IFN-γ in vitro effectively upregulates Sca-1 on HPCs without affecting expression of CD27.

<p><b>A.</b> FACS-purified steady-state HPC were cultured in various concentration of IFN-γ for 24 hrs <i>in vitro</i>. Representative histograms from three experiments are depicted. <b>B.</b> CMPs, GMP, MEP were FACS-purified from steady state HPCs and stained for expression of CD27 before (post sort, red line shows “fluorescence minus one” FMO control) and after culture with IFN-γ for 24 hrs. Histograms from one of two experiments are shown.</p

Infection-induced decrease of early myeloid progenitors is critically dependent on IFN-γ signaling.

<p><b>A.</b> Phenotype of LIN⁻ BM cells in <i>Ifngr1</i>-null mice uninfected and at day 7 after infection with <i>P. chabaudi</i> stained for indicated marker. Representative FACS plots of four experiments with each 5 mice per group are shown. <b>B.</b> Absolute numbers (per femur/pair) for <i>Ifngr1</i>-null LIN⁻ c-Kit^hi CD27⁺ cells are shown as mean ± SEM per individual subsets obtained form three experiments with 5–6 mice per group (n.s.: <i>P</i>>0.05, Mann-Whitney U-test). <b>C.</b> Absolute numbers of myeloid progenitors in radiation chimeras (Hosts: C57BL/6, left and middle; <i>Ifngr1</i>-null, right chart) reconstituted either with B6.Rosa26^eYFP or <i>Ifngr1</i>-null and 8–9 weeks after transplantation infected with <i>P. chabaudi</i>. Two different sets of chimeras were infected and 5–9 animals per time point each were analyzed. Data represent mean ± SEM of cell number per femur/pair (*: <i>P</i>≤0.05; **: <i>P</i>≤0.01, Mann-Whitney U-test).</p

Compilation of absolute numbers of CMP and GMP of bone marrow cells in steady state and at day 7 after infection with <i>Plasmodium chabaudi</i> in C57BL/6 mice and various mutant mouse strains.

<p><b>CMP:</b> LIN⁻ c-Kit⁺ CD27⁺ CD34⁺ CD16/32⁻.</p><p><b>GMP:</b> LIN⁻ c-Kit⁺ CD27⁺ CD34⁺ CD16/32⁺.</p

Contraction of lineage negative cell and Sca-1 upregulation in malaria are dependent on IFN-γ signaling.

<p><b>A.</b> Reduction of LIN⁻ and c-Kit (CD117) positive cells in the BM during infection of C57BL/6 mice with <i>P. chabaudi</i>. Significant contraction of absolute numbers was recorded at day 4 and day 7 after infection for LIN⁻ cells and at day 7 for c-Kit⁺ cells. <i>Ifngr1</i>-null mice did not undergo any significant loss of LIN⁻ or c-Kit⁺ BM cells during acute malaria. Data represent mean ± SEM of cell number per femur/pair obtained from 15 infection experiments (C57BL/6) or four experiments (<i>Ifngr1</i>-null mice) each with 4–5 mice per group (*:<i>P</i>≤0.05; **: <i>P</i>≤0.01, Mann-Whitney U-test). <b>B.</b> LIN⁻ BM cells from uninfected controls and malaria-infected animals at day 7 of infection with <i>P. chabaudi</i> were co-stained with c-Kit and Sca-1. Of note is the lack of Sca-1 upregulation on <i>Ifngr1</i>­null cells during acute malaria. Data are representative for 19 infection experiments (C57BL/6) or four experiments (<i>Ifngr1</i>-null mice) each with numbers in individual plots indicating the frequency (in %) of the respective population.</p

Changes in chemokines and chemokine receptor expression during acute malaria.

