Volume 138, Issue 3, Pages 1123-1133.e3 (March 2010) Cell-Type Specific Gene Expression Signature in Liver Underlies Response to Interferon Therapy in Chronic Hepatitis C Infection  Limin Chen, Ivan Borozan, Jing Sun, Maha Guindi, Sandra Fischer, Jordan Feld, Nitasha Anand, Jenny Heathcote, Aled M. Edwards, Ian D. McGilvray  Gastroenterology  Volume 138, Issue 3, Pages 1123-1133.e3 (March 2010) DOI: 10.1053/j.gastro.2009.10.046 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Hierachial clustering analysis of 78 CHC liver samples based on expression levels of 18 previously defined signature genes. Hierarchial clustering analysis was performed based on the expression levels of 18 previously defined genes.16 The analysis broadly separates patients into 2 groups: responders (R) and nonresponders (NR). Gastroenterology 2010 138, 1123-1133.e3DOI: (10.1053/j.gastro.2009.10.046) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Multivariate ANOVA analysis of genes altered by chronic HCV infection. (A) Response status excluded: The bar graph shows the number of genes significantly associated with each factor (genotype/disease [D], gender [G], viral load [V], fibrosis [F], and inflammation [I]). Genotype was associated with the most genes (17). The box plots below show the distribution of fold differences in gene expression between the levels of each factor for significant genes associated with each factor. (B) Response status included: In this analysis, response status (R) was considered in addition to the 5 previous factors. Response status was associated with the most genes (24). Gastroenterology 2010 138, 1123-1133.e3DOI: (10.1053/j.gastro.2009.10.046) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Hierarchical cluster plot of genotype 1 patients segregated by “genotype”-specific genes. The 17 genes shown on the right of the plot were significantly associated with “genotype/disease” using the ANOVA multivariate model. There are 2 main clusters, with nonresponders being significantly enriched in one and responders in the other (see Results section). Gastroenterology 2010 138, 1123-1133.e3DOI: (10.1053/j.gastro.2009.10.046) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 ISG activation in different cell types in pretreatment R and NR liver. (A) Quantitation of hepatocyte and macrophage expression of ISG15 protein in pretreatment liver biopsy specimens of R (n = 15) and NR (n = 16) patients, evaluated as per Patients and Methods section. Data are mean ± SD; *P < .005 and **P < .0001 between R and NR samples. (B) Quantitation of hepatocyte and macrophage expression of MxA protein in pretreatment liver biopsy specimens of R (n = 15) and NR (n = 16) patients as in A. Data are mean ± SD; *P = .0036 and **P < .001 between R and NR samples. (C) Representative ISG15 immunohistochemistry. (a) Representative liver biopsy from NR patient immunostained for ISG15 showing predominantly hepatocellular pattern of immunoreactivity (original magnification, 50×); (b) liver biopsy from same NR patient as in a, immunostained for ISG15, showing detail of hepatocellular staining at high power (original magnification, 400×); (c) representative liver biopsy from R patient, immunostained for ISG15, showing macrophage pattern of immunoreactivity (original magnification, 50×); (d) liver biopsy from same R patient as in c, immunostained for ISG15, showing detail of macrophage staining in high power (original magnification, 400×). (D) Macrophage-specific staining. (a) Liver biopsy from R patient, immunostained for ISG15, showing typical macrophage pattern of immunoreactivity in sinusoidal lining cells (original magnification, 400×); (b) liver biopsy from the same patient as in a, immunostained for smooth muscle actin, showing no reaction in sinusoidal lining cells (original magnification, 400×); (c) liver biopsy from same patient, immunostained for CD68, showing immunoreactivity in sinusoidal lining cells similar in morphology and location to the ISG15 immunoreactive cells in a (original magnification, 400×). Gastroenterology 2010 138, 1123-1133.e3DOI: (10.1053/j.gastro.2009.10.046) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 ISG15-specific immunohistochemical staining. (A) Recombinant ISG15 protein attenuates ISG15 antibody binding in vitro in a dose-dependent manner. Shown is a Western blot for ISG15: 10 ng of separated, purified human ISG15 was transferred to each of 6 nitrocellulose membrane lanes. Individual lanes were incubated with a mixture of mouse anti-human ISG15 polyclonal antibody (1:300) and ISG15 purified protein (ng/mL) 0 (lane 1), 2 (lane 2), 20 (lane 3), 200 (lane 4), 2000 (lane 5), and 4000 (lane 6) for 2 hours. The ISG15 signal was attenuated (quenched by) the higher amounts of purified ISG15 protein. (B) Confirmation of ISG15-specific staining in immunohistochemical analysis: (a) Liver biopsy from R patient, immunostained using mAb for MxA, showing typical macrophage pattern of immunoreactivity (original magnification, 200×); (b) liver biopsy from the same R patient as in a, first quenched with ISG15 protein (2 μg/mL) and then immunostained with the MxA antibody, showing persistence of immunoreactivity and indicating no cross-reaction between MxA antibody and ISG15 protein (original magnification, 200×); (c) liver biopsy from NR patient, immunostained for ISG15, showing typical hepatocellular pattern of immunoreactivity (original magnification, 200×); (d) liver biopsy from same NR patient as in c, first quenched with ISG15 protein (2 μg/mL) and then immunostained for ISG15, showing loss of immunoreactivity and indicating no cross-reaction between the ISG15 antibody and MxA or other protein (original magnification, 200×). Gastroenterology 2010 138, 1123-1133.e3DOI: (10.1053/j.gastro.2009.10.046) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 Real-time PCR of indicated genes in normal liver tissues (n = 10, open), pretreatment liver tissues from HCV treatment responders (n = 10, shaded), pretreatment liver tissues from HCV treatment nonresponders (n = 10, solid). All HCV liver biopsy specimens were taken prior to initiation of treatment with PegIFN and ribavirin. Responder and nonresponder liver tissue had similar viral loads, and all HCV biopsy specimens were obtained from genotype 1 patients. Gastroenterology 2010 138, 1123-1133.e3DOI: (10.1053/j.gastro.2009.10.046) Copyright © 2010 AGA Institute Terms and Conditions