Effects Of Malnutrition On Lymphocyte Subsets And The Changes Of Regulatory T Cells

PART ?:Characteristics of lymphocyte subsets in elderly malnourished patientsObjective:Malnutrition is a common Geriatric syndrome that can cause a series of adverse consequences such as decreased immune function and susceptibility to infectious diseases in the elderly.However,at present,there is a lack of research on the changes of lymphocyte subsets in Chinese elderly malnourished patients.The purpose of this study is to systematically describe the effects of malnutrition on T lymphocyte subsets and NK cell related parameters in the hospitalized elderly population.Methods:Patients aged ?65y in the Geriatric ward of our hospital from December 2014 to November 2017 were participated in this cross-sectional study.The excluding criteria including patients with tumors,infectious diseases,autoimmune diseases and the use of drugs that affect immune function.Flow cytometry was used to detect the proportion of peripheral blood lymphocyte subsets,including:the ratio of lymphocytes occupying nuclear cells,T lymphocytes,NK cells,NKT lymphocytes,CD4+helper T lymphocytes(Th),CD8+inhibition/cytotoxicity T cells(Ts)and CD4+CD25+CD127dim/-regulatory T cells(Treg)account for lymphocytes ratio,CD4/CD8 ratio,the ratio of naive and memory Th cells,naive and memory Ts cells,Th or Ts cells expressing the second receptor(CD28+),Th or Ts cells lacking the second receptor(CD28-)to T lymphocytes.All patients were screened for nutritional risk using the NRS2002 scale,and those with malnutrition risk(NRS2002 ?3 points)were further evaluated for malnutrition assessment.According to the results,the patients were divided into malnourished and normal nutrition groups.The baseline data including the diseases,nutrition-related indicators and lymphocyte subsets were collected and compared between the two groups.Results:A total of 172 elderly patients finally completed the survey.The average age of the enrolled patients was 80.94±9.02 years(between 65-102 years)and the average body mass index was 24.01±3.78 kg/m2.The average number of comorbidities was 7 and the average hospital stay was up to 18 days.The results showed that 53 cases(30.81%)were at risk of malnutrition,39 patients were malnourished,the incidence of malnutrition was 22.67%,of which moderate malnutrition accounted for 8.14%and severe malnutrition accounted for 14.53%.The incidence of malnutrition risk,malnutrition,and severe malnutrition in the studied population increased with age(P<0.05).Compared with the patients in the normal nutrition group,patients in malnourished group were older(P<0.001),had lower body mass index(P<0.001)and longer hospital stay(P=0.004),the prevalence of respiratory tract infections and chronic obstructive pulmonary disease was higher(P<0.001,P=0.002),the prevalence of hypertension was lower(P=0.019),however,there was no statistical difference in the gender distribution and the number of comorbidities.The study found that gender affects some lymphocyte subsets.The ratio of lymphocytes and CD4+CD28+T cells in women was higher,while the NK cells in men was higher(P<0.05).The values of Th lymphocytes,CD4/CD8 ratio,Treg,naive Th cells,CD3+CD28+T cells,CD4+CD28+T cells,CD8+CD28+T cells were lower in malnutrition patients,while the ratio of memory Ts cells,CD8+CD28-T cells were higher,with statistical difference(P<0.05).Conclusion:The incidence of malnutrition is high among the elderly hospitalized patients studied.With the increase of age,the incidence of malnutrition risk,malnutrition and severe malnutrition in the study population gradually increased.The effect of gender on lymphocytes,CD4+CD28+T cells and NK cells suggests that these subsets changes may be related to the longevity of women.Changes in the Th cell,CD4/CD8 ratio,Treg and other subsets of malnourished patients suggest that they may play a key role in the occurrence and development of malnutrition and related complications.Intervention of these changing subsets may be a target for the treatment of malnutrition.PART ?:Immunological risk phenotypes of elderly patientsObjective:The immunological risk phenotypes(IRP)is a set of indicators to evaluate the body's immune function,which can predict the occurrence of diseases.The elderly are often accompanied by a decline in immune function and often accompanied by malnutrition,which affects their quality of life and prognosis.This study aims to discover the IRP of elderly patients and provide a basis for delaying immune aging,also for nutrition and immune intervention.Methods:A prospective observation cohort study was conducted on elderly patients aged?65 years in our hospital's geriatric ward from December 2014 to November 2017,excluding patients with tumors,infectious diseases,autoimmune diseases and the use of drugs that affect immune function.Clinical data and laboratory parameters reflecting nutritional status and immune function,as well as serum cytomegalovirus antibodies were collected at baseline.Flow cytometry was used to detect the proportion of peripheral blood lymphocyte subsets,including:lymphocyte occupancy nuclear cell ratio,the ratio of T lymphocytes,NK cells,NKT lymphocytes,Th cells,Ts cells and Tregs to lymphocytes,CD4/CD8 ratio,the ratio of naive and memory Th cells,naive and memory Ts cells,T cells expressing second receptors,Th cells and Ts cells expressing second receptors,Th cells and Ts cells lacking the second receptor to T lymphocytes.All patients were followed up for 2 years,and the end point of the follow-up was all-cause death.Nutritional and immune indexes were compared between the death group and survival group,and the predictive value of all-cause death was judged by ROC curve and survival analysis.Results:The enrolled patients were followed up by telephone and clinical follow-up for 24 months,and 