SHIP-1 Mediated Migration Of CD4+ T Cells And Their Effects On Herpes Simplex Keratitis

BackgroundHerpes simplex viral keratitis(HSK)is mainly caused by the Herpes simplex virus type I,and is one of the most common blinding eye diseases both at home and abroad.If patients with HSK were not treated timely and effectively,it may cause corneal stromal opacity,corneal scarring,corneal ulcer perforation,and even blindness,eyeball losing and other serious complications.HSK is characterized by the involvement of immune factors,long courses,easy to delay and have recurrent attacks.The pathological damage of HSK mainly results from the persistent immune response of the cornea that caused by virus infection.Various inflammatory cells such as polymorphonuclear leukocytes(PMN),T lymphocytes and macrophages are all involved.It is currently believed that the persistent presence of CD4+T lymphocytes in the inflammatory site is strongly linked to the protracted course of HSK.Therefore,the regulation of T lymphocytes migration in HSK corneal tissues is of great value in exploring new HSK treatment methods.SHIP-1(SH2-containing inositol 5 ’-phosphatase 1)is a 5’ inositol phosphatase 1that contains SH2 structure.SHIP-1 can hydrolyze the phosphatidyl inositol triphosphate(PI(3,4,5)P3),the key material of phosphatidyl inositol kinase(PI3K)signal pathway.The PI3 K signaling pathway can significantly influence the cell proliferation,differentiation,migration and apoptosis,suggesting the great potential of SHIP-1 in regulating the development,differentiation and steady state of T lymphocytes.Studies have found that the phosphorylation of SHIP-1 can negatively regulate the PI3 K signaling pathway,thereby blocking the chemotaxis of CXCL11 on T lymphocytes and restricting the migration of T lymphocytes towards inflammatory sites.This effect is of great significance for immune escape that occurred during the course of tumor disease.One of the most important mediators of homeostatic trafficking of T lymphocytes to secondary lymphoid organs is the chemokine receptor CCR7.Binding of its ligands,CCL19 and CCL21,mediates the trans endothelial migration of T lymphocytes across high endothelial venules into secondary lymphoid organs,which significantly affects the emigration of T lymphocytes from peripheral tissue and the homing movement.However,whether SHIP-1 can regulate the emigration of CD4+T lymphocytes from the inflammatory site that mediated by CCL19、21-CCR7 has not been clarified.Meanwhile,it is also unknown whether the expression of CD4 + T lymphocytes and SHIP-1 in diseased cornea is related to the disease severity in clinical HSK patients.Therefore,our study mainly focused on the influence of SHIP-1 on the CCL19、21-CCR7 mediated T lymphocytes migration,as well as the expression of CD4 + T lymphocytes and SHIP-1 in the diseased cornea of HSK patients,and the relationship between their expression and the severity of HSK.PurposesTo explore the effects and mechanisms of SHIP-1 on the emigration of CD4+T lymphocytes that mediated by CCL19、21,and to analyze the relationship between the expression of CD4 + T lymphocytes,SHIP-1 in the diseased cornea and the disease severity in clinical HSK patients.MethodsIsolate and culture the activated primary CD4+T lymphocytes.24-hole Transwell system was used to analyze the changes in the migration ability of CD4+T lymphocytes to CCL19 and 21 after treated with different concentrations of SHIP-1 agonists and antagonists.Ibi Di2 D chemotactic system and live cell imaging were used to further observe and compare the differences in migration speed and distance of CD4+T lymphocytes towards CCL19 and 21 after treated with SHIP-1 agonists and antagonists.Immunofluorescence staining was used to detect the regional difference of CCR7 expression after different treatment.Western blot was used to detect the CCR7 protein expression level.The relationship between the changes in chemokine capacity and the PI3K-PI(3,4,5)P3-Akt-NF-κB signaling pathway,transcription factor KLF2 was also detected by Western blot.Graph Pad Prism 6.0 software and SPSS 24.0 software were used for data statistical analysis.Immunofluorescence staining was used to observe the expression of CD4+T lymphocytes and SHIP-1 in the diseased cornea of HSK patients after corneal transplantation,and to explore the relationship between CD4+T lymphocytes infiltration,SHIP-1 expression and the disease condition of HSK.ResultsX1.The Transwell and Ibi Di2 D chemotactic living cell imaging system showed that SHIP-1 antagonists(10 and 30 n M)could significantly inhibit CCL19 and 21 mediated CD4+T lymphocytes migration,while SHIP-1 agonists(30n M)promoted the targeted migration.Fluorescence microscopy found the SHIP-1 antagonists treatment can cause internalization and phosphorylation of CCR7 on CD4+T lymphocytes,while SHIP-1agonists can cause the surface polarization of CCR7.Western blot method also found a decreased CCR7 protein expression in cells treated with SHIP-1 antagonists,while the agonists treatment group showed the opposite.2.Western blot showed that CD4 + T lymphocytes that treated with SHIP-1agonists had an increased expression of transcription factors KLF2,while the expression of PI(3,4,5)P3,phosphorylated Akt and phosphorylation NF-ΚB decreased.In groups that treated with SHIP-1 antagonists,they showed the opposite.3.Fluorescence microscopy showed the obvious co-staining of SHIP-1 and CD4+T lymphocytes in the corneal tissues of HSK patients in inflammatory stage.However,patients with leukoma and HSK patients in stable phase did not have co-staining phenomenon.Conclusions1.Increase the expression of SHIP-1 in CD4 + T lymphocytes can promote the CCL19,21-CCR7 mediated T lymphocytes emigration by enhancing the expression of chemokine receptor CCR7,while inhibiting intracellular SHIP-1 phosphorylation can inhibit this migration process.2.The regulation of SHIP-1 on CCL19,21-CCR7 mediated T lymphocytes emigration is achieved by affecting the PI3K-PI(3,4,5)P3-Akt-NF-κB signaling pathway,thus influencing the transcription factor KLF2.3.The infiltrated CD4+T lymphocytes and SHIP-1 expression in corneal tissues may be involved in the disease development of clinical HSK patients.