Lyme disease pathogenesis results from a complex interaction between Borrelia burgdorferi and the host immune system. The intensity and nature of the inflammatory response of host immune cells to B. burgdorferi may be a determining factor in disease progression. Gene array analysis was used to examine the expression of genes encoding cytokines, chemokines, and related factors in the joint tissue of infected C3H/HeJ mice and in a murine macrophage-like cell line in response to a disseminating or attenuated clinical isolate of B. burgdorferi. Both isolates elicited a robust proinflammatory response in RAW264.7 cells characterized by an increase in transcript levels of genes encoding CC and CXC chemokines, proinflammatory cytokines, and TNF superfamily members. Transcription of genes encoding IL-1β, IL-6, MCP-1, MIP-1α, CXCR4, and TLR2 induced in RAW264.7 cells by either live or heat-killed spirochetes did not differ significantly at any time point over a 24-h period, nor was there a difference in the protein levels of IL-10, TNF-α, IL-6, and IL-12p70 in culture supernatants. Thus, induction of host macrophage expression of proinflammatory mediators by host macrophages does not contribute to the differential pathogenicity of different B. burgdorferi strains.

Lyme disease, the most prevalent arthropod-borne infection in the U.S. (1), is a multisystemic disorder caused by infection with the tick-transmitted spirochete Borrelia burgdorferi (2). Approximately 70–80% of patients develop a characteristic rash, erythema migrans (EM),5 at the site of inoculation (3). The EM lesion is characterized by an influx of immune cells, predominantly T lymphocytes, macrophages/monocytes, and dendritic cells (4), which in most cases eradicate the infection (5). In patients who develop more serious long-term sequelae, the spirochete migrates from the skin via hematogenous dissemination to a variety of secondary sites, most commonly the skin, heart, joints, and nervous system (6). Pathological manifestations are of an inflammatory nature and include carditis, arthritis, and neurological disorders (6).

Elucidation of spirochetal virulence factors may be facilitated by identification of B. burgdorferi isolates with different pathogenic properties. Clinical isolates of B. burgdorferi sensu stricto can be classified by a variety of typing procedures, including restriction fragment length polymorphism (RFLP) analysis of the 16S–23S ribosomal DNA spacer (7, 8). Ribosomal spacer type (RST)1 isolates are associated with a significantly higher percentage of positive blood cultures as well as an increased incidence of multiple EM in patients, suggesting a greater capacity for hematogenous dissemination relative to RST3 isolates (9). These clinical observations correlate with experimental findings obtained using a murine model of Lyme borreliosis, where infection of C3H/HeJ mice with RST1 strains resulted in significantly higher spirochete loads in tissue, as well as more severe arthritis and aortitis, than did infection with RST3 isolates (10, 11).

The host factors contributing to disease pathogenesis have been studied using strains of mice with differing degrees of susceptibility to B. burgdorferi infection (12). Genetically susceptible C3H/HeJ mice develop severe arthritis when infected with as few as 200 spirochetes, whereas C57BL/6N mice develop mild arthritis even at an infectious dose of 2 × 105 spirochetes (13). These distinctly different host responses may depend in part upon the qualitative and quantitative differences in cytokine expression elicited by the spirochete.

A dual role for pro- and antiinflammatory (Th1/Th2) cytokines in host defense and disease pathogenesis has been established for Lyme disease (14). However, the ratio of these groups of cytokines at different stages of infection appears to be a determining factor in disease outcome. Levels of IFN-γ, IL-4, and IL12p70 produced by B. burgdorferi-stimulated whole blood and PBMCs, or in EM blister fluids, differ between patients with localized or disseminated B. burgdorferi infection (4, 15, 16, 17). Similar observations connecting the nature of the cytokine response with disease outcome have been made using murine models of Lyme borreliosis (18, 19). Together, these studies indicate that a strong proinflammatory response early in infection mediates host protection. In contrast, a sustained and dominant Th1 cytokine response in serum or in infected target tissues is associated with severe inflammation-induced pathology both in mouse models (20) and in Lyme disease patients (17, 21, 22).

Macrophages have been implicated in the development and progression of Lyme disease pathogenesis. In susceptible laboratory mouse strains, macrophages are the most abundant cell type in the inflammatory infiltrate of Lyme carditis (23, 24) and secrete increased levels of the proinflammatory cytokines IL-1β, TNF-α, and IL-12 relative to macrophages taken from a site without active disease (25). In several rodent models, macrophages are present in the inflamed synovium of ankle joints and have been shown to be key mediators of severe arthritis (26, 27, 28). Additionally, highly activated macrophages are a predominant component of the cellular infiltrate of EM lesions in Lyme disease patients (4), thereby placing the macrophage among the first cell types to encounter and respond to the invading pathogen.

The nature and/or intensity of the macrophage response to different B. burgdorferi genotypes, as measured by the production of cytokines, may be a decisive factor in survival at the initial inoculation site and subsequent disease progression. Although a number of studies have focused on the expression of selected cytokines and chemokines in various tissues and cell lines in response to B. burgdorferi (4, 29, 30, 31, 32), only one study has characterized the global cytokine expression profile elicited by B. burgdorferi (33). We used cytokine gene arrays to assess the B. burgdorferi-induced global cytokine transcriptional profile in the joints of Lyme disease-susceptible mice and compared these results to the cytokine profiles induced in the RAW264.7 γNO(−) mouse macrophage-derived cell line in response to two clinical isolates of B. burgdorferi associated with distinctly different disease outcomes.

Murine macrophage-like cell line RAW264.7 γNO(−) was obtained from the American Type Culture Collection (CRL-2278). RAW264.7 γNO(−) cells do not produce NO upon treatment with IFN-γ alone, but they require LPS for full activation. This property makes such behavior more like that of normal macrophages from some commonly used murine models of Lyme borreliosis (e.g., C3H/HeN). Cells were grown in RPMI 1640 medium supplemented with 2 mM l-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate (American Type Culture Collection cat. no. 30–2001), and 10% FCS (American Type Culture Collection, 30–2021). Cells were maintained in T-75 cm2 tissue culture flasks (Corning) at 37°C in a humidified 5% CO2 incubator.

Low-passage (passages 3–5) B. burgdorferi clinical isolates BL206, B515, and B479, representing the disseminating RST1 genotype, and B356, B331, and B418, representing the nondisseminating RST3A genotype, were used in these studies (10, 11). B. burgdorferi was cultured at 33°C in Barbour-Stoenner-Kelley (BSK)-H medium (Sigma-Aldrich) supplemented with 6% rabbit serum. Spirochetes were grown to late-logarithmic phase and examined for motility by dark-field microscopy. Organisms were quantitated by fluorescence microscopy after mixing 10 μl aliquots of culture material with 10 μl of an acridine orange solution (100 μg/ml). Bacteria were harvested by centrifugation of the culture at 7000 × g for 15 min, washed twice with sterile PBS (pH 7.4), and diluted in the specified medium to required concentration. Heat-killed B. burgdorferi cells were prepared as described above except for heating at 56°C for 30 min before dilution.

All animal experiment protocols were reviewed and approved by the Institutional Animal Care and Use Committee of New York Medical College. Four-week-old specific pathogen-free C3H/HeJ mice of either sex were purchased from The Jackson Laboratory and maintained in separate cages in the Department of Comparative Medicine at New York Medical College following the National Institutes of Health guidelines for care and use of laboratory animals. Two groups of 5 mice were inoculated intradermally on the shaved back with 0.1 ml PBS containing 1 × 104B. burgdorferi BL206 or with PBS alone. Mice were euthanized by exposure to CO2 on day 14 after inoculation (11), and samples of ear biopsy and joint tissue were collected aseptically for culture and total RNA extraction. Culture of B. burgdorferi from ear biopsy was performed as previously described (10, 11).

