Gene expression profile data (GSE38941 and GSE65359) were downloaded from GEO: https://www.ncbi.nlm.nih.gov/geo (34). Student’s t-tests, hierarchical clustering, and heat map analysis were performed using MultiExperiment Viewer (MeV) (35). Gene set enrichment analysis (GSEA) was performed using the GSEA software (36).

Twenty patients with chronic hepatitis B were selected randomly from the cohort studied in our lab previously (37). The patients received continuous antiviral treatment of 1.5 μg/kg Peg-IFNα-2b (PegIntron, Schering-Plough, Kenilworth, NJ, USA) weekly or Peg-IFNα-2b weekly combined with 10 mg adefovir dipivoxil (ADV, Hepsera, Gilead Sciences, Foster City, CA, USA) daily for 48 weeks. Patient sera collected at 0 and 48 weeks were tested for CCL5 concentration by BD Cytometric Bead Array.

All serum samples of HBV patients were collected at the Department of Infectious Diseases, the First Affiliated Hospital of Anhui Medical University from June 2012 to July 2014. Written informed consent was obtained from all patients donating blood samples. The study was approved by the ethics committee of the First Affiliated Hospital of Anhui Medical University (Grant No. K2010003) and was carried out in accordance with the approved guidelines. This clinical research was also enrolled in the Chinese Clinical Trial Registry (Clinical trial registration number: ChiCTR-TRC-12002226). The characteristics of the HBV patients are listed in Table 1.

The cell lines SK-Hep-1 and Huh 7 were provided by the Cell Bank of the Chinese Academy of Sciences. The cell lines HepG2, HepG 2.2.15, and 293A were maintained in our laboratory. The cells were cultured in DMEM containing 10% FBS (Sigma or Gibco) and antibiotics (100 U/ml penicillin, 80 μg/ml streptomycin). Human sinusoidal endothelium cells (HSECs) were purchased from Sciencell. The HSECs were cultured with Endothelial Cell Medium (Sciencell) at 37°C with 5% CO₂, and the culture medium was renewed every other day.

The pAAV-HBV1.2 plasmid was provided by Pei-Jer Chen (National Taiwan University, Taipei) and maintained in our laboratory. The pAAV plasmid was subcloned from pAAV-HBV1.2.

The HBV subtype adw sequence was subcloned from the rAAV-HBV1.3 virus (FivePlus Molecular Medicine Institute, Beijing, China). The pAAV-HBV1.3 plasmid was constructed by ligating 1.3 copies of the HBV sequence (subtype “adw,” NsiI + EcoRI-cleaved fragment, and EcoRI + BglII-cleaved fragment) into the pAAV plasmid. The pAAV-HBV1.3 plasmid was digested using NsiI, and the HBV sequence fragment was cleaved off and then ligated into the NsiI-linearized pAAV-HBV1.3 plasmid to construct the pAAV-HBV2.3 plasmid.

CCL3 promoter (2 kbp upstream CCL3) was cloned and inserted to replace the CMV promoter of pEGFP plasmid. Then, luciferase reporter sequence was cloned downstream the CCL3 promoter to construct pCCL3p-luc-eGFP plasmid.

Plasmid transfection of SK-Hep-1, Huh 7, and HepG 2 cells was performed using Lipofectamine 2000 or Lipofectamine 3000 (Invitrogen). HSECs were transfected with FuGENE HD (Promega). siRNAs were synthesized by GenPharma, Shanghai, China, and then transfected into cells with RNAiMAX (Invitrogen). Cells were plated 24 h before transfection. All of the transfection experiments were performed according to the manufacturer’s instructions. Briefly, 1 μg/ml plasmid was transfected using 4 μl Lipofectamine 2000/3000 or 2 μl FuGENE HD. The 20 pmol siRNA was transfected with 4 μl RNAiMAX in 24-well plate. Fresh culture medium with 10% FBS was added 24 h post-transfection.

SK-Hep-1 cells were transfected with pCCL3p-luc-eGFP plasmid and selected with G418 (Gibco) for stable expression. Then, the cells were challenged with different stimulants. Forty-eight hours later, 150 μg/ml of firefly luciferin was added to the cell culture supernatant and bioluminescence was determined immediately.

Cell culture supernatant was collected and centrifuged at 16,000 g for 10 min to remove cell fragments, and serum samples were prepared by spinning coagulated blood and collecting the supernatant. Tissue samples were grinded with cell lysis buffer on ice centrifuged at 16,000 g for 10 min, and then the supernatant was collected. CCL3 and CCL5 enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D. Experiments were carried out according to the protocols of the manufacturer’s instructions.

