Lehrke, Michael Jonathan

Abstract Introduction The mitochondrial transporter ABCB7 is essential for exporting iron-sulfur (Fe-S) clusters, a function known to be critical in B cells. This study was conducted to investigate the broader, previously unknown roles of ABCB7 in regulating the overall lymphocyte development and homeostasis. Methods The study utilized conditional knockout (cKO) mouse models (CD2-iCre and Mb1-Cre) to create a specific deficiency of ABCB7 in certain immune populations. To determine if the observed effects were due to ABCB7 deletion in myeloid cells, two further models (CD2i-Cre ABCB7 cKO/C/EBPα and CD2-iCre LSL TMT ABCB7 cKO) were developed. The effects of blocking the cytokine G-CSF were also examined to understand its role in the observed myelopoiesis. Results ABCB7 deficiency was found to dramatically impair erythropoiesis while simultaneously driving emergency myelopoiesis. We confirmed that the myeloid expansion was not a direct result of ABCB7 loss in myeloid cells. A significant autoimmune phenotype also developed, characterized by hyperactivated T cells and a sharp decrease in peripheral regulatory T cells (Tregs). This Treg instability was linked to decreased thymic expression of key stability genes (like CD28 and PD-1) and reduced O-GlcNAc modification in T cells, a process crucial for maintaining Treg lineage. Conclusion The findings establish ABCB7 as a pivotal regulator of hematopoietic balance, linking mitochondrial function to both myeloid expansion and Treg stability. Funding Source T32AI170478 Topic Categories Hematopoiesis and Immune System Development (HEM)

Abstract ABCB7 exports iron-sulfur (Fe-S) clusters from the mitochondria into the cytoplasm. Conditional deletion of ABCB7 revealed that it is required for peripheral T cell homeostasis. CD4-cre ABCB7 cKO mice have normal thymic development, but a severe reduction in peripheral T cell numbers. A majority of the peripheral T cells in the ABCB7 cKO mice were recent thymic emigrants (RTEs), indicating a loss of more mature T cells in the periphery. T cell maturation markers were found to be normal and stimulation of ABCB7 cKO RTEs revealed normal levels of TNFα production. These data indicate that the loss of peripheral T cells in CD4-cre ABCB7 cKO mice was not due to a defect in development or maturation but a defect in peripheral homeostasis. ABCB7-deficient T cells did not have evidence of elevated apoptosis. We demonstrated iron accumulation was occurring in ABCB7-deficient peripheral T cells using the metal-specific dye Phen Green SK. Additionally, lipid peroxides levels were elevated in the ABCB7 cKO T cells. Lipid peroxides are a hallmark of ferroptosis, an iron-dependent form of regulated cell death. Expression of GPX4, the master regulator of ferroptosis, was normal in ABCB7 cKO T cells. In addition, levels of glutathione, which counteracts lipid peroxides were found to be normal, indicating the ferroptosis protection pathway was intact and that the iron overload was causing ferroptosis. Interestingly, treatment with the iron chelator deferoxamine did not rescue peripheral T cells in the CD4-cre ABCB7 cKO mice. Vitamin E, a potent antioxidant, also failed to rescue peripheral T cell homeostasis. Thus, ABCB7 is required to maintain iron homeostasis and its loss leads to peripheral T cell ferroptosis.

Abstract Iron-sulfur (Fe-S) clusters are cofactors essential for the activity of numerous enzymes including DNA polymerases, helicases, and glycosylases. They are synthesized in the mitochondria as Fe-S intermediates and are exported to the cytoplasm for maturation by the mitochondrial transporter ABCB7. Here, we demonstrate that ABCB7 is required for bone marrow B cell development, proliferation, and class switch recombination, but is dispensable for peripheral B cell homeostasis in mice. Conditional deletion of ABCB7 using Mb1-cre resulted in a severe block in bone marrow B cell development at the pro-B cell stage. The loss of ABCB7 did not alter expression of transcription factors required for B cell specification or commitment. While increased intracellular iron was observed in ABCB7-deficient pro-B cells, this did not lead to increased cellular or mitochondrial reactive oxygen species, ferroptosis, or apoptosis. Interestingly, loss of ABCB7 led to replication-induced DNA damage in pro-B cells, independent of VDJ recombination, and these cells had evidence of slowed DNA replication. Interestingly, ABCB7 appeared to be dispensable for splenic B cell homeostasis, as CD23-cre ABCB7 cKO mice had normal splenic B cell populations. However, stimulated ABCB7-deficient splenic B cells from CD23-cre ABCB7 cKO mice had a striking loss of proliferation and a defect in class switching. Thus, ABCB7 is essential for early B cell development, proliferation, and class switch recombination. Supported by 1R21 AI157328-01 to V.S.S., T32AI007425 to M.J.L.

Abstract ABCB7 exports iron-sulfur (Fe-S) cluster intermediates from the mitochondria to the cytoplasm. Loss of ABCB7 results in mitochondrial iron accumulation in some cell types. We found that ABCB7 is required for peripheral T cell homeostasis, as CD4-cre ABCB7 cKO have normal thymic T cell development but a severe reduction in peripheral T cells, with a majority of peripheral T cells being recent thymic emigrants. The metal-specific dye Phen Green SK indicated iron accumulation was occurring in the absence of ABCB7. We hypothesized that ABCB7-deficient T cells are eliminated by ferroptosis, an iron-dependent form of regulated cell death that is marked by generation of lipid peroxides. Both Bodipy-C11 and Liperfluo, dyes specific for lipid peroxides, were elevated in ABCB7 cKO T cells. Interestingly, expression of GPX4, the master regulator of ferroptosis, was found to be normal in ABCB7-deficient T cells. A high vitamin E diet, which can reduce ferroptosis in GPX4-deficient T cells, did not restore peripheral T cell numbers in CD4-cre ABCB7 cKO mice. Additionally, treatment with the iron chelator deferoxamine did not restore peripheral T cell numbers. Thus, more than one mechanism may be involved in regulating T cell survival in the absence of ABCB7.

