Recruitment Of Precursors Research Articles

Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

Current prophylactic human immunodeficiency virus 1 (HIV-1) vaccine research aims to elicit broadly neutralizing antibodies (bnAbs). Membrane-proximal external region (MPER)-targeting bnAbs, such as 10E8, provide exceptionally broad neutralization, but some are autoreactive. Here, we generated humanized B cell antigen receptor knock-in mouse models to test whether a series of germline-targeting immunogens could drive MPER-specific precursors toward bnAbs. We found that recruitment of 10E8 precursors to germinal centers (GCs) required a minimum affinity for germline-targeting immunogens, but the GC residency of MPER precursors was brief due to displacement by higher-affinity endogenous B cell competitors. Higher-affinity germline-targeting immunogens extended the GC residency of MPER precursors, but robust long-term GC residency and maturation were only observed for MPER-HuGL18, an MPER precursor clonotype able to close the affinity gap with endogenous B cell competitors in the GC. Thus, germline-targeting immunogens could induce MPER-targeting antibodies, and B cell residency in the GC may be regulated by a precursor–competitor affinity gap.

Phosphatidylserine-incorporated exosome mimetics encapsulating CXCR3 antagonist alleviate osteoporosis.

Exosomes derived from mesenchymal stem cells are an active area of research due to their therapeutic potential in treating osteoporosis. To further harness their therapeutic performance in modulating bone resorption, we have equipped exosomes with osteoclast-targeting moieties on their surface as well as chemokine receptor antagonists blocking osteoclast recruitment. Phosphatidylserine (PS), a membrane lipid exerting immunosuppressive and phagocytic signals, was incorporated in the membrane of exosome mimetics (EMs) to achieve a marked affinity for osteoclast precursors and potential anti-resorptive effects. We also aimed to tackle a CXCL9-CXCR3 ligand-receptor axis, a critical signaling axis in regulating osteoclast precursor recruitment and differentiation at bone resorption sites, by encapsulating a chemical antagonist of CXCR3, AMG487, in the PS-incorporated EMs (PS-EMs). The osteoclast-targeting PS-EMs loaded with AMG487 effectively protected against bone loss in an ovariectomized mouse model. Our findings demonstrate the great promise of PS-EMs as anti-resorptive nanotherapies for alleviating osteoporosis.

The cholera toxin B subunit induces trained immunity in dendritic cells and promotes CD8 T cell antitumor immunity.

Innate immune training is a metabolic, functional, and epigenetic long-term reprogramming of innate cells triggered by different stimuli. This imprinting also reaches hematopoietic precursors in the bone marrow to sustain a memory-like phenotype. Dendritic cells (DCs) can exhibit memory-like responses, enhanced upon subsequent exposure to a pathogen; however, whether this imprinting is lineage and stimulus-restricted is still being determined. Nevertheless, the functional consequences of DCs training on the adaptive and protective immune response against non-infectious diseases remain unresolved. We evaluated the effect of the nontoxic cholera B subunit (CTB), LPS and LTA in the induction of trained immunity in murine DCs revealed by TNFa and LDH expression, through confocal microscopy. Additionally, we obtained bone marrow DCs (BMDCs) from mice treated with CTB, LPS, and LTA and evaluated training features in DCs and their antigen-presenting cell capability using multiparametric cytometry. Finally, we design an experimental melanoma mouse model to demonstrate protection induced by CTB-trained DCs in vivo. CTB-trained DCs exhibit increased expression of TNFa, and metabolic reprogramming indicated by LDH expression. Moreover, CTB training has an imprint on DC precursors, increasing the number and antigen-presenting function in BMDCs. We found that training by CTB stimulates the recruitment of DC precursors and DCs infiltration at the skin and lymph nodes. Interestingly, training-induced by CTB promotes a highly co-stimulatory phenotype in tumor-infiltrating DCs (CD86+) and a heightened functionality of exhausted CD8 T cells (Ki67+, GZMB+), which were associated with a protective response against melanoma challenge in vivo. Our work indicates that CTB can induce innate immune training on DCs, which turns into an efficient adaptive immune response in the melanoma model and might be a potential immunotherapeutic approach for tumor growth control.

