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The Journal of Immunology, 2003, 171: 2453-2460.
Copyright © 2003 by The American Association of Immunologists

Adenoviral Infection Decreases Mortality from Lipopolysaccharide-Induced Liver Failure Via Induction of TNF-{alpha} Tolerance 1

Timur O. Yarovinsky2, Linda S. Powers, Noah S. Butler, Mary A. Bradford, Martha M. Monick and Gary W. Hunninghake

Department of Internal Medicine, Division of Pulmonary, Critical Care, and Occupational Medicine, University of Iowa and Veteran Affairs Medical Center, Iowa City, IA 52242


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Effects of adenoviral infection on in vivo responses to LPS mediated by TNF-{alpha} were evaluated in a murine model. Adenovirus-infected mice showed decreased mortality from fulminant hepatitis induced by administration of LPS or staphylococcal enterotoxin B in the presence of D-galactosamine. Importantly, TNF-{alpha} resistance genes within adenoviral E3 region were not required, because E1,E3-deleted vectors showed similar effects. Adenovirus-infected mice exhibited higher TNF-{alpha} levels after LPS stimulation, no difference in TNFR1 expression, and similar mortality from Fas-induced fulminant hepatitis. Decreased production of IL-6 and KC in response to exogenous TNF-{alpha}, in addition to protection from TNF-{alpha}, suggested that adenoviral infection results in TNF-{alpha} tolerance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
It has been well established in a number of epidemiological studies and animal models that concurrent viral infections increase severity and mortality of secondary bacterial infections. Staphylococcal infections of the lower respiratory tract and toxic shock syndrome have been recognized as complications of influenza (1). Lethal synergism between influenza A virus and staphylococcal enterotoxin B (SEB)3 has been demonstrated in a mouse model (2). Lymphocytic choriomeningitis virus has also been shown to increase sensitivity of mice to LPS and SEB (3, 4, 5). Adenoviruses are known to increase the occurrence and severity of bacterial infections (6, 7, 8, 9, 10, 11). We have recently reported that prior exposure of mice to first-generation adenoviral vectors worsens the outcome of bacterial sepsis (12). These associations suggest that prior adenoviral infections attenuate the host responses to bacteria or bacterial products.

The aim of the present study was to evaluate the effects of adenoviral infection on in vivo responses to LPS mediated by TNF-{alpha}. It has been well established that TNF-{alpha} is necessary for initiation of inflammation and clearance of adenoviral and bacterial infections (13, 14, 15, 16, 17). Adenoviral protein E1A has been shown to sensitize infected cells to TNF-{alpha}-induced apoptosis and enhance production of proinflammatory factors in LPS-stimulated cells (18, 19). In contrast, several adenoviral proteins (E1B-19K, E3–14.7K, E3–14.5K, and E3–10.4K) are known to suppress apoptosis induced by TNF-{alpha} and facilitate evasion of the immune responses during adenoviral infection (reviewed in Refs. 20 and 21). It has been further shown that transgenic mice expressing adenoviral E3–14.7K protein in the lung epithelial cells under the direction of the surfactant protein C promoter have decreased lung inflammation following LPS administration (22). Constitutive overexpression of E3–14.7K protein is also capable of protecting mice from LPS-induced acute liver failure (23). This suggests that adenoviruses have potential to modulate responses to LPS mediated by TNF-{alpha}. However, the ultimate effects of wild-type adenoviral infection on in vivo responses to LPS and TNF-{alpha} have not been evaluated.

For the present study, a commonly used and well-characterized model of TNF-{alpha}-mediated fulminant hepatitis with D-galactosamine (D-Gal) sensitization was chosen to examine in vivo responses to LPS (24, 25, 26). Adenoviral infection was established in mice by i.v. delivery of human wild-type adenovirus or first-generation adenoviral vectors. Despite poor replication of human adenoviruses in mice, pathology and immune responses resemble those previously described in cotton rats, which are permissive for replication (27, 28). Although most of the adenoviral infections in humans are localized to the pharynx and eyes, adenoviruses have been cultured from the blood during fatal respiratory diseases (29). Using this model, we found that both wild-type adenovirus and recombinant E1,E3-deleted adenoviral vectors induce the state of TNF-{alpha} tolerance that protects mice from LPS, SEB, or exogenously administered TNF-{alpha}.

