Research ArticleDevelopment

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

B cell development demands ion flux

In various immune cell types, the divalent cation channel-kinase TRPM7 regulates cell survival, migration, and effector functions. Krishnamoorthy et al. found that B cell–specific deletion of TRPM7 blocked the development of mature peripheral B cells in mice. Loss of TRPM7, but not its kinase activity, increased the apoptosis of B cell precursors in the bone marrow. Supplementation with extracellular magnesium rescued the development of TRPM7-deficient B cells in vitro. These data suggest that the ion channel activity of TRPM7 is required for B cell development.

Abstract

The transient receptor potential (TRP) family is a large family of widely expressed ion channels that regulate the intracellular concentration of ions and metals and respond to various chemical and physical stimuli. TRP subfamily M member 7 (TRPM7) is unusual in that it contains both an ion channel and a kinase domain. TRPM7 is a divalent cation channel with preference for Ca2+ and Mg2+. It is required for the survival of DT40 cells, a B cell line; however, deletion of TRPM7 in T cells does not impair their development. We found that expression of TRPM7 was required for B cell development in mice. Mice that lacked TRPM7 in B cells failed to generate peripheral B cells because of a developmental block at the pro-B cell stage. The loss of TRPM7 kinase activity alone did not affect the proportion of peripheral mature B cells or the development of B cells in the bone marrow. However, supplementation with a high concentration of extracellular Mg2+ partially rescued the development of TRPM7-deficient B cells in vitro. Thus, our findings identify a critical role for TRPM7 ion channel activity in B cell development.

INTRODUCTION

Ion channels play a vital role in diverse physiological functions by regulating the entry of ions into cells. Sodium (Na+), calcium (Ca2+), potassium (K+), and magnesium (Mg2+) ions are essential in numerous metabolic and cellular processes, and a myriad of human disease pathologies are associated with ion deficiencies. Moreover, several immunodeficiencies are associated with mutations in Ca2+, Mg2+, and Zn2+ channels (15). The transient receptor potential (TRP) family of ion channels is a large family of widely expressed ion channels that regulate the intracellular concentration of Ca2+, Mg2+, and trace metal ions. These ion channels have diverse roles in cellular communication with the extracellular environment and respond to various sensory stimuli, such as taste, temperature, and pain (6).

The TRP subfamily melastatin (TRPM) is a heterogeneous group of eight channels with diverse ion selectivity and mechanisms of activation (7). Two TRPM family members, TRPM6 and TRPM7, are selective for Ca2+ and Mg2+ and regulate magnesium homeostasis. Unlike the other ion channels, TRPM6 and TRPM7 also contain a kinase domain, which may play an additional role in the direct phosphorylation of signaling substrates. TRPM6 is primarily expressed in the kidney, colon, and placenta and is essential for systemic magnesium homeostasis (8). Mutations in the gene encoding this channel are associated with hypomagnesemia with secondary hypocalcemia, a rare hereditary disease in which patients develop a profound magnesium deficiency due to a defect in intestinal Mg2+ absorption (810). By contrast, TRPM7 is widely expressed in human cells and tissues, including the immune system, and is involved in a variety of cellular functions, including cell migration and homing (11, 12), macrophage activation and polarization (13, 14), and mast cell degranulation (15). However, it is vital for cell growth and survival (16); TRPM7 is essential for embryonic development (17). Deletion of TRPM7 in transformed DT40 cells (a chicken B cell line) arrests cell growth and stimulates apoptosis, but supplementation with high concentrations of extracellular Mg2+ can rescue survival (18). However, specific deletion of TRPM7 in T lymphocytes does not impair the development of peripheral T cells (17), suggesting that TRPM7 is not essential in this cell type.

