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Pattern of Proinflammatory Cytokine Induction in RAW264.7 Mouse Macrophages Is Identical for Virulent and Attenuated Borrelia burgdorferi¹

Guiqing Wang^2,3,*, Mary M. Petzke^2,*, Radha Iyer^*, Hongyan Wu and Ira Schwartz^4,*

^* Department of Microbiology and Immunology and Department of Medicine, New York Medical College, Valhalla, NY 10595

	Abstract

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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-1, 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-, 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.

	Introduction

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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 x 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-, 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-, 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 NO(–) mouse macrophage-derived cell line in response to two clinical isolates of B. burgdorferi associated with distinctly different disease outcomes.

	Materials and Methods

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Culture of RAW264.7 NO(–) cells

Murine macrophage-like cell line RAW264.7 NO(–) was obtained from the American Type Culture Collection (CRL-2278). RAW264.7 NO(–) cells do not produce NO upon treatment with IFN- 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.

B. burgdorferi isolates

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 x 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.

Mouse infection model

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 x 10⁴ B. 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).

RNA preparation from mouse joint tissues

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.

Exposure of RAW264.7 NO(–) cells to B. burgdorferi

RAW264.7 NO(–) 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 NO(–) cells obtained from the American Type Culture Collection.

RNA preparation

Total RNA was prepared from freshly harvested RAW264.7 NO(–) 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 x 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.

Gene array hybridization and data analysis

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 (RIV_j) 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 (MIV_i) (based on the raw intensity values of 514 cytokine-related genes and control DNAs, each in duplicate) was calculated as: MIV_i = (RIV_ij)/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 MIV_i of the 12 arrays analyzed was determined (GMIV = (AIV_i)/the number of arrays). Third, a normalization factor (NF_i) was assigned for each array (NF_i = GMIV/MIV_i). Finally, the raw intensity value of each spot on an individual array was multiplied with the corresponding NF_i 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.

Real-time quantitative RT-PCR

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 C_t 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- forward (5'-ATAGCTCCCAGAAAAGCAAGC-3') and TNF- reverse (5'-CACCCCGAAGTTCAGTAGACA-3'); Mcp1, MCP-1 forward (GGAAAAATGGATCCACACCTTGC-3') and MCP-1 reverse (TCTCTTCCTCCACCACCATGCAG-3'); Mip1a, MIP-1 forward (5'-CCCAGCCAGGTGTCATTTTCC-3') and MIP-1 reverse (5'-GCATTCAGTTCCAGGTCAGTG-3'); Mip2a, MIP-2 forward (5'-TCCAGAGCTTGAGTGTGACG-3') and MIP-2 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), 20x 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.

Measurement of cytokine protein levels

The Cytokine Bead Array mouse inflammation kit (BD Biosciences) was used for simultaneous measurement of IL-6, TNF-, IL-10, IL-12, IFN-, and MCP-1 in the supernatants of RAW264.7 NO(–) 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 x 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.

	Results

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B. burgdorferi induces proinflammatory cytokine and chemokine gene expression in joints of BL206-infected mice

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 x 10⁴ B. 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-1, and MIP-3β) and receptors (CCR2 and CCR9), and IL-1β and its receptor, IL-1R.

B. burgdorferi induces proinflammatory cytokine and chemokine gene expression in RAW264.7 NO(–) cells

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 NO(–) 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-1, 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.

View larger version (12K): [in this window] [in a new window]   FIGURE 1. Differential expression of genes encoding cytokines and receptors is elicited by B. burgdorferi isolates BL206 and B356. RNA from RAW264.7 NO(–) 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.

View this table: [in this window] [in a new window]   Table II. Cytokine and related genes significantly induced or repressed in RAW264.7 NO(–) cells exposed to either B. burgdorferi isolates BL206 or B356 for 16 h

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-6R, –2.1-fold).

RT-PCR validation of microarray data

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.

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-1, CXCR-4, and TLR2 were elicited by both isolates during this period (Fig. 2). Relative transcript abundance of IL-1β, IL-6, and MIP-1 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 NO(–) 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).

View larger version (11K): [in this window] [in a new window]   FIGURE 2. Gene expression profiles of RAW264.7 NO(–) 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.

