The Significance Of Cytotoxic Activity-related Proteins And MiR-146a And MiR-30e In Secondary Hemophagocytic Lymphohistiocytosis

ObjectivePerforin is a protein expressed in the cytotoxic lymphocytes and plays a key role in the cytolytic effect on target cells.Mutation of Perforin gene is one of the most important pathogenesis of primary hemophagocytic lymphohistiocytosis(pHLH).The aim of this study were to investigate the expression of perforin protein on cytotoxic lymphocytes,and to determine the proportion of the cytotoxic lymphocyte subsets in patients with secondary hemophagocytic lymphohistiocytosis(sHLH).At the same time,the clinical significance was explored.MethodsA total of 19 patients with sHLH and 10 healthy controls were enrolled in this study.Six of the patient received complete remission.Peripheral blood was collected The expression of perforin protein on cytotoxic lymphocytes and the proportion of cytotoxic lymphocyte subsets were tested by flow cytometry.At the same time,clinical parameters were collected to explored the correlations between them.Results(1)The proportion of NK cell in the newly diagnosed sHLH group was significantly lower than that of the healthy control group(P=0.006).However,no significant difference of the proportion of CD8+T cell or CD56+T cell was found between newly diagnosed sHLH group and healthy control group(P>0.05).(2)The expression of perforin in CD56+T cell in the newly diagnosed sHLH group was significantly higher than that of the healthy control group(P=0.035).However,no significant difference of the expression of perforin in CD8+T cell or NK cell was found between newly diagnosed sHLH group and healthy control group(P>0.05).(3)The proportion of CD8+T cell in the complete remission sHLH group was significantly lower than that of the newly diagnosed sHLH group(P=0.023).However,no significant difference of the proportion of NK cell or CD56+T cell was found between the complete remission sHLH group and the newly diagnosed sHLH group(P>0.05).(4)No significant difference of the expression of perforin in the cytotoxic lymphocytes subsets was found between complete remission sHLH group and the newly diagnosed sHLH group(P>0.05).(5)The proportion of CD56+T cell in patients with sHLH was positively correlated with HB(r=0.464,P=0.045),and was negatively correlated with Age(r=-0.456,P=0.049).The proportion of NK cell in patients with sHLH was positively correlated with FIB(r=0.531,P=0.023).(6)The expression of perforin in CD8+T cell in patients with sHLH was positively correlated with sCD25(r=0.498,P=0.035).The expression of perforin in CD56+T cell in patients with sHLH was negatively correlated with ALT(r=-0.516,P=0.028).ConclusionThe proportion of NK cell was significantly decreased in the newly diagnosed sHLH group,however,the expression of perforin in CD56+T cell was significantly increased.After complete remission,the proportion of CD8+T cell in the complete remission sHLH group was significantly decreased.Therefore,detection of the proportion of cytotoxic lymphocyte subsets or of the expression of perforin in cytotoxic lymphocytes cell may have significant importance in sHLH.It revealed that decrease in the proportion of NK cell and increase in the expression of perforin in CD56+ T cell occurred in the pathogenesis of sHLH.In addition,it may be related to the absence of NK cell activity and immune hyper-activation in sHLH.The decrease in the proportion of CD8+ T cells after complete remission may be one of the mechanisms for patients that obtain clinical efficacy.Dynamic detection of the proportion of cytotoxic lymphocyte subsets and the expression of perforin in them is helpful to evaluate the curative effect of sHLH.ObjectiveHemophagocytic lymphohistiocytosis(HLH)is a life-threatening syndrome of hyper-inflammation which is caused by uncontrolled activation and proliferation of mononuclear-macrophage system.Burst release of cytokines is one of the most important pathogenesis of HLH.Most of the mutations found in the pathogenesis of HLH are involved in the release process of cytotoxic granules.Granzyme B is one of the most important components of cytotoxic granules.In this study,Cytometric Bead Array flex set were applied to detect the serum levels of Granzyme B in patients with secondary HLH(sHLH).Multiple cytokines were detected at the same time.In addition,its correlation with expression level of cytokines and clinical characters,and the prognostic value of Granzyme B in sHLH patients were explored as well.MethodsA total of 44 patients with sHLH and 15 healthy controls were enrolled in this study.Serum was collected and levels of Granzyme B and multiple cytokines,including IFN-γ,IL-1β,IL-10,MIG and IP-10 were detected by using flow Cytometric Bead Array flex set.The clinical data of sHLH patients were collected.The correlation of Granzyme B with expression level of cytokines and clinical characters were explored.And the prognostic value of Granzyme B in sHLH patients were explored as well.Results(1)The serum expression level of Granzyme B in the newly diagnosed sHLH group was significantly higher than that of healthy control group(P=0.001).(2)The serum expression level of Granzyme B in the lymphoma associated HLH group(LHLH)was significantly higher than that of non-lymphoma associated group(Non-LHLH)(P=0.043).(3)The serum expression level of Granzyme B in the newly diagnosed sHLH group was positively correlated with the the expression level of IFN-γ(r=0.664,P<0.001),was positively correlated with the the expression level of IL-10(r=0.619,P<0.001),was positively correlated with the the expression level of MIG(r=0.657,P<0.001),and was positively correlated with the the expression level of IP-10(r=0.614,P<0.001).However,no correlation was found between the serum expression level of Granzyme B and the expression level of IL-1β in the newly diagnosed sHLH group(P>0.05).(4)The serum expression level of Granzyme B in the newly diagnosed sHLH group was positively correlated with the LDH level(r=0.523,P<0.001),and was negatively correlated with WBC level(r=-0.327,P=0.037).However,no correlation was found between the serum expression level of Granzyme B and HB,PLT,SF,ALT,AST,ALB,sCD25,CRP,TG,FIB,ESR level in the newly diagnosed sHLH group(P>0.05).