Individual hindlimb ankle joints from which the skin had been removed were frozen in liquid nitrogen, wrapped in aluminum foil, pulverized with a hammer, then placed immediately into a glass tissue homogenizer containing 0.5–1 ml of lysis buffer from the RNA isolation kit (RNAzol B, Tel-Test). RNA was subsequently prepared as described above for the RAW264.7 cells.

RAW264.7 γNO(−) cells, cultured as described above, were grown to 80–90% confluence in T-75 flasks or in 24-well plates. Medium was aspirated aseptically and replaced with fresh serum-free RPMI 1640 medium containing B. burgdorferi isolates at a multiplicity of infection (MOI) of 10:1 or 1 μg/ml LPS from Escherichia coli 0127:B8 (Sigma-Aldrich, cat. no. L 3880). Medium was added to controls. Duplicate or triplicate flasks/wells of RAW264.7 cells were harvested for RNA extraction at 2, 8, 16, and 24 h after exposure. Two milliliters of supernatant from each flask was also collected and stored at −80°C for measurement of the cytokine protein level by flow cytometry. The experiment was repeated on a later date using a different lot of RAW264.7 γNO(−) cells obtained from the American Type Culture Collection.

Total RNA was prepared from freshly harvested RAW264.7 γNO(−) cells using a commercial RNA isolation kit (RNAzol B). Briefly, cells were lysed by the addition of RNAzol B (1 ml/10 cm2) to homogenize the cells; subsequently, 0.2 ml of chloroform per 2 ml of homogenate was added. After shaking for 15 s and incubation on ice for 5 min, the lysates were centrifuged at 12,000 × g at 4°C for 15 min. The aqueous phase was transferred to a fresh tube and the RNA was precipitated with isopropanol and washed with 70% ethanol. Each dried RNA pellet was resuspended in 20 μl of diethyl pyrocarbonate-treated, RNase-free water and treated further with DNase (DNase Treatment & Removal Reagents, Ambion) to remove any residual DNA. The quality and quantity of RNA samples were determined by gel electrophoresis and spectrophotometry (BioPhotometer, Eppendorf). RNA samples were frozen at −80°C until analyzed by gene array or real-time RT-PCR.

The Panorama mouse cytokine gene array, consisting of 514 different cytokine-related cDNAs printed as PCR products onto a charged nylon membrane, was purchased from Sigma-Genosys. Also included on the array are eight positive control “housekeeping” genes, mouse genomic DNA, and five negative controls. 33P-labeled cDNA probes were generated with mouse cytokine cDNA labeling primers (provided by Sigma-Genosys) using 4 μg of total RNA and hybridized with the array membrane overnight at 65°C according to the manufacturer’s protocol. After hybridization, the membrane arrays were washed and exposed to a PhosphorImager screen (Molecular Dynamics) for 16–48 h as described previously (34). Four replicate arrays were prepared for each experimental condition.

The exposed PhosphorImager screen was scanned with a pixel size of 100 μm on a Storm 840 PhosphorImager (Molecular Dynamics). The image files were analyzed with a template containing the spot layout of the array using ArrayVision software (version 8.0; Imaging Research). The raw intensity value, in pixels for each spot (RIVj) on the array, was determined after subtraction of the background intensity and was exported to Microsoft Excel for further analysis. The cytokine microarray data were normalized using a global adjustment of intensity values approach (35). First, the mean intensity value of each array (MIVi) (based on the raw intensity values of 514 cytokine-related genes and control DNAs, each in duplicate) was calculated as: MIVi = Σ(RIVij)/the number of spots on the array, where i refers to individual array and j refers to individual spot. Second, a global mean intensity value (GMIV) based on the MIVi of the 12 arrays analyzed was determined (GMIV = Σ(AIVi)/the number of arrays). Third, a normalization factor (NFi) was assigned for each array (NFi = GMIV/MIVi). Finally, the raw intensity value of each spot on an individual array was multiplied with the corresponding NFi to convert it to a normalized intensity value for statistical analysis.

For each gene, normalized intensity values were generated from two duplicate spots on each of four replicate arrays for each experimental condition. A two-tailed, unpaired Student’s t test was used to estimate the statistical significance among the experimental groups by comparison of the mean intensity values from all eight replicate spots for each gene. Significance of differential expression was determined at p < 0.05.

The expression of selected cytokine and related genes in mouse tissue and in RAW264.7 cells exposed to either isolate B356 or isolate BL206 was determined by real-time quantitative RT-PCR using SYBR Green technology with the LightCycler (Roche Applied Science) or the ABI 7900HT SDS (Applied Biosystems), as described previously (10, 34, 36). For each RNA sample, the expression of β-actin was quantified by real-time RT-PCR, and a ΔΔCt method was used to estimate the differential gene expression between samples (37). Oligonucleotide sequences of primers used for RT-PCR using SYBR Green technology were as follows: actinb, β-actin (Act) B forward (5′-TCACCCACACTGTGCCCATCTACGA- 3′) and Act B reverse (5′-GGATGCCACAGGATTCCATACCCA-3′); Il1b, IL-β forward (5′-GCCTTGGGCCTCAAAGGAAAGAATC-3′) and IL-β reverse (5′-GGAAGACACAGATTCCATGGTGAAG-3′); Il6, IL-6 forward (5′-TGGAGTCACAGAAGGAGTGGCTAAG-3′) and IL-6 reverse (5′-TCTGACCACAGTGAGGAATGTCCAC-3′); Il10, IL-10 forward (5′-GTGAAGACTTTCTTTCAAACAAAG-3′) and IL-10 reverse (5′-CTGCTCCACTGCCTTGCTCTTATT-3′); Tnfa, TNF-α forward (5′-ATAGCTCCCAGAAAAGCAAGC-3′) and TNF-α reverse (5′-CACCCCGAAGTTCAGTAGACA-3′); Mcp1, MCP-1 forward (GGAAAAATGGATCCACACCTTGC-3′) and MCP-1 reverse (TCTCTTCCTCCACCACCATGCAG-3′); Mip1a, MIP-1α forward (5′-CCCAGCCAGGTGTCATTTTCC-3′) and MIP-1α reverse (5′-GCATTCAGTTCCAGGTCAGTG-3′); Mip2a, MIP-2α forward (5′-TCCAGAGCTTGAGTGTGACG-3′) and MIP-2α reverse (5′-TCAGGTACGATCCAGGCTTC-3′); Tlr2, TLR2 forward (5′-CTCCTGAAGCTGTTGCGTTAC-3′) and TLR2 reverse (5′-GCTCCCTTACAGGCTGAGTTC-3′); Tlr4, TLR4 forward (5′-TCGCCTTCTTAGCAGAAACAC-3′) and TLR4 reverse (5′-GCCTTAGCCTCTTCTCCTTC-3′); Cox2, cyclooxygenase (COX)-2 forward (5′-TCTGGAACATTGTGAACAACATC-3′) and COX-2 reverse (5′-AAGCTCCTTATTTCCCTTCACAC-3′); Cxcr4, CXCR-4 forward (5′-GAAGTGGGGTCTGGAGACTATG-3′) and CXCR-4 reverse (5′-AGGGGAGTGTGATGACAAAGAG-3′); Icam2, ICAM2 forward (5′-TGCTGTTCTTATTTGTGACATCTG-3′) and ICAM2 reverse (5′-TGTATTGAGGCTAAAAAGGAGAGG-3′); Il2r2, IL-1R2 forward (5′-TAGTCCCGTGCAAAGTGTTTC-3′) and IL-1R2 reverse (5′-CTGTATGCAGATCCTCCCTTG-3′). The expression levels of selected chemokine, cytokine, and tissue remodeling factor genes in RAW264.7 cells exposed to B. burgdorferi BL206, B515, B479, B356, B331, and B418 were determined by real-time PCR using TaqMan gene expression assays (Applied Biosystems). Duplicate or triplicate assays were performed for the following target genes: Il1b (IL-1β, Mm00434228_m1), Gdf9 (growth differentiation factor 9, Mm00433565_m1), Mmp8 (matrix metallopeptidase 8, Mm00772335_m1), Bmpr1b (bone morphogenetic protein receptor, type 1B, Mm00432117_m1), Tnfrsf6/Fas (TNF receptor superfamily 6, Mm00433237_m1), Bmp1 (bone morphogenetic protein 1, Mm0080225_m1), Bmp9 (bone morphogenetic protein 9, Mm03024080_m1), Tie2 (endothelium-specific receptor tyrosine kinase 2, Mm01256892_m1), and Tgfbr1 (TGF-β receptor I, Mm00436971_m1). The β-actin amplicon was used as the endogenous control for normalization of data. All assays were done in 20-μl reaction mixtures containing TaqMan universal PCR master mix (Applied Biosystems), 20× TaqMan gene expression assay mix, and cDNA on an Applied Biosystems ABI 7900HT SDS system. The differential gene expression between samples was calculated as described above.

The Cytokine Bead Array mouse inflammation kit (BD Biosciences) was used for simultaneous measurement of IL-6, TNF-α, IL-10, IL-12, IFN-γ, and MCP-1 in the supernatants of RAW264.7 γNO(−) cells exposed to live or heat-killed B. burgdorferi isolates, LPS, or medium control. Fifty microliters of each sample was added to an equal volume of the cytokine bead mixture and detection reagent, followed by a 3-h incubation at room temperature in the dark. Ten additional tubes, each containing equal volumes of beads, detection reagent, and graded amounts of the six cytokines, were prepared in parallel to generate a standard curve for each cytokine. Unstained, FITC-labeled, or PE-labeled cytometer setup beads were prepared immediately before use. Following incubation, beads were washed with the buffer provided in the kit, centrifuged at 200 × g for 5 min, and the supernatants were carefully aspirated. The pellets were resuspended in 300 μl of the kit wash buffer and assayed immediately on the FACSCalibur (BD Biosciences). Cytokine concentrations were determined using the software provided.

Although the pathological manifestations of Lyme disease are characterized by inflammation, little is known about the nature of the global cytokine response during early disseminated infection. Therefore, a murine model of Lyme borreliosis was used to assess the expression of cytokine and related genes in the acutely arthritic joint tissue of disease-susceptible C3H/HeJ mice. This mouse strain develops clinically apparent arthritis ∼12 days after infection (11). All 5 mice inoculated intradermally with a dose of 1 × 104B. burgdorferi BL206 were infected, as confirmed by culture of ear biopsies collected at day 14. RNA was extracted from hindlimb ankle joints on day 14 and analyzed using the Panorama mouse cytokine gene array, which consists of 514 different cDNA transcripts representing cytokines, chemokines, and other immunomodulatory factors and their receptors. Transcript abundance levels of 46 genes were found to be significantly induced (≥2-fold, p < 0.05) in the joints of BL206-infected mice relative to the PBS-inoculated controls (Table I). The most dramatic induction was observed for CXCL13, CCR9, and IL-1β. Transcription levels of a number of proinflammatory mediators were up-regulated in the BL206-infected mouse joint tissue, including several chemokines (MCP-1, MCP-3, CCL6, MIP-1γ, and MIP-3β) and receptors (CCR2 and CCR9), and IL-1β and its receptor, IL-1R.

Table I.

Genes transcriptionally induced in joint tissue of BL206-infected C3H/HeJ mice 2 wk postinfection

FunctionGene NameFCap ValueFunctionGene NameFCap Value
Cytokines/chemokines CXCL13 108.1 <0.0001 Apoptosis SARP-1 2.4 0.0002 
 IL-1β 41.8 <0.0001 Cell adhesion Integrin-β2 4.2 <0.0001 
 CCL2/MCP-1 14.9 <0.0001  Integrin-α4 3.7 0.0093 
 CXCL6 7.4 <0.0001  Integrin-αL 3.4 <0.0001 
 CXCL9 6.8 <0.0001  ICAM-1 2.0 0.0011 
 CCL7/MCP-3 2.8 <0.0001 Cell proliferation TP1 2.6 <0.0001 
 CCL6 2.6 0.0005 Cytokine suppression CIS3/SOCS3 2.1 0.0033 
 CCL9/MIP-1γ 2.5 <0.0001 Growth/development TNFRSF1B 5.8 <0.0001 
 CCL19/MIP-3β 2.4 0.0017  Cerberus 3.0 0.0010 
 CXCL14/BRAK 2.3 <0.0001  c-fos 2.2 <0.0001 
 CXCL15/lungkine 2.0 0.0002  LTBP-2 2.1 0.0093 
 CCL3/MIP-1α 2.0 0.0058 Invasion/migration MMP-3 8.8 <0.0001 
Receptors/antagonists CCR-9 158.1 0.0020  TIMP-1 3.4 <0.0001 
 IL-1R antagonist 26.1 <0.0001 Lymphocyte activation CD45 2.9 0.0002 
 CCR-5 17.8 <0.0001  MD-1 2.7 <0.0001 
 IL-12Rβ1 10.2 0.0101 Proinflammatory Cox-2 7.2 <0.0001 
 IL-18R 4.5 0.0083  Stat1 6.5 <0.0001 
 IL-10Rα 3.7 <0.0001 Caspase-1 2.7 0.0001  
 IL-2Rγ 2.9 0.0001  Stat4 2.1 0.0015 
 CCR-2 2.9 <0.0001 TNF superfamily TNFSF6/FasL 2.7 <0.0001 
 IL-13Rα1 2.5 <0.0001  TNFRSF10B 2.2 <0.0001 
 IL-8R 2.1 0.0054     
 IL-18R AcP 2.1 0.0026     
 IL-1RI 2.0 0.0025     
FunctionGene NameFCap ValueFunctionGene NameFCap Value
Cytokines/chemokines CXCL13 108.1 <0.0001 Apoptosis SARP-1 2.4 0.0002 
 IL-1β 41.8 <0.0001 Cell adhesion Integrin-β2 4.2 <0.0001 
 CCL2/MCP-1 14.9 <0.0001  Integrin-α4 3.7 0.0093 
 CXCL6 7.4 <0.0001  Integrin-αL 3.4 <0.0001 
 CXCL9 6.8 <0.0001  ICAM-1 2.0 0.0011 
 CCL7/MCP-3 2.8 <0.0001 Cell proliferation TP1 2.6 <0.0001 
 CCL6 2.6 0.0005 Cytokine suppression CIS3/SOCS3 2.1 0.0033 
 CCL9/MIP-1γ 2.5 <0.0001 Growth/development TNFRSF1B 5.8 <0.0001 
 CCL19/MIP-3β 2.4 0.0017  Cerberus 3.0 0.0010 
 CXCL14/BRAK 2.3 <0.0001  c-fos 2.2 <0.0001 
 CXCL15/lungkine 2.0 0.0002  LTBP-2 2.1 0.0093 
 CCL3/MIP-1α 2.0 0.0058 Invasion/migration MMP-3 8.8 <0.0001 
Receptors/antagonists CCR-9 158.1 0.0020  TIMP-1 3.4 <0.0001 
 IL-1R antagonist 26.1 <0.0001 Lymphocyte activation CD45 2.9 0.0002 
 CCR-5 17.8 <0.0001  MD-1 2.7 <0.0001 
 IL-12Rβ1 10.2 0.0101 Proinflammatory Cox-2 7.2 <0.0001 
 IL-18R 4.5 0.0083  Stat1 6.5 <0.0001 
 IL-10Rα 3.7 <0.0001 Caspase-1 2.7 0.0001  
 IL-2Rγ 2.9 0.0001  Stat4 2.1 0.0015 
 CCR-2 2.9 <0.0001 TNF superfamily TNFSF6/FasL 2.7 <0.0001 
 IL-13Rα1 2.5 <0.0001  TNFRSF10B 2.2 <0.0001 
 IL-8R 2.1 0.0054     
 IL-18R AcP 2.1 0.0026     
 IL-1RI 2.0 0.0025     
a

FC indicates fold change relative to uninfected control.

As shown above, isolate BL206 can disseminate to the joints in C3H/HeJ mice. Previous studies have shown that isolate B356 cannot disseminate to the joints in the same mouse strain (10, 11). It was therefore of interest to determine whether these two B. burgdorferi isolates elicit different responses in an infected mouse. As isolate B356 is not detected in joint tissue, the murine macrophage-derived cell line RAW264.7 γNO(−) was used instead to compare the expression of cytokines and related genes induced by these two B. burgdorferi clinical isolates of differing genotype and pathogenic potential. A combined total of 63 (12.3%) genes were differentially transcribed (≥2-fold change, p < 0.05) upon exposure to the two B. burgdorferi isolates (Fig. 1). Of these, 33 genes (6.4%; 30 induced and 3 repressed) were differentially transcribed upon exposure to either BL206 or B356 (Table II). The commonly induced genes included CC and CXC chemokines and the proinflammatory cytokines IL-1α, IL-1β, and IL-6. The three commonly repressed genes included one chemokine, CCL21, a chemokine receptor, CXCR4, and CD28. Although the extent of induction for a number of these genes suggested a trend toward a stronger proinflammatory response to isolate BL206 than to B356, these differences did not reach statistical significance.

FIGURE 1.

Differential expression of genes encoding cytokines and receptors is elicited by B. burgdorferi isolates BL206 and B356. RNA from RAW264.7 γNO(−) cells, which had been cocultured for 16 h with B. burgdorferi isolates BL206 or B356 (MOI of 10:1; spirochetes-RAW264.7 cells), was analyzed by gene array. Differentially expressed transcripts had changes ≥2-fold and p < 0.05. Arrows indicate induction or repression relative to the time-matched control.

FIGURE 1.

Differential expression of genes encoding cytokines and receptors is elicited by B. burgdorferi isolates BL206 and B356. RNA from RAW264.7 γNO(−) cells, which had been cocultured for 16 h with B. burgdorferi isolates BL206 or B356 (MOI of 10:1; spirochetes-RAW264.7 cells), was analyzed by gene array. Differentially expressed transcripts had changes ≥2-fold and p < 0.05. Arrows indicate induction or repression relative to the time-matched control.

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Table II.

Cytokine and related genes significantly induced or repressed in RAW264.7 γNO(−) cells exposed to either B. burgdorferi isolates BL206 or B356 for 16 h

FunctionGene NameGenBank Accession No.BL206B356
FCap ValueFCap Value
Cell cycle regulation c-myc NM_010849 3.7 0.0118 3.3 0.0025 
Cytokines/chemokines CCL20/MIP-3α NM_016960 115.1 0.0277 78.4 0.0444 
 IL-1β NM_008361 34.0 0.0004 20.8 8.6E-06 
 IL-6 X54542 27.8 0.0001 22.7 2.96E-06 
 CXCL1/GROα NM_008176 17.1 1.07E-05 11.6 0.0022 
 CCL7/MARC/MCP-3 Z12297 16.4 0.0006 14.4 0.0004 
 CCL2/JE/MCP-1 NM_011333 13.0 0.0002 10.5 0.0002 
 CXCL2/MIP-2α/GROβ NM_009140 8.0 0.0009 6.0 0.0030 
 CCL3/MIP-1α NM_011337 5.6 0.0032 4.7 0.0019 
 IL-1α NM_010554 4.5 0.0040 3.3 0.0131 
 CCL5/RANTES M77747 4.2 0.0065 3.3 0.0026 
 CCL6/C10 NM_009139 3.9 0.0009 3.0 0.0001 
 CCL22/MDC NM_009137 3.4 0.0402 2.3 0.0072 
 CCL9/MIP-1γ NM_011338 2.5 0.0023 2.4 0.0030 
 CCL19/MIP-3β NM_011888 2.0 0.0034 2.4 0.0318 
 CCL21/C6kine AF006637 −26.9 2.04E-06 −10.8 7.13E-06 
Receptors and antagonists IL-1ra M57525 7.6 0.0001 7.5 0.0010 
 CXCR-4 NM_009911 −7.3 0.0003 −5.8 0.0006 
Cytokine suppression CIS3/SOCS3 NM_007707 3.0 0.0394 3.4 0.0095 
Growth and development Notch-1 NM_008714 5.1 0.0087 5.1 0.0032 
 BDNF NM_007540 17.7 0.0110 16.4 0.0295 
 β-NGF M35075 2.7 0.0410 2.0 0.0244 
 Angiopoietin-3/4 NM_009641 2.0 0.0396 2.3 0.0113 
Immune cell activation CD28 NM_007642 −2.2 0.0360 −2.2 0.0382 
Inflammation Cox-2 NM_011198 9.1 0.0002 9.8 0.0002 
 iNOS NM_010927 4.9 0.0291 3.7 0.0420 
 Caspase-11 NM_007609 3.8 0.0106 3.2 0.0216 
 Stat1 NM_009283 3.6 0.0173 3.1 0.0078 
TNF superfamily TNFRSF6/Fas NM_007987 39.3 0.0006 20.9 0.0245 
 TNF-α M13049 6.8 0.0029 5.2 0.0042 
 TNFRII/TNFRSF1B NM_011610 3.8 0.0010 3.5 0.0041 
 TNFRSF5/CD40 NM_011611 3.3 0.0007 2.5 0.0257 
Weight regulation MCH BB175332 3.3 0.0153 2.6 0.0489 
FunctionGene NameGenBank Accession No.BL206B356
FCap ValueFCap Value
Cell cycle regulation c-myc NM_010849 3.7 0.0118 3.3 0.0025 
Cytokines/chemokines CCL20/MIP-3α NM_016960 115.1 0.0277 78.4 0.0444 
 IL-1β NM_008361 34.0 0.0004 20.8 8.6E-06 
 IL-6 X54542 27.8 0.0001 22.7 2.96E-06 
 CXCL1/GROα NM_008176 17.1 1.07E-05 11.6 0.0022 
 CCL7/MARC/MCP-3 Z12297 16.4 0.0006 14.4 0.0004 
 CCL2/JE/MCP-1 NM_011333 13.0 0.0002 10.5 0.0002 
 CXCL2/MIP-2α/GROβ NM_009140 8.0 0.0009 6.0 0.0030 
 CCL3/MIP-1α NM_011337 5.6 0.0032 4.7 0.0019 
 IL-1α NM_010554 4.5 0.0040 3.3 0.0131 
 CCL5/RANTES M77747 4.2 0.0065 3.3 0.0026 
 CCL6/C10 NM_009139 3.9 0.0009 3.0 0.0001 
 CCL22/MDC NM_009137 3.4 0.0402 2.3 0.0072 
 CCL9/MIP-1γ NM_011338 2.5 0.0023 2.4 0.0030 
 CCL19/MIP-3β NM_011888 2.0 0.0034 2.4 0.0318 
 CCL21/C6kine AF006637 −26.9 2.04E-06 −10.8 7.13E-06 
Receptors and antagonists IL-1ra M57525 7.6 0.0001 7.5 0.0010 
 CXCR-4 NM_009911 −7.3 0.0003 −5.8 0.0006 
Cytokine suppression CIS3/SOCS3 NM_007707 3.0 0.0394 3.4 0.0095 
Growth and development Notch-1 NM_008714 5.1 0.0087 5.1 0.0032 
 BDNF NM_007540 17.7 0.0110 16.4 0.0295 
 β-NGF M35075 2.7 0.0410 2.0 0.0244 
 Angiopoietin-3/4 NM_009641 2.0 0.0396 2.3 0.0113 
Immune cell activation CD28 NM_007642 −2.2 0.0360 −2.2 0.0382 
Inflammation Cox-2 NM_011198 9.1 0.0002 9.8 0.0002 
 iNOS NM_010927 4.9 0.0291 3.7 0.0420 
 Caspase-11 NM_007609 3.8 0.0106 3.2 0.0216 
 Stat1 NM_009283 3.6 0.0173 3.1 0.0078 
TNF superfamily TNFRSF6/Fas NM_007987 39.3 0.0006 20.9 0.0245 
 TNF-α M13049 6.8 0.0029 5.2 0.0042 
 TNFRII/TNFRSF1B NM_011610 3.8 0.0010 3.5 0.0041 
 TNFRSF5/CD40 NM_011611 3.3 0.0007 2.5 0.0257 
Weight regulation MCH BB175332 3.3 0.0153 2.6 0.0489 
a

