Simultaneous detection of hepatitis C virus and interferon stimulated gene expression in infected human liver - PubMed (original) (raw)

. 2014 Jun;59(6):2121-30.

doi: 10.1002/hep.26770. Epub 2014 Apr 25.

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Simultaneous detection of hepatitis C virus and interferon stimulated gene expression in infected human liver

Stefan Wieland et al. Hepatology. 2014 Jun.

Abstract

Approximately 50% of patients with chronic hepatitis C (CHC) have ongoing expression of interferon stimulated genes (ISGs) in the liver. It is unclear why this endogenous antiviral response is inefficient in eradicating the infection. Several viral escape strategies have been identified in vitro, including inhibition of interferon (IFN) induction and ISG messenger RNA (mRNA) translation. The in vivo relevance of these mechanisms is unknown, because reliable methods to identify hepatitis C virus (HCV)-infected cells in human liver are lacking. We developed a highly sensitive in situ hybridization (ISH) system capable of HCV RNA and ISG mRNA detection in human liver biopsies and applied it to study the interaction of HCV with the endogenous IFN system. We simultaneously monitored HCV RNA and ISG mRNA using HCV isolate- and ISG mRNA-specific probes in liver biopsy sections from 18 CHC patients. The signals were quantified at the single-cell resolution in a series of random high-power fields. The proportion of infected hepatocytes ranged from 1%-54% and correlated with viral load, but not with HCV genotype or ISG expression. Infected cells occurred in clusters, pointing to cell-to-cell spread as the predominant mode of HCV transmission. ISG mRNAs were readily detected in HCV-infected cells, challenging previously proposed mechanisms of viral interference with the immune system. Conversely, infected cells and neighboring cells showed increased ISG mRNA levels, demonstrating that the stimulus driving ISG expression originates from HCV-infected hepatocytes.

Conclusion: HCV infection in human hepatocytes during CHC does not efficiently interfere with IFN induction, IFN signaling, or transcription of ISG mRNA.

© 2014 by the American Association for the Study of Liver Diseases.

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Figures

Figure 1

Figure 1. In situ HCV RNA detection in human liver tissue across a range of serum viral loads

A–C) Single-color HCV RNA detection by ISH in patients with high (A, 5’754’399 IU/ml), middle (B, 707’946 IU/ml), and low (C, 87’096 IU/ml) serum viral load. D) Liver sample from an HCV-negative patient (–CT) stained with probe set directed against B271 HCV isolate. Biopsy numbers are shown in the top right corners. The size of the bar is 25 µm.

Figure 2

Figure 2. HCV infected cells occur in clusters in human liver tissue and their numbers correlate with serum viral load

A) Boxplot showing the percentage of liver cells positive for HCV RNA in 18 patients. Patients with high ISG expression in the liver (pSVR ≤ 0.5) are shown in red and patients with low ISG expression (pSVR > 0.5) in blue. Percentage of HCV-positive cells was calculated based on the number of cells harboring at least one signal dot of HCV RNA staining in the single-color detection divided by the total number of cells counted. Each dot represents the proportion of infected cells in one randomly chosen image. For every patient, data from one to three independently stained liver biopsy sections are pooled. The blue line shows log10 serum viral load expressed in IU/ml. B) Percentage of liver cells positive for HCV RNA is associated with serum viral load. Association between the variables was evaluated using Spearman’s rank correlation coefficient (rho). Patients with high ISG expression in the liver (pSVR ≤ 0.5) are shown in red and patients with low ISG expression (pSVR > 0.5) in blue. C) Percentage of liver cells positive for HCV RNA is associated with the mean number of HCV signal dots per infected cell. Association between the variables was evaluated using Spearman’s rank correlation coefficient (rho). Patients with high ISG expression in the liver (pSVR ≤ 0.5) are shown in red and patients with low ISG expression (pSVR > 0.5) in blue. D) Infected cells are more likely to have infected neighbors than non-infected cells. Each dot shows the proportion of cells that have at least one HCV-positive adjacent neighbor. Lines connect measurements from the same patient. HCV−: non-infected cells; HCV+: infected cells; HCV++: infected cells which harbor more than two HCV signal dots. P values were calculated using paired two-tailed Student’s t-test.

Figure 3

Figure 3. Extent of ISG induction in HCV patients does not correlate with serum viral load or percentage of infected cells

A) Distribution of SVR probabilities calculated according to the 4-gene classifier(14) in a sample of 246 chronic hepatitis C patients. B) There is no association of serum viral load with probability of SVR calculated according to the 4-gene classifier in a sample of 221 chronic hepatitis C patients (for 25 patients with known pSVR the serum viral load was not available). C–F) Representative examples of 2-color ISH staining for HCV RNA (in red) and IFI27 mRNA (in green) in patients with: (C) low expression levels of IFI27 (0.084 relative to GAPDH) and high viral load (831’762 IU/ml); (D) high expression levels of IFI27 (1.8 relative to GAPDH) and high viral load (2’818’383 IU/ml); (E) low expression levels of IFI27 (0.28 relative to GAPDH) and low viral load (46’773 IU/ml); (F) high expression levels of IFI27 (2.3 relative to GAPDH) and low viral load (87’096 IU/ml). Biopsy numbers are shown in the top right corners. The size of the bar is 25 µm.

Figure 4

Figure 4. HCV infected cells and their neighbors have increased probability of high ISG expression compared to non-infected cells

A) Mean integrated intensity of IFI27 mRNA ISH staining per cell in the six patients with high expression of hepatic ISGs (pSVR ≤ 0.5) for whom double-color staining was available (B631, B749, B339, B333, B321 and B827). Every cell from each patient was grouped into one of the three categories: (i) uninfected cells without any infected adjacent neighbors, (ii) uninfected cells with at least one infected adjacent neighbor, (iii) infected cells; and the average integrated intensity of IFI27 mRNA staining per cell in each category was calculated. Lines connect measurements from the same patient. P values were calculated using paired two-tailed Student’s t-test. B) Integrated intensity of IFI27 mRNA ISH staining per cell in a selected patient with induced ISG expression (B749). Each cell was grouped into one of the three categories: (i) uninfected cells without any infected adjacent neighbors, (ii) uninfected cells with at least one infected adjacent neighbor, (iii) infected cells. P values were calculated using two-tailed Student’s t-test. C–F) Selected images of 2-color ISH staining for HCV RNA (in red) and IFI27 (green) mRNA in the liver biopsy from one representative patient (B749) with induced ISGs. The arrows indicate examples of different HCV-ISG localization patterns: (C) co-localization of HCV RNA signal dots with high levels of IFI27 mRNA in the same cell; (D) high levels of IFI27 mRNA despite the lack of HCV RNA positive cells in direct proximity; (E) very weak IFI27 signals in HCV infected cells and neighboring non-infected cells; (F) very weak IFI27 signals in an HCV-infected cell and strong IFI27 signals in surrounding cells (both HCV-negative and HCV-positive). The size of the bar is 25 µm.

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