1 Istituto Nazionale Malattie Infettive L. Spallanzani IRCCS, Rome, Italy
2 Fondazione Istituto Pasteur Cenci-Bolognetti, Dipartimento di Biotecnologie Cellulari ed Ematologia, Università La Sapienza, Rome, Italy
3 Medicina Sperimentale, Sezione Anatomia Patologica, Università La Sapienza, Rome, Italy
4 IRBM, P. Angeletti, Pomezia, Italy
Correspondence
Nicola La Monica
nicola_lamonica{at}merck.com
Marco Tripodi
tripodi{at}bce.med.uniroma1.it
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ABSTRACT |
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INTRODUCTION |
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More than 170 million people worldwide are infected with HCV, and the complications of liver cirrhosis and hepatocellular carcinoma cause significant morbidity and mortality. The virus is cleared in a minority of patients and 7080 % develop chronic infection that leads to cirrhosis within 2030 years. Although the current treatment regimens (IFN- and ribavirin) help to eliminate or reduce the virus load in some patients, these treatments often fail (Armstrong et al., 2000
; Lauer & Walker, 2001
). Thus, understanding the mechanism responsible for progressive liver disease in chronic HCV infection would be highly beneficial in designing new strategies for therapeutic intervention.
Immune mechanisms are thought to be responsible, at least in part, for the liver disease pathogenesis as well as for virus clearance (Gonzales-Peralta et al., 1994; Cerny & Chisari, 1999
; Bertoletti et al., 2000
). Acute self-limited hepatitis C is generally associated with the detection of HCV-specific T cells with a predominant production of Th1 cytokines (Gerlach et al., 1999
; Gruner et al., 2000
). A prevalent Th2 pattern of cytokine production is instead associated with virus persistence and development of chronic infection (Tsai et al., 1997
; Woitas et al., 1997
). Patients with long-term chronic HCV infection also display increased intrahepatic expression of Th1-associated cytokines that correlates with progressive liver injury (Napoli et al., 1996
). Therefore, recruitment and activation of immunocompetent cells causing hepatocyte necrosis is directly related to the pathogenesis of acute hepatitis C. Moreover, the HCV proteins may be involved in the development of pathogenesis of chronic hepatitis and of hepatocellular carcinoma (HCC) development by modifying hepatocyte gene expression.
The direct role of HCV proteins in hepatitis and liver cancer is supported by the observation that core, NS3, NS4B and NS5A transform fibroblasts, modulate gene transcription and modify the susceptibility of cultured cells to apoptotic signals (Ghosh et al., 1999; Marusawa et al., 1999
; Park et al., 2000
; Ray et al., 1996
; Ruggieri et al., 1997
; Sakamuro et al., 1995
; Tan et al., 1999
; Tanaka et al., 1996
). Additionally, as observed in hepatitis B virus-infected patients, the presence of inflammatory cell infiltrates in necrotic lesions of infected livers strongly suggests a pathogenic role of the host immune response (Cerny & Chisari, 1999
). The hypothesis that expression of HCV proteins directly injures liver cells triggering an inflammatory response is attractive but difficult to test directly in the absence of a robust tissue culture system or small animal models that mimic HCV replication and the pathological features of HCV infection in humans. As an alternative, transgenic mice represent a powerful tool to investigate the role of chronic HCV gene expression in liver disease. A transgenic mouse line expressing the entire HCV genome under the control of the A1AT promoter was generated to study the direct effect of constitutive liver expression of HCV proteins in the absence of inflammation. In our previous work we showed that stable expression of HCV RNA and core protein was evident in liver of transgenic mice. Moreover, impaired interferon-induced intracellular STAT signalling and enhanced susceptibility to lymphocytic choriomeningitis virus was detected in 714-week-old animals showing no liver damage (Blindenbacher et al., 2003
). In this study, we show that transgenic HCV animals develop with ageing extensive steatosis and limited necrosis in hepatic tissue that are reminiscent of that seen in liver biopsies from patients with hepatitis C infection. More interestingly, a consistent T cell infiltrate made up largely of CD8 T cells secreting Th2-type cytokines and a limited hepatocyte proliferation were observed. The pathogenesis of liver cell injury associated with HCV polyprotein expression is discussed.
