- Split View
-
Views
-
Cite
Cite
Tung Nguyen, Jianliang Xu, Shunsuke Chikuma, Hiroshi Hiai, Kazuo Kinoshita, Kyoji Moriya, Kazuhiko Koike, Gian Paolo Marcuzzi, Herbert Pfister, Tasuku Honjo, Maki Kobayashi, Activation-induced cytidine deaminase is dispensable for virus-mediated liver and skin tumor development in mouse models, International Immunology, Volume 26, Issue 7, July 2014, Pages 397–406, https://doi.org/10.1093/intimm/dxu040
- Share Icon Share
Abstract
Activation-induced cytidine deaminase (AID) not only promotes immune diversity by initiating somatic hypermutation and class switch recombination in immunoglobulin genes but also provokes genomic instability by introducing translocations and mutations into non-immunoglobulin genes. To test whether AID is essential for virus-induced tumor development, we used two transgenic tumor models: mice expressing hepatitis C virus (HCV) core proteins (HCV-Tg), driven by the hepatitis B virus promoter, and mice expressing human papillomavirus type 8 proteins (HPV8-Tg), driven by the Keratin 14 promoter. Both strains were analyzed in the absence and presence of AID by crossing each with AID−/− mice. There was no difference in the liver tumor frequency between the HCV-Tg/AID+/+ and HCV-Tg/AID−/− mice at 20 months of age although the AID+/+ mice showed more severe histological findings and increased cytokine expression. Furthermore, a low level of AID transcript was detected in the HCV-Tg/AID+/+ liver tissue that was not derived from hepatocytes themselves but from intra-hepatic immune cells. Although AID may not be the direct cause of HCV-induced oncogenesis, AID expressed in B cells, not in hepatocytes, may prolong steatosis and cause increased lymphocyte infiltration into HCV core protein-induced liver lesions. Similarly, there was no difference in the time course of skin tumor development between the HPV8-Tg/AID−/− and HPV8-Tg/AID+/+ groups. In conclusion, AID does not appear to be required for tumor development in the two virus-induced tumor mouse models tested although AID expressed in infiltrating B cells may promote inflammatory reactions in HCV core protein-induced liver pathogenesis.
Introduction
Activation-induced cytidine deaminase (AID) is essential for inducing DNA breaks during the somatic hypermutation and class switch recombination of immunoglobulin genes required for generating antibody diversity in activated B cells (1). AID generates physiological mutations during deliberate antibody development, but can also cause chromosomal translocations and mutations in proto-oncogenes when expressed aberrantly (2). Transgenic, ubiquitous over-expression of AID causes T-cell lymphoma and micro-adenoma in the lung (3) along with mutations in the TCR and c-myc genes. Chronic infections with micro-organisms such as helicobacter pylori (4), hepatitis C virus (HCV) (5–7), and human T-cell leukemia virus type 1 (8) induce the aberrant AID expression, which has been proposed to cause tumors by introducing translocations and somatic mutations into proto-oncogenes. In addition, AID expression is associated with chronic infections of these pathogens in human cases, in which it has also been proposed to contribute to tumor formation at least in part (4, 9). However, it has not been directly determined if virus- or bacteria-induced oncogenesis requires the action of AID.
Hepatocellular carcinoma (HCC) is the fifth most frequent cancer, and hepatitis B virus (HBV) and HCV infections are the major risk factors for developing this cancer worldwide (10). In fact the risk of developing HCC is increased 11.5- to 17-fold in HCV-infected patients; however, antiviral therapies have limited effectiveness in only a small fraction of patients. Thus, elucidation of the mechanism(s) involved in promoting liver tumorigenesis is urgently required for developing a prevention strategy. As natural infection of HCV is restricted to humans and chimpanzees, several transgenic mouse models harboring parts of the HCV polyprotein have been generated to recapitulate HCC development (11). HCV, a small RNA virus, belongs to the Flaviviridae family and contains a 9.6-kb single-stranded RNA genome. The polyprotein encoded by the HCV genome is processed into the structural proteins (including core, E1 and E2) and the non-structural (NS) proteins (NS2-NS5) required for RNA genome replication by host and viral proteases (11). Among them, the HCV core protein has unique, multifunctional roles in apoptosis, signal transduction, reactive oxygen species formation, transformation and immune modulation (such as the up-regulation of TGF-β) (10) by interacting with many cellular proteins. Out of the 14 lines of HCV-transgenic mice developed, 5 HCV core protein-containing transgenic lines and 1 non-structural (NS5A) transgenic line can give rise to HCC, after the development of severe steatosis, a characteristic pathology associated with HCV infection (11–13). Especially, a transgenic mouse model expressing HCV core protein (HCV-Tg) driven HBV regulatory elements (12), which limits HCV core protein expression strictly in hepatocytes, had the highest HCC prevalence among many HCV protein transgenic model mice (11, 13); therefore, this model mouse line seemed to be a good tool to investigate AID expression in hepatocytes and its contribution to the mechanism of HCC development.