NLRC3 attenuates osteoclastogenesis by limiting TNFα+ Th17 cell response in osteoporosis

NOD-like receptor family CARD domain containing 3 (NLRC3) is the intracellular protein belonging to NLR (NOD-like receptor) family. NLRC3 can negatively regulate inflammatory signal transduction pathways within the adaptive and innate immunocytes. However, studies need to elucidate the biological role of NLRC3 in bone remodeling. Herein, our study proved that NLRC3 prevents bone loss by inhibiting TNFα+ Th17 cell responses. In osteoporosis, NLRC3 attenuated TNFα+ Th17 cell accumulation in the bone marrow. However, osteoporosis (OP) development was aggravated without affecting bone marrow macrophage (BMM) osteoclastogenesis in NLRC3-deficient ovariectomized (OVX) mice. In this study, we transferred the wild-type and NLRC3−/− CD4+ cells into Rag1−/− mice. Consequently, we evidenced the effects of NLRC3 in CD4+ T cells on inhibiting the accumulation of TNFα + Th17 cells, thus restricting bone loss in the OVX mice. Simultaneously, NLRC3−/− CD4+ T cells promoted the recruitment of osteoclast precursors and inflammatory monocytes into the OVX mouse bone marrow. Mechanism-wise, NLRC3 reduced the secretion of TNFα + Th17 cells of RANKL, MIP1α, and MCP1, depending on the T cells. In addition, NLRC3 negatively regulated the Th17 osteoclastogenesis promoting functions via limiting the NF-κB activation. Collectively, this study appreciated the effect of NLRC3 on modulating bone mass via adaptive immunity depending on CD4+ cells. According to findings of this study, NLRC3 may be the candidate anti-OP therapeutic target.Key messagesNLRC3 negatively regulated the Th17 osteoclastogenesis promoting functions via limiting the NF-κB activation.NLRC3 may be the candidate anti-OP therapeutic target.

Exploring the Interplay Between Senescent Osteocytes and Bone Remodeling in Young Rodents.

This study identifies senescent osteocytes in the femur and tibia of young rodents and explores their role in bone remodeling. The proximity of osteoclasts to senescent osteocytes was observed, which is a new finding. Cultured osteocytes, sorted using a podoplanin antibody in FACS, exhibited osteocytic characteristics and increased senescence-related genes. Senescent osteocytes secreted cytokines associated with senescence, remodeling, and inflammation. Notably, IGF1 and MMP2 were elevated in podoplanin-positive (pdpn+) osteocytes. Migration assays demonstrated significant osteoclast precursor migration towards senescent osteocytes, further confirmed by co-culture experiments leading to osteoclast differentiation. These findings suggest that senescent osteocytes have a pivotal role in initiating bone resorption, with recruitment of osteoclast precursors during early bone remodeling stages. In conclusion, our research enhances our understanding of complicated bone remodeling mechanisms and bone homeostasis.

Multivalent antigen display on nanoparticle immunogens increases B cell clonotype diversity and neutralization breadth to pneumoviruses

Nanoparticles for multivalent display and delivery of vaccine antigens have emerged as a promising avenue for enhancing B cell responses to protein subunit vaccines. Here, we evaluated B cell responses in rhesus macaques immunized with prefusion-stabilized respiratory syncytial virus (RSV) F glycoprotein trimer compared with nanoparticles displaying 10 or 20 copies of the same antigen. We show that multivalent display skews antibody specificities and drives epitope-focusing of responding B cells. Antibody cloning and repertoire sequencing revealed that focusing was driven by the expansion of clonally distinct B cells through recruitment of diverse precursors. We identified two antibody lineages that developed either ultrapotent neutralization or pneumovirus cross-neutralization from precursor B cells with low initial affinity for the RSV-F immunogen. This suggests that increased avidity by multivalent display facilitates the activation and recruitment of these cells. Diversification of the B cell response by multivalent nanoparticle immunogens has broad implications for vaccine design.