To address what effect TRPM7 had on B cell development and survival in primary cells in vivo, we generated mice that lacked TRPM7 in the B cell lineage. Here, we report that expression of TRPM7 was an absolute requirement for B cell development. Specific deletion of TRPM7 in the B cell lineage resulted in a complete loss of mature peripheral B cells and a developmental block at the pro-B cell stage. We also found an increased frequency of neutrophils in our TRPM7-deficient mice, an observation not previously reported in murine models of B cell deficiency. We found no defects in B cell development in mice lacking TRPM7 phosphotransferase activity (19). By contrast, we found that high concentrations of extracellular Mg2+ partially rescued B cell development in an in vitro model. Thus, our findings identify TRPM7 ion channel activity as being necessary for B cell development.

RESULTS

TRPM7 is essential for B cell development

To examine the role of TRPM7 in B cell development, we generated mice that lacked TRPM7 expression in the B cell lineage. We crossed TRPM7-flox mice with mice expressing Cre-recombinase under the pan B cell CD79a (also Ig-α or Mb-1) gene promoter (20) to generate TRPM7flox/floxCD79a-Cre [wild type (WT)], TRPM7flox/+CD79a-Cre+ (TRPM7+/−), and TRPM7flox/floxCD79a-Cre+ (TRPM7−/−) mice. We observed the genotypes of 463 pups from this mating and found that mice were born at the expected Mendelian frequencies (table S1), indicating that the deletion of TRPM7 in the B cell lineage did not affect embryonic survival. The gross immune cell composition of peripheral lymphoid tissues from WT, TRPM7+/−, and TRPM7−/− mice was characterized by flow cytometry. We found a complete loss of B220+CD19+ mature B cells in the spleen, lymph nodes, and blood in the absence of TRPM7 (Fig. 1A). By contrast, peripheral B cell numbers in TRPM7+/− mice were similar to those of WT mice (Fig. 1A), suggesting that some expression of TRPM7 is sufficient to rescue B cell development.

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.

Although the proportion of CD3+ T cells was slightly increased in peripheral lymphoid tissues (table S2), the absolute number of CD3+ T cells was relatively similar in lymph node tissues from WT, TRPM7+/−, and TRPM7−/− mice (table S3). However, similar to other mouse models that lack B cells, we noted a fourfold decrease in the total number of splenic T cells in TRPM7−/− mice compared to those of WT mice (table S3) (21). When peritoneal lavage samples from WT, TRPM7+/−, and TRPM7−/− mice were characterized by flow cytometry, we found a complete loss of B220+CD23+ B2 B cells in the peritoneum of TRPM7−/− mice and a near complete loss of B220+CD23 B1 B cells (Fig. 1B). Consistent with our findings on B cells in the blood, spleen, and lymph nodes, expression of a single allele of TRPM7 was sufficient to support B1 B cell development (Fig. 1B). Similar to other models of B cell deficiency (22), TRPM7−/− mice lacked discernible intestinal Peyer’s patches (Fig. 1C). We noted similar numbers of Peyer’s patches in WT and TRPM7+/− mice, and flow cytometric analysis revealed no difference in the percentage of B cells in the Peyer’s patches in either strain (Fig. 1, C and D). We also examined nonlymphoid tissue, including the lamina propria of the small and large intestine, the lung, and the liver for the presence of B cells. Similar to our findings in the lymphoid tissue, B cell frequency was markedly reduced in all of these tissues in TRPM7−/− mice (fig. S1). When we measured the serum concentrations of immunoglobulin M (IgM), IgG, and IgA, as well as the fecal concentration of IgA, we found that they were all substantially reduced in TRPM7−/− mice when compared to WT and TRPM7+/− mice (fig. S2). Although we did not detect any serum IgM or IgG in μMT mice, which lack B cells due to deletion of an exon encoding the μ heavy chain (22), we noted low amounts of these two isotypes in TRPM7−/− mice (fig. S2). Consistent with the presence of a few B1 B cells in TRPM7−/− mice, we found 15-fold less circulating IgM in TRPM7−/− mice than in WT mice. Similar concentrations of IgA in both the serum and feces were detected in TRPM7−/− and μMT mice, within the range previously reported in μMT mice (23). Together, these findings indicate a defect in peripheral B cells in the absence of TRPM7.

