Introduction
Hepatitis B virus (HBV) chronically infects 250 million people worldwide and continues to be a global public health burden. Chronic HBV infection may lead to hepatic cirrhosis and hepatocellular carcinoma, resulting in nearly 1 million deaths annually.1 Studies in recent years have significantly improved our knowledge on understanding the mechanisms of HBV persistence. It has been shown that HBV uses multiple mechanisms to dampen host adaptive immunity, especially the anti-HBV T cell responses, to facilitate its persistence.2,3
In recent years, increased evidence has suggested that the gut microbiota, which affects local and systemic immune responses,4,5 plays an important role in determining the outcome of HBV infection. On the one hand, compositional and functional changes in the gut microbiota have been observed in HBV-replicating mouse models6 and chronically HBV-infected patients,7 and is believed to be associated with disease progression.8 On the other hand, changes in gut microbiota composition may influence the outcome of HBV infection. In the HBV hydrodynamic injection (HDI) mouse model, depletion of gut microbiota by antibiotics prior to HBV challenge results in HBV persistence and impaired anti-HBV adaptive immunity.9 Moreover, using fecal microbiota transplantation to reconstitute the gut microbiota was shown to facilitate hepatitis B e antigen (HBeAg) clearance in chronic hepatitis B (CHB) patients.10 These results indicated stabilized gut microbiota play an important role in regulating the host immune response for HBV clearance. However, the detailed mechanism by which the intrahepatic anti-HBV immunity is regulated by the gut microbiota remains largely unknown.
In this study, we used an antibiotic (ABX) mixture treatment mouse model and the HBV HDI mouse model to examine the possible mechanism of regulating liver immunity by the gut microbiota during HBV infection.
Methods
Mice
Six to eight week-old wild-type male C57BL/6 mice were purchased from Hunan Slack King Laboratory Animal Co., Ltd. (Changsha, China). All animals were bred and kept in the Animal Care Center of Tongji Medical College (Wuhan, China) under specific pathogen-free conditions. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology, China (IACUC number: 612) and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals.
HDI in mice and production of LPS mice and HBV+LPS mice
HDI was performed by using HBV plasmids pAAV/HBV1.2 to establish HBV replication in mice, as described previously.11 In brief, mice were injected with 20 µg pAAV/HBV1.2 through the tail vein within an 8-s time window, in a volume of normal saline equivalent to 0.1 mL/g of the mouse body weight. Lipopolysaccharide (LPS) mice were subjected to HDI with 1 µg LPS (InvivoGen, San Diego, CA, USA). HBV+LPS mice were subjected to HDI with 20 µg pAAV/HBV1.2, in combination with 1 µg LPS.
Production of ABX mice
ABX mice were treated with antibiotics (ampicillin, neomycin, metronidazole, and vancomycin) in drinking water, according to an antibiotic mixture protocol for gut-sterilization.9
Cell isolation
Isolation of murine Kupffer cells (KCs) was performed, as described previously.12 KCs were isolated from nonparenchymal liver cells by using CD11b+ microbeads (Miltenyi, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. Preparation of single-cell suspensions of murine splenocytes was performed as described previously.13
Detection of IL-10 and IFN-γ levels in the serum and supernatants
IL-10 levels in the serum and in the cell-free supernatants were determined by the corresponding cytokine ELISA kits (eBioscience, San Diego, CA, USA), according to the corresponding manufacturer’s instructions. Red blood cell (RBC)-depleted splenocytes were cultured at 1×106 cells per well with or without KCs in a total volume of 200 µL at a ratio of 2:1 (splenocytes: KCs). Splenocytes were stimulated with anti-CD3 (1 µg/mL) and anti-CD28 (1 µg/mL) (BD Bioscience, Franklin Lakes, NJ, USA). Interferon (IFN)-γ levels in the cell-free supernatants were determined by the corresponding cytokine enzyme-linked immunosorbent assay (ELISA) kits (eBioscience), according to the corresponding manufacturer’s instructions.
Detection of serum cytokines and LPS
Serum IL-10, IL-6, IL-12, TNF-α, MCP-1, and IFN-γ levels were determined by Cytometric Bead Array Mouse Inflammation Kit (BD Biosciences), according to the manufacturer’s instructions. Serum LPS levels were determined by End-point Chromogenic TAL Endotoxin Testing Assay (Bioendo Technology, Xiamen, China), according to the manufacturer’s instructions.
