Cellular Immunology 284 (2013) 98–103

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Cellular Immunology

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Cross-linking of CD81 by HCV-E2 protein inhibits human intrahepaticplasmacytoid dendritic cells response to CpG-ODN

0008-8749/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.cellimm.2013.07.012

⇑ Corresponding author. Address: Institute of translational medicine, The FirstHospital, Jilin University, 519 E. Minzhu Ave., Changchun 130061, China.

E-mail address: tuzhengkun@hotmail.com (Z. Tu).

Zhengkun Tu a,⇑, Ping Zhang b, Haijun Li a, Junqi Niu c, Xia Jin d, Lishan Su a,e

a Institute of Translational Medicine, The First Hospital, Jilin University, Changchun, Chinab Department of Surgery, The First Hospital, Jilin University, Changchun, Chinac Department of Medicine, The First Hospital, Jilin University, Changchun, Chinad Institute Pasteur of Shanghai, Chinese Academy of Science, Shanghai 200025, Chinae Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

a r t i c l e i n f o

Article history:Received 2 May 2013Accepted 23 July 2013Available online 2 August 2013

Keywords:Plasmacytoid dendritic cells (pDCs)Hepatitis C virus envelop protein (HCV-E)CpG oligodeoxynucleotides (CpG-ODN)Toll-like receptor 9 (TLR9)Cross-linking

a b s t r a c t

Plasmacytoid dendritic cells (pDCs) are reported to be defective in HCV-infected patients, the mecha-nisms of which remain poorly understood. We isolated liver derived mononuclear cells (LMNCs) andpDCs from normal liver tissues of benign tumor dissections and liver transplant donors. Isolated pDCsand LMNCs were cultured with precoated HCV envelop protein E2 (HCV-E2) or anti-CD81 mAb in thepresence of CpG-ODN. Our results show that cross-linking of CD81 by either HCV-E2 or anti-CD81mAb inhibits IFN-a secretion in CpG-induced pDCs; down-regulates HLA-DR, CD80 and CD86 expressionin pDCs; and suppresses CpG-ODN induced proliferation and survival of pDCs. The blockade of CD81 bysoluble anti-CD81 antibody restores pDCs response to CpG-ODN. These results suggest that HCV E2 pro-tein interacts with CD81 to inhibit pDC maturation, activation, and IFN-a production, and may therebycontribute to the impaired innate anti-viral immune response in HCV infection.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

HCV readily establishes persistent hepatic infection in humansand is a major cause of chronic liver diseases worldwide. A numberof possible factors contribute to its persistence, including impair-ment of the host immune response [1]. The precise mechanismsemployed by HCV to promote chronic infections remain poorlyunderstood.

It has been proposed that suboptimal or failed antigen presen-tation during chronic HCV infection may be responsible for de-pressed antiviral T-cell responses. Dendritic cells (DCs) are themost potent professional antigen presenting cells (APC) that playa pivotal role in the initiation and maintenance of CD4+ andCD8+ T-cell responses. There are two distinct subpopulations ofDCs: the myeloid DCs (mDCs) and the plasmacytoid DCs(pDCs).pDCs are innate immune cells that circulate in the bloodand lymphoid tissues, and are specialized to produce copiousamounts of type I IFNs, IFN-a and IFN-b, in response to stimulationby virus-associated single-stranded RNA and unmethylated CpGDNA through the engagement of the Toll-like receptors TLR7 andTLR9 within the endosomal compartment [2–4]. HCV virions havea poor capacity to stimulate TLR7 and TLR9, and additionally

impair pDC production of I FN-a in response to synthetic TLR9agonist but not TLR7 agonists [5,6].

It has been extensively reported that pDCs show a striking def-icit in HCV-infected patients [6–8], the mechanisms of which re-main to be understood. In most previous reports, dysfunction ofpDCs from chronic HCV infected patients has been demonstratedin peripheral blood [7,8]. However, since immune responses toHCV are focused in the liver, changes in blood pDCs may not reflectwhat is happening locally at the site of hepatic infection. It hasbeen demonstrated that an enrichment of DCs occurs in the liveras compared with blood in chronic HCV infected patients [7,8]. InHCV-infected livers, a combination of enhanced mDCs functionand reduced numbers of pDCs may contribute to viral persistencein the face of persistent inflammation [9].

