Kupffer Cells Interact With Hepatitis B Surface Antigen In Vivo and In Vitro, Leading to Proinflammatory Cytokine Production and Natural Killer Cell Function

MAJOR ARTICLE Kupffer Cells Interact With Hepatitis B Surface Antigen In Vivo and In Vitro, Leading to Proinflammatory Cytokine Production and Natural Killer Cell Function Arjan Boltjes,1 Nadine van Montfoort,1 Paula J. Biesta,1 Marjoleine L. Op den Brouw,1 Jaap Kwekkeboom,1 Luc J.W. van der Laan,1 Harry L.A. Janssen,1,2 André Boonstra,1 and Andrea M. Woltman1 1 Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, The Netherlands; and 2Division of Gastroenterology, Liver Clinic University Health Network, University of Toronto, Ontario, Canada Keywords. Kupffer cells; macrophages; HBV; HBsAg; innate immunity. Hepatitis B virus (HBV) can cause chronic liver disease and may elicit progressive liver injury, leading to increased Received 8 April 2014; accepted 15 October 2014; electronically published 31 October 2014. Presented in part: 24th Annual Meeting of the European Macrophage and Dendritic Cell Society, Edinburgh, United Kingdom, September 2010; Monothematic Conference on Immune-Mediated Liver Injury, European Association for the Study of the Liver, Stratford-upon-Avon, United Kingdom, January 2012; Dutch Society for Immunology Annual Meeting, Noordwijkerhout, The Netherlands, December 2012; Spring Meeting of the Dutch Society for Hepatology, Veldhoven, The Netherlands, March 2013; International Meeting on Molecular Biology of Hepatitis B Viruses, Shanghai, China, 2013. Correspondence: Andrea M. Woltman, PhD, Department of Gastroenterology and Hepatology, Erasmus MC University Medical Centre, ‘s-Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands (). The Journal of Infectious Diseases® 2015;211:1268–78 © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: . DOI: 10.1093/infdis/jiu599 1268 • JID 2015:211 (15 April) • Boltjes et al risk of developing liver cirrhosis, liver failure, and liver cancer [1, 2]. The immunological mechanisms determining the induction of effective antiviral immunity leading to self-limiting hepatitis or the lack of effective immune response toward the virus leading to chronic hepatitis B are still unclear. Although recently an HBV receptor has been identified on hepatocytes [3], the early steps in virus recognition by immune cells and the functional consequences of this interaction remain to be resolved. Kupffer cells (KCs) are the resident macrophages of the liver [4, 5] that, besides being maintained by liverderived precursors [6], have been shown to derive from monocytes [7] in a macrophage colony-stimulating factor (M-CSF)–dependent manner [5, 8]. They form, together with the sinusoidal endothelial cells, the first barrier for pathogens to enter the liver [9]. In humans, KCs have been identified by CD68 and CD14 expression Background. Based on their localization, Kupffer cells (KCs) likely interact with hepatitis B virus (HBV). However, the role of KCs in inducing immunity toward HBV is poorly understood. Therefore, the interaction of hepatitis B surface antigen (HBsAg) and KCs, and possible functional consequences, were assessed. Methods. KCs in liver tissue from patients with chronic HBV were analyzed for presence of HBsAg and their phenotype, and compared with KCs in control liver tissue. Liver graft perfusate–derived KCs and in vitro–generated monocyte-derived macrophages were investigated for functional interaction with patient-derived HBsAg. Results. Intrahepatic KCs were HBsAg positive and more activated than those from control livers. KCs internalized HBsAg in vitro, which did not change their phenotype, but strongly induced proinflammatory cytokine production. Additionally, monocyte-derived macrophages also interacted with HBsAg, leading to activation and cytokine production. Furthermore, HBsAg-exposed macrophages and KC activated natural killer (NK) cells, resulting in increased CD69 expression and interferon-γ production. Conclusions. KCs directly interact with HBsAg in vivo and in vitro. HBsAg-induced cytokine production by KCs and monocyte-derived macrophages and subsequent NK cell activation may be an early event in viral containment and may support induction of HBV-specific immunity upon HBV infection, but may also contribute to liver pathology. MATERIALS AND METHODS KC Phenotype Excess material of percutaneous needle liver biopsies obtained from 7 chronic HBV patients (Table 1) and 14 incisional biopsies obtained from donor liver during transplant was dissected, incubated in Roswell Park Memorial Institute medium (RPMI; Lonza) containing 0.5 mg/mL collagenase (Sigma-Aldrich) and 0.1 mg/mL DNAse (Roche), penicillin/streptomycin (Gibco), L-glutamine (Lonza), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Lonza) for 30 minutes, filtered through a 70-µm nylon cell strainer (BD) to acquire a cell suspension. Subsequently, cells were stained with antibodies against CD11b (ICRF44; BD Pharmingen), CD16 (CB16; eBioscience), CD14 (61D3; surface KC marker), CD40 (5C3), human Table 1. Patient Characteristics Patient No. Age, y Sex HBV Genotype Viral Load, gEq/mL HBeAg Status ALT, U/L 209 1 49 F ND 4.78 × 101 – 2 36 F D 7.01 × 103 – 53 3 4 44 26 M F C A 6.63 × 106 3.55 × 107 + – 50 50 5 19 M A 1.68 × 109 + 97 6 7 24 27 F F C B 1.05 × 109 2.86 × 107 + + 41 164 Abbreviations: ALT, alanine aminotransferase; gEq, genome equivalent; HBeAg, hepatitis B early antigen; HBV, hepatitis B virus; ND, not determined. leukocyte antigen (HLA)-DR (LN3; all eBioscience), CD45 (2D1; BD; for selection on hematopoietic/immune cells), CD80 (MAB104; Beckman Coulter), CD86 (IT2.2), and HLAABC (W6/32; both Biolegend) in phosphate-buffered saline (PBS) containing 1% human AB-serum (Lonza) and 0.02% sodium azide. Data were acquired on a FACSCanto system (BD) and analyzed with FlowJo software (Tree Star, Inc). HBsAg Staining on Patient-Derived Blood and Liver Tissue Excess material of percutaneous needle liver biopsies were obtained from 13 chronic HBV patients and 9 non-HBV liver disease patients (Table 2), collected in RPMI and filtered through a 70- µm nylon cell strainer (BD) to acquire a single-cell suspension. In parallel, peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of these patients by FicollPaque (GE Healthcare) density gradient centrifugation. Liver cells and PBMCs were stained with antibodies against CD14 (MOP9) and CD45 (SK7; both BD) in PBS/1% fetal calf serum (FCS) (Sigma)/0.02% sodium azide, fixed with 2% formaldehyde, permeabilized with 0.5% saponin (VWR), and stained with an HBsAg-specific antibody (recognizing subtypes HBsAg Ad and Ay; Acris Antibodies GmbH). Data were acquired by flow cytometry and analyzed as described above. The medical ethical committee of the Erasmus MC University Medical Center declared to have no objections against the use of excess patient biopsy material, and all live (...truncated)