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. 2013 Jul;87(14):8169-78.
doi: 10.1128/JVI.00974-13. Epub 2013 May 15.

Hepatitis C virus infection induces inflammatory cytokines and chemokines mediated by the cross talk between hepatocytes and stellate cells

Hironori Nishitsuji  1 , Kenji Funami, Yuko Shimizu, Saneyuki Ujino, Kazuo Sugiyama, Tsukasa Seya, Hiroshi Takaku, Kunitada Shimotohno
Affiliations

"V体育ios版" Hepatitis C virus infection induces inflammatory cytokines and chemokines mediated by the cross talk between hepatocytes and stellate cells

Hironori Nishitsuji et al. J Virol. 2013 Jul.

Abstract

Inflammatory cytokines and chemokines play important roles in inflammation during viral infection. Hepatitis C virus (HCV) is a hepatotropic RNA virus that is closely associated with chronic liver inflammation, fibrosis, and hepatocellular carcinoma. During the progression of HCV-related diseases, hepatic stellate cells (HSCs) contribute to the inflammatory response triggered by HCV infection. However, the underlying molecular mechanisms that mediate HSC-induced chronic inflammation during HCV infection are not fully understood. By coculturing HSCs with HCV-infected hepatocytes in vitro, we found that HSCs stimulated HCV-infected hepatocytes, leading to the expression of proinflammatory cytokines and chemokines such as interleukin-6 (IL-6), IL-8, macrophage inflammatory protein 1α (MIP-1α), and MIP-1β. Moreover, we found that this effect was mediated by IL-1α, which was secreted by HSCs. HCV infection enhanced production of CCAAT/enhancer binding protein (C/EBP) β mRNA, and HSC-dependent IL-1α production contributed to the stimulation of C/EBPβ target cytokines and chemokines in HCV-infected hepatocytes VSports手机版. Consistent with this result, knockdown of mRNA for C/EBPβ in HCV-infected hepatocytes resulted in decreased production of cytokines and chemokines after the addition of HSC conditioned medium. Induction of cytokines and chemokines in hepatocytes by the HSC conditioned medium required a yet to be identified postentry event during productive HCV infection. The cross talk between HSCs and HCV-infected hepatocytes is a key feature of inflammation-mediated, HCV-related diseases. .