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METHODS |
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Transgenic C57Bl/6 mice were generated by BRL (Basel, Switzerland) as described (Hogan et al., 1996). Genotype analysis was performed by Southern blot analysis on 10 µg of genomic DNA using an HCV cDNA fragment as probe (nt 13482916).
Animals and treatments.
Mice were maintained under specific pathogen-free conditions in either IRBM or Charles River facilities under a 12 h light/dark cycle, and provided with irradiated food and autoclaved water ad libitum. Procedures involving animals and their care were conducted in conformity with national and international laws and policies. Bromodeoxyuridine (BrdU; Sigma) was administrated for 1 week [1 mg ml1 in regular drinking water, supplemented of 1 % sucrose (Sigma)]. Mice were euthanized by CO2 asphyxiation and livers collected immediately.
RNA extraction, RT-PCR and in situ hybridization analysis.
RNA was isolated from frozen tissues using Ultraspec RNA reagent (Biotecx) according to the manufacturer's instructions. RT-PCR with a Promega kit using as oligonucleotides 5'-GGCGCACATGGCATCCTC-3' (sense) and 5'-GCCACCGCAAGGTCTCGT-3' (antisense) for HCV (nt 26133241) and 5'-ATGGATGACGATATCGCTGCG-3' (sense) and 5'-ATCTTCATGAGGTAGTCTGTCAGG-3' (antisense) for the -actin gene.
In situ hybridization was performed according to Bloch (1993). A linearized plasmid containing the region 40414840 of HCV flanked by promoter sequences for T7 and T3 RNA polymerases was used to transcribe digoxigenin-labelled sense and antisense riboprobes using Riboprobe Combination System T3/T7 (Promega), according to the manufacturer's instructions.
Ten µm tissue sections were hybridized overnight at 55 °C in 50 % deionized formamide, 4x SSC, 1x Denhardt's solution, 1 µg salmon sperm DNA ml1, 1 µg tRNA ml1 and 1·53 ng DIG-labelled cRNA probe µl1. Tissue slides were washed at 37 °C in 2x SSC, then treated with 20 µg RNaseA ml1 at 37 °C for 30 min, washed at 65 °C in 2x SSC and 0·1x SSC for 1 h, and finally analysed using AP-conjugated anti-digoxigenin Fab fragments (Roche) and NBT/BCIP. Sense cRNA probes were used as control of hybridization specificity.
Western blot analysis.
Total protein extracts were prepared by homogenizing livers in buffer A (10 mM HEPES pH 7·9, 10 mM KCl, 3 mM MgCl2, 0·1 mM EDTA, 0·1 mM EGTA, 0·5 mM DTT, 1 mM PMSF, 10 µg leupeptin ml1, 10 µg pepstatin ml1, 5 µg anti-papain ml1) containing 0·05 % NP40. The nuclei, pelleted by centrifugation, were lysed in buffer C (20 mM HEPES pH 7·9, 400 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0·5 mM DTT, 1 mM PMSF, 10 µg leupeptin ml1, 10 µg pepstatin ml1, 5 µg anti-papain ml1). Samples of 100 µg of proteins were used for Western blotting analysis as described (Esposito et al., 1995) using either anti-HCV rabbit antisera (Tomei et al., 1993
), mAbs or serum from HCV-infected patients.
Immunohistological analysis of liver tissues.
Liver samples were fixed in 10 % (w/v) buffered formalin, embedded in paraffin. For histopathological analysis 48 µm thick sections were stained with haematoxylin and eosin. For detection of HCV proteins and BrdU incorporation rabbit polyclonal Abs to E2, NS3 and NS5 (a+b) (Tomei et al., 1993) or a mAb to BrdU (Roche) were used, respectively. Antigens were retrieved by trypsin treatment (0·1 %, w/v, in a solution of 7 mM CaCl2; 50 mM Tris; 150 mM NaCl; pH 7·8; Dako) and boiling in 10 mM citrate buffer pH 6·0. Sections were incubated with primary antibodies, followed by incubation with alkaline phosphatase-conjugated goat anti-mouse or anti-rabbit IgG (Dako). Antibody binding was revealed using the Fast Red Substrate System (Dako) on haematoxylin-counterstained sections.