Biotin-labeled HBV DNA was amplified by PCR with the 5′-biotin sense primer and anti-sense primer from the pAAV-HBV2.3 plasmid, excised from the agarose gel, and then transfected into SK-Hep-1 cells for 6 h. Then, the cells were lysed with NP-40 lysis buffer (150 mM NaCl, 50 mM Tris–HCl, pH = 7.4, 1 mM freshly added PMSF). The DNA-binding proteins were pulled down with a μMACS Streptavidin kit (Miltenyi Biotec). Mass spectrometry results were obtained by APTbiotech, Shanghai, China. Briefly, protein samples were digested with trypsin for 20 h. Then, protein peptides were identified by liquid chromatography (Zorbax 300SB-C18; Agilent) and tandem mass spectrometry (Thermo Finnigan). The mass spectral data were searched against UniProt human proteomic database using the Mascot 2.2 software.

C57BL/6 mice were purchased from Vitalriver, Beijing, China. All mice were kept in a specific pathogen-free (SPF) microenvironment, receiving care in compliance with the guidelines set forth in the Guide for the Care and Use of Laboratory Animals. All experiments were approved by the Ethical Review Board of the School of Life Science, University of Science and Technology of China. Mice aged between 6 and 8 weeks were used for these experiments. For virus infection, the plasmid of recombinant adenovirus that transfers a 1.3-fold overlength HBV genome (Ad-HBV) was provided by Prof. Ulrike Protzer (Technischen Universität München). Ad-null virus was purchased from the FivePlus Molecular Medicine Institute. The virus was packaged and amplified in 293A cells, and the titer was determined by plaque formation. A total of 1 × 10⁹ pfu/200 μl virus was injected intravenously (i.v.) into each mouse. Six days later, the mice were sacrificed.

Livers were ground to pass through a 200-gauge stainless steel mesh. The precipitate was re-suspended in 40% Percoll (Sigma) and centrifuged at 2400 rpm for 10 min at room temperature. Then, the precipitate was collected and re-suspended in RBC Lysis Buffer (BioLegend), incubated at room temperature for 10–15 min, and then washed twice in PBS. Splenocytes were obtained in the same manner, except for Percoll re-suspension. Flow cytometry was performed, as previously described (38).

Samples were diluted according to the manufacturer’s instructions if necessary. Then, incubated with the mixed beads at 4°C for 30 min, washed the beads with PBS twice, and applied them on flow cytometry.

Serum ALT activity was measured using a commercially available kit (Rong Sheng, Shanghai, China).

Cells or frozen tissue sections were fixed in 4% paraformaldehyde (PFA) for 15–20 min, followed by permeabilization with 1% Triton X-100 for 15–20 min, and blocking with 5% donkey serum for 1 h at room temperature. Then, the sections were incubated with the primary antibodies at 4°C overnight. Secondary antibodies were incubated with the sections at 37°C for 3 h. The nuclei were stained with DAPI (1 ng/ml) for 3 min at room temperature. Confocal images were acquired on a Zeiss LS710 microscope, and GSD super-resolution images were acquired using the Leica 3D GSD system.

Cell or tissue mRNA was isolated with TRIzol and reverse transcribed into cDNA with M-MLV (Invitrogen). Relative quantitative real-time PCR analysis was performed on Roche LightCycler 96 using the SYBR Premix Ex Taq II (Takara). These data were analyzed using ΔΔC_t method.

Cells were lysed on ice for 30 min with cytoplasm extraction buffer (10 mM HEPES, 40nM KCl, 2mM MgCl₂, 10% glycerol, and 1mM PMSF). The cells were lysed on ice for 30 min with cytoplasm extraction buffer (10 mM HEPES, 40 nM KCl, 2 mM MgCl₂, 10% glycerol, and 1 mM PMSF). The cells were centrifuged at 2000 rpm for 5 min at 4°C, and the supernatant was collected. The supernatant was then centrifuged at 14,000 rpm for 15 min at 4°C. The supernatant was the cytosolic protein solution, and the pellet was used as the nuclear protein sample.

Cells were lysed with 500 μl NP-40 lysis buffer, and then centrifuged at 14,000 rpm for 15 min at 4°C. The supernatant was collected, added in 30 μl protein G-Agarose beads (Sangon, Shanghai) together with 1–10 μg isotype IgG antibody, and incubated at 4°C for 1 h with gentle agitation. Then, centrifuged and the beads were discarded, the supernatant added in 30 μl protein G-Agarose beads and 1–10 μg intended antibody. It was incubated at 4°C overnight with gentle agitation. Centrifuged to collect down the beads and washed three times in lysis buffer.

