Complement C5a/C5ar Pathway Engages In The Pathogenesis Of Fulminant Hepatitis

Fulminate hepatitis was one of the liver diseases in the world and it was rocked to thethird-leading cause of death for China in these years. The reasons that cause to this diseasecontains5main ways: viral hepatitis (particularly HAV and HBV), medication overdose(particular paracetamol), idiosyncratic drug reactions, ingestion of toxins and metabolicdisorders, but these etiology were far-fetched and there were still many fields that we havenot know from this disease. It is recognized that this disease was not cured when it’shappening because the process of it was so quickly that we could not take steps in a minute.As a result, to study this disease was significant to assessment of the prognosis of fulminanthepatitis and important for distinguishing patients requiring liver transplantation in casemanagement of acute liver failure. At present, studies using a FH model induced by murinehepatitis virus-3(MHV-3) infection let us know the mechanisim of the Fulminant hepatitisand provide a novel therapeutics to treat the disease. MHV-3belongs to coronavirus familywhich cause hepatocellular necrosis and mortality in susceptible BALB/c and C57BL/6mice, whereas A/J mice are completely resistant to infection. Previous study indicated thatfibrinogen like protein-2(FGL2) pro-thrombinase plays an important role in MHV-3induced fulminant hepatitis.Complement system is an important part of the innate immune defense and isimportant for the cellular integrity tissue homeostasis and modifying the adaptive immuneresponse. The complement system can be activated by three ways: classic, alternative andLectin pathway. The activated complement system can eliminate the invaders and protectour body. This system can also modify self-cells such as apoptotic particles and cellulardebris but also can regulate the cell circle by its ligands and receptors. Previous studydemonstrated that C5a and its receptor C5aR play an important role in various diseasesespecially acute infection model, such as experimental autoimmune encephalomyelitis(EAE), allergic airway inflammation and sepsis. For example, C5a get a large amount of activation in serum of sepsis mouse model or patients, and blockade of C5aR alleviate theproliferation of C5a in sepsis, indicate that C5a promote the immune response.[1][2]Previousstudy indicate that A/J mice was lack of C5gene and we verified that BALB/C miceexhibited a significant higher rate of mortality than that of C5aR-deficient (C5aRKO) micefollowing MHV-3infection. This finding implies that C5a/C5aR signal pathway participatesin the regulation of FH pathogenesis. But, the biological significant of C5a/C5aR pathwayin the pathogenesis of FH remains un-investigated.In this study, we examined the role of C5a in MHV-3-mediated FH. We first infectedBALB/C and C5aRKO mice with100PFU of MHV-3viruses. We found that all of WTmice died within3days post-infection, whereas in the C5aRKO group, over80%of themice survived after3days post-infection. Several necrosis was observed by HE staining inthe livers of WT mice, but the morphology of the liver from C5aRKO mice was mostlynormal under the same condition of infection. In agreement with the observation that fewerhepatocytes were undergoing apoptosis, C5aRKO mice exhibited lower alanineaminotransferase (ALT) compared to WT mice. In addition, we used the C5aR antagonist toconfirm the role of C5a/C5aR in the pathogenesis of FH and found WT mice pre-treatedsaline30min suffered a serious survival and necrosis in liver tissue compared micepre-treated C5aR antagonists under the same condition by HE staining following MHV-3infection.To certify whether the role of C5a/C5aR pathway engages in the FH. We assessed thecomplement activation during MHV-3infection. Immunohistochemistry showed thatincreased percentage of C5b-9and C5aR deposition was detected in the liver of Balb/c miceafter MHV-3infection for72h and also in liver of FH patients in contrast to their owncontrols. PCR and Western blot revealed that the level of C5aR was drastically increased inliver of Balb/c mice during MHV-3infection as time goes on. ELISA assay indicated thatdecreased levels of C5a in the plasma of FH patients and Balb/c mice post-infection72h, itwas speculated a result of complement exhaustion. These results suggested that thecomplement was activated in FH. Previous research showed FGL2was a critical molecule that mediated hepatocellularnecrosis.[1][2][3]We therefore measure the FGL2expression in serum of MHV-3infected72hBalb/c and C5aRKO mice. Compared with WT mice, there were much lower FGL2inserum of C5aRKO mice after MHV-3infected72h. Immunohistochemistry demonstratedthat following MHV-3infection, there was a massive of FGL2and its regulated productfibrinogen deposition in Balb/c mice, whereas only a few detectable FGL2and fibrinogendeposition in C5aRKO mice. Western blot result showed the higher expression of FGL2inBalb/c mice compared to that in C5aRKO mice after72h MHV-3infection. These resultsclearly showed that C5a/C5aR pathway, unexpectedly acting an enhancer rather than anattenuator of pathology to regulate FGL2and play an important role in the development ofFH and cause high mortality upon MHV-3infection. Mice lacking C5a/C5aR signals exhibitsignificantly reduced FGL2expression in response to MHV-3infection and have lessfibrinogen deposition, which reduces tissue damage and mortality in FH.Since the report previously verified the MAPK signal pathway was activated toregulate FGL2in the MHV-3induced FH model. We wondered whether C5a/C5aR wasinvolved in FH via MAPK signal pathway to regulate FGL2.[4]We found that thephosphorylation of MAPK signal pathway in liver of Balb/c and C5aRKO mice duringdifferent MHV-3infection time points and it appeared that the phosphorylation of ERK andP38in Balb/c mice could sustain a long time compared with the C5aRKO mice afterMHV-3infection. To further demonstrated how C5a/C5aR pathway regulates the FGL2, wetreated the RAW264.7macrophage cell line and peritoneal macrophages isolated from WTmice with C5a, MHV-3, C5a+MHV-3, C5a+MHV-3+C5aRa72h and western blots showedthat C5a could enhanced the FGL2expression after MHV-3treated RAW264.7macrophagecell line and the peritoneal macrophages isolated from WT mice for72h. Meanwhile,western blot also indicated that C5a could strengthen and sustain a long time of phosphorylation of ERK and P38signal pathway in MHV-3treated RAW264.7cell line andperitoneal macrophages isolated from WT mice. To examine whether C5a/C5aR pathwaycould regulate FGL2via MAPK signal pathway, RAW264.7cell line and peritonealmacrophages from WT mice were treated with C5a, MHV-3, C5a+MHV-3alone orcomnination with ERK and P38inhibitors and prepared for Western blot to detect theexpression of FGL2in different stimulation patterns. It is showed that ERK and P38inhibitor could significant alleviate the FGL2expression of C5a add MHV-3treatedmacrophage. These results clearly explained that C5a/C5aR pathway regulates theexpression of FGL2via MAPK signal pathway.Taken together, we conclude that the C5a/C5aR pathway plays a decisive role inregulating macrophage viability, and by thus it affects the severity of liver pathology andmortality during MHV-3infection.