Isolation of spleen and intrahepatic lymphocytes.
Spleens and livers from wild-type and transgenic mice were weighed, washed with RPMI 1640 (supplemented with 10 % heat inactivated FCS and 10 U penicillin/streptomycin ml1) and manually homogenized using a 70 µm diameter filter (Becton Dickinson). Recovered cells were washed twice and counted to assess the number of lymphocytes per mg of tissue. Mononuclear cells were obtained by FicollHypaque gradient centrifugation.
Monoclonal antibodies.
mAbs (Becton Dickinson) coupled to different fluorochromes were combined for simultaneous triple/quadruple staining. Anti-CD3 (17A2), anti-B220 (RA3-6B2), anti-NK1.1 (PK136), anti-IFN- (XMG1.2) and anti-IL-2 (JES6-5H4) were FITC-coupled. Anti-
(GL3), anti-Pan-NK (DX5), anti-CD25 (3C7), anti-IL-4 (BVD4-1D11) and anti-IL-10 (JES5-16E3) were phycoerythrin (PE)-coupled. Anti-CD8-cy5 (53-6·7) was PECy5-coupled. Anti-CD4 (RM4-5) and anti-CD44 (IM7) were allophycocyanin (APC)-coupled. A PE-conjugated control mAb (IgG1; MOPC-21) was used in all experiments.
Flow cytometry for surface antigens.
For the analysis of surface antigen expression 5x105 splenic or 3x105 intrahepatic lymphocytes were incubated for 15 min at 4 °C with the mAbs listed above. After washing in 1X PBS containing 1 % BSA and 0·1 % sodium azide, samples were fixed in 4 % paraformaldehyde and acquired by a FACScalibur flow cytometer (Becton Dickinson). Twenty thousand events were acquired for each sample and analysed with the CellQuest software (Becton Dickinson).
Single-cell analysis of cytokine synthesis.
To measure cytokine production splenic or intrahepatic lymphocytes were stimulated overnight with phorbol 12-myristate 13-acetate (PMA; 50 ng ml1) and ionomycin (5 µg ml1). Monensin (10 µg ml1) was added after 1 h from stimulation to block intracellular transport processes. Cells were stained with anti-CD8Cy5, anti-CD4APC mAbs for 15 min at 4 °C and then fixed in PBS/1 % paraformaldehyde for 10 min at 4 °C and incubated with anti-cytokine-specific mAb in the presence of 0·5 % saponin to permeabilize the cell membrane. Cells were acquired on a FACScan (Becton Dickinson), using isotype-matched mAbs as control for unspecific staining.
ELISPOT assays on splenocytes and intrahepatic lymphocytes.
Cells secreting IFN- in an antigen-specific manner were detected using a standard enzyme-linked immunospot assay on splenocytes as described (Zucchelli et al., 2000
). Cells secreting IL-4 were detected by using anti-mouse IL-4 antibodies from Pharmingen (rat anti-mouse IL-4 mAb clone 1B11 and biotinylated rat anti-mouse IL-4 mAb clone BVD6-24G2). Intrahepatic lymphocytes were prepared as described above and plated at 5x104, 2·5x104, 1x104, 5x103 cells per well in the presence of 2·5x105 per well of syngeneic antigen-presenting cells. Concanavalin A at 10 µg ml1 was used as a non-specific stimulus for T cells in all assays. Twenty-amino-acid-long peptides, overlapping by 10 residues, were used in the assay at 5 µg ml1. To facilitate the analysis the peptides were collected in pools: pools C and E encompass, respectively, the sequence of HCV Core and E2 protein from the 1a viral genotype, strain H; pools F, G, H, I and L encompass the sequence of the NS region from NS3 to NS5b (aa 10262418) from the 1b viral genotype, BK strain. Immunized mice used as a positive control were electroinjected with 5 µg of plasmid pF78E2 as described (Zucchelli et al., 2000
).
