| ObjectiveDifferent subsets of iNKT cells in tissues and organs play different roles in different diseases of the same organ and different stages of the same disease,effectively regulating the activation of different subsets of iNKT cells,and obtaining iNKT cells and subsets with specific phenotypes and functions for Cell therapy of related diseases provides basic research data.MethodsC57BL/6 male mice aged 6-8 weeks were randomly divided into groups and injectedα-Galcer intraperitoneally,subcutaneously,and intravenously.Select multiple time points to dynamically observe and compare the effects of α-Galcer on the changes of iNKT cells and subsets in different organs of mice,and dynamically observe the effects of different injection routes of α-Galcer on the changes of iNKT cells and subsets in mice.Results1.Thymus:compared with the control group,the frequency of intraperitoneal injection and subcutaneous injection of α-Galcer thymus iNKT cells was significantly reduced,and the frequency of intravenous injection of α-Galcer thymus iNKT cells was significantly increased;all three injection methods increased the level of thymus iNKT1 subgroup,intraperitoneal injection and subcutaneous injection of α-Galcer thymus iNKT2 subgroup decreased,intravenous injection of α-Galcer thymus iNKT2 subgroup reached a peak in second day,and then fluctuated around the normal level;intraperitoneal injection of α-Galcer thymus iNKT17 subgroup was lower than normal on the 6th day and the 10 th day,the 8th day is higher than normal,subcutaneous injection and intravenous injection of α-Galcer thymus iNKT17 subgroup were reduced.2.Spleen:compared with the control group,the frequency of intraperitoneal injection ofα-Galcer spleen iNKT was significantly increased,except for the 8th day;the frequency of subcutaneous injection of α-Galcer spleen iNKT showed a trend of first decline and then increase,of which the 6th day decreased to the bottom;intravenous injection of α-Galcer the frequency of spleen iNKT was reduced throughout the whole process;the spleen iNKT1 subgroup of the three injection methods were all significantly lower than the normal level,the spleen iNKT2 subgroup were higher than the normal level before α-Galcer injection(the 2nd and 3rd day),and the spleen iNKT17 subgroup were significantly lower Value(except for intravenous injection of α-Galcer 6th day).3.Liver:compared with the control group,the frequency of intraperitoneal injection ofα-Galcer liver iNKT peaked on 6th day,and the rest were significantly lower than normal;subcutaneous injection and intravenous injection of α-Galcer prophase(the 2nd and 3rd day)liver iNKT cell frequency were both the liver iNKT1 subgroup of the three injection methods were significantly lower than the normal level(except for intraperitoneal injection of α-Galcer on the 4th day);the liver iNKT2 subgroup fluctuated around the normal level after intraperitoneal injection of α-Galcer,and subcutaneous injection of α-Galcer improves liver iNKT2 subgroup.In the early stage of intravenous injection of α-Galcer(4 days ago),liver iNKT2 subgroup were higher than normal,reached a peak on the 2nd day,and then decreased in the later stage;intraperitoneal injection and intravenous injection of α-Galcer liver iNKT17 subgroup were significantly lower at normal levels(except for intraperitoneal injection ofα-Galcer for 4 days),subcutaneous injection of α-Galcer liver iNKT17 subgroup is higher than normal.4.Lymph nodes:compared with the control group,the total frequency of iNKT cells in the lymph nodes of the first(2nd day)intraperitoneal injection of α-Galcer is higher than the normal level,and the frequency of subcutaneous injection and intravenous injection ofα-Galcer lymph nodes is significantly lower than the normal level;the three injection methods of lymph nodes both iNKT1 and iNKT17 subgroups were significantly lower than normal levels.After intraperitoneal injection of α-Galcer on the 4th and 10 th day,the lymph node iNKT2 subgroup were higher than normal levels,and the rest of the time points were significantly lower than normal levels.Intravenous injection of α-Galcer can increase lymph node iNKT2 subgroup.5.Lung:compared with the control group,the frequency of intraperitoneal injection ofα-Galcer lung iNKT was higher than normal in the first stage(2nd day),and then lower than normal.The frequency of subcutaneous injection of α-Galcer lung iNKT was higher than normal on the 2nd and 3rd day,and lower after 6th day.At normal levels,the frequency of intravenous injection of α-Galcer lung iNKT decreased throughout;intraperitoneal injection and subcutaneous injection of α-Galcer lung iNKT1 subgroup were significantly reduced,and intravenous injection of α-Galcer lung iNKT1 subgroup were significantly increased(except for 6 days),abdominal cavity subcutaneous injection of α-Galcer lung iNKT2 subgroup increased,subcutaneous injection and intravenous injection of α-Galcer lung iNKT2 subgroup significantly decreased,intraperitoneal injection and subcutaneous injection of α-Galcer lung iNKT17 subgroup were significantly increased,and subcutaneous injection of α-Galcer lung iNKT17 in the later stage of the subgroup(after 4 days),it was higher than normal.6.Peripheral blood serum cytokines:on the 2nd day after α-Galcer injection,compared with the control group,the level of IL-4 in the serum of intraperitoneal injection of α-Galcer was significantly increased,and there was no significant change in the serum IL-4 of subcutaneous and intravenous injection of α-Galcer.Intraperitoneal injection and intravenous injection of α-Galcer serum IFN-γ levels significantly increased,subcutaneous injection ofα-Galcer serum IFN-γ levels significantly decreased,and serum IL-17 A levels in the three injection methods were significantly reduced;α-Galcer injection After 6 days,compared with the control group,the serum levels of IL-4 in the three injection methods were significantly reduced,IFN-γ was significantly increased after intraperitoneal injection of α-Galcer,and IFN-γ was significantly decreased after subcutaneous injection and intravenous injection ofα-Galcer.The IL-17 A levels of the three injection methods were all lower than the normal level,and the changes were more obvious with subcutaneous injection of α-Galcer;8 days afterα-Galcer injection,compared with the control group,the serum IL-4 levels of the three injection methods were all lower than the normal level,it is more obvious by subcutaneous injection.The level of IFN-γ in the serum of the three injection methods is increased,and the level of IL-17 A is significantly reduced.Conclusion1.There are differences in the effects of the same injection method on the frequency and subgroups of iNKT in different organs;the effects of different injection methods on the frequency and subgroups of iNKT in the same organ are different;different injection methods have differences in the frequency and subgroups of iNKT in different organs at the same time point.2.It is reported in the literature that α-Galcer has little effect on thymus iNKT.However,we found that different injection methods(especially intravenous injection ofα-Galcer)have an effect on the frequency and subpopulations of thymus iNKT cells,and the mechanism is worthy of further investigation.3.The liver is the main place for iNKT cells to settle.The treatment of liver-related diseases based on iNKT cells can be achieved by choosing different injection methods.4.All three injection methods can increase the frequency of iNKT17 subgroup in the lungs.Therefore,the use of α-Galcer therapy should pay close attention to the risk of autoimmune diseases that may appear in the lungs.5.α-Galcer injection causes changes in serum cytokines,suggesting that injection ofα-Galcer can affect the overall immune function of the body. |
