Host Defense Mechanisms Triggered by Microbial Lipoproteins Through Toll-Like Receptors

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Science  30 Jul 1999:
Vol. 285, Issue 5428, pp. 732-736
DOI: 10.1126/science.285.5428.732


The generation of cell-mediated immunity against many infectious pathogens involves the production of interleukin-12 (IL-12), a key signal of the innate immune system. Yet, for many pathogens, the molecules that induce IL-12 production by macrophages and the mechanisms by which they do so remain undefined. Here it is shown that microbial lipoproteins are potent stimulators of IL-12 production by human macrophages, and that induction is mediated by Toll-like receptors (TLRs). Several lipoproteins stimulated TLR-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal pathway. Activation of TLRs by microbial lipoproteins may initiate innate defense mechanisms against infectious pathogens.

Besides causing disease, mycobacteria have long been recognized for having powerful immunologic adjuvant activity, augmenting both cell-mediated and humoral immune responses. In 1972, a study of the mechanism of mycobacterial adjuvants demonstrated the induction of “soluble mediators,” now known to be cytokines, which mediated the augmentation of immune responses (1). One cytokine induced by mycobacteria is IL-12 (2), a powerful signal for the generation of T helper type 1 lymphocyte (TH1) responses (3) required to eliminate intracellular pathogens (4), including Mycobacterium tuberculosis(5). Furthermore, individuals with mutations in the IL-12 receptor (IL-12R) have increased susceptibility to mycobacterial infection (6). We therefore investigated the mycobacterial products that induce IL-12 as well as the mechanism responsible for its induction.

Mycobacterium tuberculosis H37Rv was gamma-irradiated and lysed by mechanical disruption, subcellular fractions were generated (7) and tested for the capacity to induce IL-12 with a human monocyte line, THP-1 (8). Other than the M. tuberculosis lysate, the soluble cell wall–associated proteins (SCWPs) contained most of the IL-12 p40–inducing capacity, consistent with the known adjuvant activity of mycobacterial cell walls (Fig. 1A). The combined cytosolic and membrane protein fraction, the culture filtrate, the lipoglycan from M. tuberculosis, and the mycolyl arabinogalactan peptidoglycan complex were less potent on a per weight basis (9).

Figure 1

A 19-kD M. tuberculosislipoprotein induces IL-12 from monocytes. (A) Ability ofM. tuberculosis subcellular fractions to stimulate IL-12 release from monocytes. The detergent-soluble cell wall–associated subcellular fraction (SCWP) retains most of the M. tuberculosisIL-12 p40–inducing capacity, as determined with the THP-1 human monocyte cell line (8). Values are expressed as mean ± SEM of duplicate determinations. Subcellular fractions of M. tuberculosis were isolated as previously described (7). Solprot, combined cytosolic and membrane fraction; manLAM, lipoglycan from M. tuberculosis. (B) SDS-PAGE ofM. tuberculosis proteins from isoelectric focusing fraction 4 (10). Molecular size markers are in the left lane, and sizes are indicated in kilodaltons. (C) SDS-PAGE–separated proteins stimulate IL-12 release. Methods are described in (13). (D) The 19-kD lipoprotein ofM. tuberculosis is a potent inducer of IL-12 p40 release. THP-1 cells were stimulated with M. tuberculosis lysate and purified M. tuberculosis 19-kD, 38-kD, and Ag85 complex proteins (12).

To identify the cell wall–associated proteins responsible for IL-12 p40 release, we fractionated the SCWP preparation by gel filtration chromatography and preparative isoelectric focusing and monitored the bioactivity by measuring IL-12 p40 production (10). Subsequent separation of the bioactive fraction by SDS-PAGE indicated four prominently stained proteins: three bands at 17, 21.5, and 39 kD and a doublet at 60 to 70 kD (Fig. 1B). After these regions were transferred to nitrocellulose, they were solubilized (11) and used to stimulate THP-1 cells. The band migrating to 21.5 kD induced the highest levels of IL-12 p40 release, followed by the 39-kD band, with the two other regions inducing little activity (Fig. 1C). By using monoclonal antibodies to known M. tuberculosis antigens for protein immunoblot analysis, we identified the 21.5-kD band as the 19-kD lipoprotein antigen of M. tuberculosis and the 39-kD band as the M. tuberculosis 38-kD lipoprotein antigen (PstS homolog) (12). Purified 19- and 38-kD lipoproteins ofM. tuberculosis induced IL-12 p40 release, with the 19-kD lipoprotein greater than one log more potent than whole M. tuberculosis and the 38-kD lipoprotein (Fig. 1D). The ability of the 19-kD lipoprotein to induce IL-12 was independent of tumor necrosis factor–α (TNF-α) release, because neutralizing antibodies to TNF-α (anti-TNF-α) blocked by less than 10% the induction of IL-12 p40 (13). The 19-kD lipoprotein also stimulated IL-12 p70 release from normal human monocytes (14). Thus, the M. tuberculosis 19-kD lipoprotein is a major inducer of IL-12, a cytokine that can amplify TH1 and cytolytic T cell responses and hence contribute to the adjuvant activity of mycobacteria.

We investigated the mechanism by which the 19-kD lipoprotein induces IL-12 by using an IL-12 p40 promoter CAT reporter transiently transfected into the mouse macrophage cell line RAW 264.7 (15, 16). The 19-kD lipoprotein induced IL-12 p40 promoter activity in a dose-dependent manner and at a level comparable to lipopolysaccharide (LPS) (Fig. 2A). Previous studies indicated that LPS induction of IL-12 p40 promoter activity is dependent on both C/EBP and nuclear factor kappa B (NF-κB) sites (15,17). The ability of both the 19-kD lipoprotein and LPS to induce IL-12 p40 promoter activity was reduced in substitution mutants of the C/EBP and NF-κB sites, but was not affected by a mutation in a PU.1-binding site (Fig. 2B).

Figure 2

Lipoproteins induce the IL-12 p40 promoter through NF-κB and C/EBP and are blocked by a TLR-2 dominant negative mutant. (A) The 19-kD lipoprotein induces IL-12 p40 promoter activity. RAW 264.7 cells were transiently transfected with a murine IL-12 p40 promoter CAT reporter as described (15,16). Transfectants were stimulated with LPS (Salmonella typhosa, Sigma, St. Louis, MO) or 19-kD lipoprotein, or left unstimulated for 24 hours. Activation of IL-12 p40 promoter activity was measured according to CAT activity (percent chloramphenicol acetylation) with a phosphorimager. Data were normalized to a cotransfected β-galactosidase construct for transfection efficiency. Data are representative of three experiments. No stimulation was observed with a control CAT reporter plasmid that lacked the IL-12 p40 promoter sequence. (B) IL-12 p40 promoter mutations in the NF-κB (−131/−122) and C/EBP (−95/−88) sites blocked LPS (gray, 5 μg/ml) and 19-kD lipoprotein (black, 5 μg/ml) stimulated promoter activation. RAW 264.7 cells were transiently transfected as described above with NF-κB, C/EBP, and PU.1 mutant constructs, and promoter induction levels were measured by CAT assay (15, 16). (C) A mutant form of TLR-2 (TLR-2 dn1) lacking 13 amino acids of the COOH-terminal domain inhibits LPS and lipoprotein induction of IL-12 p40 promoter activity. Data reflect at least three independent experiments and are reported as a percentage of antigen-stimulated IL-12 p40 promoter activity cotransfected with a vector control. Lipoproteins and lipopeptides were prepared as described (31). (D) Monoclonal antibody to TLR-2 blocks LPS- and lipoprotein-induced, but not CD40L trimer–induced, IL-12 p40 production from human adherent monocytes (28, 38, 41).

