Nature Medicine
8, 878 - 884 (2002)
Published online: 1 July 2002; | doi:10.1038/nm732
Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient miceLena Alexopoulou1, 5, Venetta Thomas2, 5, Markus Schnare1, 5, Yves Lobet3, Juan Anguita4, Robert T. Schoen2, Ruslan Medzhitov1, 5, Erol Fikrig2, 5
& Richard A. Flavell1, 51 Section of Immunobiology and the Howard Hughes Medical Institute, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 2 Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 3 SmithKline Beecham Biologicals, Rixensart, Belgium 4 Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 5 These authors contributed equally to this work.
Correspondence should be addressed to Erol Fikrig erol.fikrig@yale.edu or Richard A. Flavell for TLR-/-richard.flavell@yale.eduThe Lyme disease vaccine is based on the outer-surface lipoprotein (OspA) of the pathogen Borrelia burgdorferi, and 95% of vaccine recipients develop substantial titers of antibodies against OspA. Here, we identified seven individuals with very low antibody titers after vaccination (low responders). The macrophages of low responders produced less tumor necrosis factor- and interleukin-6 after OspA stimulation and had lower cell-surface expression of Toll-like receptor (TLR) 1 as compared to normal cells, but normal expression of TLR2. TLRs activate innate responses to pathogens, and TLR2 recognizes lipoproteins and peptidoglycan (PGN). After OspA immunization, mice genetically deficient in either TLR2 (TLR2-/-) or TLR1 (TLR1-/-) produced low titers of antibodies against OspA. Notably, macrophages from TLR2-/- mice were unresponsive to OspA and PGN, whereas those from TLR1-/- mice responded normally to PGN but not to OspA. These data indicate that TLR1 and TLR2 are required for lipoprotein recognition and that defects in the TLR1/2 signaling pathway may account for human hyporesponsiveness to OspA vaccination.Lyme disease, caused by the spirochete Borrelia burgdorferi and transmitted primarily by the tick Ixodes scapularis, is the most common vector-borne illness in the United States1. B. burgdorferi infection usually occurs in the Northeast, upper Midwest and northern Pacific regions of the country1. A pathognomonic skin lesion, erythema migrans, often marks early-stage Lyme disease, and persistent infection may result in symptoms involving the musculoskeletal, nervous or cardiovascular system1,
2. Mice serve as an experimental model of B. burgdorferi infection3,
4,
5,
6, and the course of the disease—initial local infection and subsequent dissemination, accompanied by arthritis and carditis—is somewhat similar in mice and humans7.
The outer-surface lipoprotein (OspA) of B. burgdorferi is one of the most abundant antigens on spirochetes cultured in vitro and is centrally involved in immunity. In 1990, either passive immunization with monoclonal antibodies against OspA or active immunization with recombinant OspA were shown to protect mice from Lyme borreliosis8,
9,
10. On the basis of extensive testing, the US Food and Drug Administration approved the use of recombinant OspA as a vaccine against Lyme disease in 1998 (refs. 11,12). Protective immunity in humans correlates with the development of high-titer antibodies against OspA, similar to that seen in the mouse model of Lyme disease11,
13.
Innate immune recognition of invading pathogens is mediated by a set of germline-encoded receptors that have evolved to recognize conserved molecular patterns shared by large groups of microbial pathogens14. Recent evidence has shown that this recognition can be attributed mainly to the TLR family15,
16,
17. To date, ten different TLRs have been identified in mammals, and some of the TLR ligands have been characterized18. TLR3 detects double-stranded RNA19, TLR4 is essential for LPS recognition20,
21, TLR5 senses bacterial flagellin22 and TLR9 recognizes bacterial DNA containing unmethylated CpG motifs23. TLR2 identifies zymosan, peptidoglycan (PGN) and lipoproteins24,
25,
26, including OspA27. TLR6 can associate with TLR2 and recognize PGN and diacylated lipopeptides, but not triacylated lipopeptides such as OspA28.
In the current study, we have identified a cohort of individuals with low titers of antibodies against OspA after vaccination. We proposed that persons responding very poorly to OspA immunization might have defects in TLR-mediated lipoprotein signaling, and therefore characterized the molecular basis of hyporesponsiveness to OspA vaccination in humans, TLR2-/- mice and newly created TLR1-/- mice. We show here that TLR1 and TLR2 both contribute to signaling by lipoproteins and that several vaccine low responders are defective in TLR1/2 signaling.
