LFA-1/ICAM-1 Mediats NK Cell Killing Effect Associated With The Pathogenesis Of Ocular Toxoplasmosis In Murine Model

Objective To investigate the relationship between leukocyte function-associated antigen-1(LFA-1)and intercellular adhesion molecule-1(ICAM-1)mediated NK cell killing and T.gondii ocular infection or its effect on retinal tissue inflammation by establishing murine model of acquired ocular toxoplasmosis with Chinese 1 dominant genotype,and to provide important clues for the pathogenesis of ocular toxoplasmosis(OT)and new methods of targeted therapy.Methods (1)C57BL/6 mice were infected perorally with 20 cysts of the Tg Ct Wh6 strain to establish murine model of acquired ocular toxoplasmosis with the predominant clonal lineage Chinese 1 strain.Hematoxylin and eosin(H&E)staining was used to detect ocular pathological changes and T.gondii load in ocular tissues was detected by PCR.The expression of interferon-gamma(IFN-γ),tumor necrosis factor-α(TNF-α)and interleukin-10(IL-10)were analyzed by quantitative real-time polymerase chain reaction(q RT-PCR)and immunohistochemical staining.The differentially expressed genes in ocular tissues were detected by RNA sequencing,and then GO enrichment and KEGG enrichment were analyzed.The percentage of NK cells and CD49a~+NK cells,their surface receptor LFA-1 and ligand ICAM-1 in ocular tissues were detected by flow cytometry.The expression of LFA-1/ICAM-1 in ocular tissues was detected by Real-time PCR and verified by WB.(2)Infected mice treated with 1%lifitegrast(LFA-1/ICAM-1 inhibitor)from Day 0 as"Lifitegrast 1"group or from Day 14(disease onset)as"Lifitigrast 2"group.A single drop(5μl/eye)was administered to both eyesthrice daily until Day 30.The percentage of NK cells and CD49a~+NK cell subsets and the expression of LFA-1/ICAM-1 in ocular tissues after topical drops of lifitegrast were observed by flow cytometry and verified by WB.H&E staining was used to detect improvements in ocular histopathology with topical drops of lifitegrast.The effects on cytokine secretion and pro-apoptotic protein caspase-3 after topical drops of lifitegrast were analyzed by immunohistochemistry and WB.Results (1)After successful establishment of the ocular toxoplasmosis model,infected mice showed severe ocular damage,positive PCR amplification of the T.gondii-specific gene ITS-1,and upregulated expression of IFN-γ,TNF-α(P<0.05)and IL-10(P<0.01)in infected ocular tissues.RNA sequencing showed that 601 genes were up-regulated and108 genes were down-regulated in infected ocular tissues.In GO enrichment analysis,the differentially expressed genes were significantly enriched in immune response and inflammatory response in biological processes.KEGG enrichment analysis showed that31 differentially expressed genes were enriched in the NK-mediated cytotoxicity pathway.The percentage of NK cells in ocular tissues of infected mice was significantly increased(P<0.01),however,the percentage of NK cells in the spleen was significantly decreased after infection(P<0.05).The percentage of CD49a~+NK cell subsets was significantly increased from both eyes and spleen after infection(P<0.01).The expression of LFA-1in NK and CD49a~+NK cell subsets(P<0.05)and ICAM-1 in ocular tissues was upregulated(P<0.05).(2)Flow cytometry results showed that lifitegrast did not decrease the percentage of NK cells in ocular tissues and spleen and CD49a~+NK cell subsets in Lifitegrast 2 group(from day 14).However,the percentage of CD49a~+NK cell subsets were significantly decreased in ocular tissues(P<0.01)and spleen(P<0.05)in Lifitegrast 1 group(from day 0).Lifitegrast inhibited the protein expression of ICAM-1(P<0.05)and LFA-1(P<0.05)in ocular tissues and LFA-1 expression in NK cell and CD49a~+NK cell subsets in the Lifitegrast 1 group of eyes(P<0.01)and spleen(P<0.05).Liftegrast treatment ameliorated the pathology of murine OT and significantly inhibited the secretion of the pro-inflammatory cytokines IFN-γ,TNF-αand the pro-apoptotic protein caspase-3.Conclusions The murine model of acquired ocular toxoplasmosis with the predominant clonal lineage Chinese 1 strain has been successfully established.After T.gondii infection,the expression of pro-inflammatory cytokines in ocular tissues increased,the percentage of CD49a~+NK cells in ocular tissues and spleen increased,and the expression of LFA-1/ICAM-1 was upregulated,which promoted the inflammatory response of OT and aggravated the damage of ocular tissues.In addition,lifitegrast treatment ameliorated the pathology of OT and reduced the inflammatory response to the retina,and the interaction between LFA-1 and ICAM-1 plays a role in the early regulation of the CD49a~+NK cell subsets proportion in OT murine model.LFA-1/ICAM-1 may be a key molecule in the pathogenesis of OT,and may provide new insights for potential immunotherapy.