Given that the 19-kD lipoprotein induced monocyte IL-12 in a manner analogous to LPS, we reasoned that the cell surface receptor that transduces the signal for the 19-kD lipoprotein may be identical to that for LPS. Because Toll-like receptors (TLRs) have been reported to activate monocyte cytokine production (18), to bind LPS, and to transduce the proper signal for LPS-stimulated gene activation in monocytes (19), we hypothesized that the 19-kD lipoprotein could induce IL-12 through TLRs. TLR family members are transmembrane proteins containing repeated leucine-rich motifs in their extracellular portions, similar to other pattern recognition proteins of the innate immune system. TLR proteins also contain a cytoplasmic domain that is homologous to the signaling domain of the IL-1 receptor and can activate a signaling pathway that includes activation of NF-κB and subsequent gene transcription (19,20). We investigated the role of TLR in 19-kD lipoprotein–induced IL-12 production by cotransfecting the RAW 264.7 macrophage cell line with a TLR-2 dominant negative mutant containing a truncation of 13 amino acids at the COOH-terminus (19), along with the IL-12 p40 promoter construct (16). Transfection of various amounts of the TLR-2 dominant negative mutant (TLR-2 dn1) inhibited both 19-kD lipoprotein–and LPS-induced IL-12 p40 promoter activation relative to a vector control (Fig. 2C). Activity was not inhibited by transfection of a vector containing a control gene, IL-1R (21).

The 19-kD M. tuberculosis lipoprotein is a member of a family of prokaryotic lipoproteins. Lipoproteins have been found extensively in both Gram-positive and Gram-negative bacteria, includingTreponema pallidum, Mycoplasma species, andBorrelia burgdorferi (22–24). Profound immunoregulatory functions have been attributed to lipoproteins, including monocyte or macrophage activation (25). The portion of lipoprotein responsible for its immunologic activity is located in the NH2-terminal triacylated lipopeptide region. Removal of this lipid element rendered the parent product nonactivating, and synthetic lipopeptides could activate B cells and macrophages (23, 24, 26). Studies of the B. burgdorferi OspA lipoprotein and the 47-kD lipopeptide of T. pallidum demonstrated lipoprotein induction of IL-12 mRNA (24, 27). We found that OspA and the NH2-terminal lipopeptide of the T. pallidum47-kD antigen activated IL-12 p40 promoter activity by a TLR-dependent mechanism (Fig. 2C), thereby providing evidence that TLRs serve to recognize a diverse family of microbial lipoproteins. A monoclonal antibody specific to human TLR-2 (28) blocked the ability of LPS and the 19-kD lipoprotein to stimulate IL-12 production from primary human monocytes, indicating the crucial role for TLR-2 in monocyte activation by these microbial molecules (Fig. 2D). Because the deacylated OspA (d-OspA) was unable to activate IL-12 production from THP-1 cells (29), the fatty acyl moiety, which is genetically and structurally conserved among microbial lipoproteins, appears to be crucial for monocyte activation through TLRs.

Having shown that TLRs are necessary for gene activation by lipoproteins, we sought to learn whether TLRs are also sufficient. Using HEK 293 cells, we transfected the NF-κB–responsive ELAM enhancer, because activated NF-κB is required for IL-12 p40 promoter activity. HEK 293 cells do not express TLR-2, nor could they be activated by LPS or microbial lipoproteins, as determined by examination of NF-κB induction (Fig. 3A) (19, 30). In contrast, in stable transfectants of HEK 293 cells expressing TLR-2, microbial lipoproteins induced NF-κB in a dose-dependent manner and at levels comparable to those induced by LPS. Activation by lipoproteins was enhanced by cotransfection with a CD14 expression vector to levels analogous to LPS induction and consistent with the role of CD14 in facilitating lipoprotein activity (30–32) (Fig. 3B). Activation through TLR-2 by lipoproteins was dependent on fatty acyl moieties because deacylated forms of OspA and T. pallidumlipopeptide (d-Tp47) had no activity (Fig. 3C). Although the data indicate that TLR-2 can mediate gene activation by microbial lipoproteins, the data do not preclude a contributory role for other TLR family members (33).

