Impact of the <i>c-Myb^E308G</i> mutation on mouse myelopoiesis and dendritic cell development

<div><p><i>Booreana</i> mice carrying the <i>c-Myb</i>^<i>308G</i> point mutation were analyzed to determine changes in early hematopoiesis in the bone marrow and among mature cells in the periphery. This point mutation led to increased numbers of early hematopoietic stem and progenitor cells (HSPCs), with a subsequent reduction in the development of B cells, erythroid cells, and neutrophils, and increased numbers of myeloid cells and granulocytes. Myelopoiesis was further investigated by way of particular subsets affected. A specific question addressed whether <i>booreana</i> mice contained increased numbers of dendritic-like cells (L-DC subset) recently identified in the spleen, since L-DCs arise <i>in vitro</i> by direct differentiation from HSPCs co-cultured over splenic stroma. The non-lethal <i>c-Myb</i> mutation in <i>booreana</i> mice was associated with significantly lower representation of splenic CD8^- conventional dendritic cells (cDCs), inflammatory monocytes, and neutrophils compared to wild-type mice. This result confirmed the bone marrow origin of progenitors for these subsets since <i>c-Myb</i> is essential for their development. Production of L-DCs and resident monocytes was not affected by the <i>c-Myb</i>^<i>E308G</i> mutation. These subsets may derive from different progenitors than those in bone marrow, and are potentially established in the spleen during embryogenesis. An alternative explanation may be needed for why there was no change in CD8⁺ cDCs in <i>booreana</i> spleen since these cells are known to derive from common dendritic progenitors in bone marrow.</p></div

Analysis of bone marrow progenitors in <i>booreana</i> (<i>boo/boo</i>) and WT mice.

<p>Bone marrow from adult mice was prepared and stained with antibodies to distinguish hematopoietic progenitors flow cytometrically. A lineage cocktail of antibodies was used to gate Lin^- subsets, and Sca-1 and c-kit staining used to identify the Lin^-c-kit⁺Sca-1⁺ (LSK) subset. Staining for Flt3 and CD150 was used to distinguish LT-HSCs and MPPs, and staining for IL-7R and Flt3 was used to distinguish CLPs. Propidium iodide (PI; 1 μg/ml) staining was used to gate live cells as PI^-. Gates were set on bivariate plots using isotype control antibodies, and numbers in gates reflect % positive cells amongst Lin^-PI^- bone marrow cells. <b>(A)</b> Staining profiles for representative individual mice are shown. <b>(B)</b> Percent cells amongst viable Lin^- bone marrow is shown for 3 individual mice, with mean shown by a bar and statistically significant results (p ≤ 0.05) boxed in red.</p

<i>Booreana</i> (<i>boo/boo</i>) mice have increased numbers of dendritic cell progenitors in bone marrow.

<p>Cells from <i>boo/boo</i> and WT mice were prepared and stained with antibodies to distinguish macrophage dendritic cell progenitors (MDP) and common dendritic cell progenitors (CDP) flow cytometrically. A lineage cocktail of antibodies was used to gate the Lin^- cells and so exclude mature cells. Sca-1 and c-kit staining was used to identify the Lin^-c-kit^hiSca-1^- and Lin^-c-kit^loSca-1^- subsets, and staining for Flt3 and CD115 used to distinguish MDP and CDP amongst these subsets. Propidium iodide (PI; 1 μg/ml) staining was used to gate live cells (PI^-). Gates were set on bivariate plots using isotype control antibodies, and numbers in gates reflect % positive cells amongst Lin^-PI^- bone marrow cells. <b>(A)</b> Profiles for representative individual mice are shown. <b>(B)</b> Percent cells amongst viable Lin^- bone marrow is shown for 3 individual mice, with mean value shown as a bar, and statistically significant results (p ≤ 0.05) boxed in red.</p

<i>Booreana</i> (<i>boo/boo</i>) mice have increased numbers of dendritic cell progenitors in bone marrow.

