Adenovirus Vector–Induced IL-6 Promotes Leaky Adenoviral Gene Expression, Leading to Acute Hepatotoxicity

Kahori Shimizu, Fuminori Sakurai, Shunsuke Iizuka, Ryosuke Ono, Tomohito Tsukamoto, Fumitaka Nishimae, Shin-ichiro Nakamura, Toru Nishinaka, Tomoyuki Terada, Yasushi Fujio and Hiroyuki Mizuguchi
J Immunol January 15, 2021, 206 (2) 410-421; DOI: https://doi.org/10.4049/jimmunol.2000830

Key Points

  • Suppression of leaky Ad gene expression in the liver attenuated acute hepatotoxicity.

  • IL-6 enhanced leaky Ad gene expression and Ad vector–induced cytotoxicity.

  • Acute hepatotoxicity and leaky Ad gene expression were reduced in IL-6 KO mice.

Abstract

Adenovirus (Ad) vector–mediated transduction can cause hepatotoxicity during two phases, at ∼2 and 10 days after administration. Early hepatotoxicity is considered to involve inflammatory cytokines; however, the precise mechanism remains to be clarified. We examined the mechanism of early Ad vector–induced hepatotoxicity by using a conventional Ad vector, Ad-CAL2, and a modified Ad vector, Ad-E4-122aT-CAL2. Ad-E4-122aT-CAL2 harbors sequences complementary to the liver-specific miR-122a in the 3′ untranslated region of E4, leading to significant suppression of leaky Ad gene expression in the liver via posttranscriptional gene silencing and a significant reduction in late-phase hepatotoxicity. We found that Ad-E4-122aT-CAL2 transduction significantly attenuated acute hepatotoxicity, although Ad-E4-122aT-CAL2 and Ad-CAL2 induced comparable cytokine expression levels in the liver and spleen. IL-6, a major inflammatory cytokine induced by Ad vectors, significantly enhanced leaky Ad gene expression and cytotoxicity in primary mouse hepatocytes following Ad-CAL2 but not Ad-E4-122aT-CAL2 transduction. Furthermore, leaky Ad gene expression and cytotoxicity in Ad-CAL2–treated hepatocytes in the presence of IL-6 were significantly suppressed upon inhibition of JAK and STAT3. Ad vector–mediated acute hepatotoxicities and leaky Ad expression were significantly reduced in IL-6 knockout mice compared with those in wild-type mice. Thus, Ad vector–induced IL-6 promotes leaky Ad gene expression, leading to acute hepatotoxicity.

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Introduction

Adenovirus (Ad) vectors are extensively used not only in clinical gene therapy but also in basic research, owing to their attractive properties as a gene delivery vehicle, including high transduction efficiency in a wide range of cells and high-titer production. However, preclinical and clinical studies have reported that Ad vectors and oncolytic Ads induce hepatotoxicity as a major adverse event following in vivo application (1–13). In mice, two peaks of serum alanine aminotransferase (ALT), an enzymatic biomarker of hepatotoxicity, occur at ∼2 and 10 days following Ad vector administration (1–3, 13). To prevent Ad-induced hepatotoxicities and to develop safer recombinant Ads, it is important to clarify the underlying mechanisms.

The hepatotoxicity during the second peak has been relatively well studied and is caused primarily by adaptive immune responses against Ad proteins (10–13). Theoretically, a replication-incompetent Ad vector, in which the E1A gene that is crucial for the transcription of other Ad genes is genetically deleted, should not express Ad genes; however, leaky Ad gene expression, which leads to the induction of cellular immune responses against Ad proteins, has been observed (10, 13–17). To suppress leaky Ad gene expression in the liver, we previously developed a novel, to our knowledge, Ad vector containing sequences complementary to the liver-specific miR-122a into the 3′ untranslated region of E4 (Ad-E4-122aT) (13). Compared with a conventional Ad vector, Ad-E4-122aT mediates efficient and long-term transgene expression and significant attenuation of late-phase Ad vector–induced hepatotoxicity owing to the efficient suppression of leaky Ad gene expression in the liver. Furthermore, Ad-E4-122aT can be easily produced at a high titer through conventional methods using HEK293 cells.

Early-phase hepatotoxicity due to Ad vector administration is generally considered to be induced by inflammatory cytokines elicited in Ad vector–induced innate immune responses (18, 19). Various types of cytokines, including IL-6, IL-12, and IFNs, are expressed, primarily in the spleen, within 6 hours of Ad vector administration (5, 18, 20); however, the mechanisms of Ad vector–induced acute hepatotoxicity remain to be clarified.

In this study, we aimed to reveal the mechanisms of Ad vector–induced acute hepatotoxicity following i.v. administration by using a conventional Ad vector and Ad-E4-122aT. Our study will contribute to the improvement of Ad vector–based gene therapy by providing ways to attenuate Ad vector–induced hepatotoxicity.

