pneumocytes. The former comprises about 96% of the alveolar surface area, while type II cells cover about 4% of the surface area but comprise 60% of all the alveolar epithelial cells [11]. Thus, the inhaled tubercle bacilli most likely encounter these epithelial cells first before they can be taken up by alveolar macrophages and dendritic cells. A number of groups have recently begun to address the active involvement of alveolar epithelial cells in the early innate host defense against M. tuberculosis [12–18].

In the 1950s Segal and Bloch showed that M. tuberculosis isolated from mouse lungs use fatty acids to stimulate respiration instead of carbohydrates used by the organism grown in liquid cultures [19]. Thus, during transmission, the tubercle bacilli in droplets released from a TB patient is likely to be comprised of population of cells in different metabolic states representing those in the lung lesions. Thus, another important factor that could determine the outcome of a new infection is the relative proportions of bacterial cells in different metabolic states at the time of transmission.

We previously demonstrated that an M. tuberculosis strain disrupted in a 13-gene operon called the mce1 (Δmce1) fails to induce a characteristic pro-inflammatory response in mice or ex vivo-infected murine macrophages [20]. The mce1 operon is a member of a family of 4 related operons (mce1–4) that have been suggested by phylogenomic analyses to encode ABC transporters [21]. Pandey and Sassetti have proposed that the mce4 operon encodes a possible cholesterol importer [22]. Santangelo et al. have suggested that the mce3 operon is a regulon involved in lipid metabolism of M. tuberculosis [23]. Dunphy et al. showed that the mce1 operon may be involved in recycling mycolic acids released from dying bacteria during latent infection [24]. They found that a functional disruption of fatty acyl-CoA synthetase encoded by fadD5 in the mce1 operon caused the mutant to be diminished in its growth in minimum medium supplied only with mycolic acid as a carbon source [24].

The mce1 operon mutant is hypervirulent in BABL/c and C57BL/6 mice [25, 26]. It causes early death and induces poorly-formed granulomas in mouse lungs with aberrant migration of inflammatory cells, resembling a Th2 type immune response [25]. Mouse peritoneal macrophages or RAW cells infected ex vivo with the mutant expressed significantly lower levels of TNFα, IL-6, and MCP-1, compared to the cells infected with the wild type strain [25]. Interestingly, the cell wall of M. tuberculosis disrupted in the mce1 operon contains more than 10-fold greater amounts of mycolic acids than that of wild type M. tuberculosis [27]. Moreover, the excess mycolic acids in the mutant are not covalently linked to these fatty acids’ canonical substrates arabinogalactan or trehalose [27, 28].

We have shown that the mce1 operon is repressed in wild type H37Rv M. tuberculosis during the first 4 weeks of infection in mice [29]. That is, the wild type strain behaves like the mce1 operon mutant at one phase of infection in vivo, and that in lung lesions, a proportion of the bacterial population may indeed be comprised of cells that express free mycolic acids in their cell wall. This mixed population of cells in expectorated droplets is what is likely to be transmitted to a new host. Here we propose that the ultimate fate of a new infection in the lungs is determined by the relative proportion bacterial cells expressing and not expressing free mycolic acids during their initial encounter with alveolar epithelial cells. To address this hypothesis, we compared the interaction of M. tuberculosis and its mce1 operon mutant with A549 epithelial and RAW 264.7 macrophage cells.

MATERIALS AND METHODS

Bacterial strains and growth conditions

Wild type M. tuberculosis H37Rv, its derivative mce1 operon mutant strain (Δmce1), and the complemented mce1 strain were grown in Middlebrook 7H9 broth (Difco Laboratories, MI) containing 10% albumin-dextrose-catalase (ADC) enrichment (Beckton-Dickinson, MD), supplemented with 0.2% glycerol (Fisher Scientific, NJ), and 0.05% Tween 80 (Fischer Scientific, NJ) (7H9-ADCT) in sterile, vented tissue culture flasks at 37°C in 5% CO2. We also examined an M. tuberculosis strain disrupted in the negative transcriptional regulator (mce1R) of the mce1 operon [30]. The construction of Δmce1, Δmce1R and their phenotype is previously reported [20, 30]. Bacterial strains were grown until the optical density (OD580nm) of the cultures reached 1 (unless otherwise stated). When necessary, above bacterial strains were also grown on solid agar medium Middlebrook 7H11 containing OADC (Beckton-Dickinson, MD), 0.5% glycerol, and 100µg/mL of anti-fungal agent cyclohexamide (Sigma, MO).

Single cell suspensions

Single cell suspensions of each M. tuberculosis strain were prepared according to a previously described method [31] and quantified by enumeration of their colony-forming units (CFUs) on solid agar medium. Briefly, a 1.0 mL aliquot of a frozen mycobacterial culture (frozen at OD580nm =1.0) was used to inoculate 30mL of sterile Middlebrook 7H9-ADCT in a 75 cm2 vented tissue culture flask. The 30mL culture was incubated at 37°C in the presence of 5% CO2. After the culture was grown to OD580nm =1.0, 10mL of the culture were placed in a sterile 50mL polypropylene tube and centrifuged at 4,000 rpm for 15 minutes. The supernatant was discarded and the pellet was suspended in 5mL of RPMI 1640 cell culture media containing 5% fetal bovine serum (FBS) (Omega Scientific, CA). The suspension was mixed thoroughly by pipetting and vortexing to dissociate clumped bacteria. The 5mL suspension was then passed through a sterile 5.0 micron pore Acrodisk syringe filter (Pall Corporation, MI) with a sterile 5mL syringe. The resulting filtrate (single cell suspension) was mixed by vortexing.

Maintenance and infection of alveolar epithelial cells

The human alveolar epithelial cell line A549 (ATCC CCL-185) was cultured and maintained in RPMI 1640 medium with 2mM L-glutamine (Gibco, NY), supplemented with 10% heat-inactivated FBS, in a 5% CO2 humidified incubator at 37°C. Cell concentration was determined with a hemocytometer. Cells were seeded at 105 cells per well in 12-well tissue culture plates and incubated overnight until the cells were approximately 70% confluent. Immediately before infection, the medium was replaced with fresh and pre-warmed RPMI 1640 medium with 5% FBS. A549 cells were always infected in triplicate with a multiplicity of infection (MOI) of 1:50 (cells: bacteria). Six hours after the infection, extracellular bacteria were removed by washing cells 3 times with warm RPMI 1640 5% FBS. After the third wash, RPMI 1640 5% FBS used to wash the cells was plated on solid medium to assess the presence or absence of extracellular bacteria. Infected monolayers were then incubated for 6, 24, 48, and 72 hours at 37°C in a 5% CO2 atmosphere. Uninfected cells served as negative controls for each experiment.

Enumeration of intracellular bacteria

At different time points after infection, the supernatants were removed, and A549 cells were lysed to enumerate the intracellular bacterial load. The infected cells were lysed by incubation with 0.5mL of 0.1% Triton X-100 (Sigma, MO) in sterile water for 20 minutes at room temperature. The resulting cell lysates were serially diluted and plated in triplicate on solid medium. Three weeks after plating, CFUs were enumerated.

Enzyme-linked immunosorbent assays (ELISA)

Infected (with M. tuberculosis or its derivative strains) or uninfected (negative control) A549 cells were incubated for up to 72 hours. Supernatants were harvested at 6, 24, 48, and 72 hours post-infection, and sterilized by passing through a 0.2 µm Acrodisc PF filter (Gelman Sciences, MI). Chemokine concentrations in supernatants were measured by ELISA, according to the manufacturer’s protocol (R&D Systems, MN). OD readings were obtained with a Microplate Reader model 450. Cytokines in the supernatants were quantified against the OD values obtained for the cytokine standards provided by the kit (R&D Systems, MN). All cytokine quantification experiments were done in triplicate.

Anti-TLR-2 antibody pretreatment and infection of alveolar epithelial cells

One set of A549 cells was treated with a mouse anti-human TLR-2 blocking antibody (clone T2.1) (eBioscience, CA). After adding 10µg/mL of antibodies, cells were incubated at room temperature for 30 minutes, as previously described [32]. The second set of A549 cells was exposed to the same conditions, except for the addition of the TLR-2 blocking antibody. Following incubation, cells were washed 3 times with pre-warmed RPMI 1640 medium and infected with the bacterial strains at an MOI of 1:50 (cells:bacteria). Six hours after the infection, extracellular bacteria were removed by washing cells 3 times with warm RPMI 1640 5% FBS. After the third wash, RPMI 1640 5% FBS used to wash the cells was plated on solid medium to assess the presence or absence of extracellular bacteria. Infected monolayers were then incubated for 72 hours at 37°C in a 5% CO2 atmosphere. Uninfected cells served as negative controls for both set of cells. As a positive control, cells from each set were incubated for 72 hours with 1µg/mL of synthetic TLR-2 agonist Pam3Cys-Ser-(Lys)4 (Pam3Cys) (Sigma, CO). All experiments were done in triplicate.

Pretreatment of alveolar epithelial cell line with mycolic acids and stimulation with TLR-2 agonist

Mycolic acids extracted from a clinical strain of M. tuberculosis were purchased from Sigma- Aldrich, CO. Lyophilized mycolic acids were resuspended in sterile DMSO (Sigma-Aldrich, CO) to a concentration of 5mg/mL. Cells were pre-incubated with either varying amounts of mycolic acids (125ng/mL, 50ng/mL, 25ng/mL) or without any mycolic acids at room temperature for 30 minutes. After the pre-incubation, cells were washed 3 times with pre-warmed RPMI 1640 medium to remove excess mycolic acids. Washed cells were incubated with 1µg/mL of TLR-2 agonist Pam3Cys (Sigma, CO) for 72 hours. These doses of mycolic acids and Pam3Cys were established in pilot dose-response studies. The ability of mycolic acids to block TLR-2-induced IL-8 and MCP-1 up-regulation in a dose-dependent manner was determined by ELISA.

RAW macrophages pretreatment with free M. tuberculosis mycolic acids and stimulation with TLR-2 agonist

The RAW 264.7 murine macrophage-like cell line (ATCC TIB-71) was cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, NY) supplemented with 10% FBS at 37°C in a 5% CO2 humidified incubator. Macrophage cell number and viability were assessed by trypan blue dye (Gibco, NY) exclusion assay. Before each experiment, 2 × 105 cells/mL were cultured in 24-well tissue culture plates for 24 h to achieve 70% confluency. Cells were pre-incubated with either varying amounts of mycolic acids (125ng/mL, 50ng/mL, 25ng/mL) or without any mycolic acids at room temperature for 30 minutes. After the pre-incubation, cells were washed 3 times with pre-warmed DMEM medium to remove excess mycolic acids. Washed cells were incubated with 1µg/mL of the TLR-2 agonist for 72 hours. The ability of mycolic acids to block TLR-2-induced TNF-α, IL-12, and MCP-1 up-regulation in a dose-dependent manner was determined by ELISA. An additional experiment was done in triplicate with RAW macrophage cells with mycolic acids alone to assess immunogenicity.

Statistical analysis

Mean values of triplicate measurements for different cell groups were compared by the Student t test.

RESULTS

Production of chemokines by A549 cells infected with M. tuberculosis H37Rv or mce1 mutant

The amounts of chemokines expressed by A549 cells infected with Δmce1 were significantly lower than those in cells infected with the WT or mce1 complemented M. tuberculosis strains. IL-8 and MCP-1 measured in the supernatants of A549 cells infected with the Δmce1 strain were approximately 3-fold lower than those measured in the supernatants of A549 cells infected with WT strain or complemented strain at 72 hours post-infection (Figs. 1A & B). Also, the mutant-infected A549 cells produced 1.8 times less IP-10 than the cells infected with WT or the complemented strain (P<0.04) (Fig. 1C). Similar differences were observed in the amounts of RANTES produced by the A549 cells; mutant-infected cells produced 2.09 times less RANTES than cells infected with the WT strain or mce1 complemented strain at 72 hours post infection (P<0.02) (Fig. 1D). None of the above M. tuberculosis strains (as high as 50 organisms per cell) induced MIP-1α in these experiments (data not shown).

Figure 1. A to D. Analysis of production of IL8 (A), MCP-1 (B), IP-10 (C), and RANTES (D) by A549 cells infected with either WT (H37Rv), mce1 mutant (Δmce1), or mce1 complemented strain (mce1 KI).

Figure 1

Figure 1

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Production of IL8 (A), MCP-1 (B), IP-10 (C), and RANTES (D) by A549 cells infected at an MOI of 1:50. The scale is different for each cytokine and chemokine. Error bars represent standard error of the mean calculated from the results of three independent assays.

In a different experiment, the induction of chemokines by Δmce1R and Δmce1 mutants in A549 cells was compared. mce1R is the negative regulator of the mce1 operon; M. tuberculosis disrupted in this gene constitutively expresses the mce1 operon genes [30]. Like the mce1 operon mutant, Δmce1R is hypervirulent in mice; it causes accelerated death in mice by inducing massive pro-inflammatory response [29]. WT or Δmce1R-infected A549 cells produced the same amounts of IL-8, while the IL-8 production in Δmce1-infected cells were 4-fold lower (Fig. 2A). MCP-1 was produced in Δmce1-infected A549 cells compared to WT or Δmce1R-infected A549 cells (Fig. 2B).

Figure 2. A and B. Analysis of production of IL8 and MCP-1 by A549 cells infected with either WT (H37Rv), mce1 mutant (Δmce1), or mce1R mutant (Δmce1R).

Figure 2

Figure 2

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Production of IL-8 (A) and MCP-1 (B) by A549 cells infected at MOI of 1:50 was measured. Asterisks indicate differences that are statistically significant between H37Rv and Δmce1 (*) and H37Rv and Δmce1R (**). Error bars represent standard error of the mean calculated from the results of three independent assays.