We examined the architecture of secondary lymphoid organs from WT, TRPM7+/−, and TRPM7−/− mice and noted an about 50% reduction in the mass of the spleen as a proportion of body mass in TRPM7−/− mice (Fig. 2, A and B). We stained tissue sections from the spleens and lymph nodes of WT and TRPM7−/− mice with antibodies against CD3, IgD, and CD11b to stain for T cells, B cells, and monocytes/granulocytes, respectively. Similar to our flow cytometry data, we detected no B cells in the spleens (Fig. 2C) or lymph nodes (fig. S3) of TRPM7−/− mice but noted a disorganized population of CD11b+ leukocytes in the spleen of TRPM7−/− mice (Fig. 2C).

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.

Loss of TRPM7 increases the frequency of myeloid cells

The CD11b+ cell population in WT, TRPM7+/−, and TRPM7−/− mice was further characterized by flow cytometry. We observed an increased frequency of granulocytes in the spleens of TRPM7−/− mice based on forward and side scatter (Fig. 3A). This population was largely composed of CD11b+Ly6G+ neutrophils, CD11b+SSChi eosinophils, and CD11b+SSClo monocytes (Fig. 3, B to E). In the absence of TRPM7, mice had a threefold increased proportion of eosinophils, a 2.6-fold increased proportion of monocytes, and a 5.6-fold increased proportion of neutrophils when compared to WT mice. By contrast, the percentage of each of these populations was normal in TRPM7+/− mice. No difference was observed in the proportion of CD11bF4/80+ red pulp macrophages or CD11b+CD11c+ dendritic cells between WT, TRPM7+/−, and TRPM7−/− mice. An increased frequency of myeloid cells has been reported in B cell–deficient models after infection or vaccination (2426); however, none of these studies examined the neutrophil population in uninfected B cell–deficient mice. We repeated our analysis in μMT mice (22) and found that the frequencies of neutrophils, eosinophils, and monocytes were increased in both the spleen and blood (fig. S4). However, we noted that our CD79a-Cre mouse strain was positive for murine norovirus (MNV), and we wondered whether this might also have influenced myeloid cell frequency in TRPM7−/− mice. To control for this possibility, we either cohoused WT and μMT mice with TRPM7−/− mice to promote cross-infection of MNV in these specific pathogen–free (SPF) mice or maintained them separately under SPF conditions. MNV infection was confirmed by polymerase chain reaction (PCR) analysis of fecal samples from cohoused mice (fig. S5). MNV infection further increased the proportion of each of these populations in μMT mice, most notably in the spleen (fig. S4). By contrast, MNV infection of WT mice increased the frequency of these myeloid populations only in the blood. These findings confirmed that myeloid populations are dysregulated in mice deficient in B cells.

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.

B cell development is arrested at the pro-B cell stage in TRPM7−/− mice

Because TRPM7 is also required for cell migration (11), we characterized the B cell populations in the bone marrow of WT, TRPM7+/−, and TRPM7−/− mice by flow cytometry. We detected a smaller population of B220+ B cells in TRPM7−/− mice compared to that in WT mice (Fig. 4A). In addition, TRPM7−/− mice completely lacked B220+IgM+ immature and transitional B cells, as well as B220+IgM+IgD+ mature B cells, whereas the proportion of these populations in TRPM7+/− was similar to that of WT (Fig. 4A). These findings indicate that B cell development was blocked in the absence of TRPM7. Bone marrow B220+IgM B cell precursors were further characterized to delineate the developmental stage that was affected by TRPM7 deficiency. We found the largest increase in the frequency of CD43+CD24 pre-pro-B cells (Fig. 4B). We also found an increase in the proportion of CD43+CD24+ pro-B cells in TRPM7−/− mice and a corresponding decrease in CD43CD24+ pre-B cells in TRPM7−/− mice (Fig. 4B). No difference was observed in the proportion of these cell populations in TRPM7+/− mice compared to WT mice. These findings suggest that CD79a promoter–driven deletion of TRPM7 leads to a developmental block at the pre-pro-B cell and pro-B cell stages, coincident with promoter activity (20).

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.

To examine whether this developmental block stimulated B cell death, we examined apoptosis by flow cytometry using annexin V and 7-amino-actinomycin D (7AAD) to distinguish early (annexin V+7AAD) and late apoptotic (annexin V+7AAD+) cells in pre-pro-B, pro-B, and pre-B cells. Whereas we found comparable proportions of apoptotic cells at the pre-pro-B cell stage in all mice, the proportion of late apoptotic cells in the pre-B cell population was statistically significantly increased in TRPM7−/− mice compared to that in WT mice (Fig. 4B). Although we noted a small increase in the proportion of early apoptotic cells in the pro-B cell population in TRPM7−/− mice, this difference was not statistically significant. These findings indicate that developing B cells likely begin to undergo apoptosis in the pro-B cell stage and are largely apoptotic by the pre-B cell stage.

The kinase function of TRPM7 is not required for B cell development

We assessed the requirement for TRPM7 phosphotransferase activity using knockin mice expressing a kinase-deficient mutant of TRPM7 (TRPM7KR/KR mice) (19). In these mice, Lys1646 was replaced with an arginine, which completely ablates TRPM7 kinase activity (27). We found no differences in the proportions of peripheral B cells in the blood, spleen, lymph nodes, or Peyer’s patches in TRPM7KR/KR mice compared to those in WT mice (Fig. 5A). We also found no differences in the proportions of B1 and B2 B cells in the peritoneal cavity (Fig. 5B) or the proportions of developing B cells in the bone marrow (Fig. 5C) in the TRPM7KR/KR mice compared to WT mice. These data demonstrated that the kinase activity of TRPM7 was not required for B cell development.

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.

Supplementation with Mg2+ partially rescues B cell development

In various cell types lacking TRPM7, cell survival can be rescued by supplementation with extracellular Mg2+ (8, 18, 28). To examine whether an increased extracellular Mg2+ concentration was sufficient to rescue B cell development, we performed experiments in vitro. We cultured bone marrow hematopoietic stem cells (HSCs) from WT and TRPM7−/− mice on OP9 stromal cells expressing interleukin-7 (IL-7), FMS-like tyrosine kinase 3 ligand (Flt3L), and stem cell factor (SCF) (OP9-R7FS). We found that, without any increased extracellular Mg2+, the OP9-R7FS stromal cells were sufficient to support the development of a smaller percentage of B220+CD19+ cells from TRPM7−/− HSCs than from WT HSCs (Fig. 6). However, the development of TRPM7−/− B220+CD19+ cells from bone marrow HSCs in vitro was greater than the proportion of B220+ cells found in vivo. The addition of Mg2+ stimulated a dose-dependent increase in the B220+CD19+ population derived from HSCs from TRPM7−/− mice, although even at 10 mM Mg2+, the development of B220+CD19+ cells from TRPM7−/− HSCs was still less than that from WT HSCs (Fig. 6). Together, these findings demonstrate that TRPM7 is essential for B cell development in vitro and that supplementation with extracellular Mg2+ could partially rescue this defect.

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.

DISCUSSION

The role of TRPM7 in B cell development that we identified is in stark contrast to its role in T cells because loss of TRPM7 results in only a minor decrease in peripheral T cell numbers and a delay in thymocyte development (17). However, T cells, but not B cells, express other Mg2+ ion channels, such as TRPM6 (29). Endogenous TRPM6 functions primarily in heteromeric TRPM6/TRPM7 channel complexes, which implies that deletion of TRPM7 in T cells may also abrogate TRPM6 function (8, 30). Alternatively, the Mg2+-permeable ion channel MagT1, which is also implicated in cellular Mg2+ homeostasis (31), may play a more important role in T cell biology. Genetic defects in MagT1 are associated with the primary immunodeficiency disease known as XMEN (X-linked immunodeficiency with magnesium defect and Epstein-Barr virus infection and neoplasia) in which patients have impaired thymic production of CD4+ T cells (5). Overexpression of MagT1 in TRPM7-deficient DT40 cells partially rescues the cell proliferation defect under physiological concentrations of Mg2+ (32). In addition, leaky expression from incomplete Cre-mediated deletion of TRPM7 may also explain the absence of a phenotype in peripheral T cell numbers (28). In our model, expression of a single allele of TRPM7 was sufficient to completely support B cell development and survival, demonstrating that incomplete knockout of TRPM7 may indeed mask evident phenotypes. However, Cre-mediated deletion of TRPM7 in megakaryocytes results in increased numbers of megakaryocytes in the bone marrow and red pulp of the spleen (33), and specific deletion of TRPM7 in the myeloid lineage does not affect macrophage differentiation from bone marrow cells ex vivo (13), suggesting that TRPM7 may be required for the development of distinct hematopoietic lineages.

We examined a murine model in which the kinase function of TRPM7 was abolished (19), where inactivation of TRPM7 kinase activity has no effect on ion channel function (19, 34). We found no difference in the proportions of peripheral B cells and developing B cells in the bone marrow in the knockin mice, demonstrating that the kinase function of TRPM7 was not required for B cell development and survival, consistent with the finding that the kinase activity of TRPM7 is not required for T cell development in the thymus (12). By contrast, using an in vitro model of B lymphopoiesis, we found a dose-dependent increase in the percentage of B220+CD19+ pro-B cells with increasing concentrations of extracellular Mg2+. Our data suggest that TRPM7-dependent Mg2+ transport is required for B cell development. This is consistent with the observation that extracellular supplementation with a high concentration of Mg2+ rescues cell growth arrest in TRPM7-deficient DT40 cells (18). Moreover, supplementation with extracellular Mg2+ restores proliferation-arrested embryonic stem cells lacking the entire kinase domain of TRPM7, which reduces channel activity (28). Analogously, trophoblast stem cells derived from TRPM7-null blastocysts proliferate only in culture medium supplemented with Mg2+ (8). However, even 10 mM extracellular Mg2+, the concentration that reverted the cell growth arrest in TRPM7-deficient cells (8, 18, 28), was insufficient to wholly revert the B cell developmental defect in our TRPM7−/− cells in vitro. This discrepancy may be due to differential expression of other Mg2+ channels in other cell types in comparison to primary HSCs (31, 32). Although our findings suggest an important role for the channel function of TRPM7, integrins are also activated by divalent cations. We cannot rule out the possibility that integrin activation may facilitate the interaction between B cell progenitors and OP9 stromal cells and enhance their ability to support B cell development. Moreover, in these coculture experiments in vitro, we cannot rule out a direct effect of increased extracellular Mg2+ on OP9 stromal cells.

In comparison to the percentage of these populations in vivo, we found an increased percentage of both B220+ pre-pro-B cells and B220+CD19+ pro-B cells developed after coculture of primary HSCs from TRPM7−/− mice with OP9-R7FS cells. It is possible that increased expression of the differentiation factors, IL-7, Flt3L, and SCF in OP9-R7FS cells in comparison to bone marrow stromal cells in vivo enhanced the development of pre-pro-B and pro-B cells. TRPM7-deficient cells exhibit cell growth arrest at the G0/G1 phase of the cell cycle (28, 35), which correlates with decreased signaling through the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway (35). Because IL-7R, Flt3, and c-Kit all signal through this pathway, overexpression of the ligands for these receptors by OP9-R7FS cells may have been sufficient to promote cell growth and survival in these populations of TRPM7-deficient developing B cells.

