Preliminary Study The Expression And Mechanism Of NLRP3Inflammsome In Hepatocytes

Inflammasomes, multiprotein complexes in cells, could initiate inflammatory responseand enhance innate and adaptive immunity through promoting the secretion of IL-1β andIL-18, and pyroptosis after senseing the pathogen-associated molecular patterns (PAMPs)and damage-associated molecular patterns (DAMPs), which contribute to immune defenseand immune homeostasis. Pattern recognition receptors, Nod-like receptor (NLR) family orAIM2-like receptor (ALR) family, are involved to the formation of scaffoldmacromolecular protein about700KD. And then pro-caspase-1is recruited to this platformand autocatalytically cleaves to its active form. Subsequently, activated caspase-1cleavespro-IL-1β/IL-18in the cytoplasm into mature IL-1β/IL-18and allows its secretion, orinduces pyroptosis, a form of cell death, finally results in inflammation.NLR (NOD-like receptor), is a major sensor involved in inflammasome formation,which contains NLRP1, NLRP3, NLRP6and NLRC4. The NLR family is characterized bythe presence of a central nucleotide-binding and oligomerization (NACHT) domain, whichis commonly flanked by C-terminal leucine-rich repeats (LRRs) and N-terminal caspaserecruitment (CARD) or pyrin (PYD) domains. LRRs are believed to function in ligandsensing and autoregulation, whereas CARD and PYD domains mediate homotypicprotein-protein interactions for downstream signaling. The NACHT domain, which is theonly domain common to all NLR family members, enables activation of the signalingcomplex via ATP-dependent oligomerization. NLRP3don’t have CARD domain, and useits PYD domain to recruit adaptor protein ASC to activate caspase-1.NLR family has many activation signals, and NLRP3has the most widely activationsignals. Many studies have focused on the role that the NLRP3inflammasome has in theinnate immune system as a sensor of pathogens (such as viruses, bacteria, and fungi) andendogenous danger signals (for example ATP, Uric cid and silicon crystal), and it involvedin inflammation related disease development, but considering the NLRP3protein molecules could not combined with such a variety of signal molecular. Therefore, they are likely toactivate NLRP3inflammasomes by some common middle way. At present, three majormodels for NLRP3inflammasome activation are favored in the field:(1) The NLRP3agonist, ATP, triggers P2X7-dependent pore formation by the pannexin-1hemichannel,allowing extracellular NLRP3agonists to enter the cytosol and directly engage NLRP3.(2)Crystalline or particulate NLRP3agonists are engulfed, and their physical characteristicslead to lysosomal rupture. The NLRP3inflammasome senses lysosomal content in thecytoplasm, for example, via cathepsin-B-dependent processing of a direct NLRP3ligand.(3)All danger-associated molecular patterns (DAMPs) and pathogen-associated molecularpatterns (PAMPs), including ATP and particulate/crystalline activators, trigger thegeneration of reactive oxygen species (ROS). A ROS-dependent pathway triggers NLRP3inflammasome complex formation. Recent evidence shows that the three different wayslead to NLRP3inflammasome activation maybe just through a common way, which is theincrease of potassium outflow.Recently, some research reveals the functions of NLRP3inflammasome complexesand discusses the aberrant of them are implicated in the pathogenesis of human diseases(such as antiviral, cardiovascular disease). In vivo, inflammasomes have been shown toparticipate in the antimicrobial innate immune response. The influenza A virus is anexample of an indirect viral NLRP3inflammasome activator. On infection, recognition ofviral RNA by means of Toll-like receptor7(TLR7) induces transcription of the NLRP3inflammasome components. Subsequently, the activity of the viral ion-channel protein M2induces pH neutralization of the trans-Golgi network, leading to potassium efflux and ROSformation, which in turn induce NLRP3inflammasome assembly. And we know