TNF-{alpha} tolerance (or the state of unresponsiveness to TNF-{alpha}) has been demonstrated after various stress factors, including heat shock, exposure to LPS, TNF-{alpha}, or IL-1 (30, 31, 32). Characteristic features of the TNF-{alpha} tolerant mice include resistance to lethal doses of TNF-{alpha} or LPS and decreased production of proinflammatory mediators, such as IL-6 and NO following TNF-{alpha} administration (32, 33, 34). Genetic factors, induction of heat shock protein-70, and acute-phase proteins have been shown to mediate TNF-{alpha} tolerance (34, 35, 36, 37), whereas a role of IL-6, NO, and heme oxygenase-1 has been ruled out (32, 38). In this study, we report for the first time that TNF-{alpha} tolerance is transiently induced during the course of adenoviral infection. Remarkably, the previously described adenoviral TNF-{alpha} resistance genes within E3 region are not required for induction of TNF-{alpha} tolerance. Finally, our study provides first evidence that TNF-{alpha} tolerance may be induced by a viral infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Adenoviral infection

Serotype 5 human adenovirus was purchased from American Type Culture Collection (ATCC, VR-5, Manassas, VA) or from Advanced Biotechnologies (Columbia, MD). Replication-deficient E1,E3-deleted adenoviral vectors expressing enhanced green fluorescence protein (Ad.GFP) or firefly luciferase were obtained from the University of Iowa Gene Transfer Vector Core. The typical titers of the vectors were in the range of 1.2 - 2.6 x 1012 particles/ml or 1 - 2 x 1010 PFU/ml, as determined by spectrophotometry or by plaque assay on 293 cells, respectively. C57BL/6 or DBA/2, 6- to 8-wk-old female mice were purchased from Harlan (Indianapolis, IN). Adenoviral infections were established by i.v. injections of the viruses to ketamine/xylazine-anesthetized mice (8.9 x 105 PFU/mouse of the wild-type and 106-109 PFU/mouse of the vectors in 100 µl vol). Sterile carrier solution (3% sucrose in PBS) was used for control injections and for dilution of the viruses. Control mice were anesthetized and received 100 µl of the carrier solution, and were usually housed in the same biohazard containment rooms as the adenovirus-infected mice.

Fulminant hepatitis models

Mice were sensitized by i.p. administration of 25 mg/mouse of D-Gal (Sigma-Aldrich, St. Louis, MO). LPS from Escherichia coli 0111:B4 (Sigma-Aldrich) was used at 50 µg/kg. Staphylococcal enterotoxin B (Toxin Technology, Sarasota, FL) was used at 2.5 mg/kg. Mouse rTNF-{alpha} (Pierce Biotechnology, Rockford, IL) with sp. act. 109 IU/µg (based on comparison with mouse TNF-{alpha} National Institute for Biological Standards and Control Standard) and endotoxin content 0.019 EU/µg was used at 0.1 µg/mouse. All reagents were diluted in sterile PBS and used immediately after sensitization with D-Gal. No azide/low endotoxin hamster anti-mouse Fas mAb Jo-2 (BD Biosciences, San Jose, CA) was used at 10 µg/mouse. Mortality rate was recorded for up to 28 h after LPS or anti-Fas treatments and for up to 72 h after SEB or TNF-{alpha} treatments with periodic observations every 1 h during initial 12 h and every 4 h thereafter. The animal protocols were approved by the University of Iowa Institutional Animal Care and Use Committee.

Analysis of liver injury

Control or Ad.GFP-infected mice were euthanized before or 6 h after treatment with a lethal dose of LPS/D-Gal. Liver lobes were excised and fixed by submersion in 4% paraformaldehyde in PBS for 2 h. Cryosections were prepared following cryoprotection in 20% sucrose and embedding in Tissue-Tek OCT compound (Sakura Finetek USA, Torrance, CA) and used for TUNEL assay for apoptosis or H&E staining. In Situ Cell Death Detection kit (TMR Red; Roche Molecular Biochemicals, Indianapolis, IN) was used to detect apoptosis by TUNEL assay, according to the manufacturer’s protocol. Random fields on at least three separate cryosections from every animal were analyzed and recorded using Leitz Diaplan fluorescent microscope and SPOT cooled CCD camera (Diagnostics Instruments, Sterling Heights, MI). The number of apoptotic nuclei was counted using ImagePro Plus software (Media Cybernetics, Silver Spring, MD). H&E staining was performed at the University of Iowa Central Microscopy Research Facility.

