| Objective: Hepatitis C virus(HCV) infection causes a serious public health problem in China and the world. Studies have confirmed that, 50~85% of HCV infection will develop to chronic infection, and further progress to liver cirrhosis and hepatocellular carcinoma. Hepatitis C virus is a single-stranded positive-sence RNA virus which gene encodes three structural proteins(core, E1 and E2 protein) and seven nonstructural proteins(p7, NS2, NS3, NS4 A, NS4 B, NS5 A, and NS5B). HCV core protein(21k Da), which is the first protein to be synthesized upon virus infection and replication, takes part in activities of virus life cycle and assembly of virus particles. It can be released from infected liver cells and exhibits multiple functions to affect host immunity through interacting with a varity of immunocytes. Researches have found that HCV core protein combining with g C1 q R can inhibit T lymphocyte proliferation by reducing IL-2 and IL-2Rα gene transcription, or decrease the expression of SOCS-1 in B lymphocytes to promote proliferation, or impair IL-12 secretion by macrophages through induction of negative immunoregulators Tim-3, PD-1 and SOCS-1. In addition, HCV core protein can induce TLR tolerance in macrophages through interacting with TLR2. It also can reduce ability of DC cells to present antigen by down-regulating the MHC-Ⅰexpression. Anyhow, HCV core protein can affect the functions of a variety of immunocytes.Liver, as the main target organ for HCV infection, composed by hepatocytes, liver stromal cells, and a varity of immunocytes which include DC cells, macrophages, NK, NKT, T and B lymphocytes. Macrophages and NK cells in the liver also perform the function of the first line defense like in the other organs. Kupffer cells, traditionally denote hepatic resident macrophages and represent up to 15~20% of the total number of liver cells, 80~90% of the total body macrophage pool. Macrophages are dependent on the tissue micro-environment and external stimuli to polarize to different subtypes with distinct functions. Our preliminary works showed that the expression of transcription factor p-STAT3 and membrane molecular CD206 were up-regulated in macrophages when co-cultured with HCV core protein and liver cells, which indicated that macrophages had the tendency to polarize to M2 type macrophages. There is no doubt that M2 macrophages are implicated in inhibiting inflammatory responses, and often linked to immunosuppression, which is adverse for HCV elimination. In other words, in the settings of persistent HCV infection, macrophage activities altered along with liver pathological micro-environment may influence the process of chronic HCV diseases. It is well known that macrophages activated not only reflect phagocytosis and kill of pathogens, but also release large amounts of cytokines to impact the strength and types of adaptive immune responses, which also results in the expression of multiple negative regulators simultaneously, such as SOCS-1, SOCS-3, TOLLIP, IRAK-M, A20, CYLD, SHP etc. These negative regulators contribute to inhibit further activation of inflammation signaling pathways, thereby protecting the host against harmful excessive-responses. Our study mainly focused on negative regulator A20 expression in macrophages after being treated with HCV core protein and analyzed the possible affect factors. Our aim is to understand the pivotal role of macrophages in HCV chronic infection through research of HCV core protein interacting macrophages. It will be beneficial to provide a novel insight for revealing mechanisms of hepatitis C pathogenesis and establish a new approach to therapy of HCV.Methods:1 HCV core protein was expressed and purified in prokaryotic expression system and identified by SDS-PAGE and Western blot. HCV core protein was dealed with Endotoxin removal kit to remove endotoxin, and then we detected endotoxin in Limulus reagents assay, and quantified core protein by Bradford method.2 Monocyte THP-1 was