<p>Cells from <i>boo/boo</i> and WT mice were prepared and stained with antibodies to distinguish macrophage dendritic cell progenitors (MDP) and common dendritic cell progenitors (CDP) flow cytometrically. A lineage cocktail of antibodies was used to gate the Lin^- cells and so exclude mature cells. Sca-1 and c-kit staining was used to identify the Lin^-c-kit^hiSca-1^- and Lin^-c-kit^loSca-1^- subsets, and staining for Flt3 and CD115 used to distinguish MDP and CDP amongst these subsets. Propidium iodide (PI; 1 μg/ml) staining was used to gate live cells (PI^-). Gates were set on bivariate plots using isotype control antibodies, and numbers in gates reflect % positive cells amongst Lin^-PI^- bone marrow cells. <b>(A)</b> Profiles for representative individual mice are shown. <b>(B)</b> Percent cells amongst viable Lin^- bone marrow is shown for 3 individual mice, with mean value shown as a bar, and statistically significant results (p ≤ 0.05) boxed in red.</p

Peripheral blood analysis of chimeras reconstituted with <i>booreana</i> fetal liver.

<p>Chimeras were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176345#pone.0176345.g001" target="_blank">Fig 1</a>. At 16-weeks post-transplant, blood composition was analyzed. Individual data points are shown for <i>boo/boo</i> and WT chimeras, with the mean value indicated by a horizontal bar. All comparisons between equivalent <i>boo/boo</i> and wild type chimeras were significantly different (p ≤ 0.05), except for reticulocyte counts in chimeras reconstituted with 1 x 10⁶ cells. HGB = hemoglobin, HCT = hematocrit, RBC = red blood cells, WBC = white blood cells. Significantly different data sets are shown by *.</p

Analysis of splenic myeloid and DC subsets in <i>booreana</i> versus WT mice.

<p><b>(A)</b> Chimeras were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176345#pone.0176345.g001" target="_blank">Fig 1</a> and sacrificed at 52 weeks. The total splenic dendritic and myeloid subset was purified via red blood cell lysis and T/B cell depletion. Cells were stained with antibody to detect CD45.2⁺ cells, and the population gated as CD11b⁺ and/or CD11c⁺ to estimate % dendritic/myeloid cells, respectively. Data represent mean ± SE (<i>n</i> = 4), and <i>p</i> values for statistically significant pairs of data shown. <b>(B)</b> Spleens of adult <i>booreana</i> and WT mice were enriched for DC and myeloid cells via red blood cell lysis and T/B cell depletion. Cells were then stained with antibodies to delineate subsets of CD8⁺ cDCs (CD11c^hiCD11b^-MHCII⁺CD8⁺), CD8^- cDCs (CD11c^hiCD11b^loMHCII⁺CD8^-), L-DCs (CD11c^loCD11b^hiLy6C^-Ly6G^-), resident monocytes (resi mono: CD11b^hiCD11c^lo/-Ly6C⁺Ly6G^-), inflammatory monocytes (infl mono: CD11b^hiCD11c^-Ly6C^hiLy6G^-), and neutrophils (neu: CD11b^hiCD11c^-Ly6C⁺Ly6G⁺). The proportional representation of each subset was calculated relative to the total dendritic and myeloid population in spleen (i.e. CD11b⁺ and/or CD11c⁺ cells, respectively). Data (mean) are shown for <i>boo/boo</i> (<i>n</i> = 3) and WT (<i>n</i> = 4) mice. Statistically significant findings (<i>p</i> ≤ 0.01) are boxed and <i>p</i> values are shown.</p

Hematopoiesis due to <i>booreana</i> (<i>boo/boo</i>) and WT bone marrow in co-cultures.

<p>Bone marrow was prepared from one-year old chimeras described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176345#pone.0176345.g001" target="_blank">Fig 1</a>. Donor-derived CD45.2⁺ bone marrow cells were sorted as a Lin^- population of <i>boo/boo</i> or WT origin, and equal numbers co-cultured over 5G3 splenic stroma. Individual co-cultures were established from 4 <i>boo/boo</i> chimeras and 2 WT chimeras. Cell production was monitored over time by staining non-adherent cells using antibodies to CD11c, CD11b, MHC-II, and CD8α, or with isotype controls. Propidium iodide (PI; 1 μg/ml) staining was used to gate live cells (PI^-). Gates were set on bivariate plots using isotype control antibodies, and numbers shown in gates reflect % positive cells. <b>(A)</b> Data shows staining of 21 day co-cultures established from one of each chimera type. L-DCs were gated as CD11c^loCD11b^hiMHC II^-CD8α^-B220^- cells, and cDC-like cells as CD11c^hiCD11b^loMHC II⁺CD8α^-B220^-. <b>(B)</b> Cell production was calculated and shown as mean ± SE for co-cultures established from <i>boo/boo</i> (<i>n</i> = 4) and wild-type (WT) (<i>n</i> = 2) chimeras. Significantly different data pairs are indicated.</p

