The Role Of HBV In Complement-Dependent Cytotoxicity And The Effect Of LPS On Cytosolic DNA Induced Immunity

Part I:The effect of HBV and its fragments HBc and HBx on CDCHBV is hard to be cleared entirely because HBV DNA often integrating into host DNA, which results in persistent virus infection--a major worldwide health problem. Till now, the pathogenesis for HBV infection and HCC development is still not well-known. The involvement of some immune factors, such as T and NK cells, was documented by other groups before. Our previous studies showed that both the whole HBV genome and its fragments HBx and PreS2 enhanced TRAIL (TNF related apoptosis inducing ligand)-induced hepatocyte apoptosis, while HBc inhibited TRAIL induced apoptosis.Some other studies have already shown that complement system plays a crucial role in HBV infection. The presence of C3 and C4 in the liver parenchyma is abnormal and may be helpful in histological evaluation in chronic hepatitis. The complement system consists of about 30 soluble and membrane bound proteins,90% of plasma complement components and their soluble regulators is synthesized in the liver (mainly hepatocytes). All complement components are activated spontaneous low rate in plasma under physical condition, which is a potential threat to the body. However, Hepatocytes gain their effective complement resistance by expressing multiple regulatory proteins. Human primary hepatocytes express CD46, CD55, CD59 and factor H. CD46,CD55, factor H function by inhibiting C3 activation, while CD59 by restricting MAC formation. Blocking CD59, but not CD46, CD55, or factor H leads to higher hepatocyte lysis rate. Thus, CD59 is considered to play a decisive role in protecting hepatocytes against complement lysis. Then what's the role of complement system in HBV infection? What's the mechanism? How about its fragments HBc and HBx?OBJECTIVES:1. To explore the effect of HBV infection on complement-dependent cytotoxicity (CDC) and its mechanism.2. To investigate the role of HBc and HBx on CDC.METHODS:1. Effects of HBV1.1 expression on gene profile by high throughout screening.1.1 Microarray analysis of HBV transgenic miceTotal liver RNA from HBV transgenic mice and control mice was extracted and hybridized on gene chip. Significant differentially expressed genes were analyzed by bioinformatics approach and some differentially expressed complement components were verified.1.2 Proteomic analysis of HBV transgenic miceTotal liver protein from HBV transgenic mice and control mice was extracted and then resolved by 2D electrophoresis. Identify the differentially expressed protein by mass spectrum and bioinformatics approach.2. The effect of HBV expression on CDC2.1 Establishment of hepatoma cell line stably-expressing HBV.Plasmid pcDNA3-HBV1.1, constructed in our lab before, or pcDNA3.0 was transfected into hepatoma cell line BEL7402 and HL7702 mediated by lipofectamine 2000, and the positive cell clones were obtained by selection with G418 and termed as BEL7402-HBV1.1, BEL7402-pcDNA3 and HL7702-HBV1.1, HL7702-pcDNA3 cells respectively.2.2 The effect of HBV on complement-dependent cytotoxicityPolyclonal anti-sera were prepared by immunizing New Zealand White Rabbit with BEL7402 or HL7702 lysates three times. BEL7402-HBV1.1, BEL7402-pcDNA3 and HL7702-HBV1.1, HL7702-pcDNA3 cells were incubated with anti-sera for 30min and then incubated with normal human serum(NHS) or heat-inactivated NHS(HI-NHS) for 30min or 1h, washed twice; then labeled by incubation with 1μg/ml propidium iodide (PI) for 10 min at room temperature and detected by flow cytometry. The CCK-8 kit (Dojindo Inc.) was also used to determine the cell viability and the percentage of dead cells.2.3 The effect of HBV on CD59 expressionHepatocyte cell line model:mRNA expression level of CD59 on BEL7402-pcDNA3,BEL7402-HBV1.1; HL7702-pcDNA3,HL7702-HBV1.1 cells were detected by semi-quantitative RT-PCR and real-time PCR, protein expression level by Western blot and flow cytometry.Mouse model:Extract mRNA of HBV transgenic mice and normal control mice after liver perfusion, CD59 expression is detected by semi-quantitative RT-PCR and real-time PCR.Clinical samples:Liver tissue samples from twelve chronic hepatitis B patients were obtained by liver centesis. Six normal liver tissue samples were obtained from patients with liver hemangioma or hepatorrhexis. Expression of CD59 was determined by immunohistochemistry.2.4 Contribution of CD59 on CDC of HBV-infected hepatocytes.After blocking CD59 function by specific blocking antibody, CDC assay was carried on and cytotoxicity rate was determined by FCM and CCK-8.3. Role of HBV fragments HBc on CDC.3.1 Establishment of hepatoma cell line stably-expressing HBc or HBxPlasmid pcDNA3-HBc or pcDNA3-HBx constructed in our lab before, was transfected into hepatoma cell line BEL7402 mediated by LF 2000, and the positive cell clones were obtained by selection with G418 and termed as BEL7402-HBc, and BEL7402-HBx, cells respectively.3.2 The effect of HBV on CD59 expressionThe mRNA level of CD59 on BEL7402-pcDNA3,pcDNA3-HBc or pcDNA3-HBx cells were detected by semi-quantitative RT-PCR and real-time PCR; The protein level of CD59 was detected by Western blot and flow cytometry. 3.3 The effect of HBc and HBx on complement-dependent cytotoxicity and its mechanismComplement-dependent lysis of BEL7402-HBc, BEL7402-HBx and BEL7402-pcDNA3 was performed through the classical pathway. The CCK-8 kit was also to determine the cell viability and calculate the percentage of dead cells.。Meantime, examine the role of CD59 in this, BRIC229 was used to block CD59 function.3.4 The regulation of CD59expression in protein level.The ubiquitin-proteasome activity in BEL7402 was blocked by 5μM MG132, and CD59 protein was detected by FCM. at different time point.RESULTS:1. Expression of HBV in hepatocytes changed the level of complement components greatly.As shown by gene microarray, the expression of about 30%of complement components changed in HBV transgenic mice.2. The effect of HBV expression on CDC2.1 HBV expression upregulated hepatocytes sensitivity to CDC.Both FCM and CCK-8 assay showed, the lysis rate of complement on BEL7402-HBV1.1 and HL7702-HBV1.1 increased significantly compared with the control cells (P<0.01)2.2 HBV expression in hepatocytes downregulated CD59 expression.Hepatoma cell model:By RT-PCR and real-time PCR, we found that the RNA level of CD59 after HBV transfection was reduced in both BEL7402 (P< 0.01) and HL7702 (P< 0.05). In addition, we also tested the CD59 expression by FCM and Western blot analysis. The protein level of CD59 was also downregulated by the HBV transfection (P< 0.05).Mouse model:RT-PCR and real-time PCR data showed, the level of CD59 mRNA decreased in HBV transgenic mice (P< 0.05).Clinical Samples:Compared with normal liver tissue, CD59 expression was significantly reduced in the livers of HBV patients by immunochemistry.2.3 Role of CD59 on CDC of HBV-transfected hepatocytes.FCM and CCK-8 assay showed, blocking CD59 by 15ng/ml specific antibody BRIC229 significantly increased cytotoxicity rate in all cell lines, but the difference between HBV transfected cells and control cells diminished (P> 0.05)3. The impact of HBV fragments HBc and HBx on CDC3.1 The effect of HBc and HBx transfection on CD59 expression.Both semi-quantitative PCR and real-time PCR data showed no significant difference of CD59 mRNA level between BEL7402-HBc or BEL7402-HBx and control cells (P> 0.05). However, the protein level of CD59 on BEL7402-HBc was much lower than control (P< 0.01). There was still no difference on CD59 protein level between BEL7402-HBx and control cells (P> 0.05).3.2 Role of HBc and HBx on lysis of hepatocytes by complementCDC assay was applied with 25%of NHS by activating the classical pathway. CCK-8 data showed, the lysis rate of BEL7402-HBc was much higher than control group(P< 0.01), while the lysis rate of BEL7402-HBx was similar with control group (P>0.05)。3.3 Regulation of CD59 by HBcThe ubiquitin-proteasome activity in BEL7402 was blocked by MG132, and CD59 protein was detected by FCM. The result displayed no change on CD59 protein level (P> 0.05), demonstrated that CD59 wasn't degraded by ubiquitinization。Our MicroRNA chip showed, some microRNAs targeting CD59 was upregulated in BEL7402-HBc, which might participate in the regulation of CD59 by HBc.CONCLUSIONS:1. HBV expression changes gene profile of hepatocytes greatly, the regulation of complement system might be one of the mechanism of HBV-related diseases.2. HBV expression can significantly sensitize hepatocytes to complement-dependent cytotoxicity. The further mechanism study demonstrates HBV functions by downregulating CD59 expression. 3. HBV fragment HBc can dramatically increase the sensitivity of hepatocytes to CDC by downregulating the protein level of CD59.4. HBx has effect on neither CD59 expression nor CDC of hepatocytes.INNOVATIONS AND SIGNIFICANCES:1. It's the first time to compare the gene profile influenced by HBV with HBV transgenic mice by both microarray and proteomic methods, which will help to understand the overall effect of HBV.2. Through establishing of HBV expressed hepatoma cell lines, the present study demonstrates that the effects of HBV on CDC and firstly reports the upregulation of CDC by HBV. This is helpful to disclose the mechanism of HBV infection.3. It's the first time to prove downregulation of CD59 expression in HBV transfected hepatocytes, HBV transgenic mice and HBV patients.4. By blocking CD59 function, we firstly show the increased sensitivity to complement of HBV expressed cells is mediated by downregulating CD59, CD59 downregulation is the key factor, which can be a new target for the treatment of HBV infection.5. Based on the study of HBV on CDC, we further report HBc can downregulte CD59 and enhance CDC, while HBx has no effect.Keywords:HBV; CD59; complement; HBc; HBxPart II:The effect of LPS pretreatment on DNA immunityThe innate immune system uses pattern recognition to sense infection, and it initiates an immune response upon pathogen detection. Recent advances have defined two broad categories of microbial stimuli. The first class includes products unique to microbes, such as lipopolysaccharide (LPS) and lipotechoic acid. The second class encompasses nucleic acids derived from pathogens, particularly viruses. Some known TLRs can detect nucleic acids, for example, TLR3 can detect double-stranded RNA, TLR9 can sense methylated CpG motifs. Recent studies show almost all cytosolic double-stranded B-form DNA can trigger immune response without activating TLRs. The sensor for it is still not clear, maybe DAI or others. Cytosolic DNA can activate TBK1 and then IRF3, promote the transcription of type I IFN.LPS is a component on Gram negative bacteria, and can be sensed by TLR4. It can induce cytokine production by activating NF-κB through MyD88-dependent and independent pathway, also induce type I IFN production by activating IRF3 through MyD88-independent pathway. When cells are stimulated with LPS twice subsequently, the induction of cytokines is mostly decreased, this phenomenon is called LPS tolerance or endotoxin tolerance. Similar phenomenon also shows up when cells are stimulated with LPS and other TLR simulators subsequently, which is called cross-tolerance. Then, what's the impact of LPS on cytosolic DNA induced immunity? What's the mechanism? Is that similar to LPS tolerance? Based on these questions, we focused on the impact of LPS on DNA immunity and have gained some periodic outcome.OBJECTIVES:To probe the impact of LPS on DNA immunity and its mechanismMETHODS:1. The effect of LPS pretreatment on DNA induced type I IFN expression.1.1 The effect of LPS pretreatment on chemically synthesized DNA induced type I IFN expressionThe impact LPS pretreatment on transfection efficiency:Stimulate RAW264.7 with 100ng/ml LPS for 24h, wash twice, incubate with fresh medium for 1h, then transfect the cells with pEGFP plasmid using Lipofectamine LTX,24h later, check the GFP positive signal by FCM. Transfection of DNA into RAW264.7 or BMDM:Pretreat RAW264.7 with LPS then transfect with DNA,4h later, extract mRNA and detect Ifnb1 and Ifna4 expression by real-time PCR.1.2 The effect of LPS pretreatment on bacteria DNA induced type I IFN expression1) Impact of LPS pretreatment on infection rate of LMReal-time PCR:Pretreat RAW264.7 with LPS for 24h, then add LM (MOI 6:1), 4h later, extract genomic DNA, detect the ratio of bacteria genome and cell genome by real-time PCR.FCM:Pretreat RAW264.7 with LPS for 24h, then add GFP-LM (MOI 6:1),4h later, detect the proportion of GFP positive cells.2) Ifnbl induction by infection of LM into RAW264.7Infect RAW264.7 cells pretreated with or without LPS with LM,4h later, detect expression of Inb1 and Ifna4 by real-time PCR.2. The mechanism for the effect of LPS pretreatment on DNA induced type I IFN production.2.1 Transciptional level Tranfect HEK293-TLR4 with luciferase plasmid containing Ifnb1 promoter for 24h, then stimulate with LPS and DNA subsequently as described above,20h later, the cells were harvested and the luciferase assay was used to detect the luciferase activity.2.2 Cellular pathwayExtract protein at different time point after DNA transfection with or without LPS pretreatment. Detect the phosphorylation of IκB, IRF3, cJun. Examine Ifnb1 expression after blocking cJun or IκB activation.3. The impact of DNA induced gene profile by LPS pretreatment.BMDM were stimulated with 100ng/mL LPS for 24h, then transfected with lug/ml DNA,4h later, extract total RNA, and analyzed by microarray.RESULTS:1. The effect of LPS pretreatment on DNA induced type I IFN expression. 1.1 LPS pretreatment has no obvious impact on transfection efficiency.After transfected with pEGFP plasmid for 24h, GFP positive RAW264.7 cells were determined by FCM, and the data showed no obvious difference with or without LPS pretreatment(P> 0.05). Similarly, there's no big difference of infection efficiency between the two groups.1.2 Blunted Ifna4 and Ifnbl expression induced by DNA after LPS pretreatmentAfter stimulating both RAW264.7 and BMDM with either synthesized DNA transfection or LM infection, the induction of Ifna4 or Ifnb1 decreased in LPS pretreated cells (P< 0.01).2. Mechanism for the effect of LPS pretreatment on DNA induced IFN production.2.1 LPS pretreatment inhibited the Ifnbl promoter activity induced by DNA.Dual-luciferase reporter assay showed LPS pretreatment significantly inhibited Ifnb1 promoter activity(P<0.01).2.2 Effect of LPS pretreatment on the transcription factors of Ifnbl promoterWestern Blot assay showed, the activation of IRF3, IκB, cJun decreased.2.3 Role of NF-κB和cJun pathway on Ifnbl inductionBlocking cJun pathway by JNK inhibitor inhibited Ifnb1 induction by DNA, but blocking NF-κB pathway by IκB inhibitor didn't change Ifnb1 level.3. The impact of gene profile induced by DNA by LPS pretreatment.Microarray data showed, almost all type I IFNs induction decreased upon LPS pretreatment.754 genes were induced by DNA, of which 276 were downregulated over 2 folds by LPS pretreatment, and 92 showed no change or even higer response. The downregulated genes were rich in transcription factor IRFs and Statl. Western Blot showed the activation of Statl decreased and SOCS1 increased in LPS pretreated group.CONCLUSIONS:1. LPS pretreatment can inhibit cytosolic DNA induced type I IFNs induction, indicates TLR4 pathway can influence DNA immunity. 2. LPS can inhibit Ifnbl expression by repressing its promoter activity. The activation of two Ifnbl transcription factors, IRF3 and cJun, are tolerized by LPS.3. DNA induced genes are not all tolerized by LPS, some are even primed. This is similar to LPS tolerance, but the profile is not the same, which means the mechanism might be different.4. The tolerizable genes in LPS-DNA immunity is largely mediated by tolerized IRFs and Statl. The increased level of negative regulator SOCS1 is also a key factor.INNOVATIONS AND SIGNIFICANCES:1. It's the first time to investigate the effect of LPS on DNA immunity. This study will help to explain mechanism of combined clinic infection, and is important for the cure of infectious disease and instruction of DNA vaccine.2. The present study firstly reports LPS pretreatment upregulates the level of SOCS1 and inhibit the phosphorylation of IRF3, cJun and Stat1, result in decreased expression of type I IFN and other tolerizable genes.3. We are the first to study the changed gene profile in DNA immunity by LPS pretreatment through microarray, which is the basic for further study on its mechanism.