Distribution of intracellular calcium during flow-induced migration of RAW264.7 cells

Cell migration is an important biological process regulated by mechanical stimulation, which leads to intracellular calcium response. Cell migration are dependent on the distribution and dynamic changes of intracellular calcium concentration. However, the temporal relation among mechanical stimulation, cell migration, and intracellular calcium distribution remains unclear. In this study, unidirectional flow and oscillatory flow were applied on osteoclast precursor RAW264.7 cells. The parameters of cell migration under fluid flow and intracellular calcium distribution along the migration or flow direction were calculated. Experimental results suggest the cells to adjust the [Ca2+]i distribution in the migration direction is independent of flow application or the reverse of flow direction, but the [Ca2+]i distribution in the flow direction is determined by the [Ca2+]i distribution-adjusting ability of cells and flow stimulation. Blocking calcium signaling pathways, namely, mechanosensitive cation-selective channels, phospholipase C, and endoplasmic reticulum, and removing extracellular calcium inhibited cell migration along the flow direction and the gradient distribution of intracellular calcium. This study provided insights into the mechanism of flow-induced cell migration and quantitative data for the recruitment of osteoclast precursors targeting the location of bone resorption.

Fate mapping reveals compartment-specific clonal expansion of mononuclear phagocytes during kidney disease

Fate mapping reveals compartment-specific clonal expansion of mononuclear phagocytes during kidney disease

The Role of Cancer and Somatic Stem Cells in the Anti-Inflammatory and Antitumor Effects of Aconitum baicalense Extract on Experimental Breast Cancer.

We studied the effects of the extract of the terrestrial part of Aconitum baicalense in BALB/c female mice at the early stages after the injection of N-methyl-N-nitrosourea (MNU). The extract reduced inflammatory activity and tumor growth in the mammary gland. The antitumor and anti-inflammatory effects of the extract are based on the inhibition of cancer stem cells, hematopoietic stem cells, and hematopoietic progenitor cells that promote inflammation. The extract of A. baicalense disrupted the recruitment of epithelial progenitor cells and angiogenesis precursors to the mammary gland preventing neovascularization and transformation of epithelial cells into tumor cells.

Membrane-bound mRNA immunogens lower the threshold to activate HIV Env V2 apex-directed broadly neutralizing B cell precursors in humanized mice.

Eliciting broadly neutralizing antibodies (bnAbs) is the core of HIV vaccine design. bnAbs specific to the V2-apex region of the HIV envelope acquire breadth and potency with modest somatic hypermutation, making them attractive vaccination targets. To evaluate Apex germline-targeting (ApexGT) vaccine candidates, we engineered knockin (KI) mouse models expressing the germline B cell receptor (BCR) of the bnAb PCT64. We found that high affinity of the ApexGT immunogen for PCT64-germline BCRs was necessary to specifically activate KI B cells at human physiological frequencies, recruit them to germinal centers, and select for mature bnAb mutations. Relative to protein, mRNA-encoded membrane-bound ApexGT immunization significantly increased activation and recruitment of PCT64 precursors to germinal centers and lowered their affinity threshold. We have thus developed additional models for HIV vaccine research, validated ApexGT immunogens for priming V2-apex bnAb precursors, and identified mRNA-LNP as a suitable approach to substantially improve the B cell response.

Impact of the host response and osteoblast lineage cells on periodontal disease

Periodontitis involves the loss of connective tissue attachment and alveolar bone. Single cell RNA-seq experiments have provided new insight into how resident cells and infiltrating immune cells function in response to bacterial challenge in periodontal tissues. Periodontal disease is induced by a combined innate and adaptive immune response to bacterial dysbiosis that is initiated by resident cells including epithelial cells and fibroblasts, which recruit immune cells. Chemokines and cytokines stimulate recruitment of osteoclast precursors and osteoclastogenesis in response to TNF, IL-1β, IL-6, IL-17, RANKL and other factors. Inflammation also suppresses coupled bone formation to limit repair of osteolytic lesions. Bone lining cells, osteocytes and periodontal ligament cells play a key role in both processes. The periodontal ligament contains cells that exhibit similarities to tendon cells, osteoblast-lineage cells and mesenchymal stem cells. Bone lining cells consisting of mesenchymal stem cells, osteoprogenitors and osteoblasts are influenced by osteocytes and stimulate formation of osteoclast precursors through MCSF and RANKL, which directly induce osteoclastogenesis. Following bone resorption, factors are released from resorbed bone matrix and by osteoclasts and osteal macrophages that recruit osteoblast precursors to the resorbed bone surface. Osteoblast differentiation and coupled bone formation are regulated by multiple signaling pathways including Wnt, Notch, FGF, IGF-1, BMP, and Hedgehog pathways. Diabetes, cigarette smoking and aging enhance the pathologic processes to increase bone resorption and inhibit coupled bone formation to accelerate bone loss. Other bone pathologies such as rheumatoid arthritis, post-menopausal osteoporosis and bone unloading/disuse also affect osteoblast lineage cells and participate in formation of osteolytic lesions by promoting bone resorption and inhibiting coupled bone formation. Thus, periodontitis involves the activation of an inflammatory response that involves a large number of cells to stimulate bone resorption and limit osseous repair processes.

CCL7 playing a dominant role in recruiting early OCPs to facilitate osteolysis at metastatic site of colorectal cancer

BackgroundChemoattractant is critical to recruitment of osteoclast precursors and stimulates tumor bone metastasis. However, the role of chemoattractant in bone metastasis of colorectal cancer (CRC) is still unclear.MethodsHistochemistry analysis and TRAP staining were utilized to detect the bone resorption and activation of osteoclasts (OCs) after administration of CCL7 neutralizing antibody or CCR1 siRNA. qRT-PCR analysis and ELISA assay were performed to detect the mRNA level and protein level of chemoattractant. BrdU assay and Tunel assay were used to detect the proliferation and apoptosis of osteoclast precursors (OCPs). The migration of OCPs was detected by Transwell assay. Western blots assay was performed to examine the protein levels of pathways regulating the expression of CCL7 or CCR1.ResultsOCPs-derived CCL7 was significantly upregulated in bone marrow after bone metastasis of CRC. Blockage of CCL7 efficiently prevented bone resorption. Administration of CCL7 promoted the migration of OCPs. Lactate promoted the expression of CCL7 through JNK pathway. In addition, CCR1 was the most important receptor of CCL7.ConclusionOur study indicates the essential role of CCL7-CCR1 signaling for recruitment of OCPs in early bone metastasis of CRC. Targeting CCL7 or CCR1 could restore the bone volume, which could be a potential therapeutical target.56ZQHTj5cSEJRbeC42N2_zVideo

The Role Of BMPs in the Regulation of Osteoclasts Resorption and Bone Remodeling: From Experimental Models to Clinical Applications.

In response to mechanical forces and the aging process, bone in the adult skeleton is continuously remodeled by a process in which old and damaged bone is removed by bone-resorbing osteoclasts and subsequently is replaced by new bone by bone-forming cells, osteoblasts. During this essential process of bone remodeling, osteoclastic resorption is tightly coupled to osteoblastic bone formation. Bone-resorbing cells, multinuclear giant osteoclasts, derive from the monocyte/macrophage hematopoietic lineage and their differentiation is driven by distinct signaling molecules and transcription factors. Critical factors for this process are Macrophage Colony Stimulating Factor (M-CSF) and Receptor Activator Nuclear Factor-κB Ligand (RANKL). Besides their resorption activity, osteoclasts secrete coupling factors which promote recruitment of osteoblast precursors to the bone surface, regulating thus the whole process of bone remodeling. Bone morphogenetic proteins (BMPs), a family of multi-functional growth factors involved in numerous molecular and signaling pathways, have significant role in osteoblast-osteoclast communication and significantly impact bone remodeling. It is well known that BMPs help to maintain healthy bone by stimulating osteoblast mineralization, differentiation and survival. Recently, increasing evidence indicates that BMPs not only help in the anabolic part of bone remodeling process but also significantly influence bone catabolism. The deletion of the BMP receptor type 1A (BMPRIA) in osteoclasts increased osteoblastic bone formation, suggesting that BMPR1A signaling in osteoclasts regulates coupling to osteoblasts by reducing bone-formation activity during bone remodeling. The dual effect of BMPs on bone mineralization and resorption highlights the essential role of BMP signaling in bone homeostasis and they also appear to be involved in pathological processes in inflammatory disorders affecting bones and joints. Certain BMPs (BMP2 and -7) were approved for clinical use; however, increased bone resorption rather than formation were observed in clinical applications, suggesting the role BMPs have in osteoclast activation and subsequent osteolysis. Here, we summarize the current knowledge of BMP signaling in osteoclasts, its role in osteoclast resorption, bone remodeling, and osteoblast–osteoclast coupling. Furthermore, discussion of clinical application of recombinant BMP therapy is based on recent preclinical and clinical studies.

TNF-α Activating Osteoclasts in Patients with Psoriatic Arthritis Enhances the Recruitment of Osteoclast Precursors: A Plausible Role of WNT5A-MCP-1 in Osteoclast Engagement in Psoriatic Arthritis.

Psoriatic arthritis (PsA) results from joint destruction by osteoclasts. The promising efficacy of TNF-α blockage indicates its important role in osteoclastogenesis of PsA. WNT ligands actively regulate osteoclastogenesis. We investigated how WNT ligands activate osteoclasts amid the TNF-α milieu in PsA. We first profiled the expression of WNT ligands in CD14+ monocyte-derived osteoclasts (MDOC) from five PsA patients and five healthy controls (HC) and then validated the candidate WNT ligands in 32 PsA patients and 16 HC. Through RNA interference against WNT ligands in MDOC, we determined the mechanisms by which TNF-α exerts its effects on osteclastogenesis or chemotaxis. WNT5A was selectively upregulated by TNF-α in MDOC from PsA patients. The number of CD68+WNT5A+ osteoclasts increased in PsA joints. CXCL1, CXCL16, and MCP-1 was selectively increased in supernatants of MDOC from PsA patients. RNA interference against WNT5A abolished the increased MCP-1 from MDOC and THP-1-cell-derived osteoclasts. The increased migration of osteoclast precursors (OCP) induced by supernatant from PsA MDOC was abolished by the MCP-1 neutralizing antibody. WNT5A and MCP-1 expressions were decreased in MDOC from PsA patients treated by biologics against TNF-α but not IL-17. We conclude that TNF-α recruits OCP by increased MCP-1 production but does not directly activate osteoclastogenesis in PsA.

Segregation of brain and organizer precursors is differentially regulated by Nodal signaling at blastula stage.

ABSTRACTThe blastula Chordin- and Noggin-expressing (BCNE) center comprises animal-dorsal and marginal-dorsal cells of the amphibian blastula and contains the precursors of the brain and the gastrula organizer. Previous findings suggested that the BCNE behaves as a homogeneous cell population that only depends on nuclear β-catenin activity but does not require Nodal and later segregates into its descendants during gastrulation. In contrast to previous findings, in this work, we show that the BCNE does not behave as a homogeneous cell population in response to Nodal antagonists. In fact, we found that chordin.1 expression in a marginal subpopulation of notochordal precursors indeed requires Nodal input. We also establish that an animal BCNE subpopulation of cells that express both, chordin.1 and sox2 (a marker of pluripotent neuroectodermal cells), and gives rise to most of the brain, persisted at blastula stage after blocking Nodal. Therefore, Nodal signaling is required to define a population of chordin.1+ cells and to restrict the recruitment of brain precursors within the BCNE as early as at blastula stage. We discuss our findings in Xenopus in comparison to other vertebrate models, uncovering similitudes in early brain induction and delimitation through Nodal signaling.This article has an associated First Person interview with the first author of the paper.

Lactic acid promotes metastatic niche formation in bone metastasis of colorectal cancer

BackgroundTo investigate the effect of lactic acid (LA) on the progression of bone metastasis from colorectal cancer (CRC) and its regulatory effects on primary CD115 (+) osteoclast (OC) precursors.MethodsThe BrdU assay, Annexin-V/PI assay, TRAP staining and immunofluorescence were performed to explore the effect of LA on the proliferation, apoptosis and differentiation of OC precursors in vitro and in vivo. Flow cytometry was performed to sort primary osteoclast precursors and CD4(+) T cells and to analyze the change in the expression of target proteins in osteoclast precursors. A recruitment assay was used to test how LA and Cadhein-11 regulate the recruitment of OC precursors. RT-PCR and Western blotting were performed to analyze the changes in the mRNA and protein expression of genes related to the PI3K-AKT pathway and profibrotic genes. Safranin O-fast green staining, H&E staining and TRAP staining were performed to analyze the severity of bone resorption and accumulation of osteoclasts.ResultsLA promoted the expression of CXCL10 and Cadherin-11 in CD115(+) precursors through the PI3K-AKT pathway. We found that CXCL10 and Cadherin-11 were regulated by the activation of CREB and mTOR, respectively. LA-induced overexpression of CXCL10 in CD115(+) precursors indirectly promoted the differentiation of osteoclast precursors through the recruitment of CD4(+) T cells, and the crosstalk between these two cells promoted bone resorption in bone metastasis from CRC. On the other hand, Cadherin-11 mediated the adhesion between osteoclast precursors and upregulated the production of specific collagens, especially Collagen 5, which facilitated fibrotic changes in the tumor microenvironment. Blockade of the PI3K-AKT pathway efficiently prevented the progression of bone metastasis caused by lactate.ConclusionLA promoted metastatic niche formation in the tumor microenvironment through the PI3K-AKT pathway. Our study provides new insight into the role of LA in the progression of bone metastasis from CRC.DasE7LVW6tcKsMdapJmhMFVideo

SARS-CoV-2 sculpts the immune system to induce sustained virus-specific naïve-like and memory B-cell responses.

ObjectivesSARS‐CoV‐2 infection induces virus‐reactive memory B cells expressing unmutated antibodies, which hints at their emergence from naïve B cells. Yet, the dynamics of virus‐specific naïve B cells and their impact on immunity and immunopathology remain unclear.MethodsWe longitudinally profiled SARS‐CoV‐2‐specific B‐cell responses in 25 moderate‐to‐severe COVID‐19 patients by high‐dimensional flow cytometry and isotyping and subtyping ELISA. We also explored the relationship of B‐cell responses to SARS‐CoV‐2 with the activation of effector and regulatory cells from the innate or adaptive immune system.ResultsWe found a virus‐specific antibody response with a broad spectrum of classes and subclasses during acute infection, which evolved into an IgG1‐dominated response during convalescence. Acute infection was associated with increased mature B‐cell progenitors in the circulation and the unexpected expansion of virus‐targeting naïve‐like B cells. The latter further augmented during convalescence together with virus‐specific memory B cells. In addition to a transitory increase in tissue‐homing CXCR3+ plasmablasts and extrafollicular memory B cells, most COVID‐19 patients showed persistent activation of CD4+ and CD8+ T cells along with transient or long‐lasting changes of key innate immune cells. Remarkably, virus‐specific antibodies and the frequency of naïve B cells were among the major variables defining distinct immune signatures associated with disease severity and inflammation.ConclusionAside from providing new insights into the complexity of the immune response to SARS‐CoV‐2, our findings indicate that the de novo recruitment of mature B‐cell precursors into the periphery may be central to the induction of antiviral immunity.

Physiological properties and characteristic reactions of hydroxyurea

As it was mentioned in the previous paper, we observed the mechanism of action the interesting drug, first synthesized back in 1869 for the first time, called Hydroxyurea. A century later, phase I and II trials began to test its safety in humans with solid tumors. It was first approved by the FDA in 1967 for the treatment of neoplastic diseases and is presently approved for the treatment of melanoma, resistant chronic myelocytic leukemia (CML), and recurrent, metastatic testicular and ovarian cancer. Sickle cell disease is a genetic disorder that decreases life expectancy by 25 to 30 years. Individuals are diagnosed with sickle cell disease if they have one of several genotypes that result in at least half of their hemoglobin being hemoglobin S (HbS). Sickle cell anemia refers specifically to the condition associated with homozygosity for the Hb S mutation (Hb SS). Several other hemoglobin mutations, when occurring with an Hb S mutation, cause a similar but often milder disease than sickle cell anemia. In addition to reduced life expectancy, patients with sickle cell disease experience chronic pain and reduced quality of life. Painful crises, also known as vaso-occlusive crises, are the most common reason for emergency department use and hospitalization, and acute chest syndrome is the most common cause of death. Prior to the approval of hydroxyurea for use in sickle cell disease, patients with this condition were treated only with supportive therapies. These measures included penicillin in children to prevent pneumococcal disease, routine immunizations, and hydration and narcotic therapy to treat painful events. Red blood cell transfusions increase the blood’s oxygen carrying capacity and decrease the concentration of cells with abnormal hemoglobin, but chronic transfusion therapy predictably leads to iron overload and alloimmunization. Therapies such as hydroxyurea that raise fetal hemoglobin (Hb F, α2γ2) levels are promising because they effectively lower the concentration of Hb S within a cell, resulting in less polymerization of the abnormal hemoglobin.Hydroxyurea’s efficacy in sickle cell disease is generally attributed to its ability to raise the levels of Hb F in the blood; however, the mechanisms by which it does so are unclear. Early studies suggested that hydroxyurea is cytotoxic to the more rapidly dividing late erythroid precursors, resulting in the recruitment of early erythroid precursors with an increased capacity to produce HbF.

Nkx2-5 defines distinct scaffold and recruitment phases during formation of the murine cardiac Purkinje fiber network

The ventricular conduction system coordinates heartbeats by rapid propagation of electrical activity through the Purkinje fiber (PF) network. PFs share common progenitors with contractile cardiomyocytes, yet the mechanisms of segregation and network morphogenesis are poorly understood. Here, we apply genetic fate mapping and temporal clonal analysis to identify murine cardiomyocytes committed to the PF lineage as early as E7.5. We find that a polyclonal PF network emerges by progressive recruitment of conductive precursors to this scaffold from a pool of bipotent progenitors. At late fetal stages, the segregation of conductive cells increases during a phase of rapid recruitment to build the definitive PF network through a non-cell autonomous mechanism. We also show that PF differentiation is impaired in Nkx2-5 haploinsufficient embryos leading to failure to extend the scaffold. In particular, late fetal recruitment fails, resulting in PF hypoplasia and persistence of bipotent progenitors. Our results identify how transcription factor dosage regulates cell fate divergence during distinct phases of PF network morphogenesis.

Macrophages Switch to an Osteo-Modulatory Profile Upon RANKL Induction in a Medaka (Oryzias latipes) Osteoporosis Model.

ABSTRACTIn mammals, osteoclasts differentiate from macrophages in the monocyte lineage. Although many factors driving osteoclast formation are known, the detailed processes underlying precursor recruitment, differentiation, and interaction of macrophages with other cell types involved in bone remodeling are poorly understood. Using live imaging in a transgenic medaka osteoporosis model, where ectopic osteoclasts are induced by RANKL expression, we show that a subset of macrophages is recruited to bone matrix to physically interact with bone‐forming osteoblast progenitors. These macrophages subsequently differentiate into cathepsin K‐ (ctsk‐) positive osteoclasts. One day later, other macrophages are recruited to clear dying osteoclasts from resorbed bone by phagocytosis. To better understand the molecular changes underlying these dynamic processes, we performed transcriptome profiling of activated macrophages upon RANKL induction. This revealed an upregulation of several bone‐related transcripts. Besides osteoclast markers, we unexpectedly also found expression of osteoblast‐promoting signals in activated macrophages, suggesting a possible non‐cell autonomous role in osteogenesis. Finally, we show that macrophage differentiation into osteoclasts is dependent on inflammatory signals. Medaka deficient for TNFα or treated with the TNFα‐inhibitor pentoxifylline exhibited impaired macrophage recruitment and osteoclast differentiation. These results show the involvement of inflammatory signals and the dynamics of a distinct subset of macrophages during osteoclast formation. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.