| ObjectiveCCIN is the most common adverse effect of chemotherapy, which reduces immunity of patients and increases the susceptibility to microbial infections. Severe CCIN patients will die of hospital-acquired infections. GCSF is a prime drug used for treatment of CCIN. However, GCSF-induced neutrophils from hematopoietic stem cells during granulopoiesis are poorly differentiated with impaired microbicidal functions. Thus, although GCSF can profoundly increase the number of immature neutrophils to reduce neutropenia duration, CCIN remains a devastating challenge with substantial infection-morbidity and mortality. As such, alternative modes of treatment are needed for CCIN. Tamibarotene (Am80), an analogue of retinoic acid, which is specific for RARa and RAR?, is used for curing APL patients. Recent research shows that differentiated neutrophils from HSC, which are driven by Am80, possess great bactericidal activity in vitro. There is no study revealing the capacity of neutrophil regeneration of Am80 and antimicrobial function of the regenerated neutrophils in vivo. And there is no evidence that the granulocytes regenerated by Am80 can meet the demand of CCIN patients for the neutrophils with high quantity and quality. Using different CCIN mice models, our study firstly tested the activities of Am80, GCSF or Am80-GCSF on regenerated neutrophils fighting against S. Aureus infection. We secondly compared the capability of Am80, GCSF or Am80-GCSF on inducing neutrophil differentiation from granulocytic precursors, evaluated the bactericidal abilities of those cells and investigated the underlying mechanism. At last, we assessed the effects of Am80, GCSF or Am80-GCSF on proliferation and differentiation in primary AML cells. Here, we are trying to develop a more effective treatment for CCIN, that Am80 efficiently differentiates GCSF-induced granulocytic precursors into mature neutrophils that can fight bacterial infection in mice with CCIN.Methods1) In neutrophil-decrease stage, the effect of Am80-GCSF on bactericidal activity of neutrophils in CCIN mice.C57BL6/J mice were used in neutrophil-decrease stage tests. Normal C57BL6/J mice were injected with 200 mg/kg cyclophosphamide (CPA) to induce CCIN. Mice were treated with different doses of Am80 and/or GCSF for 3 days. Doses for GCSF were 250,50,25 ?g/kg, and doses for Am80 were 5,1,0.5 mg/kg. On day 2, S. Aureus was used to cause transiently infection by intraperitoneal (ip) or intravenous (iv) injection for up to 16 hrs. The number of white blood cells (WBC) and neutrphils were detected by Vetscan HM5. Bacterial killing experiments were performed on the peritoneal washing fluid, peripheral blood (PB), spleen or heart. Counting of monocolonal formatting units of bacteria on blood agar plates was used for detecting bacteria killing function of neutrophils. The Ficoll and gradient Percoll solutions were used for separation of neutrophils from PB and bone marrow (BM). Neutrophil numbers were analyzed by trypan blue staining and hemocytometer counting, and morphological differentiation was detected by Wright-Giemsa staining. The Student's unpaired two-tailed t-test was used to statistically analyze.2) In neutrophil-recovery stage, the effect of Am80-GCSF on bactericidal activity of neutrophils in CCIN miceC57BL6/J mice were used in neutrophil-recovery stage tests. Normal C57BL6/J mice were injected with 200 mg/kg CPA to induce CCIN on day 0. Mice were treated with 0.5 mg/kg Am80 and/or 25 ?g/kg GCSF from day 2 to day 4. On day 4,S. Aureus was used to cause transiently infection by iv injection for up to 16 hrs. The number of WBC and neutrphils were calculated by Vetscan HM5. Bacterial killing experiments were performed in PB with 3 h or 16 h infection. Counting of monocolonal formatting units of bacteria on blood agar plates was used for testing bacteria killing function of neutrophils. Ficoll and gradient Percoll solutions were used for separation of neutrophils from PB and bone marrow (BM). Neutrophil numbers were analyzed by trypan blue staining and hemocytometer counting, and morphological differentiation was detected by Wright-Giemsa staining. The Student's unpaired two-tailed t-test was used to statistically analyze.3) The effect of Am80-GCSF on reducing the infection-related mortality of CCIN mice.C57BL6/J mice were used in survival tests. Normal C57BL6/J mice were injected with 200 mg/kg CPA to induce CCIN on day 0. Mice were treated with Am80 and/or GCSF from day 0 or day 1. Doses for GCSF were 50 and 25 ?g/kg, and doses for Am80 were 1 and 0.5 mg/kg. Administration was performed up to day 8. On day 3, S. Aureus was used to cause perpetual systemic infection by iv injection. The survival rate was calculated on day 9. Body weights were recorded every two days, and spleens were collected