(5)According to their serum expression level of Granzyme B,sHLH patient were divied into Low Granzyme B group(0 pg/mL<Granzyme B≤11.95pg/mL)and High Granzyme B group(Granzyme B>11.95pg/mL).Medium survival time of the High Granzyme B group was 26.5(14～83.25)days,and that of the Low Granzyme B group was 107(20～176.25)days.The medium survival time of the High Granzyme B group was shorter than that of the Low Granzyme B group.However,the OS analysis revealed no significant difference between them(P=0.187).ConclusionThe serum expression level of Granzyme B in the newly diagnosed sHLH group was significantly increased,especially in the LHLH group,which is important in the differential diagnosis of lymphoma associated HLH.The serum expression level of Granzyme B in the newly diagnosed sHLH group was positively correlated with the the expression level of multiple cytokines,suggesting that it may play a role in the formation of cytokine storm in the pathogenesis of sHLH.The serum expression level of Granzyme B may indicate the prognosis of sHLH.The prognosis of sHLH patients with serum level of Granzyme B higher than 11.95 pg/mL were worse than sHLH patients with serum level of Granzyme B lower than 11.95 pg/mL.ObjectiveHemophagocytic lymphohistiocytosis(Hemophagocytic lymphohistiocytosis,HLH)is a clinical syndrome which is caused by uncontrolled activation and proliferation of CD8+T cell and mononuclear-macrophage system,and abundant releasing of in inflammatory cytokines.It has been confirmed that miR-146a is widely participate in the regulation of intensity of inflammatory response,while miR-30e is related to the regulation of NK cell cytotoxicity.In this study,real-time quantitative PCR was used to detect the expression of serum miR-146a and miR-30e in patients with secondary HLH(sHLH).Their correlation with clinical data,and their diagnosis and prognosis value in patients with sHLH were explored,as well.In addition,their relation with the proportion of cytotoxic lymphocyte subsets and the expression of perforin were explored.MethodsA total of 35 patients with sHLH and 10 healthy controls were enrolled in this study.Serum was collected and expression levels of miR-146a and miR-30e were detected by using real-time quantitative PCR.The clinical data of sHLH patients were collected.The correlation of the expression level of miR-146a and miR-30e and the clinical characters were explored.The diagnosis and prognosis value of the expression level of miR-146a and miR-30e in sHLH patients were explored,as well.Results(1)The serum expression level of miR-146a in the newly diagnosed sHLH group was significantly lower than that of healthy control group(P<0.001);the serum expression level of miR-30e in the newly diagnosed sHLH group was significantly lower than that of healthy control group(P<0.001).(2)The serum expression level of miR-146a and miR-30e in the lymphoma-associated HLH group,the infection-associated HLH group,and the autoimmune-associated HLH group were compared group by group,however no significant difference was found(P>0.05).(3)ROC analysis revealed that the serum expression level of miR-146a can effectively identify sHLH patients from healthy people,with area under the curve(AUC)was 0.931,the standard error was 0.037,its 95%confidence interval was(0.859,1);the serum expression level of miR-30e can effectively identify sHLH patients from healthy people,with area under the curve(AUC)was 0.914,the standard error was 0.042,its 95%confidence interval was(0.832,0.997).(4)Correlation analysis was applied and no correlation was found between the serum expression level of miR-146a and miR-30e,and Age,WBC,HB,PLT,SF,ALB,ALT,AST,LDH,FIB,TG,ESR,sCD25,CRP level in the newly diagnosed sHLH(P>0.05).(5)According to the median of serum expression level,sHLH patients were divided in to Low expression group and High expression group.OS analysis was applied and revealed that there was no significance difference between the Low miR-146a expression group and the High miR-146a expression group(P>0.05);meanwhile there was no significance difference between the low miR-30e expression group and the high miR-30e expression group(P>0.05).However,sHLH patients were divided in to Low miR-30e/miR-146a group and High miR-30e/miR-146a group according to the median of miR-30e/miR-146a.The OS of Low miR-30e/miR-146a group was significantly longer than High miR-30e/miR-146a group(P=0.022).(6)The proportion of CD8+ T cell in Low miR-146a group was significantly lower than that in High miR-146a group(P=0.016).The serum exression level of miR-146a was corelated with the proportion of CD8+ T cell(r=0.734,P=0.007).However,the proportion of cytotoxic lymphocyte subsets in Low miR-146a group has no significant difference with that in Low miR-146a group(P>0.05);the serum exression level of miR-30e has no corelation with the proportion of cytotoxic lymphocyte subsets(P>0.05).(7)The expression of perforin in cytotoxic lymphocyte subsets in Low miR-30e group has no significant difference with that in Low miR-30e group(P>0.05);the serum exression level of miR-30e has no corelation with the expression of perforin in cytotoxic lymphocyte subsets(P>0.05).ConclusionThe serum expression level of miR-146a and miR-30e in the newly diagnosed sHLH group was significantly decreased,which is important in the differential diagnosis of sHLH.Individuals with lower serum expression level of miR-146a and miR-30e were more likely to be sHLH patients.The ratio of miR-30e and miR-146a(miR-30e/miR-146a)can indicate the prognosis of sHLH patients.The prognosis of sHLH patients with lower miR-30e/miR-146a was better than that of sHLH patients with higher miR-30e/miR-146a.The serum exression level of miR-146a was corelated with the proportion of CD8+T cell.The serum exression level of miR-146a was corelated with the proportion of CD8+T cell.Therefore,miR-146a may play a role in the deficiency of CD8+T cell in the pathogenesis of sHLH.