Induction of Direct Antimicrobial Activity Through Mammalian Toll-Like Receptors

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Science  23 Feb 2001:
Vol. 291, Issue 5508, pp. 1544-1547
DOI: 10.1126/science.291.5508.1544


The mammalian innate immune system retains fromDrosophila a family of homologous Toll-like receptors (TLRs) that mediate responses to microbial ligands. Here, we show that TLR2 activation leads to killing of intracellular Mycobacterium tuberculosis in both mouse and human macrophages, through distinct mechanisms. In mouse macrophages, bacterial lipoprotein activation of TLR2 leads to a nitric oxide–dependent killing of intracellular tubercle bacilli, but in human monocytes and alveolar macrophages, this pathway was nitric oxide–independent. Thus, mammalian TLRs respond (asDrosophila Toll receptors do) to microbial ligands and also have the ability to activate antimicrobial effector pathways at the site of infection.

The primitive immune system of Drosophila has evolved to be highly efficient at combating microbial pathogens, largely through a family of cell-surface receptors known as Toll. The activation of Toll proteins by microbial ligands triggers an intracellular signaling pathway involving nuclear factor κB (NF-κB) homologs that leads to the transcription of genes encoding antimicrobial proteins (1–3). TheDrosophila Toll system is structurally conserved and homologous to the mammalian TLR family (4). Microbial ligands, including lipopolysaccharide (LPS) and bacterial lipoproteins, have been shown to activate mammalian TLRs, facilitating transcription of genes that regulate the adaptive response, including cytokines and costimulatory molecules (4–10). It remains unclear, however, whether activation of mammalian TLRs triggers direct antimicrobial effector pathways.

We initially investigated whether activation of mouse macrophages by bacterial lipoproteins reduced the viability of intracellular Mycobacterium tuberculosis(11). The 19-kD lipoprotein of M. tuberculosis(8) or the Tp47 lipopeptide of Treponema pallidumreduced the viability of intracellular M. tuberculosis in a murine macrophage-like cell line, RAW264.7, by up to 70%, as measured by colony-forming units (CFUs) (Web fig. 1) (11, 12). The induction of inducible nitric oxide synthase (iNOS) and release of nitric oxide (NO) represent a powerful antimycobacterial defense mechanism in mice (13–15). Because bacterial lipoproteins induce iNOS promoter activity (8), we hypothesized that the antimicrobial activity observed after cellular activation with lipoproteins could be mediated through TLR-induced NO production. M. tuberculosis–infected RAW cells were stimulated with the 19-kD lipoprotein in the presence or absence of pharmacologic inhibitors of iNOS (11) and assayed for NO production (16) and antimicrobial activity. We found that the production of NO was almost completely suppressed in the presence ofl-N6 -(1-iminoethyl)-lysine (l-NIL) orl-N 6-nitro- arginine-methyl ester (l-NAME), but not the inactive enantiomerd-NAME (Fig. 1A, left). Concomitantly, inhibition of NO production abrogated the antimicrobial activity induced by the 19-kD lipoprotein (Fig. 1A, right), demonstrating the requirement for NO in TLR-mediated antimicrobial responses.

Figure 1

Microbial lipoproteins trigger growth inhibition of intracellular M. tuberculosis. (A) The 19-kD lipoprotein of M. tuberculosis mediates an NO-independent antimicrobial activity in RAW cells. The CFUs were determined 48 hours after infection of RAW cells with an MOI of 5. The inhibitors (l-NIL, 1 mM; l-NAME, 2 mM) were added to the infected cells immediately after the end of the pulse infection, together with the 19-kD lipoprotein (1 μg/ml). The figures represent the average ± SEM of three or more independent experiments. (B) Lipoprotein-induced killing ofM. tuberculosis is mediated by TLR2. Thioglycollate-elicited macrophages from control (wt) and gene-deleted (TLR2–/–) mice were infected with M. tuberculosis at an MOI of 5. Production of NO (Griess reaction) and bacterial load (plating of cell lysates) were determined 48 hours after addition of the 19-kD lipoprotein (1 μg/ml) or LPS (0.1 μg/ml). The figure shows the average of one experiment ± SEM performed in triplicate.

To establish that the lipoprotein-induced antimicrobial activity is dependent on TLR activation, we tested the ability of the 19-kD protein to activate the antimicrobial pathway of peritoneal macrophages from TLR2-deficient (TLR2−/−) or TLR4-deficient (TLR4−/−) mice (17). Experiments were performed with uninfected [Web fig. 2 (12)] and M. tuberculosis–infected (Fig. 1B) macrophages. Primary peritoneal macrophages from TLR2−/− mice responded only marginally in production of NO in response to the 19-kD protein and did not appreciably reduce the viability of intracellular M. tuberculosiscompared with wild-type macrophages (Fig. 1B). This result establishes that activation of TLR2 on murine macrophages leads to NO-dependent growth inhibition of intracellular M. tuberculosis.