cultivated into macrophage MΦ-THP-1 after being added PMA for 48 hours. Negative regulator A20 expression was detected in MΦ-THP-1 treated with different concentrations of HCV core protein(1 μg/ml, 5 μg/ml, 10 μg/ml) after 3 and 6 hours to determine the subsequent experiment concentration. The expression of A20 was detected in MΦ-THP-1 and mice bone marrow derived macrophage(BMDM) treated with HCV core protein was detected in different times.3 Monocyte THP-1 was infected with sh-A20 plasmid packaged by lentivirus to establish cell line THP-1-sh-A20. At the same time infected with sh-luciferase plasmid to establish THP-1-sh-luciferase as control cell line. Two cell lines were cultivated into macrophages under circumstances of PMA named MΦ-THP-1-sh-A20 and MΦ-THP-1-sh-luciferase. Two cell lines were treated with HCV core protein,and the expression of A20 were detected at 3 and 6 hours by Western blot to validate the effect of interference A20.4 MΦ-THP-1 cells were treated with HCV core protein for 4, 8, 12 and 24 hours, cytokines including TNF, IL-6, IL-1β and TGF-β1 in the culture supernatants were detected by CBA assay. At the same time, the same cytokines in the culture supernatants of MΦ-THP-1-sh-luciferase and MΦ-THP-1-sh-A20 cells were detected to verify the relationship between cytokines and A20.5 The interaction of HCV core protein with macrophage membrane receptors g C1 q R, TLR2 and TLR4 was verified by Pull-down assay in which Ni agarose combining with HCV core protein were mixed with MΦ-THP-1 cell lysis.6 As HCV core protein combining with g C1 q R could induce A20 expression, we analyzed the expression of A20 in MΦ-THP-1 cells treated with g C1 q R’s ligand C1q(final concentration 70 μg/ml).7 Monocyte THP-1 was infected with sh-g C1 q R plasmid packaged by lentivirus to establish cell line THP-1-sh-g C1 q R. THP-1-sh-g C1 q R induced by PMA was differentiated into macrophage MΦ-THP-1-sh-g C1 q R in which the expression of g C1 q R was detected to validate the interference effect, taking MΦ-THP-1-sh-luciferase and MΦ-THP-1 as control cells.8 MΦ-THP-1-sh-g C1 q R and MΦ-THP-1-sh-luciferase cells were treated with HCV core protein, the expression of A20 was detected in different times to make clear the relationship between A20 and g C1 q R.9 The expression of signaling molecules, including p-p38, p-JNK, p-ERK, p-NF-κB p65, p-NF-κB p105 and p-AKT were detected in MΦ-THP-1 and BMDM treated with HCV core protein in different times.10 The expression of signaling molecules p-p38, p-JNK, p-ERK, p-NF-κB p65, p-NF-κB p105 and p-AKT were detected in MΦ-THP-1-sh-g C1 q R and MΦ-THP-1-sh-luciferase cells treated with HCV core protein in different times to explicit signaling pathways involved in g C1 q R activation.11 The expression of signaling molecules p-p38, p-JNK, p-ERK, p-NF-κB p65, p-NF-κB p105 and p-AKT were detected in MΦ-THP-1 cells treated with C1 q 30 minutes to verify g C1 q R signaling pathways.12 MΦ-THP-1 cells were pretreated with chemical inhibitors in different concentrations of SB203580(p38 inhibitor), SP600125(JNK inhibitor), PD98059(ERK inhibitor), IMD 0354(IKKβ inhibitor), Wortmannin(PI3K inhibitor) and MK 2206 2HCl(AKT inhibitor) respectively for 30 minutes, and then treated with HCV core protein for another 30 minutes. The expression of p-p38, p-JNK, p-ERK, p-NF-κB p65, p-AKT were detected to determine the concentration of each inhibitors. A20 expression was detected in MΦ-THP-1 cells pretreated with inhibitors, and then treated with HCV core protein for 3 and 6 hours to indicate signaling pathways involved in A20 induction.Results:1 HCV core protein was successfully expressed and purified, and proved to be a single band by SDS-PAGE and Western blot. The protein contained endotoxin 3 EU/ml(1EU=0.1~0.2ng) and was quantified as 0.5 mg/ml by Bradford method. 