Growth of mce1 mutant and control strains in A549 cells

Similar numbers of CFUs were recovered from WT, Δmce1 mutant, or mce1 complemented strain-infected A549 cells from day 0 to the final time point of the experiment, at 72 hours post infection (Fig. 3). Also, the measurement of live A549 cells with trypan blue exclusion assay indicated that the cell counts in each well were similar. Therefore, these data indicated that the difference in above chemokine production by A549 cells infected with WT or Δmce1 was not due to differences in the number of bacteria in these cells or cell viability.

Figure 3. Intracellular growth of WT (H37Rv), mce1 mutant (Δmce1), and mce1 complemented strains (mce1 KI) in A549 cells.

Figure 3

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Error bars represent standard error of the mean calculated from the results of three independent assays.

Involvement of A549 cell TLR-2 in innate immune response to M. tuberculosis infection

The production of IL-8 and MCP-1 was equally diminished in A549 cells that had their TLR-2 blocked before exposure to the WT M. tuberculosis. There was a 3-fold decrease in IL-8 production when cells were pretreated with anti-TLR-2 antibody prior to the infection with the WT (Fig. 4A). Similarly, there was a 3-fold decrease of MCP-1 production when cells were pretreated with anti-TLR-2 prior to the infection with the WT (Fig. 4B). The treatment of cells with anti-TLR-2 antibody prior to the infection with the Δmce1 mutant did not significantly decrease the expression of IL-8 or MCP-1 in A549 cells. At 72 hours post infection, anti-TLR-2 antibody pretreated cells and infected with the Δmce1 produced low concentrations of IL-8 and MCP-1 similar to the cells that were not pretreated with the antibody but were infected with the Δmce1 (Figs. 4A & B). The production of IL-8 and MCP-1 in A549 cells (pretreated or not pretreated with anti-TLR-2 antibodies) infected with the mce1 complemented strain was comparable to WT-infected A549 cells (Figs. 4A & B). The ability of anti-TLR-2 antibody to block the TRL-2 binding was confirmed by the low amounts of IL-8 (295 pg/mL) and MCP-1 (176 pg/mL) produced by anti-TLR-2 antibody treated A549 cells in response to the TLR-2 agonist Pam3CysSerLys4 (Pam3Cys)(Fig. 4C). Recovery of CFUs from A549 cells either pretreated or not treated with anti-TLR-2 antibodies was similar (Figs. 3 & 4D).

Figure 4. A to D. Effect of anti-TLR-2 antibodies on the production of IL-8, MCP-1, and the growth of bacteria in A549 cells.

Figure 4

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Production of IL-8 (A) and MCP-1 (B) by A549 cells after 72 hours of M. tuberculosis infection, in the presence or absence of 10µg/mL of anti-TLR-2 blocking antibody. Cells were infected with either WT (H37Rv), mce1 mutant (Δmce1), or mce1 complemented strains (mce1 KI). (C) Production of IL-8 and MCP-1 was stimulated by incubating A549 cells for 72 hours with 1µg/mL of the TLR-2 agonist Pam3Cys, in the presence or absence of 10µg/mL of anti-TLR-2 blocking antibody. (D) Intracellular growth of WT (H37Rv), mce1 mutant (Δmce1), and mce1 complemented strains (mce1 KI) in anti-TLR-2 blocking antibody-treated A549 cells. Asterisks indicate differences that are statistically significant between H37Rv and Δmce1 (*) and H37Rv and mce1KI (**). Error bars represent standard error of the mean calculated from the results of three independent assays.

Effects of interaction between mycolic acids and TLR-2 in A549 cells on innate immune response

Because of the previously reported effects of M. tuberculosis products on macrophage TLR-2-modulated pro-inflammatory response, the macrophage anti-Th-1 type immune response that we observed with the mce1 operon mutant [25], and the excess amounts of unbound mycolic acids in the cell wall of Δmce1 [27], we studied the effect of purified mycolic acids on A549 cells. Free mycolic acids diminished the ability of TLR-2 agonist Pam3Cys to induce production of IL-8 and MCP-1 in A549 cells in a dose-dependent manner. A459 cells pretreated with 25, 50, and 125 ng/mL of mycolic acids produced 1.3, 5.8, and 8.3 times less IL-8 than non-treated cells. Similarly, A549 cells pretreated with 25, 50, and 125 ng/mL of mycolic acids produced 2.03, 6.7 and 7.8 times less MCP-1 than non-treated cells (Figs. 5A & B).

Figure 5. A and B. Effect of mycolic acid on production of IL-8 and MCP-1 in A549 cells.

