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T. Suzuki et al.
for BM PPARd, is a dietary component able to suppress mobilization.
PPARd-induced Angptl4 suppresses mobilization
As a downstream molecule of PPARd signaling, Angptl4 regulates blood vessel permeability leading to the modula- tion of cell migration, such as tumor metastasis.23,24 We first confirmed that G-CSF upregulated the level of Angptl4 protein in BM extracellular fluid, which showed a trend of further enhancement following treatment with the PPARd agonist GW501516 (Figure 7A). In contrast, the level of Angptl4 protein in the blood was not changed by G-CSF treatment (Online Supplementary Figure S12A). G- CSF treatment together with GW501516 significantly increased Angptl4 mRNA expression in BM myeloid cells (Online Supplementary Figure S12B). The analysis of BM samples from mice used in mobilization experiments with the FFD and GW501516, as shown in Figure 4G, revealed that Angptl4 and Cpt1α mRNA levels in BM after G-CSF were decreased by the FFD but greatly increased by GW501516 treatment (Online Supplementary Figure S12C). Thus, the induction and suppression of Angptl4 mRNA expression in BM were likely associated with the suppres-
sion and enhancement of mobilization, respectively. Indeed, this increase in Angptl4 protein in BM caused by G-CSF inhibited mobilization, because administration of the anti-Angptl4 neutralizing antibody (3F4F5)25 signifi- cantly increased mobilization efficiency, as assessed by CFU-C, with a similar, but not statistically significant, trend of increased LSK cell mobilization (Figure 7B; Online Supplementary Figure S12D). BM vascular permeability, as assessed by Evans blue dye incorporation in BM, was decreased by GW501516 and/or G-CSF (Online Supplementary Figure S13), and significantly enhanced by the addition of the anti-Angptl4 antibody to G-CSF (Figure 7C), suggesting that Angptl4 may inhibit mobilization by, at least partially, suppressing BM vascular permeability. Therefore, these results suggest that Angptl4, produced mainly by BM neutrophils and their precursors via PPARd
signaling, inhibits G-CSF mobilization.
Discussion
The functions of BM as a reservoir and consumer of orally ingested fat have not been thoroughly studied. In this study, we have demonstrated that BM fat is strongly influenced by diet. In particular, ω3-PUFA and their deriv- atives are almost exhausted by a 2-week restriction of fat contents in food. BM myeloid cells such as neutrophils and their precursors have a strong demand for ω3-PUFA, including EPA, which acts, at least partially, as a PPARd lig- and to suppress HSPC mobilization via Angptl4 produc- tion. A widely variable mobilization efficiency in response to G-CSF in healty individuals, including a certain percent- age of poor mobilizers, might partially originate from the BM fat profile in association with oral fat intake. Although it is not clear whether these findings in mice are applicable to mobilization in humans, the modulation of dietary fat might be a potential strategy to reduce the risk of poor mobilizers which could be examined in a future clinical study.
In this study, we demonstrated that neutrophils and their precursors, which are the major populations in BM, are strong consumers of ω3-PUFA, particularly after G-
CSF treatment. It was reported that dietary ω3-PUFA are rapidly incorporated into phospholipids, such as phos- phatidylethanolamine and phosphatidylcholine, of
26
ry responses, such as leukotriene B production and
human neutrophils
and inhibit these cells’ inflammato- 4
However, the signaling receptor for ω3- PUFA in this pathway is unclear. It was reported that cer- tain ω6-PUFA, 15d-PGJ2, acted as ligands for PPARγ to inhibit neutrophil chemotaxis by upregulating the sepsis- induced cytokines tumor necrosis factor-α and inter- leukin-4.29 Interestingly, a biochemical study has shown that 15d-PGJ2 can also stimulate PPARd to a similar mag- nitude as EPA.21 Based on our study in BM neutrophils and the reported strong interaction of EPA with PPARd,21,30 neutrophils in circulation may also partially utilize ω3-PUFA as PPARd ligands to diminish inflamma- tion. EPA is also reported to prevent neutrophil migration across the endothelium as a supplier of PGD , which
chemotaxis.
27,28
3 antagonizes PGD receptor DP-1 on neutrophils. This
pathway might be one of the PPARd-independent EPA functions in the suppression of mobilization. In our cur- rent study, BM lipid mediators were assessed after eight doses of G-CSF, and the transition during the G-CSF treatment was not evaluated. Although no change was observed in BM ω6-PUFA after eight doses of G-CSF, we have previously reported that the level of BM PGE2 was increased after four doses.11 These data are consistent with the transition of body temperature during G-CSF treatment, which increased after four doses and returned to normal levels at eight doses.11 Thus, the contribution of BM ω6-PUFA in mobilization cannot be excluded from the current study.
The signaling partners of the various PPAR are retinoid X receptors (RXR). PPAR-RXR are permissive het- erodimers that can be activated by either PPAR ligands or RXR ligands.32 It was reported that RXR is activated during G-CSF-induced granulopoiesis. The synthetic RXR agonist bexarotene enhanced G-CSF-induced mobilization of neu- trophils and CFU-C, but not of LSK cells, in circulation.20 In contrast, PPARd ligands in our study suppressed the mobilization of both LSK cells and CFU-C. This difference may be because apo-PPARd, i.e., the absence of ligand, has been shown to reside on DNA or function as a transre- pressor, unlike RXR.33 It is also possible that RXR may not be a major signaling partner of PPARd in BM neutrophils and their precursors with ω3-PUFA as PPARd ligands. Indeed, the promyelocytic leukemia-PPARd signaling pathway is important for HSC maintenance through the regulation of fatty acid oxidation and asymmetric divi- sion.34 Although the contribution of PPAR is not clear, a very high level of fatty acids is the critical component for the ex vivo maintenance of HSC.35 Thus, a continuous sup- ply of fatty acids from the food is critically important for the maintenance of BM hematopoiesis in several different ways. BM in patients with anorexia nervosa commonly displays hypoplasia with gelatinous transformation.36,37 This may be partially due to the lack of oral intake of fatty acids, including ω3-PUFA as PPARd ligands.
Hematopoietic Angptl4 deficiency in hyperlipidemic mice causes leukocytosis,38 which suggests a potential role of Angptl4 from hematopoietic cells in the cells’ intravasa- tion from the BM cavity into the circulation. Angptl4 is known to have two major distinct roles. First, the N-termi- nal coiled-coil region (nAngptl4) regulates lipoprotein lipase leading to the control of lipid metabolism, insulin
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