The protein levels for selected cytokines (TNF-, MCP-1, IFN-, 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- 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- 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- was included as a negative control, as this cytokine is not expressed by macrophages under most conditions, and no expression of IFN- 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-, LPS induced production of all cytokines at levels significantly higher than the medium controls (data not shown).

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Cytokine protein levels in supernatants of RAW264.7 NO(–) 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.

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.

View larger version (14K): [in this window] [in a new window]   FIGURE 4. Transcriptional expression profiles of tissue remodeling genes in RAW264.7 NO(–) 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.

	Discussion

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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-1 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-1, 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-, 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 NO(–) cells. The ability of RAW264.7 NO(–) 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.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflicts of interest.

	Footnotes

 
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.

² G.W. and M.M.P. contributed equally to this work.

³ Current address: Department of Pathology, Westchester Medical Center, Clinical Laboratories, Room 1J-04, Valhalla, NY 10595.

⁴ Address correspondence and reprint requests to Dr. Ira Schwartz, Department of Microbiology and Immunology, New York Medical College, BSB Room 308, Valhalla, NY 10595. E-mail address: schwartz{at}nymc.edu

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.

Received for publication April 19, 2007. Accepted for publication April 3, 2008.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Orlowski, K. A., E. B. Hayes, G. L. Campbell, D. T. Dennis. 2000. Surveillance for Lyme disease: United States, 1992–1998. Morbid. Mortal. Wkly. Rep. CDC Surveill. Summ. 49: 1-11.
2. Steere, A. C., R. L. Grodzicki, A. N. Kornblatt, J. E. Craft, A. G. Barbour, W. Burgdorfer, G. P. Schmid, E. Johnson, S. E. Malawista. 1983. The spirochetal etiology of Lyme disease. N. Engl. J. Med. 308: 733-740. [Abstract]
3. Steere, A. C., V. K. Sikand. 2003. The presenting manifestations of Lyme disease and the outcomes of treatment. N. Engl. J. Med. 348: 2472-2474. [Free Full Text]
4. Salazar, J. C., C. D. Pope, T. J. Sellati, H. M. Feder, Jr, T. G. Kiely, K. R. Dardick, R. L. Buckman, M. W. Moore, M. J. Caimano, J. G. Pope, et al 2003. Coevolution of markers of innate and adaptive immunity in skin and peripheral blood of patients with erythema migrans. J. Immunol. 171: 2660-2670. [Abstract/Free Full Text]
5. Georgilis, K., A. C. Steere, M. S. Klempner. 1991. Infectivity of Borrelia burgdorferi correlates with resistance to elimination by phagocytic cells. J. Infect. Dis. 163: 150-155. [Medline]
6. Steere, A. C.. 2001. Lyme disease. N. Engl. J. Med. 345: 115-125. [Free Full Text]
7. Liveris, D., G. P. Wormser, J. Nowakowski, R. B. Nadelman, S. Bittker, D. Cooper, S. Varde, F. H. Moy, G. Forseter, C. S. Pavia, I. Schwartz. 1996. Molecular typing of Borrelia burgdorferi from Lyme disease patients by PCR-restriction fragment length polymorphism analysis. J. Clin. Microbiol. 34: 1306-1309. [Abstract]
8. Liveris, D., A. Gazumyan, I. Schwartz. 1995. Molecular typing of Borrelia burgdorferi sensu lato by PCR-restriction fragment length polymorphism analysis. J. Clin. Microbiol. 33: 589-595. [Abstract]
