DDW2012: HBsAg suppresses Innate and Adaptive

[StephenCastlecrag]

Hepatitis B Virus Surface Antigen Suppresses Innate and Adaptive Immune Responses of Murine and Human Liver Cells  Mo1538 |  Min Jiang1, Martin Trippler1, Ruth Bröring1, Lena Poggenpohl1, Guido Gerken1, Mengji Lu2, Joerg F. Schlaak1

Affiliation
1Gastroenterology and Hepatology, University Hospital, Essen, Germany; 2Dept. of Virology, University Hospital, Essen, Germany

Abstract:

Background: It has recently been shown that Toll-like receptor (TLR) signaling in murine non-parenchymal (NPC) liver cells is suppressed in the presence of Hepatitis B surface antigen (HBsAg). It is not clear, however, whether this is also relevant for adaptive and human immune responses and how this effect is mediated.
Methods: Peripheral blood mononuclear cells (PBMCs) from chronic HBV infected patients were stimulated by TLR3 ligands in the absence or presence of autologous serum. Murine Kupffer cells (KC), sinusoidal endothelial cells (LSEC) and hepatocytes as well as human LSEC and hepatocytes were stimulated with TLR3 ligand Poly I:C in the presence or absence of HBsAg. Expression of cytokines and TLR3 was analyzed by quantitative rt-PCR. Mixed lymphocyte reactions (MLR) were performed to study T cell activation induced by TLR-stimulated NPC. Activation of transcription factors was assessed by western blot and reporter gene assays.
Results: Activation of T lymphocytes that was induced by TLR3-activated NPC was potently suppressed by HBsAg. This suppressive effect was associated with enhanced IL-10 production that could be reverted by IL-10 antibodies. While Poly I:C-induced TLR3 expression remained unchanged, TLR-induced activation of NFκB, IRF-3 and MAPKs was potently suppressed by HBsAg. TLR3-induced production of IFN-γ was suppressed in PBMC from patients with high HBsAg-levels (p<0.0006) compared to PBMC isolated from patients with low HBsAg-levels. In the presence of autologous serum, TLR3 induced IFN-γ production was inhibited in case of HBV-containing serum.
Conclusions: These findings may, at least in part, explain how HBV evades innate and adaptive immune responses to establish a persistent infection.

Disclosure(s):
The following people have nothing to disclose: Min Jiang, Martin Trippler, Ruth Br&#246;ring, Lena Poggenpohl, Guido Gerken, Mengji Lu, Joerg F. Schlaak

[StephenCastlecrag]

This is another one, a real gem:
Hepatitis B Virus Infection Leads to Alterations in the Repertoire of Self Lipid Antigens Presented by Hepatocytes and Is Associated With Direct and Indirect Activation of Natural Killer T Cells  3:30 | 742 |  Sebastian Zeissig1, 2, Kazumoto Murata3, Lindsay Sweet1, Jean Publicover4, Zongyi Hu3, Arthur Kaser1, Alexander Arlt2, Rainer Günther2, Jochen Hampe2, Stefan Schreiber2, Jody Baron4, D. Branch Moody1, T Jake Liang3, Richard S. Blumberg1

Affiliation
1Brigham and Women's Hospital, Boston, MA; 2University Medical Center Schleswig-Holstein Kiel, Kiel, Germany; 3National Institutes of Health, Bethesda, MD; 4University of California, San Francisco, San Francisco, CA

Abstract:

Control of HBV infection is achieved by virus-specific T and B cells but little is known about the mechanisms leading to priming of these cells. Recent observations suggest that innate immune cells respond early during infection and may contribute to the outcome of disease. Using different approaches including murine studies of adenoviral HBV delivery (Ad-HBV), transgenic HBV expression, and infection of primary human and murine hepatocytes with natural HBV isolates and Ad-HBV, we have investigated the mechanisms of early immune responses against HBV.
We demonstrate, based on lipidomic analyses, that HBV infection of hepatocytes and small HBsAg-dependent recruitment of host endoplasmic reticulum (ER) lipids for the viral envelope are associated with alterations in hepatocyte lipids leading to a marked increase in abundance of selected phospholipids including phosphatidylethanolamine. Non-antigenic phospholipids are converted to antigenic lysophospholipids by infected hepatocytes through the action of small HBsAg-induced secretory phospholipase A2 enzymes and are loaded onto the atypical MHC class I molecule CD1d in a manner dependent on microsomal triglyceride transfer protein (MTP). CD1d-restricted presentation of antigenic lysophospholipids by hepatocytes is associated with direct, IL-12-independent activation of hepatic non-invariant NKT cells expressing diverse T cell receptors (TCRs). Whereas invariant (i) NKT cells, the most frequent NKT cell population in the liver that is characterized by expression of a semi-invariant TCR, also exhibit rapid activation upon Ad-HBV infection in vivo, iNKT cells are activated in a largely IL-12-dependent, indirect manner. Consistently, studies using novel reporter mouse models that allow for selective tracking of invariant and non-invariant NKT cells demonstrate that administration of blocking anti-IL-12 antibodies prevents activation of invariant but not non-invariant NKT cells. NKT cells directly suppress viral replication through secretion of IFN-γ and contribute to the generation of adaptive HBV-specific T and B cell responses. Accordingly, mice with genetic deletion of CD1d and CD1d-restricted NKT cells as well as mice with hepatocyte-specific deletion of MTP exhibit broad defects in HBV-specific T and B cell responses, impaired control of viral replication and chronic hepatitis.
These results demonstrate a novel and unanticipated role of hepatocytes in the immune response against HBV through sensing of HBV-induced alterations in the repertoire of self lipids and MTP-, CD1d-, and NKT-cell-dependent priming of HBV-specific T and B cells.

Disclosure(s):
Jochen Hampe - Grant/Research Support: Metanomics, Bayer AG; Speaking and Teaching: Falk Foundation
Stefan Schreiber - Advisory Committees or Review Panels: Abbott, AstraZeneca Pharmaceuticals, Bayer AG, Berlex Laboratories, Bristol-Myers Squibb, Centocor, Chemocentryx, Ferring Pharmaceuticals, Otsuka Pharma., Progenika Biopharma , Genentech, Schering-Plough, Shire Pharmaceuticals Group, UCB Pharma, Novartis Pharmaceuticals, Pfizer Inc , NovoNordisk; Grant/Research Support: AstraZeneca Pharmaceuticals, UCB Pharma, Shire Pharmaceuticals Group; Speaking and Teaching: Falk Foundation
The following people have nothing to disclose: Sebastian Zeissig, Kazumoto Murata, Lindsay Sweet, Jean Publicover, Zongyi Hu, Arthur Kaser, Alexander Arlt, Rainer G&#252;nther, Jody Baron, D. Branch Moody, T Jake Liang, Richard S. Blumberg

[StephenCastlecrag]

Many thanks for the explanations. Here is another DDW2012 paper:

Intrahepatic NK Cell Activation Is Dependent on Levels of HBsAg in Chronic HBeAg-Negative Hepatitis B  3:15 | 741 |  Eric T. Tjwa, Roeland Zoutendijk, Gertine van Oord, Joanne Verheij, Paula J. Biesta, Andrea Woltman, Andre Boonstra, Harry L. Janssen

Print   Affiliation
Erasmus Medical Centre Rotterdam, Rotterdam, Netherlands

Abstract:

Background Chronic hepatitis B (CHB) is the result of a failing immune response towards the virus. Blood natural killer (NK) cells serve as first-line defence against viral intruders, and these cells are functionally impaired in blood of CHB patients. Since little is known on liver NK cells in CHB, we aimed to investigate the activation status and function of intrahepatic NK cells in both HBeAg positive and negative CHB.
Methods Liver cells were isolated from biopsy specimens of 56 CHB patients. Liver leukocytes were stimulated for 24 h with IL-12/-18. Specimens were also processed for routine histopathological grading and staging according to Ishak scoring. NK cell frequencies, activation status (CD69) and function of NK cells were analysed by flow cytometry.
Results Our cohort consisted of 21 HBeAg positive and 35 HBeAg negative CHB patients. As expected, the levels of HBV-DNA and HBsAg were significantly higher in HBeAg positive disease (p<0.01). There was a moderate to strong correlation between HBV-DNA and HBsAg in both HBeAg positive and -negative disease. Frequency of liver NK cells and activated liver NK cells, as evidenced by CD69 positivity, was resp. 1.5 and 6-fold higher than in blood (p<0.001). Upon stimulation, the frequency of CD69 positive liver NK cells increased less in liver than in blood (1.1 vs 4.5-fold) regardless of HBeAg status. In line with this, the frequency of IFN-γ expressing NK cells upon stimulation was 3 fold higher in blood than in liver (p<0.001). There was no difference between HBeAg positive and -negative CHB. Liver NK cell activation and IFN-γ expression was significantly correlated in HBeAg negative CHB (r=0.50, p<0.05). In this population, also HBsAg quantification correlated with liver NK cell activation (r=-0.60, p<0.05), but not with cytokine-expression or with levels of HBV-DNA. Liver histology (grading and staging) correlated neither with liver NK cell activation nor cytokine-expression in both HBeAg positive and -negative disease.
Conclusions Our findings demonstrate that frequency and activation of NK cells differ strongly between the liver and blood. We further show that in chronic HBV patients, IFN-γ production is
compromised despite a high NK cell activation status, which suggests exhaustion of specific functions of liver NK cells in CHB. We demonstrate for the first time that in HBeAg-negative CHB but not in HBeAg positive CHB, increasing HBsAg levels coincide with less liver NK cell activation, suggesting a role for HBsAg in inflammation but not in pathology. As a consequence, monitoring HBsAg levels during antiviral therapy may reflect modulation of the antiviral intrahepatic immune response.

Disclosure(s):
Harry L. Janssen - Consulting: Bristol-Myers Squibb Co. , Gilead Sciences Inc, Novartis Pharmaceuticals, Roche Pharma AG, Santaris, Medtronic Inc. , Kirin, Abbott, DebioPharm S.A.; Grant/Research Support: Bristol-Myers Squibb Co. , Gilead Sciences Inc, Novartis Pharmaceuticals, Roche Pharma AG, Santaris, Medtronic Inc. , Anadys, Innogenetics
The following people have nothing to disclose: Eric T. Tjwa, Roeland Zoutendijk, Gertine van Oord, Joanne Verheij, Paula J. Biesta, Andrea Woltman, Andre Boonstra

[studyforhope]

The nonparenchymal liver cells (NPCs) are the nonhepatocyte cells in the liver, mostly part of the liver resident immune cell compartment. Macrophages, Kupfer cells, dendritic cells, Tcells of all classes, Bcells,  Nk cells NKT cells and importantly also the lining of the sinusoids, the endothelial cells participate in immune responses having the capacity to present antigen just like Macrophages.

TLR3 receptors are in the endosomal compartment of cells, not on the surface. They are waiting for double stranded RNA or similar molecules to be activated. It is one of the signals to the cells of viral invasion.

The presentation basically shows  that the mechanisms for proinflammatory signaling and cytokine production effects (by analysis of mRNA for NFKB and gamma interferon) are inhibited in the presence of higher concentrations of HbSAg. as well as the stimulation of Tcells by these APCs.

This will of course impair globally all Tcell activations, but it is to be emphasized that the sheer mass effect of the surface antigen will have a major contribution to the elimination/exhaustion of surface antigen specific Tcells. Any engaged Tcell will additionally not reiceive the necessary strong activation signal since the APcs themselves will not be fully activated /stimulated as shown in this presentation..

Neverthless chronic HBV patients do not seem to have a noticeable general immune defect in the sense that they respond inadequately against common viral infections.

[StephenCastlecrag]

So what are the non-parenchymal (NPC) livers cells?
These NPC have TLR3  - are these receptors on the surface of the cells or within the cells? What normally bind to these receptors?
The rest is too confusing to understand unless T lymphocytes in the liver are NPC cells. But PBMCs do not seem to be NPCliver cells???