Characterization of vaccine recipients
As part of a multicenter phase 3 efficacy study, 492 individuals received the OspA-based Lyme disease vaccine at the Yale University School of Medicine. Over 95% of the vaccine recipients had detectable antibodies against OspA after receiving three immunizations with OspA vaccine in a 0-, 1- or 12-month regimen. High antibody titers were associated with a greater degree of protective immunity11. We defined those who responded normally to OspA vaccination, consistent with the general population, as 'normal responders'. Seven individuals had very low titers of antibodies against OspA one month after the first, second or third immunization; we defined these as 'low responders' (Fig. 1a). As well as developing antibodies against OspA, normal responders showed local reactions at the site of injection, including soreness, redness or swelling lasting up to three days11. These symptoms were less apparent in low responders, consistent with the absence of an inflammatory response to OspA lipoprotein.
 | | Figure 1. Characterization of individuals with low titers of antibodies against OspA after immunization with OspA lipoprotein. | | |  | a, Antibody titers in sera from vaccinated individuals, as determined by ELISA. Lanes 1−7, low responders; lanes 8−14, representative normal responders. b, Humoral response to diphtheria, tetanus and pertussis toxoid in low (white) and normal responders (black). c−e, Percentage expression of TNF- (c) and concentrations of IL-6 (d) and IL-10 (e) in the culture supernatants of PBMCs from 7 low (white) and 4 normal (black) responders stimulated with OspA or PGN, or left untreated. *, P < 0.05, as determined by Student's t-Test. ND, not detected.
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Low responders were healthy both before enrollment in the trial and during the vaccination period, and thus their hyporesponsiveness to OspA did not seem to be related to any observable medical condition. To determine whether the lack of response to vaccination was limited to OspA, we analyzed the antibody responses of these individuals to other vaccine antigens that they had been routinely administered in childhood, including diphtheria, pertussis and tetanus toxoid, by enzyme-linked immunosorbent assay (ELISA). Antibody responses to these vaccines were similar in OspA low responders and normal responders (Fig. 1b), indicating that low responders had a selective defect in their ability to produce antibodies in response to the OspA vaccine.
OspA hyporesponsiveness of low-responder macrophages
Lipidated OspA is a potent adjuvant that stimulates the production of tumor necrosis factor- (TNF-) and interleukin-6 (IL-6) from macrophages29,
30 through a signaling process mediated by TLR2 (ref. 27). Because the innate immune response is critical for the generation of acquired immunity18,
31,
32, we investigated the response of macrophages from OspA vaccine recipients to ex vivo stimulation with lipidated OspA. On stimulation with 10  g/ml OspA, macrophages from the low responders showed lower titers of the pro-inflammatory cytokine TNF- than did macrophages from normal responders (Fig. 1c). Stimulation with 5 or 20 g/ml OspA produced similar results (data not shown). As TLR2-mediated signaling also occurs in response to PGN24, we examined the response of low and normal responders to PGN. Macrophages from both groups elicited TNF- to a similar extent in response to PGN (Fig. 1c). Stimulation with OspA resulted in significantly lower amounts of IL-6 in cell supernatants from low-responder macrophages than in those from normal-responder macrophages (Fig. 1d). In contrast, PGN stimulation produced similar amounts of IL-6 in both groups. The amounts of the anti-inflammatory cytokine IL-10 seen after either OspA or PGN stimulation were similar in cell supernatants from both groups (Fig. 1e). Because TLR2 has been implicated in the response to lipoproteins and PGN, the ability of macrophages from low responders to develop a normal TNF and IL-6 response to PGN, but not to OspA, indicates that OspA- and PGN-induced stimulation occur by different mechanisms that both involve TLR2.
Because TLR2 participates in signaling involving lipidated OspA26,
27, we investigated whether low responders had mutations in the TLR2 gene. We did not identify mutations critical for signaling in any of the low-responder individuals (data not shown). In addition, TLR2 mRNA expression (data not shown) and cell-surface expression of TLR2 were similar (P < 0.12) in low and normal responders (Fig. 2). Thus, mutations in TLR2 and expression of TLR2 do not account for hyporesponsiveness to OspA in low responders.