Unexpectedly, we identified an increase in the percentage of neutrophils in the spleens of TRPM7−/− mice, as well as a small increase in the percentages of eosinophils and monocytes. To our knowledge, this is the first description of an increased proportion of granulocytes in mice lacking B cells. Although several studies reported an increase in the neutrophil population in μMT mice after infection (2426) or immunization (25, 36), none of these studies reported increased percentages of neutrophils before immunization, although this may be due to the tissues examined, where the population of neutrophils was not increased in μMT mice at the site of immunization or the draining lymph node before immunization (25). Thus, it remains unclear whether the increased frequency of neutrophils in pathogen-infected or immunized μMT mice is specifically induced in response to infection or whether μMT mice exhibit increased frequency of neutrophils even in the absence of immune challenge. We found an increase in the percentages of neutrophils, eosinophils, and monocytes in two B cell–deficient mouse models (22). Because our TRPM7−/− mice were MNV-positive, we cohoused WT and μMT mice with TRPM7−/− mice to induce cross-infection. We found that MNV infection increased the percentages of neutrophils, eosinophils, and monocytes in the blood and spleen of μMT mice. MNV infection also increased the percentage of these populations in WT mice but only in the blood. It is not known whether the expanded proportion of myeloid cells in the spleens of μMT and TRPM7−/− mice results from an influx of cells from the circulation or whether the absence of B cells affects the extramedullary hematopoiesis of these populations. However, the increased proportion of myeloid cells after MNV infection in B cell–deficient mice was prominently observed in the spleen, but not in the blood, suggesting that local hematopoiesis of these populations within this organ may be B cell–dependent. Because these B cell–deficient models are used for the study of primary immune deficiencies, it will be important that future studies identify the underlying mechanism for the B cell–mediated control of granulocyte populations.

MATERIALS AND METHODS

Animals

129/SvEvTac TRPM7flox/flox mice were purchased from the Jackson Laboratory and were crossed with C57BL/6 Mb1-cre mice, provided by M. Reth (Max Planck Institute für Immunbiologie und Epigenetik) (20). TRPM7flox/+Mb1-Cre+ mice from F1 progeny were then back-crossed with TRPM7flox/flox mice to generate TRPM7flox/flox (WT), TRPM7flox/+Mb1-Cre+ (TRPM7+/−), and TRPM7flox/floxMb1-Cre+ (TRPM7−/−) mice. Mice were maintained on a mixed 129/SvEvTac and C57BL/6 background. TRPM7K1646R/K1646R (TRPM7KR/KR) mice were provided by A. Ryazanov (Rutgers School of Arts and Sciences) (19). We obtained μMT mice (B6.129S2-Ighmtm1Cgn/J) from the Jackson Laboratory. For cohousing experiments, female WT and μMT mice were cohoused with MNV+ TRPM7−/− mice for 2 weeks before analysis and verification of MNV infection. Mice were 12 to 16 weeks old in all experiments. All experiments were approved by the Local Animal Care Committee at the University of Toronto Scarborough.

MNV detection by PCR analysis

To extract RNA from mouse fecal samples, fecal supernatants were obtained by centrifuging fecal suspensions (120 to 666 mg/ml) in phosphate-buffered saline (PBS) at 5000g for 5 min. RNA was extracted from 200 μl of each supernatant with a High Pure Viral RNA kit (Roche Applied Science) according to the manufacturer’s instructions. Purified RNA was used to generate complementary DNA (cDNA) with the qScript cDNA Synthesis Kit (Quanta BioScience), and cDNA was subsequently used in quantitative PCR (qPCR) reactions with PerfeCTa SYBR Green SuperMix (Quanta BioScience). The following primers were used to amplify a highly conserved 97–base pair (bp) region of the MNV viral genome: 5′-ATGGTAGTCCCACGCCAC-3′ (forward) and 5′-TGCGCCATCACTCATCC-3′ (reverse). The qPCR reactions were run on a 1% agarose tris-acetate-EDTA gel. Presence of MNV was detected as a 97-bp band.

