Cutting Edge: A Toll-Like Receptor 2 Polymorphism That Is Associated with Lepromatous Leprosy Is Unable to Mediate Mycobacterial Signaling

Cells and reagents

Human embryonic kidney (HEK)293 (ATCC no. CRL-1573; American Type Culture Collection, Manassas, VA) and Chinese Hamster Ovary (CHO) K1 (ATCC no. CCL-61; American Type Culture Collection) cells were maintained at 37°C in a 5% CO₂ incubator in DMEM (HEK) or Ham’s F12 medium (BioWhittaker, Walkersville, MD) (CHO), both containing 10% heat-inactivated FCS, 1% l-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Polymixin B was from Sigma-Aldrich (St. Louis, MO).

Irradiated M. leprae, phenolic glycolipid-1 (PGL-1), and M. leprae SDS-soluble cell wall fraction components (MLcw) were provided by P. Brennan (Colorado State University, Fort Collins, CO) through the Leprosy Research Support contract (NO1-AI-55262; National Institutes of Health, National Institute of Allergy and Infectious Diseases). M. tuberculosis cell wall fraction components (MTBcw) were obtained from J. Belisle (Colorado State University) through the TB Research Materials and Vaccine Testing contract (NO1-AI-75320; National Institutes of Health, National Institute of Allergy and Infectious Diseases). LPS from Salmonella Minnessota R595 Re was obtained from List Biological Laboratories (Campbell, CA) and Saccharomyces cerevisiae particle zymosan from Molecular Probes (Eugene, OR). Bacterial flagellin was extracted from Salmonella typhimurium TH4778 (fliB⁻, fliC⁺) as previously described (8). Synthetic tripalmitoylated lipopeptide (Pam₃CSK₄) was from EMC Microcollections (Tuebingen, Germany) and peptidoglycan (Staphylococcus aureus) from Fluka (Buchs, Switzerland). Levels of contaminating LPS as determined by the chromogenic Limulus amebocyte lysate test (BioWhittaker) were 0.02, 0.007, 0.0005, and 0.0001 endotoxin U/ml, in wells incubated with zymosan, irradiated M. leprae, MTBcw, and MLcw, respectively. These values correspond to ∼2, 0.7, 0.05, and 0.01 pg/ml endotoxin, respectively. PGL-1 had no detectable endotoxin.

Bone marrow macrophages

The mutant mouse strains deficient in TLR2 and TLR4 have been described previously (20, 21). The TLR2-deficient mice were backcrossed two times with C57BL/6. The TLR4-deficient mice have been backcrossed six times with C57BL/6 mice and wild-type (WT) littermates were used as controls. Bone marrow-derived macrophages were obtained from femoral bone marrow cells that were cultured for 6–9 days in complete DMEM with 20% L929 fibroblast (ATCC no. CCL-1; American Type Culture Collection) conditioned medium. Subsequently, cells were plated in a 96-well dish at 2 × 10⁴/well and stimulated for 24 h. The concentration of TNF-α in supernatant was determined by ELISA (R&D Systems, Minneapolis, MN). The University of Washington’s Institutional Animal Care and Use approved all animal protocols.

DNA expression vectors

hTLR2 was amplified from THP1 cDNA (ATCC no. TIB-202; American Type Culture Collection) by using primers 5′-ATGCCACATACTTT GTGG-3′ and 5′-GGACTTTATCGCAGCTCTCAG-3′ and cloned into pEF6/V5-His-TOPO (Invitrogen, San Diego, CA). The TLR2 mutation described in Korean patients (Arg⁶⁷⁷Trp) (18) and the dominant-negative mutant (Pro⁶⁸¹His) (19) were elaborated using Quick Change site-directed mutagenesis (Stratagene, La Jolla, CA). The hTLR2 gene cloned into pEF6/V5-His-TOPO (WT) was amplified by PCR using primers 5′-AAGTTGTGTCTG CACAAGTGGGACTTCATTCC-3′ and 5′-GGAAGTTCAACACAGACGTGTTCACCCTGAAGTAA-3′ to generate the Arg⁶⁷⁷Trp mutant and primers 5′-GACTTCATCCATGGCAAGTGG-3′ and 5′-CGCCCTGAAGT AGGTACCGTTCACCTAG-3′ to generate the Pro⁶⁸¹His mutant. DpnI digestion was used to remove methylated DNA before transformation into competent Escherichia coli. The template and both mutants were verified by full-gene sequencing. The CD14 and hTLR5 expression constructs have been described previously (8).

