The Role And Mechanism Of Type 1 Innate Lymphoid Cells In Acceleration Of Atherosclerosis

Part I.Identification and proportion of innate lymphoid cell population in AS.Objective:To observe the phenotypes and proportion of innate lymphoid cell(ILC)in AS of ApoE KO mice.Methods:18-week-old ApoE KO mice fed with Standard diet were used as normal control.Flow cytometry(FACS)was used to detect the ILC expression of ApoE KO mice in high fat diet(HFD))in the spleen,and then the gene expression of the cells was detected by quantitative polymerase chain reaction(q-PCR).The concentration of cytokines secreted by the cells was detected by enzyme-linked immunosorbent assay(ELISA).Results:The proportion of ILC subsets increased,especially Lin-CD127 + ST2-c-kit-cells.The expression of TH1-like transcription factor T-bet and cytokine IFN-?,IL-1? and IL-6 were higher in Lin-CD 127 + ST2-c-kit-1 innate lymphoid cells(ILCls).But the expression of transcription factors GATA3,RORyT and cytokines IL-13,IL-5 and IL-17A were very low,and but no granzyme B,CD56 and IL-15R expression.Conclusion:There is an increased group of innate lymphoid cells-Lin-CD127 +ST2-c-kit-cells in the AS.And the cytokines secreted are identical to the cytokines that TH1 acts on AS,this suggests that ILC1 similar to TH1 may be involved in the plaque development of AS.Part ?.Lin-CD127+ST2-c-kit-ILCls promote atherosclerotic plaque formation.Objective:To explore whether type 1 innate lymphoid cells(ILC1s)are involved in the formation of AS plaquesMethods:ApoE KO mice fed with standard diet were used as control group.The percentage of innate lymphoid cells in spleen of AS mice was detected by FACS.Group 1 innate lymphoid cells(ILC1s and natural killer immune cells-cNK cells)in ApoE-Rag1 DKO mice were deleted by intravenous injection of anti-NK1.1 antibody,only cNK cells from ApoE-Rag1 DKO mice were deleted by intravenous injection of anti-IL-15R antibody.The ILC1s in the spleen of ApoE-Rag1 DKO mice were obtained by flow sorting and then adoptive transferred to ApoE-Ragl DKO mice treated with anti-NKl.1 antibody.The roots of the aorta and the entire descending aorta were stained with oil red to observe the area of the plaque and then analyzed.Results:The percentage of ILC in the spleen of ApoE KO mice fed with HFD was higher than that of the standard diet of ApoE KO mice,and the percentage of ILC1 in the spleen of the ApoE KO mice was mainly increased.The plaque area of ApoE-Rag1 DKO mice,which received intravenous injection of anti-NK1.1 antibody,was significantly higher than that of anti-IL-15R-treated ApoE-Rag1 DKO mice.After adoptive transferred ILC1s obtained from ApoE-/-TLR4 +/+ mice,the plaque area of ApoE-Rag1 DKO mice that received intravenous injection of anti-NK1.1 antibody increased.Conclusion:Lin-CD127 + ST2-c-kit-ILC1s are present in the development of AS and promote AS progression.Part ?.Lin-CD127+ST2-c-kit-ILCls respond to danger signals in AS conveyed by TLR4Objective:To investigate whether Toll-like receptor 4(TLR4)mediates the role of type 1 innate lymphoid cells in AS plaques formation.Methods:The expression of TLR4 in spleen Lin-CD127 + ST2-c-kit-ILC1s,Lin-CD127 +ST2 + ILC2s and Lin-CD127 + ST2-c-kit + ILC3 cells was detected by flow cytometry.Lin-CD127 + ST2-c-kit-ILC1s were incubated with pam3CSK4(500 ng/mL,TLR2 agonist),Poly:c(10 ng/ml,TLR3 agonist),LPS(100 ng/ml,TLR4 agonist)and OxLDL(25 ug/ml)for 3 days,respectively.The concentration of secreted IL-1(3,IL-6 and IFN-?in the supernatant was detected by ELISA.Lin-CD127 + ST2-c-kit-ILC1 was isolated from ApoE KO mice or ApoE-TLR4 DKO mice and then transferred to ApoE-Rag1 DKO mice treated with anti-NK1.1.The area of aortic root and total aortic plaque was measured by oil red O staining and quantified.Results:Only TLR4 was detected in Lin-CD 127 + ST2-c-kit-ILC1s among the three subtypes of innate lymphoid cells,and the secretion of proinflammatory cytokines was promoted by LPS/OxLDL.The plaque area of ApoE-Rag1 DKO mice received anti-NK1.1 antibodies increased after adoptive transferred ILC1s from ApoE KO mice,whereas after adoptive transfer ILC1s from ApoE-TLR4 DKO mice,the plaque area had no significant changes.Conclusion:ILC1s could promote AS,and this effect depends on the expression of TLR4.Part IV.The influence and mechanism of OxLDL on ILC1 cells in AS.Objective:To investigate the influence and mechanism of OxLDL on type 1 innate lymphoid cells in AS.Methods:ApoE KO mice and ApoE-TLR4 DKO mice were used for the study.The expression of TLR4 and BACH2 was detected by Western blotting in ApoE KO mice and ApoE-TLR4 DKO mice spleen Lin-CD127 + ST2-c-kit-ILC1s after stimulating with LPS/OxLDL.The levels of IFN-?,IL-1? and IL-6 secreted by Lin-CD127 + ST2-c-kit-ILC1s after challenged with different doses of LPS/OxLDL were detected by ELISA..Meanwhile the expression of BACH2 was detected by Western blotting.Results:LPS/OxLDL could up-regulate the expression of TLR4 and down-regulate the expression of BACH2 on Lin-CD127 + ST2-c-kit-ILC1s in ApoE KO mice and promote ILC1s to secrete a certain amount of proinflammatory cytokines.But after co-culture with LPS/OxLDL.Lin-CD127 + ST2-c-kit-ILC1s of ApoE-TLR4 DKO mice didn't secret proinflammatory cytokines.High-dose LPS/OxLDL stimulation of ApoE KO mice Lin-CD127 + ST2-c-kit-ILC1s will secreted more proinflammatory factors compared with stimulated with low dose.At the same time,the expression of transcription factor BACH2 was reduced.Conclusion:OxLDL could stimulate the expression of IFN-y and proinflammatory cytokines.This role dependents on the expression of TLR4 in ILC1s,and is related to the expression of BACH2.