Cutting Edge: Inflammatory Signaling by Borrelia burgdorferi Lipoproteins Is Mediated by Toll-Like Receptor 2

Materials and Methods

Results

The human glioma cell line U373 responds vigorously to LPS, but not to OspA, as assayed by IL-6 production (Fig. 1⇓B). In addition, U373 cells produce detectable levels of transcript for TLR1, TLR3, TLR4, and TLR5 (Fig. 1⇓A). No transcript was detected for TLR2 in U373 cells (Fig. 1⇓A). Interestingly, the human monocytic line THP-1 was OspA-responsive and possessed very high levels of TLR2 transcript (data not shown), while the nonresponsive U373 cells produced none. These findings suggest a correlation between expression of TLR2 and the ability of cell lines to respond to the B. burgdorferi lipoprotein OspA.

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FIGURE 1.

Lipoprotein responsiveness correlates to TLR2 expression in U373 cells. A, RT-PCR detection of TLR expression. mRNA expression for TLR1-TLR5 was quantified in U373 cells and normalized to expression of HPRT. B, Transfection of TLR2 corrects lipoprotein-unresponsiveness in U373 cells. U373 cells were transiently transfected with expression vector alone (pFLAG) or expression vector with specific TLR construct. Cells were treated with: media with 5 μg/ml polymyxin B, LPS at 100 ng/ml with 2% human serum, recombinant OspA at 500 ng/ml with 5 μg/ml polymyxin B. Supernatants were collected after 24 h and were assayed for IL-6 production by ELISA.

To test whether the lipoprotein unresponsive phenotype could be corrected in U373 cells, expression constructs encoding four of the TLR proteins were transfected individually into this cell line, and the ability to respond to lipoproteins was measured by IL-6 production. Transfection of TLR2 conferred responsiveness to OspA in U373 cells (Fig. 1⇑B), whereas transfection of vector alone, TLR1, TLR3, or TLR4 did not. TLR4 did significantly increase IL-6 production of U373 cells when stimulated with LPS (Fig. 1⇑B), a result that agrees with previous reports suggesting that TLR4 is involved in LPS signaling (24, 25, 26, 27).

To test this effect in a second TLR2-negative cell line (22), expression constructs encoding the four TLR proteins were transiently transfected into the human embryonic kidney line 293. The TLR expression plasmids were cotransfected with a NF-κB-dependent luciferase reporter plasmid that contained the E-selectin (ELAM-1) promoter. To normalize for transfection efficiency, an RSV-β-galactocidase control plasmid was also cotransfected. Only transfection of the TLR2 expression construct conferred responsiveness to OspA in 293 cells (Fig. 2⇓, A and B) and was reflected in both luciferase and endogenous IL-8 production. As reported previously, TLR2 also conferred responsiveness to LPS (22, 23). Because TLR2 conferred responsiveness to both lipoproteins and LPS, polymyxin B was added to all lipoprotein samples to eliminate stimulation due to possible LPS contamination during experimental manipulation. Transfection of TLR4 resulted in constitutive activation, as reported by others in this cell type (22), and was not up-regulated by either LPS or OspA. None of the other constructs, pFLAG vector alone, TLR1, or TLR3, conferred responsiveness to either agonist (Fig. 2⇓, A and B).

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FIGURE 2.

Transfection of TLR2 confers responsiveness to lipoproteins in 293 cells. A, NF-κB nuclear translocation in Toll-like receptor-expressing cells. 293 cells were transiently transfected with expression vector alone (pFLAG) or expression vector with specific TLR construct, plus the ELAM-1 luciferase reporter construct. Cells were stimulated for 6 h with the indicated agonist. NF-κB nuclear translocation is indicated by luciferase units. Media with 5 μg/ml polymyxin B, LPS at 100 ng/ml, recombinant OspA at 500 ng/ml plus 5 μg/ml polymyxin B. B, Induction of IL-8 secretion in Toll-like receptor-transfected cells. Supernatants from the samples shown in A were assayed for IL-8 production by ELISA. C, Specificity of TLR2-induced responsiveness to lipoproteins. 293 cells were transiently cotransfected with the TLR2 expression plasmid and the ELAM-1-luciferase construct. Cells were stimulated for 6 h and NF-κB nuclear translocation is indicated by luciferase units. Media is media alone; Media/PB has 5 μg/ml of polymyxin B; LPS is 100 ng/ml LPS; LPS/PB is 100 ng/ml LPS plus 5 μg/ml polymyxin B. The remaining samples all contained 5 μg/ml polymyxin B and are as follows: OspA, recombinant OspA at 500 ng/ml; Bb OspA, OspA purified from strain N40 of B. burgdorferi at 100 ng/ml; OspB, recombinant OspB at 500 ng/ml; Sonicated Bb, 1 μg/ml sonicated strain N40 of B. burgdorferi; Pam3Cys, Pam₃Cys-Ser-Lys₄-OH at 200 ng/ml; and UOspA, unlipidated recombinant OspA at 500 ng/ml.