Adenoviral DNA isolation and PCR

Adenoviral DNA was isolated from 1011 particles of the wild-type adenovirus (Advanced Biotechnologies) or Ad.GFP DNA using Easy DNA kit (Invitrogen, Carlsbad, CA) and measured using PicoGreen dsDNA quantitation kit (Molecular Probes, Eugene, OR), following manufacturer’s recommendations. The amplifications were conducted using 4 ng of the adenoviral DNA as template and 2.5 U of platinum Taq in 50-µl reactions containing 1.5 mM MgCl2, 200 µM dNTPs (Roche Molecular Biochemicals), and 0.3 µM each primer. The PCR primers flanking dl309 deletion were as follows: forward, 5'-CCTCATCACTGTGGTCATCG-3' (corresponding to positions 29,936–29,955 of human adenovirus 5 genome; GenBank accession number M73260) and reverse, 5'-GATCTTTGAGACCGCACAGG-3' (positions 30,803–30,822). The same reverse primer was used along with the forward primer, 5'-CAACAGCGCATGAATCAAGA-3' (positions 30,534–30,553), that is specific for wild-type adenovirus 5 only (no annealing site in the dl309 deletion). Amplification included initial denaturing at 95°C for 3 min, and 40 cycles of denaturing at 94°C for 40 s, annealing at 60°C for 40 s, and extension at 72°C for 40 s. PCR products were separated on 2% agarose gel in Tris-borate-EDTA buffer, stained with ethidium bromide, and documented using Gel Doc 2000 gel documentation system (Bio-Rad, Hercules, CA). PCR products were purified with QIAquick PCR purification kit and sequenced at the University of Iowa DNA Facility.

Enzyme immunoassays and Western blotting

Serum and liver samples were obtained from euthanized mice at different times after treatments. Serial dilutions of the serum were assayed for mouse TNF-{alpha}, soluble TNFR1, IL-6, and KC using DuoSet ELISA development kits (R&D Systems, Minneapolis, MN), following manufacturer’s recommendations. Livers were immediately frozen in liquid nitrogen and stored at -70°C until they were homogenized in 3 vol of ice-cold lysis buffer (0.05 M Tris, pH 7.4, 0.15M NaCl, 1% Nonidet P-40) supplemented with Complete protease inhibitors (Roche Molecular Biochemicals) and 1x phosphatase inhibitors (Calbiochem, La Jolla, CA). The homogenates were centrifuged twice at 16,000 x g at 4°C for 10 min, and supernatants were assayed for total protein content using a Bio-Rad protein assay kit and for TNFR1 content using the DuoSet kit for mouse soluble TNFR1. The amounts of TNF-{alpha} bound to TNFR1 were measured using the capture Ab against mouse TNFR1 and biotinylated Ab against mouse TNF-{alpha} from the corresponding DuoSet kits. Standard curve was generated using the capture Ab against TNF-{alpha}, mouse rTNF-{alpha}, and biotinylated Ab against TNF-{alpha}. The data were normalized to protein concentrations in the samples.

Western blotting analysis was performed by separating 100 µg of liver lysates/lane in 10% SDS-PAGE gel and semidry transfer onto nitrocellulose membrane (Amersham, Arlington Heights, IL). The membranes were blocked with 5% milk in TTBS (TBS with 0.1% Tween 20) for 1 h and incubated with the rabbit anti-mouse TNFR1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. The blots were washed four times with TTBS and incubated for 1 h with HRP-conjugated secondary Abs. Immunoreactive bands were developed using a chemiluminescent substrate (ECL Plus; Amersham). Autoradiographs were obtained by exposing Kodak Biomax MR films (Eastman Kodak, Rochester, NY) to the membranes for 10 s to 5 min. The intensity of the immunoblotting signals was measured using Fluor-S MultiImager system and Quantity One software (Bio-Rad).

TNF-{alpha} bioassay

L929 cells (ATCC, CCL-1) were plated overnight at 4 x 104 cells/well in 96-well plates and incubated with serial dilutions of the serum samples in the presence of 2.5 µg/ml actinomycin D (Calbiochem) for 16 h. Mouse rTNF-{alpha} (Pierce) with sp. act. 109 IU/µg (based on comparison with mouse TNF-{alpha} National Institute for Biological Standards and Control Standard) was used to generate the standard curves. Cell viability was evaluated by spectrophotometric measurement of reduction of water-soluble tetrazolium salt (Roche Molecular Biochemicals).

Statistical analyses

Survival curves between control and adenovirus-infected mice were compared using {chi}2 log rank tests. Continuous results were expressed as means ± SEM and analyzed using one-way ANOVA test. If a significant difference was found (p < 0.05), the individual groups were compared using Bonferroni postanalysis test. All calculations were performed with GraphPad Prism software version 3.0 (GraphPad Software, San Diego, CA).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Adenoviral infection decreases LPS-induced mortality

Adenoviral infection in the C57BL/6 mice was established by i.v. injection using serotype 5 human adenovirus 2, 7, and 14 days before challenge with LPS/D-Gal. Control and adenovirus-infected mice were injected i.p. with D-Gal and LPS and monitored for survival for up to 28 h. Mortality rate in the control group was consistent with previous studies (24, 25, 26). Mice that had been infected with adenovirus for 7 days showed significantly decreased mortality rate: the first death was recorded at 12 h and 50% mice survived after 28 h (Fig. 1). Mice in all other groups died within 15 h. Adenoviral infection 2 days before LPS challenge showed slightly delayed mortality, whereas the mice that had been infected for 14 days showed mortality rate undistinguishable from the control mice. The protection at 7 days postadenoviral infection was statistically significant, as confirmed by two additional independent experiments with two groups: all control mice died within 9 h after LPS challenge; at least 50% adenovirus-infected mice survived LPS challenge and showed no signs of sickness after 28 h (p < 0.01; data not shown). Thus, systemic infection with wild-type adenovirus transiently decreases LPS-induced mortality.



