Conditional deletion of ferritin h in mice reduces B and T lymphocyte populations

Abstract

The immune system and iron availability are intimately linked as appropriate iron supply is needed for cell proliferation, while excess iron, as observed in hemochromatosis, may reduce subsets of lymphocytes. We have tested the effects of a ferritin H gene deletion on lymphocytes. Mx-Cre mediated conditional deletion of ferritin H in bone marrow reduced the number of mature B cells and peripheral T cells in all lymphoid organs. FACS analysis showed an increase in the labile iron pool, enhanced reactive oxygen species formation and mitochondrial depolarization. The findings were confirmed by a B-cell specific deletion using Fth(lox/lox) ; CD19-Cre mice. Mature B cells were strongly under-represented in bone marrow and spleen of the deleted mice, whereas pre-B and immature B cells were not affected. Bone marrow B cells showed increased proliferation as judged by the number of cells in S and G2/M phase as well as BrdU incorporation. Upon in vitro culture with B-cell activating factor of the tumor necrosis factor family (BAFF), ferritin H-deleted spleen B cells showed lower survival rates than wild type cells. This was partially reversed with iron-chelator deferiprone. The loss of T cells was also confirmed by a T cell-specific deletion in Fth(lox/lox) ;CD4-Cre mice. Our data show that ferritin H is required for B and T cell survival by actively reducing the labile iron pool. They further suggest that natural B and T cell maturation is influenced by intracellular iron levels and possibly deregulated in iron excess or deprivation.

Figure 1. Fth deleted mice show reduced number of mature B and T cells.

10–18 week old Mx-Cre transgenic Fth^lox/lox mice or non-transgenic Fth^lox/lox mice were injected 5 times with poly-IC over 8 days and analyzed on day 30. Results for Fth^lox/lox (white) or Fth ^Δ/Δ mice (grey) are shown as % of each cell population normalized to the average in Fth^lox/lox mice (100%). E. Deletion efficiency of Fth mRNA measured in bone marrow, thymus and spleen (n = 9). F–H. Suspensions of bone marrow and spleen cells were stained with antibodies, analyzed by flow cytometry and plotted as numbers in experimental versus control mice (n = 8–9). F. Bone marrow subpopulations were identified as follows: granulocytes (Ter119⁻CD11b⁺GR1^high), monocytes (Ter119⁻CD11b⁺GR1^low), nucleated erythroid cells (Ter119⁺CD4⁻CD8⁻), T cells (CD4⁺ or CD8⁺) and B cells (CD19⁺CD45⁺; pool of precursor and mature B cells). G. Bone marrow B-cell populations (CD19⁺CD45⁺) were stained with relevant antibodies and gated into prepro−/pro-B cells (IgD⁻IgM⁻), pre−/immature B cells (IgD⁻μ⁺ or IgD⁻IgM⁺) and mature B cells (IgD⁺IgM⁺) as shown in panel A. H. Splenic B-cell populations (CD19⁺CD45⁺) were stained with relevant antibodies and gated into transitional (T)1 B cells (IgD^int IgM^hi), T2 B cells (IgD^hi IgM^hi), and mature B cells (IgD^hi IgM^int) as shown in panel B. I–J. Suspensions of thymocytes were stained with antibodies, analyzed by flow cytometry and plotted as numbers in experimental versus control mice (n = 8–9). I. Analysis, of the four major thymocyte subpopulations: double-negative (DN; CD4⁻CD8⁻CD3⁻), double-positive (DP; CD4⁺CD8⁺), CD4 single positive (CD4 SP; CD4⁺CD8⁻) and CD8 single positive (CD8 SP; CD4⁻CD8⁺) as shown in panel C. % of each cell population was normalized to the average in Fth^lox/lox mice. J. Analysis of the four earliest, double negative (DN) thymocyte subsets (CD4⁻CD8⁻CD3⁻): DN1 (CD44⁺CD25⁻), DN2 (CD44⁺CD25⁺), DN3 (CD44⁻CD25⁺) and DN4 (CD44⁻CD25⁻) as shown in panel D. Results are compiled of three independent experiments with each having 2–3 mice per group. ***p<0.0005; **p<0.005; *p<0.05.

Figure 2. Increased LIP and mitochondrial depolarization in bone marrow B-cell populations of Fth deleted mice.