Cell isolation and flow cytometry

Lymphoid organs were dissociated into single-cell suspensions by mincing through a 70-μm nylon mesh. To isolate peritoneal cells, the peritoneal cavity was flushed with 5 ml of ice-cold PBS. Bone marrow was obtained by flushing femurs with PBS. Erythrocytes were lysed from spleen and bone marrow suspensions with a hypotonic lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, and 0.1 mM EDTA). For intestinal lamina propria leukocyte isolation, Peyer’s patches were removed from the small intestine, and both the small intestine and colon were rinsed briefly with ice-cold Hanks’ balanced salt solution (HBSS) without Ca2+ and Mg2+ (Gibco). Intestinal tissues were incubated at 37°C in HBSS with 5 mM EDTA and 10 mM Hepes and were extensively vortexed to remove epithelial cells. The remaining colon and small intestinal tissues were then minced and digested in HBSS containing Ca2+ and Mg2+ (Gibco) supplemented with collagenase (0.5 mg/ml) (Sigma-Aldrich) and deoxyribonuclease I (DNase I) (0.5 mg/ml) (Sigma-Aldrich) at 37°C for 25 min and passed through a 70-μm strainer. For isolation of leukocytes from the lung and liver, tissues were minced and digested in collagenase (0.5 mg/ml) and DNase I (0.5 mg/ml) (Sigma-Aldrich) in HBSS containing Ca2+ and Mg2+ at 37°C for 20 min and passed through a 70-μm strainer. Leukocytes were enriched by centrifugation in a solution of 40% Percoll (GE Healthcare), followed by red blood cells lysis. Before they were subjected to staining of cell surface markers, single-cell suspensions (5 × 105 cells) were incubated with 2 μM CD16/32 (clone 2.4G2, BD Biosciences). Cells were then stained with the appropriate antibodies in staining buffer [3% fetal bovine serum (FBS) and 0.02% NaN3 in PBS], and analysis was performed with an LSRFortessa or an LSRII flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (TreeStar).

Antibodies

The following antibodies were used for flow cytometry analysis: B220-APC (clone RA3-6B2, Thermo Fisher Scientific), B220-PE (clone RA3-6B2, Thermo Fisher Scientific), CD11b-Alexa Fluor 547 (clone M1/70, BioLegend), CD11b-FITC (clone M1/70, BioLegend), CD11c-APC (clone N418, BioLegend), CD19-APC (clone 6D5, Thermo Fisher Scientific), CD19-PE (clone 6D5, Thermo Fisher Scientific), CD23-eFluor 660 (clone B3B4, eBioscience), CD24-Qdot 605 Brilliant Violet (clone M1/69, BioLegend), CD43-APC (clone S11, BioLegend), CD3-FITC (clone 500A2, Thermo Fisher Scientific), F4/80-APC-Cy7 (clone BM8, BioLegend), IgD-Pe-Cy7 (clone 11-26c.2a, BioLegend), Ly6G-Qdot 605 Brilliant Violet (clone 1A8, BioLegend), Siglec-F PerCP-Cy5.5 (clone E50-2440, BD Biosciences), and IgM μ chain Alexa Fluor 488 Fab fragment (polyclonal; Jackson ImmunoResearch). The following antibodies were used to identify B cells in intestinal, liver, and lung tissues: CD45-APC-Cy7 (clone 104, BioLegend), B220-APC (clone RA3-6B2, BioLegend), and IgM-PerCPCy5.5 (clone RMM-1, SouthernBiotech). Viable cells were identified with the LIVE/DEAD Fixable Aqua stain (Thermo Fisher Scientific).

Tissue sectioning and immunostaining

Spleens and inguinal lymph nodes were dissected and immediately immersed in optimal cutting temperature compound (Tissue-Tek). Tissues were then frozen in an isopentane bath submerged in liquid nitrogen. Frozen tissue blocks were cryosectioned into 12-μm slices and subsequently fixed in −20°C acetone on Superfrost Plus microscope slides (VWR) for 5 min and then air-dried. After rehydration with PBS for 15 min, the sections were blocked with 5% FBS for 30 min at room temperature and then stained with various antibodies in 5% FBS for 2 hours. Sections were mounted in Fluoro-Gel with DABCO Anti-Fading Mounting Medium. Slides were imaged with a Zeiss Axio Scan.Z1 at 40× magnification (Apo Plan 40×/0.95 numerical aperture).