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  		FIGURE 1. Adenoviral infection decreases LPS/D-Gal-induced mortality. Adenoviral infection was established in C57BL/6 mice by i.v. injection of 8.9 x 105 PFU/mouse of the serotype 5 human adenovirus (ATCC VR-5) 2, 7, and 14 days before challenge with LPS and D-Gal (n = 4 mice per group).

 
TNF-{alpha}, released by activated macrophages, dendritic cells, and Kupffer cells, has been shown to be the key mediator of liver injury in the LPS model inducing massive apoptosis in the liver (24, 25, 26, 39). Adenoviral proteins E1B-19K, E3–14.7, E3-RID, and E3–6.7K are known to protect infected cells from TNF-{alpha}-induced apoptosis (21). Expression of E1B-19K protein is not likely to play any role in our system because it has been shown to protect human cells, but not mouse cells (40). However, constitutive overexpression of E3–14.7K protein has been shown to protect mice from LPS-induced acute liver failure (23). To determine whether adenoviral TNF resistance genes within E3 region mediate protection from LPS in our model, we used recombinant replication-deficient adenoviral vectors that were generated using the backbone of previously described dl309 mutant (41). The deletion/insertion in the E3 region of this mutant effectively inactivates E3–10.4K, E3–14.5K, and E3–14.7K adenoviral proteins (41). In addition, this vector is rendered replication deficient by a deletion (Ad5 bp 354-3327) of the E1A and most of the E1B genes. The aforementioned vector expressing enhanced green fluorescent protein under control of the CMV promoter (Ad.GFP) was administered to C57BL/6 mice 7 days before treatment with LPS in the following experiments. We found that Ad.GFP at a dose of 109 PFU/mouse decreased mortality rate in the LPS model (Fig. 2A).



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  		FIGURE 2. Adenovirus-induced protection from LPS/D-Gal mortality does not require adenoviral TNF-{alpha} resistance genes in the E3 region. A, Adenoviral infection was established in C57BL/6 mice by i.v. injection of 109 PFU/mouse of replication-deficient E1- and E3-deleted Ad.GFP. Control mice were injected with equivalent volume of the carrier (3% sucrose in PBS). LPS and D-Gal were administered on day 7 postinfection. Mortality rate was significantly different (p < 0.005; n = 4 mice per group). B, Ad.GFP vector has dl309 deletion and no contaminating wild-type adenovirus. DNA was isolated from wild-type adenovirus (lanes 2 and 4) and Ad.GFP (lanes 3 and 5) and amplified using primers specific for human serotype 5 adenovirus DNA (lanes 2 and 3) or primers flanking previously described dl309 deletion/insertion (lanes 4 and 5). Lane 1, 100-bp DNA ladder.

 
The effect of Ad.GFP vector was not likely due to potential presence of the wild-type adenovirus, because all preparations of the vector routinely tested negative at the Gene Transfer Vector Core for the presence of the wild-type adenovirus by plaque assay and by PCR. To be certain, we independently performed PCR analysis from the DNA isolated from Ad.GFP vector to exclude the possibility of contamination with wild-type adenovirus and to confirm the deletion in the E3 region (Fig. 2B). No amplification product was detected in the DNA isolated from Ad.GFP after 40 cycles of amplification using the primer pair specific for wild-type adenovirus, confirming that Ad.GFP vector was free of contamination with wild-type adenovirus. The amplification product from the reaction using Ad.GFP DNA template and the primers flanking dl309 deletion was 100% identical with the corresponding region of the dl309 mutant, and its size was consistent with the described deletion/insertion (41). Amplification products from the positive control reactions using DNA isolated from wild-type adenovirus were also consistent with the expected size. Thus, Ad.GFP vector used in our study contains no detectable wild-type adenovirus contamination and has the previously described dl309 deletion/insertion that inactivates E3–10.4K, E3–14.5K, and E3–14.7K (41).

These results provide evidence that adenoviral infection has transient protective effects in a mouse model of fulminant hepatitis induced by LPS/D-Gal treatment. Furthermore, previously characterized adenoviral TNF resistance genes encoded within E3 region are not required for this protection, although we cannot exclude the possibility that these genes contribute to the protection following infection with the wild-type adenovirus.