On the other hand, the human papillomavirus (HPV) family of small DNA tumor viruses, of which there are over 120 types, can cause hyper-proliferative lesions in cutaneous and mucosal epithelia (14). Among them, HPV5 and HPV8 are classified as high-risk beta-type papilloma viruses and are the two major causes of cutaneous squamous cell carcinoma (SCC) in epidermodysplasia verruciformis patients. The development of SCC by HPV8 is promoted by a series of carcinogenic events, including DNA damage, evasion of apoptosis, mutation mediated by E6 protein and enhanced proliferation after ultraviolet light B exposure, mediated by E7 protein (14). Unlike HCV infection, HPV8 does not cause severe inflammation; however, DNA damage is an important carcinogenic process associated with this virus. A transgenic mouse model in which HPV8 early genes are expressed under the control of the Keratin 14 (K14) promoter (15) exhibits significant papilloma development (up to 91% of the HPV8-Tg mice) and malignant progression (6% of the HPV8-Tg mice backcrossed to FVB/N).
To determine whether AID is aberrantly induced by HCV or HPV8 and required for virally induced tumorigenesis, we crossed HCV-Tg or HPV8-Tg mice with AID−/− mice and compared the tumorigenesis frequencies in the AID+/+ and AID−/− mice. We also examined the AID expression levels in the affected tissues. We found that HCV-Tg mice exhibited enhanced AID expression in the B cells infiltrating the liver, and that the steatosis and lymphocytic follicle formation were more severe in the HCV-Tg/AID+/+ than in the HCV-Tg/AID−/− mice. However, the HCC prevalence at 20 months of age was not remarkably different between the two groups. Similarly, the time course of papilloma development was indistinguishable between the HPV8-Tg/AID+/+ and HPV8-Tg/AID−/− mice. Furthermore, AID expression was not induced in the skin papillomatous tissues of the HPV8-Tg/AID+/+ mice. We conclude that AID is not necessary for the viral protein-induced oncogenesis in these two mouse models.
Methods
Mouse maintenance and genotyping
All the mice used in this study were maintained at the Institute of Laboratory Animals in accordance with the guidelines of the Animal Research Committee, Graduate School of Medicine, Kyoto University. AID−/− mice (16) backcrossed to C57/B6 (B6) were crossed with HCV core protein transgenic mouse line (HCV-Tg) (12, 13) and HPV8-Tg mice (on an FVB/N background) (15). AID-Cre and Rosa-RFP compound mice (17) were crossed with HCV-Tg mice to enable the detection of previous and current AID expression. The genotyping primers are described in Supplementary Table 1, available at International Immunology Online.
Western blotting
Western blotting was performed by conventional methods. Mouse organs were dissected and homogenized in RIPA buffer. The primary antibodies used were the rat monoclonal anti-mouse AID antibody 2 (MAID-2) (eBioscience, San Diego, CA, USA), anti-Tubulin antibody (Calbiochem, MERCK, Darmstadt, Germany) and anti-HCV core protein antibody (clone B2, Yes Biotech Lab, Anogen, Ontario, Canada).
Reverse transcription–PCR and quantitative reverse transcription–PCR
Mouse organs were excised and homogenized in Sepasol RNA I Super (Nacalai Tesque, Kyoto, Japan) following the manufacturer’s instructions. Reverse transcription–PCR (RT–PCR) was performed as previously described (18). ExTaq DNA polymerase (TaKaRa, Shiga, Japan) and the primers described in Supplementary Table 2, available at International Immunology Online, were used for conventional PCR. Real-time PCR was performed with the primer sets in Supplementary Table 2, available at International Immunology Online, and the Power SYBR Green PCR Master Mix (ABI, Life Technologies Japan, Tokyo, Japan) using the ABI 7900HT system (ABI). The delta-delta Ct method was used to calculate the fold change in gene expression. Error bars show the standard deviation.
Histology and immunohistochemistry
For AID immunohistochemical staining, freshly excised livers were fixed in 4% paraformaldehyde and processed for frozen section as previously described (19). AID protein was detected by MAID-2 and peroxidase-labeled donkey F(ab′)2 anti-rat IgG (Jackson ImmunoResearch, West Grove, PA, USA) and stained with diaminobenzidine. Images were captured with a DM5000B microscope (Leica; Wetzlar, Germany). Hematoxylin and eosin (H&E)-stained samples were fixed with Mildform 10N (Wako Pure Chemical Industries, Osaka, Japan), embedded in paraffin and stained by standard methods.
