View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central Journal of Viral Hepatitis, 2009, 16, 315–324 doi:10.1111/j.1365-2893.2008.01011.x A class C CpG toll-like receptor 9 agonist successfully induces robust interferon-alpha production by plasmacytoid dendritic cells from patients chronically infected with hepatitis C 1 1,2 1,2, , 1, N. A. Libri, S. J. Barker, W. M. C. Rosenberg * and A. E. Semper * 1iQur Ltd, Southampton General Hospital, Southampton, UK; and 2Liver Group, Division of Infection Inflammation and Repair, University of Southampton, Southampton General Hospital, Southampton, UK Received December 2007; accepted for publication April 2008 OnlineOpen: This article is available free online at www.blackwell-synergy.com SUMMARY. Immunomodulators that induce local endoge- nors produced substantial levels of IFN-a in response to three nous interferon-alpha (IFN-a) production by plasmacytoid of the four agonists and yet only one TLR9 agonist, a class C dendritic cells (pDCs) may offer new strategies for the CpG oligodeoxynucleotide (ODN), induced robust IFN-a treatment of patients chronically infected with the production by pDCs from cHCV patients. Proinflammatory hepatitis C virus (HCV). However, such an approach may be cytokine production and phenotypic maturation in response compromised if reports are true that IFN-a production by to all four agonists was equivalent in infected and uninfected pDCs from patients with chronic HCV (cHCV) is profoundly pDCs. These data point to a profound but selective defect in impaired. To address the question of pDC dysfunction in IFN-a production by pDCs from cHCV donors. Nonetheless, cHCV more definitively, in the present study a panel of four a class C CpG ODN successfully induced robust IFN-a prototypic synthetic agonists of toll-like receptor 7 (TLR7) or production, suggesting that this class of TLR9 agonist may TLR9 were administered in vitro to pDCs purified from cHCV have utility as a future immunotherapeutic for the treatment patients and from normal uninfected donors and their of chronic HCV infection. responses compared in terms of not only IFN-a production but also the global expression of other cytokines and phe- Keywords: CpG, dendritic cell, hepatitis C, interferon-a, notypic maturation. Plasmacytoid DCs from uninfected do- plasmacytoid, toll-like receptor. lead to cirrhosis, end-stage liver disease and hepatocellular INTRODUCTION carcinoma, poses a considerable health burden. The hepatitis C virus (HCV) is a blood-borne, hepatotrophic The mainstay of current therapies for cHCV is interferon virus which establishes chronic HCV (cHCV) infection in up to alpha (IFN-a), a type 1 interferon typically administered as 85% of cases. With an estimated 2% of the global population pegylated, single-species IFN-a2 in combination with the infected with HCV [1], the potential for chronic infection to antiviral ribavirin. IFN-a has multiple antiviral actions [2]. It induces genes that inhibit viral replication [3] and boosts the innate and adaptive immune response, activating NK cells [4] *These two senior authors contributed equally to the study. and myeloid DCs [5] respectively. IFN-a also promotes the Present address: Institute of Hepatology, 69-75 Chenies Mews, development of a polarized antigen-specific type 1 T-cell University College Medical School, London, WC1E 6HX, UK response [6,7] thought to favour resolution of HCV infection [8] and pDC-derived IFN-a supports the induction of CD8+ Abbreviations: cHCV, chronic HCV; HCV, hepatitis C virus; IFN-a, interferon-alpha; IRF-7, interferon-regulatory factor-7; MFI, mean T cell responses [9]. Nonetheless, combined pegylated IFN- fluorescence intensity; NF-jB, nuclear factor jB; NHD, normal a2 ⁄ ribavirin therapy succeeds in only 42–46% of HCV healthy donors; NR, non-responders; ODN, oligodeoxynucleotide; genotype 1 infections and 76–80% of genotype 2 ⁄ 3 infections PBMCs, Peripheral blood mononuclear cells; pDCs, plasmacytoid [2]. Furthermore, the raised systemic levels of IFN-a can lead dendritic cells; TLR7, toll-like receptor 7. to psychiatric and other adverse events necessitating pre- Correspondence: Dr Amanda E. Semper, MP 811, Level D, South- mature cessation of treatment in approximately 10% of cases ampton General Hospital, Tremona Road, Southampton SO16 6YD, [10]. Alternative novel strategies for the treatment of cHCV UK. E-mail: [email protected] are therefore required. Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd 316 N. A. Libri et al. One approach being explored is the use of immunomod- diagnosis of cHCV disease were recruited from hepatology ulators to induce endogenous, local IFN-a production by clinics run by Southampton University Hospitals NHS Trust. plasmacytoid dendritic cells (pDCs). In response to viral All gave written informed consent. Patients treated for HCV infection, pDCs are stimulated to migrate to lymph nodes within the preceding 6 months or known to be coinfected where they produce large amounts of type I interferon with another blood-borne virus were excluded from the (IFN-a, IFN-b, IFN-x and IFN-k) and a broad spectrum of study. Table 1 summarizes the clinical characteristics of the IFN-a subtypes [11–13]. Plasmacytoid DCs sense viral cHCV patients and the 25 normal healthy donors (NHD) infection through innate pattern recognition receptors of the used as controls. NHD subjects had no known risk factors for toll-like receptor (TLR) family, specifically TLR7 and TLR9 blood-borne virus infection. [14]. Upon ligation, TLR7 and TLR9 trigger a cascade of shared intracellular signalling events which culminate in Plasmacytoid DC preparation abundant IFN-a production together with enhanced pro- duction of other cytokines and pDC maturation [15,16]. Peripheral blood mononuclear cells (PBMCs) were separated Thus, TLR7 or TLR9 may be appropriate pharmacological from fresh EDTA-anticoagulated blood obtained from cHCV targets for immunomodulation of IFN-a production by pDCs, patients (typically 25–50 mL) or uninfected subjects with the diverse range of endogenous type I IFN species (100–400 mL), by Lymphoprep (Robbins Scientific, Solihull, produced offering an advantage over the administration of an exogenous single species of IFN-a. Table 1 Subject clinical characteristics The natural ligands for TLR7 are patterns within viral single-stranded RNAs [17] but synthetic ligands including Normal imidazoquinolines [18] and guanosine analogues [19] are cHCV healthy also recognized. TLR9 recognizes unmethylated CpG dinu- patients subjects Characteristics (n = 37) (n = 25) cleotide-rich sequences within bacterial and viral DNA, which can be mimicked by synthetic CpG oligodeoxynucleotides Age (years); 49 (36–75) 48 (23–75) (ODNs) [20]. Three categories of CpG-ODNs have been iden- mean (range) tified: classes A (or ÔDÕ), B (or ÔKÕ) and C. CpG-A ODNs favour Gender (M ⁄ F) 27 ⁄ 10 20 ⁄ 5 IFN-a production over pDC differentiation and are poor B-cell ALT (IU ⁄ L); 88 (18–278) N ⁄ A activators, whereas CpG-B ODNs show the converse [21]. mean (range) CpG-C ODNs combine strong IFN-a induction and efficient pDC HCV viral 4.86 · 106 N ⁄ A maturation [22,23] and efficiently stimulate B cells. load (IU ⁄ mL); (1.02 · 104 A possible hurdle to using TLR7 or TLR9 agonists as mean (range)* )3.01 · 107) inducers of endogenous IFN-a production in cHCV are reports Genotype that pDCs derived from cHCV patients are profoundly 120N⁄ A impaired in their ability to produce IFN-a upon in vitro 2 ⁄ 313 stimulation [24–31]. However, other studies have found 41 IFN-a production by cHCV–pDCs to be intact [32–35]. Non-1 3 à Therefore, the purpose of the current study was to use a panel Disease severity Mild 16 N A of prototypic synthetic TLR7 and TLR9 agonists to determine ⁄ Mild ⁄ moderate 4 if normal function can successfully be induced in pDCs taken Moderate 10 from cHCV subjects. The TLR7 agonists selected were Severe (+cirrhosis) 7 (6) loxoribine (a guanosine analogue) and imiquimod (an Treatment 24 ⁄ 13 N ⁄ A imidazoquinoline). The TLR9 agonists were the class A CpG, naı¨ve ⁄ non-responder ODN2216 [36] and the class C CpG, M362 [22]. The function of pDCs purified from chronically-HCV infected and normal, *Viral load determined for 31 patients by quantitative uninfected subjects was investigated not only in terms of PCR (Cobas Amplicor HCV Monitor test; Roche Molecular IFN-a production, but also with respect to the production of a Systems, Pleasanton, CA, USA). In the remaining six range of other cytokines and pDC maturation. patients, presence of HCV virus was confirmed using a qualitative PCR assay. MATERIALS AND METHODS HCV genotype determined using PCR-based ÔGenotyping for Treatment AssayÕ (iQur Ltd, Southampton, UK), with Study subjects exception of Ônon-1Õ samples which used a superceded assay. à Based on histological analysis of biopsies for inflammation Ethical approval was obtained from Southampton and South and fibrosis. In the absence of histological evidence, disease West Hampshire Joint Research Ethics Committee. Thirty- status was determined by clinical criteria including physical seven patients with detectable HCV RNA and a clinical examination, diagnostic imaging and laboratory indices. Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd A CpG-C induces IFN-a production by pDCs in cHCV 317 UK) density gradient centrifugation. Plasmacytoid DCs and stored on ice in the dark pending data acquisition using (89–95% pure) were isolated from PBMCs by