In the present study, we isolated liver derived mononuclearcells (LMNCs) and purified pDCs from normal liver tissues ofbenign tumor dissections and liver transplant donors. The resultswe obtained show that cross-linking of CD81 by incubation witheither HCV-E2 or anti-CD81 mAb inhibits IFN-a secretion inCpG-induced pDCs; down-regulates HLA-DR, CD80 and CD86expression in pDCs; and suppresses CpG-ODN induced prolifera-tion and survival of pDCs. The blockade of CD81 by solubleanti-CD81 antibody restores pDCs response to CpG-ODN. Collec-tively, these results reveal some potential molecular mechanismsby which HCV establishes chronic hepatic infection.

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2. Materials and methods

2.1. Human liver tissue

Human liver tissue was obtained from the Liver Unit of the FirstHospital of Jilin University. Normal human liver tissue was isolatedfrom either spare donor tissue intended for transplantation or fromnormal liver tissue resected from patients having benign hepatictumors. All human tissue samples were collected with local re-search ethics committee approval and patient consent.

2.2. Isolation of liver derived mononuclear cells (LMNCs) from humanliver tissue

Fresh liver tissues were cut into small pieces, washed repeat-edly in PBS + 10% FCS until no further blood or bile was visible inthe washings, and resuspended in RPMI-1640 with 10% FCS. Theywere then homogenized with gentleMACS processor (Miltenyi Bio-tec), filtered through 70 lm nylon mesh, washed in RPMI-1640with 10% FCS, and then layered onto Lymphoprep and centrifugedat 600g for 30 min. Mononuclear cells were aspirated from theinterface, washed and then resuspended in complete RPMI-1640medium (cRPMI-1640). LMNCs were isolated from liver transplantdonors as we have described previously [10–13].

2.3. Purification of pDCs

pDCs were purified from LMNCs by BDCA-4 microbeads (Milte-nyi) according to the manufacturer’s instructions. The purity of theisolated pDCs was verified as being >90% with BDCA-2 antibody.

2.4. Immobilized E2 and CD81 antibody, and cell culture

The effects of CD81 cross-linking were investigated using amicrotiter culture system as described previously [14]. Briefly,microtiter plates (EIA/RIA plate; Corning Inc.) were coated witheither anti-CD81 mAb (5 lg/ml) or HCV-E2 in sodium bicarbonatebuffer pH 8.2. The plates were incubated overnight at 4 �C, andwashed three times with PBS. When immobilizing HVC–E2 ontomicrotiter wells, the wells were first coated with anti-HCV-E2mAb (5 lg/ml). These wells were then washed and exposed to re-combinant HCV-E2 protein (1 lg/ml). Isolated LMNCs and purifiedpDCs were cultured in the precoated microtiter plates in the pres-ence or absence of various CpG-ODNs or a-CD81.

Fig. 1. CD81 expression in LMNCs and pDCs. LMNCs were isolated from fresh normal livepDCs (B and D). Shown is one representative experiment of six.

2.5. Enzyme-linked immunosorbent assay

Concentrations of IFN-a in culture supernatants were measuredby enzyme-linked immunosorbent assay (ELISA) according to themanufacturer’s instructions.

2.6. Flow cytometry

Cells were stained and analyzed by flow cytometry (FACScan;BD Biosciences, San Jose, CA). FITC-labeled anti-human CD81, allo-phycocyanin anti-human BDCA-2, Phycoerythrin (PE)-labeled anti-human CD123, Per-CP-labeled anti-human CD80, CD86, HLA-DRantibodies were purchased from eBioscience (San Diego, CA). Datawere analyzed using FlowJo software (Treestar, Ashland, OR).

2.7. Proliferation and cell survival assays

pDCs proliferation was assessed by 3[H] thymidine incorpora-tion using 2 � 104 cells per well in mAb-coated 96-well plates.Cultures were pulsed after 48 h with 1 Ci/well 3[H] thymidineand then incubated for an additional 16 h. The incorporation of3[H] thymidine in proliferating cells was then measured with aliquid scintillation counter. Cell survival was evaluated by PIstaining.

