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. 2017 Nov 2;7(1):14953.
doi: 10.1038/s41598-017-15023-2.

Altered B-lymphopoiesis in Mice With Deregulated Thrombopoietin Signaling

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Free PMC article

Altered B-lymphopoiesis in Mice With Deregulated Thrombopoietin Signaling

Amanda E Au et al. Sci Rep. .
Free PMC article

Abstract

Thrombopoietin (TPO) is the master cytokine regulator of megakaryopoiesis. In addition to regulation of megakaryocyte and platelet number, TPO is important for maintaining proper hematopoietic stem cell (HSC) function. It was previously shown that a number of lymphoid genes were upregulated in HSCs from Tpo -/- mice. We investigated if absent or enhanced TPO signaling would influence normal B-lymphopoiesis. Absent TPO signaling in Mpl -/- mice led to enrichment of a common lymphoid progenitor (CLP) signature in multipotential lineage-negative Sca-1+c-Kit+ (LSK) cells and an increase in CLP formation. Moreover, Mpl -/- mice exhibited increased numbers of PreB2 and immature B-cells in bone marrow and spleen, with an increased proportion of B-lymphoid cells in the G1 phase of the cell cycle. Conversely, elevated TPO signaling in Tpo Tg mice was associated with reduced B-lymphopoiesis. Although at steady state, peripheral blood lymphocyte counts were normal in both models, Mpl -/- Eµ-myc mice showed an enhanced preneoplastic phase with increased numbers of splenic PreB2 and immature B-cells, a reduced quiescent fraction, and augmented blood lymphocyte counts. Thus, although Mpl is not expressed on lymphoid cells, TPO signaling may indirectly influence B-lymphopoiesis and the preneoplastic state in Myc-driven B-cell lymphomagenesis by lineage priming in multipotential progenitor cells.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Lymphoid lineage priming in Mpl −/− LSKs. (a) Hematopoietic stem and progenitor cells per femur relative to WT in 4–5 weeks old mice. WT (n = 8), Mpl −/− (n = 6) and Tpo Tg (n = 7). Mean ± SEM. Statistical significance was generated by Student’s unpaired t-test. *p < 0.05. (b) Barcode plot of the CLP gene signature in Mpl −/− and (c) Tpo Tg LSKs. Gene set tests were performed using gene set obtained for the CLP population from the Hemopedia atlas.
Figure 2
Figure 2
Lymphoid lineage priming in Mpl −/− bone marrow and spleen. (a) Bone marrow and (b) spleen cellularity of B-cell subsets in 4–5 week old mice. WT (n = 17), Mpl −/− (n = 16) and Tpo Tg (n = 15). (c-d) Ki-67/DAPI cell cycling assessment of B-cell subsets within bone marrow and spleen of 4–5 week old WT (n = 17), Mpl −/− (n = 17) and Tpo Tg (n = 13) mice. Statistical significance was generated by Student’s unpaired t-test. Mean ± SEM. *p < 0.05; **p < 0.005; ***p < 0.001
Figure 3
Figure 3
Increased lymphoproliferation in preneoplastic Mpl deficient mice. (a) Spleen weight (b) Lymphocyte (c) Red blood cell (RBC) and (d) platelet counts in 4–5 week old mice. n = 11–18 mice per genotype. Whiskers: Min to Max. (e) Bone marrow and (f) spleen cellularity and Pro-PreB1, PreB2, immature and mature B-cells in 4–5 week old mice assessed by flow cytometry. Data are presented as mean ± SEM. n = 11–18 mice per genotype. Statistical significance was generated by Student’s unpaired t-test. *p < 0.05; **p < 0.005; ***p < 0.001
Figure 4
Figure 4
Effects of TPO-mimetic on preneoplastic phase. 1-week-old Eμ-myc mice and their wild-type littermates were injected subcutaneously every 3 days with 100 μg/kg Romiplostim or saline vehicle. At 5 weeks of age (a) platelet and (b) lymphocyte counts (c) spleen cellularity, PreB2, and immature B-cells, and (d) bone marrow cellularity, ProB PreB1, PreB2 and Immature B-cells. n = 8–14 mice per group. Data are presented as mean ± SEM. Statistical significance was generated by Student’s unpaired t-test. *p < 0.05; **p < 0.005; ***p < 0.001. BM = bone marrow.
Figure 5
Figure 5
TPO delays B-cell lymphoma development in Eµ-myc mice. (a) Survival of Eµ-myc (n = 83), Mpl −/− Eµ-myc (n = 200), Tpo Tg Eµ-myc (n = 58), WT (n = 14), Mpl −/− (n = 11) and Tpo Tg (n = 13) mice. P-values were generated with Logrank (Mantel-Cox) test. (b) Spleen weight in terminally ill mice and 1-year old control mice. Eµ-myc (n = 47), Mpl −/− Eµ-myc (n = 59), Tpo Tg Eµ-myc (n = 56), WT (n = 5), Mpl −/− (n = 7) and Tpo Tg (n = 6). Whiskers: Min to Max. Statistical significance was generated by Student’s unpaired t-test. *p < 0.05; **p < 0.005; ***p < 0.001. (c) Bone marrow disease phenotype in terminally ill mice assessed by flow cytometry. Eµ-myc (n = 31), Mpl −/− Eµ-myc (n = 36) and Tpo Tg Eµ-myc (n = 25) mice.
Figure 6
Figure 6
Increased B-cell cycling in preneoplastic Mpl −/− Eµ-myc mice. Cell cycle status of (a) bone marrow and (b) spleen B-cell subsets in 4–5 week old Eµ-myc (n = 13), Mpl −/− Eµ-myc (n = 22) and Tpo Tg Eµ-myc (n = 13) mice assessed by Ki-67/DAPI staining profiles. Statistical significance was compared to Eµ-myc. Statistical significance was generated by Student’s unpaired t-test. Mean ± SEM. *p < 0.05; **p < 0.005; ***p < 0.001.

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