<i>In vivo</i> reconstitution potential of <i>booreana</i> fetal liver HSCs.

<p>Chimeras were generated to investigate the <i>in vivo</i> reconstitution ability of <i>booreana</i> (<i>boo</i>) versus WT fetal liver. Analyses involved a quantitative and kinetic study of the lineage potential of 1 x 10⁶ (1M) or 2 x10⁵ (200K) fetal liver cells from <i>boo/boo</i> and WT mice of CD45.2 origin transplanted into lethally irradiated CD45.1 mice. Donor cell reconstitution was assessed via flow cytometric analysis of subsets in peripheral blood of chimeras at 8 and 16 weeks post reconstitution. The proportional representation of T cells (CD3⁺), B cells (B220⁺), myeloid cells (CD11b⁺) and granulocytes (Gr1⁺) was measured within the donor-derived (CD45.2) leukocyte compartment of peripheral blood. Individual data points are shown, with mean value indicated by a horizontal bar. All comparisons between equivalent <i>boo/boo</i> and wild type chimeras were significantly different (p ≤ 0.05) except for T cells at 8 weeks, and macrophages and granulocytes at 16 weeks. More complete data at 4, 8, 12 and 16 weeks are summarised in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176345#pone.0176345.s003" target="_blank">S3 Fig</a>. Significantly different data sets are shown by *.</p

Changes in hematopoiesis attributable to the <i>c-Myb</i>^<i>308G</i> mutation.

<p>The summary diagram combines analysis of stem/progenitor subsets in mutant mouse bone marrow, with subset analysis of mature cell subsets in blood and spleen of chimeras reconstituted with mutant fetal liver cells and analysed at 16 weeks after reconstitution. Significant changes in cell production in relation to wild type mice or wild type control chimeras are shown in red indicating increase, or blue indicating a decrease, in cell production. Subsets unchanged in mutant and wild type are shown in grey. The diagram is not a complete lineage map since only subsets analysed are shown. These include: LT-HSC, longterm hematopoietic stem cell; MPP, multipotential progenitor; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; MDP, myeloid dendritic progenitor; CDP common dendritic progenitor; MEP, myeloid/erythroid progenitor; GMP, granulocyte/macrophage progenitor. The lineage relationship between the MDP subset and CMP and CDP is shown as a dashed line since it is still in doubt [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176345#pone.0176345.ref045" target="_blank">45</a>]]. Similarly, the derivation of L-DC directly from bone marrow HSC and MPP is shown as a dashed line, since this has only been confirmed <i>in vitro</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176345#pone.0176345.ref028" target="_blank">28</a>].</p

<i>Booreana</i> (<i>boo/boo</i>) mice show a reduction in myeloid progenitors in bone marrow.

<p>Cells from <i>boo/boo</i> and WT mice were prepared and stained with antibodies to distinguish hematopoietic progenitors flow cytometrically. A lineage cocktail of antibodies was used to gate the Lin^- subsets, so excluding mature cells. Sca-1 and c-kit staining was used to identify the Lin^-c-kit⁺Sca-1⁺ subset. Staining for CD34 and CD16/32 was used to distinguish CMPs, GMPs, and MEPs. Propidium iodide (PI; 1 μg/ml) staining was used to gate live cells (PI^-). Gates were set on bivariate plots using isotype control antibodies, and numbers in gates reflect % positive cells amongst Lin^-PI^- bone marrow cells. <b>(A)</b> Profiles for representative individual mice are shown. <b>(B)</b> Percent cells amongst viable Lin^- bone marrow is shown for 3 individual mice, with mean value shown as a bar, and statistically significant results (p ≤ 0.05) boxed in red.</p