1. Inflammation Promotes the Loss of Adeno-Associated Virus–Mediated Transgene Expression in Mouse Liver 
Ekaterina Breous, Suryanarayan Somanathan, Peter Bell, James M. Wilson 
Gastroenterology 
Volume 141, Issue 1, Pages e3 (July 2011)
DOI: /j.gastro
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2. Figure 1
Inflammatory signals extinguish LacZ expression in a mouse model of liver-directed AAV-mediated gene transfer. (A) C57BL/6 mice were IV injected with 1011 VG of AAVLacZ and IV challenged 2 weeks later with 1010 VG of AdLacZ plus daily IP injections of LPS or CpG (TLRL) for 4 days. At 28 days after AdLacZ challenge, liver tissues were evaluated for LacZ expression by X-gal histochemistry. The percentage of liver area positive for LacZ staining was quantified using ImageJ software. (B) At 9 days after AdLacZ challenge, splenocytes were stimulated with the LacZ CD8 T-cell epitope and subjected to IFN-γ ELISPOT. Background spot-forming unit (SFU) values were subtracted before plotting. (C) At 14 days after AdLacZ challenge, mouse blood was collected to measure AST and ALT levels. Data represent groups of 3 mice in at least 3 independent experiments. A 2-tailed Student t test was used for statistical analysis. *P < .05; **P < .01; ***P < .001.
Gastroenterology  , e3DOI: ( /j.gastro )
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3. Figure 2
Inflammation extinguishes LacZ expression in the absence of substantial CTL induction to LacZ. (A) C57BL/6 mice were IV injected with 1011 VG of AAVLacZ and IV challenged 2 weeks later with 1010 VG of AdGFP plus daily IP injections of LPS or CpG (TLRL) for 4 days. At 28 days after AdGFP challenge, liver tissues were evaluated for LacZ expression by X-gal histochemistry. The percentage of liver area positive for LacZ staining was quantified using ImageJ software. (B) At 9 days after AdGFP challenge, splenocytes were stimulated with the LacZ CD8 T-cell epitope and subjected to IFN-γ ELISPOT. Background SFU values were subtracted before plotting. (C) At 14 days after AdGFP challenge, mouse blood was collected to measure AST and ALT levels. Data are representative of 3 mice per group in at least 3 independent experiments. A 2-tailed Student t test was used for statistical analysis. *P < .05; ***P < .001.
Gastroenterology  , e3DOI: ( /j.gastro )
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4. Figure 3
TNF-α response extinguishes LacZ expression in the absence of a CTL response. (A) C57BL/6 mice (n = 3 per group) were IV injected with 1011 VG of AAVLacZ and IV challenged 2 weeks later with 1010 VG of AdLacZ plus daily IP injections of IL-6 or TNF-α for 4 days. At 28 days after AdLacZ challenge, liver tissues were evaluated for LacZ expression by X-gal histochemistry. The percentage of liver area positive for LacZ staining was quantified using ImageJ software. (B) At 9 days after AdLacZ challenge, splenocytes were stimulated with the LacZ CD8 T-cell epitope and subjected to IFN-γ ELISPOT. Background SFU values were subtracted before plotting. (C) At 14 days after AdLacZ challenge, mouse blood was collected to measure AST and ALT levels. Data represent 3 independent experiments. A 2-tailed Student t test was used for statistical analysis. ***P < .001.
Gastroenterology  , e3DOI: ( /j.gastro )
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5. Figure 4
AAV VGs persist despite the loss of LacZ expression. C57BL/6 mice were IV injected with 1011 VG of AAVLacZ and IV challenged 2 weeks later with 1010 VG of AdLacZ or AdGFP plus daily IP injections of LPS, CpG, or TNF-α for 4 days. (A) At 28 days after Ad challenge, total cellular DNA was extracted from the liver and AAV2/8 VGs were quantified by real-time polymerase chain reaction. (B) At 28 days after Ad challenge, liver tissues were evaluated for LacZ expression by X-gal histochemistry. The percentage of liver area positive for LacZ staining was quantified using ImageJ software. Groups consisted of 3 mice, and the data are representative of 3 independent experiments. A 2-tailed Student t test was used for statistical analysis. *P < .05; **P < .01; ***P < .001.
Gastroenterology  , e3DOI: ( /j.gastro )
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6. Figure 5
Inflammation inhibits AAVLacZ expression driven by a liver-specific promoter. C57BL/6 mice (n = 3 per group) were IV injected with 1011 VG of AAVTBGLacZ and IV challenged 2 weeks later with 1010 VG of AdLacZ or AdGFP plus daily IP injections of LPS, CpG, or TNF-α for 4 days. (A) At 28 days after Ad challenge, liver tissues were evaluated for LacZ expression by X-gal histochemistry. The percentage of liver area positive for LacZ staining was quantified using ImageJ software. (B) At 28 days after Ad challenge, total cellular DNA was extracted from the liver and AAV2/8 VGs were quantified by real-time polymerase chain reaction. (C) At 14 days after Ad challenge, mouse blood was collected to measure AST and ALT levels. Data are representative of 3 independent experiments. A 2-tailed Student t test was used for statistical analysis. *P < .05; **P < .01; ***P < .001.
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7. Figure 6
A certain level of CTL activity is required to mediate extinction of LacZ expression. (A) C57BL/6 mice (n = 3 per group) were IV injected with 1011 VG of AAVLacZ and IV challenged 2 weeks later with 1010 VG of UVAdGFP plus daily IP injections of CpG for 4 days. At 28 days after UVAdGFP challenge, liver tissues were evaluated for LacZ expression by X-gal histochemistry. The percentage of liver area positive for LacZ staining was quantified using ImageJ software. (B) RAG−/− mice (n = 3 per group) were IV injected with 1011 VG of AAVLacZ and IV challenged 2 weeks later with 1010 VG of AdGFP plus daily IP injection of CpG for 4 days. At 28 days after AdGFP challenge, liver tissues were evaluated for LacZ expression by X-gal histochemistry. The percentage of liver area positive for LacZ staining was quantified using ImageJ software. (C) C57BL/6 mice (n = 3 per group) were IV injected with 1010 VG of AdGFP or UVAdGFP. At 10 days after Ad challenge, splenocytes were stimulated with Ad5 hexon peptide pools A–D and subjected to IFN-γ ELISPOT. Background SFU values were subtracted before plotting. (D) C57BL/6 mice (n = 3 per group) were IV injected with 1010 VG of AdGFP or UVAdGFP. At 10 days after Ad challenge, splenocytes were stimulated with Ad5 hexon peptide pools A–D and stained for CD8 and IFN-γ. Representative density plots show the percentage of CD8+ cells expressing IFN-γ. Data represent 2 independent experiments. A 2-tailed Student t test was used for statistical analysis. ***P < .001.
Gastroenterology  , e3DOI: ( /j.gastro )
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8. Figure 7
A possible mechanism of inflammation-induced extinction of AAV transgene expression. TLR stimulation leads to an enhanced CTL response to viral capsid and/or transgene that may involve engagement of cognate receptors and activation of APCs, CD8 T cells, or Tregs; in the latter case, TLR ligation relieves suppression. Activated CTLs kill a fraction of hepatocytes that present antigen (condition I and II) and nonspecifically silence AAV gene transcription through release of cytokine mediators such as TNF-α (condition III). TLR ligation may also block Treg suppressor activity, thus releasing CTLs from Treg suppression (condition IV).
Gastroenterology  , e3DOI: ( /j.gastro )
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9. Supplementary Figure 1
Inflammatory signals induce only weak CTL responses to Ad capsid. C57BL/6 mice (n = 3 per group) were IV injected with 1011 VG of AAVLacZ and IV challenged 2 weeks later with 1010 VG of AdLacZ or AdGFP plus daily IP injections of LPS or CpG for 4 days. At 9 days after Ad challenge, splenocytes were stimulated with Ad hexon peptide pools A–D and subjected to IFN-γ ELISPOT. Background SFU values were subtracted before plotting. Data represent 2 independent experiments. A 2-tailed Student t test was used for statistical analysis. *P < .05.
Gastroenterology  , e3DOI: ( /j.gastro )
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10. Supplementary Figure 2
No elevations of liver transaminase levels on days 7, 21, and 28 after Ad challenge. C57BL/6 mice (n = 3 per group) were IV injected with 1011 VG of AAVLacZ or AdGFP and IV challenged 2 weeks later with 1010 VG of AdLacZ or AdGFP plus daily IP injections of LPS, CpG, or TNF-α for 4 days. At 7, 21, and 28 days after Ad challenge, mouse blood was collected to measure AST and ALT levels. Hashed bars represent control mice that received no TLR ligands or TNF-α. Data represent 2 independent experiments.
Gastroenterology  , e3DOI: ( /j.gastro )
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11. Supplementary Figure 3
Inflammatory signals extinguish expression of a secretory protein. C57BL/6 mice (n = 3 per group) were IV injected with 1011 VG of AAVAAT and IV challenged 2 weeks later with 1010 VG of AdAAT plus daily IP injections of LPS or CpG for 4 days. Control mice received no TLR ligands. (A) At 28 days after AdAAT challenge, plasma AAT levels were tested by enzyme-linked immunosorbent assay. (B) At 9 days after AdAAT challenge, splenocytes were stimulated with the AAT CD8 epitope and subjected to IFN-γ ELISPOT. Background SFU values were subtracted before plotting. Data represent at least 3 independent experiments. A 2-tailed Student t test was used for statistical analysis. *P < .05; ***P < .001.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

12. Supplementary Figure 4
Low-dose Ad and systemic TLR ligand or TNF-α are required for extinction of AAVLacZ transgene expression. C57BL/6 mice (n = 3 per group) were IV injected with 1011 VG of AAVLacZ and IP injected simultaneously or 2 weeks later with LPS, CpG, or TNF-α, which were given for 4 consecutive days. Control mice received no TLR ligands or TNF-α. At 28 days after challenge, liver tissues were evaluated for LacZ expression by X-gal histochemistry. The percentage of liver area positive for LacZ staining was quantified using ImageJ software. Data represent at least 3 independent experiments.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions