Placental macrophages, Hofbauer cells (HBC) are the only fetal immune cell population within the stroma of healthy placenta along pregnancy. They are central players in maintaining immune tolerance during pregnancy. Immunometabolism emerged a few years ago as a new field that integrates cellular metabolism with immune responses, however, the immunometabolism of HBC has not been explored yet. Here we studied the sex-specific differences in the phenotypic, functional and immunometabolic profile of HBC.
HBC were isolated from human term placentas (N = 31, 16 from male and 15 female neonates). Ex vivo assays were carried out to assess active metabolic and endoplasmic reticulum stress pathways by flow cytometry, confocal microscopy, gene expression and in silico approaches.
HBC from female placentas displayed a stronger M2 phenotype accompanied by high rates of efferocytosis majorly sustained on lipid metabolism. On the other hand, male HBC expressed a weaker M2 phenotype with higher glycolytic metabolism. LPS stimulation reinforced the glycolytic metabolism in male but not in female HBC. Physiological endoplasmic reticulum stress activates IRE-1 differently, since its pharmacological inhibition increased lipid mobilization, accumulation and efferocytosis only in female HBC. Moreover, differential sex-associated pathways accompanying the phenotypic and functional profiles of HBC appeared related to the placental villi environment.
These results support sex-associated effects on the immunometabolism of the HBC and adds another layer of complexity to the intricate maternal-fetal immune interaction.
© 2024. The Author(s).

Environmental lipids are essential for fueling tumor energetics, but whether these exogenous lipids transported into cancer cells facilitate immune escape remains unclear. Here, we find that CD36, a transporter for exogenous lipids, promotes acute myeloid leukemia (AML) immune evasion. We show that, separately from its established role in lipid oxidation, CD36 on AML cells senses oxidized low-density lipoprotein (OxLDL) to prime the TLR4-LYN-MYD88-nuclear factor κB (NF-κB) pathway, and exogenous palmitate transfer via CD36 further potentiates this innate immune pathway by supporting ZDHHC6-mediated MYD88 palmitoylation. Subsequently, NF-κB drives the expression of immunosuppressive genes that inhibit anti-tumor T cell responses. Notably, high-fat-diet or hypomethylating agent decitabine treatment boosts the immunosuppressive potential of AML cells by hijacking CD36-dependent innate immune signaling, leading to a dampened therapeutic effect. This work is of translational interest because lipid restriction by US Food and Drug Administration (FDA)-approved lipid-lowering statin drugs improves the efficacy of decitabine therapy by weakening leukemic CD36-mediated immunosuppression.
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.

Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here, we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro, both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing β-thalassemic phenotype. In an immunodeficient female mouse model, transplanted cells edited with the non-viral strategy exhibit higher engraftment and gene correction levels compared to those edited with the viral strategy. Transcriptomic analysis reveals that non-viral DNA repair template delivery mitigates P53-mediated toxicity and preserves high levels of long-term hematopoietic stem cells. This work paves the way for TALEN-based autologous gene therapy for sickle cell disease.
© 2024. The Author(s).

Reactivation of fetal hemoglobin (HbF) expression through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated disruption of regulatory elements involved in γ-globin gene repression is a promising gene therapy strategy for the treatment of sickle cell disease (SCD). However, preclinical studies aimed at optimizing the genome editing process and evaluating the safety of the editing strategy are necessary to translate this approach to the clinics. This is particularly relevant in the context of SCD, a disease characterized by inflammation, which can affect hematopoietic stem and progenitor cells (HSPCs), the target cell population in gene therapy approaches for hematopoietic disorders. Here, we describe a genome editing strategy leading to therapeutically relevant reactivation of HbF expression by targeting the binding sites (BSs) for the leukemia/lymphoma related factor (LRF) transcriptional repressor in the HBG1 and HBG2 γ-globin promoters. Electroporation of Cas9 ribonucleoprotein and single guide RNA (sgRNA) targeting the HBG promoters in healthy donor (HD) and patient-derived HSPCs resulted in a high frequency of LRF BS disruption and potent HbF synthesis in their erythroid progeny differentiated in vitro and ex vivo after transplantation into immunodeficient mice. LRF BS disruption did not impair SCD and HD HSPC engraftment and differentiation, but was more efficient in SCD than in HD cells. However, SCD HSPCs showed a reduced engraftment and a myeloid bias compared to HD cells. Importantly, in primary HSPCs, we detected off-target activity and the intra- and inter-chromosomal rearrangements between on- and off-target sites, which were more pronounced in SCD samples (likely because of the higher overall editing efficiency), but did not impact the target gene expression. Off-target activity was observed in vitro and in vivo, thus indicating that it does not impair engraftment and differentiation of both SCD and HD HSPCs. Finally, transcriptomic analyses showed that the genome editing procedure results in the upregulation of genes involved in DNA damage and inflammatory responses in both HD and SCD samples, although gene dysregulation was more evident in SCD HSPCs. Overall, this study provides evidences of feasibility, efficacy and safety for a genome editing strategy based on HbF reactivation and highlights the need of performing safety studies, when possible, in clinically relevant conditions, i.e., in patient-derived HSPCs.  

The fetal liver (FL) is the key erythropoietic organ during fetal development, but knowledge on human FL erythropoiesis is very limited. In this study, we sorted primary erythroblasts from FL cells and performed RNA sequencing (RNA-seq) analyses. We found that temporal gene expression patterns reflected changes in function during primary human FL terminal erythropoiesis. Notably, the expression of genes enriched in proteolysis and autophagy was up-regulated in orthochromatic erythroblasts (OrthoEs), suggesting the involvement of these pathways in enucleation. We also performed RNA-seq of in vitro cultured erythroblasts derived from FL CD34+ cells. Comparison of transcriptomes between the primary and cultured erythroblasts revealed significant differences, indicating impacts of the culture system on gene expression. Notably, the expression of lipid metabolism-related genes was increased in cultured erythroblasts. We further immortalized erythroid cell lines from FL and cord blood (CB) CD34+ cells (FL-iEry and CB-iEry, respectively). FL-iEry and CB-iEry were immortalized at the proerythroblast stage and can be induced to differentiate into OrthoEs, but their enucleation ability was very low. Comparison of the transcriptomes between OrthoEs with and without enucleation capability revealed the down-regulation of pathways involved in chromatin organization and mitophagy in OrthoEs without enucleation capacity, indicating that defects in chromatin organization and mitophagy contribute to the inability of OrthoEs to enucleate. Additionally, the expression of HBE1, HBZ, and HBG2 was up-regulated in FL-iEry compared with CB-iEry, and such up-regulation was accompanied by down-regulated expression of BCL11A and up-regulated expression of LIN28B and IGF2BP1. Our study provides new insights into human FL erythropoiesis and rich resources for future studies.
Copyright © 2023 Beijing Institute of Genomics, Chinese Academy of Sciences and Genetics Society of China. Published by Elsevier B.V. All rights reserved.