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Evaluation of signal transduction pathways after transient cutaneous adenoviral gene delivery.

Steinstraesser L, Sorkin M, Jacobsen F, Al-Benna S, Kesting MR, Niederbichler AD, Otte JM, Hirsch T, Stupka J, Steinau HU, Schulte M - BMC Immunol. (2011)

Bottom Line: Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon.A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed.These results provide potential for an effective adenoviral gene delivery into immunosupressed skin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Molecular Oncology and Wound Healing, Department of Plastic Surgery, Operative Reference Centre for Soft Tissue Sarcomas, BG University Hospital Bergmannsheil, Ruhr University Bochum, Bochum, Germany. lars.steinstraesser@rub.de

ABSTRACT

Background: Adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. However, these vectors can induce immune responses that may severely affect the ability of vector re-application. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. For optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. The aim of this study was to investigate the signal transduction of the innate immunity after adenoviral DNA internalization in keratinocytes.

Methods: In vitro, keratinocytes were transfected with DNA, in the presence and absence of inhibitors for signalling molecules. In vivo, immunocompetent and athymic mice (n = 3 per group) were twice transduced with an Ad-vector.

Results: The results show an acute induction of type-I-interferon after in vitro transfection. Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon. In contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo.

Conclusion: The results suggest an induction of the innate immunity triggered by cytoplasm localised DNA which is mediated by PI3K-, p38 MAPK-, JNK-, NFkappaB-, JAK/STAT- and ERK1/2-dependent pathways. A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. These results provide potential for an effective adenoviral gene delivery into immunosupressed skin.

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Involvement of Toll-like receptor family in DNA recognition. RT-PCR analysis (45 cycles) for TLR-2, -7, -9, TRIF and MyD88 and DAI expression in HaCaT cells and HKC stimulated with DNA (5 μg DNA/ml medium) from adenovirus (Ad), Saccharomyces cerevisiae (SC), calf thymus (CT) and plasmids (P). Cells were stimulated for 6 h (HKC) and 15 h (HaCaT cells). Data from DAI expression was generated from cells transfected with 5 μg DNA/ml medium. Results were normalised to 18S ribosomal RNA (ratio: target gene/housekeeping gene) and compared to non-treated samples (vehicle control) (* = p < 0.05; ** = p < 0.005 to vehicle control).
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Figure 2: Involvement of Toll-like receptor family in DNA recognition. RT-PCR analysis (45 cycles) for TLR-2, -7, -9, TRIF and MyD88 and DAI expression in HaCaT cells and HKC stimulated with DNA (5 μg DNA/ml medium) from adenovirus (Ad), Saccharomyces cerevisiae (SC), calf thymus (CT) and plasmids (P). Cells were stimulated for 6 h (HKC) and 15 h (HaCaT cells). Data from DAI expression was generated from cells transfected with 5 μg DNA/ml medium. Results were normalised to 18S ribosomal RNA (ratio: target gene/housekeeping gene) and compared to non-treated samples (vehicle control) (* = p < 0.05; ** = p < 0.005 to vehicle control).

Mentions: Toll-like receptors (TLR) are among the most important receptor of innate immunity. For an investigation of the involvement of TLRs in adenoviral DNA detection. The expression levels of TLR-2, -7 and -9 were determined after transfection of DNA from different species. There was no significant change in expression of TLR-2, -7, -9 and TRIF in both cell types (Figure 2). An increased amount of MyD88 mRNA was detected in HaCaT cells stimulated with SC-DNA (4.2-fold; p = 0.057), Ad-DNA (3.8-fold; p = 0.041) and P-DNA (1.6-fold; p = 0.001). An increased amount of MyD88 mRNA was detected in HKC cells stimulated with SC-DNA (1.6-fold; p = 0.03) and Ad-DNA (1.2-fold; p = 0.3). Stimulation of HaCaT cells and HKC with CT-DNA and P-DNA stimulation of HKC did not show any significant differences.


Evaluation of signal transduction pathways after transient cutaneous adenoviral gene delivery.

Steinstraesser L, Sorkin M, Jacobsen F, Al-Benna S, Kesting MR, Niederbichler AD, Otte JM, Hirsch T, Stupka J, Steinau HU, Schulte M - BMC Immunol. (2011)

Involvement of Toll-like receptor family in DNA recognition. RT-PCR analysis (45 cycles) for TLR-2, -7, -9, TRIF and MyD88 and DAI expression in HaCaT cells and HKC stimulated with DNA (5 μg DNA/ml medium) from adenovirus (Ad), Saccharomyces cerevisiae (SC), calf thymus (CT) and plasmids (P). Cells were stimulated for 6 h (HKC) and 15 h (HaCaT cells). Data from DAI expression was generated from cells transfected with 5 μg DNA/ml medium. Results were normalised to 18S ribosomal RNA (ratio: target gene/housekeeping gene) and compared to non-treated samples (vehicle control) (* = p < 0.05; ** = p < 0.005 to vehicle control).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037344&req=5

Figure 2: Involvement of Toll-like receptor family in DNA recognition. RT-PCR analysis (45 cycles) for TLR-2, -7, -9, TRIF and MyD88 and DAI expression in HaCaT cells and HKC stimulated with DNA (5 μg DNA/ml medium) from adenovirus (Ad), Saccharomyces cerevisiae (SC), calf thymus (CT) and plasmids (P). Cells were stimulated for 6 h (HKC) and 15 h (HaCaT cells). Data from DAI expression was generated from cells transfected with 5 μg DNA/ml medium. Results were normalised to 18S ribosomal RNA (ratio: target gene/housekeeping gene) and compared to non-treated samples (vehicle control) (* = p < 0.05; ** = p < 0.005 to vehicle control).
Mentions: Toll-like receptors (TLR) are among the most important receptor of innate immunity. For an investigation of the involvement of TLRs in adenoviral DNA detection. The expression levels of TLR-2, -7 and -9 were determined after transfection of DNA from different species. There was no significant change in expression of TLR-2, -7, -9 and TRIF in both cell types (Figure 2). An increased amount of MyD88 mRNA was detected in HaCaT cells stimulated with SC-DNA (4.2-fold; p = 0.057), Ad-DNA (3.8-fold; p = 0.041) and P-DNA (1.6-fold; p = 0.001). An increased amount of MyD88 mRNA was detected in HKC cells stimulated with SC-DNA (1.6-fold; p = 0.03) and Ad-DNA (1.2-fold; p = 0.3). Stimulation of HaCaT cells and HKC with CT-DNA and P-DNA stimulation of HKC did not show any significant differences.

Bottom Line: Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon.A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed.These results provide potential for an effective adenoviral gene delivery into immunosupressed skin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Molecular Oncology and Wound Healing, Department of Plastic Surgery, Operative Reference Centre for Soft Tissue Sarcomas, BG University Hospital Bergmannsheil, Ruhr University Bochum, Bochum, Germany. lars.steinstraesser@rub.de

ABSTRACT

Background: Adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. However, these vectors can induce immune responses that may severely affect the ability of vector re-application. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. For optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. The aim of this study was to investigate the signal transduction of the innate immunity after adenoviral DNA internalization in keratinocytes.

Methods: In vitro, keratinocytes were transfected with DNA, in the presence and absence of inhibitors for signalling molecules. In vivo, immunocompetent and athymic mice (n = 3 per group) were twice transduced with an Ad-vector.

Results: The results show an acute induction of type-I-interferon after in vitro transfection. Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon. In contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo.

Conclusion: The results suggest an induction of the innate immunity triggered by cytoplasm localised DNA which is mediated by PI3K-, p38 MAPK-, JNK-, NFkappaB-, JAK/STAT- and ERK1/2-dependent pathways. A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. These results provide potential for an effective adenoviral gene delivery into immunosupressed skin.

Show MeSH
Related in: MedlinePlus