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The ion channel TRPM7 is required for B cell lymphopoiesis

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Science Signaling  05 Jun 2018:
Vol. 11, Issue 533, eaan2693
DOI: 10.1126/scisignal.aan2693
  • Fig. 1 TRPM7 is essential for B cell development.

    (A) Left: Flow cytometry analysis of B220+CD19+ cells from the spleen (top), lymph node (center), and blood (bottom) of WT, TRPM7+/−, and TRPM7−/− mice. Numbers inside the dot plots indicate cell percentages. Data are representative of four independent experiments. Right: Quantified data are means ± SEM from four mice of each genotype. (B) Left: Flow cytometry analysis of the indicated peritoneal B cell subsets from WT, TRPM7+/−, and TRPM7−/− mice. The frequencies of B1 (B220+CD23) and B2 (B220+CD23+) B cells were determined. Data are representative of four independent experiments. Right: Quantified data are means ± SEM from four mice of each genotype. (C) Peyer’s patches (indicated by dotted circles) from the small intestine (duodenum to ileum) from WT (top), TRPM7+/− (center), and TRPM7−/− (bottom) mice. Images are representative of three independent experiments. (D) Left: Flow cytometry analysis of B220+CD19+ cells from the intestinal Peyer’s patches of WT and TRPM7+/− mice. Data are representative of four independent experiments. Right: Quantified data are means ± SEM from four mice of each genotype. Statistical significance was determined with Kruskall-Wallis test, followed by Dunn’s multiple-comparisons test (A and B) or Mann-Whitney test (D). *P < 0.05, **P < 0.01, and ***P < 0.001; ns, not significant.

  • Fig. 2 The splenic architecture is altered in mice that lack TRPM7 expression in the B cell lineage.

    (A and B) Comparison of relative spleen size and weight from WT, TRPM7+/−, and TRPM7−/− mice. (A) Images are representative of three independent experiments. (B) Quantified data are means ± SEM from at least five mice of each genotype. Statistical significance was assessed by Kruskall-Wallis test, followed by Dunn’s multiple-comparisons test. **P < 0.01 and ***P < 0.001. (C) Epifluorescence microscopy analysis of splenic morphology in WT (top), TRPM7+/− (middle), and TRPM7−/− (bottom) mice. Cryosections were stained for IgD (cyan), CD3 (magenta), and CD11b (yellow). Scale bars, 0.5 mm. Images are representative of at least three mice of each genotype.

  • Fig. 3 The proportion of myeloid cells is increased in mice that lack TRPM7 expression in the B cell lineage.

    (A to E) Flow cytometry analysis of the indicated myeloid populations in the spleens of WT, TRPM7+/−, and TRPM7−/− mice. Left: Gating strategy used to detect (A) total granulocytes (FSChiSSChi), (B) red pulp macrophages (RPM; CD11bloF4/80hi), (C) neutrophils (CD11bhiLy6Ghi), (D) dendritic cells (CD11bhiCD11chi), (E) eosinophils (top: CD11bhiSSChi), and monocytes (bottom: CD11bhiSSClo) from the live singlet cells. Data are representative of four independent experiments. Right: Quantified data are means ± SEM from four mice of each genotype. Statistical significance was assessed by Kruskall-Wallis test, followed by Dunn’s multiple-comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.001. FSC, forward scatter; SSC, side scatter.

  • Fig. 4 B cell development is arrested at the pro-B cell stage in the absence of TRPM7.

    (A) Flow cytometry analysis of B cell precursor populations in the bone marrow of WT, TRPM7+/−, and TRPM7−/− mice. The frequencies of total B220+ cells (top), as well as of pre-pro-B cell (B220+IgDIgM), immature B cell (B220+IgDIgMlo), transitional B cell (B220+IgDIgMhi), and mature B cell (B220+IgDhiIgMhi) subsets (bottom), were determined. Data are representative of four independent experiments. Bottom right: Quantified data are means ± SEM from four mice of each genotype. (B) Top left: Flow cytometry analysis of the proportion of pre-B cells (B220+IgMCD24+CD43), pro-B cells (B220+IgMCD24+CD43+), and pre-pro-B cells (B220+IgMCD24CD43+) in the bone marrow. Bottom left: Apoptosis of B cell precursors was assessed by annexin V and 7AAD staining of pre-B cell, pro-B cell, and pre-pro-B cell populations, as indicated. Cells in early apoptosis (annexin V+7AAD) and late apoptosis (annexin V+7AAD+) were identified within each B cell subpopulation. Data are representative of four independent experiments. Right: Quantified data are means ± SEM from four mice of each genotype. Statistical significance was assessed by Kruskall-Wallis test, followed by Dunn’s multiple-comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 5 TRPM7 kinase activity is dispensable in B cell development.

    (A) Flow cytometry analysis of B cells in tissues from WT and TRPM7KR/KR mice. The frequencies of B220+CD19+cells in the spleen, lymph node, blood, and Peyer’s patches (P.P.) were analyzed. (B) The frequencies of B1 (B220+CD23) and B2 (B220+CD23+) cells in peritoneal lavage samples from WT and TRPM7KR/KR mice were analyzed by flow cytometry. (C) Left: The frequencies of total B220+ cells, pre-pro-B cell (B220+IgDIgM), immature B cell (B220+IgDIgMlo), transitional B cell (B220+IgDIgMhi), and mature B cell (B220+IgDhiIgMhi) subsets in the bone marrow of WT and TRPM7KR/KR mice were determined by flow cytometry. Data are representative of three independent experiments. Right: Quantified data are means ± SEM from three mice of each genotype. Statistical significance was determined with Kruskal-Wallis test, followed by Dunn’s multiple-comparisons test.

  • Fig. 6 Supplementation with Mg2+ partially supports B cell development.

    (A) Top: Flow cytometry analysis of HSCs enriched from the bone marrow of WT and TRPM7−/− mice. Bottom: Frequency of B220+CD19+ B cells after enrichment was quantified. (B) Top: Flow cytometry analysis of B cells that developed in vitro from HSCs purified from the bone marrow of WT and TRPM7−/− mice. Purified HSCs were cocultured with OP9-R7FS murine stromal cells either in medium alone (left) or in medium supplemented with 5 mM (middle) or 10 mM MgCl2 (right). After 9 days, the percentage of B220+CD19+ B cells in each culture was assessed by flow cytometry. Data are representative of three independent experiments. Bottom: Quantified data are means ± SEM from three mice of each genotype. Statistical significance was determined with Wilcoxon matched-pairs ranked test for (A) and Friedman’s test with Dunn’s multiple-comparisons test for (B). *P < 0.05.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/533/eaan2693/DC1

    Fig. S1. TRPM7 is essential for B cell development in the gut, liver, and lung.

    Fig. S2. Basal antibody concentrations are markedly reduced in TRPM7−/− mice.

    Fig. S3. Architecture of lymph nodes in TRPM7−/− mice.

    Fig. S4. The myeloid population is expanded in μMT mice.

    Fig. S5. Confirmation of MNV infection.

    Table S1. Conditional deletion of TRPM7 in B cells does not alter embryonic survival.

    Table S2. The proportion of T cells is increased in the peripheral lymphoid tissues of TRPM7−/− mice.

    Table S3. The number of T cells is decreased in the spleen of TRPM7−/− mice.

  • Supplementary Materials for:

    The ion channel TRPM7 is required for B cell lymphopoiesis

    Mithunah Krishnamoorthy, Fathima Hifza Mohamed Buhari, Tiantian Zhao, Patrick M. Brauer, Kyle Burrows, Eric Yixiao Cao, Vincent Moxley-Paquette, Arthur Mortha, Juan Carlos Zúñiga-Pflücker, Bebhinn Treanor*

    *Corresponding author. Email: bebhinn.treanor{at}utoronto.ca

    This PDF file includes:

    • Fig. S1. TRPM7 is essential for B cell development in the gut, liver, and lung.
    • Fig. S2. Basal antibody concentrations are markedly reduced in TRPM7−/− mice.
    • Fig. S3. Architecture of lymph nodes in TRPM7−/− mice.
    • Fig. S4. The myeloid population is expanded in μMT mice.
    • Fig. S5. Confirmation of MNV infection.
    • Table S1. Conditional deletion of TRPM7 in B cells does not alter embryonic survival.
    • Table S2. The proportion of T cells is increased in the peripheral lymphoid tissues of TRPM7−/− mice.
    • Table S3. The number of T cells is decreased in the spleen of TRPM7−/− mice.

    [Download PDF]


    © 2018 American Association for the Advancement of Science

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