Adenovirus-infected mice have higher TNF-{alpha} levels and bioactivity following LPS stimulation

We have previously reported that adenovirus-infected mice had lower levels of serum TNF-{alpha} late in sepsis induced by cecal ligation and puncture (12). Decreased production of TNF-{alpha} following LPS stimulation could potentially explain the differences in the mortality rate between control and Ad.GFP-infected mice. No TNF-{alpha} was detected in the sera of control or Ad.GFP-infected mice before LPS stimulation (sensitivity of the assay, 8 pg/ml; data not shown). However, we found significantly higher serum TNF-{alpha} levels in the Ad.GFP-infected mice relative to control mice at 90 min following LPS treatment, when the levels of TNF-{alpha} have been reported to peak (39) (Fig. 3A). No difference in the TNF-{alpha} levels was found at 3 h. Thus, adenovirus-infected mice exhibit higher levels of TNF-{alpha} in the circulation, yet remain resistant to its hepatotoxic effects.



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  		FIGURE 3. Adenovirus-infected mice have higher TNF-{alpha} levels and bioactivity than control mice following LPS/D-Gal stimulation. Control and Ad.GFP-infected mice were euthanized 90 min and 3 h after LPS/D-Gal challenge to collect sera. Serum samples were obtained from four mice per group at each time point and processed individually. ELISA was used to measure TNF-{alpha} (A) and soluble TNFR1 (B). L929 cell cytotoxicity assay was used to measure the biological activity of TNF-{alpha} in the samples relative to mouse rTNF-{alpha} with known sp. act. 109 IU/mg (C). *, p < 0.01; **, p < 0.001.

 
Soluble TNF receptors stoichiometrically bind and regulate availability and biological activity of TNF-{alpha} in the circulation (42). Soluble rTNFR1 administered i.v. or overexpressed with an adenoviral vector is capable of protecting mice from LPS (43, 44). TNF-{alpha} and a number of other LPS-inducible mediators (IL-6, IL-1{beta}, etc.) are known to induce release of soluble TNFRs via ectodomain shedding (45, 46, 47). Adenoviral infection resulted in significant increase of serum soluble TNFR1 levels (2276 ± 516 pg/ml in control mice vs 5136 ± 628 pg/ml in Ad.GFP-infected mice on day 7 postinfection, p < 0.05). We have previously reported that Ad.GFP-infected mice have dramatically higher serum levels of soluble TNFR1 than the control mice at 24 h following cecal ligation and puncture (12). For these reasons, we measured soluble TNFR1 in the serum at 90 min and 3 h following stimulation with LPS to determine whether increased shedding of soluble TNFR1 occurs in this model. The levels of soluble TNFR1 were dramatically higher at 90 min after LPS treatment and returned to the basal levels by 3 h in both groups. Ad.GFP-infected mice had significantly higher levels of soluble TNFR1 than the control mice at both time points (Fig. 3B). These data suggest that adenoviral infection primes for increased TNFR1 shedding following LPS stimulation.

The in vivo interactions between soluble TNFRs and TNF-{alpha} are complex; soluble TNFRs may serve as TNF-{alpha} antagonists when present in large excess, as TNF carrier proteins, or as stabilizers of TNF-{alpha} bioactivity (42). Moreover, additional serum factors, such as soluble TNFR2 or {alpha}2-macroglobulin, may also regulate TNF-{alpha} bioactivity. To determine the amount of biologically active TNF-{alpha} in the serum of control and adenovirus-infected mice following LPS stimulation, we performed a bioassay using TNF-{alpha}-sensitive L929 cells pretreated with actinomycin D. We found that serum collected from Ad.GFP-infected mice 90 min after LPS treatment was significantly more toxic to L929 cells than the serum from control mice (Fig. 3C). Thus, adenovirus-infected mice have higher levels of biologically active TNF-{alpha}, despite the presence of increased soluble TNFR1. Therefore, increased levels of soluble TNFR1 are not sufficient to block the systemic effects of TNF-{alpha} in our system. These data also suggest that protection of adenovirus-infected mice from LPS/D-Gal-induced liver failure cannot be explained by the presence of any other TNF-{alpha}-inhibiting factors in the serum.