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RESULTS |
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High titre anti-HCV-positive sera of chronically infected patients and polyclonal (Tomei et al., 1993) and mAb specific for the individual HCV proteins were used in Western blots and immunohistochemical analysis of transgenic and wild-type liver samples. As shown in Fig. 1(E)
, a band of 22 kDa was detected in transgenic liver extracts by anti-HCV antisera. A similar protein product was also recognized by a monoclonal antiserum specific for the core protein (data not shown). Moreover, expression of the A1AT/HCV transgene appeared to be fairly constant in mice, as indicated by the presence of similar amount of core protein in mice up to 18 months of age.
Antisera specific for other HCV proteins did not reveal additional immunoreactive polypeptides by Western blot analysis (data not shown). However, immunohistochemical analysis showed the expression of E2, NS3 and NS5a+b proteins in the transgenic livers (Fig. 2AB, DE and GH, respectively) but not in wild-type littermates (Fig. 2C, F, I
). Taken together, these data demonstrate that the transgenic mouse line carries the entire A1AT/HCV transgene and that translation of the transgenic HCV coding sequence spans the entire ORF.
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Characterization of intrahepatic lymphocytes
The cell subsets infiltrating the liver of 14 transgenic and 16 wild-type littermate animals were analysed by flow cytometry. Considering that large inflammatory infiltrates were present only in a minority of transgenic animals, mice featuring large nodular perivascular infiltrates (Fig. 3E) were excluded. The total number of lymphocytes was significantly higher in the liver of transgenic mice (2- to 3-fold increase over wild-type; Table 1
). Lymphocytes homing in the liver were both B (3·98x103 cells per mg of tissue of transgenic mice vs 2·90x103 cells per mg of tissue in wild-type littermates) and T cells (3·56x103 cells per mg of tissue of transgenic mice vs 1·59x103 cells per mg of tissue in wild-type littermates). Specifically, HCV liver lymphocytes were significantly enriched for cytotoxic cells such as CD8+ T lymphocytes (2·35-fold), NK cells (2·6-fold), NKT lymphocytes (2·1-fold) and
T lymphocytes (2·6-fold). No significant differences in cell populations between wild-type and transgenic mice were found comparing spleen lymphocytes subsets, thus indicating that HCV protein expression in the liver of transgenic mice is associated with specific lymphocyte recruitment.
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DISCUSSION |
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The liver of HCV-infected patients is enriched in Th1-type T, NK and NKT cells with cytotoxic activity (Agrati et al., 2001; Nuti et al., 1998
). The unique feature of the A1AT/HCV transgenic mouse is the correlation between transgene expression and hepatic recruitment and/or local expansion of cytotoxic lymphocyte subtypes (Table 1
). This lymphocyte recruitment is probably not induced by an antigen-mediated immune response. In fact, the self nature of the transgenic proteins is underlined by the lack of: (i) HCV-specific antibodies found in the sera of the mice; (ii) IL-4 and IFN-
production to viral antigens (Table 2
); (iii) activation markers on both splenic and intrahepatic T lymphocytes. Additionally, the observation that expression of A1AT chimeric transgenes is detectable as early as 14·5 days post-coitum embryos is also in agreement with the lack of immune recognition of the HCV proteins in these mice (Amicone et al., 1995
). Thus, the recruitment of lymphocytes to the liver may be caused by an alteration of hepatocyte gene expression induced by the viral products.
Expression of HCV proteins was noted primarily in the perivascular area, although scattered A1AT/HCV-expressing hepatocytes were also detected. In contrast, lymphocyte recruitment was mainly detected in the parenchyma, suggesting that their localization is not only related to the site of transgene expression but may reflect a more generalized alteration in the microenvironment of the liver that acts as a signal for lymphocyte recruitment. We therefore analysed the gene expression profiles of some chemokines present in the liver of wild-type and A1AT/HCV mice. No significant differences were found in mRNA levels of Rantes, eotaxin, MIP-1, MIP-1
, MIP-2, IP-10 and TCA-3, indicating that these chemokines are not responsible for lymphocyte recruitment in transgenic livers (data not shown). The molecular patterns involved in cytotoxic T cell recruitment in the liver of HCV transgenic mice and, in general, in HCV-infected patients remain to be elucidated.
