2–3 month old C57BL/6J and Per2
) male mice (Jackson ImmunoResearch Laboratories) were fed rodent chow ad libitum, maintained under constant environmental conditions and entrained to a 12 h light/12 h dark cycle (light period from 7:00 a.m. to 7:00 p.m.) for at least 2 weeks before experiments. The Per2Brdm1
mutant strain was originally developed by Dr. Allan Bradley and has a genetic background of C57BL/6 (Zheng et al., 1999
). For most daily-cycle experiments, animals were euthanized at 6:00 a.m., 10:00 a.m., 2:00 p.m., 6:00 p.m., 10:00 p.m., and 2:00 a.m. These time points correspond with Zeitgeber times (ZT) 3, 7, 11, 15, 19, and 23, respectively. For experiments under constant conditions, dark/dark (DD), animals were held in constant darkness for 3 days prior to sampling at circadian times (CT) 3, 7, 11, 15, 19 and 23. Tissues were immediately collected for further processing as described below. During the study, animal care and treatment complied with National Institutes of Health policy, were in accordance with institutional guidelines, and were approved by the Yale Institutional Animal Care and Use Committee.
Peritoneal macrophages from C57BL/6J (Jackson ImmunoResearch Laboratories), or Per2Brdm1 of the same genetic background were obtained 3–4 days after i.p. injection with 3% thioglycollate broth (REMEL) by peritoneal lavage. Macrophages were purified by adherence following standard procedures and cultured overnight in RPMI 1640 containing 10% FBS plus penicillin-streptomycin (Invitrogen Life Technologies).
In vitro entrainment
Cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% FBS and transferred to a 6-well cell culture plate (BD Biosciences) were they were incubated overnight. The following day the medium was aspirated, cells were washed with 1 ml of warm PBS, and incubated for 2 h in 2 ml of serum-free medium. At the conclusion of 2 h the cells were serum shocked with 1 ml of FBS (50% final concentration). The medium containing 50% serum was replaced with serum-free medium after 2 h. Cells were harvested every 4 h, lysed in the appropriate buffer and stored at −80° C until their later use for RNA extraction or Western blot analysis.
PAMP challenge and cytokine measurements
Thioglycollate-elicited peritoneal macrophages from WT and Per2Brdm1 mice were plated and challenged the following day with pam3Cys (500 ng/ml), lipoteichoic acid (LTA, 10 μg/ml), poly(I:C) (100 μg/ml), LPS (100 ng/ml), flagellin (5 μg/ml), imiquimod (10 μg/ml) or unmethylated CpG-DNA (10 μg/ml) - synthetic oligonucleotides containing unmethylated CpG dinucleotides in particular sequence contexts. We employed CpG ODNs from Invivogen (ODN 1826), which are type B specific for mouse TLR9. The 20 mer sequence is 5′-tccatgacgttcctgacgtt-3′. TNF-α and IL-12 levels in culture supernatants were determined by ELISA 16 h after challenge using commercially available ELISA kits (R&D Systems).
Cell isolation and purification
Spleens were collected in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FBS and placed on ice. To obtain homogeneous splenocyte suspensions, each spleen was homogenized in 1 ml of RPMI, filtered through a 70 μM nylon cell strainer (BD Biosciences) and spun down for 5 min at 1,200 rpm. The supernatant was removed and cells were resuspended in 3 ml red cell lysing buffer (Sigma) and incubated for 10 min at room temperature. 7 ml of PBS were added to the cell suspension and cells were spun down for 5 min at 1,200 rpm. The supernatant was removed, cells were resuspended in 3 ml PBS and aliquoted into separate tubes for labeling with microbeads and subsequent cell separation. CD11b, CD11c and CD19 mouse microbeads (Miltenyi Biotec) were used to label splenic macrophages, dentritic cells and B cells, respectively. The labeling was followed according to manufacturers instructions and the autoMACS™ Pro Separator (Miltenyi Biotec) was utilized for the magnetic cell separation. The purity of the enriched fractions was assessed by flow cytometry using FITC-conjugated CD11b, CD11c and CD19 antibodies (Miltenyi Biotech). The enrichment method consistently yielded a purity of approximately 90, 60 and 80% for CD11b, CD11c and CD19 positive cells, respectively. Immediately after separation, enriched cells were lysed in appropriate buffers for later RNA extraction and protein analysis. For flow cytometry, cells were fixed and permeabilized before staining with purified fluorescent monoclonal anti-TLR9 antibody from Imgenex (clone 26C593.2).
