Complement-mediated Immunopathological Injury In Fulminant Hepatic Failure: Roles And Mechanisms

Fulminant hepatitis failure (FHF) has very high mortality and the condition usually worsens quickly with poor prognosis. Despite numerous research efforts, its mechanism is still not clear. Clinically, there are a variety of induction factors for FHF, thus the mechanisms of hepatic injury are complicated. FHF induced by different factors share some common features but also differ in various aspects of pathological injury and mechanism. To investigate the mechanism of acute liver injury in FHF in depth, it is essential to establish animal models of FHF induced by different factors. Complement is in the first-line of defense of the immune system, and overactivation of complement mediates immunopathological injuries. But the role of complement in FHF has been rarely reported.The current study established two mouse FHF models, one induced by LPS/D-GalN and the other by mouse hepatic virus 3 (MHV-3) infection. With the two models, the role of complement-mediated immunopathological injury in acute liver failure was studied using C3 deficient mice (C3-/- mice), targeted complement inhibitor (CR2-FH), which blocks the alternative complement activation pathway, and by blocking the binding of C3a with its receptor C3aR. These studies will provide experimental and theoretical basis for novel clinical interventions for FHF.1.Role of complement in LPS/D-GalN induced mouse FHF①Establish LPS/D-GalN-induced mouse FHF model: C57BL/6 mice were given intraperitoneal injection of different doses of LPS/D-GalN. Their survival time and liver failure pathology were monitored and the optimal experiment dose was determined. At various time pints, hepatic tissue pathology and serum levels of ALT and inflammatory factors were evaluated. The results showed that the model displays the progressive pathological damages of acute live injury associated with FHF. This model is stable and easy to operate, and could provide ideal immunopathological injury model for experimental study of acute liver injury.②Study the mechanism of complement-mediated immunopathological injury in LPS/D-GalN induced FHF: The distribution and deposition of complement C3 in liver tissues were examined by immunohistochemistry, and the expression levels of C3a receptor and C5a receptor in liver tissues at different time were examined with Real Time PCR. The results indicated that compared with the control normal C57BL/6 mice, the LPS/D-GalN intoxicated mice showed dynamic changes in C3 deposition: increased deposition at 1 h after mainly around the central hepatic vein, reduced expression at 4 h after, and peaked again at 8 h after mainly deposited around necrotic tissues. The expression level of C3aR rose at 1h, decreased at 4h, and then peaked at 8h. C5aR expression level stepped up constantly and peaked at 8h after LPS/D-GalN injection.③Role of blocking complement activation in LPS/D-GalN induced FHF:Mice lacking complement C3 activation have lighter pathologic injury: Groups of C3-/- mice and C57BL/6 mice were given intraperitoneal injection of a single dose of LPS(50ng)/D-GalN(6mg)and were sacrificed at different time points after administration (0h and 1h, 4h, 8h) or left for observation of survival time. The C57BL/6 mice had higher expression of inflammatory factors IL-6.MCP-1 and TNF-α, and more severe parenchymal damage, and faster FHF progression than C3-/- mice at all time points examined. The C3-/- mice had higher survival rate than the C57BL/6 mice.Blocking C3a binding with its receptor C3aR can alleviate the pathologic injury in FHF: At both 45 min before and after LPD/D-GalN administration, mice were given via tail vein injection C3aR inhibitor at the dose of 40μg/injection. These mice with C3aR blockade had significantly longer survival time, slower progression of liver injury, lower expression of inflammatory cytokines, and less degree of injury, and shallower deposition of complement C3 than the control FHF mice.Blocking alternative complement pathway with Targeted complement inhibitor CR2-FH can alleviate the pathologic injury in FHF: Following i.p. LPD/D-GalN administration, mice were immediately given CR2-FH via tail vein. The CR2-FH injected mice had significantly slower liver injury progression and also lighter degree of injury than the control FHF mice2. Role of complement in MHV-3 infection induced FHF①Establish MHV-3 infection induced mouse FHF model: Mice were given i.p. injection of various doses of MHV-3, their survival time and liver failure pathology were examined, the LD50 (medial lethal dose) was derived and the optimal experiment dose was decided. In addition to the same parameters examined as in the LPS/D-GalN model, virus titer and viral load were also evaluated at different time after infection. From the data of pathology, serum ALT and inflammatory cytokines, and viral replication, the pathological progress models the presentations of acute viral hepatitis in humans and it simulates the virus replication and damages induced.②Complement-mediated immunopathologic injury in MHV-3 infection induced FHF: Compared with the control (0h) group, C3 deposition in the MHV-3 infected mice started to deepen at 6 h after and peaked at 12 h and was mainly in areas of liver tissue injury, which decreaed at 24 h but increased again at 72h. The expression levels of C3aR and C5aR showed similar pattern (up, down and up again at 72 h) but C3aR first peaked at 12 h while C5aR first peaked at 6 h.③. Role of complement activation blockade in MHV-3 infection induced FHF :Lacking complement C3 activation aggravates the pathologic injury: The C3-/- mice and C57BL/6 mice were treated with a lethal dose of MHV-3 and their presentations of FHF were compared. Although the two groups did not differ in survival time (p<0.05), but the data of pathology, ALT, inflammatory cytokines,, and virus replication indicated how that the C3-/- group had faster procession of FHF and also more severe injury at any time point than the C57BL/6 mice.Blocking C3a binding with C3aR worsened injury: The C3aR inhibitor was injected, at a dose of 40μg/injection, at 1 h before, and 12h, 36h after the C57BL/6 mice were infected with a lethal dose of MHV-3. The data showed that mice with C3aR blockade had significantly faster FHF progression and less C3 deposition than mice MHV-3 infected mice without the complement blockade. Overall from the data of histopathology, ALT, inflammatory cytokines and virus replication, the FHF progression and severity of FHF of C3aR blockade mice was between that of MHV-infected C3-/- (most severe) and MHV-infected normal C57BL/6 mice.Through the above two parts of systematic experiments, this study found that in LPS/D-GalN induced FHF, complement activation is mainly through the alternative pathway, and factors produced in the activation process promotes inflammation and also complement mediates injury and necrosis of target cells in the body. Targeted complement inhibitor CR2-FH can block to some level the activation of complement alternative pathway and thus lessen complement-mediated immunopathological injury. Therefore, complement-mediated immunopathological injury serves an important pathogenic role in LPS/D-GalN-induced mouse FHF. In contrast, in MHV-3 infection induced FHF, complement activation produces oposonins which can promote phagocytes to engulf viruses and prevent viruses to enter target cells, and inhibit virus replication and lessen injury to the body. Therefore, in viral pathogen induced FHF, complement is not a factor of pathological damage, rather it plays a protective role . This is completely different from the role of complement in LPS/D-GalN induced FHF.