2 HCV core protein could induce negative regulator A20 expression in macrophages.The expression of A20 was significantly increased in MΦ-THP-1 cells treated with HCV core protein(10μg/ml) after 3 and 6 hours(P<0.05). A20 expression began to increase after 30 minutes, and sustained high expression to the detected time point of 24 hours and 6 hours respectively in MΦ-THP-1 and BMDM treated with HCV core protein(P<0.05).3 THP-1-sh-A20 cell line was successfully established.Monocyte THP-1 was infected with sh-A20 plasmid to establish cell line THP-1-sh-A20. At the same time established control cell line THP-1-sh-luciferase. The expression of A20 in MΦ-THP-1-sh-A20 cells treated with HCV core protein after 3 and 6 hours was much lower than in control cells MΦ-THP-1-sh-luciferase(P<0.05), which indicated that interference of A20 was effective.4 HCV core protein promoted the production of cytokines in macrophages, IL-6, IL-1β and TGF-β1 were negatively regulated by A20.TNF, IL-6 and IL-1β were up-regulated at 4, 8, 12 and 24 hours while TGF-β1 were up-regulated at 8, 12 and 24 hours in the supernatants of MΦ-THP-1 cells treated with HCV core protein(P<0.05). The level of IL-6, IL-1β and TGF-β1 in the supernatants of MΦ-THP-1-sh-A20 cells treated with HCV core protein were much higher than in control cells MΦ-THP-1-sh-luciferase(P<0.05), which implied that IL-6, IL-1β and TGF-β1 were negatively regulated by A20.5 HCV core protein could induce A20 expression via combining with g C1 q RPull-down assay showed that HCV core protein could interact with g C1 q R but not TLR2 and TLR4. The expression of A20 was significantly increased in macrophages treated with g C1 q R’s ligand C1 q for 6 hours(P<0.05).Subsequently,we established cell line THP-1-sh-g C1 q R in which g C1 q R expression was decreased significantly(P<0.05). The expression of A20 in THP-1-sh-g C1 q R cells treated with HCV core protein after 3 and 6 hours was 2 HCV core protein could induce negative regulator A20 expression in much lower than in control cells MΦ-THP-1-sh-luciferase(P<0.05).6 MAPK, NF-κB and PI3K/AKT signaling pathways were activated by HCV core protein combining with g C1 q R in macrophages.The expression of signaling molecules p-p38, p-JNK, p-ERK, p-NF-κB p65, p-NF-κB p105 and p-AKT were increased in MΦ-THP-1 and BMDM treated with HCV core protein after 15 minutes, 30 minutes and 1 hours(P<0.05).The expression of p-p38, p-JNK, p-NF-κB p65, p-NF-κB p105 and p-AKT in THP-1-sh-g C1 q R cells treated with HCV core protein were significantly lower than in control cells MΦ-THP-1-sh-luciferase(P<0.05).The expression of p-p38, p-JNK, p-ERK, p-AKT in MΦ-THP-1 cells treated with C1 q 30 minutes were obviously increased, while p-NF-κBp65 and p-NF-κB p105 expression had not changed.The above results suggested that: both of C1 q and HCV core protein were ligands of g C1 q R, but the combining modes and involved signaling pathways remained differences.7 P38, JNK and NF-κB pathways played pivotal roles in the induction of A20.First,a serious of experiments were done to determine the optimum concentration of the following inhibitors:p38 inhibitor SB203580 was 30 μM; JNK inhibitor SP600125 was 20μM; ERK inhibitor PD98059 was 50 μM; IKKβ inhibitor IMD 0354 was 3 μM; PI3 K inhibitor Wortmannin was 200 n M; AKT inhibitor MK 2206 2HCl was 5μM.Results showed that: SB203580, SP600125, IMD 0354 could inhibit A20 expression in macrophages induced by HCV core protein(P<0.05), while PD98059, Wortmannin, MK 2206 2HCl could not inhibit A20 expression. The results indicated that p38, JNK, NF-κB signaling pathways played a major role in A20 induction.Conclusions:1 HCV core protein induced the expression of negative regulator A20 which could negatively regulate the secretion of IL-6, IL-1β and TGF-β1 in macrophages.2 HCV core protein induced A20 expression of macrophages via combining with g C1 q R.3 HCV core/g C1 q R engagement on macrophages triggered the activation of MAPK(p38 and JNK), NF-κB and PI3K/AKT signaling pathways, among which p38, JNK and NF-κB pathways played a major role in the induction of A20. |
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