Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Aug 31;7:335.
doi: 10.3389/fimmu.2016.00335. eCollection 2016.

Extracellular Vesicles Including Exosomes Regulate Innate Immune Responses to Hepatitis B Virus Infection

Affiliations
Free PMC article

Extracellular Vesicles Including Exosomes Regulate Innate Immune Responses to Hepatitis B Virus Infection

Takahisa Kouwaki et al. Front Immunol. .
Free PMC article

Abstract

The innate immune system is essential for controlling viral infection. Hepatitis B virus (HBV) persistently infects human hepatocytes and causes hepatocellular carcinoma. However, the innate immune response to HBV infection in vivo remains unclear. Using a tree shrew animal model, we showed that HBV infection induced hepatic interferon (IFN)-γ expression during early infection. Our in vitro study demonstrated that hepatic NK cells produced IFN-γ in response to HBV only in the presence of hepatic F4/80(+) cells. Moreover, extracellular vesicles (EVs) released from HBV-infected hepatocytes contained viral nucleic acids and induced NKG2D ligand expression in macrophages by stimulating MyD88, TICAM-1, and MAVS-dependent pathways. In addition, depletion of exosomes from EVs markedly reduced NKG2D ligand expression, suggesting the importance of exosomes for NK cell activation. In contrast, infection of hepatocytes with HBV increased immunoregulatory microRNA levels in EVs and exosomes, which were transferred to macrophages, thereby suppressing IL-12p35 mRNA expression in macrophages to counteract the host innate immune response. IFN-γ increased the hepatic expression of DDX60 and augmented the DDX60-dependent degradation of cytoplasmic HBV RNA. Our results elucidated the crucial role of exosomes in antiviral innate immune response against HBV.

Accession number: Accession number of RNA-seq data is DRA004164 (DRA in DDBJ).

Keywords: exosome; innate immunity; virus.

