Additional file 1: of Metabolic characterization of the natural progression of chronic hepatitis B

Supplementary methods. (DOCX 33 kb

Additional file 2: of Metabolic characterization of the natural progression of chronic hepatitis B

Supplementary Figures S1 and S2 and Tables S1 and S3. (DOCX 397 kb

Liver Monocytes and Kupffer Cells Remain Transcriptionally Distinct during Chronic Viral Infection

<div><p>Due to the scarcity of immunocompetent animal models for chronic viral hepatitis, little is known about the role of the innate intrahepatic immune system during viral replication in the liver. These insights are however fundamental for the understanding of the inappropriate adaptive immune responses during the chronic phase of the infection. We apply the Lymphocytic Choriomenigitis Virus (LCMV) clone 13 mouse model to examine chronic virus-host interactions of Kupffer cells (KC) and infiltrating monocytes (IM) in an infected liver. LCMV infection induced overt clinical hepatitis, with rise in ALT and serum cytokines, and increased intrahepatic F4/80 expression. Despite ongoing viral replication, whole liver transcriptome showed baseline expression levels of inflammatory cytokines, interferons, and interferon induced genes during the chronic infection phase. Transcriptome analyses of sorted KC and IMs using NanoString technology revealed two unique phenotypes with only minimal overlap. At the chronic viral infection phase, KC showed no increased transcription of activation markers <i>Cd80</i> and <i>Cd86</i>, but an increased expression of genes related to antigen presentation, whereas monocytes were more activated and expressed higher levels of <i>Tnf</i> transcripts. Although both KCs and intrahepatic IM share the surface markers F4/80 and CD11b, their transcriptomes point towards distinctive roles during virus-induced chronic hepatitis.</p></div

Unique intrahepatic transcriptomics profiles discriminate the clinical phases of a chronic HBV infection

<div><p>Chronic hepatitis B is a highly heterogeneous liver disease characterized by phases with fluctuations in viral replication and progressive liver damage in some, but not all infected individuals. Despite four decades of research, insight into host determinants underlying these distinct clinical phases—immunotolerant, immune active, inactive carrier, and HBeAg-negative hepatitis–remains elusive. We performed an in-depth transcriptome analysis of archived FFPE liver biopsies of each clinical phase to address host determinants associated with the natural history. Therefore, we determined, for the first time, intrahepatic global expression profiles of well-characterized chronic HBV patients at different clinical phases. Our data, obtained by microarray, demonstrate that B cells and NK/cytotoxic-related genes in the liver, including <i>CD19</i>, <i>TNFRSF13C</i>, <i>GZMH</i>, and <i>KIR2DS3</i>, were differentially expressed across the clinical HBV phases, which was confirmed by modular analysis and also Nanostring arrays in an independent cohort. Compared to the immunotolerant phase, 92 genes were differentially expressed in the liver during the immune active phase, 46 in the inactive carrier phase, and 71 in the HBeAg-negative phase. Furthermore, our study also revealed distinctive transcription of genes associated with cell cycle activity, NF-κB signaling, cytotoxic function and mitochondrial respiration between clinical phases. Our data define for the first time using microarray unique transcriptomes in the HBV-infected liver during consecutive clinical phases. We demonstrate that fluctuations of viral loads and liver damage coincide with fluctuations in the liver transcriptome and point to functional- immune and non-immune- components contributing to the clinical phenotype in patients.</p></div

Hepatic cytokine and interferon transcript levels increase during the peak of LCMV replication but normalize thereafter.

<p>Whole liver RNA was isolated from LCMV infected mice at regular intervals and analyzed for transcription of inflammatory cytokines <i>Tnf</i>, (A) <i>Ifng</i> (B), <i>Il6</i> (C), <i>Ifnb</i> (D) and interferon induced genes <i>Isg15</i> (E) and <i>Oas12</i> (F) using qPCR. Given values on y-axes are relative expression to GAPDH. X-axes shows days post infection. Error bars indicate mean ±SEM. Significance of each time point was assessed using one-way Anova with Dunnett’s Multiple comparison test to day 0. *p<0.05, **p<0.01, ***p<0.001.</p

Clinical Scoring of LCMV infected mice.