Adenoviral infection results in TNF-{alpha} tolerance

TNF-{alpha} released by activated T cells has also been shown to be the major mediator of liver failure and death following administration of SEB to D-Gal-sensitized mice (48). To determine whether adenoviral infection has protective effects in this model as well, we challenged control and Ad.GFP-infected mice with a lethal dose of SEB and D-Gal. DBA/2 mice were used in this experiment because C57BL/6 mice cannot efficiently present SEB to T cells due to the defect in the E{alpha} gene of the MHC class II H-2 (49, 50). We found that infection with Ad.GFP for 7 days protects mice from SEB-induced mortality (Fig. 4). Thus, regardless of the type of stimulation or the source of endogenous TNF-{alpha}, adenoviral infection confers protection from TNF-{alpha}-mediated fulminant hepatitis.



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  		FIGURE 4. Adenoviral infection protects from liver failure induced by SEB/D-Gal. DBA/2 mice were infected with Ad.GFP for 7 days and challenged with 2.5 mg/kg SEB and 25 mg/mouse D-Gal. Mortality rate was significantly different (p < 0.01; n = 4 mice per group).

 
To determine whether adenoviral infection decreases sensitivity to TNF-{alpha}, we administered exogenous TNF-{alpha} to D-Gal-sensitized mice following adenoviral infection. We found that mice that had been infected with either wild-type adenovirus or Ad.GFP for 7 days were tolerant to a dose of TNF-{alpha} (0.1 µg/mouse) that was 100% lethal for the control mice within 24 h (Fig. 5A). TNF-{alpha} tolerance induced by the vector was not dependent on the transgene expression, because another vector expressing firefly luciferase had similar protective effects (data not shown).



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  		FIGURE 5. Adenoviral infection results in TNF-{alpha} tolerance. A, Adenovirus-infected mice are protected from liver failure induced by TNF-{alpha}/D-Gal. C57BL/6 mice were injected with 8.9 x 105 PFU/mouse of wild-type adenovirus (wtAd, n = 5), 109 PFU/mouse of Ad.GFP (n = 6), or 100 µl 3% sucrose in PBS (control, n = 6) 7 days before challenge with 0.1 µg/mouse TNF and 25 mg/mouse D-Gal. B and C, Adenovirus-infected mice produce less IL-6 and KC in response to TNF-{alpha}. Serum IL-6 (B) and KC (C) were measured in control and wild-type adenovirus-infected mice following treatment with 4 µg/mouse of mouse TNF-{alpha} (without D-Gal). Three mice per group were euthanized at each time point to collect sera, and serum samples were processed individually. *, p < 0.05; **, p < 0.001.

 
The protective effects of TNF-{alpha} in bacterial infections are mediated in part by induction of proinflammatory mediators in response to TNF-{alpha}. Decreased serum level of IL-6 following treatment with high doses of TNF-{alpha} is a characteristic feature of TNF-{alpha} tolerant mice (32, 33, 34). Among other TNF-{alpha}-inducible mediators, chemokines play a prominent role in regulation of neutrophil recruitment to inflammatory sites (51). To determine the effects of adenoviral infection on proinflammatory responses to TNF-{alpha}, we measured serum levels of IL-6 and KC, a C-X-C chemokine, following i.p. administration of rTNF-{alpha}. D-Gal treatment was omitted in these experiments to avoid early mortality and interference with gene transcription in hepatocytes. No differences were found between control and adenovirus-infected mice in the basal levels of IL-6 or KC (data not shown). We found that adenoviral infection resulted in decreased peak levels of IL-6 (Fig. 5B) and KC (Fig. 5C) after TNF-{alpha} treatment. These data suggest that adenoviral infection not only inhibits TNF-{alpha}-induced apoptosis in the liver, but also down-regulates proinflammatory responses to TNF-{alpha}.

Adenoviral infection has no effect on expression of TNFR1 in the liver

Adenoviral proteins E3–10.4K and E3–14.5K form a receptor internalization and degradation complex that protects cells from apoptosis induced by signaling through Fas (52). This complex has been shown to associate with E3–6.7K protein and internalize two other death receptors (TNF-related apoptosis-inducing ligand-1 and TNF-related apoptosis-inducing ligand-2) belonging to the TNFR family (53). TNFR1 has been shown to be necessary and sufficient to mediate proapoptotic TNF-{alpha} signaling in LPS- or TNF-{alpha}-mediated fulminant hepatitis with D-Gal sensitization (25, 54). Adenoviruses are not known to decrease TNFR1 expression. However, elevated levels of soluble TNFR1 indicate increased shedding of TNFR1 in response to LPS. We evaluated expression of TNFR1 using immunoblotting analysis to exclude the possibility that adenovirus-induced tolerance to TNF-{alpha} may be mediated by decreased expression of TNFR1. No difference was found in the levels of TNFR1 between control and Ad.GFP-infected mice even after LPS treatment (Fig. 6A). Furthermore, we used an ELISA to measure TNFR1 contents in the pooled liver lysates obtained from mice following Ad.GFP infection and LPS treatment (Fig. 6B). We found that TNFR1 expression in the liver decreased following LPS treatment in both groups; however, no difference was evident between control and Ad.GFP-infected mice. These data suggest that adenoviral infection does not affect TNFR1 expression in the liver. An assay for TNF-{alpha} bound to the liver TNFR1 following LPS treatment has also revealed no significant difference between control and Ad.GFP-infected groups (data not shown). This suggests that adenovirus-induced TNF-{alpha} tolerance manifests downstream of TNFR1 signaling.



