Engraftment, Fate, and Function of HoxB8-Conditional Neutrophil Progenitors in the Unconditioned Murine Host

Cohen, Joshua T.; Danise, Michael; Hinman, Kristina D.; Neumann, Brittany M.; Johnson, Renita; Wilson, Zachary; Chorzalska, Anna; Dubielecka, Patrycja M.; Lefort, Craig T.

doi:10.3389/fcell.2022.840894

Cited by 6 publications

(10 citation statements)

References 49 publications

(84 reference statements)

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“…Vinculin is an actin-binding protein whose role in cortical actin organization is well understood in mesenchymal cells but less so in neutrophils [28, 29]. Using CRISPR/Cas9 to generate Vcl -/- progenitors which were then differentiated into neutrophils, we investigated the role of vinculin in neutrophil stiffening and transit through model capillaries.…”

Section: Resultsmentioning

confidence: 99%

“…To maintain cells in the progenitor state, cells were cultured in the presence of 100 nM 4hydroxytamoxifen (4-OHT), 50 ng/mL recombinant murine stem cell factor (mSCF), and 1 μg/mL puromycin for selection of the transduced cells. The surviving cells made up our WT progenitor cell lines [28][29][30]. In some cases, 2% conditioned media from CHO cells producing SCF was used in place of mSCF (a gift from Dr. Patrice Dubreuil).…”

Section: Hoxb8-conditional Murine Neutrophil Progenitorsmentioning

confidence: 99%

“…On day two, cells were washed and resuspended in basal media with 20 ng/mL mG-CSF for three days. Successfully differentiated neutrophils exhibit multi-lobed nuclei, expression of Ly6G, and loss of expression of CD117 (cKit) [28][29][30].…”

Section: Hoxb8-conditional Murine Neutrophil Progenitorsmentioning

confidence: 99%

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Vinculin plays a role in neutrophil stiffening and transit through model capillary segments

Neumann

Wilson

Auguste

et al. 2022

Preprint

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Neutrophils are rapidly mobilized from the circulation to sites of inflammation. The mechanisms of neutrophil trafficking in the lung are distinct from those in the periphery, in part because the pulmonary capillaries are the primary site of neutrophil emigration rather than postcapillary venules. Since the diameter of a neutrophil is greater than the width of most pulmonary capillary segments, they must deform to transit through this capillary network, even at homeostasis. Resistance to deformation is primarily due to cortical actin that is rapidly assembled when a neutrophil is exposed to a priming or activation stimulus, resulting in neutrophil stiffening and subsequent sequestration within the pulmonary capillary network. In the current study, we use a microfluidic assay to characterize neutrophil transit through model capillary-like channels. Using techniques from single-particle tracking, we analyzed the cumulative distribution of neutrophil transit times and resolve population-based effects. We found that vinculin, an actin-binding adaptor protein, plays an essential role in neutrophil stiffening in response to formyl-Met-Leu-Phe (fMLP). Vinculin-deficient neutrophils lack the development of a population with slow transit through narrow channels that was observed in both wild-type murine bone marrow neutrophils and HoxB8-conditional progenitor-derived neutrophils. Atomic force microscopy studies provide further evidence that vinculin is required for neutrophil stiffening. Consistent with these findings, we observed that neutrophil sequestration in the lungs of mice is attenuated in the absence of vinculin. Together, our studies indicate that vinculin mediates actin-dependent neutrophil stiffening that leads to their sequestration in capillaries.

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Section: Resultsmentioning

confidence: 99%

Section: Hoxb8-conditional Murine Neutrophil Progenitorsmentioning

confidence: 99%

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Vinculin plays a role in neutrophil stiffening and transit through model capillary segments

Neumann

Wilson

Auguste

et al. 2022

Preprint

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“…Gene editing can be done in HoxB8 neutrophil progenitors. HoxB8 neutrophil progenitors can differentiate into neutrophils in vitro (90)(91)(92)(93)(94) and in vivo (95,96). These will provide a powerful tool to study the involvement of mitochondrial components in mouse neutrophils.…”

Section: Discussionmentioning

confidence: 99%

Roles of mitochondria in neutrophils

et al. 2022

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Neutrophils are the most abundant leukocyte in human blood. They are critical for fighting infections and are involved in inflammatory diseases. Mitochondria are indispensable for eukaryotic cells, as they control the biochemical processes of respiration and energy production. Mitochondria in neutrophils have been underestimated since glycolysis is a major metabolic pathway for fuel production in neutrophils. However, several studies have shown that mitochondria are greatly involved in multiple neutrophil functions as well as neutrophil-related diseases. In this review, we focus on how mitochondrial components, metabolism, and related /Sungenes regulate neutrophil functions and relevant diseases.

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“…HoxB8 conditionally immortalized neutrophil progenitors were generated as previously described ( 20 – 22 ). Briefly, C57BL/6 mouse bone marrow was isolated, cultured, and transduced with a tamoxifen-inducible Hoxb8 transgene.…”

Section: Methodsmentioning

confidence: 99%

Bi-fluorescent Staphylococcus aureus infection enables single-cell analysis of intracellular killing in vivo

et al. 2023

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Techniques for studying the clearance of bacterial infections are critical for advances in understanding disease states, immune cell effector functions, and novel antimicrobial therapeutics. Intracellular killing of Staphylococcus aureus by neutrophils can be monitored using a S. aureus strain stably expressing GFP, a fluorophore that is quenched when exposed to the reactive oxygen species (ROS) present in the phagolysosome. Here, we expand upon this method by developing a bi-fluorescent S. aureus killing assay for use in vivo. Conjugating S. aureus with a stable secondary fluorescent marker enables the separation of infected cell samples into three populations: cells that have not engaged in phagocytosis, cells that have engulfed and killed S. aureus, and cells that have viable internalized S. aureus. We identified ATTO647N-NHS Ester as a favorable dye conjugate for generating bi-fluorescent S. aureus due to its stability over time and invariant signal within the neutrophil phagolysosome. To resolve the in vivo utility of ATTO647N/GFP bi-fluorescent S. aureus, we evaluated neutrophil function in a murine model of chronic granulomatous disease (CGD) known to have impaired clearance of S. aureus infection. Analysis of bronchoalveolar lavage (BAL) from animals subjected to pulmonary infection with bi-fluorescent S. aureus demonstrated differences in neutrophil antimicrobial function consistent with the established phenotype of CGD.

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Engraftment, Fate, and Function of HoxB8-Conditional Neutrophil Progenitors in the Unconditioned Murine Host

Cited by 6 publications

References 49 publications

Vinculin plays a role in neutrophil stiffening and transit through model capillary segments

Vinculin plays a role in neutrophil stiffening and transit through model capillary segments

Roles of mitochondria in neutrophils

Bi-fluorescent Staphylococcus aureus infection enables single-cell analysis of intracellular killing in vivo

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