The Changes Of Immunoproteasome In The Context Of Pseudorabies Virus Bartha Strain Infection

Upon oxidative stress or in the context of inflammation reaction,proteasomes are transformed into immunoproteasomes,which plays an important role in inflammatory diseases.Pseudorabies virus?PRV?,as a member of alpha-herpesvirus with a wide range of hosts,can establish infection in diverse host brains and cause central nervous system?CNS?inflammation.However,it is not clear whether immunoproteasome participates in and regulates PRV induced CNS inflammation.In order to study the role of immunoproteasome in PRV infection,we injected 6.8×10⁴ TCID₅₀0 PRV Bartha K61 strain subcutaneouslyof 6-week-old C57BL/6 male mice and established a brain inflammation model.The histopathological diagnosis found that the infected mice developed non-purulent encephalitis with a perivascular cuffing phenomenon in the brain accompanied by infiltration of monocytes.Real-time PCR showed that the transcription level of immunoproteasome subunits LMP2,LMP7 and LMP10 were significantly up-regulated in the mouse brain from 7 dpi?days after infection??P<0.001?.Western Blot results showed that the immunoproteasome subunits LMP2,LMP7 and LMP10 were significantly induced in the mouse brain from 7 dpi.The results of real-time PCR showed that RNA transcription levels of mouse brain pro-inflammatory cytokines IFN-?,IL-6,IL-1?and TNF-?were significantly up-regulated from 7 dpi?P<0.01?.Knock out of LMP10 did not change the PRV infection induced clinical symptoms nor pathology,but significantly decreased the viral load in mouse brain?P<0.01?.Knock out of LMP10blocked the transcription and expression of LMP2 and LMP7.The transcription level of proinflammatory factors was also significantly down-regulated?P<0.001?.In order to further verify the above experimental results,6.8×10⁴ TCID₅₀ PRV Bartha K61 was used to infect passage mouse microglia BV2cells and passage mouse neural cell N2A cells.The mRNA transcription level and protein level of immunoproteasome subunits and pro-inflammatory cytokines level were detected at 0 hpi,3 hpi,6 hpi.12hpi,24 hpi and 36 hpi.The results showed that PRV infection could up-regulate the level of immunoproteasomes in BV2 cells,but could not induce the production of immunoproteasome in N2A.When BV2 was treated with LMP7 subunit specific inhibitor ONX-0914,the replication of PRV was not changed but the transcription of IFN-?and TNF-?was significantly inhibited?P<0.01?.In conclusion,the immunoproteasome is related to the production of proinflammatory cytokines in PRV infection.In this study,we successfully established a mouse model of PRV infection.Using this animal model,we first studied the changes of immunoproteasome in PRV induced encephalitis and the effect of LMP10deletion on the pathogenicity of PRV.At the cellular level,we verified the effect of LMP7 on proinflammatory cytokines.The results show that immunoproteasome is related to the production of proinflammatory cytokines in PRV infection.All the data above indicate that PRV infection will change immunoproteasome expression level.This study shed light on the study of the pathogenesis of immunoproteasome in CNS diseases and provided new insights for the study of brain inflammatory response induced by PRV infection.