Page 170 - 2021_06-Haematologica-web

T. Suzuki et al.
Introduction
Granulocyte colony-stimulating factor (G-CSF) is
widely used in the clinic as a standard agent to induce
the mobilization of hematopoietic stem/progenitor cells
(HSPC) from bone marrow (BM) into the circulation. G-
CSF-mobilized HSPC are currently a major source of
cells for stem cell transplantation which is a curative
therapeutic option for intractable hematologic diseases.
According to the current understanding of the mecha-
nism of G-CSF-induced mobilization, in addition to the
cytokine’s pharmacological effect of expanding BM neu-
trophils, its neurotropic action through the G-CSF recep-
tor in the sympathetic nervous system (SNS) leads to the
suppression of macrophages that support HSPC niche
cell function,1-3 reduction of stromal cell synthesis of fac-
tors retaining HSPC in the BM, such as CXCL12,4-6 and
lipid mediators.18 Given that all these cells are packed at high density in the marrow, they may actively exchange many lipid mediators to stimulate each other. This unique situation in BM makes it difficult to precisely evaluate lipid mediators in BM by flushing it with phosphate- buffered saline (PBS) or following pipetting, which imme- diately changes lipid metabolic cascades. We have devel- oped a new procedure for sampling BM by flushing it directly with -20°C 100% methanol and preparing it for liquid chromatography-tandem mass spectrometry (LC- MS/MS) through which stable and precise evaluation of PGE2 in BM was achieved.11
suppression of osteolineage cells through b -adrenergic
CSF-induced mobilization. We found that mobilization efficiency can be enhanced by fat restriction in food. It also appeared that BM has a strong demand for certain ingested ω3-fatty acids, which function as ligands for peroxisome proliferator-activated receptor d (PPARd) in BM mature/immature neutrophils to suppress mobiliza- tion, at least partially, by regulating BM vascular perme- ability.
Methods
Mice
Mice were cared for in the Institute for Experimental Animals, Kobe University Graduate School of Medicine. PPARd-/- mice were generated on a C57BL/6 background as described in the Online Supplementary Methods. Because all PPARd-/- mice died in utero, PPARd+/+ and PPARd+/- littermates at ages 6 to 8 weeks were used as transplant donors to generate chimeric mice. C57BL/6- CD45.1 congenic mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and used at ages 6 to 8 weeks. Wild-type (WT) C57BL/6 mice at ages 6 to 8 weeks were purchased from CLEA Japan (Chiba, Japan) and used for experi- ments after 2 weeks of acclimatization unless otherwise indicat- ed. Male mice were used in all experiments. Mice were fed with a normal diet (ND; CE-2, CLEA Japan) consisting, on average, of 4.69% fat, 24.90% protein, and 51.00% carbohydrates, yielding a total calorie content of 3.45 kcal/g, except for fat-free diet (FFD; CLEA Japan) experiments. The FFD consisted of 0.72% fat, 17.60% protein, and 63.49% carbohydrates by weight, yielding the same total calorie value as the ND, and was started at ages 8 to 10 weeks after 2 weeks of acclimatization. Animals were maintained under specific pathogen-free conditions and on a 12 h light/12 h dark cycle. All animal studies were approved by the Animal Care and Use Committee of Kobe University.
Statistical analysis
All data were pooled from at least three independent experi- ments. All values were reported as the mean ± standard error of the mean (SEM). The statistical analyses were conducted using a two-tailed unpaired Student t-test, the Mann-Whitney U test, a one-way analysis of variance (ANOVA) test with the Tukey post-hoc procedure, and Pearson correlation coefficient. No sam- ples or animals were excluded from the analyses. Animals were randomly assigned to groups. Statistical significance was assessed with Prism (GraphPad Software, San Diego, CA, USA) and defined as P<0.05.
Detailed descriptions of the methods for the other procedures are provided in the Online Supplementary Methods.
receptors (b -AR),
HSPC from2 the microenvironment rather than their expansion or active migration. Besides the mechanism of mobilization itself, two unfavorable clinical events in G- CSF-induced mobilization have long remained unex- plained and unsolved since the clinical application of G- CSF for mobilization. First, donors/patients treated with G-CSF often complain of low-grade fever and bone pain, which can be relieved by the administration of non- steroidal anti-inflammatory drugs. Second, mobilization efficiency is widely variable, and 10% to 20% of healthy donors are poor mobilizers, such that the number of HSPC that can be harvested is insufficient for transplan- tation. As an explanation of the former problem, we have reported that low-grade fever (and likely bone pain) associated with the administration of G-CSF is due to prostaglandin E (PGE ) production from mature BM neu-
7-10
2
leading to the passive release of
trophils stimulated by the SNS through b -AR. However, our understanding of the latter problem remains unacceptably inadequate. Poor mobilization is a particularly serious problem for healthy donors for allo- geneic transplantation in the National Marrow Donor Program because they receive a certain dose of G-CSF without expected volunteer contribution to the patients. The wide range of mobilization efficiency, which occurs even in genetically identical mice, is currently unpre- dictable and uncontrollable. Mobilization efficiency may be partially determined by a balance between mobiliza- tion-promoting signals, such as SNS-mediated osteolin- eage suppression, and counteraction to mobilization, such as PGE2 from neutrophils to support osteoblast activity.11 Thus, it is clinically essential to elucidate the pathways that counteract mobilization during G-CSF treatment.
Analysis of lipid mediators in the BM lags behind that of other lipid-rich organs such as the liver and brain.12,13 BM fat has been suggested to modulate hematopoiesis.14,15 Evidence of lipid mediators of hematopoietic cells as inflammatory/resolving cells is accumulating.16,17 However, a precise evaluation of total BM fat contents had not been done before our previous report on PGE2.11 In addition to fat cells, the BM contains an enormous number of inflam- matory cells, such as neutrophils, macrophages, and their precursors, which are constantly stimulated by many mar- row factors on their way to maturation and peripheraliza- tion throughout the body. Red blood cells and their pre- cursor erythroblasts could also be a significant reservoir of
22
11 3
In this study, we have applied this original method for
including not only ω6-fatty acids/proinflammatory lipid
the comprehensive analysis of marrow fat components,
mediators such as PGE but also ω3-fatty acids during G- 2
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haematologica | 2021; 106(6)