Materials and Methods

Mice and cells

C57BL/6 mice aged 5–7 wk were obtained from Nippon SLC (Hamamatsu, Japan). Rag2/Il2rγ double-knockout (DKO) mice of a C57BL/6 background, also aged 5–7 wk, were obtained from Taconic Biosciences (Hudson, NY) (21, 22). IL-6 knockout (KO) mice of a C57BL/6 background, also aged 5–7 wk, were obtained from the RIKEN BioResource Research Center (RBRC04918; Ibaraki, Japan) (23). All animal experimental procedures were performed in accordance with the institutional guidelines for animal experiments at Osaka University (approval no. 28-3-3) and Osaka Ohtani University (approval no. 1407 and 2002). Primary mouse hepatocytes were cultured in Williams’ Medium E (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS, 10 μg/ml insulin, 4 μg/ml dexamethasone, and antibiotics. HEK293 cells were cultured in DMEM supplemented with 10% FBS and antibiotics.

Replication-incompetent Ad vector construction

Ad vectors were prepared using an improved in vitro ligation method (24, 25). pHMCA6-L was constructed by inserting the firefly luciferase gene into the multicloning sites of pHMCA6 (26). pHMCA6-L was digested with I-CeuI/PI-SceI and subsequently ligated with I-CeuI/PI-SceI–digested pAdHM4 (24) and pAdHM4-miR-122aT (13), resulting in pAdHM4-CA-Luc and pAdHM4-E4-miR-122aT-CA-Luc, respectively. HEK293 cells were transduced with pAdHM4-CA-Luc and pAdHM4-E4-miR-122aT-CA-Luc to produce Ad-CAL2 and Ad-E4-122aT-CAL2, respectively. Ad vectors were propagated and purified as previously described (24). Ad-AHA-STAT3F was prepared using pAdHM4, pHMRSV6, pBS-ApoEHCR-hAATp-hFIX-Int-bpA (27), and pEF-BOS-FLAG-STAT3 (Y705F) (28) (kindly provided by Dr. S. Akira, Osaka University, Osaka, Japan), as described above. A murine-secreted alkaline phosphatase-expressing conventional Ad vector, Ad-AHASEAP, was previously produced (13). The virus particles (VP) were quantified by spectrophotometry (29). Biological titers were measured using an Adeno-X Rapid Titer Kit (Clontech Laboratories, Mountain View, CA). The ratio of the particle/biological titer was between 6.5 and 8 for each Ad vector used in this study.

Quantitative RT-PCR

Ad vectors were i.v. administered to wild-type, Rag2/Il2rγ DKO, and IL-6 KO mice at a dose of 1 × 1011 VP per mouse via the tail vein. Total RNA was extracted from the livers and spleens at 6 h following Ad vector administration using Isogen (Nippon Gene, Tokyo, Japan). Following treatment with RNase-free DNase I (New England Biolabs, Ipswich, MA), cDNA was synthesized from 2 μg of total RNA using the Superscript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). Quantitative PCR analysis of Ad gene and cytokine gene expression in the organs was conducted using THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) and a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). The thermal cycles consisted of 60 s at 95°C, 40 cycles of 15 s at 95°C, and 60 s at 63°C. Primer sequences are available on request.

Evaluation of Ad vector–mediated hepatotoxicity in vivo

Ad vectors were i.v. administered to wild-type, Rag2/Il2rγ DKO, and IL-6 KO mice as described above. Blood samples were collected via retro-orbital bleeding at 6 and 48 h following Ad vector administration. Serum samples were obtained using centrifugation. Serum IL-6 levels were measured using a mouse IL-6 ELISA MAX Deluxe (BioLegend, San Diego, CA). Serum ALT levels were analyzed using a Transaminase Cii Test Kit (Wako Pure Chemical Industries). For the histopathological examination of liver sections, the livers were collected 48 h following Ad vector administration, washed, fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with H&E for histological analysis. For the analysis of apoptosis-related gene expression in the liver, total RNA was isolated from the liver 48 h following Ad vector administration and subjected to quantitative RT-PCR (RT-qPCR) analysis as described above. Primer sequences are available on request.

Evaluation of Ad vector–mediated cytotoxicity in primary mouse hepatocytes

Primary mouse hepatocytes were isolated from a C57BL/6 mouse using the hepatic portal perfusion technique, as described previously (30), and were seeded in a 96-well plate at 1 × 104 cells per well or in a 12-well plate at 1 × 105 cells per well. One day following isolation, the hepatocytes were transduced with Ad vectors at 1000 VP per cell. At 6 h after Ad vector transduction, recombinant mouse IL-6 protein (eBioscience, San Diego, CA) was added to each at a final concentration of 10 ng/ml, unless indicated otherwise. Twenty-four hours following transduction, the medium containing Ad vectors and IL-6 was replaced with fresh medium. At 48 h after transduction, cytotoxicity was assessed by a lactate dehydrogenase (LDH) assay using a CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI) according to the manufacturer’s instructions. At 24 h following vector transduction, mRNA levels of Ad and apoptosis-related genes were determined by RT-qPCR as described above. For inhibition of STAT3, primary mouse hepatocytes were transduced with Ad-CAL2 and Ad-AHA-STAT3F or Ad-AHASEAP at 1000 VP per cell for each Ad vector. For inhibition of JAK, primary mouse hepatocytes were treated with a JAK inhibitor (InSolution JAK Inhibitor I, 1 μM; Calbiochem, San Diego, CA). After 2 h of incubation with the JAK inhibitor, Ad vectors were added to the cells followed by the addition of rIL-6 (final concentration 10 ng/ml) 6 h postinfection. Cytotoxicity levels and mRNA levels of Ad genes were measured by an LDH assay 48 h postinfection and RT-qPCR analysis 24 h postinfection, respectively, as described above.

