Preparation and culture of bone marrow-derived macrophages from mice for functional analysis

Toda, Gotaro; Yamauchi, Toshimasa; Kadowaki, Takashi; Ueki, Kohjiro

doi:10.1016/j.xpro.2020.100246

Cited by 179 publications

(112 citation statements)

References 8 publications

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“…BMDMs rats were isolated from rat models and cultured following the methods previously [ 43 ]. Briefly, bone marrow cells were flushed via PBS from the femurs and tibias in non-diabetic and diabetic groups after 12 weeks of citrate buffer or STZ injection, respectively.…”

Section: Methodsmentioning

confidence: 99%

MiR-144-5p, an exosomal miRNA from bone marrow-derived macrophage in type 2 diabetes, impairs bone fracture healing via targeting Smad1

Zhang

et al. 2021

J Nanobiotechnol

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Background Patients with diabetes have an increased risk of nonunion and delayed union of fractures. Macrophages have been shown as a key player in diabetic complications. However, it remains obscure how diabetic milieu affects macrophage-derived exosomes and its implications on osteogenic differentiation of BMSCs. In this study, we aim to define the impact of diabetic milieu on macrophage-derived exosomes, role of extracellular vesicles in intercellular communication with BMSCs, and subsequent effects on osteogenic differentiation and fracture repair. Results The osteogenic potential and the ability of fracture repair of exosomes derived from diabetic bone marrow-derived macrophages (dBMDM-exos) were revealed to be lower, as compared with non-diabetic bone marrow-derived macrophages (nBMDM-exos) in vitro and in vivo. Interestingly, miR-144-5p levels were sharply elevated in dBMDM-exos and it could be transferred into BMSCs to regulate bone regeneration by targeting Smad1. In addition, the adverse effects of dBMDM-exos on the osteogenic potential and the ability of fracture repair were reversed through the suppression of miR-144-5p inhibition in vitro and vivo. Conclusions The results demonstrated an important role of exosomal miR-144-5p in bone regeneration, offering insight into developing new strategy for the improvement of fracture healing in patients with diabetes mellitus. Graphic Abstract

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Section: Methodsmentioning

confidence: 99%

MiR-144-5p, an exosomal miRNA from bone marrow-derived macrophage in type 2 diabetes, impairs bone fracture healing via targeting Smad1

Zhang

et al. 2021

J Nanobiotechnol

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“…Bone marrow-derived macrophages (BMDMs) were obtained from 8 to 12 week old female C57BL/6J (Jackson Laboratories) mice as previously described ( Toda et al, 2021 ). Briefly, bone marrow cells were collected from the femur and tibia of mice and maintained in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, United States) supplemented with 10 ng/mL mouse macrophage colony-stimulating factor (mMCSF; Gibco), 10% fetal bovine serum (Gibco), and 1% Pen/Strep (Gibco) at 37°C in a humidified atmosphere of 5% CO 2 for 7 days.…”

Section: Methodsmentioning

confidence: 99%

The gigA/gigB Genes Regulate the Growth, Stress Response, and Virulence of Acinetobacter baumannii ATCC 17978 Strain

et al. 2021

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Acinetobacter baumannii is an important pathogen of nosocomial infection. Recently, a group of genes, named “gig” (for Growth in Galleria), have been identified in a contemporary multi-drug resistant clinical isolate of A. baumannii—strain AB5075. Among these so-called gig genes, gigA and gigB were found to promote antibiotic resistance, stress survival, and virulence of AB5075 by interacting with the nitrogen phosphotransferase system (PTSNtr). This study aimed to investigate the roles of gigA/gigB, which appear to comprise a stress-signaling pathway (encoding for an atypical two-component system response regulator and a predicted anti-anti-sigma factor, respectively), and the involvement of ptsP (encoding the Enzyme I component of the PTSNtr) in the growth, stress resistance, and virulence of the widely studied A. baumannii strain ATCC 17978. Genetic analyses of strains harboring mutations of gigA and gigB were performed to investigate the roles of these genes in bacterial growth, stress resistance, evading macrophage defense, and killing of Galleria mellonella larva. In contrast with findings from strain AB5075 where gigA and gigB contribute to aminoglycoside resistance, the data presented herein indicate that the loss of gigA/gigB does not impact antibiotic resistance of strain ATCC 17978. Interestingly, however, we found that deletion of gigA/gigB in the ATCC 17978 background imparts a general growth in laboratory medium and also conferred growth and replication defects within murine macrophages and an inability to kill G. mellonella larvae. Importantly, studies as well as the loss of ptsP restored the phenotypes of the gigA/gigB mutant to that of the wild-type. The data presented herein indicate that in A. baumannii ATCC 17978, the gigA/gigB genes play a key role in both growth and virulence traits, but are dispensable for other stress-resistance survival phenotypes, including aminoglycoside resistance. Our findings thus highlight several similarities and also important differences between the gigA/gigB stress-signaling pathway in two commonly studied isolates of this troublesome pathogen.

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“…Bone marrow-derived macrophages (BMDMs) were prepared as described elsewhere (24). Bone marrow cells were collected from femurs and tibias.…”

Section: Cell Cultures and In Vitro Stimulationmentioning

confidence: 99%

Attenuating Effect of Chlorella Extract on NLRP3 Inflammasome Activation by Mitochondrial Reactive Oxygen Species

et al. 2021

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The NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) inflammasome has been linked to the pathogenesis of a wide variety of human diseases. Although many drugs and inhibitors have been developed to treat NLRP3-associated diseases, only limited clinical data support their efficacy and safety. Chlorella, a unicellular green alga that is widely and safely used as a food supplement, contains various antioxidants. In this study, we obtained a fat-soluble extract from Chlorella (CE) and demonstrated that it reduced NLRP3 inflammasome activation by inhibiting mitochondrial reactive oxygen species and caspase-1 activation. In addition, CE supplementation attenuated lipopolysaccharide-induced interleukin 1β transcription through activation of hypoxia-inducible factor 1α in vitro and in vivo. As Chlorella is a safe and useful food supplement, it may be a practical pharmacological approach for treating NLRP3-driven diseases.

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Preparation and culture of bone marrow-derived macrophages from mice for functional analysis

Cited by 179 publications

References 8 publications

MiR-144-5p, an exosomal miRNA from bone marrow-derived macrophage in type 2 diabetes, impairs bone fracture healing via targeting Smad1

MiR-144-5p, an exosomal miRNA from bone marrow-derived macrophage in type 2 diabetes, impairs bone fracture healing via targeting Smad1

The gigA/gigB Genes Regulate the Growth, Stress Response, and Virulence of Acinetobacter baumannii ATCC 17978 Strain

Attenuating Effect of Chlorella Extract on NLRP3 Inflammasome Activation by Mitochondrial Reactive Oxygen Species

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