170 patients reached the end of follow-up.The average baseline age of the patients was 80.84±9.02 years old,including 133 males(78.2%).The number of all-cause deaths during follow-up was 34.Surviving and dying patients had significant differences in age,BMI and length of hospitalization.The patients in the death group were older(P<0.001),lower in BMI(P<0.001)and longer in hospital(P<0.05).The death group had a higher proportion of patients with respiratory infections and chronic obstructive pulmonary disease,while the survival group had a higher proportion of patients with digestive system diseases(P<0.05).The results of nutritional screening of patients showed that the death group patients with NRS2002?3 scores were as high as 79.4%,and their nutritional condition was worse(P<0.001).Almost all patients had been infected with cytomegalovirus(97.7%)in the past,no current infection was found.Through the analysis of biological markers,it was found that the possible causes of all-cause death are:low serum albumin,low prealbumin levels,low Th cells,CD4/CD8 ratio,naive and memory Th cells,CD3+CD28+T cells,CD4+CD28+T cells(P<0.05),high Ts cells,naive and memory Ts cells and CD8+CD28-T cells(P<0.05).Grouped by the CD4/CD8 ratio,it was found that the patients in the immune risk group had a larger male proportion(86.89%vs 73.39%),older age(82.72±8.64 vs 79.78±9.10),and lower BMI values(22.91±3.53 vs 24.44±3.99)(P<0.05);increased NKT lymphocytes and CD8-associated lymphocyte subsets,including naive Ts cells,memory Ts cells,CD8+CD28+T lymphocytes and CD8+CD28-T lymphocytes(P<0.05);decreased CD3+CD28+T lymphocytes and multiple CD4-related lymphocyte subsets,including Treg,naive Th cells,memory Th cells and CD4+CD28+T lymphocytes(P<0.05);CD4+CD28-T cells also decreased,but there was no statistical difference.For the prediction of all-cause death,CD8+CD28-T cells had the best prediction effect(AUC=0.706)and had a high sensitivity(0.912);followed by the naive Ts cells(AUC=0.683),but its sensitivity and specificity was not high.Survival analysis showed that CD4/CD8<1(P=0.009),lymphocytes(P=0.043),Th cells(P=0.030),Ts cells(P=0.035),naive Ts cells(P=0.010),CD3+CD28+T cells(P=0.002),CD4+CD28+T cells(P=0.001)and CD8+CD28-T cells(P=0.004)can predict death.Conclusion:This study explored the IRP of elderly patients.The results suggest that in addition to CD4/CD8<1,CD8+CD28-T cells and naive Ts cells may be good IRP indicators,while lymphocytes,Th cells,Ts cells,CD3+CD28+T cells and CD4+CD28+T cells can also be used as IRP.Males,advanced age and malnutrition are significantly associated with immune risk.Clinically,through the detection of relevant lymphocyte subsets and the screening of malnutrition,it is possible to detect,diagnose and treat patients with immune risks early,and improve the prognosis of patients.PART ?:Changes of regulatory T cells in elderly malnourished patientsObjective:The incidence of malnutrition in the elderly is high,which can easily lead to many adverse consequences,especially various infections.It is currently believed that this is due to a decline in immunity caused by malnutrition,but the specific mechanism has not yet been fully clarified.Regulatory T cells are important immune cells in the human body and play an important role in suppressing inflammatory reactions.The purpose of this study was to investigate the changes in the number and function of regulatory T cells in elderly malnourished patients.Methods:The data of 172 elderly inpatients collected from December 2014 to November 2017 were analyzed.The patients were divided into malnutrition group and normal nutrition group,and the difference of the proportion of regulatory T cells to T lymphocytes between the two groups was analyzed.Six elderly(?65 y)malnourished patients and six gender-and age-matched nutritionally normal patients were included from April to November 2019.Patients with tumors,infectious diseases,autoimmune diseases and the use of drugs that affect immune function were excluded.The general information and serum cytokines IL-6,IL-10,TNF-? levels were recorded.Peripheral blood was collected from all patients,regulatory T cells were sorted by magnetic bead sorting and counted.The expressions of anti-apoptotic genes Bcl-2,MCL-1 and apoptotic genes Caspase-3,BID,BAX were detected by RT-PCR.The difference of the number of regulatory T cells,cytokine expression level and apoptosis level were compared between the two groups.Results:The study found that the number and the proportion of peripheral blood regulatory T cells in T lymphocytes in the malnourished group were lower than those in the normal nutrition group(P<0.05).The secretion of IL-6 and IL-10 in malnourished patients was higher than the normal reference value,and significantly higher than that in normal nutrition group(P<0.001 and P<0.01).Compared with the control group,the anti-apoptotic genes Bcl-2 and MCL-1 of regulatory T cells were expressed lower in malnourished patients(P<0.001).In addition,the expressions of pro-apoptotic genes Caspase-3 and BAX were higher in malnourished patients(P<0.01).There was no statistically significant difference in the pro-apoptotic gene BID between the two groups.Conclusion:Regulatory T cells may play an important role in the decline of immune function caused by malnutrition.The decrease in the number and ratio of Tregs in malnourished patients may be related to the decreased expression of anti-apoptotic genes Bcl-2,MCL-1 and the increased expression of apoptotic genes Caspase-3,BAX.The increase of cytokines IL-6 and IL-10 in malnourished patients indicate that the interaction between proinflammatory and anti-inflammatory processes in the body is closely related to the number and function of Tregs.Through the intervention of Tregs,it may become a therapeutic target for malnutrition and its complications.