from dead mice for analyzing. The survival mice on day 9 were infected for second time, in aim to detect the phagocytosis and bacteria clearance of neutrophil with Gram stain analysis. Transmission electron microscopy analysis was applied for demonstrating ultrastructure of mouse PB neutrophils on day 11. Ficoll and gradient Percoll solutions were used for separation of neutrophils from PB and bone marrow (BM). Neutrophil numbers were analyzed by trypan blue staining and hemocytometer counting, and morphological differentiation was detected by Wright-Giemsa staining. The Student's unpaired two-tailed t-test, the Kaplan-Meier plots and the Log-rank test were used to statistically analyze.4) The role of Am80-GCSF in neutrophil regeneration.Human hematopoietic stem cells CD34+ cells and acute promyelocytic leukemia cell line NB4 were used in this section.0.5 ?mol/L RA,25 ng/mL GCSF and/or 2.5 nmol/L Am80 were applied on CD34+cells, while 1 ?mol/L RA,25 ng/mL GCSF and/or 2.5 nmol/L Am80 were used for NB4 cells. Trypan blue staining and hemocytometer counting were used to calculate the survival ratio and to monitor the cell growth. Wright-Giemsa staining was used for the detection of nuclear morphology. Bacteria killing tests were applied for evaluating neutrophil bactericidal activities. With the stimulation of fMLP or PMA, Luminol-chemiluminescence Assay (LCL Assay) was applied for detection of neutrophil ROS production. qRT-PCR assay was used to analyze mRNA levels, such as C/EBPa, C/EBP?, CEBP?, RAR?2, CD18, p21Cip/Kip, CD66a, CD66b, CD66c and so on. Flow cytometry was performed to analyze the co-expression of cell surface antigen CD66-CD18. The Student's unpaired two-tailed t-test was used to statistically analyze.5) The effect of Am80-GCSF on primary AML specimens.Primary acute myeloid leukemia bone marrow cells were used in this section.25 ng/mL GCSF and/or 20,50,150 nmol/L Am80 were applied in primary cells. Ficoll solution was used for the separation of mononuclear cells from specimens. Trypan blue staining and hemocytometer counting were used to calculate cell survival ratio and to monitor cell growth. Wright-Giemsa staining was used for the detection of nuclear morphology. Bacteria killing tests were applied to evaluate neutrophil bactericidal activities. With the stimulation of fMLP or PMA, LCL Assay was applied for detection of neutrophil ROS production. qRT-PCR assay was used to analyze mRNA levels, such as CEBP?, RAR?2, CD11b, and CD66c. The Student's unpaired two-tailed t-test was used to statistically analyze.Results1) In neutrophil-decrease stage, Am80 alone improved immunity against S. Aureus in CCIN mice.In neutrophil-decrease stage, we tested whether different doses of GCSF, Am80 or Am80-GCSF could effectively differentiate granulocytic precursors into functional neutrophils in CCIN mice with S. Aureus infection. The results showed that no matter which dose we tested in experiments, Am80 couldn't produce a good number of neutrophils, but still significantly decreased the amount of live S. Aureus. It suggested that Am80-enhanced bacterial killing could be the result of Am80-induced effective differentiation of existing granulocytic precursors into functional neutrophils.2) In neutrophil-recovery stage, Am80-GCSF combination induced enough number of functional neutrophils in CCIN mice to fight against S. Aureus.Results obtained from neutrophil-recovery stage of CCIN mice experiments showed that Am80 had low effect on regeneration of granulocytes, while GCSF produced numerous neutrophils with uncompleted differentiation. However, smilar with Control group, neutrophils in Am80-GCSF mice possessed great bacterial killing activities. Data analysis of neutrophils from bone marrow and peripheral blood revealed that, Am80-GCSF induced enough number of well-differentiated neutrophils production in CCIN mice. It implied that Am80-GCSF coordinated myeloid expansion with granulocytic differentiation to generate large amount of functional neutrophils that reduced infection of CCIN mice.3) Am80-GCSF reduces infection-related mortality of CCIN mice.Survival tests showed that Am80-GCSF reduced infection-related mortality, and prolonged survival time of CCIN mice; but neither Am80 nor GCSF had such therapeutical effects as Am80-GCSF did. By examining myeloid production and neutrophil differentiation in survived CCIN mice, we found that the density of GCSF-induced BM myeloid cells was different from Control cells. Further ultrastructural analysis of newly generated PB neutrophils showed that, in clear contrast to neutrophils from Control or Am80-GCSF mice, neutrophils from GCSF mice displayed cellular degeneration. There were 76.9% cells showing variably sized intracytoplasmic vacuoles and 27.5% cells displaying inner nuclear membrane