The experiments in Figs. 1⇑, 2⇑A, and 2B used recombinant lipidated OspA, which was previously shown to possess the same stimulatory properties as OspA purified from B. burgdorferi (30). This was confirmed in Fig. 2⇑C, where OspA purified from B. burgdorferi was found to activate NF-κB in 293 cells transfected with TLR2. Sonicated B. burgdorferi, an abundant source of lipoproteins, was also a strong stimulant in TLR2-expressing cells. A second recombinant lipoprotein, OspB, which possesses the same lipid modification on a distinct polypeptide, also strongly activated TLR2-expressing cells. The fact that the synthetic lipopeptide Pam₃Cys-Ser-Lys₄-OH also possesses this activity further supports the requirement for the lipid modification. These findings indicate that several distinct Pam₃Cys-containing molecules induce inflammatory signaling through TLR2, and suggest that the interaction is dependent on the common lipid modification. This specificity was confirmed with unlipidated OspA, a recombinant protein that possesses the same amino acid sequence as lipidated OspA, but lacks the amino-terminal cysteine required for the Pam₃Cys modification (31). The unlipidated OspA did not activate either NF-κB translocation (Fig. 2⇑C) or IL-8 production (data not shown) in TLR2-expressing 293 cells. These results indicate that the lipid modification is required for cellular activation via TLR2, and that several lipoproteins and a synthetic lipopeptide possess this activity.

Further evidence that lipoproteins are a biologically important ligand for TLR2 was sought by comparing the dose required for NF-κB translocation and IL-8 production by LPS and OspA. Human neutrophils have been found to respond to 10-fold lower molar concentrations of LPS than OspA when adequate amounts of LPS binding protein and CD14 are present (9). In contrast, TLR2-transfected 293 cells responded to at least 10-fold lower concentrations of OspA than LPS (Fig. 3⇓, A and B). The greater sensitivity of TLR2-expressing cells to OspA than LPS strongly argues that TLR2 is important in mediating inflammatory signaling by lipoproteins in humans.

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FIGURE 3.

Comparison of the sensitivity of TLR2-dependent responses to OspA and LPS. A, NF-κB nuclear translocation in response to increasing doses of OspA or LPS. 293 cells transiently cotransfected with the TLR2 expression plasmid and ELAM-1 luciferase construct were incubated with increasing concentrations of LPS, OspA plus 5 μg/ml polymyxin B, or LPS plus 5 μg/ml polymyxin B for 6 h. Samples were assayed for NF-κB translocation by luciferase. B, IL-8 production in response to increasing doses of OspA or LPS. Supernatants from the samples shown in A were assayed for IL-8 production by ELISA.

The finding that TLR2-negative U373 cells are responsive to LPS but not lipoproteins suggests that endogenously expressed TLR2 mediates inflammatory signaling by bacterial lipoproteins. The extremely high expression of TLR2 obtained in transiently transfected 293 cells may promote responsiveness to LPS that is not observed under physiological conditions. When the amount of TLR2 construct transfected into 293 cells was reduced, responsiveness to both LPS and OspA were conferred by transfection with as little as 0.03 μg of TLR2-expressing plasmid (Fig. 4⇓). Very low concentrations of plasmid conferred maximal responsiveness in 293 cells, and luciferase units remained high at the highest concentrations of plasmid, arguing that the difference in dose response seen between LPS and OspA did not reflect differential saturation of cofactors by either agonist. This finding further suggests that the dependence of LPS responsiveness on TLR2 expression in transfected 293 cells and its independence of TLR2 in U373 cells may reflect the influence of other essential components of the signaling pathway present in these different cell types.