Luciferase expression in the liver following Ad vector administration

Wild-type and IL-6 KO mice were administered Ad-CAL2 at a dose of 1 × 1011 VP per mouse. Livers were recovered 48 h after administration and homogenized in lysis buffer (0.05% Triton X-100, 2 mM EDTA, 0.1 M Tris [pH 7.8]), using a homogenizer. After three cycles of freezing and thawing, the homogenates were centrifuged at 15,000 × g at 4°C for 10 min. Luciferase activity levels in the supernatants were measured using a luciferase assay system (PicaGene 5500; Toyo Ink, Tokyo, Japan). Protein contents were measured using a Bio-Rad Bradford Assay Kit (Bio-Rad Laboratories, Hercules, CA).

Statistical analysis

Data are presented as the mean ± SD. Means were compared by one-way ANOVA followed by Tukey–Kramer tests. A p value <0.05 was considered significant.

Results

Early-phase hepatotoxicity following Ad vector administration in mice

To examine early-phase Ad vector–induced hepatotoxicity, serum ALT levels were determined 48 h following i.v. administration of Ad-E4-122aT-CAL2 and the conventional Ad-CAL2 vector in wild-type mice. Serum ALT levels were markedly elevated by 2.5-fold in mice administered Ad-CAL2 when compared with the levels in Ad-E4-122aT-CAL2–administered mice (Fig. 1A). Histopathological analysis of liver tissue sections revealed the presence of focal necrotic changes of hepatocytes in the livers of Ad-CAL2–injected mice, whereas apparent hepatotoxicity was not observed in the livers of Ad-E4-122aT-CAL2–injected mice (Fig. 1B). Furthermore, RT-qPCR analysis demonstrated that expression levels of several apoptosis-related genes, including Bim, Bax, and mixed-lineage kinase domain-like pseudokinase (MLKL), were significantly upregulated in the livers of Ad-CAL2–injected but not Ad-E4-122aT-CAL2–injected mice (Fig. 1C). Also, Ad-E4-122a-CAL2 less strongly enhanced serum ALT levels during the late phase (10 d after administration) than did Ad-CAL2 (Fig. 1D). These results indicate that Ad vector–mediated hepatotoxicity during not only the late but also the early phase following systemic administration was significantly lower for Ad-E4-122aT-CAL2.

FIGURE 1.
FIGURE 1.

Ad vector–induced hepatotoxicity during the early phase in mice. (A) Serum ALT levels in wild-type and Rag2/Il2rγ DKO mice 48 h following Ad vector administration. Data are expressed as the mean ± SD (n = 4–6). *p < 0.05. (B) Liver sections of wild-type mice 48 h after Ad vector administration. H&E staining. Bar, 10 μm. (C) Apoptosis-related gene expression in the livers of wild-type mice 48 h following Ad vector administration. mRNA levels in the livers of PBS-injected mice were set as 1. Wild-type and Rag2/Il2rγ DKO mice were administered Ad-CAL2 or Ad-E4-122aT-CAL2 at 1 × 1011 VP per mouse. Forty-eight hours following vector administration, blood samples were collected via retro-orbital bleeding, and serum ALT levels were analyzed. Liver tissues were collected 48 h after administration and subjected to histological analysis and RT-qPCR analysis. Histological analysis was performed by H&E staining of tissue sections. Images are representative from at least two independent experiments. Data are expressed as the mean ± SD (n = 4–6). *p < 0.05. (D) Serum ALT levels in wild-type mice 10 d following Ad vector administration. Wild-type mice were administered Ad-CAL2 or Ad-E4-122aT-CAL2 at a dose of 1 × 1011 VP per mouse. Ten days following Ad vector administration, blood samples were collected via retro-orbital bleeding and used to determine serum ALT levels. The experiments were repeated at least twice. Data are expressed as the mean ± SD (n = 4–6). *p < 0.05.

To examine the involvement of immune cells in Ad vector–induced acute hepatotoxicity, serum ALT levels in Rag2/Il2rγc DKO mice, which lack T, B, and NK cells (21, 22), were measured following Ad vector administration. Significant elevations in serum ALT levels were not found in Rag2/Il2rγc DKO mice at 48 h following administration of Ad-CAL2 or Ad-E4-122aT-CAL2 (Fig. 1A), suggesting that immune cells are involved in early Ad vector–induced hepatotoxicity.

