The Effect Of Hepatitis C Virus Core On Macrophage And Their Interaction In Proliferation Of Human Hepatocytes

Objective: More and more studies showed that hepatitis C viruse, ahepatotropic virus, which frequently leads to chronic infection, can not onlyinfect hepatocytes, but also replicate in many immune cells (such as B cells,T cells, monocytes and macrophages). Furthermore, through binding withintracellular or extracellular protein of the host cells, HCV is able to effect thecellular biological activities, for example, non-structural protein NS3caninhibit innate immune response through blocking TLR3pathway or activatingthe TLR2receptor pathway to promote innate immune; Membrane protein E2can bind with CD81of NK cell surface receptor to inhibit NK cell function. Itis concluded that during the process of infection, HCV interacts with immunecells through a variety of ways, and the interactions of HCV, immune cellsand hepatocytes not only enable remove HCV, but also construct the evadingmechanism of HCV which may lead to chronic hepatitis, cirrhosis andhepatocellular carcinoma. However, the mechanisms how HCV proteinsmodulate the innate immune system to contribute to disease are unanswered.HCV core protein as an important structural protein, is the significantpart of the viral capsid, which participates in virus assembling and in a seriesof biochemical process of host cell including the signal transduction,metabolism and apoptosis: the HepG2transfected with HCV core result in theincreasing proliferation of HepG2through activating intracellularMAPK/ERK pathway by autocrine TGF-α; HCV core also occurred in HCCby inhibiting promoter activity of the tumor suppressor gene p53. Kupffercells (KCs), resident liver macrophages, are long lived and abundant,representing15to20%of the total liver cell population，play an importantrole in immune surveillance and immune regulation. However, during HCVpersistent infection, monocytes expressing HCV core receptor C1qR can be recruited to the liver, and differentiate into different subtypes of macrophagecells in the liver inflammatory microenvironment, including classicalactivation of M1macrophages、alternative activation of M2macrophages、tumor associated macrophages、myeloid derived suppressor cells and so on.The different subtypes of macrophages can secrete different cytokines, andplay different role in virus infection and tumor formation. In other words,during the course of HCV chronic infection, HCV core combined with C1qRin macrophage surface leads to what kinds of biological changes inmacrophages? And what is the relationship between these changes andchronic hepatitis caused by HCV? How to restrict each other between livercells and macrophages in the hepatic microenvironment? All above are notvery clear.The purpose of this study was to understand the changes of liver cellsand macrophages treated with HCV core. To investigate the role of HCV inHCC and provide a new perspective for research of chronic mechanism andimmunotherapy of HCV. Therefore, the following experiments will be used:1,Macrophages will be treated with HCV core from a prokaryotic expressionsystem. Supernatants and macrophages were collected respectively. Differenthepatocyte cell lines (L02, HepG2) proliferation will be analyzed afterinteraction with supernatants and macrophages;2, Following theunderstanding of supernatant of macrophage treated with HCV core canpromote liver cell proliferation, the changes of signal transduction molecule inliver cells will be analyzed after macrophages and hepatocyte cell lines (L02,HepG2) co-culture by using transwell cell culture system (non direct contactco-culture,);3, According to the above results, expression of NF-κB,STAT3，STAT1, inflammatory cytokines and cell membrane molecules inmacrophages treated with HCV core protein or co-culturing with hepatocytecells will be analyzed by RT-PCR, ELISA, Western blot, flow cytometry andthe role of HCV core protein in differentiation of macrophages will berevealing. Methods：1HCV core gene fragment was obtained from full gene plasmid ofJFH-1strain (HCV2a) by RT-PCR with designed primers.The RT-PCRproduct was inserted into pMD18-T vector and sequenced. The positiverecombinant plasmid and the prokaryotic expression plasmid pET28a were cutwith the same enzymes, BamHI and HindⅢ. Thus a recombinant prokaryoticexpression plasmid pET28a/HCV core was constructed. After induced byIPTG, Inclusion bodies were expressed highly. Then through purification,renaturation and the quantitative detection of LPS, HCV core protein wasidentified by SDS-PAGE and Western-blot.2Human monocytic cell line Thp1stimulated by PMA was differentedinto macrophages PMA-Thp1which was treated with HCV core. So as toverify the biological activity of prokaryotic expression protein HCV core, themRNA expression level of the receptor C1qR of PMA-Thp1cell was detectedby RT-PCR, PMA-Thp1treated with PBS or LPS as the control. Later theoptimal concentration of HCV core in experiment was determined.3Cell culture supernatant (10%supernatant+90%complete medium,20%supernatant+80%complete medium,40%supernatant+60%completemedium) obtained from HCV core treating PMA-Thp1cells was collected.Human liver cell line L02and HepG2were respectively cultured with thecollected cell culture supernatant. Dynamic changes of liver cell proliferationwas analyzed with CCK8method at different time points (24h,48h,72h), cellculture supernatant of PMA-Thp1treated with PBS and complete mediumcontaining only HCV core protein as control.4PMA-Thp1cells were collected after treating with HCV core proteinfor24h, and respectively co-cultured