<p><b>A.</b> Serum concentration of IFN-γ induced chemokines in C57BL/6 and <i>Ifngr1</i>-deficient mice infected with <i>P. chabaudi</i>. Data are shown as mean ± SEM of serum concentration of respective chemokine in three infection experiments with 5–6 mice per each experimental group. <b>B.</b> Cell surface expression of CD192 (CCR2) on myelo-erythroid progenitors in the BM of mice infected with <i>P. chabaudi</i> day 7 (lower part) or before infection. All data are representative of two independent experiments with 4–5 mice per group each. Black line represents signals in <i>Ccr2^−/−</i> animals for comparison.</p

Effect of acute infection with <i>P. chabaudi</i> on myeloid progenitor cells in the bone marrow.

<p>Infection of C57BL/6 female mice (6–8 weeks) with <i>P. chabaudi</i> resulted in a transient decrease of myeloid lineage progenitors in the bone marrow (BM) during acute infection. <b>A.</b> BM cells from uninfected controls and malaria-infected animals at day 7 of infection with <i>P. chabaudi</i> were co-stained with antibodies against lineage markers, Sca-1, CD117 (c-Kit), CD27, CD34, CD16/32 (FcγRII/III) and separated as indicated. Doublets were electronically excluded prior to analysis. Data are representative for 19 infection experiments with numbers in individual plots indicating the frequency (in %) of the respective population. <b>B.</b> Analysis of granulocyte-monocyte colony forming units in semi-solid media from total BM during acute malaria. Data are from two experiments with 4–5 BM samples per experiment and shown as mean ± SEM of total number of CFU-GM per femur (*: <i>P</i>≤0.05, two-tailed Student's <i>t</i>-test). <b>C.</b> Absolute cell numbers of the CD27⁺ CMP (light blue bars) and the GMP (blue bars) progenitor populations during the course of a malaria infection. Data represent mean ± SEM of cell number per femur/pair obtained from 19 infection experiments with 4–5 mice per group (**: <i>P</i>≤0.01, Mann-Whitney U-test). <b>D.</b> Single cell <i>in vitro</i> cultures that support myeloid or erythroid differentiation of FACS purified hematopoietic BM progenitors obtained at day 7 after infection with <i>P. chabaudi</i>. Data are shown as mean ± SEM of individual positive wells (in %) of three infection experiments with 5 mice per subset (N.D. = not detected).</p

Extramedullary myelopoiesis in malaria is established by mobilizing CCR2⁺ myeloid-restricted BM progenitors.

<p><b>A.</b> Expression of message for <i>Ifng</i> and IFN-γ induced chemokines in organ samples from pre-immune and mice infected with <i>P. chabaudi</i> at day 7. Three to four individual organ samples each were obtained from two independent experiments and analyzed by RT-PCR. <b>B.</b> Phenotype of LIN⁻ cells in the spleen of C57BL/6 and <i>Ccr2</i>-null mice at day 7 after infection with <i>P. chabaudi</i> stained for indicated marker. Results in <b>B.–E.</b> are obtained form three infection experiments with 4–5 mice per group each. <b>C.</b> Absolute numbers of LIN⁻ c-Kit^hi CD27⁺ CD16/32⁻ (CD27⁺ CMPs) and CD16/32⁺ (GMPs) cells in the spleen of C57BL/6 and <i>Ccr2</i>-null animals in steady state or at day 7 after infection with <i>P. chabaudi</i> as quantified by flow cytometry. Results are shown as mean ± SEM from three independent experiments with 4–5 animals per group (**: <i>P</i>≤0.01, Mann-Whitney U-test). <b>D.</b> Changes in the absolute number of clonogenic myeloid progenitors (CFU-GM) in the BM and the spleen in uninfected mice and at day 7 after infection with <i>P. chabaudi</i>. Data for <i>Ccr2</i>-null mice and controls were obtained in three experiments with 4–5 samples per experiment and shown as mean ± SEM of total number of CFU-GM per organ (*: <i>P</i>≤0.05, **: <i>P</i>≤0.01, two-tailed Student's <i>t</i>-test). <b>E.</b> Phenotype of intrasplenic myeloid precursors during acute infection with <i>P. chabaudi</i>. Lineage-negative (Ter-119, CD3, CD11c, CD19, NK1.1) cells were stained for CD11b versus CD115 (M-CSF receptor) allowing the resolution of early myeloid precursors as c-Kit^hi Ly-6C⁻. <b>F.</b> Absolute numbers of early myeloid precursors (LIN⁻ c-Kit^hi CD115⁺ CD11b⁻ Ly-6C⁻) in the spleen of uninfected mice and at day 7 after infection. Representative staining and splenic cellularity were obtained from three independent infection experiments with 3–5 animals per group. Data represent mean ± SEM of cell number per spleen (**: <i>P</i>≤0.01, Mann-Whitney U-test). <b>G.</b> Course of malaria infection in C57BL/6 and <i>Ccr2</i>-null mice inoculated with blood stages of the malaria parasite <i>P. chabaudi</i>. Parasitemia was counted on the indicated days. Results for four infection experiments with 4–5 animals per time point in each experiment are shown as mean ± SEM (*: <i>P</i>≤0.05, **: <i>P</i>≤0.01, Mann-Whitney U-test).</p

