Analysis of mice with conditional ferritin H deletion

Iron is a transitional metal required by virtually all organisms as a dietary micromineral, indispensable for cellular survival and proliferation. The management of iron absorption and distribution in the organism and inside the cell must be tightly regulated in order to avoid the deleterious consequences of free iron-induced oxidative stress. Ferritin is the major protein responsible for iron storage and release of intracellular iron. Ferritin shells are formed from two types of subunits, known as H and L. The present work investigates the effects of conditional ferritin H deletion in mice. A broad ferritin H deletion in liver, spleen, bone marrow and thymus results in an alteration of iron metabolism, characterized by increased transferrin saturation and hepcidin mRNA levels and decreased liver iron deposits. Iron loading prior to deletion leads to liver failure early after deletion, showing that ferritin H is indispensable for limiting iron toxicity through iron sequestration. A hepatocyte-specific deletion fails to reproduce the same phenotype, suggesting an important role for Kupffer macrophages in liver iron detoxification. Ferritin H is also required for B lymphocyte survival, as indicated by the loss of mature B cells in an CD19-specific ferritin H deletion mouse strain, where this population is substantially reduced because of massive reactive oxygen species generation. Conditional deletion in heart leads to fibrosis, increased oxidative stress and to a switch towards a gene expression profile characteristic for cardiac hypertrophy, while iron loading prior to deletion causes a dramatic alteration of the cardiac output function and ultimately to heart failure. An intestine-specific ferritin H deletion results in a typical hemochromatotic phenotype, characterized by increased transferrin saturation and liver iron stores, increased hepcidin expression and decreased IRP2 activity. Hepcidin-mediated ferroportin downregulation at protein level is also shown not to be sufficient to limit the intestinal iron export. An modified version of the current model of intestinal iron absorption is proposed, that includes the function of ferritin H as an iron sink in intestine. This work contributes to a better understanding of iron metabolism in general and intestinal iron absorption in particular, and it might have an impact on the development of treatments against human hemochomatosis and anemia

LAPTM4b recruits the LAT1-4F2hc Leu transporter to lysosomes and promotes mTORC1 activation

Mammalian target of rapamycin 1 (mTORC1), a master regulator of cellular growth, is activated downstream of growth factors, energy signalling and intracellular essential amino acids (EAAs) such as Leu. mTORC1 activation occurs at the lysosomal membrane, and involves V-ATPase stimulation by intra-lysosomal EAA (inside-out activation), leading to activation of the Ragulator, RagA/B-GTP and mTORC1 via Rheb-GTP. How Leu enters the lysosomes is unknown. Here we identified the lysosomal protein LAPTM4b as a binding partner for the Leu transporter, LAT1-4F2hc (SLC7A5-SLAC3A2). We show that LAPTM4b recruits LAT1-4F2hc to lysosomes, leading to uptake of Leu into lysosomes, and is required for mTORC1 activation via V-ATPase following EAA or Leu stimulation. These results demonstrate a functional Leu transporter at the lysosome, and help explain the inside-out lysosomal activation of mTORC1 by Leu/EAA

Real-time functional characterization of cationic amino acid transporters using a new FRET sensor

L-arginine is a semi-essential amino acid that serves as precursor for the production of urea, nitric oxide (NO), polyamines, and other biologically important metabolites. Hence, a fast and reliable assessment of its intracellular concentration changes is highly desirable. Here, we report on a genetically encoded Förster resonance energy transfer (FRET)-based arginine nanosensor that employs the arginine repressor/activator ahrC gene from Bacillus subtilis. This new nanosensor was expressed in HEK293T cells, and experiments with cell lysate showed that it binds L-arginine with high specificity and with a K d of ∼177 μM. Live imaging experiments showed that the nanosensor was expressed throughout the cytoplasm and displayed a half maximal FRET increase at an extracellular L-arginine concentration of ∼22 μM. By expressing the nanosensor together with SLC7A1, SLC7A2B, or SLC7A3 cationic amino acid transporters (CAT1-3), it was shown that L-arginine was imported at a similar rate via SLC7A1 and SLC7A2B and slower via SLC7A3. In contrast, upon withdrawal of extracellular L-arginine, intracellular levels decreased as fast in SLC7A3-expressing cells compared with SLC7A1, but the efflux was slower via SLC7A2B. SLC7A4 (CAT4) could not be convincingly shown to transport L-arginine. We also demonstrated the impact of membrane potential on L-arginine transport and showed that physiological concentrations of symmetrical and asymmetrical dimethylarginine do not significantly interfere with L-arginine transport through SLC7A1. Our results demonstrate that the FRET nanosensor can be used to assess L-arginine transport through plasma membrane in real time