FC indicates fold change relative to control.

A surprisingly small number of genes showed patterns of differential transcription unique to either of the two isolates. Eighteen genes were uniquely regulated in RAW264.7 cells exposed to isolate BL206 (Fig. 1 and Table III). These included induction of a number of genes involved in tissue remodeling processes, including growth differentiation factor 9 (GDF-9, 51.2-fold), bone morphogenetic protein 1 (BMP-1, 15.6-fold), bone morphogenetic protein 9 (BMP-9, 4.2-fold), matrix metalloproteinase 8 (MMP-8, 2.2-fold), and TGF-β receptor 1 (TGF-βR1, 5.4-fold). Transcript abundance of the gene encoding melanocortin receptor subtype 2 (MC2R, 18.5-fold) was also markedly increased. Transcription of five genes were uniquely suppressed by BL206: those encoding IL-18 binding protein (−7.4-fold), E-cadherin (−2.2-fold), a developmental factor (wingless-related MMTV integration site 13 (WNT-13), −2.0), an angiogenic factor (endothelium-specific receptor tyrosine kinase 2 (TIE-2), −4.8-fold), and a cytokine receptor (IL-6Rα, −2.1-fold).

Table III.

B. burgdorferi isolate-specific regulation of cytokine and related genes in the murine macrophage-derived cell line RAW264.7 gamma NO(-) after 16 hours co-culture

Gene FunctionGene NameGenbank Accession No.FCap Value
Genes uniquely regulated by BL206     
 Apoptosis A1 L16462 2.5 0.0088 
 Cell adhesion Hip AF116865 3.1 0.0474 
 Integrin-β6 X69902 2.1 0.0402 
 E-cadherin NM_009864 −2.2 0.0029 
 Cytokine receptors CCR-5 NM_009917 3.3 0.0466 
 IL-6Rα X51975 −2.1 0.0237 
 IL-18BP NM_010531 −7.4 0.0250 
 Endocrine stress response MC2R NM_008560 18.5 0.0074 
 Growth and development GDF-9 NM_008110 51.2 0.0332 
 BMP-1 L35281 15.6 0.0320 
 TGF-βRI NM_007394 5.4 0.0238 
 BMP-9 AF188286 4.2 0.0359 
 Chordin NM_009893 3.6 0.0415 
 NRG-3 NM_008734 2.1 0.0369 
 WNT-13 NM_009520 −2.0 0.0002 
 TIE-2 X71426 −4.8 0.0302 
 Invasion/migration MMP-8 NM_008611 2.2 0.0372 
 T cell regulation CTLA-4 NM_009843 2.2 0.0074 
Genes uniquely regulated by B356     
 Apoptosis Bad NM_007522 −2.7 0.0295 
 B cell development EBF NM_007897 4.2 0.0211 
 Chemokines/cytokines CCL12/MCP-5 NM_011331 5.6 0.0120 
 CX3CL1 NM_009142 −3.1 0.0008 
 IL-16 NM_010551 −3.6 0.0047 
 CCL28 NM_020279 −4.5 0.0476 
 Cytokine receptor IL-8R NM_009909 9.3 0.0310 
 Endocrine stress response CRFR1 NM_007762 2.5 0.0446 
 Growth and development TGF-β3 NM_009368 3.8 0.0132 
 FGF-1 NM_010197 −3.1 0.0373 
 BMP-RIB NM_007560 −10.2 0.0087 
 Iron uptake TransferrinR X57349 −2.0 0.0248 
Gene FunctionGene NameGenbank Accession No.FCap Value
Genes uniquely regulated by BL206     
 Apoptosis A1 L16462 2.5 0.0088 
 Cell adhesion Hip AF116865 3.1 0.0474 
 Integrin-β6 X69902 2.1 0.0402 
 E-cadherin NM_009864 −2.2 0.0029 
 Cytokine receptors CCR-5 NM_009917 3.3 0.0466 
 IL-6Rα X51975 −2.1 0.0237 
 IL-18BP NM_010531 −7.4 0.0250 
 Endocrine stress response MC2R NM_008560 18.5 0.0074 
 Growth and development GDF-9 NM_008110 51.2 0.0332 
 BMP-1 L35281 15.6 0.0320 
 TGF-βRI NM_007394 5.4 0.0238 
 BMP-9 AF188286 4.2 0.0359 
 Chordin NM_009893 3.6 0.0415 
 NRG-3 NM_008734 2.1 0.0369 
 WNT-13 NM_009520 −2.0 0.0002 
 TIE-2 X71426 −4.8 0.0302 
 Invasion/migration MMP-8 NM_008611 2.2 0.0372 
 T cell regulation CTLA-4 NM_009843 2.2 0.0074 
Genes uniquely regulated by B356     
 Apoptosis Bad NM_007522 −2.7 0.0295 
 B cell development EBF NM_007897 4.2 0.0211 
 Chemokines/cytokines CCL12/MCP-5 NM_011331 5.6 0.0120 
 CX3CL1 NM_009142 −3.1 0.0008 
 IL-16 NM_010551 −3.6 0.0047 
 CCL28 NM_020279 −4.5 0.0476 
 Cytokine receptor IL-8R NM_009909 9.3 0.0310 
 Endocrine stress response CRFR1 NM_007762 2.5 0.0446 
 Growth and development TGF-β3 NM_009368 3.8 0.0132 
 FGF-1 NM_010197 −3.1 0.0373 
 BMP-RIB NM_007560 −10.2 0.0087 
 Iron uptake TransferrinR X57349 −2.0 0.0248 
a

FC indicates fold change relative to control.