cholesterolcrystal deposition in arterial vessels has long been a pathognomonic feature ofatherosclerosis. Cholesterol crystals activate the NLRP3inflammasome throughphagolysosome destabilization in mmLDL (minimal modified low density lipoprotein)primed mouse and human macrophages. LDL-receptor-deficient mice (prone toatherosclerosis) reconstituted with bone marrow deficient in NLRP3, ASC or IL-1α/β aremarkedly resistant to the development of atherosclerosis, suggesting that NLRP3inflammasome activation and IL-1secretion from the haematopoietic compartment are keyevents in the early stages of disease. Chronic hepatitis B (CHB) is induced by liver virus HBV infection, which leading topersisting liver injury. More than half of HCC patients are attributable to persistent hepatitisB virus (HBV) infections. Although antiviral treatment could significantly improveprognosis of CHB, there is still half of patients difficult to stop the liver inflammatoryinjury and change the end of the chronic liver cirrhosis and HCC. IL-1β was reported inmany researches that it was related with the pathogenesis of liver fibrosis. Because of IL-1βis also an important downstream product of inflammasome, we highly suspect disregulatedinflammasome may play an important role in the progress of CHB. Because NLRP3inflammasome has a wide variety of activation signals, and it also plays an important rolein other chronic inflammatory diseases, so NLRP3inflammasome dysfunction may be thegreatest possibility in CHB progression. More and more research has demonstrated thatNLRP3Inflammasome not only expressed in bone marrow-derived cells (such as adipocyte,cardiac cells/cardiac fibroblasts), but also play an important role in diabetes, myocardialinfarction development. Hepatocytes are major of liver cells. In contrast to hepatocytesoccupy almost80%of the total liver volume and perform the majority of numerous liverfunctions, nonparenchymal liver cells (such as Kupffer cells (KC), sinusoidal endothelialcells (SEC) and hepatic stellate cells (HSC)), which contribute only6.5%to the livervolume. Therefore, we prepared to investigate the expression and function of NLRP3inflammasome in hepatocytes, including cell lines and primary liver tissue in order topreliminary judgment whether NLRP3inflammasome is involved in the pathogenesis ofCHB, and for further in-depth understanding of its function in CHB.To sum up, this research was divided into two parts. First, we investigated theexpression of NLRP3inflammsome components in hepatocytes and liver tissue in situ bywestern blot and immunohistochemistry (IHC)(Thp-1as the positive control). The resultsshow that NLRP3and caspase-1are expressed in hepatocytes (L02and HepG2) andprimary liver tissue, and NLRP3inflammasome is significantly up-regulated in CHB livertissues compared with normal liver tissues. Second, we detect whether the signal pathwayof NLRP3inflammasome in hepatocytes was functional. Results show that under thestimulation of endogenous danger signal ATP, the IL-1β secretion increased obviously inHepG2/L02. We verify NLRP3inflammasome in hepatocytes could initate an inflammatoryresponse by endougenous signals ATP effectively. Treated with5mM ATP6h (LPS100ng/ml priming12h), the secretion of IL-1β is assayed by ELISA (Thp-1500-1000pg/ml, HepG2300-600pg/ml, L0220-50pg/ml). Considering the hepatocytesamounts in liver, inflammatory response capacity of hepatocytes in CHB should be notignored in chronic hepatitis environment.Methods:1) Part one: the expression of NLRP3inflammsome in hepatocytes.1. We explore the NLRP3inflammsome expression of protein in L02and HepG2wasassayed by immunohistochemistry (IHC) or immunofluorescence (Thp-1as the positivecontrol).2. We detect and analysis the expression of NLRP3, caspase-1and IL-1β in CHB livertissue and normal liver tissue in situ by immunohistochemistry (IHC) orimmunofluorescence (amygdale tissue as the positive control).3. We detect the NLRP3inflammsome expression of protein in