Luciferase assay

The day before transfection, CHO and HEK293 cells were plated in a 96-well dish at 2 × 10⁴ or 6 × 10⁴ per well, respectively. Transient transfection was performed using the Polyfect transfection reagent (Qiagen, Chatsworth, CA). Cells were transfected with 0.25–0.5 μg of endothelial leukocyte molecule-1 (ELAM-1) firefly luciferase construct, 0.025–0.05 μg of the thymidine kinase Renilla luciferase (pRL-TK) vector (Promega, Madison, WI), and 0.25–0.5 μg of the expression vector(s) per well. CHO cells were washed in PBS 2–3 h after transfection. Twenty hours after transfection, cells were stimulated for 4 h. The cells were lysed in passive lysis buffer and the luciferase activity was determined using the dual-luciferase reporter assay system (Promega).

TLR2 mediates innate immune response to M. leprae

We examined the role of TLR2, TLR4, and TLR5 in the inflammatory response to M. leprae. First, we analyzed the production of TNF-α in bone marrow macrophages from WT and TLR2- and TLR4-deficient mice upon M. leprae stimulation. Bone-marrow macrophages were cultured with irradiated M. leprae, MLcw, or LPS for 24 h, and the concentration of TNF-α was measured in the supernatant by ELISA. WT and TLR4-deficient macrophages produced similar amounts of TNF-α in response to MLcw and MTBcw. In TLR2-deficient macrophages, the level of TNF-α produced upon mycobacterial stimulation was dramatically reduced (M. leprae) or not detectable (MTBcw) (Fig. 1⇓A). WT and TLR2-deficient macrophages produced similar amounts of TNF-α in response to LPS, whereas TLR4-deficient macrophages produced no detectable TNF-α (20). As a control, assays were also performed with polymixin B (at 10 μg/ml) to inhibit any possible effect of LPS contamination. However, no differences were observed when polymixin B was present.

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FIGURE 1.

Innate immune recognition of dead M. leprae cells is mediated by TLR2, but not TLR4 or TLR5. A, Bone marrow macrophages from WT and TLR2- and TLR4-deficient mice were cultured in the presence of irradiated M. leprae (ML; 80 μg/ml), MTBcw (1 μg/ml), and LPS (10 ng/ml) for 24 h. TNF-α production was measured by ELISA. The results are mean ± SD of triplicate wells and are representative of three different experiments. ND, Not detected. B, CHO cells were transiently transfected with a vector expressing hTLR5 or an empty vector (EF6), together with NF-κB reporter (ELAM-1 firefly luciferase) and transfection control (thymidine kinase Renilla luciferase). Cells were stimulated with irradiated M. leprae (80 μg/ml), MTBcw (1 μg/ml), and bacterial flagellin (FL; 5 ng/ml), and luciferase activity was measured with a luminometer. Firefly luciferase units were divided by Renilla units to obtain relative luciferase units (RLU). The results are mean ± SD of triplicate wells and are representative of four separate experiments.

The role of TLR5 in M. leprae-induced signaling was examined in transiently transfected CHO cells, a cell line that does not express functional endogenous TLR2 (22). CHO cells were transfected with a construct expressing hTLR5 together with an NF-κB reporter (ELAM-1), and a transfection control (pRL-TK). The cells were stimulated with irradiated M. leprae cells, MTBcw, and bacterial flagellin as a positive control (8). Expression of hTLR5 did not confer M. leprae nor MTBcw sensitivity (Fig. 1⇑A). As a control, flagellin responsiveness was detected in CHO cells transfected with hTLR5.