Twelve genes were differentially regulated only by B356 (Fig. 1 and Table III). These included three genes encoding chemokines (MCP-5/CCL12, 5.6-fold; neurotactin/CX3CL1, −3.1-fold; and CCL28, −4.5-fold, IL-16, −3.6-fold), a chemokine receptor (IL-8R, 9.3-fold), and two members of the TGF-β superfamily (TGF-β3, 3.8-fold; bone morphogenetic protein receptor IB (BMP-RIB), −10.2-fold).

To validate the gene array results, transcriptional activity of 13 genes was analyzed by real-time quantitative RT-PCR. The selected genes included those found to be induced, repressed, or unchanged relative to the control by gene array analysis (Table IV). A strong correlation (R2 = 0.71) was found between the fold change values obtained by gene array analysis and real-time RT-PCR.

Table IV.

A comparison of differential expression values of selected genes as determined by microarray and quantitative RT-PCR

GeneBL206B356
MicroarrayqRT-PCRMicroarrayqRT-PCR
IL-1β 34.0 24.4 20.8 21.6 
Il-6 27.8 12.5 22.7 10.5 
IL-10 1.1 0.8 1.9 
IL-1RII 1.1 1.4 −1.7 −5 
TNF-α 6.8 2.7 5.2 
CCL3/MIP-1α 5.6 5.5 4.7 5.6 
CXCL2/MIP-2α 8.0 12.6 6.0 13.7 
CCL2/MCP-1 13.0 2.3 10.5 2.2 
CXCR-4 −7.3 −7.7 −5.8 −7.3 
Cox-2 9.1 11.7 9.8 13.3 
ICAM2 1.8 −2.5 −1.7 −2.5 
TLR2 1.3 1.3 1.1 1.3 
TLR4 −1 −0.5 −1.7 −0.7 
GeneBL206B356
MicroarrayqRT-PCRMicroarrayqRT-PCR
IL-1β 34.0 24.4 20.8 21.6 
Il-6 27.8 12.5 22.7 10.5 
IL-10 1.1 0.8 1.9 
IL-1RII 1.1 1.4 −1.7 −5 
TNF-α 6.8 2.7 5.2 
CCL3/MIP-1α 5.6 5.5 4.7 5.6 
CXCL2/MIP-2α 8.0 12.6 6.0 13.7 
CCL2/MCP-1 13.0 2.3 10.5 2.2 
CXCR-4 −7.3 −7.7 −5.8 −7.3 
Cox-2 9.1 11.7 9.8 13.3 
ICAM2 1.8 −2.5 −1.7 −2.5 
TLR2 1.3 1.3 1.1 1.3 
TLR4 −1 −0.5 −1.7 −0.7 

Real-time RT-PCR was used to analyze the transcriptional expression kinetics of selected genes in response to B. burgdorferi isolates B356 and BL206 over a 24-h period. Nearly identical patterns of transcription for genes encoding IL-1β, IL-6, MCP-1, MIP-1α, CXCR-4, and TLR2 were elicited by both isolates during this period (Fig. 2). Relative transcript abundance of IL-1β, IL-6, and MIP-1α increased over time, peaking at 16 h, and then falling to 2-h levels by 24 h. This latter decrease may be attributable to an increased death rate of the RAW264.7 γNO(−) cells at 24 h of exposure, although this parameter was not quantitatively assessed. Transcript levels of MCP-1 increased steadily over 24 h to maximum increases of 3.0-fold (BL206) and 2.7-fold (B356). In contrast, transcription of CXCR-4 was repressed 2-fold by both isolates relative to the control by 2 h, and continued to decrease over time, reaching a maximal decline of −10.6 (BL206) and −7.9 (B356) at 24 h. For comparison, the transcriptional profiles of these genes in response to E. coli LPS, a potent stimulator of cytokine expression produced by Gram-negative bacteria (38), were also examined. At 16 or 24 h, transcription levels of CXCR-4, IL-6, and IL-1β were 6-, 7- and 46-fold higher, respectively, in RAW264.7 cells exposed to 1 μg/ml LPS (data not shown). There was no significant difference at any time point in the transcript abundance of these cytokines in RAW264.7 cells exposed to either live or heat-killed BL206 (data not shown).

FIGURE 2.

Gene expression profiles of RAW264.7 γNO(−) cells after exposure to B. burgdorferi clinical isolates BL206 and B356 as determined by real-time RT-PCR. RNA was isolated from the murine macrophage-like cell line after incubation for 2, 8, 16, or 24 h with either BL206 or B356 clinical isolate of B. burgdorferi (MOI of 10:1, spirochetes-RAW264.7 cells). Samples were assayed in duplicate, with the exception of 16- and 24-h time points for IL-6, for which a single value was available. ∗, p < 0.05, BL206-exposed RAW264.7 cells vs medium control (unpaired Student’s t test). #, p < 0.05, B356-exposed RAW264.7 cells vs medium control.

FIGURE 2.

Gene expression profiles of RAW264.7 γNO(−) cells after exposure to B. burgdorferi clinical isolates BL206 and B356 as determined by real-time RT-PCR. RNA was isolated from the murine macrophage-like cell line after incubation for 2, 8, 16, or 24 h with either BL206 or B356 clinical isolate of B. burgdorferi (MOI of 10:1, spirochetes-RAW264.7 cells). Samples were assayed in duplicate, with the exception of 16- and 24-h time points for IL-6, for which a single value was available. ∗, p < 0.05, BL206-exposed RAW264.7 cells vs medium control (unpaired Student’s t test). #, p < 0.05, B356-exposed RAW264.7 cells vs medium control.

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The protein levels for selected cytokines (TNF-α, MCP-1, IFN-γ, IL-6, IL-10, and IL-12p70) in the culture supernatants of RAW264.7 cells were measured after coculture with B. burgdorferi strains BL206 or B356 for 2 or 16 h (Fig. 3). Strikingly, no statistically significant differences were detected in protein levels induced by B356 or BL206 for any parameter tested. Both isolates rapidly induced a proinflammatory cytokine response by 2 h, characterized by significant production of TNF-α and MCP-1 relative to the medium controls (p < 0.01 and p < 0.05, respectively). Levels of these cytokines increased substantially by 16 h. Production of the proinflammatory cytokine IL-6 was delayed relative to that of TNF-α and MCP-1; protein was not detected at 2 h but was measured at 1100 pg/ml by 16 h (p < 0.01 vs control). The antiinflammatory cytokine, IL-10, was not detected at 2 h, and by 16 h was produced at levels that differed significantly from the control only in RAW264.7 cells that had been cocultured with B356. However, there was no significant difference in IL-10 levels induced by either isolate B356 or BL206. Neither of the B. burgdorferi isolates induced protein expression of IL-12p70 at levels that differed significantly from the medium control at either of the two time points tested (data not shown). IFN-γ was included as a negative control, as this cytokine is not expressed by macrophages under most conditions, and no expression of IFN-γ was observed, as expected (data not shown). As a positive control, RAW264.7 cells were incubated with 1 μg/ml E. coli LPS. With the exceptions of IL-12p70 and IFN-γ, LPS induced production of all cytokines at levels significantly higher than the medium controls (data not shown).

