1. Volume 15, Issue 2, Pages 378-385 (February 2007)
Toll-like Receptor 9 Triggers an Innate Immune Response to Helper-dependent Adenoviral Vectors 
Vincenzo Cerullo, Michael P Seiler, Viraj Mane, Nicola Brunetti-Pierri, Christian Clarke, Terry K Bertin, John R Rodgers, Brendan Lee 
Molecular Therapy 
Volume 15, Issue 2, Pages (February 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Macrophages directly sense HD-Ad vectors through TLR9 and respond by secreting pro-inflammatory cytokine. Macrophages were harvested from the peritoneal cavities of C57BL/6 and TLR9CpG1/CpG1 mice as described. Infections were performed with 100 μl of vector (HD-AdLacZ) diluted in PBS. (a) 1 × 106 cells were infected with 10,000 VP/cell with or without a selective TLR9 blocker (CpG ODN-2088) at a concentration of 50 μM. (b) 1 × 106 cells were infected with 100 VP/cell with or without calcium phosphate co-precipitation (CaPi:HD). IL-6 levels were measured 6 h after the infection. *P<0.02.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
The nature of the expression cassette does not affect the cytokine response specified by TLR9 signaling in primary macrophages. Macrophages were harvested from the peritoneal cavity of C57BL/6 as well as TLR9 mice as described in Materials and Methods. 1 × 106 cells were infected with 250 μl of indicated vectors at a concentration of 100 VP/cell co-precipitated with calcium phosphate. (a) IL-6 and (b) TNF-α were measured in cell media 24 h after the transduction. LacZ=HD-AdCMVLacZ; Ad(0)=HD-Ad empty; FVIII=HD-AdpepckBDDFVIII; EC=empty capsid; HV=first-generation helper virus (NG163R). The results are reported in fold change over PBS-treated cells and all the conditions were tested in triplicate. *P< (c) 1 × 106 cells were infected with 250 μl of HD-AdCMVLacZ at a concentration of 10 and 100 VP/cell co-precipitated with calcium phosphate. β-Galactosidase assay was performed 24 h after the infection.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
TLR9 deficiency attenuates innate immune response to HD-Ad5 in vivo. Two groups of TLR9CpG1/CpG1 were injected with two different doses (5 × 1012 and 1 × 1013 VP/kg) of HD-Ad expressing LacZ under the control of CMV promoter. C57BL/6 mice were injected with the same doses as WT control. Six hours after the injection, plasma samples were collected and cytokine levels were assayed. (a) IL-6 levels were measured by FACSARRAY as described in Materials and Methods. *P<0.05. (b) IL-12 levels were measured by enzyme-linked immunosorbent assay. *P<0.05. (c, d) MCP-1 and KC from mice injected with the higher dose (1 × 1013 VP/kg) were measured as described in Materials and Methods.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Similar hepatocyte transduction following intravenous injection of HD-AdLacZ in C57BL/6 and TLR9CpG1/CpG1 mice. X-Gal staining of liver from WT mice following (a) PBS, (b) 5 × 1012 VP/kg, and (c) 1 × 1013 VP/kg injection. X-Gal staining of the liver from TLR9CpG1/CpG1 mice following (d) PBS, (e) 5 × 1012 VP/kg, and (f) 1 × 1013 VP/kg injection.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Selective TLR9 blockers reduce the acute inflammatory response to HD-Ad5. Two groups of C57BL/6 mice (N=5) were injected with two different doses (5 × 1012 and 1 × 1013 VP/kg) of HD-Ad expressing LacZ under the control of CMV promoter with or without a selective TLR9 blocker (ODN-2088, 100 μg). Six hours after the injection, plasma samples were collected and cytokine levels were assayed. (a) IL-6 levels were measured by FACSARRAY as described in Materials and Methods. *P<0.05. (b) IL-12 levels were measured by enzyme-linked immunosorbent assay. *P<0.05.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Adenoviral capsid stimulates TLR9 gene expression. Macrophages were harvested from the peritoneal cavity of TLR9CpG1/CpG1 mice as described in Materials and Methods. The infections were performed with 100 μl of vector (HD-AdLacZ) diluted in PBS. (a) 1 × 106 cells were infected with different concentration of HD-AdLacZ (103, 104, and 105 VP/cell). Three hours after infection, cells were collected for total mRNA isolation. cDNA was generated by RT and measured by QPCR with TLR9-specific primers. PBS and lipopolysaccharide were used as negative and positive controls, respectively (lipopolysaccharide was used at a concentration of 10 μg/ml). (b) 1 × 106 cells were infected with HD-AdLacZ at a dose of 104 VP/cell. Total mRNA was collected at different time points (1, 3, 6, and 24 h). cDNA was generated by RT and assayed by QPCR with TLR9-specific primers. The signal was normalized to mouse β-actin. (c) 1 × 106 cells were infected with HD-AdLacZ at a dose of 104 VP/cell with or without a selective inhibitor of NF-κB (pyrrolidine dithiocarbamate at a concentration of 15 μM). Three hours after infection, total mRNA was collected. cDNA was generated by RT and measured by QPCR with TLR9-specific primers. PBS and lipopolysaccharide were used as control (lipopolysaccharide was used at a concentration of 10 μg/ml).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions