The Effects Of HBV On NLRP3Inflammasome Activation And Related Mechanism

BackgroundThe hepatitis B virus (HBV) genome is a3.2-kb uncomplicated double stranded DNA. The HBV genome has4overlapping open reading frames (ORFs) coding coated antigen (HBs),core protein (HBc), X protein (HBx) and DNA polymerase respectively. HBV is a hepatotropic virus. HBV infection could induce chronic hepatitis, even progress to liver fibrosis, cirrhosis, and eventually to hepatocellular carcinoma (HCC). It is estimated that over400million people are chronically infected with HBV worldwide.And there are more than100million people died of liver disease every year. HBV-related liver disease and its complications result in approximately40%deaths out of the world. Therefore, HBV is harmful to human health and is one of the major killers to people. However, immunological liver injury induced by HBV infection is not very clear so far, its pathogenic mechanism is urgent to be discussed.For a long time, most scholars believed that the success of HBV clearance was associated with strong specificity of cellular immune responses, however, a growing evidence suggests that innate immunity plays a significant role in the process of HBV infection and pathogenesis.It is found that HBV is recognized by nonparenchymal cells of the liver, mainly by liver macrophages (Kupffer cells). KCs are macrophages residing in the liver. KCs occupy80%to90%out of the total number of macrophages, which is the largest group of macrophages in the body.KCs are the major cells responsible for the inflammatory reaction and cytokines in the liver and involved in a variety of chronic liver disease in the process of the occurrence and development of the liver damage by producing inflammatory factors such as IL-1β, IL-18.The maturation of IL-1β and IL-18depends on the activation of inflammasome. Tschopp group discovered and reported inflammasome for the first time in2002. The first identified inflammasome is NLRP1inflammasome. The inflammasome is a molecular platform for IL-1β and IL-18production by triggering activation of inflammatory caspase-1.IL-1β is synthesized as pro-IL-1β which is further processed to its mature form by activated caspase-1. So far, several inflammasome complexes have been identified, including NLRP3inflammasome, NLRP1inflammasome, NLRC4inflammasome, AIM2inflammasome, RIG-I inflammasome et al. The NLR family, and pyrin domain-containing3(NLRP3) inflammasome is the best-characterized. The components of the NLRP3inflammasome are involved in sensing multiple danger signals. Recent studies have indicated that both DNA viruses such as adenovirus, varicella-zoster virus and RNA viruses such as influenza virus, hepatitis c virus (HCV) can stimulate the NLRP3inflammasome.It is reported that small DNA virus without or with a coating of macromolecular DNA viruses can activate NLRP3inflammasome.The activation of NLRP3inflammasome involves proteolytic activation of caspase-1, promoting mature and release of IL-1β or IL-18, leading to inflammation.It is reported that the promoter polymorphism of IL-18, IL-1β gene and HBV infection and prognosis is closely related. HBx and HBc protein can induce the generation of IL-18. IL-1β level is increased in patients with chronic hepatitis B, and IL-1β levels are positively correlate with HBV DNA. However, related reports about the mechanism of HBV regulating production of IL-1β and IL-18have not been found.This paper aims to evaluate the regulatory effect of HBV on NLRP3inflammasome for the first time.Tim (T-cell immunoglobulin domain and mucin domain) family was firstly discovered and identificated by McIntire in2001, which was a gene familiy associated with asthma and allergic disease. Murine Tim genes are located on chromosome11B1.1. There are eight members in mouse Tim family, which includes genes encoding Tim-1to Tim-4proteins and Tim-5to Tim-8hypothetical genes. Human Tim family includes only three genes, located on chromosome5q33.2, coding Tim-1, Tim-3, Tim-4protein respectively. Tim protein is a type I transmembrane glycoprotein with a common motif containing immunoglobulin (IgV) domain, mucins (Mucin) domain, transmembrane region and intracellular region. Except for Tim-4, intracellular regions of Tim-1to Tim-3molecules contain tyrosine kinase phosphorylation site, so these molecues are involved in the intracellular signal transduction directly. Tim-4plays an important role in immune regulation and maintaining homeostasis. Tim-4is a natural ligand of Tim-1, mainly expressed on the surface of activated antigen presenting cells, especially on macrophages and dendritic cells.Tim-4and Tim-1can provide costimulatory signal for T cell activation, which is important for T cells activation. However, the regulation of Tim-4on T cell activation is a two-edged sword. Tim-4shows negative regulation on macrophages. Our previous results showed that overexpress of Tim-4can inhibit IL-1β production from macrophages induced by LPS. In mice hepatitis model induced by ConA, transfer macrophages with Tim-4overexpression inhibited the production of IL-1β, indicating that Tim-4may inhibit the activation of inflammasome. In addition, it is found that Tim-4expression in PBMCs from the patients with chronic hepatitis B or macrophages from HBV transgenic mice were significantly lower than those of controls. Whether HBV regulates the expression of Tim-4by affecting the promoter activity of Tim-4is not clear. It should be clarified that whether HBV affects NLRP3inflammasome through regulating Tim-4expression. This study is performed to solve these related questions.AimsTo investigate the effects of HBV on NLRP3inflammasome activity and explore the related mechanisms. To construct the report vector of Tim-4gene promoter and identify the promoter activity. To explore the effects of Tim-4on NLRP3inflammasome activity.MethodsPartⅠ:The effects of HBV on NLRP3inflammasome1. Clinical samples(1)Collection of plasma from CHB patients and healthy controlsHeparin anticoagulant whole blood from56patients with chronic hepatitis B without treatment and20healthy controls were collected. Plasma were collected after centrifugation and saved at-20℃. (2)Detection of IL-1β by ELISAThe plasma IL-1β levels were measured by ELISA kits according to the instructions.2. Animal experiments(1) HBV transgenic mice and normal controls35HBV transgenic mice with Balb/c background and12Balb/c mice were included in this study.(2) Detection the HBV DNA and antigenThe whole blood was taken from HBV transgenic mice.The serum was separated and used to detect HBV DNA copies by real time PCR, and HBsAg by ELISA.(3)Detection of IL-1β by ELISAIL-1β levels in the serum were detected using ELISA kits.(4) Detection of Caspase-1activation in liver tissue by Western blotWe chose14cases of HBV transgenic mice, and liver proteins were extracted to measure Caspase-1by western blot.2. Cell models(1) Screen and identification of the Caspase-1, ASC, NLRP3siRNASmall interfering RNA targeting human ASC, caspase-1and NLRP3were designed and synthesized respectively.In HEK293cells, co-transfection with siRNA and plasmid DNA was performed.48h later, western blot was used to verify the interference effect. The effect of siRNA was also assayed with THP-1cells induced by PMA.(2) THP1cells were stimulated with200μl culture supernatants of HepG2.2.15or HepG2cells, and supernatants were collected at indicated time points for IL-1β detection.(3) HepG2.2.15cells or HepG2cells were cocultured with THP1cells in transwell Chambers. THP1cells were plated in12culture plates at the density of4×105/ml, and HepG2.2.15/HepG2cells were plated in the upper chamber with different ratio.48hours later, THP-1cells and supernatants were collected for detection.(4) Detection of IL-1β:Same as above (5) Identification of NLRP3infammasome:THP1cells were stimulated with HepG2.2.15or HepG2cell culture supernatants.48h later, stimulated THP-1cells were collected and fixed with4%paraformaldehyde. Rabbit anti-human Caspase-1and rabbit anti-human NLRP3monoclonal antibodies were used to do IFA, and the fluorescence was observed under fluorescence microscopy.4. NLRP3inflammsome reconstitution(1) Reconstitution of NLRP3inflammasome system According to the references, NLRP3inflammasome system was reconstituted in HEK293cells by cotransfection with pDsRed-monomer-Cl-hASC, pDsRed-monomer-Cl-hCaspase-1,pCR4-TOPO-h-NLRP3, pDsRed-monomer-Cl-hIL-1β.48hours after transfection, the supernatants were collected to detect IL-1β by ELISA.