Detection of serological HBV antigens and DNA
Serum was collected from the retro-orbital sinus of the mouse at the indicated time points. Serum hepatitis B surface antigen (HBsAg) and HBeAg levels were measured by the corresponding ELISA kits (Kehua, Shanghai, China), according to the manufacturer’s instructions. Serum HBV DNA copies were measured by a diagnostic kit for HBV DNA (Sansure, Changsha, China) using quantitative real-time PCR, according to the manufacturer’s instructions.
Flow cytometry
For surface staining, KCs were stained with CD80 (eBioscience), CD86 (BioLegend, San Diego, CA, USA), and PD-L1(eBioscience) antibodies for 15 m at 4°C in darkness. Freshly isolated cells were used for all assays, and approximately 20,000–40,000 T cells were acquired for each sample using a FACS Canto II flow cytometer (BD Biosciences). Data analysis was performed using FlowJo software V10.0.7 (Tree Star, Ashland, OR, USA). Cell debris and dead cells were excluded from the analysis based on scatter signals and Fixable Viability Dye eFluor 506.
IL-10 blockade and KC depletion
Purified rat anti-mouse IL-10 receptor (IL-10R) monoclonal antibodies (Clone 1B1.3a) were purchased from Bio X Cell (Lebanon, NH, USA). For IL-10 blockade, mice were intraperitoneally injected with 250 µg anti-mouse IL-10 receptor antibody at 1, 24 and 96 h before HBV and LPS HDI. For KC depletion, mice were injected via the tail vein with 200 µL of clodronate-liposome (5 mg/mL) or a phosphate-buffered saline (PBS)-liposome (LIPOSOMA BV, Netherlands) suspension at 24 h before HBV and LPS HDI.
Statistical analysis
Nonparametric t-test was used when comparing two groups. One-way analysis of variance was used with a Tukey post hoc test (GraphPad Prism software; GraphPad, San Diego, CA, USA) when more than two groups were compared. Statistical significance was considered when the p value was less than 0.05.
Results
Gut microbiota sterilization by antibiotics results in increased serum LPS levels and enhanced IL-10 production and T cell suppression of KC
LPS is the main component of the outer membrane of Gram-negative bacteria and is an endotoxin mainly released by Enterobacteriaceae. Therefore, we first investigated the influence of gut microbiota sterilization on serum LPS concentration. Adult C57BL/6 mice were gut-sterilized using a well-established antibiotic (ABX) mixture protocol9 and serum LPS levels were monitored by ELISA every other day for a week from the ABX treatment. The ABX treatment resulted in statistically significant increases in serum LPS concentrations up to 70% on 3 days and 5 days post-treatment in mice compared to untreated controls (Fig. 1A).
KCs are the largest population of tissue-resident macrophages, and LPS stimulation induces their activation and secretion of immunosuppressive mediators, such as IL-10.14 We next examined how gut microbiota sterilization influences the IL-10 production and suppressive function of KCs for T cell activation. KCs were purified from the liver of mice 30 days post-continuous ABX treatment, and were examined for IL-10 secretion by ELISA. KCs of ABX-treated mice showed significantly increased IL-10 production compared to those of control mice (Fig. 1B). KCs were also cocultured with anti-CD3/anti-CD28-activated T cells and the IFN-γ production by T cells was measured after 24, 48 and 72 h by ELISA. Consistent with our previous report,15 KCs separated from untreated control mice strongly suppressed the IFN-γ production of TCR-activated T cells. Compared to control KCs, KCs from ABX mice showed a significantly enhanced ability in suppressing the IFN-γ production of TCR-activated T cells (Fig. 1C). Taken together, these results showed that gut microbiota sterilization by antibiotics resulted in increased serum LPS levels and enhanced IL-10 production and T cell suppression of KCs.
LPS stimulation induces strengthened suppressive phenotype and T cell suppression of KC in HBV-replicating mice
Next, we investigated the impact of LPS stimulation on KC phenotype and function in the context of intrahepatic HBV replication. C57BL/6 mice were subject to HDI with normal saline (NS), LPS, HBV-replicating plasmid pAAV/HBV1.2 in combination with LPS (HBV+ LPS) or not (HBV), and KCs were purified from the liver of those mice 3 h after HDI. Flow cytometry analysis demonstrated that KCs separated from LPS mice showed significantly decreased CD80 and CD86 expressions and increased PD-L1 expression compared to those of NS HDI mice (Fig. 2A). Consistently, the expression of CD80 and CD86 on KCs was significantly decreased and the PD-L1 expression was significantly increased in HBV+LPS mice compared with those of HBV mice (Fig. 2A). Moreover, KCs of HBV+LPS mice secreted a significantly increased amount of IL-10 than HBV mice (Fig. 2B). Consistently, KCs of HBV+LPS mice showed a significantly enhanced ability in suppressing the IFN-γ production of TCR-activated T cells than KCs from HBV mice (Fig. 2C).
