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. 2010 Nov;38(11):1036-1046.e1-4.
doi: 10.1016/j.exphem.2010.07.004. Epub 2010 Jul 18.

Interferon regulatory factor-8-driven myeloid differentiation is regulated by 12/15-lipoxygenase-mediated redox signaling

Michelle Kinder  1 , James E Thompson, Cong Wei, Suresh G Shelat, Ian A Blair, Martin Carroll, Ellen Puré
Affiliations

Interferon regulatory factor-8-driven myeloid differentiation is regulated by 12/15-lipoxygenase-mediated redox signaling

Michelle Kinder et al. Exp Hematol. 2010 Nov.

Abstract

Objective: Several transcription factors determine the cell fate decision between granulocytes and monocytes, but the upstream signal transduction pathways that govern myelopoiesis are largely unknown. Based on our observation of aberrant myeloid cell representation in hematopoietic tissues of 12/15-lipoxygenase (12/15-LOX)-deficient (Alox15) mice, we tested the hypothesis that polyunsaturated fatty acid metabolism regulates myelopoiesis.

Materials and methods: Multicolor flow cytometric analysis and methylcellulose assays were used to compare myelopoiesis and the differentiative capacity of progenitors from Alox15 and wild-type mice. Furthermore, we elucidated the mechanism by which 12/15-LOX is involved in regulation of myelopoiesis.

Results: Granulopoiesis in Alox15 mice is increased while monopoiesis is reduced. Moreover, there is an accumulation of granulocyte-macrophage progenitors that exhibit defective differentiation. Mechanistically, we demonstrate that transcriptional activity of interferon regulatory factor-8 (Irf8), which regulates myelopoiesis, is impaired in Alox15 progenitors and bone marrow-derived macrophages due to loss of 12/15-LOX-mediated redox regulation of Irf8 nuclear accumulation. Restoration of redox signaling in Alox15 bone marrow cells and granulocyte-macrophage progenitors reversed the defect in myeloid differentiation.

Conclusions: These data establish 12/15-LOX-mediated redox signaling as a novel regulator of myelopoiesis and Irf8.

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Conflict of interest statement

Conflict-of-interest disclosure

authors have no conflicting financial interests.