2.8. Statistical analysis

All data were analyzed and found to be significant using theD’Agostino and Pearson omnibus normality test. Mean valueswere compared using either a paired t test (2 groups) or ANOVA(>2 groups), followed by Bonferroni correction for multiplecomparison test. P values <0.05 were considered significant. Allstatistical tests were performed with Prism software (GraphPad,San Diego, CA).

3. Results

3.1. Human hepatic pDCs express CD81

It has been reported that CD81 molecules are required for HCVentry into hepatoma cell lines [15–17]. CD81 acts as both an HCVattachment receptor with high affinity for HCV-E2 and as a nativefunctional component of signal transduction complexes on the cellsurface of lymphocytes [17–19]. We first evaluated whether

r tissue. CD81 expression was analyzed by flow cytometry on LMNCs (A and C) and

100 Z. Tu et al. / Cellular Immunology 284 (2013) 98–103

freshly isolated human intrahepatic cells including pDCs expressthis receptor. As shown in Fig. 1, most LMNCs and pDCs expressCD81 molecules on their surface.

3.2. Cross-linking of CD81 by HCV-E2 or specific antibody inhibitsA-type CpG-induced pDCs IFN-a secretion

To investigate whether HCV-E2 protein can modulate pDCs inHCV infection, we immobilized HCV-E2 and anti-CD81 mAb sepa-rately on a microtiter plate culture system as previously described[14]. LMNCs and purified pDCs were separately cultured in theprecoated microtiter plates for 24 h in the presence of an A-typeCpG-ODN, CpG-2216, or the control CpG-2243. As shown inFig. 2A, CpG-2216 induces LMNCs to produce an elevated amountof IFN-a, whereas the CpG-2243 control does not. Cross-linkingof CD81by either HCV-E2 or anti-CD81 mAb inhibits IFN-a produc-tion in these CpG-2216 induced LMNCs. Blockade of CD81 with sol-uble anti-CD81 antibody (a-CD81) restores IFN-a production byCpG-2216 induced LMNCs. As shown in Fig. 2B, pure pDCs producean elevated amount of IFN-a (10180 ± 1650 pg/ml) in response toCpG-2216. Cross-linking of CD81 by either HCV-E2 or anti-CD81mAb significantly inhibits IFN-a production in the pDCs(2706 ± 234 and 3167 ± 165 pg/ml, respectively, ⁄⁄p < 0.01). Solu-ble a-CD81 blocks HCV-E2 cross-linking to partially restore IFN-a

Fig. 2. CD81 cross-linking inhibits induction of IFN-a release in LMNCs and purified pDCsCpG-2216 or CpG-2243 control in pre-coated microtiter wells containing either immobilizof blocking by exposure to soluble a-CD81 antibody. The supernatants were assayed fopDCs were cultured for 24 h in the presence of 20 lg/ml of either CpG-2216 or CpG-2243or immobilized HCV-E2 to induce cross-linking and one instance of blocking by exposulevels. The figure shows median levels and standard error from 6 donors (⁄p < 0.05, ⁄⁄p <

production in the CpG-2216 induced pDCs (7632 ± 898 pg/ml,⁄p < 0.05).

3.3. Cross-linking CD81 with HCV-E2 or specific antibody inhibits CpG-OGN induced pDCs maturation

To explore the effect of HCV-E2 on pDCs maturation, purifiedpDCs were cultured in precoated microtiter plates in the presenceof a B – type CpG-ODN, CpG-2006, for 48 h. Surface markers of DCmaturation were analyzed by flow cytometry. As shown in Fig. 3Aand B, CpG-2006 induced HLA-DR (MFI: 214 ± 65), CD86 (MFI:78 ± 16), and CD80 (MFI: 35 ± 7.8) expression on pDCs. Cross-link-ing CD81 with CD81 mAb or HCV-E2 significantly inhibit HLA-DR(MFI: 134 ± 25 and 165 ± 34, respectively, ⁄p < 0.05), CD86 (MFI:39 ± 13 and 27 ± 8.0, respectively, ⁄p < 0.05), and CD80 (MFI:19 ± 4.3 and 15 ± 2.5, respectively, ⁄p < 0.05). Soluble a-CD81partially restores HLA-DR (MFI:198 ± 45), CD86 (MFI:41 ± 6.0),and CD80 (MFI:33 ± 6.6) expression on pDCs.

