The Effect Of HBeAg On TLR3 And TLR4 Expression In Human Placental Trophoblast Cells And Its Role In Mother-infant Transmission

Backgroud & ObjectiveHepatitis B virus (HBV), a hepatotropic DNA virus, severely threats human health. HBV may vertically transmit into fetus from placenta in pregnant women infected by HBV, and this is one of important routes of HBV transmission. HBV e antigen (HBeAg) is an accessory protein not required for viral replication and assembly. HBV may establish persistent infection in pregnant women HBeAg-positive for chronic hepatitis B(CHB), which probably results in HBV intrauterine infection. It is shown that HBeAg inhibits TLR2 expression and TLR2-mediated innate immunity response in hepatocyte, peripheral blood monocyte and kupffer cells (KCs). Similarly, the expression of TLR3 in dendritic cells (DCs) of peripheral blood is attenuated. This suggests that HBeAg may play roles in immune-regulation which in turn to promote viral persistent infection.Toll-like receptors (TLRs), identified as pattern recognition receptors in recent years, are capable of recognizing and responding to the pathogen-associated molecular patterns(PAMPs), and thus play important roles in defending microorganisms.The innate immunity system, in which the TLRs are key components, represents the first defense line against invading pathogens in organism.TLRs are widely expressed in mammalian tissues and cells. Up to now,10 TLRs have been characterized in human being, and all of them express in human placenta tissues whereas TLR3 and TLR4 mainly express in human placenta trophoblast cells. There are 2 signal pathways related to TLRs based upon its dependence or independence on MyD88. TLR3 belongs to the MyD88-independent but TRIF-dependent pathway, whereas TLR4 pathways are both MyD88-and TRIF-dependent. Inhibition of HBV replication are mediated by TLR3 and TLR4 in hepatocytes and peripheral blood dendritic cells TLR3 recognize virus double strands RNA leading to immune response. Stimulated with TLR3 ligands polyI:C, trophoblast cells will produce the typeⅠinterferon(IFN-α/β), and IFN-P is the key cytokine from the TLR3-induced cells. However, trophoblast cells treated with TLR4 ligands LPS will result in up-regulation of proinflammatory cytokines(TNF-a) and chemotatic factor (IL-8), indicating that the placenta trophoblast cells play a key role in recognizing PAMPs to trigger anti-virus responses.As a physical and immunologic barrier, placenta is important for defending the diverse array of microorganisms invaded. Trophoblast cells, directly connecting with mother blood, are the first cell layer in placental barrier and are able to recognized and respond to pathogenic microorganisms. Therefore, trophoblast cells may be key players in the prevention of intrauterine infection by HBV.However, the mechanisms by which the pregnant women HBeAg-positive for chronic hepatitis B(CHB) have persistent infection of HBV are poorly understood. Bewo cells, derived from a human choriocarcinoma, are widely used to study the functions of trophoblast cells.In this study, we have constructed eukaryotic expression plasmids of HBeAg and 1.3 fold of HBV genome, separately, and have established the Bewo cells overexpressing HBV or HBeAg. Using these cells, we have investigated regulation of TLR3 and TLR4 expression and biological effect by HBeAg in human placental trophoblast cells, and have further explored the potential mechanisms for persistent infection or vertical transmission of HBV. It will be helpful to prevent and/or cure HBV intrauterine infection.Methods1. TLR3 and TLR4 expression in placenta of pregnant women with HBeAg- positive chronic hepatitis B and its significance46 Pregnant women with chronic hepatitis B (CHB), including 22 HBeAg-positive and 24 HBeAg-negative,and 25 normal pregnant women were chosen from November 2008 to May 2009. The mRNA and protein levels of TLR3 or TLR4 in placentas were detected by real-time quantitative RT-PCR(qRT-PCR) and immunohistochemitry (IHC), respectively. The levels of HBV DNA and HBeAg were assayed by real-time fluorescence quantitative PCR(FQ-PCR) and microparticle enzyme immunoassay (MEIA),respectively.2. The effect of TLR3-and TLR4-mediated innate immune on HBV replicationThe 1.3-fold HBV genome DNA was constructed from a recombinant plasmid called pMD18T-HBV and was sub-cloned into the eukaryotic expression plasmids pCDNA3.1(+).The recombinant vector was confirmed by restriction enzyme digestion,PCR and sequencing, then was transfected into Bewo cells.The extracellular and intracellular HBsAg,HBeAg expression and HBV DNA level was detected by Western blotting,MEIA and FQ-PCR, respectively. The mRNA of TRIF and MyD88 in Bewo cells were determined by RT-PCR, whereas the levels of IFN-P and TNF-a in supernatant were detected by ELISA.3. HBeAg affects TLR3 and TLR4 expression and biological effect in Bewo cellHBeAg gene was amplified from the pMD18T-HBV vector by PCR and then was cloned into the pcDNA3.1(+),the recombinant vector was confirmed by restriction enzyme digestion, PCR and sequencing, and then was transfected into Bewo cells.The extracellular and intracellular HBeAg expression was detected by Western blotting and MEIA, and the same methods as described above were applied for mRNA levels of TLR3 and TLR4 in Bewo cells and IFN-βand TNF-αlevel in supernatant. NF-κB p65 protein nuclear translocation was detected by immunofluorescence (IF).Results1. The