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. 1998 Nov;72(11):9267-77.
doi: 10.1128/JVI.72.11.9267-9277.1998.

Inhibition of NF-kappaB activation in combination with bcl-2 expression allows for persistence of first-generation adenovirus vectors in the mouse liver

A Lieber  1 , C Y He, L Meuse, C Himeda, C Wilson, M A Kay
Affiliations

Inhibition of NF-kappaB activation in combination with bcl-2 expression allows for persistence of first-generation adenovirus vectors in the mouse liver

A Lieber et al. J Virol. 1998 Nov.

Abstract

NF-kappaB is a key regulator of the innate antiviral immune response, due in part to its transcriptional activation of cytokines and adhesion molecules, which, in turn, function in chemotaxis and activation of inflammatory cells. We reported earlier that viral gene expression in hepatocytes transduced with first-generation (E1-deleted) adenoviruses induced NF-kappaB activation, elevation of serum cytokines, and hepatocellular apoptosis during the first days postinfusion. These events did not occur in mice infused with an adenovirus vector deleted for E1, E2, E3, and late gene expression. In the present study, we used an adenovirus expressing an IkappaBalpha supersuppressor (Ad.IkappaBM) and bcl-2 transgenic mice to unravel the role of virus-induced NF-kappaB activation and apoptosis in the clearance of recombinant adenovirus vectors from the liver. The combined action of IkappaBM and Bcl-2 allowed for vector persistence in livers of C57BL/6 x C3H mice. In the absence of Bcl-2, IkappaBM expression in mouse livers significantly reduced NF-kappaB activation, cytokine expression, leukocyte infiltration, and the humoral immune response against the transgene product; however, this was not sufficient to prevent the decline of vector DNA in transduced cells. Infusion of Ad.IkappaBM caused extended apoptosis predominantly in periportal liver regions, indicating that NF-kappaB activation may protect transduced hepatocytes from apoptosis induced by adenovirus gene products. To confer vector persistence, bcl-2 transgene expression was required to block virus-induced apoptosis if NF-kappaB protection was inactivated by IkappaBM. Expression of gene products involved in early stages of apoptotic pathways was up-regulated in response to virus infusion in bcl-2 transgenic mice, which may represent a compensatory effect. Our study supports the idea that the suppression of innate defense mechanisms improves vector persistence.

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Figures

FIG. 1
FIG. 1
Hepatic NF-κB activity after adenovirus infusion. bcl-2tg−/− and bcl-2tg+/− mice were injected with 4 × 109 PFU of Ad.Co (without transgene) or 4 × 109 PFU Ad.IκBM together with an equal dose of Ad.hAAT, and nuclear extracts were analyzed by EMSA at day 3 p.i. Reticulocyte lysate (RL) was used as the marker to determine the position of the p50-p65 complex. Positions of the NF-κB p50-p65 complex and the p50 homodimers were identified in supershift assays with specific antibodies (30). Each lane represents an individual animal.
FIG. 2
FIG. 2
Concentrations of cytokine RNAs after adenovirus infusion. Adenovirus was administered to mice as described for Fig. 1. At days 4 and 40 p.i., 2.5 μg of total liver RNA was quantified for RNAs specific for the inflammatory cytokines TGF-β1, IFN-γ IL-6, and TNF-α by RPA using the mCK-3b Multi-Probe Template Set (PharMingen) for mouse cytokine gene expression. Noninfused bcl-2tg−/− mice were used as controls. Cytokine mRNA expression in these mice was the same as in naive bcl-2tg+/− animals. Signals from protected fragments were quantified on a PhosphorImager. Represented are the average mRNA intensities normalized to the signals of the housekeeping genes (mL32 and mGAPDH) and expressed as arbitrary units. Results are means ± standard deviations for at least three animals. (IL-6 mRNA was not detectable by RPA in any of the groups and therefore was not included in the graphic.)
FIG. 3
FIG. 3
TUNEL analysis of liver sections. Adenovirus was administered to mice as described for Fig. 1. Liver sections were obtained at days 1, 2, 5, and 40 p.i. and analyzed for apoptotic cell death by the TUNEL technique (counterstaining with hematoxylin and eosin). (a) Naive mouse; (b) bcl-2tg−/−/Ad.Co, day 1; (c) bcl-2tg−/−/Ad.IκBM, day 1; (d) bcl-2tg−/−/Ad.IκBM, day 2; (e) bcl-2tg+/−/Ad.Co, day 1; (f) bcl-2tg+/−/Ad.IκBM, day 1; (g) bcl-2tg−/−/Ad.Co, day 5; (h) bcl-2tg−/−/Ad.IκBM, day 5; (i) bcl-2tg+/−/Ad.Co, day 5; (j) bcl-2tg+/−/Ad.IκBM, day 5. Note the neutrophil infiltration in panels g and i. Magnification, ×60.
FIG. 4
FIG. 4
Expression of proapoptotic genes after adenovirus infusion. Adenovirus was administered to mice as described for Fig. 1. (A) At days 4 and 40 p.i., total liver RNA from all four experimental groups was quantified by RPA for RNAs specific for gene products involved in apoptotic pathways. (B) Analysis of gene expression in bcl-2tg+/− mice at days 1, 2, 3, and 4 after infusion of Ad.IκBM. Signals from protected bands were quantified on a PhosphorImager. The signals from specific mRNAs were normalized to signals from housekeeping genes (mL32 and mGAPDH) run on each lane to adjust for loading differences and expressed as arbitrary units. Apoptotic gene expression in the experimental groups is represented in comparison to that of normal animals that did not receive rAd. Results are means ± SD for at least three animals.
FIG. 5
FIG. 5
Serum hAAT levels after Ad.hAAT infusion in relation to bcl-2 expression and NF-κB inhibition by IκBM. A dose of 4 × 109 PFU of Ad.Co or 4 × 109 PFU of Ad.IκBM together with an equal dose of Ad.hAAT (first-generation vector) was injected via tail vein into bcl-2tg−/− and bcl-2tg+/− mice. In one group of bcl-2tg+/− animals that received Ad.IκBM (bcl-2tg+/−/Ad.IκBM∗), bcl-2 expression was induced only during the first 7 days after adenovirus infusion by ZnSO4. All the other animals were under ZnSO4 for the full duration of the experiment. Serum was analyzed periodically for hAAT by ELISA. Each line represents an individual animal.
FIG. 6
FIG. 6
Southern blot analysis of adenovirus vector (Adv) DNA in transduced mouse livers. A dose of 4 × 109 PFU of Ad.Co or 4 × 109 PFU of Ad.IκBM together with an equal dose of Ad.hAAT (first-generation vector) was injected into bcl-2tg−/− and bcl-2tg+/− mice. Mice were sacrificed at day 100 p.i. Ten micrograms of BamHI-digested genomic liver DNA was loaded on each lane. Blots were hybridized with a labeled hAAT probe; Exposure was for 4 days. The fragment specific for Ad.hAAT is 2.0 kb. The analysis was performed with animals used for hAAT studies represented in Fig. 5.
FIG. 7
FIG. 7
Hypothetical mechanisms for the roles of NF-κB and Bcl-2 in vector persistence. Expressed adenovirus proteins can induce early apoptosis. (1) NF-κB has an antiapoptotic role. Bcl-2 blocks virus-induced apoptosis if NF-κB protection is inactivated by IκBM. (2) NF-κB transactivates cytokine expression, which is a key element in recruitment and activation of immune cells. Hence, NF-κB promotes cytokine/CTL-mediated cell death. Bcl-2 is required to block residual NF-κB activity, which was not inhibited by IκBM.
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