The intrahepatic CD8+ T lymphocytes of transgenic mice were found to produce more cytokines, mainly IL-10, when stimulated with PMA and ionomycin (Fig. 4). IL-10 is a multifunctional cytokine known for its crucial role in regulating immune and inflammatory responses. Among its activities, IL-10 regulates growth and/or differentiation of B cells, NK cells, cytotoxic and helper T cells (reviewed by Moore et al., 2001
; Akdis & Blaser, 2001
). It is tempting to speculate that IL-10 may play a role in the control of immune responses and tolerance against HCV in the liver. In accordance with this hypothesis, Nelson et al. (2003)
recently reported that long-term therapy of HCV patients with rIL-10 decreased disease activity and led to increased HCV viral burden via alterations in immunological viral surveillance.
Several transgenic mouse lines expressing portions of the HCV ORF or the entire coding sequence have been reported to be associated with a variety of phenotypes including steatosis, necrosis, sensitivity to Fas-induced apoptosis and HCC (reviewed by Koike, 1999; Fimia et al., 2003
). The hepatic alterations observed in the A1AT/HCV mice are partially in agreement with these published reports. Although positional effects of the exogenous A1AT/HCV gene cannot be determined based only on results from one mouse line, the data collected support the validity of the A1AT/HCV transgenic mouse line and point to the direct involvement of HCV proteins in liver damage. We cannot exclude the possibility that the fusion between the 32 aa of A1AT and the core protein of HCV may have contributed, at least in part, to the histological alterations noted in the transgenic mice. Nonetheless, the involvement of HCV proteins in liver damage is also supported by the observation that hepatic steatosis and lymphocyte recruitment have not been observed so far with other transgenic mouse lines expressing either the full-length A1AT gene (Ruther et al., 1987
) or other genes of interest driven by the A1AT gene (Amicone et al., 1997
).
The immunohistochemistry data indicate that expression of the entire ORF takes place in transgenic hepatocytes, although no direct evidence could be provided for correct processing of the nonstructural region. The expression vector utilized for the development of this HCV mouse line is based on a human genomic fragment comprising the A1AT gene, and shown to be suitable for transgenic liver expression (Amicone et al., 1995). Thus, the lack of detection of the NS proteins by Western blot can probably be ascribed to factors such as instability of viral proteins and low antigen affinity of the antisera used for the immunodetection. Additionally, in view of the histological alterations noted in the transgenic mice, it is possible that NS enzymes may be harmful to mouse development and this may allow establishment of mice only with low levels of transgene expression. This conclusion is also supported, at least in part, by the observation that in transgenic mouse lines carrying the full-length HCV cDNA under the control of a mouse albumin enhancer/promoter protein expression could not be confirmed by sensitive immunohistochemical techniques, yet significant hepatic histological alterations were observed (Lerat et al., 2002
).
The extensive steatosis noted in the transgenic mice, in agreement with expression of HCV core protein (Moriya et al., 1997), declined with age and no development of neoplastic or cancerous lesions occurred in liver from A1AT/HCV mice by the age of 20 months. Additionally, development of steatosis and liver cancer has been reported in mice carrying the full-length HCV cDNA under the control of a liver-specific promoter (Lerat et al., 2002
). Why the extent of steatosis varied over time and neoplastic changes did not appear might be related to the extent of liver injury and/or HCV protein expression in A1AT/HCV mice. Additionally, oxidative stress, associated with steatosis and core gene expression, may determine, at least in part, the onset of hepatocarcinogenesis (Moriya et al., 1997
, 2001
; Okuda et al., 2002
). Although we have not performed any studies to characterize the mechanism of steatosis in the A1AT/HCV mice, the lack of a sustained steatosis phenotype and of neoplasia may be connected with a reduced oxidative stress in these mice. Finally, although the differing phenotypes associated with transgenic mice expressing various portions of the HCV genome may be difficult to reconcile, these observations suggest the possible presence of multiple pathways of liver damage and oncogenesis that are affected by the constitutive expression of the viral proteins.
The A1AT/HCV mice allow the analysis of the effects of HCV genome expression in the absence of humoral or cellular immune responses to the viral polypeptides. The analysis of the molecular mechanisms involved in this animal model may contribute to an understanding of the complex interactions between HCV and the host immune system, with particular focusing on innate pathways.
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ACKNOWLEDGEMENTS |
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Received 16 October 2003;
accepted 3 February 2004.
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