levels of TLR3 and TLR4 were significantly lower in the CHB pregnant women HBeAg-positive than those HBeAg-negative or normal pregnant women (P=0.000). But the serum level of HBeAg and HBV DNA were significantly higher in the CHB pregnant women HBeAg-positive than those HBeAg-negative or normal pregnant women (P=0.000). Moreover, strong staining of TLR3 was observed by IHC in cytomembrane and endochylema of syncytiotrophobalst (SCTs) and villous cytotrophoblast. In addition,TLR4 mainly localized to cytomembrane of these extravillous trophoblasts. The average levels of TLR3 and TLR4 were significantly lower in the CHB pregnant women HBeAg-positive than those HBeAg-negative or normal pregnant women (P=0.000). There was a reverse correlation between both of TLR3 and TLR4 mRNA in placentas and the levels of HBeAg in serum (r=-0.495,P <0.05,r=-0.450,P<0.05), while there was no relationship between TLR3 and HBV DNA.2. The 1.3-fold HBV genome DNA inserted into pCDNA3.1 was confirmed by the restriction enzyme digestion,PCR and sequencing, and Bewo cells transfected with this vector are able to express HBsAg and HBeAg proteins in cells or supernatant, and high level of HBV DNA were detected in supernatant.2μg or 8μg HBV recombinant vector was transfected into Bewo cells,compared with control,polyI:C and LPS could significantly suppressed HBV replication (P<0.01), but the inhibitory rate of polyI:C and LPS were strikingly lower after transfecting 8μg HBV recombinant vector into Bewo cells (P<0.01).The suppressive effect of polyI:C and LPS on HBV replication was reversed by neutralizing antibodies against human TLR3 and TLR4 and IFN-β, but anti-IFN-βantibody did not inhibit the antiviral effect of LPS (P>0.05). Compared with control, polyI:C could significantly induce the production of IFN-P and TNF-αin Bewo cells (P<0.05 or 0.01),whereas LPS only obviously up-regulated TNF-αexpression, but not IFN-P (P<0.05 or 0.01), in time-and dose-dependent manners.Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, strongly inhibited polyI:C-and LPS-indued TNF-a production, not IFN-P in Bewo cells(P<0.01).The mRNA levels of TRIF or MyD88 were significantly induced by polyI:C and LPS in the Bewo cells transfected with this recombinant vector (P<0.01). 3. The HBeAg gene inserted into the pcDNA3.1(+) was confirmed by restriction enzyme digestion,PCR and sequencing. Bewo cells transfected with this vector are able to express HBeAg protein in cells or supernatant. Compared with control,polyI:C and LPS could significantly induce TLR3 and TLR4 mRNA expression in Bewo cells (P<0.05 or 0.001),in time-and dose-dependent manners. IFN-βsignificantly induced TLR3, not TLR4 mRNA in Bewo cells(P<0.01). However,2μg or 8μg HBeAg recombinant vector was transfected into Bewo cells, then the cells were treated with polyI:C and LPS,TLR3 mRNA level and IFN-βand TNF-αproduction in transfecting 8μg HBeAg recombinant vector group were obviously lower than those in control (P <0.01). Exogenous HBeAg remarkably suppressed TLR3 mRNA level and both of IFN-βand TNF-αproduction induced by polyI:C in Bewo cells (P<0.05 or 0.01),and TLR4 mRNA and TNF-a production induced by PLS were also inhibited by exogenous HBeAg (P<0.05 or 0.01) in a dose-dependent manner. PolyI:C or LPS induced NF-κB p65 translocation into nuclei (NF-κB activation) in Bewo cells measured by IF (P<0.01),which was abolished by endogenous or exogenous HBeAg (P<0.01).Conclusions1. There was a persistently high level of HBeAg in serum of HBeAg-positive CHB pregnant women,furthermore, its rate of intrauterine infection rised significantly than normal and HBeAg-negative CHB pregnan women,but the mRNA level and protein expression of TLR3 and TLR4 were lower in HBeAg-positive CHB pregnant women than that in other 2 groups. There was a reverse correlation between both of TLR3 and TLR4 mRNA in placentas and the levels of HBeAg in serum.This may imply that the low expression of TLR3 and TLR4 have close relationships with HBV intrauterine infection of pregnant women,and it plays a negative role in the expression of TLR3 and TLR4 in placentas.2. The 1.3-fold HBV genome DNA inserted into pCDNA3.1 was constructed successfully, setting up the foundation for future research on intrauterine HBV infection. TLR3-and TLR4-mediated innate defence responses could suppress HBV replication in Bewo cells via its ligands-induced production of IFN-P and TNF-a. But with the increase of HBV infectious dose, their suppressive effect on HBV replication was strikingly decreased.TLR3-mediated suppression HBV replication was TRIF-dependent,while TLR4 mainly depended on regulatory protein MyD88. This indicated that TLR3 and TLR4 activation could represent a powerful and novel therapeutic approaches against HBV intrauterine infection.3. The HBeAg gene inserted into the pcDNA3.1(+) was constructed successfully, allowing for future research on the effect of intracellular HBeAg on the expression of Toll-like receptors in Bewo cells.polyI:C and LPS could significantly induce TLR3 and TLR4 mRNA expression in Bewo cells. TLR3 expression was induced through IFN-βautocrine feedback. HBeAg could impair the immune function by down-regulating TLR3 and TLR4 mRNA level, abrogating NF-κB activation, and decreasing the production of cytokines in Bewo cells. This may imply that HBeAg diminished TLR3 and TLR4 mRNA expression and biological effect in placenta trophoblasts to lead to HBV intrauterine infection.