<p>Clinical Scoring of LCMV infected mice.</p

Kinetics of distinct F4/80+ cell populations from LCMV infected mouse livers.

<p>Mice were infected with LCMV Cl13 and were sacrificed at day 15, 22 or 39 p.i.. Distinct cell populations were determined using FACS analysis gating strategy of Live/CD45⁺F4/80^highCD11b^int and Live/CD45⁺F4/80^lowCD11b^highLy6c^high (A). Arrow indicates follow-up gate for the selection of Live/CD45⁺F4/80^lowCD11b^highLy6c^high (A). Quantification of F4/80^highCD11b^int (black bars) and F4/80^lowCD11b^highLy6c^high (gray bars) cells as log cells per gram liver (B) isolated from mouse livers (n = 4–6). Statistical significance was assessed using one-way Anova with Dunnett’s Multiple comparison test to day 0. **p<0.01</p

Transcriptomic changes during chronic LCMV-induced hepatitis in liver derived monocytes and Kupffer cells.

<p>Liver derived CD45⁺F4/80⁺CD11b^int KC and CD45⁺F4/80^intCD11b^intLy6c^high IM were sorted and purity of sorted cells was assessed by FACS analyses and overlay graph (A). RNA was isolated from sorted cells at baseline (day 0) and after LCMV infection during the early chronic phase (day 15), chronic phase (day 22) and at time of viral clearance (day 41). Gene expression was measured using the nCounter GX Mouse Immunology Kit. Principal component 1 and 2 comprise 65% of the variance between samples (B). Transcription of myeloid cell defining markers for F4/80⁺CD11b^int (Left) and F4/80^intCD11b^intLy6c^high (right) cells (C). Gene legend is indicated on the right side (C). Y-axis shows days post infection and X-axis indicates relative RNA counts (C). # 29297, 23963, 32090 relative RNA counts for <i>Csf1r</i> day 0, 22, and 41, respectively (C). $ 22720 relative RNA counts for <i>Marco</i> at day 41 (C).</p

Evident LCMV-induced hepatitis with overt clinical symptoms, rise in serum cytokines and intensified F4/80+ cell staining.

<p>During the course of LCMV Cl13 infection mice were weighed (A) and clinically scored using predefined criteria (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166094#pone.0166094.t001" target="_blank">Table 1</a>) (B). At regular intervals mice were sacrificed to determine the intrahepatic LCMV viral load (C). Serum was collected at different time points to measure ALT (D) and cytokines TNF, IFNγ, CXCL1, and IL10 (D) using a multiplex assay. X-axis shows days post infection (A-D). Error bars indicate mean ± SEM (A-D). ND, not determined (C). F4/80 IHC staining was performed at indicated time points to characterize the presence, morphology and localization of F4/80+ cells within the liver (E). Insert at day 0 shows non-primary control staining of mouse spleen (E). Scale bar indicates 100 μm (E).</p

Schematic presentation of patient selection.

<p>(A) Total RNA from liver samples of 94 chronic HBV patients were examined by DASL microarray in 2 batches. A quality-based selection excluded 20 arrays from further analysis. Based on HBV DNA and ALT levels, 52 out of 74 cases were further selected as the core cohort, which have a well-defined clinical HBV phase, and a fibrosis grade of 2 or less, and no evidence of NASH or other complications. Significance Analysis of Microarray, K-means pattern analysis, and modular repertoire analysis were performed to get expression signatures of chronic HBV clinical phases. For validating the expression profiling, NanoString was employed to studied the expression of immune genes in an independent liver FFPE cohort and immunohistochemical staining was applied on 38 paired FFPE samples to evaluating the functional status of altered gene expression. (B) Baseline characteristics of chronic HBV patients divided into four clinical phases based on HBV-DNA and ALT levels. ULN: upper limit of normal (40 U/L).</p