Project description:Extravasated RBCs and their main component heme could function as specific microenvironmental drivers of TAM differentiation. We established two subcutaneous matrigel plug models of tissue microhemorrhage, which allow post-experimental recovery of invading cells at high quality for downstream analysis.
Project description:Bulk RNA sequencing revealed that heme exposure induced the phenotype of Hmox1high, Marcohigh, and MHC class IIlow macrophages, demonstrating a strong expression of NRF2-regulated. However, these cells showed weak expression of the TAM marker genes Arg1 and Spp1, indicating that heme treatment alone was insufficient to induce the full heme-TAM phenotype.
Project description:Heme-activation of NRF2 is a strong anti-inflammatory signal in macrophages, and analyses in this study indicated that the expressed transcriptome in heme-TAMs was consistently enriched for NRF2 target genes. We have therefore delineated the role of NRF2 in a series of experiments with Nrf2 knockout BMDMs, leading to a locked NRF2-inactive state, and macrophages with a knockout of the cytoplasmic NRF2 capture protein KEAP1, leading to a locked NRF2-active state, irrespective of the presence or absence of heme. To demonstrate that activated NRF2 is sufficient to drive heme-TAM transformation, we analyzed Keap1 knockout macrophages.
Project description:Macrophages are central in regulating iron homeostasis. Transcription repressor Bach2 regulates by heme. Here we investigated the relationship between heme-regulated Bach2 and macrophage in spleen. We found that gene expression were not many change between WT and Bach2 knock out mice in red-pulp macrophage.Our results suggest that the function of the red-pulp macrophage is not dependent on according to expression of Bach2.
Project description:Heme is an erythrocyte-derived toxin that drives disease progression in hemolytic anemias. During hemolysis, specialized bone marrow-derived macrophages with a high heme-metabolism capacity orchestrate disease adaptation by removing damaged erythrocytes and heme-protein complexes from the blood and supporting iron recycling for erythropoiesis. Here, we performed single-cell RNA sequencing with RNA velocity analysis of GM-CSF-supplemented mouse bone marrow cultures to assess myeloid differentiation under heme stress. We found that heme-activated NRF2 signaling shifted the differentiation trajectories of cells towards antioxidant, iron-recycling macrophages at the expense of dendritic cells, as these cells were selectively deficient in heme-exposed bone marrow cultures. Heme eliminated the capacity of GM-CSF-supplemented bone marrow cultures to activate antigen-specific T cells. The generation of functionally competent dendritic cells was restored by NRF2 loss. The heme-induced phenotype was reproduced in hemolytic mice with sickle cell disease and spherocytosis and associated with reduced dendritic cell functions in the spleen. Our data provide a novel mechanistic underpinning how hemolytic stress may provoke hyposplenism-related secondary immunodeficiency, which is a critical determinant of mortality in patients with genetic hemolytic anemias.
Project description:Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by luminal cytotoxicity and reactive oxygen species. This surface injury is overcompensated by hyperproliferation and hyperplasia of crypt cells. Transcriptome analysis of mucosa of heme-fed mice showed, besides stress- and proliferation-related genes, many upregulated lipid metabolism-related PPARα target genes. The aim of this study was to investigate the role of PPARα in heme-induced hyperproliferation and hyperplasia. Male PPARα KO and WT mice received a purified diet with or without heme. As PPARα is proposed to protect against oxidative stress and lipid peroxidation, we hypothesized that the absence of PPARα leads to more surface injury and crypt hyperproliferation in the colon upon heme-feeding. Heme induced luminal cytotoxicity and lipid peroxidation and colonic hyperproliferation and hyperplasia to the same extent in WT and KO mice. Transcriptome analysis of colonic mucosa confirmed similar heme-induced hyperproliferation in WT and KO mice. Stainings for alkaline phosphatase activity and expression levels of Vanin-1 and Nrf2-targets indicated a compromised antioxidant defense in heme-fed KO mice. Our results suggest that the protective role of PPARα in antioxidant defense involves the Nrf2-inhibitor Fosl1, which is upregulated by heme in PPARα KO mice. We conclude that PPARα plays a protective role in colon against oxidative stress, but PPARα does not mediate heme-induced hyperproliferation. This implies that oxidative stress of surface cells is not the main determinant of heme-induced hyperproliferation and hyperplasia.
Project description:Heme is an erythrocyte-derived toxin that drives disease progression in hemolytic anemias. During hemolysis, specialized bone marrow-derived macrophages with a high heme-metabolism capacity orchestrate disease adaptation by removing damaged erythrocytes and heme-protein complexes from the blood and supporting iron recycling for erythropoiesis. Here, we performed single-cell RNA sequencing with RNA velocity analysis of GM-CSF-supplemented mouse bone marrow cultures to assess myeloid differentiation under heme stress. We found that heme-activated NRF2 signaling shifted the differentiation trajectories of cells towards antioxidant, iron-recycling macrophages at the expense of dendritic cells, as these cells were selectively deficient in heme-exposed bone marrow cultures. Heme eliminated the capacity of GM-CSF-supplemented bone marrow cultures to activate antigen-specific T cells. The generation of functionally competent dendritic cells was restored by NRF2 loss. The heme-induced phenotype was reproduced in hemolytic mice with sickle cell disease and spherocytosis and associated with reduced dendritic cell functions in the spleen. Our data provide a novel mechanistic underpinning how hemolytic stress may provoke hyposplenism-related secondary immunodeficiency, which is a critical determinant of mortality in patients with genetic hemolytic anemias.
Project description:Heme is an erythrocyte-derived toxin that drives disease progression in hemolytic anemias. During hemolysis, specialized bone marrow-derived macrophages with a high heme-metabolism capacity orchestrate disease adaptation by removing damaged erythrocytes and heme-protein complexes from the blood and supporting iron recycling for erythropoiesis. Here, we performed single-cell RNA sequencing with RNA velocity analysis of GM-CSF-supplemented mouse bone marrow cultures to assess myeloid differentiation under heme stress. We found that heme-activated NRF2 signaling shifted the differentiation trajectories of cells towards antioxidant, iron-recycling macrophages at the expense of dendritic cells, as these cells were selectively deficient in heme-exposed bone marrow cultures. Heme eliminated the capacity of GM-CSF-supplemented bone marrow cultures to activate antigen-specific T cells. The generation of functionally competent dendritic cells was restored by NRF2 loss. The heme-induced phenotype was reproduced in hemolytic mice with sickle cell disease and spherocytosis and associated with reduced dendritic cell functions in the spleen. Our data provide a novel mechanistic underpinning how hemolytic stress may provoke hyposplenism-related secondary immunodeficiency, which is a critical determinant of mortality in patients with genetic hemolytic anemias.