Fe-59-distribution in conditional ferritin-H-deleted mice

The objective was to explore how ferritin-H deletion influences Fe-59-distribution and excretion-kinetics in mice. Kinetics of Fe-59-release from organs, whole-body excretion, and distribution-kinetics of intravenously injected Fe-59 trace amounts were compared in iron-deficient and iron-replete mice with (Fth(Delta/Delta)) and without (Fth(lox/lox)) conditional Mx-Cre-induced ferritin-H deletion. Fe-59 was released from spleen and liver beginning on day 2 and day 5 after ferritin-H deletion, respectively, but was not excreted from the body. Plasma-Fe-59 was cleared significantly faster in iron-deficient Fth(Delta/Delta)-mice than in iron-adequate Fth(lox/lox)-controls. Fe-59-distribution showed a transient peak (e.g., in heart, kidney, muscle) in Fth(lox/lox) control mice, but not in ferritin-H deleted Fth(Delta/Delta) mice 24 hours after Fe-59 injection. Fe-59 uptake into the liver and spleen was significantly lower in iron-deficient Fth(Delta/Delta) than in Fth(lox/lox) mice 24 hours and 7 days after injection, respectively, and rapidly appeared in circulating erythrocytes instead. The rate of Fe-59 release after ferritin-H deletion supports earlier data on ferritin turnover in mammals; released Fe-59 is not excreted from the body. Instead, Fe-59 is channeled into erythropoiesis and circulating erythrocytes significantly more extensively and faster. Along with a lack of transient interim Fe-59 storage (e.g., in the heart and kidney), this finding is evidence for ferritin-related iron storage-capacity affecting rate and extent of iron utilization. (C) 2014 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc

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

<p>Lymphocytes of thymus and spleen of 5–7 weeks old <i>Fth^+/+</i>;CD4-Cre<i>⁺</i> or <i>Fth^lox/lox</i> control mice (<i>white</i>) and <i>Fth</i>^Δ/Δ mice (<i>grey</i>) 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 <i>Fth^+/+</i>;CD4-Cre mice and 3 <i>Fth^lox/lox</i> control mice, and data were pooled. <b>A.</b> Total viable cell number in thymus and spleen. <b>B.</b> Number of cells in T cell subsets in thymus and spleen of Fth^Δ/Δ mice relative to control mice, set as 100%. <b>C.</b> % cells with low calcein staining due to quenching by high LIP. <b>D.</b> % 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.</p

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

<p>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 <i>Fth^lox/lox</i> (<b>A–E</b>) and <i>Fth</i>^Δ/Δ mice (<b>F–J</b>). <b>C–E</b> and <b>H–J</b> 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 <i>Fth^lox/lox</i> mice representing a low LIP (<b>A</b>). Only about 10% of cells with polarized mitochondria showed low calcein staining, which was unquenched by the iron chelator deferiprone (<b>B</b>). In contrast, in <i>Fth</i>^Δ/Δ mice about 80% of cells with polarized mitochondria showed a low calcein staining representing a high LIP (<b>F</b>) that was unquenched by deferiprone (<b>G</b>). Double staining with mitochondrial depolarization marker TMRM showed a similar average staining in <i>Fthl^ox/lox</i> and <i>Fth</i>^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 (<b>C–E</b> and <b>H–J</b>), only cells with polarized mitochondria (red zone of <b>A</b> and <b>F</b>) or sub-fractions thereof with low LIP (above the blue line) or high LIP level (below the blue line) were analyzed. <b>K.</b> Percent thymocytes with polarized mitochondria with a high LIP in total T cells or T-cell subsets of <i>Fth^lox/lox</i> (<i>white</i>) and <i>Fth</i>^Δ/Δ mice (<i>grey</i>). <b>L.</b> Percent cells with a low TMRM fluorescence indicating depolarization in each subset of <i>Fth^lox/lox</i> (w<i>hite</i>) and <i>Fth</i>^Δ/Δ mice (<i>grey</i>). <b>M.</b> Graphical representation of all subset data obtained in <b>C–E</b> and <b>H–J</b>. T cells in each subset expressed as % of T cells with polarized mitochondria in the low LIP (<i>white</i>), total (<i>medium grey</i>) or high LIP (<i>dark grey</i>) fraction of <i>Fth^lox/lox</i> and <i>Fth</i>^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.</p