Cytokine expression in the spleen following Ad vector administration

To examine whether Ad vector–induced innate immune responses are involved in early-phase hepatotoxicity, mRNA levels of several cytokine genes, including IL-6, IL-12, IFN-γ, and IFN-β, in the spleens of wild-type and Rag2/Il2rγc DKO mice were determined 6 h following i.v. administration of Ad-CAL2 or Ad-E4-122aT-CAL2. Both vectors significantly elevated the mRNA levels of all cytokines examined to similar levels in the spleen (Fig. 2A). mRNA levels of these cytokines in the liver were also induced to comparable levels in Ad-CAL2– and Ad-E4-122aT-CAL2–injected mice (Fig. 2B); however, they were substantially lower than those in the spleen (data not shown). Serum IL-6 levels were increased to similar levels in mice that received Ad-CAL2 and those injected with Ad-E4-122aT-CAL2 (Fig. 2C). Spleen mRNA levels of all cytokines examined were significantly lower in Rag2/Il2rγc DKO mice than in wild-type mice at 6 h following administration of Ad-CAL2 or Ad-E4-122aT-CAL2 (Fig. 2A), likely because Rag2/Il2rγc DKO mice lack lymphocytes and NK cells. The functions of dendritic cells, which are largely involved in Ad vector–mediated inflammatory cytokine production (18, 31), are significantly impaired in Rag2/Il2rγc DKO mice (32). mRNA levels of IL-6, IFN-β, and IFN-γ in the spleens of Rag2/Il2rγc DKO mice following Ad-CAL2 or Ad-E4-122aT-CAL2 administration were not significantly different from the basal levels (Fig. 2A). These results suggest that, whereas Ad vector–induced cytokines are associated with early-phase Ad vector–mediated hepatotoxicity, they are not the sole determinant of early hepatotoxicity.

FIGURE 2.
FIGURE 2.

Cytokine levels following i.v. administration of Ad vectors. Cytokine mRNA levels in the spleen (A), liver (B), and (C) serum IL-6 levels following i.v. administration of Ad-CAL2 or Ad-E4-122aT-CAL2 in wild-type mice. Wild-type and Rag2/Il2rγ DKO mice were administered Ad-CAL2 and Ad-E4-122aT-CAL2 at 1 × 1011 VP per mouse. Cytokine mRNA levels in the spleen and liver and serum IL-6 levels were measured 6 h following vector administration by RT-qPCR and ELISA, respectively. Cytokine mRNA levels in wild-type mice following administration of Ad-CAL2 were set as 1. The experiments were repeated at least twice. Data are expressed as the mean ± SD (n = 4–6). N.S., not significant.

Leaky Ad gene expression in the liver during the early phase

The above results indicated that, whereas Ad-CAL2 and Ad-E4-122aT-CAL2 induced comparable levels of cytokine expression in the spleen and liver, early-phase hepatotoxicity induced by Ad-E4-122aT-CAL2 was significantly lower than that induced by Ad-CAL2. These findings led us to hypothesize that factors other than Ad vector–induced cytokine expression are involved. Therefore, we next focused on leaky Ad gene expression in the liver. E2A and E4 expression levels from Ad-E4-122aT-CAL2 in the liver were 7–9-fold lower than those from Ad-CAL2 at 48 h after administration in wild-type mice (Fig. 3A). Wild-type mice injected with Ad-E4-122aT-CAL2 showed ∼2-fold and 100-fold lower pIX and hexon mRNA levels in the liver than those injected with Ad-CAL2.

FIGURE 3.
FIGURE 3.

Leaky Ad gene expression levels in the liver following Ad vector administration. (A) Comparison of Ad gene expression levels in the liver between Ad-CAL2– and Ad-E4-122aT-CAL2–transduced wild-type mice. Ad gene mRNA levels in the livers of Ad-CAL2–treated mice were set as 1. (B) Comparison of Ad gene expression levels in the liver between Ad-CAL2–injected wild-type and Rag2/Il2rγ DKO mice. Ad gene mRNA levels in the livers of Ad-CAL2–treated wild-type mice were set as 1. Wild-type and Rag2/Il2rγ DKO mice were administered Ad-CAL2 and/or Ad-E4-122aT-CAL2 at 1 × 1011 VP per mouse. Liver tissues were collected 48 h after administration and subjected to RT-qPCR analysis. The experiments were repeated at least twice. Data are expressed as the mean ± SD (n = 4–6). *p < 0.05.

To determine whether Ad vector–induced cytokine levels are associated with leaky Ad gene expression levels, we examined the Ad gene expression levels in Rag2/Il2rγc DKO mice following Ad-CAL2 administration. Rag2/Il2rγc DKO mice exhibited significantly lower leaky Ad gene expression levels in the liver following i.v. administration of Ad-CAL2 than did wild-type mice. mRNA levels of E2A and hexon in the livers of Ad-CAL2–injected Rag2/Il2rγc DKO mice were 16–20-fold lower than those in wild-type mice, and those of E4 and pIX were 2.5–3.5-fold lower (Fig. 3B). We also examined the leaky Ad gene expression levels from Ad-E4-122aT-CAL2 in the livers of wild-type mice and Rag2/Il2rγc DKO mice. Rag2/Il2rγc DKO mice exhibited significantly lower leaky Ad gene expression levels (2–4-fold) in the liver following i.v. administration of Ad-E4-122aT-CAL2 than did wild-type mice (Supplemental Fig. 1). These results suggest that leaky Ad gene expression in the liver following systemic administration is correlated with the Ad vector–induced hepatotoxicity level during the early phase.

