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T. Suzuki et al.
S5 and S6). Without depletion, the absolute numbers of mature and immature neutrophils were comparable in steady-state BM, while the number of immature neu- trophils was greatly increased and the number of mature neutrophils decreased by G-CSF treatment (Online Supplementary Figure S6A). The vast majority of increased neutrophils in peripheral blood following G-CSF treat- ment were also immature neutrophils (Online Supplementary Figure S6B). With selective depletion of mature neutrophils, the number of immature neutrophils in the BM was greatly increased without G-CSF treatment and the mobilization of both immature neutrophils and HPC (LSK cells and CFU-C) by G-CSF was slightly decreased (Online Supplementary Figure S6A-H), with no significant alteration of BM PPARd mRNA (Online Supplementary Figure S6I). Thus, the cell population that mediated the increase of BM PPARd mRNA in response to G-CSF was not restricted to mature neutrophils.
in vitro using the neutrophil precursor cell line 32D. As
reported previously, BM is richly innervated with sympa-
expression of major downstream genes of PPARd signaling such as carnitine palmitoyltransferase-1α (Cpt1α) and angiopoietin-like protein 4 (Angptl4) in BM was significant- ly increased by GW501516, suggesting that the PPARd ago- nist worked directly in BM cells (Figure 4B). Conversely, the administration of the PPARd antagonist GSK3787 enhanced G-CSF-induced mobilization through the inhibition of PPARd signaling in BM cells (Figure 4C and D; Online Supplementary Figure S9B). Furthermore, chimeric mice gen- erated by the transplantation of BM cells from PPARd het- erozygous deficient mice into lethally irradiated WT mice showed significantly increased mobilization and lower mRNA expression of Cpt1α and Angptl4 in BM cells (Figure 4E and F; Online Supplementary Figure S9C). GW501516 also significantly inhibited the enhanced mobilization of CFU-C by the FFD (Figure 4G; Online Supplementary Figure S9D). These results suggest that PPARd signaling in BM cells is indeed a negative regulator of mobilization.
Certain ω3-fatty acids are PPARd ligands
We have previously reported an original method of
sampling BM in which lipids in the marrow can be stably and precisely evaluated.11 Using this method combined with LC-MS/MS, a series of ω3- and ω6-polyunsaturated fatty acids (PUFA) in BM were enumerated in mice fed with the ND or FFD in G-CSF mobilization. In Figure 5A, ω3-PUFA, such as eicosapentaenoic acid (EPA), docosa- hexaenoic acid (DHA), and their derivatives, were drasti- cally decreased by eight doses of G-CSF and/or FFD, whereas ω6-PUFA, including arachidonic acid and associ- ated pro-inflammatory lipid mediators, were unchanged (Figure 5B). These observations suggest that BM requires a continuous supply of ω3-fatty acids from diet, and that G- CSF treatment likely triggers strong consumption of ω3- fatty acids in BM. Indeed, similarly to the PPARd agonist GW501516, EPA- and DHA-induced PPARd signaling in 32D cells upregulated Cpt1α and Angptl4 mRNA expres- sion (Online Supplementary Figure S10A and B). This effect, particularly with EPA, was significantly inhibited by the PPARd antagonist GSK3787 (Online Supplementary Figure S10C). Among sorted BM myeloid cells, EPA, but not DHA, significantly upregulated PPARd mRNA expression in mature/immature neutrophils in vitro (Online Supplementary Figure S10D). EPA, and to a lesser extent also DHA, upregulated Cpt1α and Angptl4 mRNA expres- sion in these cells, and this effect was inhibited by GSK3787 (Figure 6A). These results suggest that EPA (and/or its metabolites) may be a functional fatty acid lig- and for PPARd in neutrophils and their precursors.
In concordance, EPA administration in vivo to normal mice partially attenuated the enhanced mobilization induced by a FFD (Figure 6B; Online Supplementary Figure S11A). We repeated the same experiment in chimeric mice with PPARd+/+ or PPARd+/- BM. Consistently, in PPARd+/+ BM chimera, EPA administration showed a trend to partial reduction in CFU-C mobilization (Figure 6C; Online Supplementary Figure S11B). In PPARd+/- BM chimera, how- ever, mobilization efficiency in the FFD condition was fur- ther enhanced, and this effect was greatly inhibited by EPA (Figure 6C; Online Supplementary Figure S11B). These results suggest that BM in the FFD condition still contains lipid mediators that function as PPARd ligands, and that EPA may also use pathways other than PPARd to inhibit mobilization.
In addition to myeloid cell fractions, the upregulation of --
PPARd mRNA was assessed in sorted BM CD45 Ter119 cells (non-hematopoietic [stromal] cells), LSK cells, B220+ B lymphocytes, and CD3+ T lymphocytes. These investi- gations suggested that some nonmyeloid cell fractions, such as stromal cells and T cells, might contribute partially to the increase of BM PPARd mRNA by G-CSF treatment (Online Supplementary Figure S7).
We assessed the signals that increase PPARd expression
thetic nerves that regulate mobilization via suppression
of the osteoblastic microenvironment through b2-AR
stimulation by catecholamines and a marrow lipid medi-
ator from mature neutrophils through b -AR stimula-
3
tion. The pan-b-AR agonist isoproterenol, but not G-
7-11
CSF, was an inducer of PPARd mRNA (Online
Supplementary Figure S8A). Among all three b-AR (b , b , 12
and b -AR) agonists, the b -AR agonist dobutamine reca- 31
pitulated the effect of isoproterenol, significantly increas-
ing PPARd mRNA, and the b -AR agonist clenbuterol also 2
showed a trend to induce PPARd mRNA, albeit to a lesser
extent (Online Supplementary Figure S8B). This observation
was further confirmed at the protein level by flow cyto-
metry (Online Supplementary Figure S8C). The increase of
PPARd mRNA by b /b -AR agonists was also confirmed 12
in sorted BM mature/immature neutrophils and mono- cytes/macrophages (Figure 3E).
Thus, marrow PPARd expression strongly correlates with mobilization efficiency and is enhanced mainly in myeloid cells, particularly in neutrophil lineage cells, by G-CSF-induced high sympathetic tone, likely through b1/b2-AR.
Marrow PPARd signaling negatively regulates mobilization efficiency
Because FFD-G-CSF resulted in the upregulation of both BM PPARd expression and mobilization efficiency (Figure 2E, orange dots), greater mobilization was likely achieved via reduced PPARd activity due to the lack of natural fat lig- ands in the BM. In other words, marrow PPARd signaling might be a negative regulator of mobilization. We next sought to explore whether the modulation of PPARd signal- ing regulates HPC mobilization. The administration of the PPARd agonist GW501516 inhibited G-CSF-induced mobi- lization with no alteration in BM HPC (Figure 4A; Online Supplementary Figure S9A). In G-CSF-treated mice, mRNA
Thus, a certain ω3-fatty acid, partially as a natural ligand
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