Figure 3

TLR proteins are sufficient for the induction by lipoproteins of NF-κB activation, and induction is enhanced by the presence of CD14. (A) Stable expression of TLR-2 in HEK 293 cells confers lipoprotein responsiveness. HEK 293 TLR-2 and HEK 293 vector control cells were transiently transfected with a luciferase reporter gene driven by the NF-κB responsive enhancer of the E-selectin gene (19, 30). Luciferase activity (RLU) was measured with a luminometer (arbitrary units). Tp47 concentrations are corrected by 6.25 × 10−3. (B) CD14 enhances lipoprotein activation of NF-κB through TLR-2. HEK 293 TLR-2 and HEK 293 vector control cells were cotransfected with ELAM-luc (0.5 μg), along with CD14 expression plasmid (1 μg) or vector control (1 μg) (30). Twenty-four hours after transfection the transfectants were activated with a titration of LPS or lipoproteins for 6 hours. Activity was measured by luciferase assay. Data are representative of three independent experiments. (C) Fatty acyl moieties are required for TLR-dependent activation of NF-κB. The HEK 293 TLR-2 stable clone and HEK 293 control cells were transiently transfected with the ELAM-luciferase construct and activated with the OspA lipoprotein, the Tp47 lipopeptide, or deacylated forms of both antigens (31). NF-κB activation was measured by luciferase assay.

To determine whether the TLR signaling pathway stimulated by microbial lipoproteins could be linked to a known macrophage antimicrobial mechanism, we investigated whether the M. tuberculosis lipoprotein could activate gene transcription for inducible nitric oxide synthase (iNOS), given the critical role of iNOS in the production of nitric oxide from macrophages, currently the only effective macrophage mycobactericidal mechanism known in vitro and in vivo (34). Analysis of gene-disrupted mice revealed that this mechanism was necessary for protection against M. tuberculosis (35). The 19-kD and OspA lipoproteins induced iNOS promoter activity in the RAW 264.7 macrophage cell line (16, 36). Again, activation was dependent on the fatty acyl moieties, because d-OspA had no activity (Fig. 4A). Cotransfection with the TLR-2 dominant negative mutant inhibited the ability of lipoproteins to induce the iNOS promoter (Fig. 4B), thereby suggesting a role for TLRs in the activation of iNOS by microbial pathogens. Stimulation of monocytes with the 19-kD antigen also induced production of nitric oxide (37).

Figure 4

Microbial lipoproteins induce iNOS gene transcription through TLRs. (A) Microbial lipoproteins induce iNOS promoter activity in RAW 264.7 cells. RAW 264.7 cells were transiently transfected with the iNOS promoter construct as described previously (16). (B) A dominant negative mutant of TLR-2 (TLR-2 dn1) inhibits LPS and lipoprotein induction of iNOS promoter activity. Data reflect at least two independent experiments and are reported as a percentage of antigen-stimulated iNOS promoter activity in cells not transfected with TLR-2 dn1 expression plasmid (16). Media controls were comparable between vector control and TLR-2 dn1 transfectants.

The presence of Toll in Drosophila indicates that Toll proteins represent a host defense mechanism that has been conserved over hundreds of millions of years of evolution. In mammals, TLRs provide the innate immune system with the ability to react to a spectrum of microbial pathogens expressing lipoproteins and lipopolysaccharides. Animals with altered TLRs manifest increased susceptibility to infection (33). Our data indicate that TLRs can activate innate immune responses including the generation of NO, a direct microbicidal mechanism, and provide an early signal for induction of IL-12, which functions as a biologic adjuvant amplifying acquired T cell responses to pathogens. Under certain conditions, the TLR signaling pathway can lead to apoptosis of the target cells resulting in down-regulation of the immune response or pathology to the host (28). It should be possible, however, to develop strategies to stimulate TLRs in order to activate antimicrobial defense mechanisms and to amplify immune responses to a variety of antigens in vaccines.

  • * These authors contributed equally to this work.

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


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