TRIM21 Aggravates Herpes Simplex Virus Epithelial Keratitis By Regulating The Secretion Of Type ? Interferon And Pro-inflammatory Cytokines

Objetive:Herpes simplex virus-1?HSV-1?is a neurotropic double-stranded DNA virus,which is the leading cause of infectious blindness in the developed world.Epidemiology shows that HSV-1 has been recognized as a ubiquitous human pathogen,infecting 50–90%of the world population.Eye disease caused by HSV-1 infection usually presents as epithelial keratitis,which accounts for 50%to 80%of ocular herpes.Although HSV epithelial keratitis is self-limiting within one week,without adequate treatment,it may progress to stromal keratitis,leading to progressive corneal opacity.Primary infection is usually caused by direct infection of the cornea by HSV-1.After infection,HSV-1replicates in the corneal epithelial cells and then triggers innate immune signaling through the production of cytokines and chemokines.At present,antiviral medicines has been used to cure HSV epithelial keratitis.However,controversies persist about the side effects of antiviral drugs.The treatment objective of HSV epithelial keratitis is to inhibit viral replication and to prevent the active viral infection of corneal cells.The tripartite motif?TRIM?protein family consists of up to 100 members and is the largest group of E3 ubiquitin ligases in mammals.Many TRIMs proteins have been reported to have direct or indirect antiviral activity.TRIM21,also known as Ro52/SS-A or RNF81,has a B30.2 domain encoded in the C-terminal region,which comprises a combination of a PRY motif followed by a SPRY motif.B30.2 domain can be recruited to incoming viral cores and determines antiviral specificity.Recent studies have shown that TRIM21 can recruit proteasomes and savagely break down capsids so that the exogenous viral genomes are exposed prematurely to promote the activity of PRRs.HSV-1 belongs to an enveloped virus.Thus we suspect that TRIM21 may affect HSV-1infection.In this study,we have investigated the role of TRIM21 in the host defense against HSV-1 infection in a murine model of HSV epithelial keratitis and explored its underlying mechanism in human corneal epithelial cells.Research method:Development of HSV epithelial keratitis in C57BL/6J mice was studied with HSV-1 McKrae strain.To explore the potential role of TRIM21 in HSV epithelial keratitis,its expression was measured in corneas of epithelial keratitis mice by Western Blotting at 0,2 and 4 days post-HSV-1 infection?dpi?.To further confirm whether TRIM21 was involved in the pathological process of HSV epithelial keratitis,we detected the localization of TRIM21 in corneas by immunofluorescence.Subsequently,to further ascertain the role of TRIM21 in HSV epithelial keratitis,siRNA transfection was used to limit TRIM21 expression in the corneas before establishing the HSV epithelial keratitis mice model.After that,corneas were examined to score the severity of HSV epithelial keratitis by hand-held slit lamp microscope and histopathology showed that corneal histopathology changed after TRIM21 was silenced.TCID₅₀ assay was used to investigate the effect of TRIM21 on virus replication,combined with qPCR and ELISA to detect the production of pro-inflammatory factors and interferon,the mechanism of TRIM21 in the acute phase of HSV-1 infected cornea was initially explored.Next,we explored the mechanism of TRIM21 affecting HSV-1 replication in human corneal epithelial cells?HCE cells?.To interfere with TRIM21 expression,lentivirus-TRIM21?LV-TRIM21?,LV-control,siRNA-TRIM21,and siRNA-control were used to transfect HCE cells,respectively.In order to explore the mechanism of TRIM21 on the production of IFN-a in HSV-1 infected HCE cells,Western Blotting was used to detect the STING/IRF3 signaling pathway.Results:Compared with uninfected corneas,HSV-1 infected corneas displayed abnormally elevated TRIM21 level.Overall,as the course of HSV epithelial keratitis progressed,the expression of TRIM21 continued to increase.Immunofluorescence results showed that TRIM21 was predominantly expressed in the corneal epithelium at 0dpi,while the expression of TRIM21 was increased significantly at 3 dpi.TRIM21mainly expressed in the cytoplasm of corneal epithelial cells.Next,si RNA-TRIM21 was used to silence the expression of TRIM21 in the cornea.After that,corneas were infected with HSV-1 and then examined to score the severity of HSV epithelial keratitis by hand-held slit lamp microscope in a blinded manner every day.We found that the clinical scores and the degree of corneal opacity in the siRNA-TRIM21group were significantly lower than that in the siRNA-control group?p<0.05?.Results showed that the number of corneal lesion score of?2 in the siRNA-TRIM21 group?8/8?was more than the number in the si RNA-control group?1/8?.Compared with histopathology in the siRNA-TRIM21 treated corneas,corneal epithelial cells in the siRNA-control treated corneas showed obvious proliferation and growth disorders.Corneal epithelial layer showed vacuole-like changes,and a small number of inflammatory cells infiltrated into the superficial layer of the corneal stroma.These results indicated that silencing TRIM21 can reduce the severity of HSV epithelial keratitis.TCID₅₀0 assay results showed that silencing TRIM21 controlled the virus particle release at 1,3,and 5 dpi significantly.The qPCR results showed that the IFN-a expression in the siRNA-TRIM21 treated corneas was approximately twice that of the siRNA-control treated corneas.To determine the effects of TRIM21 on pro-inflammatory cytokines in HSV epithelial keratitis,corneas from the siRNA-TRIM21 and siRNA-control treatment groups were pooled to obtain one sample for analysis of IL-6and TNF-a expression levels by ELISA.Compared with the siRNA-control treated corneas,the expression of these two pro-inflammatory cytokines in the siRNA-TRIM21treated corneas was significantly suppressed at 3 dpi.Finally,we explored the mechanism of TRIM21 affecting HSV-1 replication in(HCE cells.HSV-1 infected the pretreated cells at MOI of 10.qPCR results showed that the expression of IFN-a was enhanced in si RNA-TRIM21 pretreated cells and suppressed in LV-TRIM21 pretreated cells.Western Blotting results showed that the expression of STING and phosphorylated IRF3 was enhanced in siRNA-TRIM21 pretreated cells after HSV-1 infection 12 hours.Conversely,the expression of STING and phosphorylated IRF3 is reduced in LV-TRIM21 pretreated cells.When we pretreated HCE cells with both siRNA-TRIM21 and si RNA-STING,qPCR results showed that the level of IFN-a production decreased significantly compared to siRNA-TRIM21 pretreated cells alone.Conclusion:TRIM21 is constitutively expressed in normal corneas and is abnormally high expressed in HSV epithelial keratitis.Highly expressed TRIM21 enhances the replication of HSV-1 in corneal epithelial cells via suppressing the production of type ? IFN by inhibiting STING/IRF3 signaling.It also promotes the production of pro-inflammatory cytokines IL-6 and TNF-a to the point of synergistically exacerbating the severity of HSV epithelial keratitis.