9. Wormser, G. P., D. Liveris, J. Nowakowski, R. B. Nadelman, L. F. Cavaliere, D. McKenna, D. Holmgren, I. Schwartz. 1999. Association of specific subtypes of Borrelia burgdorferi with hematogenous dissemination in early Lyme disease. J. Infect. Dis. 180: 720-725. [Medline]
10. Wang, G., C. Ojaimi, R. Iyer, V. Saksenberg, S. A. McClain, G. P. Wormser, I. Schwartz. 2001. Impact of genotypic variation of Borrelia burgdorferi sensu stricto on kinetics of dissemination and severity of disease in C3H/HeJ mice. Infect. Immun. 69: 4303-4312. [Abstract/Free Full Text]
11. Wang, G., C. Ojaimi, H. Wu, V. Saksenberg, R. Iyer, D. Liveris, S. A. McClain, G. P. Wormser, I. Schwartz. 2002. Disease severity in a murine model of Lyme borreliosis is associated with the genotype of the infecting Borrelia burgdorferi sensu stricto strain. J. Infect. Dis. 186: 782-791. [Medline]
12. Wooten, R. M., J. J. Weis. 2001. Host-pathogen interactions promoting inflammatory Lyme arthritis: use of mouse models for dissection of disease processes. Curr. Opin. Microbiol. 4: 274-279. [Medline]
13. Ma, Y., K. P. Seiler, E. J. Eichwald, J. H. Weis, C. Teuscher, J. J. Weis. 1998. Distinct characteristics of resistance to Borrelia burgdorferi-induced arthritis in C57BL/6N mice. Infect. Immun. 66: 161-168. [Abstract/Free Full Text]
14. Weis, J. J.. 2002. Host-pathogen interactions and the pathogenesis of murine Lyme disease. Curr. Opin. Rheumatol. 14: 399-403. [Medline]
15. Glickstein, L., B. Moore, T. Bledsoe, N. Damle, V. Sikand, A. C. Steere. 2003. Inflammatory cytokine production predominates in early Lyme disease in patients with erythema migrans. Infect. Immun. 71: 6051-6053. [Abstract/Free Full Text]
16. Oksi, J., J. Savolainen, J. Pene, J. Bousquet, P. Laippala, M. K. Viljanen. 1996. Decreased interleukin-4 and increased interferon production by peripheral blood mononuclear cells of patients with Lyme borreliosis. Infect. Immun. 64: 3620-3623. [Abstract]
17. Widhe, M., S. Jarefors, C. Ekerfelt, M. Vrethem, S. Bergstrom, P. Forsberg, J. Ernerudh. 2004. Borrelia-specific interferon- and interleukin-4 secretion in cerebrospinal fluid and blood during Lyme borreliosis in humans: association with clinical outcome. J. Infect. Dis. 189: 1881-1891. [Medline]
18. Zeidner, N., M. Dreitz, D. Belasco, D. Fish. 1996. Suppression of acute Ixodes scapularis-induced Borrelia burgdorferi infection using tumor necrosis factor-, interleukin-2, and interferon-. J. Infect. Dis. 173: 187-195. [Medline]
19. Zeidner, N., M. L. Mbow, M. Dolan, R. Massung, E. Baca, J. Piesman. 1997. Effects of Ixodes scapularis and Borrelia burgdorferi on modulation of the host immune response: induction of a TH2 cytokine response in Lyme disease-susceptible (C3H/HeJ) mice but not in disease-resistant (BALB/c) mice. Infect. Immun. 65: 3100-3106. [Abstract]
20. Brown, J. P., J. F. Zachary, C. Teuscher, J. J. Weis, R. M. Wooten. 1999. Dual role of interleukin-10 in murine Lyme disease: regulation of arthritis severity and host defense. Infect. Immun. 67: 5142-5150. [Abstract/Free Full Text]
21. Gross, D. M., A. C. Steere, B. T. Huber. 1998. T helper 1 response is dominant and localized to the synovial fluid in patients with Lyme arthritis. J. Immunol. 160: 1022-1028. [Abstract/Free Full Text]
22. Harjacek, M., S. Diaz-Cano, B. A. Alman, J. Coburn, R. Ruthazer, H. Wolfe, A. C. Steere. 2000. Prominent expression of mRNA for proinflammatory cytokines in synovium in patients with juvenile rheumatoid arthritis or chronic Lyme arthritis. J. Rheumatol. 27: 497-503. [Medline]