Protein samples were applied to SDS-PAGE, and then transferred to PVDF membrane (0.45 μm, Millipore). It was incubated with the primary antibody at 4°C overnight or 3 h at room temperature. The membrane was washed in TBST for three times, 5 min each, followed by incubation of HRP-conjugated secondary antibody at room temperature for 1 h. The membrane was washed four times in TBST, and then developed it with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher).

Ku70, Ku80, PARP1, IRF1, cGAS, STING primary antibodies, anti-HBsAg, and anti-HBcAg were purchased from Abcam. IRF3, IRF7, RNA pol III, CCL3, and CCL5 antibodies were purchased from Santa Cruz. Secondary antibodies labeled with Alexa Flour were all purchased from Invitrogen. Mouse antibodies were all purchased from BD Bioscience, except for CD8 primary antibody from eBioscience. Refer to Table S1 in Supplementary Material for detailed information.

For detailed information on PCR primers and siRNA sequences, see Table S1 in Supplementary Material.

We used Student’s t-tests to compare the mean values between groups. Bars represent the SD of each group (*p < 0.033, **p < 0.002, ***p < 0.0002, and ****p < 0.0001).

To identify HBV infection-related factors, we searched HBV-associated gene expression profiles in GEO. In dataset GSE38941, which consisted of liver specimens from patients with HBV-associated acute liver failure (ALF) and healthy donors (39), we noticed that most of the chemokines were significantly upregulated in the livers of patients compared with healthy donors (Figure 1A). A GSEA plot of the chemokine activity gene set also showed a strong enrichment of chemokines in the livers of patients (Figure 1B). In particular, CCL5 had a top-ranked p-value among genes that were significantly different between patients and donors (Figure 1C). The absolute expression of CCL5 in HBV-associated ALF patients was also much higher than that in healthy donors (Figure 1D). In another dataset, GSE65359 (40), which consisted of which consisted of liver specimens from HBV-infected patients in the immune tolerance, immune clearance, and chronic phases, some chemokines were also highly expressed in patients in the immune clearance phase (Figure S1A in Supplementary Material). Of these chemokines, CCL5 showed a very significant difference and high expression levels in immune clearance group (Figures S1B,C in Supplementary Material). Considering the symptoms of hepatitis in patients with HBV-associated ALF and in patients of immune clearance phase, these data suggest that CCL5 is important in hepatitis B. Next, we tested the serum of HBV patients receiving continuous antiviral treatment (Table 1). As expected, when the hepatitis-related symptoms of ALT level and HBV DNA copy number decreased, the CCL5 concentration in the patient serum also decreased (Figure 1E).

We used an Ad-HBV infection model of acute viral hepatitis in C57BL/6 mice (41) to determine whether inflammation-associated chemokines were present at higher levels during HBV-mediated hepatitis. As previously reported, mice infected with Ad-HBV showed a high level of serum ALT, which indicated severe liver damage. Meanwhile, the serum CCL5 level was significantly increased in Ad-HBV infection group (Figure 2A). When we examined the expression of CCL3 and CCL5 in the liver, both protein secretion and mRNA expression increased dramatically (Figures 2B,C). CCL3 is another ligand for CCR5, and because CCL3 and CCL5 possess strong chemotactic activity for CCR5+ circulating cells, a large number of immune cells infiltrated the liver in response to the increased expression of these chemokines (Figure 2D). In comparison, mononuclear cells in the spleen exhibited no changes. Analysis of the subtypes of infiltrating lymphocytes revealed a major population of CD8+ T cells (Figure 2E), which tended to be HBV specific and constituted the main cause of hepatitis, according to a previous publication (41). In addition, the accumulation of NK and CD4+ T cells was also substantial, although not as large as that of CD8+ T cells (Figure 2F).

We next investigated chemokine and immune cell localization using frozen liver sections. As expected, the livers of Ad-HBV-infected mice showed robust lymphocyte infiltration, and the hepatocytes with HBV replication secreted a large amount of CCL3 (Figure 2G). These data reveal that CCL3 and CCL5 are hepatitis-associated immune factors.