(2)Co-transfection of HBV and NLRP3inflammasome As above, NLRP3inflammasome was reconstituted together with pcDNA3.0-HBV1.1or control vector in HEK293, HepG2, HeLa or Huh7cells.48h later, the cells and supernatants were collected.(3) Detection of IL-1β by ELISA IL-1β was detected using ELISA kits.(4) Detection of Caspase-1activation by Western blot After cotransfection, HEK293cell protein was extracted to detect Caspase-1activation by western blot.5. Set up the stable cell line with ASC overexpressionOptimal concentration of G418was screened with HEK293cells. HEK293cells were set up at1000cells/ml, and G418was added with final concentration at400μg/ml,600ug/ml,800μg/ml or1000μg/ml. Culture supernatants were changed with fresh mediun containing the same concentration of G418every1to2days. After two to three weeks, the lowest concentration of G418leading to HEK293cell death was used for screening.pDsRed-monomer-Cl-hASC or control vecter plasmid DNA were transfected into HEK293cells using liposome.48h after transfection,800μg/ml of G418was added to transfected HEK293cells, and fresh medium was changed every1-2days. After two weeks, cells were observed under fluorescence microscopy. Western blot was used to assay ASC expression.PartⅡ:Constuction of report vector of TIM-4promoter and identification(1) Construction of the report vector of TIM-4promoterAfter analysis with software, the human TIM-4gene promoter region was located at-1887/+65, and a series of truncated promoters were designed according to the binding site of transcription factors.Human PBMCs genome DNA was used as template to amplify TIM-4gene promoter fragment by PCR amplification.TIM-4gene promoter fragment was linked to T-esay vector by T4Iigase, and positive clone was screened by blue white spot screening. Then the plasmid was digested with Sac Ⅰ and Xho Ⅰ and linked to pGL3. After transformation, positive recombinant plasmid was identified by PCR, enzyme digestion and sequencing.pGL3-TIM-4(-1887/+65) plasmid DNA was used as the template, other truncated promoters including pGL3-TIM-4(1682/+65),-1307/+65,-1162/+65,-887/+65,-662/+65,-392/+65,-162/+65,-42/+65were constructed as above.(2)Detection of promoter activityThe activity of the report plasmid TIM-4promoter was assay by double luciferase method. A549cells were planted in48well plate at the density of2×105/ml,500ng of promoter plasmid DNA and10ng of pGL-TK were cotransfected using liposome.48h later, cells were washed with PBS for three times, then PBL lysis buffer was used to lyse cells completely. Fluorescent counting instrument was used to detect promoter luciferase activity and internal pGL-TK. Renilla luciferase activity.PartⅢ:The effect of TIM-4on NLRP3inflammasome(1) Reconstitution of NLRP3inflammasome systemAs above, NLRP3inflammasome was reconstituted together with pcDNA3.0-hTIM-4or control vector in HEK293or HeLa cells.48h later, the supernatants were collected for IL-1β assay.(2) Construction the expression vector of murine Tim-4Recombinant plasmids containing murine Tim-4with different domain deletion were constructed.These plasmids included pEGFP-mTim4(full length), pEGFP-mTim4(ΔC), pEGFP-mTim4(ΔTC), pEGFP-mTim4(ΔTC)-DAF. The positive recombinant plasmids were identified by PCR, enzyme digestion and sequencing. Then plasmids were transfected into CHO cells, and the fluorescence was observed under fluorescence microscope.ResultsPartⅠ:The effect of HBV on NLRP3inflammasome1. Enhanced IL-1β in chronic hepatitis B patients or HBV transgenic miceIL-1β levels in plasma from patients with chronic hepatitis B or serum from HBV transgenic mice were significantly higher than those of normal controls. However, the levels of IL-1β were relatively low, no significant correlation of IL-1β with HBV DNA or antigen was found.2. Increased IL-1β in HBV stimulated monocyte/macrophagesTHP-1cells were cocultured with different ratio of HepG2.2.15cells for48hours. The supernatants were used to detect IL-1β. The results showed that IL-1β levels were increased in THP-1cells after coculture with HepG2.2.15cells. Supernatants from