IL-6 exacerbates Ad vector–induced cytotoxicity in primary mouse hepatocytes by elevating leaky Ad gene expression

We hypothesized that Ad vector–induced cytokines promoted leaky Ad gene expression in hepatocytes, leading to Ad protein-induced hepatotoxicity during the early phase. To test this hypothesis, primary mouse hepatocytes were transduced with Ad-CAL2 or Ad-E4-122aT-CAL2 and then treated or not with rIL-6. IL-6 is an inflammatory cytokine that is strongly induced following Ad vector administration and is involved in liver toxicity (33–36). The concentrations of IL-6 added to primary mouse hepatocytes were comparable to or slightly higher than those in the serum of Ad vector–administered mice in this and previous studies (5, 18, 33). In the absence of IL-6, we found a significant elevation in the secreted LDH levels following transduction with Ad-CAL2 when compared with those in the mock group (Fig. 4A). Addition of IL-6 to the medium significantly increased LDH release by 1.8-fold in Ad-CAL2–transduced cells. LDH release levels gradually increased as the IL-6 concentration increased (Fig. 4B). IL-6 alone did not induce a significant elevation in LDH release. Ad-E4-122aT-CAL2 did not induce a significant elevation in LDH release in primary mouse hepatocytes, regardless of the presence of IL-6. The expression levels of several apoptosis-related genes, including Bim, Noxa, and p53-upregulated modulator of apoptosis (PUMA), were significantly higher in primary mouse hepatocytes treated with IL-6 and Ad-CAL2 than in mock-treated cells and those treated with IL-6 and Ad-E4-122aT-CAL2 (Fig. 4C). Neither IFN-β nor IFN-γ significantly exacerbated Ad vector–mediated cytotoxicity in primary hepatocytes following transduction with Ad-CAL2 at the concentrations used in this study (Supplemental Fig. 2). Furthermore, depletion of NK cells, which mainly produce IFN-γ (37, 38), by administration of anti–asialo GM1 Ab following Ad vector administration in mice did not reduce the serum ALT levels 48 h after administration of Ad-CAL2 when compared with control IgG treatment (Supplemental Fig. 3). This indicates that IL-6 but not IFN-β or IFN-γ exacerbate Ad vector–mediated cytotoxicity in hepatocytes.

FIGURE 4.
FIGURE 4.

Cytotoxicity and Ad gene expression in primary mouse hepatocytes following treatment with Ad vectors and rIL-6. (A) LDH levels in the culture medium following transduction with Ad vectors in primary mouse hepatocytes in the presence or absence of IL-6 (10 ng/ml). LDH levels in the culture medium of mock-treated cells in the absence of IL-6 were set as 100%. (B) Effects of IL-6 concentrations in the medium on Ad vector–mediated cytotoxicity in primary mouse hepatocytes. LDH levels in the culture medium of mock-treated cells in the absence of IL-6 were set as 100%. (C) Apoptosis-related gene expression in the primary mouse hepatocytes treated with IL-6 and Ad vectors. mRNA levels in mock-treated cells were normalized to 1. (D) Ad gene expression levels in the cells following transduction with Ad-CAL2 in the presence or absence of IL-6 (10 ng/ml). Ad gene expression levels in the cells treated with Ad-CAL2 in the absence of IL-6 were set as 1. Primary mouse hepatocytes were transduced with Ad-CAL2 and Ad-E4-122aT at 1000 VP per cell. rIL-6 was added to the culture medium 6 h after the addition of Ad vectors to the cells. Forty-eight hours following Ad vector transduction, LDH levels in the culture medium were determined. Expression levels of Ad genes and apoptosis-related genes were determined 24 h posttransduction using RT-qPCR. The experiments were repeated at least twice. Data are expressed as the mean ± SD (n = 4). *p < 0.05.

Next, leaky Ad gene expression levels in the presence or absence of IL-6 in primary mouse hepatocytes were examined following Ad vector transduction. Leaky expression of E2A, E4, pIX, and hexon from Ad-CAL2 was ∼2–3-fold elevated upon the addition of IL-6 (Fig. 4D). Ad-E4-122aT-CAL2 induced substantially lower E2A, E4, and hexon mRNA levels in primary hepatocytes than did Ad-CAL2. Treatment with IL-6 did not apparently upregulate Ad gene expression in Ad-E4-122aT-CAL2–transduced cells, although pIX expression was slightly enhanced by IL-6. IFN-β and IFN-γ did not significantly promote leaky Ad gene expression in primary hepatocytes following transduction with Ad-CAL2 at the concentrations used in this study (Supplemental Fig. 4). Mean Ad gene expression levels in cells treated with Ad-CAL2 and IFN-β were lower than those in cells treated with Ad-CAL2 alone. These results indicate that IL-6 promotes leaky Ad gene expression from a conventional Ad vector in hepatocytes, leading to hepatotoxicity, but not from Ad-E4-122aT-CAL2.