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  		FIGURE 6. Adenoviral infection has no effects on TNFR1 expression in the liver. A, TNFR1 expression in the livers from control and Ad.GFP-infected mice was evaluated by Western blotting at day 7 postinfection and 90 min after LPS/D-Gal treatment. Fifty micrograms of pooled liver lysates (4 mice per group) were separated on 10% SDS-PAGE and probed with rabbit polyclonal Ab against TNFR1. Densitometric analysis revealed no difference between the groups. B, ELISA was used to measure TNFR1 content in the pooled liver lysates (n = 4 mice per group) following LPS/D-Gal treatment. The data were normalized to protein concentrations in the samples.

 
Adenoviral infection-induced TNF-{alpha} tolerance decreases liver injury following LPS treatment

To determine whether decreased mortality from LPS-induced liver failure correlates with the degree of liver injury, we analyzed liver sections prepared from control and Ad.GFP-infected mice (day 7 postinfection) before and after 6-h treatment with lethal dose of LPS/D-Gal (Fig. 7A). No apoptosis was detected by TUNEL assay in livers from control group. Extensive apoptosis was found in the liver sections of mice treated with LPS/D-Gal, which is consistent with previous studies using the models of LPS-induced fulminant hepatitis (26, 36). Few apoptotic nuclei were found in the liver sections from Ad.GFP-infected mice without treatment. LPS/D-Gal treatment resulted in increased apoptosis in the livers from Ad.GFP-infected mice. Importantly, the extent of apoptosis in Ad.GFP-infected mice was significantly lower than in control mice after LPS/D-Gal treatment, as determined by the mean number of apoptotic nuclei per field (Fig. 7B). H&E staining of liver sections revealed noticeable leukocyte infiltration in the livers of Ad.GFP-infected mice, which is not observed in normal livers (data not shown). Treatment of control mice with LPS/D-Gal resulted in extensive hemorrhage and necrosis in the livers typical for LPS-induced fulminant hepatitis (26, 36). The extent of liver hemorrhage and necrosis was substantially lower, albeit not eliminated in Ad.GFP-infected mice following LPS/D-Gal treatment (data not shown).



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  		FIGURE 7. Adenoviral infection decreases liver injury following LPS/D-Gal administration. Liver cryosections were prepared from control and Ad.GFP-infected mice (day 7 postinfection) before and after 6 h of LPS/D-Gal treatment. A, TUNEL assay for apoptosis. Representative sections and the mean numbers of apoptotic nuclei per field are shown. B, The mean number of apoptotic nuclei (apoptotic index) in the livers of Ad.GFP-infected mice treated with LPS is significantly lower than in control mice treated with LPS (p < 0.001; several randomly chosen fields were quantified from at least three separate sections per animal; average numbers were compared; 5 mice per group were analyzed). Representative sections are shown (6 mice per group were analyzed). Scale bar represents 100 µm.

 
In summary, the livers of adenovirus-infected mice display leukocyte infiltration and a low level of apoptosis. Hemorrhage and increased apoptotis were evident in Ad.GFP-infected mice after LPS/D-Gal treatment, although their extent was significantly lower relative to LPS-treated control mice. Thus, adenoviral infection only partially decreases the extent of liver injury mediated by LPS-induced TNF-{alpha}; nevertheless, this may be sufficient to decrease mortality in this model.

Adenoviral infection does not protect from liver failure induced by Fas Ab

Adenoviruses are known to inhibit cellular apoptosis at several levels, including internalization of the death receptors, interactions with Bax and Bak, and inhibition of p53 (21). Agonistic Abs against Fas receptor are known to trigger apoptosis in the liver and fulminant hepatitis similarly to TNF-{alpha} (55). Proapoptotic signaling via TNFR1 and Fas receptor converges at the level of recruitment of procaspase-8 to Fas-associated death domain protein (56). To determine whether adenovirus-induced TNF-{alpha} tolerance is mediated by inhibition of apoptosis, we treated control and adenovirus-infected mice with the well-characterized Fas agonist Ab Jo-2 (Fig. 8). Control mice displayed 67% mortality following Jo-2 administration. Infection with the wild-type adenovirus slightly delayed mortality; none of these mice died within the first 8 h after the treatment, but all succumbed by 24 h. The mortality rate in the group of mice infected with Ad.GFP vector was similar to the control. Thus, it is not likely that inhibition of apoptosis explains protection from TNF-{alpha} following adenoviral infection.



