Innate Immune Responses in Viral Hepatitis: the role of Kupffer cells and liver-derived monocytes in shaping intrahepatic immunity in mice using the LCMV infection model

__Abstract__ This study was performed to elucidate the immunological role of the liver in viral hepatitis. The immune functions of the liver are shaped by the intrahepatic cells present during steady state condition, as well as the recruited immune cells during liver inflammation. Liver resident Kupffer cells, by performing endocytosis, determine the functionality of the liver as a filtering organ. Additionally, Kupffer cells produce IL-10. This function, regulated by Ctcf, suggests the potential of Kupffer cells to perform immunoregulation. Monocytes patrol the liver in the steady state, but accumulate in the liver during viral hepatitis. They show functional versatility as demonstrated in the distinct polarization towards TNF-producing and endocytic cells in LCMV-infected and LPS-treated livers, respectively. During LCMV-induced hepatitis, fractions of both Kupffer cells and inflammatory monocytes alter their F4/80 expression, posing a challenge in immunological studies using flow cytometry. Furthermore, in this study we describe distinct clinical responses induced by TLR7 treatment at different phases of LCMV infection, which are associated with distinct states of immune activation. Results from our study contribute to better understand the regulation of intrahepatic immune responses in the steady state condition and during viral hepatitis. Better insights in the functions of Kupffer cells and inflammatory monocytes will open up their potential to be targeted by HBV and HCV therapy. Furthermore, this study emphasizes the importance of characterizing the intrahepatic immune responses during chronic viral hepatitis to understand the mechanisms for the induction of adverse side-effects by TLR7 treatment. This information is valuable in order to prevent or predict the clinical outcome of TLR7-based treatment of HBV or HCV patients. Although this need to be validated in more detail, our findings suggest the significance of evaluating the TLR7 expression levels, either intrahepatic or systemic, in chronically infected patients prior to TLR7 treatment to minimalize the occurrence of adverse side-effects

Liver monocytes and kupffer cells remain transcriptionally distinct during chronic viral infection

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 cli

Kupffer cells express a unique combination of phenotypic and functional characteristics compared with splenic and peritoneal macrophages

The immunostimulatory role of Kupffer cells in various inflammatory liver diseases is still not fully understood. In this study, phenotypic and functional aspects of Kupffer cells from healthy C57BL/6 mice were analyzed and compared with those of splenic and peritoneal macrophages to generate a blueprint of the cells under steady-state conditions. In the mouse liver, only one population of Kupffer cells was identified as F4/ 80highCD11blow cells. We observed that freshy isolated Kupffer cells are endocytic and show a relatively high basal ROS content. Interestingly, despite expression of TLR mRNA on Kupffer cells, ligation of TLR4, TLR7/8, and TLR9 resulted in a weak induction of IL-10, low or undetectable levels of IL-12p40 and TNF, and up-regulation of CD40 on the surface. Kupffer cells and splenic macrophages show functional similarities, in comparison with peritoneal macrophages, as reflected by comparable levels of TLR4, TLR7/8, and TLR9 mRNA and low or undetectable levels of TNF and IL-12p40 produced upon TLR ligation. The unique, functional characteristics of Kupffer cells, demonstrated in this study, suggest that Kupffer cells under steady-state conditions are specialized as phagocytes to clear and degrade particulates and only play a limited immunoregulatory role via the release of soluble mediators

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

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

Kupffer cells and infiltrating monocytes exhibit a distinctive viral antigen associated gene expression.

<p>Hierarchical cluster of complete transcriptome of sorted KC and IM (A). Gene upregulation (left) and down regulation (right) associated with presence of viral antigens in the liver of KC (B) and IM (C). Viral antigen plots show simplified <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166094#pone.0166094.g001" target="_blank">Fig 1C</a> (B, C). Dark red indicates row max value, dark blue indicates row min value (B, C).</p

Clinical Scoring of LCMV infected mice.

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

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

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