The question of whether human macrophages exert antimicrobial activity through NO remains highly controversial, and we sought to ascertain whether they did so after activation of TLRs. Activation of human monocytes with bacterial lipoproteins reduced the viability of intracellular M. tuberculosis to an extent comparable in magnitude to that found in activated mouse macrophages (Web fig. 3) (12, 18). The killing of intracellularM. tuberculosis in human monocytes was clearly dependent on lipoprotein activation via TLR2, because a monoclonal antibody against TLR2 (8, 9) blocked the reduction in CFUs by greater than 90% (Fig. 2A). In striking contrast to mouse macrophages, the addition of the iNOS inhibitor, l-NIL, failed to block the 19-kD lipoprotein–induced killing of intracellular M. tuberculosis. This correlated with the failure of the 19-kD lipoprotein to induce production of NO in human monocytes (Fig. 2B). This observation was extended by use of another potent stimulus known to induce NO in mouse macrophages, the combination of tumor necrosis factor–α (TNF-α) and interferon-γ (IFN-γ) (19) (Fig. 2B). In mouse RAW cells, TNF-α plus IFN-γ induced the production of NO and reduced the viability of intracellularM. tuberculosis to an extent equivalent to the effect of the 19-kD lipoprotein. In contrast, in human monocyte-derived macrophages, the combination of TNF-α plus IFN-γ neither induced detectable NO production nor exerted any antimicrobial effect, but did induce cytokine release. TNF-α has been shown to induce growth inhibition of avirulent M. tuberculosis in human macrophages (20), but we found it to support the growth of virulentM. tuberculosis in these cells (21). Furthermore, the addition of antibodies against TNF-α did not abrogate the antimicrobial activity of the 19-kD lipoprotein against the virulent form used in this study (Fig. 2C). Taken together, these data indicate that TLR2 activation in human monocytes induces a powerful antimicrobial activity that is independent of NO and TNF-α.

Figure 2

Primary human cells kill M. tuberculosis via a TLR2-dependent, but NO-independent, pathway. (A) Inhibition of mycobacterial growth in human monocytes is inhibited in the presence of a blocking antibody to TLR2 (αTLR2), but is independent of NO production. Infected monocytes (MOI 5) were cultured in the presence of an antibody against TLR2 (10 μg/ml) or l-NIL (1 mM) for 48 hours, and intracellular growth was determined by plating serial dilutions of cell lysates in duplicates. (B) TNF-α and IFN-γ induce the production of NO and antibacterial activity in mouse macrophages, but not in human monocytes. TNF-α and IFN-γ (both at 10 ng/ml) were added to infected murine macrophages (RAW) or human monocytes. NO release and bacterial viability were determined after 48 hours. The figures show the average of four independent experiments ± SEM using cells from different donors. (C) Antimycobacterial activity of the 19-kD lipoprotein is TNF-independent. The 19-kD lipoprotein (2 μg/ml) and antibodies against TNF (anti-TNF-α, 20 μg/ml) were added as indicated. Cells were lysed after 48 hours, and the number of CFUs were determined.

The ability of alveolar macrophages (AMs) to kill M. tuberculosis represents a critical local defense mechanism in determining the outcome of tuberculosis infection in the lung. Alveolar macrophages were isolated from bronchoalveolar lavage, infected with M. tuberculosis (22), and stimulated with the M. tuberculosis 19-kD lipoprotein, then survival of the bacteria was determined. Stimulation of infected AMs with lipoprotein induced an antimicrobial response that was independent of NO release, but dependent on TLR2 signaling (Fig. 3A). Consistent with the failure of the specific iNOS inhibitor l-NIL to inhibit antibacterial activity, AMs infected with mycobacteria failed to secrete detectable amounts of NO in vitro. Although it has been reported that AMs can express iNOS (23), these results further indicate that a mechanism independent of NO contributes to their microbicidal activity. The presence of TLR2 on cells of the monocyte/macrophage lineage in lesions of tuberculosis infection indicates that activation of TLR2 could contribute to host defense at the site of disease activity (Fig. 3B and Web figs. 4 and 5) (24).

Figure 3

TLR2 is expressed at the site of infection in human tuberculosis. (A) Activation of AMs by the 19-kD lipoprotein reduces the viability of intracellular M. tuberculosis by a TLR2-dependent, NO-independent mechanism. AMs were infected with M. tuberculosis (MOI 2) for 4 hours, and the 19-kD lipoprotein (1 μg/ml) or the inhibitors were added. Bacterial growth was determined after 48 hours. The graph shows one representative experiment of four using cells from different donors and are expressed as CFUs ± SEM. (B) TLR2 expression in tuberculous lymphadenitis by immunoperoxidase. Photomicrograph (40× objective) demonstrates the presence of TLR2 (red label) on large ovoid cells within granulomas, typical of cells of the monocyte/macrophage lineage.

Throughout millions of years of evolution, the immune system, from insects to mammals, has retained the structure of Toll receptors, as well as an NF-κB signaling pathway, as an innate mechanism to respond to threats from microbial pathogens. The present results indicate that the mouse and human TLR pathway has similarly retained the ability to activate direct antimicrobial effector mechanisms, although the pathways are distinct. Whereas, in mice, TLR activation leads to an NO-dependent antimicrobial pathway, in humans the TLR-activated antimicrobial pathway is NO-independent. InDrosophila, activation of Toll leads to the induction of a variety of antimicrobial peptides, including metchnikowan, defensins, cecropins, and drosomycin (1–3). The present study suggests that a detailed investigation of the TLR2-dependent antimicrobial effector mechanisms in human cells of the monocyte/macrophage lineage is warranted. The clues fromDrosophila suggest that exploring induction of antimicrobial peptides in these cells could be fruitful (25). Knowledge arising from such investigation should provide new insights into mechanisms of innate immunity in humans.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: rmodlin{at}


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