Double-Stranded RNA-Mediated TLR3 Activation Is Enhanced by CD14 Hyun-Ku Lee, Stefan Dunzendorfer, Katrin Soldau, Peter S. Tobias  Immunity  Volume 24, Issue 2, Pages 153-163 (February 2006) DOI: 10.1016/j.immuni.2005.12.012 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Impaired Responses to pIpC in CD14-Deficient Macrophages (A–D) Bone marrow-derived macrophages (BMDMs) from wild-type (wt) and CD14−/− mice (A and C) as well as MyD88−/− and TLR4−/− mice (B and D) were stimulated with pIpC (20 μg/ml), LPS (0.2 μg/ml), MALP-2 (10 ng/ml), or CpG (10 μg/ml) in the presence or absence of 50 μg/ml polymyxin B (PB). The nuclear translocation of NF-κB was determined by EMSA at the indicated time points. Abbreviation: NS, nonspecific bands. (E–H) BMDMs were stimulated with LPS (0.2 μg/ml) (E and G) and pIpC (20 μg/ml) (F and H). At the indicated time points, IL-12p40 (E and F) and IL-6 (G and H) in the culture supernatants were measured by ELISA. Data are shown as mean ± SD of three different experiments. ∗p < 0.05 versus wt at the corresponding time points (Mann-Whitney U test). Immunity 2006 24, 153-163DOI: (10.1016/j.immuni.2005.12.012) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 pIpC-Induced IL-12p40 Production Is Decreased in CD14−/− Mice, and CD14 Enhances pIpC-Mediated TLR3 Activation (A and B) Wt, CD14−/−, and TLR3−/− mice were injected intraperitoneally (ip) with 10, 25, and 50 μg pIpC. Sera were taken 2 hr (A) and 6 hr (B) later, and serum levels of IL-12p40 were measured by ELISA. Results are shown as the mean ± SD of sera samples from three mice. ∗p < 0.05 versus wt at the corresponding time points (Mann-Whitney U test). Abbreviation: C, saline control. (C and D) CD14-dependent enhancement of pIpC-mediated ISRE and IFN-β promoter activities in HEK293. Cells were transiently transfected with control vector (0.1 μg/ml), mCD14 plasmid (0.1 μg/ml), TLR3 plasmid (0.1 μg/ml), or TLR3 plasmid plus increasing amounts (0.01, 0.05, 0.1, 0.25, and 0.5 μg/ml) of mCD14 plasmid. Each sample was cotransfected with pSV-β-galactosidase vector plus pISRE-Luc plasmid (C) or p125-Luc plasmid (D) (0.075 μg/ml each). After 24 hr, cells were stimulated with pIpC (20 μg/ml) or LPS (0.2 μg/ml) for 6 hr. Thereafter, cells were lysed and the luciferase activity was measured. All the luciferase activity was normalized with β-galactosidase activity. Data are shown as mean ± SD of three different experiments. ∗p < 0.05 versus TLR3 alone (Mann-Whitney U test). (E) Soluble CD14 rescues the defect of TLR3 activity in CD14-deficient macrophages. BMDMs from wt and CD14−/− mice were stimulated with pIpC (10 μg/ml) plus polymyxin B (PB, 50 μg/ml) in the presence of increasing amounts of sCD14 (lane 1, none; lane 2, 0.05 μg/ml; lane 3, 0.5 μg/ml; and lane 4, 5.0 μg/ml) for 1 hr. The nuclear translocation of NF-κB was determined by EMSA. Abbreviations: C, control; NS, nonspecific bands. (F) pIpC-induced STAT1 phosphorylation is decreased in CD14-deficient macrophages. BMDMs from wt or CD14−/− mice were stimulated with pIpC (20 μg/ml), LPS (0.2 μg/ml), or IFN-γ (100 U/ml) for different time periods as indicated. Phosphorylated STAT1 (p-STAT1) and residual STAT1 were determined by Western blotting using anti-p-STAT1 or anti-STAT1 antibody. Immunity 2006 24, 153-163DOI: (10.1016/j.immuni.2005.12.012) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Purified pIpCsf Mediates TLR3-Dependent Cell Activation (A) Production of homogeneous 200 bp-short pIpCsf. Crude pIpC was partially digested with RNase III and fractionated into discrete size polymers and purified by gel filtration using Sephadex G-75. Crude pIpC and pIpCsf (1 μg each) were run on a 1% agarose gel. (B) pIpCsf activity. BMDMs from wt or TLR3−/− mice were stimulated with pIpC (20 μg/ml), pIpCsf (20 μg/ml), or LPS (0.2 μg/ml) for 1 hr. The nuclear translocation of NF-κB was determined by EMSA. Abbreviation: NS, nonspecific bands. (C) pIpCsf-induced nuclear translocation of NF-κB p65 subunit. CHO stable cell lines, CHO/CD14, CHO/CD14/TLR4, CHO/CD14/TLR3, and CHO/TLR3 were stimulated with pIpC (20 μg/ml), AF488-labeled-pIpCsf (20 μg/ml), LPS (0.2 μg/ml), or TNFα (0.05 μg/ml) for 2 hr, stained for NF-κB p65 subunit (red), and analyzed by fluorescence microscopy (green, intracellular AF488-pIpCsf; blue, DAPI nuclear stain). The arrows indicate internalized AF488-pIpCsf. The bar represents 10 μm. Immunity 2006 24, 153-163DOI: (10.1016/j.immuni.2005.12.012) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 pIpCsf Uptake Is Dependent upon CD14, and pIpCsf Directly Binds to CD14 (A) Various CHO stable cell lines were incubated with 0.1 μg/ml [32P]-pIpCsf for various time intervals in serum-free RPMI. The cells were washed three times with PBS and treated with 20 μg/ml proteinase K for 1 hr at 37°C to remove cell surface