These data suggest that the innate immune recognition of irradiated M. leprae cells is mediated by TLR2, but not TLR4 or TLR5. To confirm the role for hTLR2 in M. leprae-induced signaling, we transiently transfected CHO cells with a construct expressing hTLR2, together with a luciferase reporter as described previously. The transfected cells were then stimulated with irradiated M. leprae cells, MLcw, or PGL-1, a specific M. leprae Ag that was recently reported to be directly involved in the neuropathogenesis of leprosy (23, 24). Expression of hTLR2 was sufficient to confer responsiveness to M. leprae and MLcw, but not to PGL-1 (Fig. 2⇓A). Because CD14 is required for the recognition of certain TLR2 ligands (25, 26, 27), we transfected HEK293 cells with hTLR2 together with a construct expressing CD14 as shown in Fig. 2⇓B. HEK293 cells were stimulated with MLcw, PGL-1, and yeast particles (zymosan). Expression of hTLR2 was sufficient to confer responsiveness to MLcw in HEK293 cells, and coexpression of CD14 together with hTLR2 facilitated this response. As a control, coexpression of CD14 was required to activate NF-κB production upon zymosan stimulation. Expression of hTLR2 with or without CD14 did not confer PGL-1 responsiveness.

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FIGURE 2.

Expression of hTLR2 is sufficient to mediate M. leprae and MLcw-induced NF-κB activation. A, CHO cells were transiently transfected with a vector expressing hTLR2 or an empty vector (EF6) as described in Fig. 1⇑B. Cells were stimulated with M. leprae (80 μg/ml), MLcw (1 μg/ml), and PGL-1 (1 μg/ml). The results expressed in RLU are mean ± SD of triplicate wells and are representative of four different experiments. B, HEK293 cells were transiently transfected as described in Fig. 2⇓. Expression of hTLR2 together with CD14 was compared with expression of hTLR2 alone. The total amount of DNA per well was normalized by addition of EF6. Cells were stimulated with MLcw (1 μg/ml), PGL-1 (1 μg/ml), and the yeast particle zymosan (ZYM, 400 ng/ml). The results are expressed in RLU and represent mean ± SD of triplicate wells. Similar results were obtained in three different experiments.

Polymorphism in hTLR2 affects the innate immune response to mycobacteria

To evaluate the relevance of the hTLR2 SNP described in Korean patients with lepromatous leprosy (Arg⁶⁷⁷Trp), we transfected HEK293 cells with constructs expressing WT and mutant (Arg⁶⁷⁷Trp) hTLR2. As a control, we used a construct expressing another hTLR2 mutant (Pro⁶⁸¹His) analogous to a mouse TLR2 dominant-negative mutant (19). Transiently transfected HEK293 cells were stimulated with escalating doses of MTBcw (Fig. 3⇓A) and MLcw (Fig. 3⇓B). HEK293 cells transfected with WT hTLR2 induced NF-κB activation in a dose-dependent fashion in response to MLcw and MTBcw. In contrast, NF-κB activation was abolished in HEK293 cells transfected with the Korean (Arg⁶⁷⁷Trp) and dominant-negative (Pro⁶⁸¹His) hTLR2 mutants. All the transfectants expressed equivalent amounts of V5 epitope-tagged hTLR2 as revealed on Western blot (Fig. 3⇓C). Transfection of CD14 together with hTLR2 Arg⁶⁷⁷Trp did not confer responsiveness to MLcw (Fig. 3⇓D). Of note, Arg⁶⁷⁷Trp and Pro⁶⁸¹His hTLR2 mutants also failed to activate NF-κB in response to Pam₃CSK₄, peptidoglycan, and zymosan.

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FIGURE 3.

Arg⁶⁷⁷Trp polymorphism in hTLR2 affects NF-κB activation in response to M. leprae and M. tuberculosis. HEK293 cells were transiently transfected with constructs expressing WT, mutant (Arg⁶⁷⁷Trp; Pro⁶⁸¹His) hTLR2 (0.5 μg each), or an empty vector (EF6) together with an NF-κB reporter as described in Fig. 1⇑B. Cells were stimulated with escalating doses of MTBcw (A) and MLcw (B), as indicated. Results are expressed in RLU and represent mean ± SD of triplicate wells. Similar results have been observed in two separate experiments. C, Anti-V5 Western blot of the lysates shown in A and B. Equal volumes of lysate were loaded on a gel and blotted with anti-V5 Abs. D, HEK293 cells were transiently transfected as described in A and B. In addition, CD14 was added in each well. Cells were stimulated with Pam₃CSK₄ (PAM3; 400 ng/ml), zymosan (ZYM; 400 ng/ml), peptidoglycan (PGN; 50 μg/ml), and MLcw (1 μg/ml). Results, expressed in RLU, are mean ± SD of triplicate wells and are representative of three separate experiments.