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, 12 (9), e0184127
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Hepatitis C Virus Mediated Chronic Inflammation and Tumorigenesis in the Humanised Immune System and Liver Mouse Model

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Hepatitis C Virus Mediated Chronic Inflammation and Tumorigenesis in the Humanised Immune System and Liver Mouse Model

Zhiqiang Zheng et al. PLoS One.

Abstract

Hepatitis C is a liver disease caused by infection of the Hepatitis C virus (HCV). Many individuals infected by the virus are unable to resolve the viral infection and develop chronic hepatitis, which can lead to formation of liver cirrhosis and cancer. To understand better how initial HCV infections progress to chronic liver diseases, we characterised the long term pathogenic effects of HCV infections with the use of a humanised mouse model (HIL mice) we have previously established. Although HCV RNA could be detected in infected mice up to 9 weeks post infection, HCV infected mice developed increased incidences of liver fibrosis, granulomatous inflammation and tumour formation in the form of hepatocellular adenomas or hepatocellular carcinomas by 28 weeks post infection compared to uninfected mice. We also demonstrated that chronic liver inflammation in HCV infected mice was mediated by the human immune system, particularly by monocytes/macrophages and T cells which exhibited exhaustion phenotypes. In conclusion, HIL mice can recapitulate some of the clinical symptoms such as chronic inflammation, immune cell exhaustion and tumorigenesis seen in HCV patients. Our findings also suggest that persistence of HCV-associated liver disease appear to require initial infections of HCV and immune responses but not long term HCV viraemia.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Longitudinal serum ALT and human albumin levels in HIL mice post HCV infection.
3 cohorts of HIL mice were either mock infected or infected with a 106 or 107 ffu dose of HCV. For mice that survived more than 8 weeks, serum taken from each mouse every month were analysed (Cohort 1: mock n = 8, 106 ffu HCV n = 10, 107 ffu HCV n = 10. Cohort 2: mock n = 10, 107 ffu HCV n = 13. Cohort 3: mock n = 3, 107 ffu HCV n = 11). (A, C, E) Serum ALT and (B, D, F) HSA levels were measured at various time points for a period of 27–28 weeks post HCV infection.
Fig 2
Fig 2. Liver abnormalities detected in HCV infected mice at 27 weeks post infection.
(A-H) Livers were harvested from mice in the mock and HCV infected groups, paraffin-embedded and processed for staining with (B-G) haematoxylin and eosin or (H) Sirius red/fast green stains. (A) Representative image showing gross appearance of livers obtained from either mock or HCV infected mice at 27–28 weeks post infection. Representative image of H&E stains showing (B) normal liver from mock infected mice and (C) steatosis, (D) hepatocellular adenoma, (E) hepatocellular carcinoma, (F) granulomatous inflammation, (G) focal nodular hyperplasia and (H) liver fibrosis from livers harvested from HCV infected mice.
Fig 3
Fig 3. Majority of dividing cells in liver tumours are of human origin.
(A-C) H&E stain of a hepatocellular adenoma containing liver section. (B&C) Nodular growth, loss of normal architecture with irregular growth pattern and compression of the surrounding parenchyma. (D-F) Serial section stained using an antibody specific against HSA showing 80–90% human albumin positivity within the liver tumours. (G) HSA expressing hepatocytes of mock (n = 3), non-tumour (n = 2) and tumour (n = 2) regions within liver sections were counted and expressed as a percentage of total hepatocytes. Percentages represent averaged counts of HSA expressing hepatocytes against total hepatocytes from five randomly selected 500 x 250 μm regions per sample. Error bars represent standard error of the means. (H-I) Representative images of mitotically active hepatocytes that (H) express HSA or (I) do not express HSA.
Fig 4
Fig 4. Human immune cell infiltration and inflammation in HCV infected HIL mice.
Liver sections from (A-B) mock infected or (C-F) HCV infected HIL mice 27 weeks post infection were stained for human CD45 and counterstained with haematoxylin. Representative images showing (A) liver from mock infected HIL mice, (B) high magnification of an area in mock infected mice (Squared region), (C) liver from HCV infected HIL mice, (D-E) high magnification of areas in a hepatocellular adenoma from HCV infected mice, and (F) high magnification of an area in a non-tumour region from HCV infected mice. (G-I) Representative images showing human (G) CD68, (H) CD3 and (I) CD20 stains within hepatocellular adenoma in HCV infected mice. (J-K) Plasma samples at 27–28 weeks post infection were quantified for the presence of human cytokines: (J) IL10, IL12p70, IL17A, IL23 (K) MCP-1, IL6, IL8, IL18 and IFN-γ. Mock n = 10, 106 ffu HCV n = 5, 107 ffu HCV n = 16.
Fig 5
Fig 5. Human immune cell composition and phenotyping in HCV infected HIL mice.
Leukocytes were prepared from livers and spleens of mock infected or mice infected with 107 ffu of HCV 27 weeks post infection and analysed by flow cytometry. Shown are absolute numbers of various immune cell populations in (A) livers or (B) spleens of mock or HCV infected mice. Error bars represent standard error of the means. Mock n = 5, 107 ffu HCV n = 12.
Fig 6
Fig 6. Deposition of human T cells and macrophages in non-tumour and tumour regions in the livers of HCV infected HIL mice.
Livers sections from mock infected and HCV infected HIL mice 27 weeks post infection were stained for human CD3, PD1, CD68, IL6 and IL10. Representative images are shown for the stains of (A) CD3 and PD1, (B) CD68 and IL6 and (C) CD68 and IL10. Scale bars represent 50 μM. (D) Total number of CD3+PD1+, CD68+IL6+ or CD68+IL10+ cells were counted from five randomly selected 310 x 310 μm regions from mock liver sections, non-tumour or tumour regions from HCV infected HIL mice. Error bars represent standard error of the means.

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References

    1. Choo Q-L, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science. 1989;244(4902):359–62. - PubMed
    1. Averhoff FM, Glass N, Holtzman D. Global burden of hepatitis C: considerations for healthcare providers in the United States. Clinical Infectious Diseases. 2012;55(suppl 1):S10–S5. - PubMed
    1. Dustin LB, Cashman SB, Laidlaw SM. Immune control and failure in HCV infection—tipping the balance. Journal of leukocyte biology. 2014;96(4):535–48. doi: 10.1189/jlb.4RI0214-126R - DOI - PMC - PubMed
    1. Afdhal NH, editor. The natural history of hepatitis C. Seminars in liver disease; 2004: Copyright© 2004 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA.
    1. Freeman AJ, Dore GJ, Law MG, Thorpe M, Von Overbeck J, Lloyd AR, et al. Estimating progression to cirrhosis in chronic hepatitis C virus infection. Hepatology. 2001;34(4):809–16. - PubMed

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Grant support

“This study was supported by Joint Council Office Development Programme 1334k00082, the Agency for Science, Technology and Research (A*STAR), Singapore. Qingfeng Chen is also supported by National Research Foundation Fellowship Singapore NRF-NRFF2017-03. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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