23. Armstrong, A. L., S. W. Barthold, D. H. Persing, D. S. Beck. 1992. Carditis in Lyme disease susceptible and resistant strains of laboratory mice infected with Borrelia burgdorferi. Am. J. Trop. Med. Hyg. 47: 249-258. [Abstract/Free Full Text]
24. Ruderman, E. M., J. S. Kerr, S. R. Telford, III, A. Spielman, L. H. Glimcher, E. M. Gravallese. 1995. Early murine Lyme carditis has a macrophage predominance and is independent of major histocompatibility complex class II-CD4⁺ T cell interactions. J. Infect. Dis. 171: 362-370. [Medline]
25. Montgomery, R. R., X. M. Wang, S. E. Malawista. 2001. Murine Lyme disease: no evidence for active immune down-regulation in resolving or subclinical infection. J. Infect. Dis. 183: 1631-1637. [Medline]
26. Brown, C. R., V. A. Blaho, C. M. Loiacono. 2003. Susceptibility to experimental Lyme arthritis correlates with KC and monocyte chemoattractant protein-1 production in joints and requires neutrophil recruitment via CXCR2. J. Immunol. 171: 893-901. [Abstract/Free Full Text]
27. Du Chateau, B. K., D. M. England, S. M. Callister, L. C. Lim, S. D. Lovrich, R. F. Schell. 1996. Macrophages exposed to Borrelia burgdorferi induce Lyme arthritis in hamsters. Infect. Immun. 64: 2540-2547. [Abstract]
28. DuChateau, B. K., J. R. Jensen, D. M. England, S. M. Callister, S. D. Lovrich, R. F. Schell. 1997. Macrophages and enriched populations of T lymphocytes interact synergistically for the induction of severe, destructive Lyme arthritis. Infect. Immun. 65: 2829-2836. [Abstract]
29. Ebnet, K., M. M. Simon, S. Shaw. 1996. Regulation of chemokine gene expression in human endothelial cells by proinflammatory cytokines and Borrelia burgdorferi. Ann. NY Acad. Sci. 797: 107-117. [Medline]
30. Habicht, G. S., G. Beck, J. L. Benach, J. L. Coleman, K. D. Leichtling. 1985. Lyme disease spirochetes induce human and murine interleukin 1 production. J. Immunol. 134: 3147-3154. [Abstract]
31. Sprenger, H., A. Krause, A. Kaufmann, S. Priem, D. Fabian, G. R. Burmester, D. Gemsa, M. G. Rittig. 1997. Borrelia burgdorferi induces chemokines in human monocytes. Infect. Immun. 65: 4384-4388. [Abstract]
32. Wooten, R. M., T. B. Morrison, J. H. Weis, S. D. Wright, R. Thieringer, J. J. Weis. 1998. The role of CD14 in signaling mediated by outer membrane lipoproteins of Borrelia burgdorferi. J. Immunol. 160: 5485-5492. [Abstract/Free Full Text]
33. Crandall, H., D. M. Dunn, Y. Ma, R. M. Wooten, J. F. Zachary, J. H. Weis, R. B. Weiss, J. J. Weis. 2006. Gene expression profiling reveals unique pathways associated with differential severity of Lyme arthritis. J. Immunol. 177: 7930-7942. [Abstract/Free Full Text]
34. Ojaimi, C., C. Brooks, S. Casjens, P. Rosa, A. Elias, A. Barbour, A. Jasinskas, J. Benach, L. Katona, J. Radolf, et al 2003. Profiling of temperature-induced changes in Borrelia burgdorferi gene expression by using whole genome arrays. Infect. Immun. 71: 1689-1705. [Abstract/Free Full Text]
35. Yang, Y. H., T. Speed. 2003. Normalization. D. Bowtell, III, and J. Sambrook, III, eds. DNA Microarrays, A Molecular Cloning Manual 536-543. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
36. Wang, G., Y. Ma, A. Buyuk, S. McClain, J. J. Weis, I. Schwartz. 2004. Impaired host defense to infection and Toll-like receptor 2-independent killing of Borrelia burgdorferi clinical isolates in TLR2-deficient C3H/HeJ mice. FEMS Microb. Lett. 231: 219-225. [Medline]
37. Livak, K. J., T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–C(T)) method. Methods 25: 402-408. [Medline]
38. Ulevitch, R. J., P. S. Tobias. 1999. Recognition of Gram-negative bacteria and endotoxin by the innate immune system. Curr. Opin. Immunol. 11: 19-22. [Medline]
39. Miller, L. C., S. Isa, E. Vannier, K. Georgilis, A. C. Steere, C. A. Dinarello. 1992. Live Borrelia burgdorferi preferentially activate interleukin-1 β gene expression and protein synthesis over the interleukin-1 receptor antagonist. J. Clin. Invest. 90: 906-912. [Medline]
40. Miller, L. C., E. A. Lynch, S. Isa, J. W. Logan, C. A. Dinarello, A. C. Steere. 1993. Balance of synovial fluid IL-1 β and IL-1 receptor antagonist and recovery from Lyme arthritis. Lancet 341: 146-148. [Medline]
41. Rupprecht, T. A., H. W. Pfister, B. Angele, S. Kastenbauer, B. Wilske, U. Koedel. 2005. The chemokine CXCL13 (BLC): a putative diagnostic marker for neuroborreliosis. Neurology 65: 448-450. [Abstract/Free Full Text]
42. Eberhard, Y., S. Ortiz, L. A. Ruiz, R. Kuznitzky, H. M. Serra. 2004. Up-regulation of the chemokine CCL21 in the skin of subjects exposed to irritants. BMC Immunol. 5: 7-14. [Medline]
43. Serra, H. M., Y. Eberhard, A. P. Martin, N. Gallino, J. Gagliardi, C. E. Baena-Cagnani, A. R. Lascano, S. Ortiz, A. L. Mariani, M. Uguccioni. 2004. Secondary lymphoid tissue chemokine (CCL21) is upregulated in allergic contact dermatitis. Int. Arch. Allergy Immunol. 133: 64-71. [Medline]
44. Diterich, I., L. Harter, D. Hassler, A. Wendel, T. Hartung. 2001. Modulation of cytokine release in ex vivo-stimulated blood from borreliosis patients. Infect. Immun. 69: 687-694. [Abstract/Free Full Text]
45. Straubinger, R. K., A. F. Straubinger, B. A. Summers, H. N. Erb, L. Harter, M. J. Appel. 1998. Borrelia burgdorferi induces the production and release of proinflammatory cytokines in canine synovial explant cultures. Infect. Immun. 66: 247-258. [Abstract/Free Full Text]
46. Giambartolomei, G. H., V. A. Dennis, B. L. Lasater, M. T. Philipp. 1999. Induction of pro- and anti-inflammatory cytokines by Borrelia burgdorferi lipoproteins in monocytes is mediated by CD14. Infect. Immun. 67: 140-147. [Abstract/Free Full Text]
47. Murthy, P. K., V. A. Dennis, B. L. Lasater, M. T. Philipp. 2000. Interleukin-10 modulates proinflammatory cytokines in the human monocytic cell line THP-1 stimulated with Borrelia burgdorferi lipoproteins. Infect. Immun. 68: 6663-6669. [Abstract/Free Full Text]
48. Radolf, J. D., L. L. Arndt, D. R. Akins, L. L. Curetty, M. E. Levi, Y. Shen, L. S. Davis, M. V. Norgard. 1995. Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides activate monocytes/macrophages. J. Immunol. 154: 2866-2877. [Abstract]
49. Hirschfeld, M., C. J. Kirschning, R. Schwandner, H. Wesche, J. H. Weis, R. M. Wooten, J. J. Weis. 1999. Cutting edge: Inflammatory signaling by Borrelia burgdorferi lipoproteins is mediated by Toll-like receptor 2. J. Immunol. 163: 2382-2386. [Abstract/Free Full Text]
50. Cabral, E. S., H. Gelderblom, R. L. Hornung, P. J. Munson, R. Martin, A. R. Marques. 2006. Borrelia burgdorferi lipoprotein-mediated TLR2 stimulation causes the down-regulation of TLR5 in human monocytes. J. Infect. Dis. 193: 849-859. [Medline]
51. Hu, L. T., M. A. Eskildsen, C. Masgala, A. C. Steere, E. C. Arner, M. A. Pratta, A. J. Grodzinsky, A. Loening, G. Perides. 2001. Host metalloproteinases in Lyme arthritis. Arthritis Rheum. 44: 1401-1410. [Medline]
52. Zhao, Z., H. Chang, R. P. Trevino, K. Whren, J. Bhawan, M. S. Klempner. 2003. Selective up-regulation of matrix metalloproteinase-9 expression in human erythema migrans skin lesions of acute Lyme disease. J. Infect. Dis. 188: 1098-1104. [Medline]
53. Rosenberger, C. M., M. G. Scott, M. R. Gold, R. E. Hancock, B. B. Finlay. 2000. Salmonella typhimurium infection and lipopolysaccharide stimulation induce similar changes in macrophage gene expression. J. Immunol. 164: 5894-5904. [Abstract/Free Full Text]

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