| BackgroundHepatitis B virus (HBV) infection leads to a wide spectrum of liver disease ranging from acute to chronic viral hepatitis, which may progress to liver cirrhosis and development of hepatocellular carcinoma (HCC). More than 360 million people are chronically infected worldwide and about 1 million people die per year due to HBV-associated liver pathologies. Until now, HBV infection remains as a globe public health problem.Host immune system plays an important role in control of chronic HBV infection. Persistence of chronic HBV infection is mainly due to the successful establishment of a complex network of virus-host interactions permitting the virus to evade the antiviral response of the host. It has been clearly identified that adaptive immune response is required for effective control of HBV infection. The deletion of HBV-specific T cells or their functional inactivation (exhaustion) leads to deficiency in eliminating HBV in chronic patients. However, our knowledge of anti-HBV innate immune responses is still hampered by model system limitations. Studies in acute infected patients and chimpanzees documented a lack of type I IFN induction and proinflammatory cytokine production in acute HBV infection. In contrast to these observations, recent in vitro studies indicated that the intracellular innate immune signaling pathway of hepatocytes may sense the infection and limit the spread of HBV. Moreover, a modest activation of IFN-stimulated genes was also observed in human hepatocytes in chimeric mice model challenged with HBV. Recently, increasing evidences showed that HBV was not a’stealth virus’, since it could be sensed by host innate immune system while HBV had developed mechanisms to evade the innate immune responses.Toll-like receptors (TLRs) are able to recognize a set of evolutionary highly conserve structures, so called pathogen-associated molecular patterns (PAMPs), and subsequently elicit innate antiviral immune responses. Activation of TLR system leads to the expression of antiviral genes as well as proinflammatory cytokines, such as type I IFNs (IFN-α/β), TNF-αand IL-6. TLR signal pathways play an important role in antiviral innate immunity, as well as in HBV infection.TLR2 is a receptor for bacterial, fungal, viral and parasitological PAMPs. Thompson et al. showed in vitro that TLR2 activation led to reduction of HBV replication and capsid formation in recombinant HBV baculovirus transduced hepatoma cells. Our previous results also demonstrated that TLR2-activated intracellular MAPK/ERK and PI3k/Akt pathways in hepatocytes are required for the suppression of HBV replication. Recently, different lines of evidence showed that HBV could modulate the expression of TLR2 and/or inhibit TLR2 signaling cascades, suggesting that HBV might use this strategy to escape the innate immune response. Visvanathan et al. reported that expression of TLR2 in peripheral blood CD 14+ monocytes, hepatocytes and Kupffer Cells was downregulated at the presence of HBV precore protein (HBeAg) in CHB. TLR2 expression and function were also examined in the woodchuck model. Consistently, our results showed TLR2 expression in woodchuck Peripheral blood mononuclear cells (PBMCs) and liver tissue was down regulated in the chronically WHV-infected woodchuck. Interestingly, TLR2 expression in PBMCs from chronically WHV-infected woodchucks returned to a level comparable with that in PBMCs from naive animals after over-night culture, suggesting that viral products in sera may inhibit TLR2 expression in PBMCs. In addition, TLR2-mRNA levels were inverse correlation with viral load in sera during acute and chronic WHV infection. The interplay between HBV and TLR2-mediated innate immune responses suggests that restoration of TLR2 functions might be a new target for therapeutic option.TLR7 is predominately expressed on plasmacytoid dentritic cells (pDCs), B cells and monocytes/macrophages. It can initiate the MyD88-dependent pathway in response to viral single-stranded RNA, culminating in synthesis of type I IFNs and proinfiammatory mediators via activation of IRF7 and NF-κB. Stimulation of TLR7 results in activation of natural killer (NK) cells and cross-priming of cytotoxic lymphocytes, thereby orchestrating both innate and adaptive immune responses. Mouse hepatitis virus (MHV) could be recognized by TLR7, pDCs controlled the fast replicating MHV through the immediate production of type I IFNs in the mouse model. Furthermore, tail-vein injection of TLR7 ligand into HBV transgenic mice showed that TLR7 inhibited HBV replication in an IFN-α/β-dependent manner. Recently, the