3.4. Cross-linking CD81 with HCV-E2 or specific antibody inhibits CpG-induced pDCs proliferation and survival

IL-3 is the key cytokine for promoting the generation of pDCsfrom high IL-3R-expressing plasmacytoid precursor cells, and it en-sures their proliferation and survival [20]. In order to evaluate

. (A) Freshly isolated LMNCs were cultured for 24 h in the presence of serially diluteded anti-CD81 mAb or immobilized HCV-E2 to induce cross-linking, and one instance

r IFN-a levels by ELISA. Shown is one representative experiment of six. (B) purifiedcontrol in pre-coated microtiter wells containing either immobilized anti-CD81 mAbre to soluble a-CD81 antibody. The supernatants were assayed by ELISA for IFN-a0.01).

Fig. 3. CD81 cross-linking inhibits CpG-ODNinduced pDC maturation. Purified pDCs were incubated with 50 lg/ml CpG 2006 for 48 h in presence or absence of CD81 cross-linking with one instance of blocking by exposure to soluble a-CD81 antibody. Surface expression of the indicated molecules was determined by flow cytometry. (A) Shown isone representative experiment of six. (B) Histograms represent the mean and standard error of median fluorescence intensities of 6 experiments from 6 independent donors(⁄⁄p < 0.05).

Z. Tu et al. / Cellular Immunology 284 (2013) 98–103 101

whether HCV-E2 inhibits pDCs proliferation and survival, purifiedpDCs were cultured with IL-3 and CpG-2006 for 3 days. As shownin Fig. 4A, CpG-2006 synergizes with IL-3 to induce pDCs prolifer-ation (CPM � 1000: 3.19 ± 0.195). However, cross-linking CD81with anti-CD81 mAb (CPM � 1000: 1.25 ± 0.171, ⁄⁄p < 0.01) orHCV-E2 (CPM � 1000: 1.09 ± 0.129, ⁄⁄p < 0.01) inhibited CpG-2006 and IL-3 induced pDCs proliferation. a-CD81 blockade ofCD81 cross-linking by HCV-E2 partially restores CpG-2006 andIL-3 induced pDCs proliferation (CPM � 1000: 2.27 ± 0.189,⁄p < 0.05). As shown in Fig. 4B, both IL-3 and CpG-2006 inducepDCs survival (% living cells: 74.5 ± 2.81% and 79.35 ± 2.41%,respectively) by either promoting pDCs proliferation or inhibitingpDCs apoptosis. The results in Fig. 4B also indicate that CpG-2006 induced pDCs survival is inhibited by cross-linking CD81with anti-CD81 mAb (% live cells: 28.95 ± 1.30%, ⁄⁄p < 0.01) orHCV-E2 (% live cells: 27.37 ± 2.73%, ⁄⁄p < 0.01). Soluble a-CD81blockade of CD81 cross-linking by HCV-E2 partially restores CpG-2006induced pDCs survival (% live cells: 66.27 ± 1.98%, ⁄p < 0.05).

4. Discussion

HCV entry into hepatocytes is the first step of a virus life cyclethat leads to persistent viral infection. It is a multistep processmediated by HCV envelop glycoproteins E1 and E2 and several hostcell surface receptors. The cell surface receptors mainly include tet-raspanin protein CD81, SR-BI, LDLR and CLDN1 which is a tightjunction protein. Human CD81 is the first receptor identified as

being necessary for HCV entry into host cells. CD81 is a widely dis-tributed cell-surface tetraspanin that participates in differentmolecular complexes on various cell types, including hepatocytes,B lymphocytes, T lymphocytes, natural killer cells and dendriticcells. Consistent with this, we show that both human liver-derivedLMNCs and intrahepatic pDCs express a high level of CD81.

Dendritic cells bridge innate and adaptive immunity and play acentral role in anti-viral defense. Human pDCs are especiallyimportant in viral infections because of their capacity to produceIFN-a in larger amounts than any other cell types. HCV has conse-quently developed strategies to evade the IFN system. The mecha-nisms by which HCV is able to modulate the IFN-a response ofintrahepatic pDCs has been unclear.