L02/HepG2、CHB livertissue and normal liver tissue. We use GAPDH as an internal control, and analyze thedifferential expression of NLRP3inflammsome in CHB liver tissue and normal liver tissueby Quanity One.2) Part two: the mechanism of NLRP3inflammsome in hepatocytes.1. In order to know the mRNA expression of caspase-1and IL-1β in hepatocytes whenresponse to inflammatory stimulus. So the mRNA expression of caspase-1and IL-1β in L02and HepG2cells treated with LPS or not was assayed by RT-PCR and RT-real time PCR.2. In order to verify whether NLRP3inflammasome in hepatocytes could initiate aninflammatory response to endougenous danger signals ATP effectively. So the NLRP3expression of protein in L02and HepG2and the activation of caspase-1in L02(primingwith LPS) and HepG2(priming with LPS) treated with ATP was assayed by western blot.Inorder to know whether the ATP promote secretion of IL-1β and IL-18in hepatocytesdependent on caspase-1. So the secretion of IL-1β in L02/HepG2/Thp-1treated with LPS,LPS/ATP or LPS/ATP/Y-VAD-fmk was assayed by ELISA.Results:1. Immunohistochemistry (IHC) and immunofluorescence results show that NLRP3 and caspase-1are not significant expressed in hepatocytes (L02and HepG2). NLRP3inflammasome is significant up-regulated when LPS stimulus around24h. And, not onlyNLRP3inflammasome and its components in normal liver tissues and CHB liver tissues,but also the expression of NLRP3, caspase-1and IL-1β were significant up-regulated inhepatic parenchymal cells in CHB liver tissues compared with normal Liver tissues. We cannot detect the expression of caspase-3in CHB liver tissue by IHC.2. RT-PCR and RT-real time PCR results show that the mRNA expression of caspase-1and IL-1β treated with LPS18h were up-regulated in L02and HepG2cells compared withno stimulus, but the mRNA expression of caspase-1and IL-1β in L02/HepG2was increaseda little when LPS short-term stimulus around4h. So the two strain liver cells may not besensitive enough response to LPS stimulation, and increase inflammasome componentsneed a long-term stimulus.3. Western blot results show that NLRP3inflammasome is expressed in hepatocytesL02/HepG2, CHB liver tissue and normal liver tissue. Quanity One analysis show thatNLRP3inflammasome is significant up-regulated in CHB liver tissues compared withnormal Liver tissues. And it was clear that damage associated signal ATP promoteactivation of NLRP3inflammasome, and pro-caspase-1is recruited to the platform and thenautocatalytically cleaved to its active form (P10substrate).4. ELISA results show that the secretion of IL-1β and IL-18in hepatocytes ispromoted by endougenous danger signals ATP, and caspase-1blocker Y-VAD-fmk cansignificant down-regulate the secretion of IL-1β and IL-18when L02and HepG2cellsstimulated by ATP. So the secretion of IL-1β and IL-18in hepatocytes is promoted byendougenous danger signals ATP dependent on the activation of caspase-1.Conclusion:1. NLRP3inflammasome is expressed in hepatocytes L02/HepG2, CHB liver tissueand normal liver tissue. Damage associated signal ATP promote activation of NLRP3inflammasome in hepatocyte. The secretion of IL-1β and IL-18in hepatocytes is dependenton the activation of caspase-1. So NLRP3inflammasome in hepatocytes can initiate aninflammatory response to endougenous danger signals.2. Compared with bone marrow cell line Thp-1, hepatocytes inflammatory response ability is weak. Upon the similar stimulating intensity, concentration of IL-1β in thesupernatant of L02is only1/50-1/10of Thp-1, but HepG2can reach the1/3-1/2of Thp-1.Considering the ratio between hepatocytes and macrophages, dendritic cells in the localliver tissue, we suspect that NLRP3inflammasome in hepatocytes may be involved ininflammatory response of CHB, but it needs further data.3. The expression of NLRP3, caspase-1and IL-1β were up-regulated in hepaticparenchymal cells in CHB liver tissues compared with normal Liver tissues. Which implyNLRP3inflammasome play a role in the development of CHB.