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

<p>Cells of <i>Fth^+/+</i>;CD19-Cre<i>⁺</i> (<i>white</i>) and <i>Fth</i>^Δ/Δ (<i>grey</i>) mice were identified as B220⁺, CD19⁺ and EYFP⁺. <b>A.</b> Cells in S and G₂/M phases of the cell cycle were analyzed by FACS. <b>B.</b> Mice were exposed to BrdU <i>in vivo</i> 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.</p

Fth deletion blocks BAFF-supported survival of spleen B cells <i>in vitro</i>.

<p>B cells were isolated from either <i>Fth^lox/lox</i> and <i>Fth</i>^Δ/Δ mice at 15–20 weeks (w) or CD19-Cre<i>⁺</i>;<i>Fth^+/+</i> and <i>Fth</i>^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 <i>in vitro</i> in absence (<i>white</i>) or presence (<i>grey</i>) of BAFF (20 ng/ml) for 72 h. <b>A.</b> 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>B.</b> EYFP⁺ B cells in the viable cell population (<b>A</b>) were measured 72 h after BAFF addition and plotted as % EYFP⁺ among viable B cells. <b>C.</b> % of viable CD19⁺ cells that harbor the Fth deletion as determined by genomic PCR at time 0 h and 72 h of cell culture. <b>D.</b> % of viable CD19⁺ B cells from CD19-Cre<i>⁺</i>;Rosa-EYFP;<i>Fth^+/+</i> and CD19-Cre<i>⁺</i>;Rosa-EYFP;<i>Fth</i>^Δ/Δ that are EYFP⁺ at time 0 h and 72 h of cell culture. <b>E.</b> Viability of CD19⁺ B cells in absence or presence of iron chelator deferiprone and BAFF after 24 h of culture. <b>F.</b> 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 <i>Fth^lox/lox</i> littermates without CD19-Cre, while the 50–70 w old control mice had a <i>Fth^+/+</i>;CD19-Cre<i>⁺</i> 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.</p

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

<p>10–18 week old Mx-Cre transgenic <i>Fth^lox/lox</i> mice or non-transgenic <i>Fth^lox/lox</i> mice were injected 5 times with poly-IC over 8 days and analyzed on day 30. Results for <i>Fth^lox/lox</i> (<i>white</i>) or <i>Fth</i>^Δ/Δ mice (<i>grey</i>) are shown as % of each cell population normalized to the average in <i>Fth^lox/lox</i> mice (100%). <b>E.</b> Deletion efficiency of Fth mRNA measured in bone marrow, thymus and spleen (n = 9). <b>F–H.</b> 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). <b>F.</b> 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). <b>G.</b> 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 <b>A</b>. <b>H.</b> 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>B</b>. <b>I–J.</b> Suspensions of thymocytes were stained with antibodies, analyzed by flow cytometry and plotted as numbers in experimental versus control mice (n = 8–9). <b>I.</b> 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 <b>C</b>. % of each cell population was normalized to the average in <i>Fth^lox/lox</i> mice. <b>J.</b> 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 <b>D.</b> Results are compiled of three independent experiments with each having 2–3 mice per group. ***p<0.0005; **p<0.005; *p<0.05.</p

Metabolic Adaptation to Tissue Iron Overload Confers Tolerance to Malaria

Disease tolerance is a defense strategy that limits the fitness costs of infection irrespectively of pathogen burden. While restricting iron (Fe) availability to pathogens is perceived as a host defense strategy, the resulting tissue Fe overload can be cytotoxic and promote tissue damage to exacerbate disease severity. Examining this interplay during malaria, the disease caused by Plasmodium infection, we find that expression of the Fe sequestering protein ferritin H chain (FtH) in mice, and ferritin in humans, is associated with reduced tissue damage irrespectively of pathogen burden. FtH protection relies on its ferroxidase activity, which prevents labile Fe from sustaining proapoptotic c-Jun N-terminal kinase (JNK) activation. FtH expression is inhibited by JNK activation, promoting tissue Fe overload, tissue damage, and malaria severity. Mimicking FtH's antioxidant effect or inhibiting JNK activation pharmacologically confers therapeutic tolerance to malaria in mice. Thus, FtH provides metabolic adaptation to tissue Fe overload, conferring tolerance to malaria