Imaging, myeloid precursor immortalization, and genome editing for defining mechanisms of leukocyte recruitment <i>in vivo</i>

Gran, Sandra; Honold, Lisa; Fehler, Olesja; Zenker, Stefanie; Eligehausen, Sarah; Kuhlmann, Michael; Geven, E.J.; Bosch, M. van den; Lent, P. van; Spiekermann, Christoph; Hermann, Sven; Vogl, Thomas; Schäfers, Michael; Roth, Johannes

doi:10.7150/thno.23632

Cited by 25 publications

(30 citation statements)

References 60 publications

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“…Myh9‐deficient NLC had severe migration defects in 2D and 3D environments that were restored by viral transduction of EGFP‐Myh9, further validating the use of this cellular model for critical rescue experiments. Two other studies showed that dye‐labeled HoxB8‐derived cells injected intravenously are recovered at inflammatory sites, validating the use of HoxB8‐derived neutrophils for adoptive transfer experiments. The Mc Donald et al.…”

Section: Discussionmentioning

confidence: 81%

“…The HoxB8 system was recently used to establish the role of the non‐muscle myosin Myh9 in neutrophil trafficking in vitro and to monitor neutrophil trafficking during inflammation in vivo . These studies validate the use of this cellular model to study neutrophil trafficking in vitro and in vivo and highlight its advantages for rescue experiments or adoptive transfer of cells bearing mutations resulting in lethal phenotypes in mice.…”

Section: Introductionmentioning

confidence: 78%

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Signaling and functional competency of neutrophils derived from bone-marrow cells expressing the ER-HOXB8 oncoprotein

Saul

Castelbou

Fickentscher

et al. 2019

Journal of Leukocyte Biology

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Neutrophils play a central role in immunity and inflammation via their intrinsic ability to migrate into inflamed tissue, to phagocytose pathogens, and to kill bacterial and fungi by releasing large quantities of superoxide anions and lytic enzymes. The molecular pathways controlling neutrophil microbicidal functions are still unclear, because neutrophils have a short half‐life and are resistant to genetic manipulation. Neutrophil‐like cells (NLC) can be generated from myeloid progenitors conditionally immortalized with the ER‐HoxB8 oncoprotein, but whether these cells can replace neutrophils in high‐throughput functional assays is unclear. Here, we assess the ability of NLC derived from ER‐HoxB8 progenitors to produce ROS and to perform chemotaxis and phagocytosis. We compare the Ca2+ responses and effector functions of NLC to primary murine neutrophils and document the molecular basis of their functional differences by mRNA profiling. Pro‐inflammatory cytokines enhanced the expression by NLC of neutrophil surface markers and transcription factors. Ca2+ elevations evoked in NLC by agonists, adhesion receptors, and store depletion resembled the physiological responses recorded in primary neutrophils, but NLC expressed reduced amounts of Ca2+ signaling proteins and of chemotactic receptors. Unlike their myeloid progenitors, NLC produced H2O2 when adhered to fibronectin, migrated toward chemotactic peptides, phagocytosed opsonized particles, and generated intracellular ROS. NLC phagocytosed as efficiently as primary neutrophils but produced 50 times less ROS and migrated less efficiently toward chemoattractant. Our data indicate that NLC can replace neutrophils to study Ca2+ signaling and phagocytosis, but that their incomplete granulocytic differentiation limits their use for chemotaxis and ROS production assays.

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Section: Discussionmentioning

confidence: 81%

Section: Introductionmentioning

confidence: 78%

Signaling and functional competency of neutrophils derived from bone-marrow cells expressing the ER-HOXB8 oncoprotein

Saul

Castelbou

Fickentscher

et al. 2019

Journal of Leukocyte Biology

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“…. Moreover, the assay is suitable for mouse bone marrow-derived macrophages or macrophages derived from conditionally immortalized myeloid precursor cells 39,40 . We have previously used polytetrafluoroethylene (PTFE) bags with luer adapters to culture bone marrow cells and obtain macrophages…”

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confidence: 99%

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Bos

Walbaum

Horsthemke

et al. 2020

JoVE

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Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 µm/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopybased macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis. Video Link The video component of this article can be found at https://www.jove.com/video/60750/ 17. At that time, the molecular identities of chemotactic factors were unknown. In the 1960s, Stephen Boyden 18 recognized that techniques to study the chemotactic activity of soluble substances were lacking. He devised a chamber, subsequently known as the Boyden chamber, with two compartments separated by a filter paper membrane. A cell

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“…This could allow to define the macrophage migration mode within a large collection of healthy or pathological tissues isolated from different mouse models. The use of ER-Hoxb8 monocytes injected in blood circulation to study tissue recruitment of leukocytes from blood has been recently described (Gran et al, 2018). A future objective will be to examine by intravital microscopy the migration of macrophages in tumors (Gui et al, 2018) by labeling genetically modified Hoxb8-macrophages and directly injecting them within tumors generated in dorsal window chambers.…”

Section: Discussionmentioning

confidence: 99%

Genetic engineering of Hoxb8-immortalized hematopoietic progenitors – a potent tool to study macrophage tissue migration

Accarias

Sanchez

Labrousse

et al. 2020

Journal of Cell Science

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Tumor-associated macrophages (TAMs) are detrimental in most cancers. Controlling their recruitment is thus potentially therapeutic. We previously found that TAMs perform protease-dependent mesenchymal migration in cancer, while macrophages perform amoeboid migration in other tissues. Inhibition of mesenchymal migration correlates with decreased TAM infiltration and tumor growth, providing rationale for a new cancer immunotherapy specifically targeting TAM motility. To identify new effectors of mesenchymal migration, we produced ER-Hoxb8-immortalized hematopoietic progenitors (cells with estrogen receptor-regulated Hoxb8 expression), which show unlimited proliferative ability in the presence of estrogen. The functionality of macrophages differentiated from ER-Hoxb8 progenitors was compared to bone marrow-derived macrophages (BMDMs). They polarized into M1-and M2-orientated macrophages, generated reactive oxygen species (ROS), ingested particles, formed podosomes, degraded the extracellular matrix, adopted amoeboid and mesenchymal migration in 3D, and infiltrated tumor explants ex vivo using mesenchymal migration. We also used the CRISPR/Cas9 system to disrupt gene expression of a known effector of mesenchymal migration, WASP (also known as WAS), to provide a proof of concept. We observed impaired podosome formation and mesenchymal migration capacity, thus recapitulating the phenotype of BMDM isolated from Wasp-knockout mice. Thus, we validate the use of ER-Hoxb8-immortalized macrophages as a potent tool to investigate macrophage functionalities.

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Imaging, myeloid precursor immortalization, and genome editing for defining mechanisms of leukocyte recruitment in vivo

Cited by 25 publications

References 60 publications

Signaling and functional competency of neutrophils derived from bone-marrow cells expressing the ER-HOXB8 oncoprotein

Signaling and functional competency of neutrophils derived from bone-marrow cells expressing the ER-HOXB8 oncoprotein

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Genetic engineering of Hoxb8-immortalized hematopoietic progenitors – a potent tool to study macrophage tissue migration

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