Wnt4 Enhances Murine Hematopoietic Progenitor Cell Expansion Through a Planar Cell Polarity-Like Pathway

Background: While the role of canonical (b-catenin-mediated) Wnt signaling in hematolymphopoiesis has been studied extensively, little is known of the potential importance of non-canonical Wnt signals in hematopoietic cells. Wnt4 is one of the Wnt proteins that can elicit non-canonical pathways. We have previously shown that retroviral overexpression of Wnt4 by hematopoietic cells increased thymic cellularity as well as the frequency of early thymic progenitors and bone marrow hematopoietic progenitor cells (HPCs). However, the molecular pathways responsible for its effect in HPCs are not known. Methodology/Principal Findings: Here we report that Wnt4 stimulation resulted in the activation of the small GTPase Rac1 as well as Jnk kinases in an HPC cell line. Jnk activity was necessary, while b-catenin was dispensable, for the Wnt4-mediated expansion of primary fetal liver HPCs in culture. Furthermore, Jnk2-deficient and Wnt4 hemizygous mice presented lower numbers of HPCs in their bone marrow, and Jnk2-deficient HPCs showed increased rates of apoptosis. Wnt4 also improved HPC activity in a competitive reconstitution model in a cell-autonomous, Jnk2-dependent manner. Lastly, we identified Fz6 as a receptor for Wnt4 in immature HPCs and showed that the absence of Wnt4 led to a decreased expression of four polarity complex genes. Conclusions/Significance: Our results establish a functional role for non-canonical Wnt signaling in hematopoiesis throug

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness.

International audienceThe gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture

Persistent Cutaneous <i>Leishmania major</i> Infection Promotes Infection-Adapted Myelopoiesis

Hematopoietic stem/progenitor cells (HSPC) are responsible for the generation of most immune cells throughout the lifespan of the organism. Inflammation can activate bone marrow HSPCs, leading to enhanced myelopoiesis to replace cells, such as neutrophils, which are attracted to inflamed tissues. We have previously shown that HSPC activation promotes parasite persistence and expansion in experimental visceral leishmaniasis through the increased production of permissive monocytes. However, it is not clear if the presence of the parasite in the bone marrow was required for infection-adapted myelopoiesis. We therefore hypothesized that persistent forms of Leishmania major (cutaneous leishmaniasis) could also activate HSPCs and myeloid precursors in the C57Bl/6 mouse model of intradermal infection in the ear. The accrued influx of myeloid cells to the lesion site corresponded to an increase in myeloid-biased HSPCs in the bone marrow and spleen in mice infected with a persistent strain of L. major, together with an increase in monocytes and monocyte-derived myeloid cells in the spleen. Analysis of the bone marrow cytokine and chemokine environment revealed an attenuated type I and type II interferon response in the mice infected with the persistent strain compared to the self-healing strain, while both strains induced a rapid upregulation of myelopoietic cytokines, such as IL-1β and GM-CSF. These results demonstrate that an active infection in the bone marrow is not necessary for the induction of infection-adapted myelopoiesis, and underline the importance of considering alterations to the bone marrow output when analyzing in vivo host-pathogen interactions

Infection-adapted emergency hematopoiesis promotes visceral leishmaniasis

<div><p>Cells of the immune system are derived from hematopoietic stem cells (HSCs) residing in the bone marrow. HSCs become activated in response to stress, such as acute infections, which adapt the bone marrow output to the needs of the immune response. However, the impact of infection-adapted HSC activation and differentiation on the persistence of chronic infections is poorly understood. We have examined here the bone marrow outcome of chronic visceral leishmaniasis and show that the parasite <i>Leishmania donovani</i> induces HSC expansion and skews their differentiation towards non-classical myeloid progenitors with a regulatory phenotype. Our results further suggest that emergency hematopoiesis contributes to the pathogenesis of visceral leishmaniasis, as decreased HSC expansion results in a lower parasite burden. Conversely, monocytes derived in the presence of soluble factors from the infected bone marrow environment are more permissive to infection by <i>Leishmania</i>. Our results demonstrate that <i>L</i>. <i>donovani</i> is able to subvert host bone marrow emergency responses to facilitate parasite persistence, and put forward hematopoiesis as a novel therapeutic target in chronic infections.</p></div

<i>Fzd6</i>^-/- T lymphocytes are functionally indistinguishable from their <i>Fzd6</i>^+/+ counterparts.