FIGURE 3.

Cytokine protein levels in supernatants of RAW264.7 γNO(−) cells exposed to B. burgdorferi isolates BL206 or B356 for 2 or 16 h. RAW264.7 cells grown to 80–90% confluence were incubated with live spirochetes (MOI of 10:1, spirochetes-RAW264.7 cells). Cytokine proteins were measured by flow cytometry using the Cytokine Bead Array, as described in Materials and Methods. Values represent the average of two samples ± SD. ∗, p < 0.05 relative to control (unpaired Student’s t test). ∗∗, p < 0.01 relative to control.

FIGURE 3.

Cytokine protein levels in supernatants of RAW264.7 γNO(−) cells exposed to B. burgdorferi isolates BL206 or B356 for 2 or 16 h. RAW264.7 cells grown to 80–90% confluence were incubated with live spirochetes (MOI of 10:1, spirochetes-RAW264.7 cells). Cytokine proteins were measured by flow cytometry using the Cytokine Bead Array, as described in Materials and Methods. Values represent the average of two samples ± SD. ∗, p < 0.05 relative to control (unpaired Student’s t test). ∗∗, p < 0.01 relative to control.

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Microarray analysis identified 18 genes uniquely regulated in RAW264.7 cells by isolate BL206 (Table III). Notably, many of these genes have annotated functions in growth and development or invasion/migration. To determine whether the ability to effect changes in the extracellular matrix is associated with invasive potential, RAW264.7 cells were cocultured with B. burgdorferi clinical isolates BL206, B515, or B479, which have been shown to disseminate in C3H/HeJ mice (11), or with isolates B356, B331 or B418, which do not disseminate. Total RNA collected after 2, 16, and 24 h was analyzed by real-time RT-PCR for the expression genes encoding TGF-βR1, MMP-8, and TNFRSF6/Fas. Surprisingly, transcriptional expression profiles were strikingly similar for all isolates (Fig. 4). Transcript levels of TGF-βR1 and MMP-8 were significantly repressed at 16 and 24 h upon exposure to either disseminating or attenuated isolates. The difference between the two groups of isolates was statistically significant only for TGF-βR1 at 16 h (p = 0.045). Transcript levels of GDF-9 were significantly induced by 2 h, and then they declined. Transcript levels of TNFRSF6/Fas were significantly induced by either disseminating and attenuated isolates by 2 h and reached maximum increase at 16 h with mean fold changes of 9.7 and 12.0, respectively. As a control, transcription of IL-1β was also measured and was found to be significantly induced at all time points by all six isolates (data not shown). Transcription of BMP-1, BMP-9, BMP-RIB and TIE-2 was also explored but could not be quantitated, as the expression levels of these genes were below the detection threshold even after 40 amplification cycles.

FIGURE 4.

Transcriptional expression profiles of tissue remodeling genes in RAW264.7 γNO(−) cells exposed to disseminating (RST1) or non-disseminating (RST3A) B. burgdorferi isolates. RNA was isolated from the murine macrophage-like cell line after incubation for 2, 16, or 24 h with RST1 (BL206, B515, B479) or RST3A (B356, B331, B418) clinical isolates of B. burgdorferi (MOI of 10:1, spirochetes-RAW264.7 cells). Samples were assayed in triplicate. Mean fold-change values between groups were compared. ∗, p < 0.05, RST1-exposed RAW264.7 cells vs medium control. #, p < 0.05, RST3A-exposed RAW264.7 cells vs medium control. §, p < 0.05, RST1-exposed RAW264.7 cells vs RST3A-exposed RAW264.7 cells.

FIGURE 4.

Transcriptional expression profiles of tissue remodeling genes in RAW264.7 γNO(−) cells exposed to disseminating (RST1) or non-disseminating (RST3A) B. burgdorferi isolates. RNA was isolated from the murine macrophage-like cell line after incubation for 2, 16, or 24 h with RST1 (BL206, B515, B479) or RST3A (B356, B331, B418) clinical isolates of B. burgdorferi (MOI of 10:1, spirochetes-RAW264.7 cells). Samples were assayed in triplicate. Mean fold-change values between groups were compared. ∗, p < 0.05, RST1-exposed RAW264.7 cells vs medium control. #, p < 0.05, RST3A-exposed RAW264.7 cells vs medium control. §, p < 0.05, RST1-exposed RAW264.7 cells vs RST3A-exposed RAW264.7 cells.

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B. burgdorferi clinical isolates BL206 and B356 represent genotypes that are associated with distinctly different disease profiles in both humans and mice; BL206 infection results in severe, disseminated disease in C3H/HeJ mice, whereas B356 does not disseminate from the inoculation site (10, 11). Gene expression profiling of the acutely arthritic joint tissue of BL206-infected C3H/HeJ mice during the early disseminated stage (14 days postinfection) revealed strong transcriptional induction of genes encoding proinflammatory cytokines, CC and CXC chemokines and receptors, inflammatory mediators such as COX-2, and tissue remodeling factors MMP-3 and tissue inhibitor of metalloproteinase 1 (TIMP-1). Transcription of genes encoding both IL-1β and its receptor antagonist, IL-1ra, was induced by 41.8- and 26.1-fold, respectively (Table I), which is consistent with previously reported findings connecting a higher IL-1β/IL-1ra ratio with Lyme arthritis (39, 40).

While these experiments were in progress, a study was published that used array analysis to profile the global gene expression in the B. burgdorferi-infected joints of arthritis-susceptible C3H/HeNCr mice and arthritis-resistant C57BL/6 mice (33). Despite the differences in the mouse strains (C3H/HeJ vs C3H/HeNCr), the array methodologies (cytokine-specific membrane array vs Affymetrix whole gene array) and the B. burgdorferi strains (BL206 vs N40) used, the induction of a similar subset of cytokines and chemokines was observed in the present study. By 2 wk postinfection, a number of transcripts encoding factors involved in host defense and inflammation were induced in the joint tissue of both mouse strains. These included chemokines and cytokines (CXCL13, CCL9, CXCL14, CCL2, and IL-1β) and factors involved in invasion and migration (MMP-3, TIMP-1). The gene encoding CXCL13, a proposed diagnostic marker for Lyme neuroborreliosis (41), which was induced 108-fold in joint tissue by isolate BL206 in the present study, was found by Crandall et al. (33) to be associated with greater numbers of spirochetes in the joints. One notable difference between the studies was the complete absence of differentially regulated chemokine and cytokine receptor genes observed in the joints of N40-infected C3H/HeNCr mice, whereas 10 of these genes were induced in the joints of C3H/HeJ mice by isolate BL206 in the present study. Crandall et al. also observed induction of a number of type I and/or type II IFN-responsive genes and concurrent repression of genes involved in epidermal differentiation in the joints of N40-infected C3H/HeNCr mice at 2 wk postinfection. Transcriptional activity of these IFN-responsive and epidermal differentiation genes could not be assessed in the current study because the Panorama mouse cytokine gene array does not contain transcripts for these genes.

