| ObjectiveTo explore the role of cofilin 1 in the immune pathogenesis in severe aplastic anemia(SAA)and to provide theoretical basis for seeking novel therapeutic methods by studying the expression level of cofilin 1 in myeloid dendritic cell(mDC)of SAA patients and its effect on mDC and downstream effector T lymphocytes.Methods1,The protein levels of cofilin 1 was detected in 30 SAA patients(15 untreated and15 with complete remission)and 15 healthy controls by FACS and WB.Correlation analysis was made between the levels of cofilin 1 in patients with SAA and their blood routine and immune indexes.The relative m RNA expression levels of cofilin 1was detected by q RT-PCR.2,Cofilin 1 in mDC in SAA patients was knocked down by RNA interference.The following indexes before and after transfection were compared.Cell proliferation was detected by CCK-8.Cell apoptosis was detected by FACS.Phagocytosis was detected by FACS and immunofluorescence.Cell migration was detected by transwell.The expression levels of co-stimulators CD80 and CD86 were detected by FACS.F-actin was observed by immunofluorescence.3,Myeloid dendritic cell before and after cofilin 1 knockdown were co-cultured with CD4~+T lymphocytes,respectively.The expression levels of Th1 and Th2-related cytokines in the co-culture system were detected by FACS,and the expression levels of foxp3 in Treg cells were also detected by FACS.4,Myeloid dendritic cell before and after cofilin 1 knockdown were co-cultured with CD8~+T lymphocytes,respectively.The proliferation of CD8~+T lymphocytes in each group was detected by CFSE,and the levels of perforin and granular enzyme B were detected by FACS.Results1,FACS results showed that cofilin 1 in mDC in untreated SAA patients[(70.37±22.70)%]was significantly higher than that in healthy controls[(39.65±23.43%),P=0.006]and that in complete remission SAA patients[(43.97±21.23)%,P=0.002].There was no statistical difference between the complete remission group and the healthy control group.The level of cofilin 1 in mDC in SAA patients was negatively correlated with the white blood cell count(r=-0.57,P=0.0026),the absolute value of neutrophils(r=-0.49,P=0.0134),the platelet count(r=-0.57,P=0.0028),the hemoglobin level(r=-0.47,P=0.0192),the absolute value of reticulocytes(r=-0.4089,P=0.0424)and the CD4~+/CD8~+ratio(r=-0.62,P=0.0010).It was positively correlated with the concentration of IL-2(r=0.56,P=0.0037)and IFN-?(r=0.56,P=0.0037).WB detection showed that the expression level of cofilin 1in mDC in untreated SAA patients was higher than that in complete remission SAA patients and healthy controls.q RT-PCR results indicated that the relative m RNA expression level of cofilin 1 in mDC in untreated SAA group(9.13±10.32)was significantly higher than that in the complete remission group(2.91±3.08,P=0.049)and the healthy control group(1.74±1.70,P=0.020).2,It was verified by q RT-PCR and WB that the level of cofilin 1 in mDC was successfully knocked down,and the protein knockdown level was 31.6%.FACS results showed that the phagocytosis ability of mDC from the cofilin 1-si RNA group was significantly lower than that from the scrambled si RNA group[(22.64±12.53)%vs(40.07±11.90)%,P=0.000],which was supported by immunofluorescence assay.Cell migration of mDC from the cofilin 1-si RNA group was significantly lower than that from the scrambled si RNA group(45.08±31.98 vs 67.75±38.07,P=0.044).The expression level of CD86 on mDC from the cofilin 1-si RNA group was significantly lower than that from the scrambled si RNA group[(73.80±17.18)%vs(77.26±14.39)%,P=0.034].There were no significant differences in mDC proliferation,apoptosis,and CD80 expression levels between the cofilin 1-si RNA group and the scrambled si RNA group.Immunofluorescence detection results showed that in the cofilin 1-si RNA group F-actin content increased,cell protrusion density increased and significant remodeling occurred.3,The concentration of Th1-related cytokines was detected by FACS.Compared with the scrambled si RNA group,the concentration of IL-2 in the cofilin 1-si RNA group decresed[(179.48±180.52)pg/ml vs(216.32±203.24)pg/ml,P=0.024],the concentration of TNF-?decreased[(178.08±146.00)pg/ml vs(232.48±157.75)pg/ml,P=0.017],and the concentration of IFN-?decreased[(2499.71±2051.73)pg/ml vs(3020.96±2340.99)pg/ml,P=0.023].Among the Th2-related cytokines,only the concentration of IL-6 decreased[(357.19±237.02)pg/ml vs(435.74±325.01)pg/ml,P=0.047].There was no statistical difference in the concentrations of IL-4 and IL-10between cofilin 1-si RNA group and scrambled si RNA group.4,The mean fluorescence intensity of CFSE in the cofilin 1-si RNA group was significantly higher than that in the scrambled si RNA group.The perforin level in CD8~+T lymphocytes from the cofilin 1-si RNA group was significantly lower than that from the scrambled si RNA group[(29.39±15.51)%vs(31.71±14.99)%,P=0.023].The granzyme B level in CD8~+T lymphocytes from the cofilin 1-si RNA group was significantly lower than that from the scrambled si RNA group[(15.49±10.89)%vs(23.35±10.56)%,P=0.019].Conclusion1.The levels of cofilin 1 protein in mDC in SAA patients were higher than those in complete remission SAA patients and healthy controls.The level of cofilin 1 in mDC of SAA patients is closely related to the immune status and disease severity of the patients.The higher the level of cofilin 1,the more severe the immune disorder and the worse the disease.The increase of cofilin 1 occurs at the transcription level.2.The knockdown of cofilin 1 by RNAi can reduce the phagocytosis,migration and CD86 expression of mDC in SAA patients.This might be achieved by F-actin remodeling.Myeloid DC proliferation,apoptosis and CD80 expression were not affected.3.Cofilin 1 can increase the ability of mDC in SAA patients to stimulate CD4~+T lymphocytes to secrete Th1-related cytokines(IL-2,TNF-?,IFN-?)and Th2-related cytokines(IL-6),with Th1 prominent.There was no effect on Foxp3 expression in Treg.4.Cofilin 1 can increase the ability of mDC in SAA patients to stimulate the proliferation of CD8~+T cell and their secretion of perforin and granzyme B.5,Cofilin 1 may be a new indicator for the diagnosis and evaluation of SAA.Cofilin1 intervention may be a new method for the treatment of SAA. |
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