<p><b>(A-B)</b> Numbers of CD4⁺ and CD8⁺ T cells in BM <b>(A)</b> and spleen <b>(B)</b> of naïve and infected mice on day 28. <b>(C-D)</b> Cytokine production by bone marrow and spleen CD4⁺ T cells isolated from infected mice and stimulated <i>ex vivo</i> with parasite-pulsed bone marrow dendritic cells. Representative flow cytometry data are shown in <b>(C)</b>. Graph in <b>(D)</b> shows compiled results from a representative experiment. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s010" target="_blank">S10 Fig</a>. All bar graphs represent mean + SEM with 7 mice per group for day 28. <b>(E)</b> Macrophages derived from naïve <i>Fzd6</i>^-/- (KO) and <i>Fzd6</i>^+/+ (WT) bone marrow were either left untreated, stimulated with IFN-γ alone or first primed with IFN-γ and then infected with PKH26-labeled <i>L</i>. <i>donovani</i> amastigotes. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s010" target="_blank">S10 Fig</a>. Imaging flow cytometry analysis of macrophages infected with fluorescent <i>Leishmania</i> parasites, showing multiple parasites within both <i>Fzd6</i>^-/- (KO) and <i>Fzd6</i>^+/+ (WT) macrophages. <b>(F)</b> Histograms represent percentage of infected macrophages and <b>(G)</b> parasite numbers in infected macrophages 72h post-infection. Low = 1–3, medium = 4–10, high = >10 parasites / cell. Bar graphs represent mean + SEM from two independent experiments.</p

Diminished myeloid output in <i>Fzd6</i>^-/- mice correlates with a reduced parasite burden during the chronic phase of infection.

<p><b>(A)</b> Analysis of bone marrow myeloid subsets infected <i>Fzd6</i>^-/- (KO) and <i>Fzd6</i>^+/+ (WT) mice. Mean percentage for each cell subset is indicated within flow cytometry plots. Graphs show numbers of granulocytes and monocytes on day 14 and 28. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s004" target="_blank">S4</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s006" target="_blank">S6</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s007" target="_blank">S7</a> Figs. <b>(B)</b> Numbers within flow cytometry plots indicate mean percentage of Ly6C^lo/- F4-80⁺ bone marrow macrophages. Histograms show total numbers of macrophages and percent F4-80⁺ within Ly6C^hi monocytes (mean + SEM from seven mice per group). <b>(C)</b> Ly6C and CCR expression (MFI) on Ly6C^hi monocytes at day 28pi. <b>(D)</b> Percentage of CXCR4⁺, Sca-1⁺ and MHC-II⁺ cells within Ly6C^hi monocytes on day 28pi. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s008" target="_blank">S8 Fig</a>. <b>(E)</b> Percentage of Arginase-1 (Arg-1) and IL-10 expressing cells within Ly6C^hi monocytes. <b>(F)</b> NOS2 expression (MFI) on Ly6C^hi monocytes and Ly6C^lo/- F4-80⁺ bone marrow macrophages at day 28pi. <b>(G)</b> Parasite burden determined by the limiting dilution assay in WT and KO bone marrow at day 28. Data shown were pooled from two independent infections with 10 mice per genotype. <b>(H-I)</b> Pearson's correlation coefficient was used to assess correlation between bone marrow parasite burden and bone marrow HSCs <b>(H)</b> and Ly6C^hi monocytes <b>(I)</b> during the course of the infection. Data for correlation were pooled from C57BL/6, <i>Fzd6</i>^+/+ and <i>Fzd6</i>^-/- mice at day 14, 21 or 28. All bar graphs represent mean + SEM with 6 mice per group for day 28 and 3 mice per group for day 14 unless otherwise noted. Similar results were obtained from three independent experiments for day 28. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

Infected bone marrow microenvironment directly promotes HSPC expansion and the generation of permissive monocytes.

