Effects of dexamethasone and meloxicam on Borrelia burgdorferi-induced inflammation in glial and neuronal cells of the central nervous system

B. burgdorferi B31 clone 5A19 spirochetes, passage 1–3, were grown in Barbour-Stoenner-Kelly (BSK) medium, supplemented with 6% rabbit serum (Sigma, St. Louis, MO) and antibiotics (rifampicin at 45.4 μg/mL, phosphomycin at 193 μg/mL, and amphotericin at 0.25 μg/mL) to late logarithmic phase under microaerophilic conditions. Spirochetes were pelleted, washed using sterile phosphate-buffered saline (PBS), and resuspended in the working medium at the desired density, as previously described [12].

Freshly harvested frontal cortex tissues were collected at necropsy from three rhesus macaques (Macaca mulatta) that were scheduled for euthanasia due to chronic idiopathic diarrhea or had undergone trauma. Animals were euthanized in accordance with the recommendations of the American Veterinary Medical Association’s Panel on Euthanasia. The frontal cortex was sliced into 2-mm sections, and each section was placed in separate wells of 12-well plates. Each well contained 2 mL of RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10% FBS, as previously described [14]. Tissue sections were exposed to medium alone or to medium with added Bb (1 × 10⁷ bacteria/mL) in the presence or absence of either 150 μM dexamethasone (Sigma) or 100 μM meloxicam (Sigma). Controls with no spirochetes were also included. Prior to stimulation with live Bb, FC explant cultures were incubated with the respective concentrations of dexamethasone or meloxicam for 1 h in a humidified 5% CO₂ incubator at 37 °C, after which they were washed and then incubated in fresh growth medium containing the appropriate concentrations of dexamethasone or meloxicam and live Bb for an additional 6 h at 37 °C. The above concentrations of dexamethasone and meloxicam were the highest concentrations that were found to be safe as evaluated by the trypan blue exclusion assay (Sigma) in previous studies with cultures of human Schwann cells and rhesus dorsal root ganglia cultures [13]. At the end of the incubation times, culture supernatants were collected for evaluation of inflammatory mediators. Culture supernatants were centrifuged at 4 °C at 2000 × g to remove any suspended bacteria, and the supernatant was aliquoted and stored at −70 °C until used, as previously reported [13]. Tissue explants in respective wells were fixed using 2% paraformaldehyde and cryopreserved for apoptosis assays.

Primary rhesus astrocyte and microglial cultures were established following previously described protocols [15]. Briefly, after the meninges and blood vessels from fresh frontal cortex tissue were removed, tissue was mechanically dissociated, treated with 0.25% trypsin, 0.38 g/mL EDTA (Invitrogen), and 0.1% DNase (Sigma-Aldrich), and incubated at 37 °C for 40 min with intermittent shaking. After incubation, the dissociated tissue was centrifuged for 10 min at 425 × g and cells filtered through a Nitex filter (20 μm) and resuspended in glial culture medium, which was composed of DMEM-F-12 with l-glutamine and HEPES buffer, 10% fetal bovine serum (HyClone), 0.5 ng/mL of granulocyte-macrophage colony-stimulating factor (Sigma-Aldrich), 100 U/mL penicillin, and 100 μg/mL streptomycin. After 14 to 21 days in culture at 37 °C, dislodged microglia were collected after vigorously tapping the flasks, and resuspended in glial culture medium.

To obtain purified astrocytes, glial cells were incubated for 90 min in 10 mM l-leucine methyl ester (LME) (Sigma-Aldrich). This concentration has been shown to be effective in ensuring maximal microglial lysis with minimal toxicity to astrocytes [15]. Thereafter, astrocytes were washed thoroughly and resuspended in glial culture medium. Purity of astrocytes and microglial cultures was assessed by staining with a specific microglial marker anti-IBA antibody at a concentration of 1:50 (Wako Chemicals, Richmond, VA) and was routinely of 99%. Astrocyte cultures were stained with astroglial marker anti-GFAP antibody at a dilution of 1:200 (Sigma) after permeabilizing fixed astrocyte cultures with a mixture of chilled ethanol-acetic acid mixture (2:1), for 5 min at −20 °C, for detection of intracellular phenotypic markers as previously described [16]. Confocal microscopy was performed using a Leica TCS SP8 confocal microscope (Leica Microsystems, Exton, PA).

The human oligodendrocyte cell line MO3.13 obtained from CELLutions Biosystems Inc. (Burlington, Ontario, Canada) was revived and established following manufacturer’s instructions as previously described [17]. Differentiated oligodendrocytes were obtained after maintaining cultures in differentiation medium (DM), consisting of DMEM, P/S, and phorbol 12-myristate 13-acetate (Sigma), at a concentration of 100 nM and devoid of serum.

Glial and Bb cultures were washed in their respective media, devoid of antibiotics. Controls with no spirochetes were also included. Prior to stimulation with live Bb, glial cell cultures were incubated with various concentrations of dexamethasone 5 μM, 15 μM and 150 μM (Sigma) or meloxicam 1 μM, 10 μM, 50 μM and 100 μM (Sigma) for 2 h at 37 °C, after which they were washed and then incubated in fresh growth medium containing the respective concentrations of dexamethasone or meloxicam and live Bb at a multiplicity of infection (MOI) of 10:1 at 37 °C. The effect of the carrier substance of dexamethasone (2-hydroxypropyl)-β-cyclodextrin), at the respective molar concentrations accompanying dexamethasone, was assessed by incubating rhesus astrocytes and microglial cultures in the presence and absence of Bb and carrier alone at 15, 45, and 450 μM, respectively, as previously described for oligodendrocyte cultures [16]. After 48 h, culture supernatants were collected and processed for evaluation of inflammatory mediators. The drug concentrations were chosen based on previous reports where dexamethasone (5–150 μM) had been shown to inhibit the production of CCL2 in mice microglia [18], and meloxicam (1–100 μM) had been shown to be effective in inhibiting COX-2 in vitro [19].

The concentrations of cytokines and chemokines present in the culture supernatants from rhesus astrocyte and microglial cultures and rhesus FC explant cultures were quantified using the MilliPlex MAP Non-Human Primate Cytokine Magnetic Bead Panel-Premixed 23 Plex, Cytokine-Chemokine Array kit (Millipore), following the manufacturer’s instructions. The concentrations of cytokines and chemokines present in the culture supernatants from human oligodendrocytes were quantified using the Human 14-plex Cytokine-Chemokine Array kit (Millipore), following the manufacturer’s instructions. The multiplex plate was read using a Bio-Plex 200 Suspension Array Luminex System (Bio-Rad, Hercules, CA, USA).

Tissue sections from rhesus FC explants were incubated with anti-NeuN or anti-S-100 antibodies and then subjected to the TUNEL-ApopTagPlus fluorescein in situ apoptosis assay (Chemicon, Temecula, CA) as previously described [14]. The percentage of apoptotic neurons and the percentage of apoptotic oligodendrocytes in brain sections were evaluated by counting at least 500 cells, followed by the percentage of cells that showed co-localization of both the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) signal and NeuN or S-100 expression, respectively. Apoptotic cells from ten fields were counted from each section for the various incubation conditions. All counts were made by viewing slides under a fixed magnification of ×63 (corresponding to an area of 0.05 mm²) using the Leica TCS SP8 confocal microscope. The identity of the oligodendrocytes that stained with the anti-S-100 antibody was confirmed by their morphology as previously described [14].

The two-way ANOVA and Tukey’s multiple comparison test was used to evaluate the statistical significance between means of data sets, using Graphpad Prism software (Graph Pad Software Inc.) version 5.