HCV-E2 has previously been shown to bind to the mainextracellular loop of CD81, which leads to inhibition of NK cellsactivation [14,21]. To investigate the outcome of an interactionbetween HCV-E2 and CD81 on the IFN-a response of human intra-hepatic pDCs, we developed an assay system in which LMNCs orpurified pDCs are cultured in microtiter plates coated with eitherimmobilized HCV-E2 or anti-CD81 mAb. Human pDCs expressTLR9, which is responsible for recognition of CpG motifs presentin bacterial DNA or ODNs. It is now clearly established that theA-type CpG-ODNs can efficiently stimulate IFN-a production bypDCs [22]. In the present study, we stimulated pDCs withCpG-2216 to induce IFN-a production, and demonstrated thatcross-linking CD81 with either immobilized HCV-E2 or anti-CD81mAb, but not soluble HCV-E2 protein, inhibits IFN-a production

Fig. 4. CD81 cross-linking inhibits CpG-ODN induced pDC proliferation andsurvival. Purified pDCs were cultured with IL-3 and/or CpG-2006 for 3 days in thepresence or absence of CD81 cross-linking and one instance of blocking by exposureto soluble a-CD81 antibody. (A) 3[H] thymidine incorporation assay was used tomeasure cell proliferation. (B) PI staining was used to analyze cell survival. Thefigure shows median levels of experiments from 6 donors (⁄⁄p < 0.01, ⁄p < 0.05).

102 Z. Tu et al. / Cellular Immunology 284 (2013) 98–103

by CpG-2216induced pDCs. In agreement with our findings, Zhangs et al. recently reported a novel mechanism for recognition of HCVin infected hepatoma cells by pDCs which involves the formationof membrane microdomains by CD81- and CD9-associated tetra-spanin webs and lipid rafts [23]. Therefore, our above results withrespect to IFN-a production may provide a potential mechanism bywhich HCV is able to evade the IFN driven immune response.

B-type CpG-ODNs elicited only low levels of type I IFN but in-duced rapid pDCs maturation with increased surface expressionof MHC class II and costimulatory molecules (CD80, CD86, CD40)[22]. Maturated pDCs mixed with activated allogeneic T cellspotentially produce large amounts of IL-12, including its bioactiveform IL-12 p70, to promote Th1 responses [24]. In the presentstudy, the B-type CpG-ODN, CpG-2006, was used to induce pDCsmaturation, and we demonstrated that cross-linking CD81 witheither immobilized HCV-E2 or immobilzed anti-CD81 mAb, butnot soluble HCV-E2 protein, inhibits CpG-2006 induced HLA-DR,

CD80 and CD86 expression on pDCs. In addition to their potencyfor stimulating innate immunity, pDCs can polarize adaptive im-mune responses against exogenous particulate Ag [25]. Theseobservations suggest that monovalent binding between HCV-E2and CD81 per se is insufficient to induce signals that cause defec-tive IFN-a production in pDCs.

CpG-ODNs alone can substitute for the cytokine requirementfor survival of primary human DCs. CpG-ODNs are even superiorto GM-CSF in promoting pDCs survival in tissue culture [26]. Ithas been reported that the numbers of pDCs were significantlylower in HCV infected patients than in healthy controls [7,27–29], and successful antiviral therapy was able to normalize thenumber of pDCs in patients [30]. The present study shows thatcross-linking of CD81 by either HCV-E2 or anti-CD81 mAb inhib-ited CpG-2006 and IL-3 induced proliferation and survival of pDCs,and thus may help to explain the treatment success mentionedabove.

In summary, HCV may employ envelop protein HCV-E2 to inhi-bit, maturation, proliferation, survival and IFN-a production inpDCs. This may contribute to impaired innate and adaptive im-mune response during HCV infection. As yet, we do not know theexact mechanism as to how HCV-E2 interacts with CD81 to inhibitTLR9 signaling in pDCs. It may be that viral particles interact withregulatory receptors on pDCs to impair TLR7 and TLR9 signalingand to also downregulate TLR9 gene expression [31–33]. The lim-ited responsiveness of pDCs may be contributing to reduced innateimmune responses during chronic HCV infection and oncogenesis,and the mechanisms revealed above may represent potential tar-gets for new therapeutic interventions.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgment

We would like to thank Dr. Andrew Kenedy (keenidea@hotmail.com) for editorial assistance.

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