<p>Freshly isolated lineage-depleted <i>Fzd6</i>^+/+ (WT) BM cells were cultured in complete medium supplemented with 30% BM supernatant as indicated. <b>(A)</b> Representative flow cytometry data show the gating strategy for CD11b⁺, LSK and GMP populations. Graphs show numbers of cell recovered per 5x10⁵ cells seeded for each subset. Lin^- <i>Fzd6</i>^+/+ (WT) BM cells cultured with BM supernatant obtained from infected <i>Fzd6</i>^-/- (KO) and <i>Fzd6</i>^+/+ (WT) mice. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s011" target="_blank">S11 Fig</a>. Data were pooled from three independent experiments. <b>(B)</b> Percentage of Ly6C⁺ monocytes in differentiation cultures following infection on day 4, and <b>(C)</b> the proportion of infected Ly6C⁺ monocytes after 24h and 72h. <b>(D-E)</b> Imaging flow cytometry analysis of monocytes infected with fluorescent Leishmania parasites, showing multiple parasites at <b>(D)</b> 24h and <b>(E)</b> 72h post-infection. Histograms represents parasite uptake in infected macrophages. Low = 1–3, medium = 4–10, high = >10 parasites respectively. Bar graphs represent mean + SEM from three independent experiments. *<i>P</i><0.05; **<i>P</i><0.01.</p

Bone marrow HSCs switch their differentiation towards non-classical myeloid progenitors.

<p><b>(A)</b> Representative flow cytometry data and gating strategy of granulocyte-monocyte progenitors (GMPs) in the bone marrow. Steady state myeloid progenitor (MP) cells were first gated on Lin^-Sca1^-c-kit^hi and then subdivided according to the expression of CD41, CD150 and CD16/CD32. GMPs were identified as CD16/CD32⁺ CD41^- CD150^-. Due to the inflammation-induced shift in Sca1 expression, the total Lin^- c-Kit^hi HSPC population was included for analysis during infection. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s002" target="_blank">S2 Fig</a>. <b>(B)</b> Graphs show percentage and absolute numbers of GMPs at various time points. Data are pooled from three independent experiments, with each individual dot representing one mouse. Horizontal lines represent the sample mean. <b>(C)</b> Percentage of GMPs in S/G2/M phases of cell cycle. <b>(D)</b> Percentage of Sca-1⁺ emergency GMPs within all GMPs. <b>(E)</b> Numbers of cells within Sca-1⁺ and Sca-1^- GMP subsets. <b>(F)</b> Intracellular active β-catenin levels (MFI) in GMPs.</p

Leishmania parasite expansion promotes myeloid output in the bone marrow.

<p><b>(A)</b> Representative flow cytometry data to demonstrate gating strategy for myeloid cell subsets in the bone marrow. Graphs show percentage and numbers of granulocytes (GR1^hi SSC^hi), mature monocytes (Ly6C^hiCD11b⁺) and remaining immature/resident myelo-monocytes (Ly6C^lo/- GR1^lo/- CD11b⁺). Data are pooled from three independent experiments, with each individual dot representing one mouse. Horizontal lines represent the sample mean. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s003" target="_blank">S3 Fig</a>. <b>(B)</b> Giemsa-stained infected bone marrow monocytes, myeloblast-like cells and macrophages bearing intracellular Leishmania amastigotes (100 X under oil immersion lens). Scale bar = 5μm. <b>(C)</b> Sca-1 and MHC-II expression on Ly6C^hi and Ly6C^lo/- monocyte subsets. <b>(D)</b> Galectin and Ly6C expression (MFI) on Ly6C^hi monocytes. All bar graphs represent mean + SEM with 4 mice per group coming from one single infection. Similar results were obtained in a second, independent experiment. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p