Figure 5

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Stimulation of IL-8 (A) and MCP-1 (B) with 1µg/mL of the TLR-2 agonist Pam3Cys in A549 cells pretreated or non-pretreated with M. tuberculosis-derived mycolic acids. IL-8 and MCP-1 were measured 72 hours after the infection. Error bars represent standard error of the mean calculated from the results of three independent assays.

Similar to A549 cells, the response of RAW macrophages to the TLR-2 agonist Pam3Cys was also affected by the mycolic acids pretreatment. The effect of pretreatment of RAW macrophages with 25ng/mL of mycolic acids on IL-12, TNF-α, and MCP-1 production was small, i.e. the decrease in cytokine or chemokine production between pretreated and non-pretreated was 0.4%, 17.5% and 3.9 % respectively. However, RAW cells pretreated with 50 ng/mL of mycolic acids produced 4.5 times (75.9%), 4.8 times (81.4%), and 4 times (73.3%) less IL-12, TNF-α, and MCP-1, respectively, than untreated cells. RAW macrophages pretreated with 125 ng/mL of mycolic acids produced 9.2 times (89.1%), 7.4 times (86.5%), and 14 times (93%) less IL-12, TNF-α, and MCP-1, respectively, than non-treated cells (Figs. 6A, B & C).

Figure 6. A to C. Effect of mycolic acid on production of IL12, TNF-alpha, and MCP-1 in RAW macrophages.

Figure 6

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Stimulation of IL-12 (A), TNF-alpha (B), and MCP-1 (C) with 1µg/mL of the TLR-2 agonist Pam3Cys in RAW macrophages pretreated or nonpretreated with M. tuberculosis-derived mycolic acids. IL-12, TNF-alpha, and MCP-1 were measured 72 hours after the infection. The scale for figure 6B is different from the others. Error bars represent standard error of the mean calculated from the results of three independent assays.

DISCUSSION

We previously showed that M. tuberculosis disrupted in the mce1 operon proliferates unchecked in BALB/c mouse lungs, but the same pattern of bacterial proliferation was not observed in less susceptible C57BL/6 mice [26]. Nevertheless, C57BL/6 mice infected with the Δmce1 succumbed to the infection faster than the WT- infected mice [26]. These observations suggested that rather than the bacterial burden, products of the mce1 operon may directly or indirectly modulate the host immune response.

The mce1 operon of M. tuberculosis belongs to a family of 4 related operons (mce1–4) that have been suggested by phylogenomic analyses to comprise ABC transporters involved in lipid importation [21, 22, 33]. Dunphy et al showed that the mce1 operon may serve to recycle mycolic acids released from dying M. tuberculosis inside granulomas [24]. The operon contains a gene fadD5 that encodes fatty acyl-CoA synthetase. They showed that a mutant functionally disrupted in fadD5 is diminished in growth in minimum medium supplied only with mycolic acid as a carbon source; the mutant grew normally in minimum medium supplied with glycerol or other fatty acids as a carbon source [24]. More recently, we showed that the mce1 operon mutant contained nearly 11-fold more mycolic acids in the cell wall compared to the wild type H37Rv strain [27]. The excess mycolic acids were not bound to arabinogalactan or trehalose. The mutant and wild type strains contained similar amounts of bound mycolic acids. These observations led us to examine the possible role of mycolic acids in the interaction of M. tuberculosis with epithelial cells.

Uchida et al showed that during the first 4 weeks of infection in mice, the mce1 operon of wild type M. tuberculosis is not expressed [29]. Indeed, Casali et al have shown that the operon is negatively regulated by mce1R, which is active only when M. tuberculosis is intracellular [30]. Thus, during a natural course of infection, wild type M. tuberculosis may undergo a physiologic state exhibited by the mce1 operon mutant—that is, it may make excess amounts of mycolic acids. If a new transmission occurs with a population of bacterial cells mostly in this physiologic state, they could down-modulate the innate immune response of a contact host’s alveolar epithelial cells as well as dendritic and macrophage cells, which may allow M. tuberculosis to establish persistence. If transmission occurs when most of the cells are not expressing free mycolic acids, the infection may be eliminated by the contact host at this initial encounter.

In this study, we chose A549 cells as the primary cell line to represent early cellular responses to mycolic acids and Δmce1. A549 cells are an immortalized cell line derived from human alveolar epithelial cells, which are represented by type I and II pneumocytes. Much of the surface area of the alveolar space is covered by type I pneumocytes [11]. Type II pneumocytes are more numerous and they synthesize and secrete a variety of surface active products, including surfactant proteins A and D (known as collectins), which play an important role in innate immune protection against pathogens, including against M. tuberculosis [18, 34–36]. Thus, alveolar epithelial cells are more likely to serve as the first line of innate defense against inhaled pathogens, even before alveolar macrophages and dendritic cells set in.

