MyD88 Exacerbates Immunological Pathology In Experimental Viral Fulminant Hepatitis

Objective Viral fulminant hepatitis（FH）is a serious liver disease with high mortality due to excessive inflammation of the liver tissue.Lack of understanding of inflammatory signaling pathways in FH disease seriously hinders the implementation of clinical disease interventions.Myeloid differentiation primary response gene 88（MyD88）,a key adaptor protein for most TLR,IL-1R,and IL-18R-dependent inflammatory signaling pathways,plays essential roles in mediating the primary inflammatory responses against pathogen invasions in the host.Hyper-activation of this signaling pathway can exacerbate certain disease conditions which can be fatal in nature.However,it is not clear whether and how MyD88 signaling participates in the pathogenesis of viral fulminant hepatitis.This study aims to provide new clues and experimental evidence for the treatment strategy of FH disease by revealing the relationship between MyD88-mediated signaling pathway and FH.Methods Age matched C57BL/6（WT）and congenic MyD88^-/-mice were infected with MHV-3（100 PFU）,the survival rate was monitored for a total of 20 days.Liver tissues were isolated from virus infected WT and congenic MyD88^-/-mice at different time points,the morphology was analyzed by H&E staining,cells undergoing apoptosis was analyzed by TUNEL staining.The expression of Bgp1 in livers at 24h and 72h post-infection was analyzed by western-blotting.The virus titers in livers at 72h post-infection were analyzed by plaque assay,and results were compared by statistical analysis.Cells were isolated from virus infected livers.Liver recruitment of CD11b⁺/F4/80⁺monocytes/macrophages,Gr-1^highigh CD45⁺neutrophils of infection was measured by flow cytometry.Liver infiltration of Lin^-Thy1.2⁺ILCs of MHV-3 infection was detected by flow cytometry.The secretion of IL-17,TNF-αand IL-1βfrom NKp46^-Lin^-Thy1.2⁺Roγt⁺ILC3 was analyzed by flow cytometry.The macrophage cell line,Raw264.7 cells,were infected with MHV-3-infected（MOI=1）,HMGB1 localization before and after 24h of infection was monitored by immunofluorescent confocal microscopy,the accumulation of HMGB1 in the supernatants was detected by ELISA,the expression of HMGB1protein in normal and MHV-3-infected liver tissues was detected by immunohistochemistry,serum concentration of HMGB1 in MyD88^-/-and WT mice was detected by ELISA,n=5 per group,liver concentration of HMGB1 in MyD88^-/-and WT mice was detected by western-blot,N=4 per group.Results We found that all the WT,Rag-1^-/-and Trif^-/-mice died within 8 days of infection,whereas over 72.7%（8/11）of the MyD88^-/-group still survived after 20 days（P≤0.0001）.Hematoxylin and eosin（H&E）staining showed the morphology of MyD88^-/-livers was mostly normal at 48h,and the necrosis area also was dramatically reduced at 72h.Additionally,fewer cells were found to be apoptotic in MHV-3 infected MyD88^-/-livers at72h of infection.The expression of Bgp1 appeared to be significantly lower in the virus infected MyD88^-/-livers comparing to that in the WT controls,concurring with the plaque assay data showing limited virus entry and amplification in the MyD88^-/-livers at 72h post-infection.RT-qPCR showed that liver fgl2 mRNA transcription was induced by MHV-3,while its level was dramatically reduced in MyD88^-/-livers.The reducing FGL2protein in virus infected MyD88^-/-livers was also lower as confirmed by Western-blot,and substantial lower level of serum accumulation of FGL2 in MyD88^-/-mice at 72h of infection.Therefore,MyD88^-/-mice responded with limited fibrinogen formation,leading to a reduced liver coagulation and necrosis.Moreover,the Fgl2^-/-mice are total resistant to MHV-3 mediated mortality.The expression of some proinflammatory cytokines including Ifn-γ,Tnf-α,proIl-1βand Il-6 was significantly reduced than that from virus-infected WT littermates by qRT-PCR.These results were also confirmed at the protein levels by Western-blot and immunohistochemistry.Finally,we showed that both Tnf-α^-/-and IL-1R^-/-mice are resistant to MHV-3 mediated mortality.The change in both CD11b⁺/F4/80⁺monocytes/macrophages and Gr-1^highCD45⁺neutrophils was not statistically significant,and flow cytometry showed that in the liver-tissue samples at 24h and 48h post-infection,the infiltration of ILCs（Lin^-Thy1.2⁺）was significantly higher in the WT livers than that in the MyD88^-/-littermates.Additionally,statistical analysis showed that these NKp46^-Lin^-Thy1.2⁺Roγt⁺ILC3 were severely impaired in MHV-3infected MyD88^-/-liver tissues.Furthermore,these ILC3 have the capacity to produce proinflammatory mediators,like proIL-1β,TNF-α,and IL-17.Immunofluorescent confocal microscopy visualized HMGB1 localization and results showed that HMGB1localized in the nucleus of the mock-infected Raw264.7 cells as opposed to distribution in both the nucleus and cytoplasm in MHV-3-infected counterparts.Furthermore,a time-dependent increase of HMGB1 accumulation in the supernatants was also seen during72h of infection.Immunohistochemistry showed that HMGB1 protein was localized in nucleus of hepatocytes/macrophages from normal liver tissues,whereas it was mostly found within the cytoplasm of MHV-3-infected hepatocytes,especially within the necrotic liver tissues.The infected tissue damage also results from high level of HMGB1protein accumulated in MHV-3 infected serum.Moreover,liver accumulation of HMGB1was dramatically reduced in Myd88^-/-mice post MHV-3 infection.Conclusion We successfully constructed a viral fulminant hepatitis model,clarifying that MyD88^-/-mice can reduce the pro-inflammatory response by reducing the expression and secretion of HMGB1 in the liver.Compared with WT mice,the liver damage of MyD88^-/-mice was significantly reduced,the secretion of pro-inflammatory cytokines was reduced,and the survival rate of mice was significantly prolonged post MHV-3 infection.And the results suggest that attenuation of viral FH by MyD88 deficiency at least partly could be due to limited proinflammatory NKp46^-Lin^-Thy1.2⁺Roγt⁺ILC3 infiltrate into the livers.In summary,MyD88 gene will aggravate the immunopathology of experimental viral severe hepatitis.We can achieve the goal of treating human viral FH and other severe inflammatory diseases by properly regulating the MyD88 signaling pathway and blocking other inflammatory factors.