Responses of TLR2-/- mice to OspA
In mice, lipidated OspA can stimulate antibody production in the absence of adjuvant, and adjuvant can enhance the antigenicity of the unlipidated form of OspA30,
33. To investigate the role of TLR2 in the generation of antibodies against the lipidated form of OspA, we immunized wild-type and TLR2-/- mice with lipidated OspA in the absence of adjuvant. TLR2-/- mice generated significantly lower titers of antibodies against OspA than did wild-type controls, confirming the importance of TLR2-mediated pathways in the response to OspA lipoprotein (Fig. 3a). However, immunization with lipidated OspA in the presence of complete Freund's adjuvant (CFA) resulted in similar titers of antibodies against OspA in TLR2-/- and wild-type mice (Fig. 3a). Infection of TLR2-/- and wild-type mice with B. burgdorferi resulted in similar titers of antibodies against B. burgdorferi flagellin B (FlaB) protein (41G) and two lipoproteins, decorin-binding protein (DbpA) and OspA, as assessed by ELISA and immunoblotting (Fig. 3b and c). These data indicate that general stimulation of the innate immune system, either through multiple TLRs during immunization with a potent adjuvant such as CFA or by infection with a live organism, can initiate an immune response against spirochete lipoproteins independent of TLR2.
| | Figure 3. Responses of wild-type (WT) and TLR2-/- mice to OspA immunization and B. burgdorferi infection. | | |  | Black, WT; white, TLR2-/-; gray, normal mouse serum. a, Titers of antibodies against OspA in sera from mice immunized with lipidated OspA in PBS or CFA 4 times over an 8-week period. Sera were obtained 2 weeks after the final boost and tested by ELISA using OspA as antigen. b, Titers of antibodies to whole Borrelia lysate, B. burgdorferi FlaB (41G), DbpA and OspA in wild type (black) and TLR2-/- (white) mice following 8 weeks of infection. c, B. burgdorferi lysates were probed with sera from wild-type and TLR2-/- mice (n = 3 per group) infected with B. burgdorferi. d, IL-6 concentrations in the culture supernatant of whole splenocytes from wild-type and TLR2-/- mice stimulated with OspA or PGN or left untreated, as measured by ELISA.
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Because the TLR2-/- mice produced lower antibody titers than did normal mice in response to OspA immunization (Fig. 3a), and OspA is known to activate macrophages to produce IL-6 (refs. 29,34), we assessed the production of IL-6 in response to OspA ex vivo. After stimulation with OspA, macrophages from wild-type mice produced significant amounts of IL-6, whereas those from TLR2-/- mice did not (Fig. 3d). Stimulation with PGN produced similar results (Fig. 3d). The absence of response to PGN in the TLR2-/- mice contrasted with the normal response to PGN in human low responders, indicating that OspA and PGN induce TLR2-mediated signaling by different pathways.
We next examined the course of Lyme disease in the TLR2-/- mice by infecting wild-type and TLR2-/- mice with B. burgdorferi. Both groups showed a similar degree of mild arthritis and carditis two weeks after infection, and in both the disease resolved after eight weeks. At two weeks, the spirochete burden, as determined by quantitative PCR of the skin, was much greater in TLR2-/- mice than in control mice (7.7 + 0.8 ng as compared to 0.6 + 0.2 ng B. burgdorferi DNA per g skin, respectively).
Contribution of TLR1 to OspA responsiveness
TLR2 may cooperate with either TLR1 or TLR6 for signaling35,
36, and PGN signaling involves TLR2 and TLR6 (ref. 28). PGN stimulation of macrophages in low and normal responders seemed to be intact (Fig. 1c and d), implying that the defect in OspA signaling might be associated with TLR1. 293 T cells express endogenous TLR1, TLR6 and TLR10 but not TLR2 (ref. 37). Transient transfection of TLR2 reconstituted the responsiveness to OspA in 293 T cells, as indicated by its activation of NF- B-dependent luciferase reporter gene (Fig. 4a), consistent with previous observations38. We next tested whether cotransfection of various TLRs along with TLR2 could enhance the TLR2-mediated response to OspA. Of all the TLRs analyzed, only TLR1 enhanced NF-kB activation mediated by TLR2 after OspA stimulation (Fig. 4a).
| | |
We also constructed mutant, dominant-negative forms of TLR1, TLR2, TLR6 and TLR10 and assessed their ability to inhibit inhibited the TLR2-mediated response to OspA. The dominant-negative TLR1 and TLR2 each inhibited the response to OspA in a dose-dependent manner and with approximately equal efficiency (Fig. 4b). Neither dominant-negative TLR6 (Fig. 4b) nor dominant-negative TLR10 (data not shown) inhibited TLR2 activation by OspA.