<i>Ccr2</i>-null mice induce IFN-γ-dependent chemokines but do not mobilize early myeloid progenitors.

<p><b>A.</b> Serum concentration of IFN-γ induced chemokines in <i>Ccr2</i>-null mice infected with <i>P. chabaudi</i>. Data are shown as mean ± SEM of serum concentration of respective chemokine in three infection experiments with 4–5 mice per each experimental group. <b>B.</b> Phenotype of LIN⁻ BM cells in <i>Ccr2</i>-null mice uninfected and at day 7 after infection with <i>P. chabaudi</i> stained for indicated marker. <b>C.</b> Absolute numbers (per femur/pair) for <i>Ccr2</i>-null LIN⁻ c-Kit^hi CD27⁺ cells are shown as mean ± SEM per individual subsets during infection with <i>P. chabaudi</i>.</p

Blockade of IL-12 and IFNγ during co-infection preserves Th2 responses.

<p>A-C). C57BL/6 mice were orally infected with 200 <i>H</i>. <i>polygyrus</i> larvae. 6 days post-infection, mice were infected with 10⁵<i>P</i>. <i>chabaudi</i>. At day 8-post infection with <i>P</i>. <i>chabaudi</i> (d14 <i>H</i>. <i>polygyrus</i>), mice were harvested. Mice were treated with 0.5 mg of anti-IL-12 and anti-IFNγ i.p. at days 0, 5, and 11. B). Total numbers of CD4⁺CD44^hi<i>Il4</i>^gfp+ cells in the mesenteric lymph nodes. C). IgE measured in the serum by ELISA. Data are representative of 2 independent experiments with 6 mice per group. * denotes P<0.05.</p

<i>H</i>. <i>polygyrus/ P</i>. <i>chabaudi</i> co-infection leads to impaired Th2 responses.

<p>A-C). Triple reporter mice were orally infected with 200 <i>H</i>. <i>polygyrus</i> larvae. 6 days post-infection, mice were infected i.p. with 10⁵<i>P</i>. <i>chabaudi</i>. At day 8 of <i>P</i>. <i>chabaudi</i> infection (d14 <i>H</i>. <i>polygyrus</i>), mice were harvested. B). Total numbers of CD4⁺CD44^hi<i>Il4</i>^gfp+ cells in the mesenteric lymph nodes. Data are representative of 5 independent experiments with 2–4 mice per group. C). IgE measured in the serum by ELISA from 3 pooled experiments. D and E). C57BL/6 mice were infected with 200 <i>H</i>. <i>polygyrus</i> larvae, treated on 2 consecutive days (days 14–15) with pyrantel pamoate (5 mg), infected with 10⁵<i>P</i>. <i>chabaudi</i>, and re-infected with <i>H</i>. <i>polygyrus</i>. Adult worms in intestine were counted on day 51. Data are representative of 4 independent experiments with 6–7 mice per group. * denotes P<0.05.</p