Chemokine expression can be regulated in various ways in different types of cells. However, whether HBV directly stimulates liver cells to express CCL3 and CCL5 remains to be determined. To imitate HBV genomic DNA presentation in the cytoplasm, we transfected the pAAV-HBV1.2 plasmid into liver-derived cells, i.e., SK-Hep-1, HepG 2, Huh 7, and primary HSECs. Forty-eight hours later, we evaluated the changes in mRNA expression encoding several inflammation-associated cytokines (Figure 3A). The PCR bands revealed that the chemokines CCL3 and CCL5 were strongly increased in HSEC and SK-Hep-1 cells, despite other weak changes. Further real-time qPCR showed that, although some other cytokines such as IL-6 were significantly upregulated, the fold changes in CCL3 and CCL5 were much higher (Figures 3B,D). Meanwhile, CCL3 and CCL5 secretion into HBV plasmid-transfected SK-Hep-1 cell supernatants increased robustly (Figure 3E), confirming the results of mRNA level. Notably, IFN-β, a cytokine that is widely involved in cytosolic innate immune responses, showed an increased mRNA level after HBV plasmid transfection (Figures 3A,D). However, IFN-β was barely detectable in the cell culture supernatant (data not shown), which is similar to HBV infection in vivo. This transfection model showed that HBV DNA stimulation directly upregulates CCL3 and CCL5 expression in liver-derived cells.

We next constructed two additional types of HBV plasmid containing 1.3 or 2.3 copies of HBV genomic DNA (Figure S2A in Supplementary Material). In this transfection model, the induction of CCL3 and CCL5 did not depend on the HBV DNA insertion length of the plasmids; all three plasmids efficiently stimulated SK-Hep-1 cells (Figure S2B in Supplementary Material). However, chemokine upregulation depended on the stimulant dosage (Figure S3A in Supplementary Material) and stimulation duration (Figure S3B in Supplementary Material), and we chose 1 μg/ml and 48 h as optimal conditions for subsequent experiments.

Although CCL3 and CCL5 are hepatitis associated, precisely how their expression is regulated remains unclear. Upon observing that cells respond quite rapidly to HBV DNA, within 24 h (Figure S3B in Supplementary Material), and considering that an autocrine feedback mechanism might not be sufficient, we hypothesized that this response was due to direct nucleic acid sensing. As the backbone vector pAAV contained no open reading frames (ORFs) and did not stimulate the cells in comparison to the HBV plasmids (Figure 3; Figure S2 in Supplementary Material), the inserted HBV DNA in the vector was likely the factor that elicited a response. However, it remained unclear whether HBV DNA itself or the transcribed HBV RNAs mediated the observed chemokine upregulation. To address this question, we inserted a luciferase sequence downstream of the CCL3 promoter region and stably expressed the construct in SK-Hep-1 cells. Then, total RNAs and cytosolic DNAs were isolated from HepG 2.2.15 cells, an HBV replication-competent cell line (42), and used for transfection as an HBV nucleic acid stimulator. Although the relationship between HBV RNAs and RIG-I has been identified (17), our data comparing the relative luciferase activity indicated that HBV DNA, rather than RNA, functioned more efficiently in the chemokine upregulation response (Figure 4A).

We next labeled HBV DNAs with 5′-biotin, transfected them into SK-Hep-1 cells, and then pulled down the DNA-binding proteins 6 h later. Through subsequent mass spectrometry analysis (Table S2 in Supplementary Material), we found that the most abundant HBV DNA-binding proteins were the Ku70/80 complex. The Western blot results also confirmed this observation (Figure 4B). Classically responsible for DNA repair reactions, the Ku70/80 complex is mainly localized into the nucleus. However, most of the HBV DNAs were cytosolic 6 h after transfection. To directly examine the localization of the sensing reaction in the cells, we labeled the Ku70/80 complex by immunofluorescence. The confocal images revealed that without transfection stimulation, almost all of the complex molecules remained in the nucleus, whereas a substantial amount was transferred to the cytoplasm and colocalized with HBV DNAs 6 h after transfection (Figure 4C). Moreover, in HepG 2.2.15 cells, we also observed colocalization of Ku80 and HBcAg in the cytoplasm (Figure 4D). With total internal reflection fluorescence (TIRF) microscopy, colocalization of the HBcAg and Ku70 proteins was also visualized near the cell membrane (Figure 4E). Because HBV genomic DNA is reverse transcribed within the assembling core particle during replication (16), this colocalization suggests that HBV DNA could be recognized by the Ku70/80 complex in the cytoplasm.

Next, we investigated whether HBV DNA sensing by the Ku70/80 complex resulted in chemokine upregulation signals. We used RNA interference (RNAi) targeting Ku70 in SK-Hep-1 cells (Figure 5A) and re-transfected the cells with HBV plasmid. Both the mRNA synthesis and protein secretion of CCL3 and CCL5 were reduced as a consequence of Ku70 knockdown (Figures 5B,C). These data show that HBV DNA sensing by the Ku70/80 complex leads directly to CCL3 and CCL5 upregulation.