Liver cell fractionation and FACS analysis
The isolation of intra-hepatic immune cells (IHICs) from the liver of HCV-Tg mice was performed as previously described, with some minor modifications (20). The composition of IHIC cells was assessed by staining with the following antibodies: PE-labeled anti-mouse B220 for B cells, allophycocyanin (APC)-conjugated anti-mouse CD11b for macrophages and FITC-labeled anti-mouse CD8 and APC-anti-mouse CD4 for T cells. The stained cells were analyzed on a FACSCalibur (BD Japan, Tokyo).
ELISA
TNF-α, IL-1β and TGF-β were detected using ELISA kits specific for each cytokine (BioSource, Life Technologies), according to the manufacturer’s instructions.
Statistical analysis
The Mann–Whitney U-test was used to calculate the statistical differences in AID expression (Fig. 1B). Fisher’s exact test was used to determine significant differences in tumor incidence (Table 3). Student’s t-test was used to determine significant differences in cytokine expression (Fig. 3B and C), and Welch’s t-test was used for pathological severity validation (Table 2). P values < 0.05 were considered statistically significant.
. | Genotype of AID . | Severity score . | ||||
---|---|---|---|---|---|---|
0 . | 1 . | 2 . | 3 . | Average . | ||
Steatosis in HCV(+) | ||||||
12 months | −/− | 0 | 0 | 0 | 3 | 3 |
+/+ | 0 | 0 | 0 | 2 | 3 | |
16 months | −/− | 2 | 8 | 2 | 0 | 1.00* |
+/+ | 0 | 8 | 4 | 0 | 1.33 | |
20 months | −/− | 9 | 7 | 1 | 4 | 1.00* |
+/+ | 3 | 12 | 2 | 4 | 1.33 | |
Lymphoid follicle in HCV(+) | ||||||
16 months | −/− | 9 | 3 | 0 | 0 | 0.25* |
+/+ | 5 | 7 | 0 | 0 | 0.58 | |
20 months | −/− | 4 | 13 | 1 | 3 | 1.14* |
+/+ | 2 | 11 | 4 | 4 | 1.48 |
. | Genotype of AID . | Severity score . | ||||
---|---|---|---|---|---|---|
0 . | 1 . | 2 . | 3 . | Average . | ||
Steatosis in HCV(+) | ||||||
12 months | −/− | 0 | 0 | 0 | 3 | 3 |
+/+ | 0 | 0 | 0 | 2 | 3 | |
16 months | −/− | 2 | 8 | 2 | 0 | 1.00* |
+/+ | 0 | 8 | 4 | 0 | 1.33 | |
20 months | −/− | 9 | 7 | 1 | 4 | 1.00* |
+/+ | 3 | 12 | 2 | 4 | 1.33 | |
Lymphoid follicle in HCV(+) | ||||||
16 months | −/− | 9 | 3 | 0 | 0 | 0.25* |
+/+ | 5 | 7 | 0 | 0 | 0.58 | |
20 months | −/− | 4 | 13 | 1 | 3 | 1.14* |
+/+ | 2 | 11 | 4 | 4 | 1.48 |
The severity of steatosis and lymphoid follicle formation is classified as: 0 (none), 1 (mild), 2 (moderate) or 3 (severe). Values are the numbers of mice with each score.
*AID−/− versus AID+/+, P > 0.05 by Welch’s t-test.
. | Genotype of AID . | Severity score . | ||||
---|---|---|---|---|---|---|
0 . | 1 . | 2 . | 3 . | Average . | ||
Steatosis in HCV(+) | ||||||
12 months | −/− | 0 | 0 | 0 | 3 | 3 |
+/+ | 0 | 0 | 0 | 2 | 3 | |
16 months | −/− | 2 | 8 | 2 | 0 | 1.00* |
+/+ | 0 | 8 | 4 | 0 | 1.33 | |
20 months | −/− | 9 | 7 | 1 | 4 | 1.00* |
+/+ | 3 | 12 | 2 | 4 | 1.33 | |
Lymphoid follicle in HCV(+) | ||||||
16 months | −/− | 9 | 3 | 0 | 0 | 0.25* |
+/+ | 5 | 7 | 0 | 0 | 0.58 | |
20 months | −/− | 4 | 13 | 1 | 3 | 1.14* |
+/+ | 2 | 11 | 4 | 4 | 1.48 |
. | Genotype of AID . | Severity score . | ||||
---|---|---|---|---|---|---|
0 . | 1 . | 2 . | 3 . | Average . | ||
Steatosis in HCV(+) | ||||||
12 months | −/− | 0 | 0 | 0 | 3 | 3 |
+/+ | 0 | 0 | 0 | 2 | 3 | |
16 months | −/− | 2 | 8 | 2 | 0 | 1.00* |
+/+ | 0 | 8 | 4 | 0 | 1.33 | |
20 months | −/− | 9 | 7 | 1 | 4 | 1.00* |
+/+ | 3 | 12 | 2 | 4 | 1.33 | |
Lymphoid follicle in HCV(+) | ||||||
16 months | −/− | 9 | 3 | 0 | 0 | 0.25* |
+/+ | 5 | 7 | 0 | 0 | 0.58 | |
20 months | −/− | 4 | 13 | 1 | 3 | 1.14* |
+/+ | 2 | 11 | 4 | 4 | 1.48 |
The severity of steatosis and lymphoid follicle formation is classified as: 0 (none), 1 (mild), 2 (moderate) or 3 (severe). Values are the numbers of mice with each score.
*AID−/− versus AID+/+, P > 0.05 by Welch’s t-test.
Age . | 16 months . | 20 months . | ||
---|---|---|---|---|
AID genotype | −/− | +/+ | −/− | +/+ |
Male/Female | 0/15 | 0/15 | 21/0 | 21/0 |
Tumour | 0 | 0 | 3 | 4 |
Malignancy | 0 | 0 | 2 | 4* |
Age . | 16 months . | 20 months . | ||
---|---|---|---|---|
AID genotype | −/− | +/+ | −/− | +/+ |
Male/Female | 0/15 | 0/15 | 21/0 | 21/0 |
Tumour | 0 | 0 | 3 | 4 |
Malignancy | 0 | 0 | 2 | 4* |
*AID−/− versus AID+/+, P > 0.05 by Fisher’s exact test.
Age . | 16 months . | 20 months . | ||
---|---|---|---|---|
AID genotype | −/− | +/+ | −/− | +/+ |
Male/Female | 0/15 | 0/15 | 21/0 | 21/0 |
Tumour | 0 | 0 | 3 | 4 |
Malignancy | 0 | 0 | 2 | 4* |
Age . | 16 months . | 20 months . | ||
---|---|---|---|---|
AID genotype | −/− | +/+ | −/− | +/+ |
Male/Female | 0/15 | 0/15 | 21/0 | 21/0 |
Tumour | 0 | 0 | 3 | 4 |
Malignancy | 0 | 0 | 2 | 4* |
*AID−/− versus AID+/+, P > 0.05 by Fisher’s exact test.
Results
Increased AID transcripts in the liver of HCV-Tg mice
To generate the observation groups, HCV-Tg mice were crossed with AID−/− mice. Then HCV-Tg/AID+/−(HCV(+)AID+/−) mice from the first filial generation were again crossed each other and the obtained HCV(+)AID−/− and HCV(+)AID+/+ mice of the second filial generation were compared as the observation groups. The expression level of the HCV core protein in the liver was similar in the HCV(+)AID−/− and HCV(+)AID+/+ mice (Supplementary Figure 1, available at International Immunology Online). Because HCV-Tg mice develop severe steato-hepatitis within 9 months after birth (12), and this chronic inflammation is supposed to reproduce the similar cytokine environment to the TNF-α-stimulated hepatocyte cell lines that express AID (9), we examined AID expression in the liver. AID transcripts were detected in the liver from 16-month-old HCV(+)AID+/+ mice, but not from wild-type B6 mice (Fig. 1A, Supplementary Figure 2, available at International Immunology Online). However, the AID expression detected in the HCV(+)AID+/+ liver was comparable to the low levels observed in primary unstimulated spleen cells, which included B and T lymphocytes. Transcripts for CD19, a specific marker for B lymphocytes, were also higher in the HCV(+) compared with the B6 liver, suggesting that the HCV(+) liver may contain a considerable number of B lymphocytes, which could contribute to the increased AID expression. Real-time PCR analysis revealed that the AID transcript level in the liver from 16-month-old HCV(+)AID+/+ females was 15-fold greater than that from the liver of similarly aged B6 mice and of 12-month-old HCV(+)AID+/+ males (Fig. 1B).
The AID protein levels were measured in the same liver samples by western blotting (Fig. 1C). However, using MAID-2, no protein signal could be detected in the same samples that contained AID transcripts (Fig. 1A). To quantify the limitation of AID protein detection by MAID-2, we used spleen cells as a control (Supplementary Figure 3, available at International Immunology Online). We assigned one arbitrary unit of AID mRNA to the q-PCR signal detected from 500ng of naive spleen cell RNA. Extracts prepared from the same number of spleen cells contained 8.6 μg protein, which did not elicit a detectable AID signal in the western blot. Since the AID transcript level from the HCV(+)AID+/+ samples in Supplementary Figure 2, available at International Immunology Online, was lower than that in naive spleen cells, we conclude that the AID protein signal in the HCV(+)AID+/+ liver was below the level detected by MAID2.
We next explored the possibility that the AID protein expression was limited to a specific location in the liver, such as the immune cells in the hepatic blood vessels. We therefore performed an immunohistochemical analysis of AID (Fig. 1D). Although positive controls including liver tissue from CAG-promoter-driven AID transgenic mice (3) and Peyer’s patches from B6 wild-type mice showed clear brownish signals, there was no signal detected in any part of the HCV(+)AID+/+ liver tissue samples.
AID transcripts are detected in IHICs, but not in hepatocytes from HCV-Tg mice
We next explored the possibility that the low level of AID transcripts was contributed by B cells infiltrating the HCV-Tg liver. Liver cells from three HCV(+)AID−/− or HCV(+)AID+/+ mice at 16 months of age were fractionated to separate the IHICs from the hepatocytes (Fig. 2A and B). RNA was purified from both fractions of each genotype, and the AID, CD19 and albumin transcripts were analyzed to confirm the purity of these fractions and to identify the cellular origin of the AID mRNA (Table 1). CD19 transcripts were detected almost exclusively in IHICs, while albumin transcripts were mostly in hepatocytes, validating the fractionation procedure. The level of AID transcripts detected in the HCV(+)AID+/+ IHICs was comparable to the level observed in splenic B cells (132.6±11.0 versus 338.6±11.7, respectively), while the level in hepatocytes was much lower, indicating that the source of AID transcripts in the liver was not the hepatocytes themselves but the IHICs. Cell surface marker analysis by FACS revealed that the IHICs consisted of B220+ B cells (25–29%), CD4+ or CD8+ T cells (~45%) and CD11b+ cells (7–10%), and that this composition was not notably changed by the presence of the HCV transgene or the AID genotype at 16 months of age (Fig. 2A, Supplementary Table 3, available at International Immunology Online).
. | CD19/18s rRNA . | Albumin/18s rRNA . | AID/18s rRNA . | |||||
---|---|---|---|---|---|---|---|---|
IHICs . | Hepatocytes . | Spleen B cells . | IHICs . | Hepatocytes . | IHICs . | Hepatocytes . | Spleen B cells . | |
B6 | 326.6 | 3.3 | 920.0 | 1.0 | 1171.3 | 7.0±0.30 | 1.0 | 338.6±11.7 |
HCV(+)AID−/− | 306.1 | 1.3 | 0.27 | 1082.0 | 0.0 | 0.0 | ||
HCV(+)AID+/+ | 360.6 | 1.0 | 10.6 | 1027.2 | 132.6±11.0 | 3.2±0.63 |
. | CD19/18s rRNA . | Albumin/18s rRNA . | AID/18s rRNA . | |||||
---|---|---|---|---|---|---|---|---|
IHICs . | Hepatocytes . | Spleen B cells . | IHICs . | Hepatocytes . | IHICs . | Hepatocytes . | Spleen B cells . | |
B6 | 326.6 | 3.3 | 920.0 | 1.0 | 1171.3 | 7.0±0.30 | 1.0 | 338.6±11.7 |
HCV(+)AID−/− | 306.1 | 1.3 | 0.27 | 1082.0 | 0.0 | 0.0 | ||
HCV(+)AID+/+ | 360.6 | 1.0 | 10.6 | 1027.2 | 132.6±11.0 | 3.2±0.63 |
Relative AID/18s rRNA expression in B6 hepatocyte is set as 1.0, relative CD19/18s rRNA in HCV(+)AID+/+ as 1.0 and relative Albumin/18s rRNA in B6 as 1.0. Zero means undetectable signal by q-PCR.
. | CD19/18s rRNA . | Albumin/18s rRNA . | AID/18s rRNA . | |||||
---|---|---|---|---|---|---|---|---|
IHICs . | Hepatocytes . | Spleen B cells . | IHICs . | Hepatocytes . | IHICs . | Hepatocytes . | Spleen B cells . | |
B6 | 326.6 | 3.3 | 920.0 | 1.0 | 1171.3 | 7.0±0.30 | 1.0 | 338.6±11.7 |
HCV(+)AID−/− | 306.1 | 1.3 | 0.27 | 1082.0 | 0.0 | 0.0 | ||
HCV(+)AID+/+ | 360.6 | 1.0 | 10.6 | 1027.2 | 132.6±11.0 | 3.2±0.63 |
. | CD19/18s rRNA . | Albumin/18s rRNA . | AID/18s rRNA . | |||||
---|---|---|---|---|---|---|---|---|
IHICs . | Hepatocytes . | Spleen B cells . | IHICs . | Hepatocytes . | IHICs . | Hepatocytes . | Spleen B cells . | |
B6 | 326.6 | 3.3 | 920.0 | 1.0 | 1171.3 | 7.0±0.30 | 1.0 | 338.6±11.7 |
HCV(+)AID−/− | 306.1 | 1.3 | 0.27 | 1082.0 | 0.0 | 0.0 | ||
HCV(+)AID+/+ | 360.6 | 1.0 | 10.6 | 1027.2 | 132.6±11.0 | 3.2±0.63 |
Relative AID/18s rRNA expression in B6 hepatocyte is set as 1.0, relative CD19/18s rRNA in HCV(+)AID+/+ as 1.0 and relative Albumin/18s rRNA in B6 as 1.0. Zero means undetectable signal by q-PCR.
To detect both current and past AID expression, transgenic reporter mice expressing tdRFP under the control of BAC-AID-Cre (17) were crossed to HCV-Tg mice (Fig. 2B and C, Supplementary Table 3, available at International Immunology Online). This mouse line reveals tdRFP fluorescence in any cells that have (or had) expressed AID. Using this approach, tdRFP+ cells were found to represent 3–4% of the total IHICs in both HCV-Tg and no HCV-Tg (HCV(−)) mice at 3 months of age (Fig. 2B). tdRFP was not detected in hepatocytes from 3-month-old HCV(+) mice that had already developed mild steato-hepatitis (12). Analysis of three mice of each genotype (HCV(+) and HCV(–)) revealed that the tdRFP+ cells represented 5.8±0.45% versus 6.2±1.0% of the B220+ population, respectively (Fig. 2C), indicating that the presence of the HCV transgene did not affect the AID gene expression in mice during the first 3 months of life.
AID deficiency reduces the severity of histopathological phenotypes and cytokine expression profiles in the liver of HCV-Tg mice
It is reported that 14–30% of HCV-Tg male mice develop HCC between 16 and 19 months of age (13). We therefore analyzed the histopathological phenotypes of H&E-stained liver sections from HCV(+)AID−/− and HCV(+)AID+/+ mice at 12 (male), 16 (female) and 20 months (male) of age (Figs 3A and 4A and Table 2). The severities of steatosis and lymphocyte infiltration were graded from 0 to 3, and the average scores for each group were calculated and tested by Welch’s t-test (Table 2).
At 12 months of age, both HCV(+)AID−/− and HCV(+)AID+/+ male mice had developed severe steatosis. Unexpectedly, the steatosis was milder in both HCV(+)AID−/− and HCV(+)AID+/+ female mice at 16 months of age than HCV(+)AID−/− and HCV(+)AID+/+ male mice at 12 months of age. The steatosis in the HCV(+)AID+/+ mice appeared to be more severe than in HCV(+)AID−/− mice, but the difference was not significant (P > 0.05). This decrease in steatosis severity may have been due to the female composition of the mice because females of this HCV-Tg line did not show tumor development (13), and human HCV-infected cirrhotic females develop HCC less frequently (21). In addition, lymphoid follicle formation was apparent at 16 months of age and was more frequent in the HCV(+)AID+/+ than the HCV(+)AID−/− mice (Fig. 3A; Table 2). Consistent with the previous report (13), the fibrotic or regenerative nodular changes were very mild at 16 months of age.
However, the liver samples from both HCV(+)AID−/− and HCV(+)AID+/+ mice at 20 months of age revealed marked progressive changes, with nuclear atypia detected in 10 out of 21 of each genotype, and liver cell degeneration or regenerative changes detected in 10 and 11 out of 21 HCV(+)AID−/− and HCV(+)AID+/+ mice, respectively (Fig. 4A). Interestingly, both the steatosis and lymphoid follicle severity scores appeared to be higher in the HCV(+)AID+/+ than the HCV(+)AID−/− 20-month-old mice (1.33 versus 1.00 for steatosis and 1.48 versus 1.14 for lymphoid follicles, respectively) although the observed differences were not statistically significant (Table 2).
We next examined whether the more advanced inflammatory histological phenotypes observed in the HCV(+)AID+/+ mice were associated with increases in cytokine expression. Since cytokine production levels are altered in chronic HCV hepatitis (22) and in HCV-Tg mouse (23), we used q-PCR to measure representative pro-inflammatory Th1 and Th2 cytokine expression levels in the livers of 16-month-old female mice (Fig. 3B). The presence of the HCV transgene was associated with significantly higher levels of IL-1β, TNF-α and TGF-β mRNA, and HCV(+)AID+/+ mice exhibited higher TNF-α levels than HCV(+)AID−/− mice. Consistent with the q-PCR results, the protein levels of IL-1β and TGF-β were also elevated by the presence of the HCV transgene, and the TNF-α protein production level was dependent on the presence of AID, suggesting that TNF-α production may have led to the aggressive pathological findings observed in HCV(+)AID+/+ mice (Fig. 3C).
Similar tumor incidence in HCV(+)AID−/− and HCV(+)AID+/+ mice
The incidence of tumor formation was carefully examined by histopathological and macroscopic evaluation. None of the 15 HCV(+)AID−/− or 15 HCV(+)AID+/+ 16-month-old female mice showed evidence of liver tumor formation, consistent with a previous report (Table 3) (13). Further analysis of the 20-month-old male groups, including 21 HCV(+)AID−/− and 21 HCV(+)AID+/+ mice, revealed that 4 out of 21 HCV(+)AID+/+ mice carried macroscopic tumors, all of which were determined to be malignant by histological examination (Fig. 4A, right 4 panels). Similarly, 3 out of 21 HCV(+)AID−/− mice bore macroscopic tumors, two of which were judged to be malignant (Fig. 4A, left 3 panels). These results suggest that AID is not essential for HCV-induced carcinogenesis. Consistent with these findings, the AID transcript level in the tumor region of an AID+/+ mouse (#241) was equivalent to the level detected in a non-tumor area (Fig. 4B). Comparison of the AID expression in the tumor and non-tumor areas from the two tumor-bearing mice of the HCV(+)AID−/− and the two of HCV(+)AID+/+ indicated that the tumor tissues did not contain elevated AID expression levels (Fig. 4C). The AID protein levels were not detectable by western blotting in either the tumor or non-tumor areas (Fig. 4D).
Dispensability of AID for the development of skin tumors in HPV8-Tg mice
To examine AID’s involvement in the development of HPV8-induced skin tumors, HPV8-Tg (HPV(+)) mice were crossed with AID−/− mice. Then HPV8(+)AID+/− mice of the first filial generation were again crossed each other and the obtained HPV8(+)AID−/− and HPV8(+)AID+/+ mice of the second filial generation were compared as the observation groups. Both genotypes developed skin tumors, and tumor samples from 6-month-old mice were tested for AID expression by RNA and protein analyses (Fig. 5A and B). Neither the AID protein nor its RNA was detectable in the samples analyzed. After 6 months, the final skin tumor prevalence was ~30% in both mouse populations (15 out of 49 HPV8(+)AID−/− and 16 out of 51 HPV8(+)AID+/+) (Supplementary Table 4, available at International Immunology Online), and the frequency and time course of tumor development in the two groups were almost indistinguishable (Fig. 5C). The histological examination of the skin tumor did not show any difference between HPV8(+)AID+/+ and HPV8(+)AID−/− mice (Fig. 5D). The lower papilloma prevalence (~30%) compared to the original report describing the HPV8(+) mice (15) may be due to the mixed genetic background of FVB/N and B6 in both groups (the HPV8(+)AID−/− and HPV8(+)AID+/+ mice), since mice with an FVB/N genetic background are reported to have more severe papilloma progression than those with a B6 background (15). Although the malignant progression to SCC in these skin tumors was not examined, AID expression was absent, and tumor development was equivalent in the AID−/− and AID+/+ mice. We thus conclude that AID is not involved in HPV8-induced skin tumorigenesis.
Discussion
In this study, we investigated the requirement of AID for virally induced tumorigenesis by using compound mice that were generated by crossing mice transgenic for either HCV core proteins or HPV8 early proteins with either AID wild-type or knockout mice. Our results indicated that AID was expressed in neither hepatocytes of HCV-Tg nor skin tissue of HPV8-Tg. Thus, AID was not shown to be required for the development of both HCV- and HPV8-promoted tumorigenesis. We could not conclude that the frequency of the liver malignancy is statistically different between HCV(+)AID+/+ (4 out of 21 mice) and HCV(+)AID−/− (2 out of 21 mice), partly because the frequency of the liver malignancy was unexpectedly lower than that in the original report (13). Studies on 5 times the number of mice may allow us to obtain statistically significant conclusions about the frequency of the liver malignancy between HCV(+)AID+/+ and HCV(+)AID−/− mice. We note, however, that the HCV(+)AID+/+ mice exhibited higher levels of TNF-α production with more severe histological phenotypes than the HCV(+)AID−/− mice.
AID is reported to be expressed in HCC and in the surrounding non-cancerous liver tissues (6, 7). In addition, the hepatoma-derived cell lines HepG2, Hep3B and Huh-7 have all been shown to express AID in response to HCV core protein-induced NF-κB signaling (9). It therefore has been assumed that AID is induced in HCV-Tg mice and contributes to liver carcinogenesis. However, AID expression was not detected in HCV core protein-positive hepatocytes, and the low levels of AID transcripts in liver tissues were attributable to infiltrating B cells in this study. The different AID expression levels observed in the present mouse model and HCV-infected patients could be due in part to pathogenic differences between the two systems. HCV-Tg mice lack cirrhotic changes (12, 13), which may be involved in inducing AID expression. In contrast, the natural course of human HCV infection leads to chronic hepatitis development, with bile duct damage and steatosis in the majority of patients (24), and the failure of virus eradication leads to liver cirrhosis and/or HCC (25). In human cases, 80–90% of the HCC develops from cirrhotic liver tissues (21), indicating that chronic inflammatory reactions generally contribute to carcinogenesis.