with human liver cell line L02andHepG2(the ratio of liver cells and PMA-Thp1cells were25:1,50:1) Bymeans of CCK8method, dynamic changes of liver cell proliferation wasanalyzed at different time points (24h,48h,72h), human liver cell lineco-cultured with or without PMA-Thp1cells treated with PBS as control.5HCV core and PMA-Thp1cells respectively co-cultured with humanliver cell line L02, HepG2(non-direct contact co-culture) in24h using tanswell cell culture system. MAPK pathway proteins (ERK, JNK, p38)activity were detected by Western blot in liver cells, liver cells groupco-cultured with PBS and PMA-Thp1，liver cells group treated with HCV coreprotein and live cells group as the control group.6After treated with HCV core protein, proinflammatory andanti-inflammatory cytokines (IL-1β, TNF-α, IL-12, IL-8, TGF-β) secreted byPMA-Thp1was detected for the mRNA level changes; TGF-β in supernatantwas detected by ELISA; the expression of cell surface membrane moleculars(CD14, CD16, CD206, CD86) were detected by flow cytometry，PMA-Thp1treated with PBS or LPS or LPS+HCV core as the control group; cellulartranscription factors (NF-κB, STAT3, STAT1) activity were detected byWestern blot.Results：1A prokaryotic expression plasmid pET28a/HCVcore is successfullyconstructed and expressed. The content of LPS in prokaryotic expressionprotein was0.9EU/ml by Limulus reagents assay. SDS-PAGE electrophoresisand Western blot showed that a single band protein of21KD and specifichybridization with mouse anti-human HCV core monoclonal antibody wereobserved.2By detection of cell surface molecules (CD11b, CD11c) through cellmorphology and flow cytometry, it is indicated that Thp1was differented intomacrophages PMA-Thp1successfully by treating with40ng/ml phorbol ester(PMA) after48h.3C1qR mRNA levels of PMA-Thp1treated with HCV core in6h,12hwere significantly higher than other groups (P<0.05) by Real-time PCR. Theoptimal concentration of HCV core was10μg/ml in experiment.4Cell proliferation curve shows the effect of supernatant fromPMA-Thp1treated with HCV core on the proliferation of L02cells: comparedto the negative control group, different concentrations of cell culturesupernatants all lead to L02cell proliferation, and20%cell culturesupernatant shows significant proliferation of L02(P<0.05). 5Cell proliferation curve shows that effect of supernatant fromPMA-Thp1treated with HCV core on the proliferation of HepG2cells:HepG2growth were greater than the control group cultured in10%cellculture supernatant on24h,48h,72h, and were significantly different on48h,72h(P<0.05). However,20%supernatant group and40%supernatant groupon the proliferation of HepG2was not obvious.6No obvious effect on promoting proliferation was observed after livecells were co-cultured with PMA-Thp1cells following treated with HCV core.The data suggest that macrophage membrane molecule has no effect on theproliferation of liver cells.7After liver cells co-cultured with PMA-Thp1cells following treatedwith HCV core, intracellular phosphorylated ERK expression weresignificantly increased in L02cells, while phosphorylated JNK and p38expression have no change. As for HepG2cells, the phosphorylation of JNKexpression was only significantly increased.8PMA-Thp1treated with HCV core for different time:(1) RT-PCRresults showed that the mRNA levels of IL-1β, TNF-α, IL-12, IL-8, TGF-βsignificantly changed（P<0.05）, compared to control group;(2) ELISA resultsshowed that the expression of TGF-β in cells culture supernatant wassignificantly higher than control group (P<0.05), and the amount of TGF-β insupernatant was168pg/ml.(3) Flow cytometry results showed that theexpression of cell surface molecule CD14, CD16, CD206had no differencescompared to other three groups, and the expression of CD86was nosignificant difference between the experimental group and PBS group.(4)Western blot results showed that the expression of phosphorylated NF-κB65and NF-κ B105increased in1h and4h, but there were no obviousdynamic change for the expression of phosphorylated STAT3and STAT1.9Expression and activation of transcription factors of macrophagefollowing HCV core protein, liver cells and PMA-Thp1co-cultured:(1)Phosphorylation of STAT3was significantly incressed after coculturing withL02cells and core prote, howere, there was no obvious change after co-culturing with core protein and HepG2cells;(2) The expression ofphosphorylated STAT3was higher after co-culturing with L02cells and coreprotein, at the same time, the expression of phosphorylated STAT1was lowerthan the control group of PMA-Thp1cells co-cultured with L02cells.Conclusions:1HCV core protein with biological activity was constructed andexpressed successfully.2The proliferation of liver cells(L02and HepG2) may correlate thesoluble factor secreted by PMA-Thp1which was treated with HCV core, butno direct association with surface molecules of PMA-Thp1cells.3Liver cells proliferation was promoted by PMA-Thp1after beingtreated by HCV core. It may be associated with the up-regulation theexpression of phosphorylated ERK in L02, and the expression ofphosphorylated JNK in HepG2. At the same time, L02and HCV core mayalso effect PMA-Thp1biological function through up-regulating theexpression of phosphorylated STAT3in PMA-Thp1.4The activity of transcription factors and the secretion of cytokines ofPMA-Thp1appeared dynamic changes after being treated with HCV core, butthe results of the interaction between different liver cells and PMA-Thp1arenot exactly the same. The relationship between liver cells and macrophagetreated with HCV core can affect each other with the normal biologicalfunction.