C. burnetii Purification and Antiserum Production
C. burnetii Nine Mile phase I (RSA493) was propagated in African green monkey kidney (Vero) fibroblasts and purified by Renografin density gradient centrifugation [18]. Purified bacteria were suspended in K-36 buffer (0.1 M KCl, 0.015 M NaCl, 0.05 M K2HPO4, 0.05 M KH2PO4, pH 7.0). Murine anti-Coxiella antiserum was prepared by immunizing C57Bl/6 mice i.p. three times at 2 wk intervals with 108 fixed C. burnetii. C. burnetii were fixed with 4% paraformaldehyde for 24 h at 4°C. The bacteria were washed three times with phosphate buffered saline (PBS, Invitrogen, Carlsbad, CA) and resuspended in PBS at the desired concentration. Immune sera were harvested 2 wks after the final immunization and pooled. Serum from unvaccinated mice was harvested as naïve (control) serum. Human serum from a chronic Q fever patient was generously provided by Ted Hackstadt, Rocky Mountain Laboratories, NIAID, NIH.
Cell Culture
Human monocyte-derived DC were prepared as previously described [19]. Briefly, human PBMC were isolated from buffy coats by centrifugation through a Ficoll-Paque Plus (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient. Cells were enriched for monocytes (CD14+ cells) using a RossetteSep Monocyte Enrichment Kit (Stem Cell Technologies, Vancouver, Canada). Monocytes were resuspended at 106 cells per ml in DC medium (RPMI +Glutamax [Invitrogen], 5% FBS, 15 mM Hepes, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin) containing IL-4 (10 ng/ml) and GM-CSF (10 ng/ml) (Peprotech, Rocky Hill, NJ) and cultured for 6 days with replacement of half of the medium and addition of fresh cytokines every other day. Non-adherent DC were harvested by centrifugation and suspended in DC medium without antibiotics. The resulting cells were determined to be >95% CD11c+/CD209+ by flow cytometry. Monocyte-derived macrophages were generated by culturing monocytes in MΦ medium (RPMI, 10% FBS, and 50 ng/ml M-CSF [Peprotech]) for 7 days. Adherent MΦ were harvested by washing with cold PBS and gentle scraping.
The method used to prepare bone marrow derived DC (BMDC) was adapted from methods described by Inaba et al. [29]. Briefly, bone marrow cells were isolated from C57Bl/6 or FcR k/o mouse femurs and cultured in DC medium containing 20 ng/ml murine GM-CSF (Peprotech) at 37°C, 5% CO2. After 3 days, the culture medium and non-adherent cells were removed and replaced with fresh DC medium containing fresh GM-CSF. The cells were cultured with replacement of half of the medium and addition of fresh cytokines every third day. After 6–8 days of culture, BMDC were harvested as non-adherent cells. These cells were determined to be >80% CD11c+/CD11b+ by flow cytometry.
For C. burnetii infection experiments, DC were plated in 24-well plates at a concentration of 5 × 105 per ml in DC medium. Purified C. burnetii were added directly to the culture medium at a multiplicity of infection (MOI) of 100. Unless otherwise indicated, the inoculum was not removed. LPS from E. coli serotype O111:B4 (Sigma, St. Louis, MO) was used at 500 ng/ml as a positive control for DC stimulation/maturation. Cells were mock-infected with K-36 buffer. For in vitro experiments in which C. burnetii were Ab opsonized, approximately 20 μl of a C. burnetii suspension (approximately 108 bacteria) were mixed with 50 μl of naïve or immune serum (human or mouse) and incubated for 30 min at room temperature. Bacteria were washed twice with PBS (Invitrogen) and resuspended at the desired concentration in PBS.
Mouse Strains
C57Bl/6 and Fc receptor common gamma chain k/o (Fcer1g) [30] mice on a C57Bl/6 background were obtained from Taconic Farms (Germantown, NY). Breeding colonies of C2/factorB double k/o [31] mice were maintained at Taconic Farms. MBL/C1q double k/o, MBL/factorD double k/o and C1q/factorD double k/o mice were generated by interbreeding MBL k/o [32], C1q k/o [33], and factorD k/o [34] mice and maintained at Taconic Farms. All complement knockout mice were on the C57Bl/6 background (backcrossed at least 10 generations).
Passive Immunization Experiments
On day -1, mice were injected with 300 μl naïve or immune serum via the intraperitoneal (i.p.) route. On day 0, mice were challenged i.p. with 105 C. burnetii. Mice were sacrificed on day 14, spleens were harvested and blood was collected. Severity of infection was determined by measuring spleen weight and bacterial genomes per spleen. All procedures and animal protocols used in this study were approved by the Biosafety and IACUC committees at Rocky Mountain Laboratories. Work with viable C. burnetii was conducted in either BSL-3 or ABSL-3 laboratories.
Quantitative PCR
C. burnetii replication during infection of MΦ was quantified using TaqMan quantitative PCR (QPCR) of genome equivalents as previously described [35]. Briefly, MΦ were incubated with C. burnetii (MOI = 100) for 24 h at 37°C, 5% CO2. Non-adherent bacteria were removed by washing MΦ with Hank's balanced salt solution and the medium replaced. This point was considered 0 h post-infection. MΦ were harvested by scraping and DNA was isolated with an UltraClean microbial DNA isolation kit (MoBio Laboratories, Carlsbad, CA). For determination of bacterial genome numbers in the spleen, a small portion of spleen tissue (<50 mg) was excised from the spleen, weighed, and DNA extracted using the same DNA isolation kit. The primers/probe set used was designed with PrimerExpress software (Applied Biosystems, Foster City, CA) and is specific for the C. burnetii dotA gene. The forward and reverse primers are GCGCAATACGCTCAATCACA and CCATGGCCCCAATTCTCTT, respectively, and the probe sequence is CCGGAGATACCGGCGGTGGG. Purified plasmid DNA containing the C. burnetii dotA gene was used as template to generate standard curves ranging form 103 to 108 plasmid copies.
Flow Cytometry
Human DC were harvested by incubation for 5 min in cold 0.1 M EDTA in PBS, followed by gentle scraping and centrifugation at 500 × g for 5 min at 4°C. The following monoclonal antibodies were used to characterize DC phenotypes: antiCD83-APC, antiCD86-PE, antiCD209-PerCP-Cy5.5, antiCD80-FITC, and antiCD11c-PE (BD PharMingen, San Diego, CA). Approximately 5 × 105 DC were stained with antibodies in FACS Wash (PBS, 2% FBS) for 15 min on ice, washed twice with FACS Wash and fixed with 4% paraformaldehyde. Samples were run on a FACS-Canto II flow cytometer (Becton Dickinson, San Jose, CA) and analyzed using FlowJo software (TreeStar, Ashland, OR).
Cytokine Assay
The concentrations of mouse IL-6 and TNF or human IL-1β, IL-6, IL-12p70 and TNF in culture supernatants were determined by BioPlex X-plex multiplex cytokine assay (BioRad, Hercules, CA) according to the manufacturers instructions.
Statistics
Statistical analysis was conducted to evaluate the significance between differences. The Student's t-test was used to compare the difference between groups. A p value of <0.05 was regarded as statistically significant.