As mentioned earlier, the Ku70/80 complex is known for its DNA repair activities, which also require DNA-PKcs (28). Recently, a DNA-sensing function was reported for Ku70/80, in which DNA-PKcs is involved, independently of its kinase activity (32). In our research, DNA-PKcs also co-precipitated with Ku70 after HBV DNA transfection (Table S3 in Supplementary Material). DNA-PKcs participated in CCL3 and CCL5 regulation in a kinase activity-independent manner because inhibition of its kinase activity by Nu7026 had no effect on subsequent CCL3 and CCL5 upregulation (Figure 6A), while knockdown of DNA-PKcs inhibited this response (Figure 6B). Another DNA repair-related molecule, PARP1, was also a Ku70-co-precipitated protein (Table S3 in Supplementary Material; Figure 6C). Consistent with the Ku70/80 complex, PARP1 demonstrated significant cytoplasmic translocation after HBV DNA transfection (Figure 6D). Moreover, the colocalization of PARP1 and Ku70 was observed by super-resolution microscopy (Figure 6E). When we knocked down PARP1 in SK-Hep-1 cells, the upregulation of CCL3 and CCL5 in response to HBV DNA transfection was affected significantly (Figure 6F). Thus, we believe that the Ku70/80 complex elicits both DNA-PKcs and PARP1 to activate the intracellular signaling cascade that upregulates the chemokines CCL3 and CCL5.

Because there are different DNA-sensing pathways in the cytoplasm, we investigated whether Ku70/80 sensing interacts with multiple DNA-sensing pathways. In addition to inflammasome-associated DNA-sensing pathways, which usually result in IL-1β and/or IL-18 production, the RNA polymerase III/RIG-I and cGAS-STING pathways are crucial methods of cytosolic DNA sensing (13). For this reason, we investigated whether members of these two pathways were recruited to the Ku70/80–DNA-sensing complex by immunofluorescence. However, confocal images showed neither RNA polymerase III nor cGAS colocalized with cytosolic Ku70 proteins (Figure S4A in Supplementary Material), which ruled out the possibility of their direct participation in the HBV DNA response. Because STING functions as a DNA-sensing adapter capable of activating both the IRF3 and NF-κB pathways, on which many cytosolic nucleic acid-sensing pathways rely, we examined the behavior of the STING protein post-transfection. The results suggested that STING did not assemble to the extent of activation and that IRF3 protein nuclear transfer did not occur (Figure S4B in Supplementary Material). Moreover, native PAGE and Western blotting showed that IRF3 proteins remained as inactive monomers (Figure S4C in Supplementary Material). Furthermore, we found that inhibition of the NF-κB pathway did not affect the chemokine upregulation signal (Figure S4D in Supplementary Material). These results indicate no direct roles for typical DNA-sensing pathways in Ku70/80 DNA sensing.

Immunofluorescence data from a previous publication clearly revealed the translocation of IRF3 to the nucleus after stimulation with DNA (32). However, in our model, IRF3 did not seem to be crucial, as it demonstrated no significant nuclear translocation after HBV plasmid transfection (Figures S4B,C in Supplementary Material). In another study, IRF1 and IRF7 were identified as the key transcription factors for activating type III interferon expression (31). Thus, we examined whether these transcription factors played a role in the Ku70/80-mediated chemokine upregulation response. We observed that IRF1 was assembled to DNA sites in the cytoplasm, and nuclear translocation occurred 6 h after transfection, while IRF7 remained cytosolic (Figure 7A). Moreover, in GSD super-resolution images, cytosolic colocalization of Ku70 and IRF1 was observed upon HBV plasmid stimulation (Figure 7B), which implied a direct interaction. To confirm this result, we immunoprecipitated IRF1 and found increased binding of IRF1 to Ku70/PARP1 (Figure 7C). Meanwhile, the IRF1 protein abundance decreased significantly in the cytoplasm, compared to a moderate increase observed in the nuclear (Figure 7D). This response clearly indicated the activation and nuclear transfer of IRF1. Next, we knocked down IRF1 in SK-Hep-1 cells and then stimulated the cells with HBV DNA. As expected, CCL3 and CCL5 mRNA expression decreased dramatically (Figure 7E). In contrast, knockdown of IRF7 decreased only CCL3 expression (Figure 7F). From the above results, we can conclude that IRF1 plays a crucial role as a key transcription factor in the Ku70/80 sensing-mediated chemokine upregulation pathway.