deficient in various components of the type I IFN system in the context of an L. monocytogenes infection. Materials and Methods Mice. 6–12-wk-old C57BL/6 (The Jackson Laboratory) mice were used as wild-type controls in all experiments, unless noted. MyD88 / mouse lines were provided by S. Akira (Osaka University, Osaka, Japan). To obtain IFNAR1 / , IFNAR1 / , and matched littermate wild-type controls on a C57BL/6 genetic background, A129 mice (B&K Universal Limited) were back- crossed with C57BL/6 (The Jackson Laboratory) mice for five generations. All mice, including F9 C57BL/6 IRF3 / mice, were maintained and bred under specific pathogen-free condi- tions in the UCLA-DLAM mouse facility and experiments were conducted within the parameters of our approved protocol. Bacteria. L. monocytogenes strain 10403S was used for the in vitro portions of this study and all in vivo L. monocytogenes infec- tion experiments were performed using L. monocytogenes express- ing an immunodominant H-2Db–restricted epitope from the lymphocytic choriomeningitis virus–NP protein. The epitope was embedded in an hly fusion protein that was integrated into the L. monocytogenes chromosome using a genetic system pro- vided by P. Lauer and R. Calendar (University of California, Berkeley, Berkeley, CA). Although we found this particular strain of L. monocytogenes to be approximately five times less viru- lent than wild-type L. monocytogenes as determined by LD50, IRF3 / mice were also more resistant to infection by wild-type L. monocytogenes compared with control mice (unpublished data). L. monocytogenes was grown in brain heart infusion (BHI) broth with 200 g/ml streptomycin. Bacteria were grown to midloga- rithmic phase, pelleted, washed three times using PBS, and stored as glycerol stocks at 80 C in small aliquots until use. The con- centration of L. monocytogenes was quantified by plating serial di- lutions on BHI agar plates and counting colonies after growth at 37 C for 24–36 h. Cell Culture. Murine bone marrow–derived macrophages (BMMs) were differentiated from adherent bone marrow cells from mice as described previously (18). In brief, bone marrow cells were isolated and maintained in macrophage differentiating media (1 DMEM [Mediatech Inc.], 10% fetal bovine serum, 100 U/ml penicillin, 100 g/ml streptomycin, and 30% L929 conditioned medium) for 7 d. Purity was assayed to be 94–99% CD11b and negative for several contaminating markers. Immunoblotting. Cell fractionation and nuclear and cytoplas- mic immunoblotting were performed as described previously (6). Anti-IRF3 was obtained from Zymed Laboratories. Anti– upstream stimulating factor (USF)-2 and anti-STAT1 were ob- tained from Santa Cruz Biotechnology, Inc. Antibodies specific to the phosphorylated forms of STAT1 were obtained from Cell Signaling Technologies. Cell lysates were quantified and 40 g of nuclear extract or 25 g of cytoplasmic extract was loaded in each lane of an acrylamide gel and separated by SDS-PAGE. Gels were transferred to Immobilon-P membranes (Millipore) and im- munoblotted according to the antibody manufacturer’s recom- mended instructions. ELISA. To assay cytokine levels, serum was isolated from in- fected and uninfected mice as indicated. IFN- and IL-6 levels were assessed using the mouse IFN- (AN-18) and IL-6 ELISA sets from BD Biosciences according to the manufacturer’s protocol. Quantitative Real-Time PCR (Q-PCR). RNA was isolated using TRIzol Reagent (Invitrogen) according to the manufac- turer’s protocol. RNA was quantitated and 2 g RNA was used to make cDNA templates using Superscript II (Invitrogen) ac- cording to the manufacturer’s instructions and oligo-dT primers. Q-PCR analyses were performed using the iCycler thermocycler (Bio-Rad Laboratories). Q-PCR was conducted in a final vol- ume of 20 l containing the following: Taq polymerase, 1 Taq buffer (Stratagene), 125 M dNTP, SYBR™ Green I (Molecu- lar Probes), and Fluorescein (Bio-Rad Laboratories), using oligo- dT cDNA as the PCR template. Amplification conditions were as follows: 95 C for 3 min, 40 cycles of 95 C for 20 s, 55 C for 30 s, and 72 C for 20 s. Primers were used to amplify a specific 100–120-bp fragment corresponding to these respective genes. Primer sequences for L32, IFN- , Mx1, IL-6, and IP10 are the same as those published previously (6, 7). Primers for TRAIL, TGTP, PML, Daxx, PKR, and IRF1 are as follows: TRAIL: for- ward, ACCTCAGCTTCAGTCAGCACTTC, reverse, TGTA- AGTCACAGCCACAGACACAG; TGTP: forward, CGATT- TCCCCATTTGTTTGC, reverse, TGGCCCACCAGTAAC- TGAAGA; PML: forward, CAGGCTCTTAGCACCCAC- ATG, reverse, AGGCATCCCTTACTTTCAGCTTT; Daxx: forward, TTCGGGAAAAT CGAACCTTG, reverse, CCTCA- GTCTTGTCTTGCATCATTG; PKR: forward, GGAGCAC- GAAGTACAAGCGC, reverse, GCACCGGGTTTTGTAT- CGA; and IRF1: forward, TCCAAGTCCAGCCGAGACA, re- verse, TGCTGAGTCCATCAGAGAAAGTGT. L32 expression measurements were conducted in tandem with the gene of inter- est. All data is presented as relative expression units after normal- ization to the average L32 value to control for loading of total RNA. Measurements were conducted in triplicate. Infections. For in vitro infections, bacteria were diluted from glycerol stocks into media (1 DMEM, 7% fetal bovine serum, no antibiotics), added onto cells, and incubated at 37 C, 10% CO2, until harvest. Concentration of bacteria in the infectious stimulus was verified by plating serial dilutions on the appropriate agar plates. For in vivo infections, L. monocytogenes was grown to midlogarithmic phase, pelleted, washed, resuspended in PBS three times, and quantified by visible spectrometry readings at 600 nm. For intravenous injection, bacteria were diluted in pyrogen-free saline and 200 l was injected into the lateral tail vein. Concentra- tion of bacteria in the infectious dose was controlled by plating se- rial dilutions on BHI/streptomycin agar plates. For determination of bacterial recovery, mice were killed and the livers and spleens were homogenized in 1% Triton-X solution. Serial dilutions of homogenates were plated on BHI/streptomycin agar plates and colonies were counted after growth at 37 C for 24–36 h. Histology. Wild-type, IRF3 / , and IFNAR / mice were inoculated with 5 105 CFUs of L. monocytogenes intravenously. Livers were harvested 3 d later, fixed in formalin, and embedded in paraffin. Sections were cut at 4 m and mounted onto poly-l- lysine–coated slides (Fisher Scientific). Serial sections were then processed for hematoxylin and eosin staining according to standard protocols. TdT-mediated dUTP Nick-End Labeling (TUNEL) Assays. Spleens were removed from infected mice and snap-frozen in OCT (Sakura Finetek). 4- m sections were cut and mounted onto poly-l-lysine–coated slides. Splenic tissue was fixed using 4% paraformaldehyde, permeabilized using 0.1% Triton and 0.1% sodium citrate, and then double stranded DNA breaks were la- beled using an in situ Cell Death Kit (Roche) according to the manufacturer’s protocol. Sections were then counterstained using DAPI and analyzed using a Leica DM IRBE fluorescent micro- scope at a magnification of 10. Openlab software (Improvision) was used for image acquisition. 438 Type I Interferons Enhance Listeria monocytogenes Infection