The direct effect of LPS stimulation on inducing KC IL-10 production was also examined in vitro. KCs were purified from the liver of untreated mice and stimulated by LPS in vitro for 24 h and monitored for IL-10 production up to 7 days. As shown in Figure 2D, significant up-regulation of IL-10 production by LPS-stimulated KCs was observed in the first day when LPS was presented in the medium. However, no IL-10 production by LPS-stimulated KCs was detected from days 2 to 7 in the supernatant when LPS was washed away. These results suggested that in the context of intrahepatic HBV replication, LPS stimulation could induce strengthened suppressive phenotype and T cell suppression of KCs in mice.
LPS stimulation induces potent proinflammatory cytokine production in HBV-replicating mouse and results in delayed HBV clearance
We further detected proinflammatory cytokine levels in the sera of HBV and HBV+LPS mice at 3 and 6 h post-HDI. The concentrations of IL-10, IL-6, TNF-α, MCP-1, IFN-γ, and IL-12 were maintained at low levels in the sera of HBV mice at the detected early time points post-HDI (Fig. 3A–F). In a sharp contrast, HBV+LPS mice showed significant increases in serum IL-10, IL-6, TNF-α, and MCP-1 levels at both 3 and 6 h post-HDI (Fig. 3A–D). In general, the serum IFN-γ and IL-12 levels were comparable between the two groups, and there was only a slight increase of IFN-γ levels in HBV+LPS mice observed at 6 h post-HDI (Fig. 3E–F).
Next, the impact of LPS stimulation on HBV clearance was examined by monitoring serum HBsAg and HBV DNA. As shown in Figure 4A, HBV+LPS mice showed higher serum HBsAg levels than HBV mice starting at 21 days post-HDI (dpi). At 42 days post-injection (dpi), serum HBsAg remained positive in 50% of HBV+LPS mice, but in only 10% of HBV mice (Fig. 4B). Moreover, HBV+LPS mice also showed higher serum HBV DNA levels than HBV mice. LPS treatment resulted in a 13.56-fold increase in serum HBV DNA levels at 35 dpi (Fig. 4C). Taken together, these results suggested that LPS stimulation in HBV-replicating mice induced potent proinflammatory cytokine production and resulted in a delay of HBV clearance.
IL-10 functional blockade and KC depletion results in accelerated HBV clearance in LPS treated HBV-replicating mice
We then examined whether the LPS-induced delay of HBV clearance in mice is mediated by IL-10 production and KCs. First, mice were treated with IL-10 receptor (IL-10R) blocking antibody (anti-IL-10R) prior to HBV and LPS HDI. As shown in Figure 5A, HBsAg clearance was significantly accelerated in anti-IL-10R-treated mice compared to untreated control mice. All anti-IL-10R-treated mice became serum HBsAg-negative at 4 dpi, whereas all control mice remained serum HBsAg-positive at least at 26 dpi (Fig. 5A). Next, mice were injected with clodronate-liposome to deplete KCs16,17 prior to HBV and LPS HDI. KC depletion resulted in significant decreases in serum IL-10 levels at both 3 and 6 h post-HBV HDI in clodronate-liposome treated mice compared to PBS-liposome treated mice (Fig. 5B), suggesting KCs were the main source of IL-10 production in LPS-treated HBV-replicating mice. Moreover, KC depletion also resulted in accelerated HBsAg clearance in mice, as 80% of PBS-liposome treated control mice remained serum HBsAg-positive, but only 20% of clodronate-liposome treated mice were so at 26 dpi (Fig. 5C). These results suggested that LPS-induced HBV persistence in mice is mediated by IL-10 and KCs.
Discussion
In a previous study, Chou et al.9 observed that adult C3H/HeN mice rapidly cleared HBV, while their young counterparts showed HBV persistence in the pAAV/HBV1.2 HDI mouse model. Sterilization of gut microbiota using antibiotics prevented adult mice from rapid HBV clearance.
In contrast, depleting KCs or abolishing the Toll-like receptor (TLR)4 signaling pathway in young mice prior to HBV HDI resulted in rapid viral clearance. The authors thus suggested that the gut microbiota sterilization-induced HBV immune tolerance in mice was highly probably mediated by KCs via the activation of TLR4 by its ligand LPS. Based on these observations and speculations, in the current study we further demonstrated that sterilizing gut microbiota by antibiotics results in an increase of serum LPS level in mice. Consistently, KCs from both gut-sterilized mice and LPS-treated HBV-replicating mice produce a significantly increased amount of IL-10 and show enhanced ability in suppressing T cell activation. This LPS-induced functional change of KCs results in delayed HBV clearance in mice.