Figures

Figure 1
Figure 1
HBV induces hepatic IFN-γ expression. (A) Experimental procedure for infection and sampling. Tree shrews were infected intravenously with the HBV infectious particles. The livers were isolated on the day indicated. (B,C) Hierarchical clustering analysis of gene expression in the liver at 0, 1, and 3 days post-infection. Total RNA was extracted, and then RNA-seq analysis was performed using a next-generation sequencer. The data are representative of at least three independent experiments. The heat map of all genes was shown in (B). The clustered regions, in which the expression of genes was upregulated (left panel), downregulated (center pane), and not changed by infection (right panel), were shown in (C). (D) The gene expression levels in the liver of HBV-infected tree shrew were determined using RT-qPCR and was normalized against that of β-actin. Data are presented as mean ± SD (n = 4).
Figure 2
Figure 2
The response of human hepatic cells to HBV. (A) HuH-7 cells were transfected with pHBV plasmid, and RT-qPCR analysis was performed to determine the expression levels of HBV RNA, IFN-β, IFN-γ, IFN-λ1, and DDX60. Data are presented as mean ± SD (n = 3). (B) HuH-7 and HuH-7.5 cells were transfected with a plasmid carrying 1.4× HBV genome for 24 h. IFN-λ1 expression was determined using RT-qPCR and normalized to GAPDH. Data are presented as mean ± SD (n = 3). (C) Human primary hepatocytes were infected with HBV for 24 h, and the expression of the genes were determined by RT-qPCR. Data are presented as mean ± SD (n = 4). (D) Human primary hepatic stellate cells were infected with HBV for 24 h, and the expression of the genes was determined by RT-qPCR. Data are presented as mean ± SD (n = 4).
Figure 3
Figure 3
Exosomes containing viral RNA induce NKG2D ligand expression in macrophages. (A) HepG2 cells were seeded onto a 24-well plate and cultured for 24 h. EVs were isolated from 0.5 ml of cell culture medium using a polyethylene glycol method with exosome isolation kit (see Isolation of Exosomes in Experimental Procedures) and suspended with 150 μl of 1× SDS sample buffer. The 150 μl of whole cell extract (WCE) were prepared from cultured cells. The 10 μl of EVs and 10 μl of WCE were subjected to SDS-PAGE. CD9 and β-actin proteins were detected by western blotting with anti-CD9 antibody and anti-β-actin antibody. (B) Extracellular vesicles (EVs) released from HepG2 (left) or HuH-7 (right) transfected with pHBV were collected, and the total RNA was extracted. The RNA levels of HBV RNA and GAPDH mRNA were determined by RT-qPCR and normalized against that of U6 RNA. Data are presented as mean ± SD. (C) Exosomes were isolated from EVs released from HepG2 cells using anti-CD81 antibody beads. WCE, EVs, and 10× concentrated exosomes were subjected to SDS-PAGE, and the proteins were detected by western blotting. (D) The CD81+ exosomes were isolated from EVs with anti-CD81 microbeads. The RNA levels were determined as described in (B). Data are presented as mean ± SD (n = 3). (E) Hepatic F4/80+ cells and hepatic NK cells were co-cultured with normal HepG2 cells or HepG2-T23 cells (HepG2-HBV), which stably express HBV, for 1 day. IFN-γ levels in the culture supernatants were determined using ELISA (n > 3). (F) EVs released from HuH-7 or HepG2 with or without HBV were added to PMA-treated THP-1 cells (THP-1 macrophages) for 24 h. The expression of mRNA in THP-1 macrophages was determined by RT-qPCR and normalized to GAPDH (n = 3). (G) HepG2 cells in six-well plates were transfected with mock or pHBV and were cultured for 24 h. EVs were isolated from 5 ml of culture medium and were suspended with 100 μl of PBS. The 5 μl of EVs were mixed with 5 μl of 2× SDS sample buffer and were subjected to SDS-PAGE. The proteins were detected by western blotting with anti-CD63 antibody. (H) EVs released from HepG2 with or without HBV were added to mouse hepatic F4/80+ cells for 24 h. The expression of mRNA in hepatic F4/80+ cells was determined by RT-qPCR and normalized to GAPDH (n = 3). (I) HepG2-NTCP cells were infected with HBV for 9 days and were subsequently co-cultured with THP-1 (transwell co-culture) for 3 days. The expression of mRNA in THP-1 macrophages was determined by RT-qPCR.
Figure 4
Figure 4
The role of exosomes in the activation of PRRs. (A) EVs released from HepG2 cells transfected with mock or pHBV was treated with or without anti-CD81 antibody beads (α-CD81 beads) and was subsequently added to THP-1 macrophages for 24 h. ULBP1 and ULBP2 mRNA expression in THP-1 macrophages were determined by RT-qPCR and normalized to GAPDH. Data are presented as mean ± SD (n = 3). (B) THP-1 macrophages were transfected with siRNA for mock, TICAM-1, VISA, MyD88, and PYCARD and were subsequently treated with EV from HepG2 with or without HBV for 24 h. ULBP1 and ULBP2 mRNA expression in THP-1 macrophages were determined by RT-qCR and normalized to GAPDH. Data are presented as mean ± SD (n = 3).
Figure 5
Figure 5