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FIGURE 4.

Dose dependence of TLR2 conferred responsiveness to OspA. 293 cells were transiently cotransfected with the indicated amount of TLR2 expression plasmid plus the ELAM-1 luciferase construct. Luciferase activity was measured in cells collected 6 h following stimulation. Media, media with 5 μg/ml polymyxin B; LPS, 100 ng/ml LPS; OspA, recombinant OspA at 500 ng/ml plus 5 μg/ml polymyxin B.

OspA activation of human endothelial cells, neutrophils, monocytes, and mouse monocytes has been shown to be facilitated by the LPS coreceptor CD14 (20, 21). Although 293 cells do not express CD14 on their surface (22), human serum present in the culture medium provides soluble CD14. OspA activation of 293 cells transfected with the TLR2 expression plasmid was diminished in the presence of the CD14-blocking mAb, 60bca, when compared with an isotype control (Fig. 5⇓). The diminished activity in the presence of blocking Ab is consistent with the 10- to 20-fold inhibition previously reported in endothelial cells, monoctyes, and neutrophils (20, 21). These findings indicate that TLR2-mediated lipoprotein signaling can be enhanced by soluble CD14 in a manner consistent with that exhibited by cell types naturally responsive to bacterial lipoproteins.

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FIGURE 5.

CD14 facilitates inflammatory activation by OspA. 293 cells were transfected with 0.03 μg of the TLR2 expression plasmid in the presence of the ELAM-1 luciferase construct. Transfected cells were then cultured in DMEM with 2% human serum for 36 h followed by the addition of increasing concentrations of OspA in the presence of 5 μg/ml of polymyxin B and 5 μg/ml of either a CD14 blocking Ab (60bca) or its isotype control (mopc 21). LPS, 100 ng/ml of LPS. NF-κB translocation to the nucleus was measured by luciferase.

Discussion

Innate recognition of microbial products forms the initial defense against pathogen infection. In Drosophila, activation of Toll controls the induction of the antifungal peptide drosomycin in response to fungal infection (32), while two of the mammalian homologues, TLR2 and TLR4, have been associated with LPS responses. Our results demonstrate that TLR2 is required for bacterial lipoprotein signaling in two different TLR2-negative cell lines, U373 and 293, and that signaling requires the lipid modification. Although both LPS and lipoproteins can induce signaling through TLR2, transfected 293 cells responded to at least 10-fold lower concentrations of lipoprotein than LPS, supporting the physiological relevance of the lipoprotein interaction. Additionally, the defect in TLR4 that has been associated with the inability to C3H/HeJ mice to respond to LPS while responding normally to B. burgdorferi lipoproteins suggests that additional regulatory pathways are present in mammalian cells.

Other properties have been described for the Pam₃Cys modification of bacterial lipoproteins, including that of immunological adjuvant (31). Animal studies of the OspA-based Lyme vaccine have demonstrated that its immungenicity is dependent on this lipid modification (31). The identification of TLR2 as a critical component of Pam₃Cys signaling may provide a mechanism by which lipoproteins stimulate humoral immune responses without additional adjuvant.

Lyme arthritis, the most common manifestation of late Lyme disease, is associated with the presence of B. burgdorferi in the joint (3, 4). The abundantly expressed lipoproteins are intricately involved in the pathology of Lyme disease. These lipoproteins possess potent stimulatory properties for inflammatory cells that are associated with affected joints: namely neutrophils, mononuclear cells, and endothelial cells (6, 7, 8, 9, 15, 16). Thus, understanding the molecular basis of the signaling events caused by lipoproteins will lead to a greater understanding of the inflammatory events associated with Lyme arthritis in general. In addition, lipoproteins are broadly expressed by numerous pathogen species, many of which have been associated with inflammatory pathology (33, 34, 35). Thus, an understanding of lipoprotein-induced signaling events will have widespread implications in the understanding of the diverse pathologies caused by these microbial species.