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Fig 1
Fig 1
Conditioned medium from LX2 induces MIP-1β expression in JFH1-infected Huh7.5 cells. (A) Huh7.5 cells (1 × 105 cells) and JFH1-infected Huh7.5 (Huh7.5/JFH1) cells (1 × 105 cells) were cultured alone or in the presence of LX2 cells (1 × 105 cells) for 24 h. The level of MIP-1β was measured by qRT-PCR. Quantitative analysis of the PCR data was performed using the 2−ΔΔCT method, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) CT values were used for normalization. The fold changes are relative to values for Huh7.5 cells. (B) Huh7.5 cells and JFH1-infected Huh7.5 cells (Huh7.5/JFH1) were cultured alone or in the presence of LX2, 293T, HeLa, or THP-1 cells for 24 h. The expression level of MIP-1β was measured by qRT-PCR as described for panel A. (C) Huh7.5 cells and JFH1-infected Huh7.5 cells were treated with conditioned medium from Huh7.5 (Huh7.5 CM) or LX2 (LX2 CM) cells for 24 h. The expression level of MIP-1β was measured by qRT-PCR as described for panel A. (D) Huh7.5, JFH1-infected Huh7.5 (Huh7.5/JFH1), Huh7/NNC, Huh7.5/TNS2J1, and Huh7.5/SGR-JFH1 cells were treated with CM from Huh7.5 or LX2 cells for 24 h. The expression level of MIP-1β was measured by qRT-PCR as described for panel A. (E) Huh7.5 and JFH1-infected Huh7.5 (Huh7.5/JFH1) cells were treated with Huh7.5 CM, LX2 CM, or LI90 CM for 24 h. The expression level of MIP-1β was measured by qRT-PCR as described for panel A. (F) LX2 cells were treated with 2.5 ng/ml TGF-β1 for 24 h, and then the level of collagen mRNA in these cells was determined by qRT-PCR (left). Huh7.5 or JFH1-infected Huh7.5 cells were treated with LX2 CM or TGF-β1-stimulated LX2 CM for 24 h. The expression level of MIP-1β was measured by qRT-PCR as described for panel A (right). The results are representative of three independent experiments, and the error bars represent the standard deviation of the means.
Fig 2
Fig 2
Conditioned medium from JFH1-infected Huh7.5 cells treated with LX2-conditioned medium induces chemotaxis of NP-2-CCR5 cells. Huh7.5 CM or LX2 CM was concentrated 60-fold using a 100,000-molecular-weight-cutoff membrane filter. Huh7.5 and Huh7.5/JFH1 cells were treated with each concentrated medium (S). After 24 h of treatment, the medium was changed to serum free DMEM for 24 h. The chemotactic activity of each conditioned medium (C) was determined by using 5 nM maraviroc (CCR5 inhibitor) and NP-2-CCR5 cells, as described in Materials and Methods. The results are representative of three independent experiments, and the error bars represent the standard deviations of the means. DMSO, dimethyl sulfoxide.
Fig 3
Fig 3
Identification of a factor(s) in the LX2 conditioned medium that is responsible for induction of MIP-1β expression. (A) Huh7.5 CM or LX2 CM was concentrated using a 100,000-molecular-weight-cutoff membrane filter. Huh7.5 and JFH1-infected Huh7.5 (Huh7.5/JFH1) cells were treated with each unconcentrated conditioned medium, with the flowthrough fraction (100K-FT), or with the trap fraction (100K-T). The MIP-1β expression level was analyzed by qRT-PCR as described for Fig. 1A. (B) LX2 CM was concentrated using a 100,000-molecular-weight-cutoff membrane filter. A cytokine antibody array (RayBiotech) was used for the simultaneous detection of 507 inflammatory factors. The antibody-coated membrane was incubated with the trap fraction (100K-Trap) or with the flowthrough fraction (100K-Flow through). Representative spots (TSG-14, MCP-1, MCP-3, and IL-1α) are shown.
Fig 4
Fig 4
IL-1α induces MIP-1β expression in JFH1-infected Huh7.5 cells. (A) Huh7.5 and JFH1-infected Huh7.5 (Huh7.5/JFH1) cells were treated with recombinant MCP-2 (10 ng/ml), recombinant MCP-3 (rMCB-3; 10 ng/ml), recombinant TSG14 (10 ng/ml), or recombinant IL-1α (10 pg/ml) for 24 h. The level of MIP-1β expression was analyzed by qRT-PCR as described for Fig. 1A. (B) Huh7.5 and JFH1-infected Huh7.5 (Huh7.5/JFH1) cells were treated with various amounts of recombinant IL-1α (0, 0.1, 1.0, 10, or 100 pg/ml) for 24 h. The level of MIP-1β expression was analyzed by qRT-PCR as described for Fig. 1A. (C) JFH1-infected Huh7.5 cells were treated with Huh7.5 CM or LX2 CM along with an isotype control (0.2 μg/ml), anti-IL-1α (0.2 μg/ml), or IL-1RA (100 μg/ml) for 24 h. The level of MIP-1β expression was analyzed by qRT-PCR as described for Fig. 1A. (D) Huh7.5 and Huh7.5/JFH1 cells were transfected with 50 nM control siRNA (siControl) or 50 nM IRAK1 siRNA (siIRAK1). At 48 h after transfection, cells were treated with Huh7.5 CM or LX2 CM. The level of MIP-1β expression was analyzed by qRT-PCR as described for Fig. 1A. The results are representative of three independent experiments, and the error bars represent the standard deviations of the means.
Fig 5
Fig 5
Conditioned medium from LX2 induces C/EBPβ-mediated proinflammatory gene expression in JFH1-infected Huh7.5 cells. (A) The indicated cells were treated with Huh7.5 CM or LX2 CM for 24 h. The level of C/EBPβ expression was analyzed by qRT-PCR as described for Fig. 1A. (B and C) Huh7.5 cells and JFH1-infected Huh7.5 cells were transfected with 50 nM control siRNA or siC/EBPβ. After 24 h of transfection, the cells were treated with Huh7.5 CM or LX2 CM for 24 h. The levels of C/EBPβ (B) and MIP-1β (C) expression were analyzed by qRT-PCR as described for Fig. 1A. (D) Huh7.5 cells and JFH1-infected Huh7.5 cells were treated with Huh7.5 CM or LX2 CM for 24 h. The levels of the indicated cytokines and chemokines were measured by qRT-PCR, as described for Fig. 1A. The results are representative of three independent experiments.
Fig 6
Fig 6
HCV entry is required for induction of MIP-1β expression by LX2 CM. (A) Huh7.5, JFH1-infected Huh7.5 (Huh7.5/JFH1), and JFH1-CL3B-infected Huh7.5 (Huh7.5/JFH1-CL3B) cells were treated with Huh7.5 CM or LX2 CM for 24 h. The level of MIP-1β expression was analyzed by qRT-PCR as described for Fig. 1A. (B) Huh7.5 cells were treated with Huh7.5 CM or LX2 CM. After 24 h of treatment, cells were infected with JFH1 for 2, 4, or 6 h. The level of MIP-1β expression was analyzed by qRT-PCR as described for Fig. 1A. (C) Huh7.5 cells were treated with Huh7.5 CM or LX2 CM in the presence of an isotype control IgG or anti-CD81 antibody (1 μg/ml). After 24 h of treatment, the cells were infected with JFH1 in the presence of an isotype control IgG or anti-CD81 antibody (1 μg/ml) for 2, 4, or 6 h. The level of MIP-1β expression was analyzed by qRT-PCR as described for Fig. 1A. (D) HCV JFH1 was UV irradiated and used to infect Huh7.5 cells. At 24 h after infection, cells were treated with Huh7.5 CM or LX2 CM. The level of MIP-1β expression was analyzed by qRT-PCR as described for Fig. 1A. (E) Huh7.5 cells expressing the indicated HCV proteins were treated with Huh7.5 CM or LX2 CM for 24 h. The levels of MIP-1β expression were analyzed by qRT-PCR as described for Fig. 1A. The results are representative of three independent experiments, and the error bars represent the standard deviations of the means.
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References

