Study On The Effect And Mechanism Of Retinoid Agonist AM80-enhanced Neutrophil Bactericidal Activity Arising From Granulopoiesis

Objective:Neutropenia is a granulocyte disorder characterized by an abnormally low number of neutrophils. It is a potentially fatal complication of chemotherapy. The incidence of neutropenia among patients with solid tumors was10%. A study showed in-hospital mortality of neutropenia was9.5%. Despite advances in the prophylactic use of recombinant granulocyte colony-stimulating factor (G-CSF) to promote granulopoiesis of human hematopoietic stem cells (HSCs), there are no benefits consistently on reducing infection and infection-related mortality or all-cause mortality. In that case, additional study and investigation is desirable to enable further development of new and more therapeutic or convenient agents. Retinoic acid (RA) activated transcription factor RARa to regulate granulocytic differentiation of both leukemic myeloblasts and HSCs. AM80is a specific RARa agonist. An earlier study showed Am80is approximately10-fold more efficient, with lower toxicity, than RA to induce differentiation of acute promyelocytic leukemia(APL). Currently, Am80has been approved for the treatment of relapsed or refractory APL in Japan. First, we compared the ability of AM80and G-CSF to induce neutrophil differentiation and immunity development. Second, we evaluated the bactericidal activities of Am80-induced neutrophils (AIN) and G-CSF-induced neutrophils (GIN) either in vitro or in neutropenic mouse model. Last, we investigate the mechanism of the enhanced bacterial killing by AIN. Our findings herein suggest a molecular rationale for developing new therapy against neutropenia using Am80as a cost-effective treatment option. Methods and Results:We first compared the efficiencies of AM80, G-CSF and other retinoid agonists in mediating granulopoiesis from CD34+cells. The results showed that2.5nM of Am80(2.5nM) stimulated proliferation of CD34+cells while preventing cell death by the sixth day, which is similar to G-CSF (25ng/ml). By analysis of morphologic differentiation which is assessed by Giemsa staining, we found Am80is more effective than G-CSF in inducing granulocyte differentiation. Neutrophils induced for6days from CD34+cells by G-CSF or Am80were analyzed by transmission electron microscopy. The ultrastructural images showed that, compared with GIN, AIN contained increased numbers of primary and secondary-like granules at the segmented neutrophil level, though their numbers were considerably less than those in PBN. During neutrophil differentiation, heterogeneous populations of granule proteins are produced sequentially and stored in cytoplasm for first-line defense against different pathogens. We thus investigated whether Am80-enhanced neutrophil maturation is associated with increased granule production. We tested the degranulation ability of GIN, AIN on bacterial stimuli. Western blotting was used to determine the protein level of production and secretion of MPO, lactoferrin, LL37, MMP9. The results showed that MPO, lactoferrin, and LL-37are effectively produced and/or secreted by AIN but not GIN. Phagocytosis and bacterial killing experiment was used to evaluate the ability of GIN and AIN to kill Escherichia coli DH5a (E.coli DH5a) and Staphylococcus aureus (S.aureus). We found thst AIN possess significantly higher phagocytotic and bactericidal activities than do GIN. To confirm that AIN possess greater bactericidal activity than do GIN, we examined bacterial killing in situ using confocal microscopy combined with LIVE/DEAD BacLight Viability Kit. The results demonstrated that significantly more surviving E.coli were retained in GIN samples, where we found much less dead bacteria in contrast to observations in AIN or PBN samples. Furthermore, ultrastructural images of E.coli infection by electron microscope showed that numerous intact/surviving bacteria were retained in GIN; whereas similar to PBN, only a few intact/surviving bacteria were identified in AIN. To find the signaling regulation against bacterial infection which developed during neutrophil differentiation, we analyzed the coexpression level of CD66-CD11b and CD66-CD18. Flow cytometric analyses showed that coexpression of CD66a-CD18and CD66a-CD11b surface markers in GINs was significantly lower than in AINs or PBNs samples. To test whether higher levels of CD66-CD18signaling correlated with the significantly greater bactericidal activity of AIN, PBN versus GIN, we disrupted CD66-CD18signaling by neutralization of CD18function with anti-CD18antibody. The results showed that anti-CD18antibody neutralizes Am80-enhanced neutrophil bactericidal activity. Using a neutropenic mouse model induced by a single dose of cyclophosphamide, we tested whether in vivo mobilized neutrophils by Am80indeed possess the same greater neutrophil immunity against bacterial infection than those by G-CSF. The results demonstrated that neutrophils mobilized by Am80in neutropenic mice display greater bactericidal activity than those by G-CSF.Conclusion:These studies demonstrate that Am80is more effective than G-CSF in promoting neutrophil differentiation and bactericidal activities, probably through coordinating the functional interaction of CD66with CD18to enhance the development of neutrophil immunity during granulopoiesis. Our findings herein suggest a molecular rationale for developing new therapy against neutropenia using Am80as a cost-effective treatment option.