Abstract ABCB7 is a mitochondrial transporter that exports iron-sulfur (Fe-S) cluster intermediates from the mitochondria to the cytoplasm. Using CD2-iCre ABCB7 cKO mice, we found ABCB7 is required for early B cell development. These mice have a severe block in pro-B cell development at the Fr. C to Fr. C′ transition, with reduced expression of intracellular μ and pre-BCR. CD19-cre and CD23-cre ABCB7 cKO mice have normal B cell development and peripheral B cell populations, indicating ABCB7 is required only during early B cell development. We observed reduced expression of Runx1, Ikaros, Foxo1, E47 (E2A), and Pax5 in CD2-iCre ABCB7 cKO pro-B cells. Interestingly, we also observed decreased BACH2 expression, which indicates elevated heme synthesis is occurring in the absence of ABCB7. We hypothesize that in the absence of ABCB7, developing B cells enhance heme synthesis to dispose of accumulating mitochondrial iron. Using a hen egg lysozyme BCR transgene, we were able to rescue B cell development in CD2-iCre ABCB7 cKO mice. Intriguingly, at the age of 5–8 weeks old, CD2-iCre ABCB7 cKO mice become lethargic and hunched. CBCs reveal an increase in peripheral blood neutrophils. Both the spleen and bone marrow become highly infiltrated by CD11b+Ly6Glow/+Ly6C− cells that are immature and mature neutrophils. As other knockout mice with pro-B cell blocks do not have neutrophilia, we considered if increased iron or heme is responsible for our observation. As heme can stimulate TLR4, we propose that excess heme from developing ABCB7-deficent B cells activates macrophages. In support of this, hemin induced GM-CSF production in BMDM cultures. We are currently exploring the mechanism by which excess heme production can stimulate the production of neutrophils.

Iron-sulfur (Fe-S) clusters are cofactors essential for the activity of numerous enzymes including DNA polymerases, helicases, and glycosylases. They are synthesized in the mitochondria as Fe-S intermediates and are exported to the cytoplasm for maturation by the mitochondrial transporter ABCB7. Here, we demonstrate that ABCB7 is required for bone marrow B cell development, proliferation, and class switch recombination, but is dispensable for peripheral B cell homeostasis in mice. Conditional deletion of ABCB7 using Mb1-cre resulted in a severe block in bone marrow B cell development at the pro-B cell stage. The loss of ABCB7 did not alter expression of transcription factors required for B cell specification or commitment. While increased intracellular iron was observed in ABCB7-deficient pro-B cells, this did not lead to increased cellular or mitochondrial reactive oxygen species, ferroptosis, or apoptosis. Interestingly, loss of ABCB7 led to replication-induced DNA damage in pro-B cells, independent of VDJ recombination, and these cells had evidence of slowed DNA replication. Stimulated ABCB7-deficient splenic B cells from CD23-cre mice also had a striking loss of proliferation and a defect in class switching. Thus, ABCB7 is essential for early B cell development, proliferation, and class switch recombination.

Hematopoietic cell homing after hematopoietic cell transplant (HCT) is governed by several pathways involving marrow niche cells that are evoked after pre-HCT conditioning. To understand the factors that play a role in homing, we performed expression analysis on the zebrafish marrow niche cells following conditioning. We determined that the non-collagenous protein extracellular matrix related protein dermatopontin (Dpt) was upregulated seven-fold in response to irradiation. Studies in mice revealed DPT induction both with radiation and lipopolysaccharide exposure. Interestingly, we found that co-incubation of zebrafish or murine hematopoietic cells with rDPT impedes hematopoietic stem and progenitor cell homing by 50% and 86%, respectively. Similarly, this translated into a 24% reduction in long term engraftment (versus control, p = 0.01). We found DPT to interact with VLA-4 and block hematopoietic - endothelial cell adhesion and transendothelial migration. Finally, a DPT knockout mouse displayed a 60% increase in homing of hematopoietic cells versus wildtype (p = 0.03) with slight improvement in long-term LSK-SLAM engraftment (2-fold, p = 0.04). These data show that the extracellular matrix (ECM)-related protein DPT increases with radiation and transiently impedes the transendothelial migration of hematopoietic cells to the marrow.

Abstract NKAP is a multifunctional nuclear protein that associates with the histone deacetylase HDAC3. Although both NKAP and HDAC3 are critical for hematopoietic stem cell (HSC) maintenance and survival, it was not known whether these two proteins work together. To assess the importance of their association in vivo, serial truncation and alanine scanning was performed on NKAP to identify the minimal binding site for HDAC3. Mutation of either Y352 or F347 to alanine abrogated the association of NKAP with HDAC3, but did not alter NKAP localization or expression. Using a linked conditional deletion/re-expression system in vivo, we demonstrated that re-expression of the Y352A NKAP mutant failed to restore HSC maintenance and survival in mice when endogenous NKAP expression was eliminated using Mx1-cre and poly-IC, whereas re-expression of wild type NKAP maintained the HSC pool. However, Y352A NKAP did restore proliferation in murine embryonic fibroblasts when endogenous NKAP expression was eliminated using ER-cre and tamoxifen. Therefore, Y352 in NKAP is critical for association with HDAC3 and for HSC maintenance and survival but is not important for proliferation of murine embryonic fibroblasts, demonstrating that NKAP functions in different complexes in different cell types.