Inhibition of JAK/STAT signaling suppresses Ad vector–induced hepatotoxicity in mouse primary hepatocytes

To further examine the effects of IL-6 on Ad vector–induced hepatotoxicity, primary mouse hepatocytes were treated with Ad-CAL2 and IL-6 in the presence of a JAK inhibitor. JAK/STAT signaling is the major signal transduction pathway of cytokines, including IL-6 (35, 39, 40). As shown above, LDH release following transduction with Ad-CAL2 was significantly elevated upon addition of IL-6 (Fig. 5A). The JAK inhibitor significantly suppressed LDH release in the culture medium of Ad-CAL2–transduced cells in the presence of IL-6 when compared with that in mock-treated cells. No significant changes in LDH release levels were observed following treatment with IL-6 or IL-6/JAK inhibitor in Ad-E4-122aT-CAL2–transduced mouse primary hepatocytes.

FIGURE 5.
FIGURE 5.

JAK inhibition attenuates cytotoxicity and Ad gene expression in primary mouse hepatocytes following transduction with an Ad vector in the presence of IL-6. (A) LDH levels in the culture medium of cells pretreated with a JAK inhibitor (InSolution JAK Inhibitor I) following transduction with Ad-CAL2. LDH levels in the culture medium of mock-treated cells in the absence of JAK inhibitor or IL-6 were set as 100%. (B) Ad gene expression levels in cells pretreated with the JAK inhibitor following transduction with Ad-CAL2. Primary mouse hepatocytes were treated with the JAK inhibitor at a final concentration of 1 μM for 2 h. The cells were then transduced with Ad-CAL2 at 1000 VP per cell. rIL-6 was added to the culture medium at a final concentration of 10 ng/ml 6 h following the addition of Ad vectors to the cells. LDH levels in the culture medium were determined 48 h following Ad vector transduction. Ad gene expression levels were determined 24 h posttransduction using RT-qPCR. The experiments were repeated at least twice. Data are expressed as the mean ± SD (n = 4). *p < 0.05.

Next, we examined the effects of JAK inhibition on leaky Ad gene expression in primary mouse hepatocytes following transduction with Ad-CAL2. As shown above, expression levels of E2A, E4, pIX, and hexon in the presence of IL-6 were ∼3–9-fold higher than those in the absence of IL-6 (Fig. 5B). The JAK inhibitor significantly suppressed the expression levels of these genes. Ad gene expression levels in Ad-CAL2–transduced primary hepatocytes in the presence of IL-6 and the JAK inhibitor were comparable with those in cells treated with Ad-CAL2 in the absence of IL-6. These results indicate that inhibition of IL-6 signaling by a JAK inhibitor significantly suppresses leaky Ad gene expression in Ad vector–transduced primary mouse hepatocytes, leading to attenuation of Ad vector–mediated cytotoxicity.

To further examine the involvement of JAK/STAT signaling in Ad vector–mediated hepatotoxicity and leaky Ad gene expression, a dominant-negative form of STAT3 (STAT3F) was overexpressed in primary mouse hepatocytes. LDH release levels following cotransduction of a STAT3F-expressing Ad vector, Ad-AHA-STAT3F, and Ad-CAL2 were approximately one third of those following cotransduction of Ad-AHASEAP and Ad-CAL2 in primary hepatocytes (Fig. 6A). E2A and E4 mRNA levels in primary hepatocytes cotransduced with Ad-AHA-STAT3F and Ad-CAL2 were more than 30-fold lower than those in cells cotransduced with Ad-AHASEAP and Ad-CAL2 (Fig. 6B). These results indicate that inhibition of IL-6/JAK/STAT3 signaling suppresses leaky Ad gene expression in Ad vector–transduced hepatocytes, leading to reduction in Ad vector–mediated cytotoxicity.

FIGURE 6.
FIGURE 6.

Inhibition of STAT3 attenuates cytotoxicity and Ad gene expression in primary mouse hepatocytes and mouse liver following transduction with an Ad vector. (A) LDH levels in the culture medium 48 h following cotransduction with Ad-CAL2 and Ad-AHA-STAT3F. LDH levels in the culture medium of mock-treated cells in the absence of IL-6 were set as 100%. (B) Ad gene expression levels in primary mouse hepatocytes following cotransduction with Ad-CAL2 and Ad-AHA-STAT3F. Primary mouse hepatocytes were cotransduced with Ad-CAL2 and Ad-AHA-STAT3F or Ad-AHASEAP at 1000 VP per cell for each Ad vector. rIL-6 was added to the culture media at a final concentration of 10 ng/ml 6 h following the addition of Ad vectors to the cells. Ad gene expression levels were determined 24 h after transduction using RT-qPCR. The experiments were repeated at least twice. Data are expressed as the mean ± SD (n = 4). *p < 0.05.