antiviral effect of the TLR7 ligand GS-9620 was investigated in the woodchuck and chimpanzee model in vivo. In chronically WHV-infected woodchucks, a 4-week treatment with GS-9620 resulted in a sustained and marked reduction of serum WHV DNA and WHsAg levels and induction of anti-WHs antibody response, as well as a markedly decreased incidence of hepatocellular carcinoma. In chronically HBV-infected chimpanzees, a reduction in HBV viral load and serum HBsAg was observed in all three animals treated with GS-9620. Additionally, GS-9620 induced a dose-dependent increase in serum IFN-a and triggered interferon-stimulated genes in PBMCs and livers. Considering these exciting observations, the TLR7 might be used as the therapeutic target for CHB patients.The natural history of CHB is generally regarded as consisting of four phases: immune tolerace, HBeAg-positive CHB (immune clearance), inactive carrier (low or non-replicative) and recurrence phase (immune escape). In the natural history of chronic HBV infection, there is 2 to 15 percent people emerging spontaneous HBeAg seroconversion, and most of them become the inactive carriers (IC), who show the well control of HBV replication and inflammation. Compared with HBeAg positive patients, they have lower risk of developing cirrhosis and HCC. REVEAL Study found that HBV replication was the independent predictor for cirrhosis and HCC through long-term follow-up. The development of cirrhosis and HCC were reduced in inactive carrier. Antiviral therapy has been considered as the most important program. The sustained suppression of HBV DNA and accompanied HBeAg seroconversion is the substitution goal of antiviral therapy. Patients who have successfully undergone this process usually become the inactive HBsAg carrier.Though TLR2 and-7 mediated innate immune responses might be involved in control HBV infection, the related clinical data in CHB patients were limited at present. The expression and antiviral effects of TLR2 and TLR7 in different phases of chronic HBV infection and during CHB treatment remain largely unknown.AimsThe expression profiles of TLR2 and TLR7-mRNA in different phase of chronic HBV infection were investigated through a cross-section study. Two clinical trials with telbivudine or Peg-IFN-a-2a antiviral therapy were included. TLR2 and TLR7-mRNA levels in PBMC were evaluated longitudinally during treatment. And experiments in vitro were carried out to explore the antiviral effects of TLR2 and TLR7 in chronic HBV infection.Materials and MethodsSubjectsSeventy-three patients with chronic HBV infection were recruited at Nanfang Hospital and Guangzhou the Eighth Hospital (Guangzhou, China) for the cross-sectional study. The subjects were classified into immune tolerant carrier (IT, n=24), HBeAg positive chronic hepatitis B (CHB, n=27) and inactive carrier (IC, n=22) group according to Chinese Clinical Practice Guideline. IT was defined as being HBsAg-positive on at least three occasions in one year, HBeAg-positive and having normal ALT levels. IC was defined as being HBsAg-positive on two occasions at least six months apart, HBeAg-negative, anti-HBe-positive with persistently normal ALT levels and HBV DNA<10,000 copies/mL. CHB was defined as being HBsAg-positive, HBeAg-positive and anti-HBe-positive, with HBV DNA higher than 100,000 copies/mL, and having a flare ALT levels (>1 ULN). Meanwhile, twenty-one healthy controls (HC) were enrolled. Ten ml of heparinzed blood and 5 ml of serum were collected.Two multi-center clinical trials with Telbivudine (EFFORT, NCT00962533) or Peg-IFN-a-2a antiviral therapy (EXCEL, NCT01086085) were included. Forty-one patients with telbivudine(LdT) therapy and 10 patients with Peg-IFN-a-2a antiviral therapy participate in this study. All patients were HBsAg-positive and had been HBeAg-positive for more than 6 months. The criteria for inclusion in the trial were an alanine aminotransferase (ALT) greater than the upper limit of normal (ULN) and an HBV DNA level greater than 100,000 copies/ml. Twenty milliliter of heparinzed blood was taken at Nanfang Hospital for immune studies and 1 ml of serum was obtained from subjects at both hospitals for measurement of cytokine concentrations. These samples were taken at baseline,12 weeks,24weeks, and 52 weeks after starting telbivudine treatment, or were taken at baseline,4weeks,12 weeks,24weeks, and 48 weeks during Peg-IFN-a-2a treatment. The subjects were classified into either a complete response (CR) group or a non-complete response (NCR) group. Patients in CR group have undergone ALT normalization, HBeAg seroconversion and achieved serum HBV DNA level less than 300copies/ml by week 52 or 48. While serum HBV DNA could be detected higher than 300copies/ml or HBeAg remained positive by week 52 or 48, which were defined NCR.Furthermore,10 HC,10 CHB patients and 5 IT patients were recruited for experiments in vitro as described previously.The study was conducted according to the Declaration of Helsinki guidelines, and was approved by the Ethical Committee of Nanfang Hospital. Written and informed consent was obtained from all subjects.Serological assays and HBV-DNA assaysThe normal range for serum ALT level is 0 to 40U/L. The HBV-DNA level was quantified using the Roche Diagnostics Cobas Taqman 48 (Meylan, France), which has a detection limit of 300 HBV-DNA copies/ml. The presence of HBsAg, anti-HBs, HBeAg, anti-HBe and anti-HBc was determined using commercial AxSYM MEI kits (Abbott Laboratories, North Chicago, IL).PBMC Isolation and cell sortingPBMCs were separated on Ficoll-Histopaque (BD) density gradients, and cryopreserved as routinely described. Cryopreserved and thawed PBMCs were used for the longitudinal study, and fresh PBMCs were used for the cross-sectional study. CD 14+monocytes and T cells were sorted from fresh PBMC isolated from eight healthy individuals or 8 patients with CHB, respectively. Freshly isolated CD 14+ monocytes were obtained from PBMC by positive selection using CD 14 MicroBeads and MACS separation columns from Miltenyi (Miltenyi Biotech), followed by T cells sorting through negative selection using Pan T MicroBeads (Miltenyi Biotec). This purification protocol resulted in>95% purity of selected cells, as determined by flow cytometry with immunofluorescence staining using anti-CD 14 APC-conjugated monoclonal antibody (BD), and anti-CD3 PE-conjugated monoclonal antibody (BD). The rest of cells were considered as Non-monocyte-T cells.Real-time RT-PCRFrozen PBMCs were thawed and washed twice with PBS. Total cellular RNA was isolated from 2×106 PBMC using either RNeasy Mini kit (Macherey-Nagel) or miRNeasy Mini Kits (Qiagen). cDNA synthesis was performed with 500ng of total RNA using QuantiTect Reverse Transcription Kit (Qiagen). The expression of different cellular genes was determined by quantification of specific mRNAs using commercial QuantiTect Primer Assays (Qiagen, primer sequences not available). The real-time RT-PCR was performed by a two-step method using QuantiTect SYBR Green RT-PCR Kit (Qiagen) on a Light Cycler (Roche Diagnostics) as described. For each sample, RT-PCR was performed in duplicate. The expression levels of each gene are presented as values normalized against 103 copies of β-actin transcripts.Cells culture, stimulation and analysis of HBV replicationFreshly isolated PBMC were cultured at 5×106 cells/ml in RPMI 1640 (Life Technologies) supplemented with 10% fetal calf serum,2 mM L-glutamine (Invitrogen) and antibiotics (Penicillin-streptomycin, Life Technologies). PBMC were stimulated with three distinct ligands:TLR2 ligand (Pam3CSK4,5μg/ml), TLR7 ligand (Imiquimod,5μg/ml), and TLR8 ligand (ssRNA40/LyoVecTM,0.5μg/ml). Culture supernatants were collected after stimulation for 48h.HepG2.2.15 cells were maintained in Dulbecco’s modified Eagle’s medium with 10% heat-inactivated fetal bovine serum, supplemented with 2mmol/L L-glutamine, 1% nonessential amino acids,50 IU/ml of streptomycin,500 g/ml ciprofloxacin 500mg/L of G418, grown in 5%CO2 at 37℃ and passed at 90% confluency. The cells were seeded at a density 7.5×105 cells at 6-well plate for 16-20h before treated with various aforementioned supernatant for 4d at a gradient concentration (The radio of supernatant to media is from 0,1:40,1:20,1:10 to 1:5). Meanwhile, neutralizing antibody including anti-IFN-a, anti-IFN-p, anti-IFN-y or anti-TNF-a (Final concentration is from 0.1μg/ml, 1μg/ml to 10μg/ml) were added to block corresponding cytokine, to explore which pathway was involved in antiviral effect. Thereafter, the media was collected and DNA was extracted for further analysis.HBV replicative intermediates (HBV RI) from intracellular core particles and HBV transcripts were extracted from HepG2.2.15 cell lines and detected by southern blot and quantified by real-time PCR respectively, according to the published protocols.Flow cytometry analysisIntracellular cytokine staining (ICS) was carried out as described elsewhere, but with minor modifications. TNF-a, IFN-y and CD 107a production by T cells or NK cells was tested by ICS. Freshly isolated PBMCs were plated at 2×105 cells/well with brefeldin A (Sigma-Aldrich, St Louis, Missouri, USA) in 96 well flat bottom plates. After 5-hour incubation at 37℃, cells were harvested, washed with phosphate-buffered saline (PBS), and stained for 30 minutes at 4℃ with directly conjugated Live/Dead-APC-Cy7, anti-CD3-PerCP-Cy5.5 and anti-CD56-FITC, or anti-CD4-FITC and anti-CD8-APC antibodies. Next, PBMCs were washed, fixed, and permeabilized by BD Cytofix/Cytoperm Solution (BD Biosciences) according to the manufacturer’s instructions and stained with anti-IFN-y-APC or anti-IFN-y-PE, anti-TNF-a-PerCy7 or CD107a PE-conjugated antibodies (BD Biosciences). A minimum of 200,000 lymphocytes were collected, and analyzed with BD FACSDiva (BD Bioscience, San Jose, CA) and FlowJo (Tree Star Inc., Ashland, OR) software.Statistical analysisContinuous data are expressed as the median (minimum-maximum). The Mann-Whitney U test or the Chi square test was used to compare two groups, and Walloon’s Sign Rank test to compare matched paired groups. The Kruskal-Wallis H test was used in multiple group comparisons. The general linear model repeated measures procedure was used for interclass comparisons of repeated sample data, and the P values for the conservative Greenhouse-Geisser corrections are reported for univariate effects. Multiple comparisons of variance among different times were performed with a LSD-/test and comparisons of variance between groups were performed with a Folded F test. Receiver operating characteristic curves were constructed to predict a complete response to LdT treatment. All statistical analyses were based on two-tailed hypothesis tests with a significance level of P<0.05.ResultsTLR2 and TLR7 expression on PBMC were suppressed in Chronic HBV infectionThere was a significantly lower TLR2-mRNA expression on PBMC in patients with chronic HBV infection than health controls (P<0.001). A similar trend was showed for TLR7-mRNA expression on PBMC, though not reaching statistical significance (P=0.141). For a detailed analysis, we then assessed TLR2 and TLR7 expression in different phase of chronic HBV infection. The median TLR2-mRNA and TLR7-mRNA levels were lower for both IT and IC groups (TLR2:HC vs IT, P<0.001, HC vs IC, P<0.001; TLR7:HC vs IT, P=0.028, HC vs IC, P=0.001), while getting an obvious increase in CHB with liver inflammation (TLR2:CHB vs IT, P=0.002, CHB vsIC, P=0.012; TLR7:CHB vs IT, P=0.003, CHB vs IC, P<0.001). Interestingly, no difference of TLR2 or TLR7 expression was observed for patients with CHB group compared to health controls (P=0.088, P=0.135). In summary, these data suggested that TLR2 and TLR7 mediated innate immunity were suppressed in chronic HBV infection but could be restored partly in CHB with inflammatory activity.TLR2 and TLR7 expression could not yet restore completely in CHB patientsAs described above, TLR2 and TLR7 levels were recovered to a certain degree in CHB patients during phase of liver inflammation activity. To better investigate whether TLR2 and TLR7 expression on all subsets of PBMC could restore completely in CHB patients, we further sorted fresh PBMC into three subgroups, including CD14+ monocytes, T cells, and Non-Mon-T cells by magnetic bead selection. Both TLR2 and TLR7 expressed mainly on monocytes, being far higher on T cells and Non-Mon-T cells, no matter in CHB group or HC group. TLR2 was weakly detected on T cells, while TLR7 expression was hardly detected. There were no significant differences in TLR2 and TLR7 levels on Non-Mon-T cells between CHB and HC groups (P=0.161, P=0.442). However, we observed that expression of TLR2 and TLR7 still decreased on CD 14+monocytes in CHB patients(P=0.0499, P=0.021). The results showed that TLR2 and TLR7 mRNA levels on monocytes could not restore to normal levels, despite of the obvious improvement on other subsets of PBMC found in CHB patients.Higher TLR2 and TLR7 levels on PBMC at baseline were associated with complete response to LdT treatmentTo explore whether TLR2 and TLR7 mediated innate immunity could affect treatment response of CHB patients, we prospectively assessed the TLR2 and TLR7-mRNA expression on PBMC in CHB patients with HBeAg-positive who received LdT therapy. We included forty-one patients participating in a clinical trial of Telbivudine as described above. Age, gender, serum ALT, HBV genotype, quantitative HBsAg and serum HBV-DNA level at baseline were all comparable between CR(n=18) and NCR(n=23) patients, for excluding other influence factor, so to confirm the independent effect of TLR2 and TLR7. We observed a reduced expression of TLR2 and TLR7 in NCR compared with CR patients at baseline of LdT therapy (P=0.029, P=0.038).A ROC curve was generated which indicated that expression of TLR2 or TLR7 on PBMC at baseline was predictive of complete response during 52 weeks of LdT treatment (P=0.029, P=0.038, respectively).The results implied that when in hepatitis activity TLR2 and TLR7 mediated innate immunity could partly restore, might contribute to control HBV.Ten patients in the longitudinal study during 48 weeks therapy of Peg-IFN-a-2a were also followed prospectively, including CR (n=5) and NCR (n=5) group. However, there was no significant difference of TLR2 or TLR7 expression at starting Peg-IFN-a-2a therapy in CR patients compared NCR (P=0.690,P=0.548)Different dynamic models of TLR2 and TLR7 expression during LdT or Peg-IFN-a-2a antiviral therapyAs aforementioned, we observed the association between expression of TLR2, TLR7 on PBMC and treatment response, which prompted us to evaluate the effects of antiviral therapy on the kinetics of TLR2 and TLR7 expression. Twenty patients with sufficient PBMC from at specific 4 time points (0,12,24 and 52weeks) were followed prospectively in the longitudinal study during 52 weeks therapy of LdT including CR group(n=10) and NCR(n=10). Compared to baseline, there was a progressive decline in both TLR2 and TLR7 expression on PBMC of all patients starting at week 12 on therapy, accompanying with serum ALT levels decreased to approximate normal value. Whereas, it was interesting that a statistically significant recover was shown on TLR2 and TLR7 expression during week 12 to week 24, with serum HBV-DNA maintaining a continuous decline. Collectively, partly restored TLR2 and TLR7 expression in liver inflammation activity phase displayed a trend of down-regulation during early 12 weeks antiviral therapy of LdT, following with normalization of ALT levels. These data suggested there might be a correlation between liver inflammation and TLRs expression. Of note, TLR2 and TLR7 expression got recovery along with remove of inflammation and continuous suppression of HBV-DNA, which implied conversely HBV might inhibit the expression of TLRs to evade innate immunity to some extent. In general, there were significantly difference in TLR7-mRNA dynamic changes among 4 time points, but not in TLR2 expression (P=0.034, P=0.564). Comparison of TLR2 and TLR7-mRNA levels showed no significantly difference between CR and NCR patients in various times during LdT therapy (TLR2:F=0.228, P=0.639; TLR7:F=3.156, P=0.093).For a detailed analysis, we compared dynamic profiles of TLR2 and TLR7 during the whole 52 weeks course of LdT treatment in CR and NCR patients. Obviously, both TLR2 and TLR7 expression were higher in CR patients than that of NCR at baseline, in spite of no significant difference for small size (TLR2:P=0.639; TLR7:P=0.093). Both these two TLRs exhibited consistent trends of decline during the first 12 weeks therapy in CR patients, while no apparent changes in NCR. From week 12 to week 24, both CR and NCR patients had obvious recovery for TLR2 and TLR7 expression, except for almost no shift of TLR7 in CR patients. Afterwards, both two TLRs seemed to decline slightly but no apparent difference happened in CR and NCR patients until 52 weeks treatment.Notably, it was worth mentioning that we also evaluated the effects of Peg-IFN-a-2a therapy on the kinetics of TLR2 and TLR7 expression meanwhile. Ten patients in the longitudinal study during 48 weeks therapy of Peg-IFN-a-2a were followed prospectively, including CR group(n=5) and NCR(n=5), with regular blood samples at 5 time points (0,4,12,24 and 48weeks). Comparison of TLR2 and TLR7-mRNA levels showed no significantly difference between CR and NCR patients in various times during Peg-IFN-a-2a therapy (TLR2:F=0.03, P=0.866; TLR7:F=1.551, P=0.247). TLR2 expression showed a gradual but not significantly decrease during the whole course in all patients (P=0.123). Conversely, the expression of TLR7 increased obviously during first 12 weeks treatment in both two subgroups, accompanying with continuous decline of serum HBV-DNA. General line model analysis showed that the dynamic changes of TLR7-mRNA in CR group were significantly different among 5 time points, while NCR group had not reached significant difference (CR:P=0.004,NCR:P=0.067). It was totally different from dynamic shifts during LdT treatment. Most interestingly, there was no remarkable difference on TLR7 expression at baseline (P=0.548), however, it reached approximately significantly higher in CR patients as compared to NCR at end of 48 weeks Peg-IFN-a treatment (P=0.056).TLR7 ligand stimulation on PBMC could induce antiviral effects through secretion of IFN-a/y, but that not of TLR2 ligandAs mentioned above, higher