We hypothesized that the differences in pathogenic potential of disseminating and attenuated isolates may be attributable, at least in part, to differences in the nature of the inflammatory response induced in host cells. However, as strain B356 and other RST3A isolates do not disseminate to joint tissue, an in vitro model was used to test this hypothesis. Host macrophages are prolific producers of cytokines and chemokines and are among the first cell types to encounter the spirochete at the site of inoculation (4). The expression of 514 cytokine and related genes by the murine macrophage RAW264.7 cell line was analyzed by gene array after 16 h of coculture with the B. burgdorferi isolates B356 and BL206. The results yielded several interesting observations. Remarkably, more than half (33/63) of the total number of genes differentially regulated transcriptionally in response to B. burgdorferi were commonly regulated by both genotypes. This group consisted primarily of genes encoding potent mediators of inflammation, including CC and CXC chemokines and their receptors, and the proinflammatory cytokines IL-1α and IL-1β. Transcription of three genes was commonly repressed in RAW264.7 cells by both B. burgdorferi isolates. The most striking repression was observed for the gene encoding CCL21. CCL21 is reportedly up-regulated in different skin inflammatory conditions and is proposed to function in the recruitment of dendritic cells and T lymphocytes to inflammatory foci (42, 43). Down-regulation of CCL21 expression by B. burgdorferi may therefore be a means of suppressing its clearance by skin dendritic cells.

Real-time quantitative RT-PCR analysis of selected genes over a 24-h time course of coculture with isolates B356 and BL206 revealed that levels of transcriptional expression of IL-1β, IL-6, MCP-1, MIP-1α, and CXCR-4 did not significantly differ between the two strains. These data support the initial observation of similar cytokine induction by both genotypes at 16 h and expand it to include indistinguishable expression kinetics of key inflammatory mediators from as early as 2 h after pathogen contact. No difference in transcription of these cytokines upon exposure to live or heat-killed BL206 was observed, which is consistent with other studies that found no difference in the production of selected cytokines elicited by live spirochetes or by heat-killed or sonicated B. burgdorferi extracts in human (44) and canine cells (45). This suggests that the elicited host cytokine responses are most likely induced by cell membrane components rather than by secreted or cytosolic factors. Although it is possible that incubation in serum-free medium may have affected the expression of spirochetal virulence factors that distinguish the two isolates, we think that this is unlikely, as B. burgdorferi cultures were grown in BSK medium and only exposed to serum-free RPMI for a maximum time of 24 h. Even after only 2 h of incubation in the serum-free medium, there were no differences in cytokine mRNA or protein levels elicited by the two isolates, suggesting that the similar cytokine responses of RAW264.7 cells to these isolates are not due to the effects of serum starvation on the expression of B. burgdorferi virulence factors.

Measurement of the protein levels of selected cytokines by RAW264.7 cells yielded results that were consistent with the transcriptional data. Both B. burgdorferi isolates stimulated rapid and robust production of TNF-α, MCP-1, and IL-6. There was no significant difference in protein production between isolates BL206 and B356 at any time point. The barely significant induction of IL-10 by B356 that was observed is not entirely consistent with published data that report B. burgdorferi-stimulated production of IL-10 by human PBMCs, a human monocytic cell line, and murine macrophages (20, 46, 47). However, while the protein concentration of induced IL-10 in several of these studies was comparable to that observed here, the background production of IL-10 was very low. In the current study, high background production of both IL-10 (71 pg/ml) and MCP-1 (14,101 pg/ml) was observed at 16 h for RAW264.7 cells exposed to medium alone. This has not been reported with other cell types and may be a characteristic of RAW264.7 γNO(−) cells. The ability of RAW264.7 γNO(−) cells to secrete IL-10 in response to a stimulus was established by coculture with E. coli LPS, which resulted in IL-10 production at levels significantly higher than the control (164.9 ± 18.5 pg/ml; p < 0.05; data not shown).

B. burgdorferi has been shown to induce cytokine production in monocytes/macrophages (48, 46) primarily by signaling through TLR2 (32, 49). We did not observe any differential expression of the gene encoding TLR2 during a 24-h period of exposure to either B. burgdorferi isolate (Fig. 2), although transcript levels of genes encoding proinflammatory cytokines and chemokines increased significantly. This contrasts with other reports of increased TLR2 protein expression in B. burgdorferi-stimulated human monocytes and PBMCs (4, 50). Several factors may account for this apparent disparity. We used a macrophage-derived cell line, while other studies used samples obtained from human subjects and likely contained populations of activated cells. Second, Cabral et al. measured surface protein expression of TLR2 after 48 h (50), whereas we did not measure protein levels but quantitated mRNA transcript abundance for up to 24 h. However, both Cabral et al. (50) and the present study concur in detecting significantly elevated levels of IL-1β and IL-6.

The strikingly similar chemokine and cytokine gene expression profiles of the B356- and BL206-stimulated RAW264.7 cells suggests that modulation of other host factors by B. burgdorferi contributes to the development and severity of clinical disease. The array results indicated that the disseminating isolate BL206 uniquely induced transcription of a number of genes involved in tissue remodeling processes. These genes consisted of MMP-8 and members of the TGF-β superfamily, including BMP-1, BMP-9, GDF-9, and TGF-βRI. The role of host MMPs in the pathogenesis of Lyme arthritis has been well established (51), and their presence in EM skin lesions has been hypothesized to facilitate spirochete dissemination through the extracellular matrix (52). BMPs cleave collagen and other extracellular matrix components and have been implicated in Salmonella pathogenesis (53). We therefore examined the transcriptional expression of these genes by real-time RT-PCR in RAW264.7 cells cocultured with additional disseminating and attenuated isolates. In all samples, transcript levels of BMP-1 and BMP-9 were too low to be detected even after 40 PCR cycles. With the exception of one time point, there was no significant difference in the mean fold changes in transcript levels of GDF-9, MMP-8, and TGF-βRI elicited by the RST1 or RST3 isolates. The apparent disparity between the array and RT-PCR results may be explained by the degree of variability between the replicate samples of RNA used in array analysis. Our criteria for differential regulation of a gene consisted of both a fold change ≥2 and p < 0.05 using a two-tailed, unpaired Student’s t test. Many of the genes meeting these criteria for one isolate also displayed a fold change of similar magnitude by the other isolate but, due to sample variability, had a p value >0.05. These genes were therefore considered uniquely induced by either BL206 or B356. In contrast, RNA used for RT-PCR analysis of these genes was obtained from a separate experiment using multiple isolates assayed in triplicate. The larger sample size resulted in greater statistical robustness and, consequently, significant differences in expression relative to the control for both experimental groups.

In summary, transcriptional profiling of a murine macrophage cell line cocultured with disseminating or nondisseminating clinical isolates of B. burgdorferi revealed no differences in the pro- and antiinflammatory cytokine and chemokine responses. Furthermore, there was no difference in the expression of selected factors involved in remodeling of the extracellular matrix. We conclude that the differential pathogenicity of disseminating and nondisseminating isolates of B. burgdorferi does not result from differences in the induction or repression of host macrophage-mediated inflammation.

The authors have no financial conflicts of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grant AI45801 and Grant 5UO1CI000160 from the Centers for Disease Control and Prevention.

5

Abbreviations used in this paper: EM, erythema migrans; BMP, bone morphogenetic protein; BMP-RIB, bone morphogenetic protein receptor IB; COX, cyclooxygenase; GDF, growth differentiation factor; MMP, matrix metalloproteinase; MOI, multiplicity of infection; MMP, matrix metalloproteinase; RST, ribosomal spacer type; TIE, endothelium-specific receptor tyrosine kinase; TIMP, tissue inhibitor of metalloproteinase; TNFSF6, TNF receptor superfamily 6.

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