Here, we showed that Δmce1 M. tuberculosis and purified mycolic acids can inhibit chemokine and cytokine response in A549 cells as well as in a macrophage-like cell line in a TLR-2-dependent manner. Free mycolates blocked the effect of the TLR-2 agonist Pam3Cys. Birchler et al. have demonstrated that the A549 alveolar epithelial cell line constitutively expresses TLR-2 on its apical surface [37]. Armstrong et al. detected TLR-2 in situ on alveolar epithelial cells by immunohistochemistry [38]. Thus, it is conceivable that, in vivo, mycolic acids exert an inhibitory effect on alveolar pneumocytes via TLR-2.

The above observations demonstrate the need to better understand the interaction of M. tuberculosis with host cells at a stage much earlier than what is traditionally studied. We also need to better understand the effect on outcomes of infection due to physiologic differences in bacterial populations that serve as the infectious inoculum, rather than assuming that all the bacterial cells exert a similar effect on their target cells once they enter the lungs.

Acknowledgment

This study was supported in part by the National Institute of Allergy and Infectious Diseases of the National Institute of Health (R01AI035266) and Ellison Medical Foundation under the program on Senior Scholar in Global Infectious Disease Program (ID-SS-0356-02).

References

  • 1.Global tuberculosis control: a short update to the 2009 report. [ http://www.who.int/tb/publications/global_report/2009/update/en/index.html] [Google Scholar]
  • 2.Tailleux L, Schwartz O, Herrmann JL, Pivert E, Jackson M, Amara A, Legres L, Dreher D, Nicod LP, Gluckman JC, et al. DC-SIGN is the major Mycobacterium tuberculosis receptor on human dendritic cells. J Exp Med. 2003;197(1):121–127. doi: 10.1084/jem.20021468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Ehlers S. DC-SIGN and mannosylated surface structures of Mycobacterium tuberculosis: a deceptive liaison. Eur J Cell Biol. 89(1):95–101. doi: 10.1016/j.ejcb.2009.10.004. [DOI] [PubMed] [Google Scholar]
  • 4.Herrmann JL, Lagrange PH. Dendritic cells and Mycobacterium tuberculosis: which is the Trojan horse? Pathol Biol (Paris) 2005;53(1):35–40. doi: 10.1016/j.patbio.2004.01.004. [DOI] [PubMed] [Google Scholar]
  • 5.Wolf AJ, Linas B, Trevejo-Nunez GJ, Kincaid E, Tamura T, Takatsu K, Ernst JD. Mycobacterium tuberculosis infects dendritic cells with high frequency and impairs their function in vivo. J Immunol. 2007;179(4):2509–2519. doi: 10.4049/jimmunol.179.4.2509. [DOI] [PubMed] [Google Scholar]
  • 6.Means TK, Jones BW, Schromm AB, Shurtleff BA, Smith JA, Keane J, Golenbock DT, Vogel SN, Fenton MJ. Differential effects of a Toll-like receptor antagonist on Mycobacterium tuberculosis-induced macrophage responses. J Immunol. 2001;166(6):4074–4082. doi: 10.4049/jimmunol.166.6.4074. [DOI] [PubMed] [Google Scholar]
  • 7.Scanga CA, Bafica A, Feng CG, Cheever AW, Hieny S, Sher A. MyD88-deficient mice display a profound loss in resistance to Mycobacterium tuberculosis associated with partially impaired Th1 cytokine and nitric oxide synthase 2 expression. Infect Immun. 2004;72(4):2400–2404. doi: 10.1128/IAI.72.4.2400-2404.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Pai RK, Pennini ME, Tobian AA, Canaday DH, Boom WH, Harding CV. Prolonged toll-like receptor signaling by Mycobacterium tuberculosis and its 19-kilodalton lipoprotein inhibits gamma interferon-induced regulation of selected genes in macrophages. Infect Immun. 2004;72(11):6603–6614. doi: 10.1128/IAI.72.11.6603-6614.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Fulton SA, Reba SM, Pai RK, Pennini M, Torres M, Harding CV, Boom WH. Inhibition of major histocompatibility complex II expression and antigen processing in murine alveolar macrophages by Mycobacterium bovis BCG and the 19-kilodalton mycobacterial lipoprotein. Infect Immun. 2004;72(4):2101–2110. doi: 10.1128/IAI.72.4.2101-2110.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Harding CV, Boom WH. Regulation of antigen presentation by Mycobacterium tuberculosis: a role for Toll-like receptors. Nat Rev Microbiol. 2010;8(4):296–307. doi: 10.1038/nrmicro2321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Castranova V, Rabovsky J, Tucker JH, Miles PR. The alveolar type II epithelial cell: a multifunctional pneumocyte. Toxicol Appl Pharmacol. 1988;93(3):472–483. doi: 10.1016/0041-008x(88)90051-8. [DOI] [PubMed] [Google Scholar]
