Mechanism Of Abnormal Neutrophil Function In Myelodysplastic Syndromes

Objective:In this study,we examined the chemotaxis,phagocytosis and reactive oxygen species(ROS)production in neutrophils from myelodysplastic syndrome(MDS)patients,as well as expression patterns of associated genes in MDS hematopoietic stem and progenitor cells(HSPCs)to explore the origin of aberrant neutrophil function.Furthermore,using a single-cell culture system,we studied the proliferation and differentiation defects of HSPCs from patients with MDS.These findings provide potential mechanisms for the granulocytic defects of MDS and might lead to effective therapeutic strategies for treating neutropenia and infections in MDS.Methods:The neutrophil chemotaxis assay was performed using TAXIScan-FL.Neutrophil phagocytosis of opsonized zymosan was assessed by confocal microscopy.ROS in neutrophils was measured by peroxidase-dependent luminol-amplified chemiluminescence.Gene Ontology(GO)-terms associated with neutrophil chemotaxis,phagocytosis and ROS were selected to reveal their expression patterns in HSPCs from MDS patients and controls.Fluorescence activated cell sorting was performed with a FACS AriaⅢ flow cytometer.Single hematopoietic stem or progenitor cell was sorted onto round bottom 96-well plates containing following human cytokines:stem cell factor(SCF,20ng/ml),FMS-related tyrosine kinase 3-ligand(Flt3-L,20ng/ml),interleukin-3(IL-3,20ng/ml),thrombopoietin(TPO,50ng/ml)with/without granulocyte colony stimulating factor(G-CSF,100ng/ml).The diameter of colonies and the morphology of cells was assessed to evaluate the proliferation and differentiation defects of HSPCs in MDS.Results:The absolute neutrophil count(ANC)of MDS patients was(2.25±1.53)x 109/L,which was lower than that in control(P<0.001).The LTB4-induced migration speed of neutrophils from MDS patients was 13.60μm/min and 26.40μm/min in controls,the migration speed of neutrophils in MDS was lower than that in control(P<0.001).The directionality of neutrophil migration was 0.85 for controls and 0.55 for patients with MDS(P<0.01).The upward directionality was distributed over a range of lower values for MDS cells than that for control cells(0.70 vs.1.30,P<0.01).The percentage of phagocytosis neutrophils was 79.50%for controls and 24.50%for patients with MDS(P<0.05).ROS production was significantly reduced in neutrophils from the MDS patient(by～50%,P<0.05).The phagocytosis and ROS were both impaired in neutrophils from MDS patients.The frequency of Lin-CD34+CD38-CD90-CD45RA-CD49f hematopoietic multipotent progenitor(MPP)within CD34+population was higher in MDS patients than controls(4.60%vs.0.97%,P<0.05).The percentages of Lin-CD34+CD38+CD135+CD45RA-common myeloid progenitor(CMP)within CD34+ population in the controls,low risk MDS,intermediate risk MDS and high risk MDS were 42.90%,17.90%,8.75%and 1.14%,respectively.The CMPs were significantly reduced in high risk MDS patients(P<0.05).In addition,the reduced frequency of Lin-CD34+CD38+CD135+CD45RA+ granulocyte-monocyte progenitor(GMP)within CD34+ population in intermediate risk MDS(P<0.01)or high risk MDS(P<0.001)was significant.The colonies of MPP harvested from patients with MDS were substantially smaller in size(P<0.05).The harvested cells from the MDS MPP condition were promyelocyte,while the control were morphologically myelocyte.The reduction of growth was increasingly significant in MDS GMPs over the course of cultivation(P<0.05).The diameter of colonies harvested from patients with MDS GMP were substantially smaller in size(566μm vs.847μm,P<0.05).The harvested cells from the MDS GMP were still myelocyte,and the control were already in metamyelocyte stage.Moreover,MDS GMPs were poor response to G-CSF.We then specifically focused on the expression of genes annotated to regulation of neutrophil function in the GMP compartment and discovered that about 70%of the MDS samples clustered together,demonstrating that these genes are dysregulated at the GMP stage.Conclusion:The expansion and differentiation capacities of HSPCs in MDS patients were impaired since the stage of MPPs which leads to granulocytic defects.The aberrant regulation of neutrophil functions might have occurred at the stage of GMPs.The defects in the GMP compartment of MDS patients might be responsible for the downstream neutrophil impairment in chemotaxis,phagocytosis and ROS production.