Exosomes with miR attenuates the IL-12p35 mRNA expression. (A) CD81+ exosomes were isolated from HepG2 with pHBV. Mock or 5 μg of exosomal RNA was added to THP-1 macrophages for 6 h, and then cells were transfected with mock or 1 μg of exosomal RNA and 1 μg of DNA for 24 h. ULBP2 mRNA expression was determined by RT-qPCR and normalized to GAPDH. (B) PMA-treated THP-1 cells were stimulated with 500 μM of ODN2216, 1 μg/ml of CL097, and/or polyI:C [transfection (2 μg/ml) and addition (100 μg/ml)] for 24 h. The expression of mRNA was determined by RT-qPCR. (C,D) RNA was extracted from EVs and CD81+ exosomes released from HepG2 with or without HBV (C) or HBV-infected HepG2-NTCP cells (D), and the expression of miR was determined by RT-qPCR and normalized to U6 RNA level. Fold increase of miR expression was calculated by dividing miR level of HBV sample by that of mock. (E) THP-1 macrophages were treated with IFN-γ together with mock or EVs that were isolated from 1 ml of HepG2 cell culture medium for 24 h in a 24-well plate and were subsequently stimulated with 1 μg/ml of CL097 and polyI:C [transfection (2 μg/ml) and addition (100 μg/ml)] for 24 h. IFN-β and IL-12p40 mRNA expression was determined by RT-qPCR and normalized to GAPDH. (F) EVs were isolated from 1 ml of cell culture medium of HepG2 with or without pHBV. THP-1 macrophages were treated with EVs and were then simulated with 500 μM of ODN2216, 1 μg/ml of CL097, and polyI:C [transfection (2 μg/ml) and addition (100 μg/ml)] for 24 h in a 24-well plate. The expression of IFN-β and IL-12p35mRNA in THP-1 macrophages was determined by RT-qPCR and normalized to GAPDH. (G) EVs were isolated from 1 ml of cell culture medium of HepG2 cells. THP-1 macrophages were treated with EVs from HepG2 cells with or without pHBV for 24 h in a 24-well plate. Cells were washed twice with PBS, and total RNA was extracted from THP-1 cells. The expression of miR in THP-1 macrophages was determined by RT-qPCR. (H) HepG2 cells were transfected with miR-29a for 1 day, and EVs were subsequently isolated from HepG2 cell culture supernatant. THP-1 macrophages were treated with EVs for 1 day and then stimulated with 1 μg/ml of CL097 and polyI:C [transfection (2 μg/ml) and addition (100 μg/ml)] for 24 h. IL-12p40 mRNA expression was determined by RT-qPCR. Data are presented as mean ± SD (n ≥ 3).
Figure 6
Figure 6
DDX60 promotes cytoplasmic HBV RNA degradation. (A) HuH-7 cells transfected with pHBV were treated with 10 ng/ml of IFN-γ for 1 day. Cells were treated with actinomycin D (ActD), and then HBV RNA degradation was determined by RT-qPCR and normalized to GAPDH. (B) HuH-7 and HepG2 cells were stimulated with 10 ng/ml of IFN-γ. Total RNA was extracted at indicated time points, and mRNA levels were determined by RT-qPCR and normalized to GAPDH. Data are presented as mean ± SD (n = 3). (C) HepG2-T23 cells were transfected with pHBV for 24 h. Cells were stimulated with 10 ng/ml of IFN-γ, and the expression of DDX60 was determined by RT-qPCR. Data are presented as mean ± SD (n = 3). (D) HepG2-NTCP cells were stimulated with 10 ng/ml of IFN-γ, and whole cell extract was prepared at indicated time points. The proteins were subjected to SDS-PAGE and were detected by western blotting with anti-DDX60 and anti-β actin antibodies. (E) HepG2-NTCP cells were fixed and labeled with anti-DDX60 antibody. Cells were stained with DAPI and Alexa Fluor-488 secondary antibody and were observed using confocal microscopy. The scale bar represents 10 μm. (F–J) HuH-7 cells transfected with pHBV together with an empty vector or a DDX60 expression vector (F) or siRNA for control or DDX60 (G–J) were treated with ActD. Total RNA (F–H), nuclear RNA (I), and cytoplasmic RNA (J) were extracted at indicated time points. HBV RNA levels were determined using RT-qPCR and normalized to GAPDH (n = 3). (K–M) HuH-7 cells transfected with the combination of pHBV and either an empty vector or a DDX60 expression vector (K,M) or with pHBV and an siRNA (as a negative control) or DDX60 (L). Four days after transfection, total RNA was extracted, and HBV RNA and DDX60 mRNA levels were determined by RT-qPCR and normalized to GAPDH (K,L). HBsAg levels in the culture medium 2 days after transfection were determined using ELISA (M). (N) siRNA for control or DDX60 was transfected into HuH-7 cells with the HBV plasmid. Two days after transfection, cells were washed with fresh medium and subsequently treated with or without 10 ng/ml IFN-γ for 2 days. HBsAg levels in culture medium were determined by ELISA. (O) HepG2-NTCP cells were infected with infectious HBV particles for 6 days. Total RNA was extracted from the HBV-infected cells. HBV RNA and DDX60 mRNA levels were determined using RT-qPCR and normalized to GAPDH. Data are presented as mean ± SD (n ≥ 3).

Similar articles

See all similar articles

Cited by 30 articles

See all "Cited by" articles

References

    1. Kawai T, Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity (2011) 34:637–50.10.1016/j.immuni.2011.05.006 - DOI - PubMed
    1. Loo YM, Gale M, Jr. Immune signaling by RIG-I-like receptors. Immunity (2011) 34:680–92.10.1016/j.immuni.2011.05.003 - DOI - PMC - PubMed
    1. Li XD, Wu J, Gao D, Wang H, Sun L, Chen ZJ. Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science (2013) 341:1390–4.10.1126/science.1244040 - DOI - PMC - PubMed
    1. Schoggins JW, MacDuff DA, Imanaka N, Gainey MD, Shrestha B, Eitson JL, et al. Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity. Nature (2014) 505:691–5.10.1038/nature12862 - DOI - PMC - PubMed
    1. Unterholzner L, Keating SE, Baran M, Horan KA, Jensen SB, Sharma S, et al. IFI16 is an innate immune sensor for intracellular DNA. Nat Immunol (2010) 11:997–1004.10.1038/ni.1932 - DOI - PMC - PubMed

LinkOut - more resources

Feedback