Low response to OspA in TLR1-/- mice
Given that TLR1 can affect TLR2 signaling, we decided to assess the biological function of TLR1 directly, by generating TLR1-/- mice through homologous recombination in embryonic stem (ES) cells (Fig. 5a−d; see Methods) and examining the responses of TLR1-/- macrophages to various microbial molecules. In response to OspA, macrophages from wild-type mice produced IL-6 in a dose-dependant manner, whereas the ability of TLR1-/- macrophages to produce IL-6 was significantly impaired (Fig. 6a). In contrast, wild-type and TLR1-/- macrophages cultured with either 10 g/ml Staphylococcus aureus PGN, 1 ng/ml Salmonella enteritidis LPS, 100 g/ml Saccharomyces cerevisiae zymosan or 50 g/ml poly (I:C) did not produce significantly different amounts of IL-6 (Fig. 6b). The IL-10 response of macrophages from TLR1-/- mice to OspA was similar to the response of control mice, whereas the response of TLR2-/- macrophages was significantly lower than that of controls (Fig. 6c).
| | Figure 5. Generation of TLR1-/- mice. | | |  | a, Schematic representation of the targeting procedure. The wild-type TLR1 locus, the targeting construct and the predicted disrupted gene are shown, along with the locations of the external 3' and internal 5' probes (probes A and B, respectively). Arrows show transcriptional orientation of the genes; solid bar, TLR1 coding exon; closed triangles, loxP sites. Restriction enzyme sites: A, AflII; B, BamHI; N, NdeI. b, Southern blot analysis of NdeI-digested genomic DNA from wild-type (+/+) and heterozygous (+/-) ES cell clones. c, Southern blot analysis of BamHI-digested genomic DNA from wild-type (+/+), heterozygous (+/-) and homozygous (-/-) mice. d, Expression of TLR1 and HPRT by bone marrow−derived macrophages from wild-type or TLR1-/- mice, either untreated (-) or stimulated with LPS, as determined by RT-PCR.
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| | Figure 6. TLR1 is critical to the recognition of OspA. | | |  | a and b, Concentrations of IL-2 in culture supernatants of macrophages from wild-type (WT; black) or TLR1-/- mice (white) stimulated with OspA (a) or with PGN, LPS, zymosan (Zym.) and poly (I:C) (b), as measured by ELISA (average  s.d., n = 4 per group). ND, not detected; Med., medium; *, P < 0.01. c, Concentrations of IL-10 in the culture supernatants of macrophages from WT (black), TLR1-/- (white) or TLR2-/- mice (gray) stimulated with OspA, as measured by ELISA (average s.d., n = 4 per group). *, P < 0.01. d, Titers of antibodies against OspA in sera from wild-type (black) and TLR1-/- (white) mice immunized with lipidated OspA in PBS or CFA 3 times over 6−8 weeks. Sera was collected 2 weeks after the final boost and tested by ELISA using OspA as antigen. *, P < 0.001. e, Titers of antibodies in sera from the wild type (black) and TLR1-/- (white) mice to whole Borrelia lysate, B. burgdorferi FlaB (41G), DbpA and OspA following 8 weeks of infection. f, Immunoblot of B. burgdorferi lysates were probed with sera from wild-type and TLR1-/- mice (n = 5 per group) infected with B. burgdorferi. g, Expression of TLR1 surface protein in low (red) and normal (black) responders or isotype control (gray, filled). Cells were analyzed as described in Fig 2.
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To verify the functional participation of TLR1 in OspA signaling, we immunized TLR1-/- mice with lipidated OspA in the presence or absence of CFA. The outcome was similar to that in TLR2-/- mice. As compared to their wild-type counterparts, TLR1-/- mice produced significantly lower titers of antibodies against OspA in the absence of adjuvant, but comparable titers in the presence of adjuvant (Fig. 6d). Infection of wild-type and TLR1-/- mice with B. burgdorferi resulted in similar titers of antibodies against whole B. burgdorferi extracts, DbpA, OspA and 41G as assessed by ELISA (Fig. 6e) and immunoblotting (Fig. 6f), indicating that the defect in lipoprotein signaling in TLR1-/- mice can be overcome by infection. In addition, the course of disease was similar in TLR1-/- and control mice: in both groups, mild arthritis and carditis developed at two weeks and the disease resolved eight weeks. However, the spirochete burden at two weeks was much higher in TLR1-/- mice than in control mice (8.2 + 1.2 ng as compared to 0.7 + 0.3 ng B. burgdorferi DNA per g skin, respectively).
