TLR2 Pathway Impairment In Hepatitis B Virus Related Acute Liver Failure

Background and aim:Efficient immune control plays a pivotal role in determining the outcome of HBV infection.Based on the genetic hypothesis of rare variant-rare phenotype,the extremely rare 2-week-onset form of HBV-ALF was chosen as rare phenotype for the host deficient pathway enrichment followed by functional analysis.Methods: HBV-ALF cases were collected.The genetic impairment were screened using whole exomic sequencing,compared and enriched at pathway level.HBV mutations were determined.TLR2 mutant was studied in vitro.312 Non-ALF outcomes post HBV exposure were chosen as disease controls(124 asymptomatic self-limiting HBV infection,65 acute symptomatic hepatitis B,93 chronic hepatitis B and 30 acute-on-chronic liver failure patients).HBV transfection mice model was constructed using TLR2 knockout mice hydrodynamically injected with wide type or BCP/pre C mutated HBV containing plasmids,while recombinant adenovirus was used in transdunction mice.These two models were established to study TLR2’s imput in the viral clearance,hepatic necroinflammation and regulatory T cells(Tregs)proliferation.Additionally,the impact of a case specific mutant of TIRAP,a TLR2 adaptor molecule,on its protein and NF-κB signaling was studied in three cells lines.Results:The enrichment of impaired TLR2 pathway in 10 HBV-ALF cases was prominent.Two cases shared an identical TLR2 F679 I heterozygous mutation.Wildtype TLR2 displayed significant NF-κB activation upon stimulation in vitro,whereas F649I-transfected cells displayed almost zero activation.Interestingly,when wildtype and F649 I mutants were co-transfected at a ratio of 1:1,the level of activation decreased by 40-50%.None of the HBV-ALF rare mutation was found in 312 disease controls cases.In both heterologous and homologous TLR2 knockout(KO)mice,injection of HBV replicon plasmid resulted in more prominent serum alanine aminotransferase(ALT)elevations and hepatic necroinflammation than in wild type mice,while no significant difference noticed in viral clearances.Mechanistic analyses demonstrated reduced Tregs percentages in post-exposure TLR2 KO mice.The inflammations were more prominant in recombinant HBV adenovirus experiments.In both models,inflammations in heterozygous TLR2 KO mice transfected with BCP/pre C mutant were the severest.HBV-ALF case specific TIRAP P149 S located within the TIR domain,highly homologous to HBe Ag specific sequence.The mutation resulted in a weakened protein expression.Upon Pam3CSK4 stimulation,no significant difference in terms of NF-κB activation was noticed.Conclusion:These data suggested an involvement of TLR2 signaling in FHB pathogenesis,partially through directly or indirectly regulating Tregs levels,the abundance of TIR-containing genes was also highlighted.Certain HBV mutations apparently also contributed towards modifying post-injection host responses in coordination with TLR2 locus status.Models based on the mutations identified will facilitate the identification of molecules capable of exerting efficient immune control.