Experimental Study On Interferon Alpha-induced Apoptosis Of Keratinocytes Via T Cell Differentiation

Oral lichen planus(OLP)is a T lymphocyte-mediated chronic inflammatory disease whose pathogenesis is still unclear.OLP is a typical T cell immune disease.According to its histopathological features,it is widely believed that the T lymphocytes infiltrating the mucosa lamina propria are the main cause of pathological death of epithelial keratinocytes,but the upstream induction mechanism of T cell differentiation and its initiation of attacking keratinocytes is currently unclear.The previous study of our group showed that the increase of interferon alpha in OLP lesions was positively correlated with the occurrence and development of OLP disease,which may be an early immune response event leading to disease.Does interferon alpha promote an immune response between keratinocytes and T cells? Is this immune response based on the induction of differentiation by T cells and the production of related effector molecules,which ultimately leads to apoptosis in keratinocytes? Explaining these questions will be an important basis for revealing the pathogenic mechanism of interferon alpha in OLP.This study intends to use interferon alpha to stimulate keratinocytes,T cells and their co-culture systems,to detect and evaluate the effect of interferon alpha on keratinocyte activity,and to promote the expression of T cell immune-related molecules in keratinocytes.Ability;detection of interferon alpha promotes the functional effects of T cell subsets to Th1 phenotype differentiation;and simultaneously detects its effect on keratinocyte-T lymphocyte co-culture system to initially evaluate the mechanism of action of interferon alpha in OLP,To provide a basis for explaining the immune response effect mechanism of T cells in OLP.Objective:(1)To evaluate the effect of interferon alpha on the expression of T lymphocyte immune-related molecules in keratinocytes,including antigen-presenting molecules(HLA-I,HLA-II molecules)and costimulatory molecules on the surface of keratinocytes(CD40),CD80,CD86)and its secreted T lymphocyte chemotactic factor(CXCL10);and evaluated the ability and mechanism of interferon alpha to induce keratinocyte apoptosis.(2)To explore the ability of interferon alpha to induce differentiation of T lymphocytes into Th1 direction;and the changes of proapoptoticassociated cytokines derived from T lymphocytes in culture supernatants.(3)Establish a co-culture model of T lymphocytes and keratinocytes,and directly observe the ability of interferon alpha to induce T lymphocytes to polarize in the Th1 type and induce apoptosis of keratinocytes.Methods:(1)Human immortalized oral keratinocytes(HOK cells)and human immortalized epithelial cells(HaCaT cells)were treated with interferon alpha(1000 U/mL),and PBS was used as a negative control group.Flow cytometry was used for quantification.The expression levels of HLA-I,HLA-DR,CD40,CD80 and CD86 on keratinocytes were analyzed.The level of CXCL10 in cell supernatant was detected by ELISA.The proportion of apoptosis in HOK cells was detected by flow cytometry and Western technique.Apoptosis-related molecular changes(2)Interferon ?(1000 U/mL)was used to treat total T lymphocytes isolated from normal human peripheral blood,and PBS group was used as a negative control.Flow cytometry was used to detect Th1 subset in T lymphocytes.The proportion of subpopulations accounted for CD4+ T lymphocytes;the changes of apoptosis-related molecules(TNF-?,FasL,IFN-?)were detected by ELISA;(3)The co-culture of T lymphocytes and keratinocytes was established(mixed culture and Transwell culture).In the system,the co-culture system was stimulated with interferon alpha,and PBS was used as a control to observe and evaluate the effect of interferon alpha on the induction of T cell differentiation and keratinocyte apoptosis in the co-culture system.(4)Interferon ?(10000 U/mL)was injected subcutaneously into C3 H mice every 2 days for 2 weeks.The mice were sacrificed to obtain tissue samples,and HE staining and CD3 immunohistochemical staining were performed to evaluate the interference in vivo.The ability of alpha alpha to induce non-specific infiltration of T lymphocytes under the epithelium.Results:(1)Interferon alpha increased the proportion of HLA-I+ cells in HOK cells(before:18.15%±0.64%,after:53.58%±0.99%)and fluorescence intensity(before:1410.00±16.52,after:1663.00±8.75),and increased the proportion of HLA-I+ cells in HaCaT cells(before:38.67%±6.85%,after:72.62%±2.81%),but the fluorescence intensity did not increase significantly;interferon alpha increased the proportion of CD40+ cells in HOK cells(before:1.15%±0.13%,after:7.07% ± 0.31%)and fluorescence intensity(before:1255±112,after:1331 ± 13),increasing the proportion of CD40+ cells in HaCaT cells(before:13.51%±1.65%,after:33.77 ± 1.47),However,the fluorescence intensity did not increase significantly;interferon alpha could not increase the expression of MHC class II molecules and CD80 and CD86 molecules in HOK and HaCaT cells.Compared with the PBS group(24h: 5.63 ± 0.50 pg / mL;48h: 109.60 ± 6.48 pg / mL),interferon alpha can significantly increase the secretion of CXCL10 by HOK cells(24h: 6.30 ± 0.25 pg / mL;48h: 217.60 ± 7.14pg/mL);compared with PBS group(5.99%±0.74%);48h after interferon ? treatment of HOK cells,the proportion of apoptosis(10.49%±0.77%)was significantly increased.Interferon alpha induced STAT1/Caspase3 expression in HOK cells after 6 h stimulation,but did not cause its activation;(2)Interferon alpha induced a significant increase in the proportion of CD4+ T lymphocytes in total T lymphocytes isolated from human peripheral blood.(Before induction: 75.93%±0.39%;after induction: 78.48%±0.33%),and induced T lymphocytes to secrete IFN-?(341.90±84.37 pg/mL)and inhibit the secretion of TNF-?(3.16±0.95 pg/mL)(3)IFN-? induced pro-apoptotic cytokines in IFN-?(PBS group: 2.09±0.22pg/mL;co-culture system: 10.85±1.67 pg/mL)in co-culture system of T lymphocytes and HOK cells.mL),TNF-?(PBS group: 0.03±0.007 pg/mL;co-culture system: 0.99±0.08 pg/mL),FasL(PBS group: 0.39±0.16 pg/mL;co-culture system: 8.10±0.17 pg/ mL))significantly increased and led to a large number of apoptosis in HOK cells in the co-culture system(PBS group: 2.20% ± 0.12%;co-culture system: 17.98% ± 1.50%);(4)C3H mice were continuously injected with interferon-? subcutaneously.After 14 days of injection,HE staining showed a large amount of lymphocyte infiltration under the skin;Immunohistochemical staining of CD3 also showed an increasing outcome.Conclusion:(1)Interferon alpha enhances the expression of MHC-I and CD40 molecules on keratinocytes,and also induced apoptosis of keratinocytes independently;(2)Interferon alpha may also induce T lymphocytes differentiate into Th1 type cells to form a Th1 type immune pattern;(3)Interferon alpha can induce T lymphocytes to secrete a large number of pro-apoptotic factors,which can induce higher apoptosis of keratinocytes by T lymphocytes;Subcutaneous injection of interferon alpha in C3 H mice can result in non-specific infiltration of subcutaneous T lymphocytes.