proteins/receptors. The cells were than harvested, washed three times with PBS, and lysed in 200 μl of 2% SDS and 50 mM EDTA, pH 7.4. The lysates were assayed in scintillation fluid for radioactivity. Data are shown as mean ± SD of three different experiments. (B) Soluble CD14-enhanced pIpCsf uptake. CHO cells were incubated with 0.1 μg/ml [32P]-pIpCsf or 0.1 μg/ml [32P]-pIpCsf/sCD14 (20 μg/ml) complex for various time intervals in serum-free RPMI. [32P]-pIpCsf and sCD14 were preincubated to form a complex for 30 min at 37°C. Other procedures were as described in (A). (C–E) Ligand binding was performed with [32P]-pIpCsf and purified soluble CD14. [32P]-pIpCsf (10 ng/reaction of 20 μl) was mixed with increasing concentrations of sCD14 protein (0.05, 0.5, 1.0, and 2.0 μM) (C) or in the presence of various cold competitors (5× and 50×, each) as noted (D). The reactions were incubated at room temperature for 30 min and run on a 4% native acrylamide gel followed by autoradiography. Anti-CD14 antibody (63D3) (5 μg/ml) was added to the reaction 10 min before termination (E). Immunity 2006 24, 153-163DOI: (10.1016/j.immuni.2005.12.012) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Intracellular TLR3 Colocalizes and Coimmunoprecipitates with CD14 (A) Intact or permeabilized CHO/CD14/TLR3 cells were stained with anti-TLR3 or anti-CD14 antibody (bold line) and analyzed by FACS; dotted line indicates a control IgG staining. (B and C) CHO/CD14/TLR3 cells were fixed, permeabilized, and stained for TLR3 or CD14 (green) (B) or both (red or green as noted) (C). pIpC (20 μg/ml) was added to the cells 2 hr before fixation (C2). F-actin was stained with fluorescent phallotoxin (red) (B) and nuclei with DAPI (blue) (B and C). Similarly, CHO/TLR3/TLR9 and CHO/CD14/TLR9 cells were fixed, permeabilized, and stained for TLR9 (red), TLR3 (green), and CD14 (green) (C3 and C4). Images were acquired by deconvolution confocal microscopy. The colocalization coefficient for red against green was 0.931 (C1), 0.953 (C2), 0.361 (C3), and 0.162 (C4) analyzed by NIH image analysis software packages, Image J (v. 1.33). The bars represent 10 μm. (D) HEK293 cells were transiently cotransfected with CD14 and TLR3 plasmids (0.5 μg/ml each). After 48 hr, cells were lysed and immunoprecipitated with anti-Myc antibody. pIpC (20 μg/ml) was added to the cells 6 hr before lysis. Western blotting was done with anti-Myc or anti-TLR3 antibody as noted. Abbreviations: IP, immunoprecipitation; WB, Western blotting. Immunity 2006 24, 153-163DOI: (10.1016/j.immuni.2005.12.012) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 TLR3 and CD14 Colocalize with Internalized AF488-pIpCsf, and pIpCsf Locates to the Lysosomes via the Endosomes (A) Internalized AF488-pIpCsf colocalizes with TLR3 and CD14. CHO/CD14/TLR3 cells were incubated with AF488-pIpCsf (20 μg/ml) in serum-free RPMI for 2 hr. Cells were washed with PBS, fixed/permeabilized, and stained for TLR3 or CD14 (red) followed by DAPI nuclear staining (blue). Images were acquired by deconvolution confocal microscopy. The colocalization coefficient for green against red was 0.82 (A1) and 0.712 (A2), as analyzed by NIH image analysis software packages, Image J (v. 1.33). The bars represent 10 μm. (B) TLR3 activation by pIpC requires endosomal maturation. Mouse BMDMs were preincubated with or without 5 μM chloroquine for 30 min and stimulated with pIpC (10 μg/ml), CpG DNA (10 μg/ml), or LPS (0.2 μg/ml) for 1 hr. The nuclear translocation of NF-κB was determined by EMSA. Abbreviations: C, control; Chlrq, chloroquine; and NS, nonspecific bands. (C) AF488-pIpCsf locates to the lysosomes via the endosomes. CHO/CD14/TLR3 cells were incubated with AF488-pIpCsf (20 μg/ml) in serum-free RPMI for 2 hr. Cells were either further incubated with 50 μM LysoTracker reagent (red) for 1 hr or fixed/permeabilized to be stained for EEA1 or LAMP-1 (red). Nuclei were stained with DAPI (blue). The colocalization coefficient for green against red was 0.52 (C1), 0.869 (C2), and 0.62 (C3), as analyzed by NIH image analysis software packages, Image J (v. 1.33). The bars represent 10 μm. Immunity 2006 24, 153-163DOI: (10.1016/j.immuni.2005.12.012) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 pIpC-Dependent Lysosomal Localization of TLR3 and CD14 CHO/CD14/TLR3 cells were incubated with or without pIpC (20 μg/ml) for 2 hr. Cells were washed with PBS, fixed/permeabilized, and double stained for TLR3 or CD14 (green) plus LAMP-1 (red) as noted. Nuclei were stained with DAPI (blue). The colocalization coefficient for red against green was 0.12 (1), 0.72 (2), 0.211 (3), and 0.61 (4), as analyzed by NIH image analysis software packages, Image J (v. 1.33). The bars represent 10 μm. Immunity 2006 24, 153-163DOI: (10.1016/j.immuni.2005.12.012) Copyright © 2006 Elsevier Inc. Terms and Conditions