TLR2 and TLR7 expression on PBMC at baseline contributes to complete response of LdT therapy. Therefore, we further investigated whether it could enhance to control HBV through stimulation on TLR2 or TLR7 signal response in vitro. Five healthy volunteers and 5 CHB patients were recruited, 20ml heparinzed blood samples were collected simultaneously. Fresh PBMC were isolated and stimulated for 48h by TLR2 ligand (Pam3CSK4,5μg/ml), TLR7 ligand (Imiquimod,5μg/ml), and TLR8 ligand (ssRNA40,0.5μg/ml), respectively. Then supematants of CHB or HC were harvested and pooled separately. The supematants from TLR7-ligand treatment group led to suppression of HBV replication in HepG2.2.15 cell line, as compared to mock controls on day 4. The amount of HBV replicative intermediates decreased most significantly through TLR8-ligand stimulation, while TLR2-ligand stimulation did not induce antiviral effects of PBMC. The supematants stimulation on HepG2.2.15 cells displayed similar results from both CHB and HC. Subsequently, we explored which factors may play the main role in inhibition of HBV replication by using corresponding neutralizing antibodies to blocking probable cytokines. Consequently, suppression of HBV replication were reversed evidently when neutralizing IFN-a or IFN-y at lOug/ml concentration, but not for other two cytokines. It was reasonable to speculate that TLR7-ligand stimulation on PBMC could promote to IFN-a/y secretion which led to inhibit HBV replication, and the response to TLR7-ligand of PBMC was not hampered seriously in CHB patients compared to healthy individuals.TLR2 ligand stimulation on PBMC could enhance production of TNF-aThough the supematants of TLR2 ligand stimulation of PBMC had not exhibited significant suppression of HBV replication, we found that fold increase of TNF-a production were driven. As we known, TLR2 mediated innate immunity could induce a direct antiviral state in infected hepatocytes and activate immune cells by producing proinflammatory cytokines (such as TNF-a, IL-1, IL-6) and chemokines. TNF-a was shown to play an important role in the control of viral infections. It has a direct antiviral effect and is essential for the proliferation of HBV-specific cytotoxic T cells to coordinate with adaptive antiviral immunity. Corresponding with lower TLR2 expression, base levels of TNF-a production were suppressed in Immune Tolerance patients as compared to Healthy Controls(P=0.032). Interesting, the response to TLR2 ligand stimulation remained to drive multifold increase of TNF-a production whether in IT or HC (P=0.043, P=0.043), despite TNF-a levels still were lower in IT than HC (P=0.056). So, higher TLR2 expression might represent better response to TLR2 ligand, with more TNF-a production, contributed to control HBV infection.TLR2 and TLR7 ligand stimulation made minor changes on function of T cell and NK cell in CHB patientsRecently, some studies presented ideas that TLRs could induce NK cell or T cell activation, and coordinate adaptive immunity through cell-cell cross talk. Therefore, it is necessary to explore whether TLR2 or TLR7 ligand stimulation could induce adaptive immunity or lead to activation of NK cell function. Unfortunately, neither TLR2 nor TLR7 ligand could induce obvious changes of T cells and NK cells, except for TLR8 ligand stimulation on PBMCs in HC or CHB patients showed moderate increase in IFN-y secretion of NK cell(P=0.043, P=0.043). A further design was needed to explore the contradiction.Conclusions1. TLR2 and TLR7 expression on PBMC were suppressed in chronic inactive HBV infection, and could be partly restored with CHB in inflammation activity. While not all subsets of PBMC could restore completely, TLR2 and TLR7 expression were still suppressed on CD 14+monocytes in CHB patients.2. Higher TLR2 and TLR7 levels on PBMC at baseline were associated with complete response of LdT antiviral treatment, but it was not applicable to Peg-IFN-a-2a therapy. Peg-IFN-a-2a therapy could induce up-regulation of TLR7 levels on PBMC, higher TLR7 expression were showed in complete response patients in week 48 comparing to NCR. TLR7 mediated immune response may be involved in antiviral effect of Peg-IFN-a-2a.3. The in vitro experiments showed that TLR7 ligand stimulation on PBMC in CHB patients could induce antiviral effects through secretion of INN-α/γ, but that not of TLR2 ligand. While TLR2 ligand stimulation on PBMC in chronic HBV infection could enhance production of TNF-α. The results suggested restoring of TLR2 and TLR7 contribute to control HBV infection. |
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