Analysis Of Frequency,phenotype And Functionalactivity Of CD56^BRIGHT And CD56^DIM Natural Killer Cells In Peripheral Blood Of Children With Hand,food And Mouth Disease

Objectives:Hand,foot and mouth disease is an acute infectious disease caused by a variety of enteroviruses,and its pathogenesis is still unclear.The natural immune system is the leading force in children’s defense against viral infections,and natural killer cells play a very important role in the immune response to antiviral infection.At present,the role of NK cells in HFMD is still unclear.This study was to investigate the changes of frequency,phenotype and functional activity of two important subpopulations of NK cells,CD56^brightright and CD56^dimim NK cells,in HFMD patients with different conditions and EV71 infection.Methods:1.In strict accordance with the inclusion and exclusion criteria,55children with acute HFMD and 39 Healthy Children who underwent outpatient physical examination at the same time were randomly included.2.Flow cytometry was used to detect the frequencies,phenotypes（NKp46,NKG2D and NKG2A）and cytotoxic substance or cytokine secretion function（CD107a,GB,GL or TNF-α,IFN-γ,IL-10）of CD56^brightright and CD56^dimim NK cells in all subjects.3.The levels of cytokines and chemokines（IL-2,IL-6,IL-10,TNF-α,IFN-γand MCP-1）in plasma were measured by enzyme-linked immunosorbent assay.4.Experimental data were expressed as mean±standard error（?X±SEM）and statistical analysis was performed using SPSS v.23.0（SPSS Inc,USA）software.Based on the validity of the normal hypothesis and the homogeneity of the variance,the difference between the mean values of multiple sets of continuous variables was examined using one-way analysis of variance or Kruskal Wallis test.Correlation between the two sets of statistical variables was assessed using the Spearman correlation coefficient.And P<0.05 was considered statistically significant.Results:1.Compared with HC,the expression of NKp46 receptor in CD56^dimim NK cells was significantly decreased in mild HFMD case（P<0.01）,while the expression of CD107a,GB and GL was significantly increased（P<0.05）,and CD56^brightright NK cells expressed NKG2A receptor and IFN-γlevels were significantly decreased（P<0.01）while GB,GL and IL-10expression was significantly increased（P<0.05）.Compared with HC,in severe HFMD cases,the number and frequency of total NK or CD56^dimim NK cells were significantly decreased（P<0.01）,and CD56^dimim NK cells expressed NKG2D receptors levels decreased（P<0.01）while NKG2A receptor levels increased（P<0.05）,and CD56^brightright NK cells expressed NKG2D,NKG2A receptor and IFN-γlevels decreased（P<0.05）,while TNF-αwas significantly increased（P<0.001）.Compared with mild cases,the expression of CD107a,GB,and GL in CD56^dimNK cells of severe cases decreased,and GL（P<0.01）decreased significantly.2.Compared with HC,in EV71⁺HFMD cases,the number and frequency of total NK cells or CD56^dimim NK cells were significantly decreased（P<0.05）,and the expression of NKG2A in CD56^dimNK cells was significantly increased（P<0.05）,and CD56^brightright NK cells expressed IL-10 and TNF-α（P<0.01）was significantly increased.Compared with HC,in EV71^-HFMD cases,the expression of NKp46and NKG2D receptors in CD56^dimim NK cells was significantly decreased（P<0.05）,while the expression of NKG2A receptor and GB was increased（P<0.05）;CD56^brightright NK cells expressed NKG2D,NKG2A receptor and IFN-γmolecules were decreased（P<0.05）while TNF-αmolecules were increased（P<0.05）.Compared with EV71⁺HFMD cases,the proportion of IFN-γexpressed by CD56^brightright NK cells was significantly decreased in EV71^-HFMD cases（P<0.05）.Compared with HC,the expression of GB（P<0.05）in CD56^dimim NK cells was significantly increased and GL（P>0.05）was decreased in severe EV71⁺HFMD cases,while the expression of TNF-αand IL-10 in CD56^brightright NK cells was significantly increased（P<0.05）;The proportion of IFN-γexpressed by CD56^brightright NK cells in severe EV71^-HFMD cases was significantly decreased（P<0.01）while TNF-αwas significantly increased（P<0.01）.3.Compared with HC,mild HFMD cases had no significant changes in inflammatory factors except TNF-α（P<0.001）.While plasma concentrations of IL-6,IFN-γ,TNF-αand MCP-1 were significantly increased in severe HFMD cases（P<0.05）.And compared with mild cases,IL-10 levels were significantly higher in severe cases（P<0.05）.Compared with HC,plasma IFN-γ,TNF-αand IL-6 levels were elevated in EV71⁺HFMD cases（P<0.05）,and plasma IFN-γ,TNF-α,IL-10and MCP-1 levels were elevated in EV71^-HFMD cases（P<0.01）.Compared with EV71⁺HFMD cases,plasma IL-10 levels were significantly higher in EV71^-HFMD cases（P<0.05）.There was no significant difference in plasma IL-2 levels between the groups（P>0.05）.4.The PCT level was negatively correlated with the percentage of GB⁺CD56^dimim NK（P<0.05）,but positively correlated with the plasma concentration of IFN-γ（P<0.01）;The LYM%was positively correlated with the percentage of NKG2D⁺and GB⁺CD56^dimim NK cells（P<0.05）,but negatively correlated with IL-10 plasma concentration（P<0.01）;MCP-1plasma concentration was positively correlated with the percentage of IL-10⁺CD56^dimim NK（P<0.05）;There was a positive correlation between IL-2 plasma concentration and CD56^brightright NK cell count（P<0.05）.Conclusions:1.Peripheral blood NK cells were significantly reduced in severe and EV71⁺patients,mainly due to the sharp decrease of CD56^dimim NK cells,but no significant changes in CD56^brightright NK cells.2.The cytotoxic function（CD107a,GL and GB）of CD56^dimim NK cells in peripheral blood increased when HFMD was mild,which might be related to virus clearance.With the aggravation of the disease,the expression of inhibitory receptor（NKG2A）increased while the killer molecules decreased,showing cell depletion or dysfunction,which may be associated with severe HFMD.3.CD56^brightright NK cells increased the secretion of inflammatory cytokines and anti-inflammatory factors in children with EV71 infection,and the secretion function was severely disordered,which may be involved in the severe HFMD of EV71 infection.4.The number,phenotype and immune function of NK cells and their subpopulations in HFMD are closely related to the occurrence and development of HFMD diseases.