In vitro B cell development assay

OP9 stromal cells (37) stably expressing IL-7, Flt3L, and SCF (OP9-R7FS; provided by J.C.Z.-P) were cultured as a monolayer in α–minimum essential medium supplemented with 5% FBS and 1% penicillin/streptomycin (all Gibco, Life Technologies) at 37°C with 5% CO2. HSCs were enriched from murine bone marrow with the EasySep Mouse Hematopoietic Progenitor Cell Isolation kit (STEMCELL Technologies) according to the manufacturer’s protocol. OP9-R7FS cells were seeded in a six-well plates 24 hours before coculturing with 1.0 × 105 HSCs from WT or TRPM7−/− mice. Cultures were additionally supplemented with 0, 5, or 10 mM MgCl2. Five days later, differentiated cells were washed off the monolayer and replated onto a fresh confluent monolayer of OP9-R7FS cells. Three days later, differentiated cells were collected from the monolayer, and the cell surface expression of B220 and CD19 was assessed by flow cytometry.

Apoptosis assay

Cells were stained with the appropriate antibodies, as detailed previously, and were washed once with staining buffer. Cells were subsequently stained for 15 min at 20°C with annexin V–Pacific Blue (8 μg/ml; BioLegend) in Annexin V Binding Buffer (BioLegend) and were washed with staining buffer before being resuspended in 7AAD (4 μg/ml) in staining buffer. Cells were then analyzed by flow cytometry.

Determination of antibody concentrations

Blood and feces were collected from 2- to 5-month-old WT, TRPM7+/−, TRPM7−/−, and μMT mice. Blood samples were collected by saphenous vein bleed and allowed to coagulate for 30 min before they were centrifuged to collect serum. Feces (0.1 g) were dissolved in 100 μl of PBS and incubated at room temperature for 15 min before the samples were centrifuged and the supernatant was collected. Mouse serum IgM, IgA, and IgG and fecal IgA were quantified with the Mouse IgM, IgA, or IgG ELISA Quantitation Set (Bethyl Laboratories Inc.) according to the manufacturer’s recommended protocol. Serum was diluted 1:500 to measure IgA and IgM titers but was diluted 1:1000 to measure IgG titers. Fecal supernatant was diluted 1:50 to measure IgA titers to achieve absorbance values within the range of the standard curve.

Statistical analysis

Statistical significance was assessed by the Kruskal-Wallis test, followed by Dunn’s multiple-comparisons test, Mann-Whitney test, χ2 test, Wilcoxon matched-pairs ranked test, or Friedman’s test with Dunn’s multiple comparisons with Prism software (version 6.01, GraphPad).

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.

REFERENCES AND NOTES

Acknowledgments: We thank M. Reth (MPI für Immunbiologie und Epigenetik) for the CD79a-Cre mouse strain, A. Ryazanov (Rutgers School of Arts and Sciences) for the TRPM7K1646R/K1646R mouse strain, and P. M. Brauer and J.C.Z.-P. (Sunnybrook Research Institute) for the OP9-R7FS cells. Funding: This work was supported by funding from the Canadian Institutes of Health Research (MOP-136808), Natural Sciences and Engineering Research Council of Canada (NSERC; RGPIN 418756-12), and Canada Research Chair (905-231134) to B.T., and NSERC graduate student fellowships to M.K. and T.Z. Author contributions: M.K. and B.T. designed the research study. M.K., F.H.M.B., T.Z., V.M.-P., K.B., E.Y.C., and A.M. conducted the experiments and data analysis. P.M.B. and J.C.Z.-P. generated the OP9-R7FS cells. M.K. and B.T. wrote the manuscript, and all authors commented on the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: CD79a-Cre mouse strain requires a material transfer agreement (MTA) from M. Reth (MPI für Immunbiologie und Epigenetik), and OP9-R7FS cells requires an MTA from J.C.Z.-P. (Sunnybrook Research Institute). All other data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.
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