TLR1 expression in human low and normal responders
We sequenced the TLR1 gene in the human OspA low and normal responders and did not find any mutations or deletions in the 7 low responders. We detected TLR1 mRNA in all 7 low responders by polymerase chain reaction with reverse transcription (RT-PCR; data not shown). Flow cytometric analysis showed, however, that the cell-surface expression of TLR1 protein was significantly lower in low responders than in normal responders (P < 0.04) (Fig. 6g). In 6 of the 7 low responders, the amounts of TLR1 protein detected were indistinguishable from that of an isotype control antibody, meaning that they were below the level of detection.
Discussion
TLR2 is essential for the recognition of OspA lipoprotein27, but the importance of OspA in bacterial recognition during active infection is not clear. Our data show that the acquired humoral response to infection with B. burgdorferi, a spirochetal agent bearing numerous lipoproteins and other molecules on its outer surface, is normal in TLR2-/- mice. Administration of B. burgdorferi cells or whole-cell lysates, or of selected recombinant B. burgdorferi antigens prominently expressed during infection, such as FlaB and the lipoprotein DbpA, elicits similar antibodies in TLR2-/- and control mice. B. burgdorferi cells do not express abundant OspA during infection of mice. Mice challenged with more than 104 spirochetes develop detectable amounts of antibodies against OspA, however, and our data showed that TLR2-/- and control mice developed similar titers of antibody against OspA. These data indicate that acute infection with live B. burgdorferi stimulates immune responses through multiple pathways, perhaps including several TLRs and other receptors, and that the selective absence of TLR2 does not result in a gross alteration in the production of B. burgdorferi−specific antibodies. In addition, the absence of TLR2 did not alter Lyme arthritis or carditis in the mice, but did result in a larger spirochete load. This increase in pathogen number in the absence of TLR2 is consistent with previous findings, except that greater ankle swelling was seen in the TLR2-/- mice in the earlier work40. The differences in disease severity could result from the strain of B. burgdorferi used, the inoculum dose or the assessment interval. However, active immunization of TLR2-/- mice with OspA lipoprotein does not produce an acquired antibody response to OspA. This defect in acquired immunity can be overcome by administering the OspA in CFA. As in infection, stimulation of the immune response through more than one pathway is sufficient to compensate for the deficiency.
Our data also demonstrate a fundamental role for TLR1 in lipoprotein signaling, and further delineate the separate pathways used for lipoprotein and PGN recognition by TLR2. Macrophages from TLR1-/- mice showed normal IL-6 response to PGN but no response to OspA, in contrast to those from TLR2-/- mice, which are unresponsive to both molecules. Our transfection studies showed that TLR1 facilitates TLR2-mediated lipoprotein responsiveness. In addition, the responses of the TLR1-/- and TLR2-/- mice after immunization with OspA and after infection with B. burgdorferi were very similar. These experiments indicate that TLR1 is required for lipoprotein recognition and for the development of an acquired immune response to OspA.
Most human vaccines are administered in aluminum hydroxide, which functions primarily as an inert carrier, in contrast to adjuvants such as CFA routinely used in mice, which can induce profound inflammation. The mouse studies reported here show that deletion of either TLR1 or TLR2 can result in ineffective responses to OspA lipoprotein. Our assessment of individuals who received the Lyme disease vaccine further shows that 7 individuals (approximately 1% of subjects) were hyporesponsive to OspA vaccination. The macrophage response of these low responders was similar to that in TLR1-/- mice, in that the cells responded normally to PGN but poorly to OspA. In addition, macrophages from TLR2-/- mice produced significantly lower levels of IL-10 after OspA stimulation, whereas cells from both TLR1-/- mice and human low responders had IL-10 responses to OspA that were similar to those of controls. Thus, the phenotype in low responders is more closely associated with TLR1 than TLR2. These data also imply that different TLR-mediated signaling pathways may result in the selective induction of anti-inflammatory or pro-inflammatory cytokines.