RNA extraction and quantitative PCR
Total RNA was isolated using the RNeasy mini kit (Qiagen). cDNA was synthesized using the high capacity cDNA reverse transcription kit (Applied Biosystems). Relative quantitation of mRNA levels was performed by quantitative PCR via TaqMan Gene Expression Assays (Applied Biosystems) using a real-time PCR 7500 Fast System (Applied Biosystems). Analyses were performed using the standard curve method with β-actin as the normalizing endogenous control. RNA from enriched cells was isolated using the RNeasy Plus-Micro kit (Qiagen). Due to low expression levels and a limited amount of cDNA, the TaqMan PreAmp Master Mix Kit (Applied Biosystems) was used in conjunction with TaqMan Gene Expression Assays (Applied Biosystems) to examine mRNA levels of selected genes from individual cell types. The procedure was followed according to manufacturers instructions.
Protein isolation and Western blot analysis
For protein analysis, spleen tissue and cells were lysed in T-PER and M-PER Protein Extraction Reagent (Thermo Scientific), respectively. Protein samples were subjected to standard SDS-PAGE electrophoresis, transferred to a nitrocellulose membrane, and probed with TLR9 and beta Actin polyclonal Ab (Imgenex). After chemiluminescence detection using ECL Western blot detection kit (Amersham), densitometry analyses were performed using ImageJ64 software.
ChIP, EMSA, and si-RNA-mediated knockdown of Clock
ChIP was performed using EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore) and EMSA was performed using the LightShift® Chemiluminescent EMSA Kit (Thermo Scientific). The procedures were followed according to manufacturers instructions. Clock expression was knocked down using Clock siRNA (Santa Cruz Biotechnology) and the transfection protocol was followed according to manufacturers instructions (Santa Cruz Biotechnology). Clock antibody (Santa Cruz Biotechnology) was used to verify knockdown in CLOCK protein levels.
Mice entrained to a 12 h light/12 h dark cycle were injected intraperitoneally with 10 μg OVA and 30 μg CpG ODNs (InvivoGen) at either ZT7 or ZT19. Two weeks later, mice received a boost (10 μg OVA and 30 μg CpG ODNs) at their corresponding time. Twenty-eight days after the initial OVA CpG ODN challenge, mice were sacrificed and lymph nodes were pooled, according to time of immunization or mouse strain, in cell culture medium (RPMI supplemented with 10 % FBS). Lymph nodes were homogenized and passed through a 70 μM nylon cell strainer (BD Biosciences). Lymphocytes were spun down, resuspended in 10 ml of medium and counted using a hemocytometer. 96 well U-bottom tissue culture plates (BD Biosciences) were seeded with 105 or 106 cells per well for lymphocyte proliferation or IFN-γ production, respectively, in 200 μl of medium. Cells were allowed to incubate for 1 h in a 37° C CO2 incubator and then cells were either challenged with 20 μg of OVA, 1 μg of Concanavalin A (Sigma), or were left unstimulated. Lymphocyte proliferation was determined using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega). IFN-γ was determined using the Mouse IFN-γ High Sensitivity ELISA (eBioscience).
CLP and related measurements
CLP was performed as previously described(Rittirsch et al., 2009
) (75% of the cecum was ligated and pierced with a 21G needle). Mice received 1 ml warmed saline s.c. post-operatively and buprenorphine analgesia at 12h intervals. Surface temperature was determined with an infrared thermometer. Blood was collected by retro-orbital bleed and serum was prepared in microtainer tubes (Becton Dickinson, San Jose, CA). ELISA kits for IL-6, IL-12/23p40, and MCP-1 (eBiosciences, San Diego, CA) and the CK reagent set (Pointe Scientific, Canton, MI) were used per manufacturer’s instructions. Peritoneal lavage was plated on plated on BBL agar plates to determine bacterial CFU.
Data were analyzed using GraphPad Prism 4.0b (GraphPad, San Diego, CA). For parametric comparisons, we employed a two-way T test for pair-wise comparisons and the analysis of variance test for repeated measures. For nonparametric comparisons, we employed Mann-Whitney for pairwise comparisons and Kruskal-Wallis for comparison of three or more groups. Survival curves were compared by the Logrank test. *P < 0.05 was considered to be statistically significant, **P < 0.01 highly significant. Unless otherwise stated, P values result from the statistical comparison of mean values obtained from independent experiments.