HepG2.2.15cells also stimulated THP-1or RAW264.7cells to produce IL-1β.3. HBV promoted IL-1β production in a Caspase-1dependent wayELISA results showed that HBV induced IL-1β production in THP-1cells treatment with Caspase-1inhibitor was significantly lower than those of the control group.4. NLRP3played a role in HBV stimulated IL-1β production4.1The effect of siRNAAfter cotransfection with plasmid DNA and siRNA in HEK293cells,the effect of siRNA was assayed by western blot.The results showed that siNLRP3-978,2545could inhibit NLRP3expression. For ASC,the three siRNAs all took effect, while siCaspase-1-788,1094showed better effect of knock down.4.2HBV promoted IL-1β production through activating NLRP3inflammasome The results showed that HBV induced IL-1β production in NLRP3siRNA transfected THP-1cells was significantly lower than that of siRNA control group,indicating that NLRP3inflammasome is responsible for HBV stimulated IL-1β secretion.4.3HBV promoted the NLRP3inflammasome assemblyIn order to further identify the role of NLRP3inflammasome in HBV induced IL-1β production,NLRP3inflammasome system was reconstituted successfully by IL-1β assay and RT-PCR detection of NLRP3or other gene mRNA.Then we reconstituted NLRP3inflammasome together with pcDNA3-HBV1.1in HEK293, HepG2, HeLa, or Huh7cells, and the results showed that cotransfection with plasmid containing HBV could promote IL-1β production, and Caspase-1activation was also increased significantly. In addition, we found that NLRP3inflammasome activation could inhibit the production of HBV antigen.4.4We detected the expression of NLRP3and Caspase-1in THP-1cells stimulated with supernatants containing HBV. IFA results showed that expression of NLRP3and Caspase-1in THP-1cells treated with supernatants containing HBV was higher than that of control group.5. Establish of ASC overexpression cell line and construction of pcDNA3-hASC-HA5.1After G418screening, the cells could expression the red fluorescence fusion protein which could be observed under fluorescence microscopy. In addition, ASC protein could also be detected in HEK293cells with ASC overexpression, indicating that the stable cell line with ASC overexpression was established successfully.5.2pcDNA3.0-hASC-HA was contructed regularly. After identification with PCR, digestion and sequencing, the recombinant plasmid was verifed correctly.6. HBV activated NLRP3inflammasome through RaclThe results showed that Racl siRNA treatment could reduce IL-1β production in HBV stimulated THP-1cells, indicating that Racl signal pathway was important for HBV induced NLRP3inflammasome activation.PartⅡ:Constuction of the report vector of TIM-4promoterA series of report vectors of full length or trancated TIM-4promoter were constructed and verified by PCR, digestion or sequencing.The activity of these TIM-4promoters were assayed in A549cells, however the activity was relatively low.ParⅢ:The effect of TIM-4on NLRP3inflammasomeIn the reconstituteion system, cotransfection with plasmid containing TIM-4could inhibit IL-1β production significantly.Then a series of vectors of murine Tim-4with different domain deleteon were constructed successfully.The recombinant plasmids were transfected into CHO cells, and the green fluorescence could be observed under the fluorescence microscope.Conclusion1. HBV could promote IL-1β production, which was verified with chronic hepatitis B patients and HBV transgenic mice.These data indicate that HBV infection might be involved in inflammasome activation.2. HBV induces IL-1β production in a Caspase-1dependent manner.4. Rac1is involved in activation of NLRP3inflammasome induced by HBV.5. TIM-4reduces the activation of NLRP3inflammasome.Innovation points and significanceIn the current study, we found that HBV activated NLRP3inflammasome and TIM-4inhibited NLRP3inflammasome activation for the first time. In addition, we found Rac1was involved in activation of NLRP3inflammasome by HBV infection. These data indicate that HBV infection leads to liver injury partially through activating NLRP3inflammasome, which would provide novel theory for the pathogenesis of hepatitis B and provide new targets for intervene of HBV related liver diseases.