Notably, the cell-surface expression of TLR2 was similar in low and normal responders, whereas that of TLR1 was significantly lower in low responders (Fig. 6g). These data indicate that the defect in the low responders resides within the TLR1-mediated signaling pathway. The factors contributing to altered TLR1 surface expression are not yet known.
The ability of the host to respond to multiple ligands of a pathogen makes it unlikely that defects in a single TLR will drastically alter the response to a particular pathogen. Indeed, both TLR2-/- and TLR1-/- mice develop a response to B. burgdorferi infection that is similar to that in controls, and individuals hyporesponsive to OspA vaccination do not seem to have an abnormally high susceptibility to infection in general. Deficiencies in some of the TLRs may be noted only in artificial situations, such as vaccination, when an antigen is introduced in such a manner that only one TLR can be effectively stimulated. These studies provide insight into the importance of TLR1 in lipoprotein recognition and demonstrate that defects in TLR1 or TLR2 can abrogate the response to vaccination with OspA in mice. Our studies in human OspA low responders demonstrate that these individuals have altered cell-surface expression of TLR1 protein and may have an interruption in the TLR1-mediated lipoprotein signaling pathway. These studies provide a link between TLR1, TLR2 and acquired humoral immune responses, and may lead to new approaches to improving the efficacy of vaccination.
Methods
Cells and mice.
Peripheral blood mononuclear cells (PMBCs) were purified by Ficoll-Hypaque gradient centrifugation and plated at 2  106 cells/ml. Human embryonic kidney (HEK) 293 cells were obtained from the American Type Culture Collection (Manassas, Virginia). TLR2-/- mice24 (F2 interbred from 129/SvJ C57/BL6) were a gift from S. Akira (Osaka University, Osaka, Japan). Low responders were healthy as determined by a routine medical history and physical exam. The studies were approved by the animal and human investigator committees at Yale University. Informed consent was obtained from the human subjects.
Detection of intracellular TNF- from PBMCs.
Intracellular staining for TNF- was done with the Cytofix/CytoPerm Plus kit from BD PharMingen (San Diego, California). PBMCs (2 106 cells/ml) were stimulated with 10 g/ml lipidated OspA (99% pure, endotoxin free and identical to the material provided to humans as a vaccine11; SmithKline Beecham Biological, Rixensart, Belgium) or 10 g/ml PGN from Staphylococcus aureus (Fluka, St. Louis, Missouri) for 40 h, and brefeldin A was added for an additional 4 h. Cells were treated with mouse IgG1 to block FC receptors and surface stained with a Cy-Chrome-conjugated antibody specific for CD4, fixed, permeabilized and stained using an antibody against human TNF- conjugated to phycoerythrin (PE). They were then analyzed using a flow cytometer with the CellQuest software package (Becton Dickinson, Franklin Lakes, New Jersey).
Measurement of IL-6 and IL-10.
Mouse splenocytes (1 106 cells/ml) or human PBMCs (2 106 cells/ml) were stimulated with 10 g/ml of lipidated OspA or PGN for 16−20 h. Culture supernatants were collected and analyzed for IL-6 (antibodies were from BD PharMingen) and IL-10 (DuoSet ELISA, R& D Systems, Minneapolis, Minnesota) by ELISA.
DNA expression vectors.
Human CD14, TLR1-6 and TLR10 were cloned into the pcDNA3.1 expression vector (Invitrogen, Carlsbad, California). Dominant-negative TLR1, TLR2, TLR6 and TLR10 were generated by site-directed mutagenesis to insert point mutations resulting in proline-to-histidine conversions: TLR1, P675H; TLR2, P681H; TLR6, P680H; TLR10, P674H.
Transient transfection.
HEK 293 cells stably transfected with large T antigen from SV40 (293 T cells) were plated at 2 105 cells/well overnight at 37 °C. Cells were transiently transfected with 100 ng of the pBIIXluc NF-B luciferase reporter plasmid, 50 ng human CD14 and 100 ng human TLR2 with or without 100 ng of other TLRs using Lipofectamine 2000 (Gibco-BRL, Rockville, Maryland). The total amount of DNA in each transfection was kept constant by adding pcDNA3.1 plasmid. After 16 h, the cells were stimulated with 100 ng/ml of OspA for 6 h and lysates were processed with luciferase substrate (Promega, Madison, Wisconsin) to measure luciferase activity. In the inhibition experiments, cells were transfected and stimulated as described above except that the dominant-negative constructs were also added.