Leaky Ad gene expression in the liver and Ad vector–mediated hepatotoxicities during the early phase are significantly reduced in IL-6 KO mice

To examine the involvement of IL-6 in leaky Ad gene expression in the liver and Ad vector–mediated hepatotoxicities in mice, IL-6 KO mice were i.v. administered Ad-CAL2. Serum ALT levels in IL-6 KO mice were 2.7-fold lower than those in wild-type mice 48 h following i.v. administration of Ad-CAL2 (Fig. 7A), indicating that IL-6 KO significantly reduced Ad-CAL2–induced hepatotoxicity during the early phase. RT-qPCR analysis demonstrated that leaky expression levels of the E2A and hexon genes in the livers of IL-6 KO mice were 11–25-fold lower than those in the livers of wild-type mice (Fig. 7B). mRNA levels of E4 and pIX also tended to be lower, albeit not significantly, in IL-6 KO mice. We did not find significant differences in luciferase expression in the livers of wild-type and IL-6 KO mice (Fig. 7C). These data indicate that IL-6 significantly induced leaky Ad gene expression in the liver, leading to hepatotoxicities during the early phase (Fig. 8).

FIGURE 7.
FIGURE 7.

Reduction in leaky Ad gene expression in the liver and Ad vector–mediated hepatotoxicities in IL-6 KO mice following i.v. Ad vector administration. (A) Serum ALT levels in wild-type and IL-6 KO mice 48 h following Ad vector administration. (B) Comparison of Ad gene expression levels in the liver between Ad-CAL2–injected wild-type and IL-6 KO mice. Ad gene mRNA levels in the livers of Ad-CAL­–treated wild-type mice were set as 1. (C) Luciferase expression levels in the livers of wild-type and IL-6 KO mice following i.v. administration of Ad-CAL2. Wild-type and IL-6 KO mice were administered Ad-CAL2 at 1 × 1011 VP per mouse. Forty-eight hours after vector administration, blood samples were collected via retro-orbital bleeding for the measurement of serum ALT levels. Liver tissues were collected 48 h after administration and subjected to RT-qPCR analysis. Luciferase expression levels in the livers were measured 48 h after administration. The experiments were repeated twice. Data are expressed as the mean ± SD (n = 4–5). *p < 0.05.

FIGURE 8.
FIGURE 8.

Schematic representation of the mechanism of early-phase Ad vector–mediated hepatotoxicity.

Discussion

This study demonstrated that the Ad-E4-122aT-CAL2 vector caused significantly lower hepatotoxicity during not only the late but also the early phase than a conventional Ad vector, Ad-CAL2, despite inducing comparable levels of cytokine expression following i.v. administration. The conventional Ad vector induced significantly lower cytokine expression in the spleen, hepatotoxicity, and leaky Ad gene expression in the liver in Rag2/Il2rγc DKO mice than in wild-type mice. rIL-6 promoted leaky Ad gene expression and Ad vector–mediated cytotoxicity in Ad-CAL2–transduced hepatocytes. Leaky Ad gene expression in the liver and hepatotoxicity during the early phase were reduced in IL-6 KO mice when compared with wild-type mice following Ad-CAL2 administration. Based on these findings, we suggest the following mechanisms (Fig. 8): 1) conventional Ad vectors induce IL-6 production via the activation of innate immune responses, primarily in the spleen, and IL-6 upregulates leaky Ad gene expression from the vector genome in the liver, resulting in hepatotoxicity during the early phase; 2) Ad-E4-122aT-CAL2, whereas inducing a similar level of IL-6 expression in the spleen as a conventional Ad vector, induces significantly lower leaky Ad gene expression in the liver (even in the presence of IL-6) because of miR-122a–mediated posttranscriptional gene silencing, resulting in less vector-mediated hepatotoxicity in the early phase. A helper-dependent Ad vector that lacked nearly all viral genes but possessed the dsDNA genome and capsid proteins reportedly induced similar levels of inflammatory cytokine expression in mice as a conventional vector (20, 41), which is in line with our findings. Nonetheless, helper-dependent Ad vector–induced serum ALT levels during the early phase were lower than those induced by a conventional Ad vector (3, 20, 42, 43). These findings indicate that Ad vector–induced inflammatory cytokines are not the sole cause of early Ad vector–mediated hepatotoxicity.

Among the various types of cytokines induced by Ad vector–induced innate immune responses, we focused on the effects of IL-6 on Ad vector–mediated hepatotoxicity. IL-6, a multifunctional cytokine with pleiotropic effects on inflammation, immune responses, and hematopoiesis (36), is an inflammatory cytokine that is primarily induced via innate immune responses following Ad vector transduction (5, 33). Previous studies have reported that various Ad components, including Ad genome and capsid proteins, were involved in Ad-induced IL-6 production via innate immunity following in vivo application (31, 44). We have demonstrated that IL-6 significantly enhanced leaky Ad gene expression and Ad vector–mediated cytotoxicity in primary mouse hepatocytes following transduction with a conventional Ad vector (Fig. 4C). Leaky Ad gene expression levels in the liver and hepatotoxicities following administration of a conventional Ad vector were lower in IL-6 KO mice than in wild-type mice (Fig. 7). These data indicated that IL-6 is crucial for Ad vector–induced hepatotoxicity during the early phase. Previous studies have reported that IL-6 enhanced E1B, E2A, and penton base gene expression from E1A-deleted Ad (45) and replication and progeny production of an E1A-deleted Ad in tumor cell lines (46). An IL-6–regulated human NF of the C/EBP family (NF-IL6) transactivated E1A-responsive promoters (47). Although both E1A and E1B were deleted from the Ad vectors used in this study, NF-IL6 would induce Ad gene transcription in an E1-independent manner.

