Alteration Of Circulating Innate Lymphoid Cells In Patients With Atherosclerotic Cerebral Infarction

Background:Atherosclerosis(AS)is a chronic inflammatory injury disease characterized by classic inflammation such as degeneration,exudation and hyperplasia.It is an important risk factor for the development of ACI,in which lymphocytes play an important immune role.In particular,innate and adaptive immune responses are essential in the development of atherosclerosis.It is reported that inflammatory reactions exist in all stages of atherosclerotic lesions,and atherogenic factors may cause atherosclerosis via inflammatory response.Acute cerebral infarction(ACI)is an acute cerebral infarction caused by vascular obstruction,which leads to the interruption of cerebral blood supply,and cerebral tissue necrosis caused by ischemia.Most of them have the pathological basis of atherosclerosis and plaque rupture,which can be fatal.It has a high incidence in the elderly population,which seriously affects the health level of the elderly,and atherosclerosis(AS)plaque rupture is its main pathological basis.ACI is a common cardiovascular disease with high morbidity,high disability rate and high mortality around the world,posing a serious threat to human health.This disease is more common in the elderly population,with the acceleration of China's aging process,the incidence of acute cerebral infarction increased.There are mainly inflammatory substances released and lymphocyte activation caused by local inflammation,and hypoxic ischemia caused by brain cell edema,cell apoptosis and other pathological and physiological changes.Atherosclerosis(AS)is one of the main causes of acute cerebral infarction.Oxidized low-density lipoprotein(ox-LDL)can damage endothelial cells themselves,promote the adhesion and migration of macrophages under the endothelium,absorb oxidized low-density lipoprotein to form foam cells and deposit them under the endothelium,and promote the occurrence and development of the pathological process of atherosclerosis.The formation and deposition of foam cells are the early pathological manifestations of atherosclerosis and also the key links leading to atherosclerosis.Innate Lymphoid Cells is a newly discovered lymphocyte population,which originated from common lymphocyte progenitor(CLP).Unlike T and B lymphocytes,proper lymphocytes do not express antigen-specific recognition receptors(TCR or BCR)and belong to the lymphocyte lineage in development and morphology.According to the killing ability,ILC can be divided into two categories:one is NK cells with strong killing activity,and the other is auxiliary ILC without or with weak killing ability.According to the difference of secreted cytokines and expressed transcription factors,the ILC subgroup can be divided into three categories:ILC1s,ILC2s and ILC3s.Innate Lymphoid Cells play an important role in the immune system.Although they are innate immune cells,they can mediate acquired immune functions.Such cells between innate and acquired immunity are likely to fill the gap between the two systems,further blur the boundary between innate and adaptive immunity,and become an important bridge between the two immune systems.Innate Lymphoid Cells and Th1,Th2 and Th17 are"mirror cells"to each other and play an important role in the protective immunity against pathogens and immune/inflammatory diseases.Th1,Th2,Th17,etc.have been reported in atherosclerosis for a long time.However,the expression and role of ILC in ACI are unknown.Oxidized low-density lipoprotein(ox-LDL)is a key factor in the occurrence and development of AS,and leads to endothelial dysfunction and plaque instability through a variety of mechanisms.It is also unclear whether oxidized LDL has a direct effect on ILCs.Therefore,in this study,ILCs expression in peripheral blood of healthy people and ACI patients was detected by flow cytometry,and the effect of ox-LDL on ILCs expression was further studied through cell experiments.The mechanism of ILC imbalance in ACI was preliminarily discussed,and new indicators for early diagnosis and prognosis of ACI were provided.Objective:To investigate the expression of ILC in ACI patients by peripheral blood,to clarify the imbalance of peripheral blood ILC subgroups in ACI patients,and to explore its mechanism preliminarily.Methods:(1)We first chose the subjects including 63 healthy people and 60 ACI patients.Surfaceandcytoplasmaantigenswerelabelledbymulticolor immunofluorescence labeling method,and the frequency of peripheral ILCs in different groups was detected by multiparameter flow cytometry,and the expressions of ILC1,ILC2 and ILC3in ILCs from different groups were analyzed.The serum levels of ox-LDL were examined by enzyme-linked immunosorbent assay(ELISA).Correlations of the levels of ILC1 and ILC2 and ILC1/ILC2 ratio to ox-LDL,LDL-C,and biochemical markers expression levels were analyzed.Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the key cytokines IL-18 and IL-23,which induce the production of ILCs in peripheral blood mononuclear cells(PBMCs).IL-33expression level.(2)Ox-ldl induced PBMCs in vitro.The in vitro incubation of peripheral blood mononuclear cells(PBMC)by health people with ox-LDL of different concentrations were cultured together for 24h.The frequency of peripheral ILCs was detected by multiparameter flow cytometry.The effects of different concentrations of ox-LDL on ILCs were analyzed.10?g/mL ox-LDL,which lead to the most obvious alteration in ILCs,with PBMC of healthy people and ACI patients cultured together for 24h in vitro.The frequency of peripheral ILCs was detected by multiparameter flow cytometry.The expression of ILCs from different groups was analyzed.Results:(1)The basic data and blood lipid results of each group showed that compared with the control group,the fasting blood glucose(FBG)of the ACI group was significantly increased,and the difference was statistically significant(P<0.01).In addition,the total cholesterol(TC)and LDL-C in the ACI group were significantly higher than those in the control group,and the difference was statistically significant(P<0.05).(2)Flow cytometry in human peripheral blood identified ILC1,ILC2 and ILC3:according to the expression of CD294(CRTH2)and CD117,CD127~+ILC can be divided into three subgroups:ILC1(CD117~–CD161~+CD294~–),ILC2(CD294~+)and ILC3(CD294~–CD117~+)cells.(3)ILC2 cells were decreased and ILC1 cells were increased in ACI patients.The expression rate of peripheral ILC1 cells in ACI patients(40.58±8.10%)was significantly higher than that in the control group(20.12±8.44%),and the difference was statistically significant(P<0.01).The expression rate of peripheral ILC2 cells in ACI patients(23.34±6.12%)was significantly lower than that in the control group(41.78±11.11%),and the difference was statistically significant(P<0.01).In addition,there was no significant difference in the ILC3 expression rate between the ACI patients(28.11±8.80%)and the control group(24.95±10.06%)(P>0.05).In addition,the ILC1/ILC2 ratio(1.72±0.35)in ACI patients was significantly higher than that in the control group(0.58±0.24),and the difference was statistically significant(P<0.01).(4)Serum concentrations of ox-LDL,LDL-C and inflammatory markers(HCY,Lp(a)and CRP)in ACI patients were significantly higher than those in healthy people(all P<0.05).(5)Notably,the levels of ILC1 and ILC2 and ILC1/ILC2ratio showed a significant correlation with the levels of ox-LDL,LDL-C,and biochemical markers in the two groups.The level of ILC1 positively correlated with the serum levels of ox-LDL,LDL-C,Hcy,Lp(a),and CRP(P<0.01 and r=0.894,0.467,0.672,0.561,and 0.643,respectively),while the level of ILC2 negatively correlated with the serum levels of five markers(P<0.01 and r=–0.663,–0.548,–0.606,–0.497,and–0.514,respectively).In addition,the ILC1/ILC2 ratio also positively correlated with the levels of ox-LDL,LDL-C,Hcy,Lp(a),and CRP(P<0.01 and r=0.828,0.321,0.664,0.439,and 0.649,respectively).(6)Expression of Interleukin 18(IL-18),IL-33 and IL-23 in PBMCs from ACI.IL-18 mRNA levels in PBMCs were significantly higher in ACI patients than in control subjects(P<0.01),while IL-33mRNA levels were markedly lower in ACI patients than in control subjects(P<0.01).With respect to IL-23 mRNA levels,there were no significant differences between ACI and control groups(P>0.05,n=10 for each gorup).(7)Effects of ox-LDL on ILCs in vitro:The effects of various levels of ox-LDL on the number of ILCs in controls were examined.The level of ILC2 decreased while the level of ILC1 increased with increased levels of ox-LDL.However,no difference was observed in the level of ILC3 in cultured PBMCs derivedfromtheox-LDL-treatedcontrols.Thesensitivityofthe ox-LDL-mediated decrease in ILC2s and increase in ILC1s in patients with ACI and controls were investigated.PBMCs from the control and ACI groups were incubated with 10?g/mL ox-LDL for 24 h.Significant changes were noted in the number of ILC1s and ILC2s in ox-LDL-treated patients with ACI(approximately 39.2%reduction in the levels of ILC2s and 41.7%increase in the levels of ILC1s)than in controls(approximately 19.3%reduction in the levels of ILC2s and 28.7%increase in the levels of ILC1s).However,no difference was observed in the numbers of whole ILC cells in cultured PBMCs derived from controls and ox-LDL-treated patients with ACI.Compared with the control group,the range of ILC1s and ILC2s in ACI patients was larger and more obvious.There was no significant difference in the number of CD45~+and lin-cd127~+ILC cells between the two groups.Conclusions:(1)ILC2 cells were decreased and ILC1 cells were increased in ACI patients.The change trend of related cytokines was highly consistent with ILC.The level of ILC1 positively correlated with the serum levels of ox-LDL,LDL-C,Hcy,Lp(a),and CRP,while the level of ILC2 negatively correlated with the serum levels of five markers.ILC may be a new sensitive indicator for early diagnosis and prognosis of ACI.Ox-LDL,an important pro-as factor,can cause ILC imbalance.(2)Ox-LDL up-regulates ILC1 expression and down-regulates ILC2 expression with concentration dependent.The effect of ox-LDL on the expression of ILC1/ILC2 in ACI patients was more significant.In summary,significant changes in ILC1 and ILC2 occur in patients with ACI.Changes in the expression of ILC1s and ILC2s are a new sensitive indicator for early diagnosis of ACI.ox-LDL directly affects the changes of ILC1s and ILC2s,leading to the occurrence of ACI.