Figures

Figure 1
Figure 1
Enhanced granulopoiesis at the expense of monopoiesis of Alox15 progenitors in methylcellulose assays. (A) B6 and Alox15 BM cells were plated in methylcellulose in the presence of IL-3, IL-6, SCF and Epo. After 10 days, colonies were enumerated using light microscopy. Shown are the ratio of CFU-G/CFU-M and the numbers of types of identifiable colonies. (B) Cells from methylcellulose plates were recovered and analyzed for monocytic markers, Ly6C, F4/80 and CD115, and the granulocytic marker Ly6G by flow cytometry. (C) Wright-Giemsa stain from cytospin of representative B6 and Alox15 methylcellulose colonies. 20X magnification. (D) B6 and Alox15 BM cells were cultured in methylcellulose in the presence of GM-CSF. Shown are a summary of three independent experiments as a ratio of CFU-G/CFU-M and numbers of colonies CFU-M (black), CFU-G (checkered) and CFU-GM (white) (E) B6 and Alox15 BM cells were cultured in methylcellulose in the presence of M-CSF or G-CSF as indicated on the x-axis. Shown are numbers and phenotypes of colonies. n=6 in 3 independent experiments for all data shown. *p<.05, **p<.01. Error bars represent ± Standard Error of the Mean (SEM).
Figure 2
Figure 2
Accumulation of GMP in Alox15 mice. BM cells from 5–8 week (A), 12–16 week (B, E), 20+ week (C) and spleens from 12–16 week (D) old B6 and Alox15 mice were isolated and subjected to multicolor flow cytometric analysis to determine the relative frequency (A–D) and absolute number (E) of myeloid progenitors in tibias and fibulas of each mouse: LSK (Lin−Sca1+cKit+), CMP (Lin−Sca1−cKit+CD34+CD16/32lo), GMP (Lin−Sca1−cKit+CD34+CD16/32hi), and MEP (Lin−Sca1−cKit+CD34−CD16/32−) n=3 independent experiments for all data shown. *p<.05, **p<.01. Error bars represent ± SEM.
Figure 3
Figure 3
Alox15 GMP exhibit defective differentiation. B6 and Alox15 BM were isolated and sorted for myeloid progenitors. LSK (A), CMP (B) and GMP (C) were plated in methylcellulose in the presence of IL-3, IL-6, SCF and Epo. Shown are a summary of three experiments as colonies enumerated and the CFU-G/CFU-M ratio. (D) B6 and Alox15 GMP were plated in the presence of M-CSF or G-CSF in methylcellulose assays. n=6 in three independent experiments for all data shown*p<.05, **p<.01 Error bars represent ± SEM. (E) Diagram of GMP differentiation in Alox15 mice. There is an accumulation of Alox15 GMP which exhibit reduced monocyte differentiation and enhanced granulopoiesis.
Figure 4
Figure 4
Decreased Irf8 nuclear accumulation and transcriptional activity in Alox15 myeloid progenitors. (A) Decreased accumulation of Irf8 in nuclear lysates of Alox15 Lin−cKit+ myeloid progenitors determined by immunoblot. Shown are a representative experiment and a summary of 3 experiments as Irf8/Retinoblastoma protein (Rb) relative to wild-type (B) Immunoblot of total lysates from B6 and Alox15 Lin−cKit+ cells demonstrating decreased levels of Irf8. Shown are a representative blot and a summary of 3 experiments as Irf8/Actin relative to wild-type. (C) Real time analysis of Irf8 mRNA in B6 and Alox15 Lin−cKit+ cells (n=3 independent experiments). (D) Real time analysis of enriched B6 and Alox15 CMP and GMP demonstrate that Alox15 myeloid progenitors exhibit decreased expression of the Irf8-mediated transcripts Nf1 and Egr1 and similar levels of Pu.1, C/ebpα and Irf8. Gene expression was normalized by Gapdh. (n=3 independent experiments for all experiments shown). *p<.05, **p<.01. Error bars represent ± SEM.
Figure 5
Figure 5
ROS-signaling regulates Irf8 nuclear accumulation in Alox15 cells. (A) Alox15 BMM have decreased expression of Nf1 and similar levels of Irf8 compared to B6 by real time analysis normalized to Gapdh (n=3 independent experiments). (B–C) Total and nuclear lysates were isolated from B6 and Alox15 BMM and immunoblotted for Irf8 demonstrating (B) Alox15 exhibit comparable expression of total Irf8 protein but (C) decreased expression of nuclear Irf8 protein (n=3 independent experiments.) (D) Lipid product formation in B6 and Alox15 BMM after stimulation with 50 μM AA demonstrating decreased 12(S)HETE and increased 5(S)HETE in Alox15 BMM compared to B6. (n=4 in two independent experiments). (E) Flow cytometric analysis of B6 and Alox15 BMM loaded with the ROS sensitive dye H2DCFDA demonstrate decreased levels of ROS in Alox15 BMM. Shown are a representative experiment and a summary of 5 experiments. (F) Addition of 10 mM Tiron, a ROS scavenger, to B6 BMM overnight decreases Nf1 transcription while addition of 50 μM BSO, which increases ROS, to Alox15 BMM increases Nf1 transcription measured by real time analysis. n=3 independent experiments. (G) Modulation of ROS regulates Irf8 nuclear accumulation. Nuclear lysates were isolated from B6 BMM in the presence and absence of 10 mM Tiron and Alox15 BMM cultured alone or in the presence of 50 μM and 100 μM BSO overnight. Protein expression of IRF-8 was determined by immunoblot. Shown is an immunoblot from a representative experiment in which an irrelevant lane was removed and a summary of three experiments. *p<.05, **p<.01. Error bars represent ± SEM.
Figure 6
Figure 6
ROS-signaling rescues myeloid differentiation in Alox15 BM and GMP. B6 and Alox15 BM (A–B) and sorted GMP (C) were plated in methylcellulose in the presence and absence of 0.025 μM BSO. (A, C) Shown are colony number and ratio CFU-G/CFU-M. (B) The percentage of Ly6G+ cells were determined by flow cytometry. n=6 in three independent experiments for all data shown *p<.05, **p<.01. Error bars represent ± SEM.
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References

    1. Akashi K, Traver D, Miyamoto T, Weissman IL. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature. 2000;404:193–197. - PubMed
    1. Friedman AD. Transcriptional control of granulocyte and monocyte development. Oncogene. 2007;26:6816–6828. - PubMed
    1. Tamura T, Nagamura-Inoue T, Shmeltzer Z, Kuwata T, Ozato K. ICSBP directs bipotential myeloid progenitor cells to differentiate into mature macrophages. Immunity. 2000;13:155–165. - PubMed
    1. Koenigsmann J, Rudolph C, Sander S, Kershaw O, Gruber AD, Bullinger L, Schlegelberger B, Carstanjen D. Nf1 haploinsufficiency and Icsbp deficiency synergize in the development of leukemias. Blood. 2009;113:4690–4701. - PubMed
    1. Scheller M, Foerster J, Heyworth CM, Waring JF, Lohler J, Gilmore GL, Shadduck RK, Dexter TM, Horak I. Altered development and cytokine responses of myeloid progenitors in the absence of transcription factor, interferon consensus sequence binding protein. Blood. 1999;94:3764–3771. - PubMed

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