Addition of rIL-6 to Ad-CAL2–transduced mouse primary hepatocytes resulted in significant transcriptional upregulation of all Ad genes examined, indicating that IL-6 was involved in leaky Ad gene expression in the hepatocytes (Fig. 4D). Meanwhile, there were no significant differences in the leaky expression levels of E4 or pIX in the livers of IL-6 KO and wild-type mice, despite the significant reductions in E2A and hexon mRNA levels in IL-6 KO mice (Fig. 7B). Not only IL-6 but also other inflammatory cytokines would be involved in leaky expression of E4 and pIX in the liver.

In addition to IL-6, several types of cytokines, including IFN-γ and IL-12, are induced via innate immune responses following Ad vector transduction (6, 18, 20, 33, 48–50). Initially, we hypothesized that IFN-γ might play a crucial role in early vector-induced hepatotoxicity because it has been found to be involved in several types of hepatotoxicity in previous studies (51–53). IFN-γ is a member of type II IFN class and is mainly secreted by T and NK/NKT cells (54). In particular, NK cells are crucial for IFN-γ production in mice following systemic administration of an Ad vector (37). However, NK cell depletion using anti–asialo GM1 Ab did not suppress serum ALT levels following the administration of a conventional Ad vector when compared with control IgG treatment (Supplemental Fig. 3). In addition, IFN-γ did not enhance LDH release in Ad-CAL2–transduced primary mouse hepatocytes (Supplemental Fig. 2). These findings indicate that IFN-γ contributes less to early-phase Ad vector–induced hepatotoxicity than does IL-6.

The conventional Ad vector induced significant cytotoxicity in primary mouse hepatocytes in the presence of IL-6, suggesting that the early-phase hepatotoxicity was directly induced by Ad gene products, including E2A and E4, not by immune-cell attack against the hepatocytes. Previous studies have reported that leaky expression of Ad proteins, including E4, directly induced cytotoxicity in Ad vector–transduced cells (10, 13–17). Ad-CAL2 transduction resulted in higher expression levels of several apoptosis-related genes in the mouse liver (Fig. 1C) and in primary mouse hepatocytes treated with IL-6 (Fig. 4C) than Ad-E4-122aT-CAL2 transduction, indicating that apoptosis is involved in Ad protein-induced cytotoxicity. Apoptosis involvement has been also reported in previous studies (10, 19, 20, 55); however, the precise mechanism of Ad protein-mediated hepatotoxicity remains to be elucidated.

In summary, we have examined how Ad-E4-122aT-CAL2 causes less hepatotoxicity during the early phase following Ad vector transduction than does a conventional Ad vector. The findings in this study indicated that, whereas the expression levels of several cytokines, including IL-6, were comparable between Ad-E4-122aT-CAL2– and Ad-CAL2–transduced mice, in the latter, IL-6, which is elicited via Ad vector–induced innate immunity, induced leaky Ad gene expression in hepatocytes in a JAK/STAT3 signaling-dependent manner, leading to acute hepatotoxicity, whereas in the former, leaky Ad gene expression induced by IL-6 was substantially lower, leading to low hepatotoxicity. This study indicates that leaky Ad gene expression is involved in Ad vector–mediated hepatotoxicity during not only the late but also the early phase and that Ad-E4-122aT is a promising vector that induces less hepatotoxicity during both the early and the late phase for gene therapy and basic research. This study provides important contributions to the improvement of Ad vector–based gene therapy.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Kazuo Ohashi, Dr. Emiko Kasahara, Sayuri Okamoto, and Eri Hosoyamada (Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan) for help. The plasmid encoding a dominant-negative form of STAT3 (STAT3F) (pEF-BOS-FLAG-STAT3 [Y705F]) was gifted by Dr. Shizuo Akira (Osaka University, Osaka, Japan). pBS-ApoEHCR-hAATp-hFIX-Int-bpA was a gift of Dr. Mark A. Kay (Stanford University, Stanford, CA). The human cytomegalovirus immediate-early enhancer/β-actin promoter with β-actin intron (CA promoter) was provided by Dr. Jun-ichi Miyazaki (Osaka University, Osaka, Japan).

Footnotes

  • This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI (grants 23689010, 17H00863, JP15K18939, and JP18K14964), the Japan Agency for Medical Research and Development (Grant 17am0101084j0001), grants from the Ministry of Health, Labor and Welfare of Japan, Osaka Ohtani University, the Sumitomo Electric Industries Group Corporate Social Responsibility Foundation, and Takara Bio. K.S. and S.I. were Research Fellows of the JSPS.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    Ad
    adenovirus
    ALT
    alanine aminotransferase
    DKO
    double-knockout
    KO
    knockout
    LDH
    lactate dehydrogenase
    RT-qPCR
    quantitative RT-PCR
    VP
    virus particle.

  • Received July 23, 2020.
  • Accepted November 4, 2020.

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