The liver is not in direct contact with gut microbiota. However, it constantly encounters bacterial components, such as LPS generated from Enterobacteriaceae, translocated from the gut into the portal vein. It has been shown that LPS can disrupt expression of the tight junction protein OZ-1 in the intestinal tract, which increases the permeability of the intestinal mucosa.18 This in turn allows more LPS to enter the blood flow through the portal venous system and leads to low-grade endotoxemia.19 Therefore, in cases such as gut microbiota sterilization or dysbiosis, increased LPS release into the intestinal tract may result in increased intrahepatic LPS stimulation. Moreover, it has also been shown recently by Guo et al.20 that ABX treatment in mice results in translocation of live commensal gut bacteria and their components, including LPS into the liver. Therefore, gut microbiota sterilized mice may have an increased intrahepatic LPS stimulation, with even the serum LPS concentrations in these mice returning to physiological levels. LPS is mainly sensed by TLR4, which is constitutively expressed on the surface of macrophages.21–23
KCs are the largest population of liver-resident macrophages, and activation of the TLR4 signaling pathway by LPS induces the production of proinflammatory cytokines, including IL-10, by KCs.14,24–26 IL-10 is a cytokine with pleiotropic functions, and in many cases, it has been considered as an immunosuppressive cytokine that can attenuate inflammatory responses and suppress T cell activation and proliferation via the IL-10/IL-10R pathway.27 During HBV infection, elevated serum IL-10 level has been observed in both the acute phase28 and chronic phase.29 It is believed that KCs are one of the main sources of IL-10 production during CHB, and this KC-derived IL-10 plays an important role in maintaining liver immune tolerance and participates in HBV persistence.30 We have recently reported that the HBV particle directly stimulates KCs to produce IL-10 via activation of the TLR2 signaling pathway.15 This effect strengthens liver tolerance by promoting KC-mediated CD8 T cell suppression.15 In line with these findings, blocking the function of IL10 or depleting KCs in HBV-carrier mice leads to rapid HBV elimination and improved anti-HBV CD8 T cell responses.31,32
In the current study, we further demonstrate that gut microbiota dysbiosis may also trigger IL-10 production by KCs in the context of HBV infection, which plays a key role in mediating HBV persistence. Our finding has implications for the understanding of mechanisms of regulating intrahepatic immune response by the gut microbiota and their relationship with HBV infection.
Abbreviations
- CD:
cluster of differentiation
- dpi:
days post-injection
- ELISA:
enzyme-linked immunosorbent assay
- HBeAg:
hepatitis B e Antigen
- HBsAg:
hepatitis B surface antigen
- HBV:
hepatitis B virus
- HDI:
hydrodynamic injection
- IFN:
interferon
- IL:
interleukin
- LPS:
lipopolysaccharide
- KCs:
Kupffer cells
- MCP:
monocyte chemoattractant protein
- NS:
normal saline
- PBS:
phosphate-buffered saline
- PC:
positive control
- PD-L:
programmed death-ligand
- RBC:
red blood cell
- SPLs:
splenocytes
- TCR:
T cell receptor
- TNF:
tumor necrosis factor
- TLR:
Toll-like receptor
Declarations
Data sharing statement
The raw data of the manuscript are deposited in the database of the Department of Infectious Diseases, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, and can be provided to inquirers upon reasonable requests.
Funding
This work was supported by grants from the National Science and Technology Major Project for Infectious Diseases of China (Nos. 2017ZX10202203-007-006, 2017ZX10202202-001-009, 2017ZX10202202-002-008, 2017ZX10202201-002-003), the National Natural Science Foundation of China (Nos. 81461130019, 91642118, 91742114, 8181101231), the Integrated Innovative Team Project for Major Human Diseases Program of Tongji Medical College, Huazhong University of Science and Technology (HUST), the Double First-Class Disciplines Program of HUST (International Joint Laboratory for Infection and Immunity), the International Cooperation Base of Hubei Province for Infection and Immunity, and Deutsche Forschungsgemeinschaft (Transregio TRR60).
Conflict of interest
The authors have no conflict of interests related to this publication.
Authors’ contributions
Study concept and design (JL), acquisition of data (WZ, JzL, XX, SY, DZ, HH), analysis and interpretation of data (WZ, JzL, JL), drafting of the manuscript (WZ, JzL, XX), critical revision of the manuscript for important intellectual content (WZ, JL), administrative, technical, and material support (JL, DY), and study supervision (JL, DY).