    1. Bartosch B, Thimme R, Blum HE, Zoulim F. 2009. Hepatitis C virus-induced hepatocarcinogenesis. J. Hepatol. 51:810–820 - VSports最新版本 - PubMed
    1. Bowen DG, Walker CM. 2005. Adaptive immune responses in acute and chronic hepatitis C virus infection. Nature 436:946–952 - "V体育官网" PubMed
    1. Liaw YF, Lee CS, Tsai SL, Liaw BW, Chen TC, Sheen IS, Chu CM. 1995. T-cell-mediated autologous hepatocytotoxicity in patients with chronic hepatitis C virus infection. Hepatology 22:1368–1373 - PubMed
    1. Shields PL, Morland CM, Salmon M, Qin S, Hubscher SG, Adams DH. 1999. Chemokine and chemokine receptor interactions provide a mechanism for selective T cell recruitment to specific liver compartments within hepatitis C-infected liver. J. Immunol. 163:6236–6243 - PubMed
    1. Harvey CE, Post JJ, Palladinetti P, Freeman AJ, Ffrench RA, Kumar RK, Marinos G, Lloyd AR. 2003. Expression of the chemokine IP-10 (CXCL10) by hepatocytes in chronic hepatitis C virus infection correlates with histological severity and lobular inflammation. J. Leukoc. Biol. 74:360–369 - PubMed

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