OspA immunization and B. burgdorferi infection.
TLR1-/-, TLR2-/- and wild-type mice were immunized intraperitoneally with 2 g lipidated OspA in 100 l sterile PBS or CFA. Mice were given booster immunizations every 14 d for 2 months. In the infection studies, mice were infected with 106 B. burgdorferi N40 by intradermal injection in the midline of the back. The optical density of ELISAs was measured at 450 nm (OD450) and immunoblots were done as previously described8,
41.
Generation and genetic analysis of TLR1-deficient mice.
The genomic DNA of the TLR1 gene was isolated from a 129SvJ mouse genomic library (Stratagene, La Jolla, California) using a human TLR1 probe. A targeting vector was constructed from a 3.0-kb NotI−EcoNI and a 4.4-kb AflII−AflII fragment containing the 5' and 3' ends of TLR1, respectively. The TLR1 gene was replaced by a neomycin-resistance gene (derived from the plasmid pMC1neo poly A, Stratagene) flanked by two loxP sites, and a thymidine kinase (TK) gene was used for negative selection of clones. The targeting construct was transfected into ES cells (W9.5) using a standard protocol42. Homozygous recombinants were identified by Southern blot analysis of NdeI-digested DNA, using a 495-bp NcoI−SalI fragment as the 3' external probe (probe A) (Fig. 5b). Chimeric mice were generated by microinjecting TLR1+/- ES cells from 1 correctly targeted clone into C57BL/6 blastocysts. A line of these chimeric mice successfully transmitted the disrupted TLR1 gene through the germline (Fig. 5b and c). Heterozygous F1 mice were intercrossed to produce homozygous TLR1-/- and wild-type mice. The homozygous mice were then interbred for the current studies. The mice had normal appearance, growth, size, fertility and life span and no obvious behavioral abnormalities. Flow cytometric analysis showed that the expression of CD3, B220, CD4 and CD8 in thymocytes and splenocytes were not altered in TLR1-/- mice as compared with the wild-type controls (data not shown).
DNA was analyzed by Southern blot analysis of BamHI-digested DNA, using a 335-kb XbaI−NcoI fragment as 5' probe (probe B) (Fig. 5c). Absence of TLR1 mRNA expression was confirmed by RT-PCR. The RNA was isolated from macrophages using TRIzol reagent (Gibco-BRL) and contaminant DNA was removed with DNasin (Ambion, Austin, Texas). RNA was reverse transcribed and the cDNA was amplified using the primers 5'-GCCAAACGCAAACCTTACCAGAGTG -3' and 5'-ACGGACACATCCAGAAGAA AACGG-3' for TLR1, and 5'-GTTGGATACAGGCCAGACTTTGTTG-3' and 5'-TCGGATT CCGGTCGGATGGGAG-3' for HPRT (control for the amount of RNA). TLR1 mRNA was absent from untreated and LPS-stimulated TLR1-/- macrophages but was present in wild-type mice (Fig. 5d).
FACS staining for TLR1 and TLR2.
PBMCs were stained with a combination of biotin-conjugated antibody against CD4 (PharMingen), PE-conjugated antibody against human TLR1 clone GD2.F4 and fluorescein isothiocyanate (FITC)-conjugated antibody against human TLR2 clone TL2.1 (eBioscience, San Diego, California). CD4 was labeled with streptavidin-conjugated Cy-Chrome (PharMingen). Cells were stained with isotype controls for TLR1 (mouse IgG1−PE, Sigma) and TLR2 (mouse IgG2a−FITC, PharMingen). Cells gated by size and CD4 expression were analyzed by flow cytometry with CellQuest software (Becton Dickinson).
Received 15 April 2002; Accepted 6 June 2002; Published online: 1 July 2002.
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Acknowledgments
We thank T. Deshefy-Longhi, S. Samanta and D. Beck for assistance, and F. Manzo for assistance with manuscript preparation. This work was supported by grants from the US National Institutes of Health, and an Arthritis Foundation Biomedical Science Grant (RAF). L.A. received a Human Frontier Science Program postdoctoral long-term fellowship, E.F. received a Clinical-Scientist Award in Translational Research from the Burroughs Wellcome Fund, R.M. is supported by a Searl Scholarship, and R.M. is an Assistant Investigator and R.A.F. is an Investigator of the Howard Hughes Medical Institute.
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