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  		FIGURE 8. Adenoviral infection does not protect from Fas-induced liver failure. C57BL/6 mice were infected with 8.9 x 105 PFU/mouse of wild-type adenovirus (wtAd), 109 PFU/mouse of Ad.GFP, or 100 µl 3% sucrose in PBS (control) 7 days before challenge with 10 µg/mouse of anti-Fas Ab Jo-2 (n = 6 mice per group).

 
In summary, our study shows that adenoviral infection primes for increased production of TNF-{alpha} and shedding of TNFR1 in response to LPS, but induces tolerance to hepatotoxic effects of TNF-{alpha}. This TNF-{alpha} tolerance protects mice from fulminant hepatitis triggered by LPS, SEB, or exogenous TNF-{alpha}. Another novel observation is that the effects of adenovirus are not dependent on previously described TNF-{alpha} resistance genes E1B-19K, E3–14.7K, E3–10.4K, and E3–14.5K, because E1- and E3-deleted adenoviral vector renders mice TNF-{alpha} tolerant. Importantly, adenovirus-infected mice were protected from LPS despite increased production and serum bioactivity of TNF-{alpha} following LPS stimulation. This demonstrates that adenoviral infection does not result in endotoxin tolerance, which is characterized by poor responses to LPS, such as decreased production of TNF-{alpha} (57). Furthermore, this separates adenovirus from influenza virus and lymphocytic choriomeningitis virus, which have been previously shown to increase mortality from LPS- and/or SEB-mediated shock (2, 3, 4, 5).

It is well established that excessive TNF-{alpha} signaling may be harmful by inducing shock (58). Overexpression of soluble TNFR1 or TNFR1 deletion in transgenic mice protects against lethal effects of LPS (14, 15, 44). In contrast, TNF-{alpha} has been shown to be necessary for antibacterial host response because defects in TNF-{alpha} production or TNFR1 signaling lead to increased sensitivity to bacterial infections (14, 15, 16, 17, 44). Our previous study demonstrated that adenovirus-infected mice have higher mortality from sepsis induced by cecal ligation and puncture (12). The fact that adenovirus-infected mice had significantly higher bacterial load in the blood, liver, and lungs in that study suggests that antibacterial host response was impaired. This is further supported by our present data showing decreased production of IL-6 and KC in adenovirus-infected mice following TNF-{alpha} administration. We believe that adenovirus-induced TNF-{alpha} tolerance may contribute to the lethal synergy between adenoviral infection and sepsis, although a number of additional mechanisms may be implicated. It remains to be determined whether adenovirus-induced TNF-{alpha} tolerance inhibits protective functions of TNF-{alpha} during bacterial infections and sepsis.

Further studies will be necessary to precisely determine the mechanism of adenovirus-induced TNF-{alpha} tolerance. A number of instances and mechanisms of TNF-{alpha} tolerance have been reported previously. Low doses of TNF-{alpha}, IL-1, or LPS; exposure to heat shock; or genetic defects may render mice TNF-{alpha} tolerant (30, 31, 32, 35, 36). We found no evidence of decreased bioactivity of TNF-{alpha} in the serum or decreased TNFR1 expression. Moreover, proapoptotic signaling downstream of Fas-associated death domain protein, shared by Fas receptor, appears to be intact in adenovirus-infected mice. It is also not likely that protection is mediated by NF-{kappa}B-mediated antiapoptotic signaling via TNFR1 or TNFR2, because D-Gal sensitization prevents gene transcription in liver cells (24, 25). It is reasonable to suggest that adenovirus-induced TNF-{alpha} tolerance leads to a defect in the proximal TNFR1 signaling.


   Acknowledgments
 
We are grateful to John F. Engelhardt for great discussions and useful comments. We also thank Maria L. Scheel at the Gene Transfer Vector Core for helpful consultations on the adenoviral vectors, and Timothy J. Ahlers for technical assistance.


   Footnotes
 
1 This work was supported by grants from National Institutes of Health (NIH-HL-60316 and ES-09607) and VA Merit Review (to G.W.H.). Back

2 Address correspondence and reprint requests to Dr. Timur O. Yarovinsky, University of Iowa, 100 EMRB, Iowa City, IA 52242. E-mail address: timur-yarovinsky{at}uiowa.edu Back

3 Abbreviations used in this paper: SEB, staphylococcal enterotoxin B; Ad.GFP, adenoviral vector expressing enhanced green fluorescent protein; D-Gal, D-galactosamine. Back

Received for publication February 19, 2003. Accepted for publication July 9, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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