a) HEK293 cells stably transfected with a NF-κB luciferase reporter (HEK293) or the reporter together with plasmids encoding human TLR2 and human CD14 (HEK293-TLR2) were stimulated with UV-inactivated MCMV or vaccinia virus (VV) at the indicated MOIs. Pam₃SK₄ (Pam3) served as a positive control. Luciferase activity was measured 10 hours after activation. Fold activation was calculated relative to unstimulated cells. b & c) Bone marrow-derived DCs (b) or macrophages (c) were derived from the indicated mouse strains, stimulated with the indicated TLR ligands or viruses, and intracellular TNFα was measured by flow cytometry. Plots of stimulated cells (black lines) overlaid on plots of unstimulated cells (shaded) are shown. Plots of DCs are gated on CD11c-positive cells. The data presented are representative of at least 3 experiments.

a) TLR2-dependent type I IFN production requires MyD88 but not Trif. Bone marrow cells from the indicated mice were stimulated as indicated and type I IFN was measured in the supernatants 24 hours after stimulation by bioassay. b & c) TLR2-dependent type I IFN production requires IRF3 and IRF7 but not TLR3, TLR7, or TLR9. Bone marrow cells from the indicated mice were stimulated as indicated and type I IFN production was measured by bioassay. d) TLR2 and TLR9 induce type I IFN with different kinetics. Transcripts for IFNβ and IFNα4 were measured by real-time PCR in bone marrow cells stimulated with vaccinia virus (VV) or CpG oligos. The data presented are representative of at least 2 experiments.

a) IFNβ transcripts were measured in bone marrow cells treated for 12 hours with the bacterial TLR2 ligand Pam₃SK₄ (Pam3) or vaccinia virus (VV). b) Bone marrow or splenocytes from MOB mice were stimulated as indicated for 20 hours, and YFP production was measured by flow cytometry. The percentage of YFP-positive cells is indicated for each plot. All data are representative of at least 3 experiments.

a) Bone marrow cells from C57BL/6 or TLR2-deficient mice were MACS sorted into CD11b-positive and CD11b-negative populations. The sorted cells were stimulated as indicated, and type I IFN was measured after 24 hours by bioassay. b) The same experiment was performed as described in (a), except that MACS sorting was based on CD11c. c) Splenocytes were harvested from CD11b-DTR transgenic mice 24 hours after injection with saline (PBS) or diptheria toxin. The resulting cells were stimulated as indicated and type I IFN production was measured after 24 hours by bioassay. All data are representative of at least 3 experiments.

a) Bone marrow or splenocytes from MOB mice were stimulated with CpG or vaccinia virus (VV) for 20 hours, stained with antibodies against B220 or CD11c, and analyzed by flow cytometry. Comparisons between YFP-gated cells (orange line) versus total ungated cells (shaded) are shown. b) Cells were treated as in (a) but stained with antibodies against Ly6C, CD11b, or Ly6G. Comparisons between YFP-gated cells (orange line) versus total ungated cells (shaded) are shown. c) TLR2 is expressed on inflammatory monocytes. Bone marrow or splenocytes from C57BL/6 or TLR2-deficient mice were stained with antibodies specific for CD11b, Ly6C, and TLR2. d) TLR2 on inflammatory monocytes mediates type I IFN production in response to vaccinia virus. Bone marrow cells from MOB mice were cultured in the presence or absence of an anti-TLR2 blocking monoclonal antibody and stimulated as indicated. The percentage of YFP⁺ cells was determined by flow cytometry. e) Purified inflammatory monocytes are sufficient to produce type I IFN when stimulated with vaccinia virus. Bone marrow cells from C57BL/6 or TLR2-KO mice were sorted based on CD11b and Ly6C as shown. The plots shown have excluded B220⁺ and CD11c⁺ cells. Post-sort populations are shown and the percentage of inflammatory monocytes is indicated. The positive and negative populations were stimulated as indicated, and type I IFN production was measured after 24 hours by bioassay. All data are representative of at least 3 experiments.

a) Deletion of inflammatory monocytes in CD11b-DTR mice. Splenocytes were harvested from CD11b-DTR transgenic mice 24 hours after intravenous injection of diptheria toxin or PBS followed by staining with antibodies against Ly6C, CD11b, or Ly6G. b) Inflammatory monocytes are required for production of type I IFN in response to vaccinia virus. CD11b-DTR transgenic mice where injected with diptheria toxin or PBS 24 hours before infection with 1×10⁶ PFU of vaccinia virus. Serum was collected 24 hours post infection and type I IFN were quantified by bioassay. c) Depletion of inflammatory monocytes impairs viral clearance. CD11b-DTR transgenic mice were infected with 1×10⁶ PFU of vaccinia virus 24 hours after intravenous injection of diptheria toxin or PBS. PFU were determined in the liver 48 hours after infection. The data presented are representative of at least 2 experiments.

a) Bone marrow from MOB mice was incubated with 15μM chloroquine, 1μM cytochalasin D, or left untreated prior to stimulation with vaccinia virus (VV) or CpG. The percentages of YFP+ cells were measured 20h later by flow cytometry. b) Cells were treated as in (a) except that intracellular TNFα was measured in inflammatory monocytes cells by intracellular cytokine staining and flow cytometry. The data presented are representative of at least 2 experiments.