  • 12.Bermudez LE, Sangari FJ, Kolonoski P, Petrofsky M, Goodman J. The efficiency of the translocation of Mycobacterium tuberculosis across a bilayer of epithelial and endothelial cells as a model of the alveolar wall is a consequence of transport within mononuclear phagocytes and invasion of alveolar epithelial cells. Infect Immun. 2002;70(1):140–146. doi: 10.1128/IAI.70.1.140-146.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Rivas-Santiago B, Contreras JC, Sada E, Hernandez-Pando R. The potential role of lung epithelial cells and beta-defensins in experimental latent tuberculosis. Scand J Immunol. 2008;67(5):448–452. doi: 10.1111/j.1365-3083.2008.02088.x. [DOI] [PubMed] [Google Scholar]
  • 14.Rivas-Santiago B, Hernandez-Pando R, Carranza C, Juarez E, Contreras JL, Aguilar-Leon D, Torres M, Sada E. Expression of cathelicidin LL-37 during Mycobacterium tuberculosis infection in human alveolar macrophages, monocytes, neutrophils, and epithelial cells. Infect Immun. 2008;76(3):935–941. doi: 10.1128/IAI.01218-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Lee HM, Yuk JM, Shin DM, Jo EK. Dectin-1 is inducible and plays an essential role for mycobacteria-induced innate immune responses in airway epithelial cells. J Clin Immunol. 2009;29(6):795–805. doi: 10.1007/s10875-009-9319-3. [DOI] [PubMed] [Google Scholar]
  • 16.Kohwiwattanagun J, Kawamura I, Fujimura T, Mitsuyama M. Mycobacterial mammalian cell entry protein 1A (Mce1A)-mediated adherence enhances the chemokine production by A549 alveolar epithelial cells. Microbiol Immunol. 2007;51(2):253–261. doi: 10.1111/j.1348-0421.2007.tb03897.x. [DOI] [PubMed] [Google Scholar]
  • 17.Roy S, Sharma S, Sharma M, Aggarwal R, Bose M. Induction of nitric oxide release from the human alveolar epithelial cell line A549: an in vitro correlate of innate immune response to Mycobacterium tuberculosis. Immunology. 2004;112(3):471–480. doi: 10.1046/j.1365-2567.2004.01905.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Torrelles JB, Azad AK, Henning LN, Carlson TK, Schlesinger LS. Role of C-type lectins in mycobacterial infections. Curr Drug Targets. 2008;9(2):102–112. doi: 10.2174/138945008783502467. [DOI] [PubMed] [Google Scholar]
  • 19.Segal W, Bloch H. Pathogenic and immunogenic differentiation of Mycobacterium tuberculosis grown in vitro and in vivo. Am Rev Tuberc. 1957;75(3):495–500. doi: 10.1164/artpd.1957.75.3.495. [DOI] [PubMed] [Google Scholar]
  • 20.Shimono N, Morici L, Casali N, Cantrell S, Sidders B, Ehrt S, Riley LW. Hypervirulent mutant of Mycobacterium tuberculosis resulting from disruption of the mce1 operon. Proc Natl Acad Sci USA. 2003;100:15918–15923. doi: 10.1073/pnas.2433882100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Casali N, Riley LW. A phylogenomic analysis of the Actinomycetales mce operons. BMC Genomics. 2007;8:60. doi: 10.1186/1471-2164-8-60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Pandey AK, Sassetti CM. Mycobacterial persistence requires the utilization of host cholesterol. Proc Natl Acad Sci U S A. 2008;105(11):4376–4380. doi: 10.1073/pnas.0711159105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.de la Paz Santangelo M, Klepp L, Nunez-Garcia J, Blanco FC, Soria M, Garcia-Pelayo MC, Bianco MV, Cataldi AA, Golby P, Jackson M, et al. Mce3R, a TetR-type transcriptional repressor, controls the expression of a regulon involved in lipid metabolism in Mycobacterium tuberculosis. Microbiology. 2009;155(Pt 7):2245–2255. doi: 10.1099/mic.0.027086-0. [DOI] [PubMed] [Google Scholar]