B220⁺ B cells in bone marrow were selected with PE-Cy7-conjugated anti-B220 antibody. Cells were stained with TMRM to assess their mitochondrial polarization and with calcein AM for cell viability and LIP content. Using FACS gating (A), cells with polarized mitochondria (red zone) were analyzed with APC-conjugated anti-CD93 and PE-conjugated anti-CD43 to distinguish B-cell subsets (B). C. Typical FACS profile of total B cells of Fth^lox/lox mice with high calcein staining indicating a low LIP. D. The number of low-LIP cells was virtually unaltered by the iron-chelator deferiprone. In contrast, most cells of Fth ^Δ/Δ mice showed low calcein staining and hence a high LIP (G) that was strongly reduced by deferiprone (H). TMRM showed a similar average staining in Fth^lox/lox (C) and Fth ^Δ/Δ mice (G). However, the number of cells with depolarized mitochondria (low TMRM) was higher in Fth ^Δ/Δ mice correlating with a high LIP (G). E. Adding the protonophore CCCP depolarized mitochondria in all cells. F. B220⁺ B cells with polarized mitochondria of Fth^lox/lox mice (red zone in C) were analyzed for B cell subsets. I. B220⁺ B cells with polarized mitochondria of Fth ^Δ/Δ mice (red zone in G) were analyzed for B cell subsets. J–K. The same cells were subdivided into cells with low and high LIP (above or below blue line in the red zone) to determine the subset composition of each fraction. L. Percent cells in each B-cell subset of Fth ^Δ/Δ mice that show a high LIP. Few high LIP cells (<10%) were detected in B-cell subsets of Fth^lox/lox control mice, and are not reported. M. Percent cells with low TMRM fluorescence indicating mitochondrial depolarization in each B-cell subset of Fth^lox/lox (white) and Fth ^Δ/Δ mice (grey). N. Graphical representation of all data gathered by F and I–K. B cells in each subset are expressed as % of cells analyzed in Fth^lox/lox (white), Fth ^Δ/Δ mice (medium grey), or Fth ^Δ/Δ mice with low LIP (light grey) or high LIP (dark grey). Subsets for each color add up to 100%. Results are average values of 8 mice ± SD. ***p<0.0005; **p<0.005; *p<0.05.

Figure 3. Increased LIP and mitochondrial depolarization in thymocytes of Fth deleted mice.

Thymocytes were stained with Pacific Blue-conjugated anti-CD4 and Alexa Fluor 700-A conjugated anti-CD8α to analyze their state of T-cell differentiation, followed by TMRM for mitochondrial depolarization and calcein AM for cell viability and LIP content. FACS analysis was carried out on cells from Fth^lox/lox (A–E) and Fth ^Δ/Δ mice (F–J). C–E and H–J show a representative FACS gating used to distinguish double-negative cells in lower left zone (DN; CD4^−/CD8α^–), double-positive cells in upper right zone (DP; CD4⁺/CD8α⁺), single-positive cells for CD4 in upper left zone (CD4 SP; CD4⁺/CD8α^–), and single-positive cells for CD8α in lower right zone (CD8 SP; CD4^−/CD8α⁺). Most T cells showed a high calcein staining in Fth^lox/lox mice representing a low LIP (A). Only about 10% of cells with polarized mitochondria showed low calcein staining, which was unquenched by the iron chelator deferiprone (B). In contrast, in Fth ^Δ/Δ mice about 80% of cells with polarized mitochondria showed a low calcein staining representing a high LIP (F) that was unquenched by deferiprone (G). Double staining with mitochondrial depolarization marker TMRM showed a similar average staining in Fthl^ox/lox and Fth ^Δ/Δ mice in spite of a very different LIP. Only a small fraction of cells showed depolarized mitochondria. Adding the protonophore CCCP depolarized mitochondria in all cells (not shown). For the analysis of T cell subsets (C–E and H–J), only cells with polarized mitochondria (red zone of A and F) or sub-fractions thereof with low LIP (above the blue line) or high LIP level (below the blue line) were analyzed. K. Percent thymocytes with polarized mitochondria with a high LIP in total T cells or T-cell subsets of Fth^lox/lox (white) and Fth ^Δ/Δ mice (grey). L. Percent cells with a low TMRM fluorescence indicating depolarization in each subset of Fth^lox/lox (white) and Fth ^Δ/Δ mice (grey). M. Graphical representation of all subset data obtained in C–E and H–J. T cells in each subset expressed as % of T cells with polarized mitochondria in the low LIP (white), total (medium grey) or high LIP (dark grey) fraction of Fth^lox/lox and Fth ^Δ/Δ mice. Subsets for each color and separate genotype add up to 100%. Results are average values of 7 or 8 mice ± SD. ***p<0.0005; **p<0.005; *p<0.05.

Figure 4. Reduced mature B-cell number in mice with a B-cell specific Fth deletion.