The involvement of B and T cells in liver injury via autoimmune antibody may be a possible reason for the strong inflammatory response in some part of natural HCV infection cases (26–28). CD81, an HCV-binding molecule, is expressed on the surface of B and T cells and both type of lymphocyte may be infected by HCV (28, 29). HCV infection of B cells causes the development of non-organ-specific auto-antibodies (NOSAs) and cryoglobulinemia (26, 30). In the various NOSAs, some antibodies add auto-antibody-mediated liver injury to viral hepatitis (30). For example, anti-liver/kidney microsomal antibody type 1 (LKM1) and anti-smooth muscle antibody (SMA) are also found in autoimmune hepatitis, and anti-microsomal antibody (AMA) is closely related to primary biliary cirrhosis (31). These liver-targeting antibodies of NOSAs are proposed to be produced based on the mimicry of autoantigens by HCV polyproteins including NS3, 4 and 5 in addition to core proteins (32). In the current HCV-Tg mouse model, auto-antibody-dependent liver injury is less likely because only the core of the HCV polyprotein is expressed and HCV infection in B cells is absent. The absence of this extra-hepatic complication partly explains the reasons why the inflammation is weak in this study compared to natural HCV infection and why AID is not expressed in the hepatocytes of HCV-Tg mouse.
The discrepancy of AID expression between the current animal study and the natural HCV infection could be also due to different regulation of AID expression between mouse and human hepatocytes. The transcriptional regulation of AID expression in B lymphocytes has been extensively examined both in vitro and in vivo and shown to depend on B-cell-specific and environmental stimulus-specific factors (33). The latter include Stat6 and NF-κb, which are activated by viral infection. AID was activated by transfected HCV core protein responding to the NF-κB signaling pathway (9) in human cell lines; however, NF-κB is not activated in the current HCV-Tg model whereas the upstream TNF-α signal is increased (23). Although the difference in the AID promoter between human and mice is not known, we cannot totally exclude this possibility to explain the different AID expression response.
Although HCV-Tg had weaker inflammatory responses than natural HCV infection, the inflammation observed in HCV(+)AID+/+ mice tended to be more severe than that in HCV(+)AID−/− mice. The HCV core protein expression induced infiltration of IHICs including T, B and probably NK cells, and AID+/+ B cells enhanced TNF-α production more than AID−/− B cells. The mechanism of TNF-α up-regulation by AID is unknown; however, higher levels of TNF-α production are likely to affect the pathogenesis or prognosis. TNF-α and IL-1β were increased in the whole liver lysates of 16-month-old HCV-Tg mice, as previously reported (23). Clinically, increased TNF-α production from liver-infiltrating monocytes in Non-A, Non-B hepatitis has also been reported (34). Furthermore, TNF-α was shown to activate the AP-1 pathway (23), which promotes cell proliferation (35).
The HCC development without severe ‘cirrhotic’ findings may be due to carcinogenic properties associated with the HCV core protein, including suppression of apoptosis (by interacting with p53 and pRb), promotion of proliferation (by up-regulating the Wnt/b-catenin and Raf/MAPK pathways) and induction of reactive oxygen species (10, 36).
Stat3 is essential for HPV8-induced skin carcinogenesis (37). The Stat6 and Stat3 DNA recognition motifs are not completely identical, but share partial homology (38). We suspected that HPV8 induces AID through Stat and NF-κB signaling pathways and examined AID’s involvement in the HPV8-Tg mouse model. Contrary to our expectation, AID expression was not induced in the HPV8-Tg mouse model, which developed papilloma at a frequency of 30%. Since we observed these mice only up to 6 months before SCC development, we cannot rule out the possibility that AID in the skin squamous cells is induced later to convert papilloma to SCC.
In conclusion, our results suggest that AID may not be essential for either HCV-induced liver carcinogenesis or HPV8-induced papillomagenesis. We were unable to detect AID expression in transgenic cells expressing viral oncogenic proteins in either model, contrary to expectations. These results indicate that the expression of AID is strictly regulated in both hepatocytes and cutaneous keratinocytes at least in the mouse models used here.
Funding
Ministry of Education, Culture, Sports, Science and Technology of Japan Grant-in-Aid for Specially Promoted Research (17002015 to T.H.); Grant-in-Aid for Scientific Research (C) (25440007 to M.K.).
Acknowledgements
We thank Prof. Tsutomu Chiba for helpful discussions, Prof. Hitoshi Nagaoka for useful suggestions, Dr Le Thi Huong for technical assistance and Mrs Mikiyo Nakata for providing HepG2 cells.
References