dilation or separation from outer nuclear membrane. Meanwhile, GCSF cells had fewer granules than Am80 and Control cells. Moreover, the increased spleen size, as well as neutrophil number in Am80-GCSF mice on day 9, dropped to the level similar to those in Control mice on day 13. Altogether, these results demonstrated that Am80-GCSF synergizes myeloid expansion with effective granulocytic differentiation to generate sufficient numbers of functional neutrophils without causing myeloid overexpansion.4) Neutrophil regeneration induced by Am80-GCSF.We compared granulocytic differentiation of RA, Am80 and GCSF in CD34+ and NB4 cells. The results revealed that RA didn't induce CD34+ cells to produce ROS with fMLP stimulation, while GCSF wasn't able to promote NB4 cells differentiation and ROS production. However, Am80 did improve granulocytic differentiation and ROS production on both CD34+ and NB4 cells.To test the effect of Am80-GCSF on regeneration of neutrophils, we tested 2.5 nmol/L Am80,25 ?g/mL GCSF and Am80-GCSF combination in CD34+ cells. Am80-GCSF didn't inhibit proliferation of CD34+ cells, but induced significantly larger amount of neutrophils with better morphologic differentiation (differentiated cells ratio was 32.8%), increased bacterial killing, as well as enhanced ROS production stimulated with neutrophil activator fMLP or PMA. Results obtained from qRT-PCR revealed that, compared to Am80 in the early and late differentiation induction stages, Am80-GCSF induced significantly higher expression of RA-target genes. In addition, transcription of CD66a and CD66b is significantly upregulated on day 3. Further flow cytometry analysis illustrated that in Am80-GCSF group, the pecentage of cells with co-expression of CD66 and CD18 surface markers was 47%. These results demonstrated that Am80-GCSF generated significantly larger amount of functional neutrophils from CD34+ cells which was mediated by C/EBP??C/EBPs and RAR?2. The significantly increased co-expression of CD66-CD18 induced by Am80-GCSF accelerated the development of neutrophil innate immunity.Proliferation test showed Am80-GCSF inhibited NB4 growth, and promoted granulocytic differentiation ratio from 20% to higher than 50%. Enhanced ROS production stimulated with fMLP or PMA appeared in Am80-GCSF treated cells. qRT-PCR data demonstrated that compared to Am80, Am80-GCSF induced significantly higher expression of RA-target genes in the early differentiation induction stages. This suggested GCSF took part in up-regulating mRNA of RA-target genes in improving the activity of Am80-GCSF.5) The influence of Am80-GCSF in primary AML specimens.20 nmol/L and 50 nmol/L of Am80,25 ?g/mL GCSF or combinations were used to treat mononuclear cells isolated from primary AML specimens. Compared to GCSF promoting or sustaining cell proliferation, Am80-GCSF inhibited cell growth. The data from Luminol-chemiluminescence assay demonstrated that Am80-GCSF enhanced ROS production. Results obtained from qRT-PCR displayed Am80-GCSF significantly upregulated transcription of RAR?2?C/EBPs and CDllb, and sustained those mRNAs at a high level.We further evaluated 150 nM Am80 in suppressing proliferation and inducing functional granulocytic differentiation when combined with GCSF. With treatment for up to 12 days, both Am80 and Am80-GCSF inhibited leukemic growth on #S062615 cells. But Am80-GCSF promoted more granulocytic differentiation than other groups. Moreover, Am80-GCSF promoted the highest neutrophil ROS production, bacterial killing, RA-target gene expression, and morphologic differentiation at day 12. These results implied the regulation of proliferatin and granulocytic differentiation by Am80-GCSF could be related to upregulation transcriptional level of RA-target genes in primary AML cells.ConculsionOur study revealed Am80 together with GCSF synergistically promoted neutrophil differentiation in vivo and in vitro. Am80 promoted effective granulocytic differentiation, but hardly induced HSC proliferation; while GCSF produced a great number of neutrophils, which were deficient in fighting bacterial infection. Am80-GCSF combination promoted both myeloid expansion of HSC and effective differentiation of neutrophils, meeting the demand for fighting microbial infection in CCIN. The underlying mechanism of the neutrophil regeneration induced by Am80-GCSF was probably mediated by RAR?2?C/EBP? and C/EBP?. Well-developed CD66-CD18 signaling contributed to the immune function of Am80-GCSF induced neutrophils. Thus, our findings suggested a novel Am80-GCSF synergistic therapy for effective treatment of CCIN, as Am80 sufficiently differentiated GCSF-induced granulocytic precursors into mature neutrophils to reduce infection and infection-related mortality. |
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