Fth^+/+;CD19-Cre⁺ (white) and Fth ^Δ/Δ (grey) mice carried the CD19-Cre allele for B-cell specific deletion and Rosa-EYFP allele as a marker for cells where CD19-Cre is active. B cells were in all instances identified as B220⁺, CD19⁺ and EYFP⁺. A. Viable B cells among total cells were determined in different lymphoid tissues. B. B cells were stained with dihydroethidium to determine the % cells with ROS activity above background. C. Bone marrow B cells were divided into 3 subsets with antibodies against CD93 and CD43 as shown in Fig. 2B: CD93⁺/CD43⁺, prepro−/pro-; CD93⁺/CD43⁻, pre−/immature; and CD93⁻/CD43⁻, mature B cells. Spleen B cells were divided into 4 subsets with antibodies against CD21, CD23, and IgM, separating transitional stages 1 and 2, follicular, and marginal zone B cells. D. Staining with TMRM was used to determine mitochondrial depolarization for each subset. Values represent the % of the parent population (gating as shown in Fig. 2A). Results are average values of 8 mice ± SD. ***p<0.0005; **p<0.005; *p<0.05.

Figure 5. Proliferation and cell division of B cells in bone marrow of mice with CD19-Cre mediated Fth deletion.

Cells of Fth^+/+;CD19-Cre⁺ (white) and Fth ^Δ/Δ (grey) mice were identified as B220⁺, CD19⁺ and EYFP⁺. A. Cells in S and G₂/M phases of the cell cycle were analyzed by FACS. B. Mice were exposed to BrdU in vivo for 12 h prior to the isolation of bone marrow B cells. BrdU was detected by FACS. Results are average values of 3 mice ± SD. ***p<0.0005; **p<0.005.

Figure 6. Fth deletion blocks BAFF-supported survival of spleen B cells in vitro.

B cells were isolated from either Fth^lox/lox and Fth ^Δ/Δ mice at 15–20 weeks (w) or CD19-Cre⁺;Fth^+/+ and Fth ^Δ/Δ mice at 50–70 w, and compared in their response to BAFF. For this, CD19⁺ splenocytes were separated on magnetic beads and cultured in vitro in absence (white) or presence (grey) of BAFF (20 ng/ml) for 72 h. A. Cell viability was determined by FACS based on scatter 72 h after BAFF addition and expressed as % survival compared to plated cells. The additional strain secreting TACI-Fc blocking BAFF was used as a negative control for BAFF regulation and analyzed in duplicate. B. EYFP⁺ B cells in the viable cell population (A) were measured 72 h after BAFF addition and plotted as % EYFP⁺ among viable B cells. C. % of viable CD19⁺ cells that harbor the Fth deletion as determined by genomic PCR at time 0 h and 72 h of cell culture. D. % of viable CD19⁺ B cells from CD19-Cre⁺;Rosa-EYFP;Fth^+/+ and CD19-Cre⁺;Rosa-EYFP;Fth ^Δ/Δ that are EYFP⁺ at time 0 h and 72 h of cell culture. E. Viability of CD19⁺ B cells in absence or presence of iron chelator deferiprone and BAFF after 24 h of culture. F. Viability of 15–20 w old EYFP⁺ B cells in absence or presence of 300 µM deferiprone and BAFF (20 ng/ml) after 24 h of culture. In experiments A, B, E, and F, the 15–20 w old control mice were Fth^lox/lox littermates without CD19-Cre, while the 50–70 w old control mice had a Fth^+/+;CD19-Cre⁺ genotype. All cell cultures were analyzed in duplicates. Results are average values of 3 to 5 independent experiments ± SD. ***p<0.0005; **p<0.005; *p<0.05.

Figure 7. CD4-Cre mediated Fth deletion induces a reduction of T cells in thymus and spleen concomitant with high LIP and mitochondrial depolarization.

Lymphocytes of thymus and spleen of 5–7 weeks old Fth^+/+;CD4-Cre⁺ or Fth^lox/lox control mice (white) and Fth ^Δ/Δ mice (grey) were stained with calcein AM and TMRM, and in addition with anti-CD4 and anti-CD8α antibodies as detailed in Fig. 3. They were further separated into high- and low-level CD24 expressing cells. No significant differences were visible between 3 Fth^+/+;CD4-Cre mice and 3 Fth^lox/lox control mice, and data were pooled. A. Total viable cell number in thymus and spleen. B. Number of cells in T cell subsets in thymus and spleen of Fth^Δ/Δ mice relative to control mice, set as 100%. C. % cells with low calcein staining due to quenching by high LIP. D. % cells with low TMRM staining that is a sign of mitochondrial depolarization. Results are average values of 6 mice ± SD. ***p<0.0005; **p<0.005; *p<0.05.