  • 24.Dunphy KY, Senaratne RH, Masuzawa M, Kendall LV, Riley LW. Attenuation of Mycobacterium tuberculosis functionally disrupted in a fatty acyl-coenzyme A synthetase gene fadD5. J Infect Dis. 2010;201(8):1232–1239. doi: 10.1086/651452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Shimono N, Morici L, Casali N, Cantrell S, Sidders B, Ehrt S, Riley LW. Hypervirulent mutant of Mycobacterium tuberculosis resulting from disruption of the mce1 operon. Proc Natl Acad Sci U S A. 2003;100(26):15918–15923. doi: 10.1073/pnas.2433882100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Lima P, Sidders B, Morici L, Reader R, Senaratne R, Casali N, Riley LW. Enhanced mortality despite control of lung infection in mice aerogenically infected with a Mycobacterium tuberculosis mce1 operon mutant. Microbes Infect. 2007 doi: 10.1016/j.micinf.2007.05.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Cantrell SA, Leavell MD, Marjanovic O, Iavarone AT, Leary JA, Riley LW. Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis. J Microbiol. 2013 doi: 10.1007/s12275-013-3092-y. [DOI] [PubMed] [Google Scholar]
  • 28.Barry CE LR, 3rd, Mdluli K, Sampson AE, Schroeder BG, Slayden RA, Yuan Y. Mycolic acids: structure, biosynthesis and physiological function. Prog Lipid Res. 1998;37(2–3):143–179. doi: 10.1016/s0163-7827(98)00008-3. [DOI] [PubMed] [Google Scholar]
  • 29.Uchida Y, Casali N, White A, Morici L, Kendall LV, Riley LW. Accelerated immunopathological response of mice infected with Mycobacterium tuberculosis disrupted in the mce1 operon negative transcriptional regulator. Cell Microbiol. 2007;9(5):1275–1283. doi: 10.1111/j.1462-5822.2006.00870.x. [DOI] [PubMed] [Google Scholar]
  • 30.Casali N, White AM, Riley LW. Regulation of the Mycobacterium tuberculosis mce1 operon. J Bacteriology. 2006;188:441–449. doi: 10.1128/JB.188.2.441-449.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Grover AA, Kim HK, Wiegeshaus EH, Smith DW. Host-parasite relationships in experimental airborne tuberculosis. II. Reproducible infection by means of an inoculum preserved at −70 C. J Bacteriol. 1967;94(4):832–835. doi: 10.1128/jb.94.4.832-835.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Asai Y, Jinno T, Ogawa T. Oral treponemes and their outer membrane extracts activate human gingival epithelial cells through toll-like receptor 2. Infect Immun. 2003;71(2):717–725. doi: 10.1128/IAI.71.2.717-725.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Dassa E, Bouige P. The ABC of ABCS: a phylogenetic and functional classification of ABC systems in living organisms. Res Microbiol. 2001;152(3–4):211–229. doi: 10.1016/s0923-2508(01)01194-9. [DOI] [PubMed] [Google Scholar]
  • 34.Hall-Stoodley L, Watts G, Crowther JE, Balagopal A, Torrelles JB, Robison-Cox J, Bargatze RF, Harmsen AG, Crouch EC, Schlesinger LS. Mycobacterium tuberculosis binding to human surfactant proteins A and D, fibronectin, and small airway epithelial cells under shear conditions. Infect Immun. 2006;74(6):3587–3596. doi: 10.1128/IAI.01644-05. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Sano H, Kuroki Y. The lung collectins, SP-A and SP-D, modulate pulmonary innate immunity. Mol Immunol. 2005;42(3):279–287. doi: 10.1016/j.molimm.2004.07.014. [DOI] [PubMed] [Google Scholar]
  • 36.Ferguson JS, Voelker DR, McCormack FX, Schlesinger LS. Surfactant protein D binds to Mycobacterium tuberculosis bacilli and lipoarabinomannan via carbohydrate-lectin interactions resulting in reduced phagocytosis of the bacteria by macrophages. J Immunol. 1999;163(1):312–321. [PubMed] [Google Scholar]
  • 37.Birchler T, Seibl R, Buchner K, Loeliger S, Seger R, Hossle JP, Aguzzi A, Lauener RP. Human Toll-like receptor 2 mediates induction of the antimicrobial peptide human beta-defensin 2 in response to bacterial lipoprotein. Eur J Immunol. 2001;31(11):3131–3137. doi: 10.1002/1521-4141(200111)31:11<3131::aid-immu3131>3.0.co;2-g. [DOI] [PubMed] [Google Scholar]
  • 38.Armstrong L, Medford AR, Uppington KM, Robertson J, Witherden IR, Tetley TD, Millar AB. Expression of functional toll-like receptor-2 and -4 on alveolar epithelial cells. Am J Respir Cell Mol Biol. 2004;31(2):241–245. doi: 10.1165/rcmb.2004-0078OC. [DOI] [PubMed] [Google Scholar]

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