Related Experiment Video
Updated: Jul 23, 2025
Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line
Published on: August 2, 2021
Macrophages polarize to the pro-inflammatory phenotype and delay neutrophil efferocytosis to augment Aggregatibacter
Koren Hashai1,2, Fairuz Abadi1,2, Dana Clyman1,2
1Faculty of Dental Medicine, The Hebrew University, Jerusalem, Israel.
Objective:
The study examines how neutrophils cross-talk with macrophages during JP2 Aggregatibacter actinomycetemcomitance infection and factors that are involved in inflammatory resolution and efferocytosis.
Background:
Although sub-gingival bacteria constitute the primary initiating factor in the pathogenesis of molar-incisor pattern periodontitis (MIPP), the non-resolved host response has a major role in tissue destruction. While evidence links neutrophils to MIPP pathogenesis, their clearance during inflammatory resolution, governed by macrophages, is poorly understood.
Methods:
Human neutrophils (differentiated from HL60 cells) and macrophages (differentiated from THP1 cells) were inoculated with JP2. The supernatants were collected and exposed to naïve neutrophils or macrophages with or without exposure to JP2. Reactive oxygen species (ROS) were measured with 2'-7'-dichlorofluorescein-diacetate and a fluorescent plate reader. Immunofluorescence labeling of CD47 and cell vitality were examined using flow cytometry. Macrophage polarization was tested by immunofluorescence staining for CD163 and CD68 and a fluorescent microscope, and TNFα and IL-10 secretion was tested using ELISA and RT-PCR. Efferocytosis was examined by pHrodo and carboxyfluorescein succinimidyl ester staining and fluorescent microscopy. In vivo, macrophages were depleted from C57Bl/6 mice and neutrophil CD47 levels were tested using the subcutaneous chamber model.
Results:
Neutrophils exposed to macrophage supernatant show increased ROS, mainly extracellularly, that increased during JP2 infection. Macrophages showed pro-inflammatory M1 phenotype polarization during JP2 infection, and their supernatants prolonged neutrophil survival by inhibiting CD47 down-expression and reducing neutrophil necrosis and apoptosis. Also, the macrophages delay neutrophil efferocytosis during JP2 infection which, in turn, enhanced JP2 clearance. Depletion of macrophages in mice mildly prevented neutrophils CD47 reduction and reduced JP2 clearance. The JP2 infection in mice also led to macrophage M1 polarization similar to the in vitro results.
Conclusions:
As shown in this study, neutrophil efferocytosis potentially may be reduced during JP2 infection, promoting JP2 clearance, which may contribute to the inflammatory-mediated periodontal tissue damage.
Insights
During Aggregatibacter actinomycetemcomitance (JP2) infection, macrophages delay neutrophil efferocytosis, promoting bacterial clearance but potentially increasing periodontal tissue damage. This neutrophil-macrophage cross-talk is key to understanding inflammatory resolution in periodontitis.
Area of Science:
- Investigates the complex interplay between immune cells in the context of periodontal disease.
- Focuses on neutrophil and macrophage interactions during infection with Aggregatibacter actinomycetemcomitance (JP2).
Background:
- Molar-incisor pattern periodontitis (MIPP) is initiated by sub-gingival bacteria, with unresolved host responses contributing to tissue destruction.
- Neutrophils are implicated in MIPP pathogenesis, but their clearance by macrophages during inflammatory resolution is not well understood.
Purpose of the Study:
- To examine neutrophil-macrophage cross-talk during JP2 infection.
- To identify factors involved in inflammatory resolution and efferocytosis in this context.
Main Methods:
- Utilized differentiated human neutrophils and macrophages (from HL60 and THP1 cells, respectively) exposed to JP2.
- Measured reactive oxygen species (ROS), CD47 expression, cell vitality, macrophage polarization (CD163, CD68), cytokine secretion (TNFα, IL-10), and efferocytosis.
- Employed in vivo models with C57Bl/6 mice, including macrophage depletion and assessment of neutrophil CD47 levels.
Main Results:
- JP2 infection increased extracellular ROS. Macrophages exhibited M1 polarization and prolonged neutrophil survival by inhibiting CD47 down-expression.
- Macrophages delayed neutrophil efferocytosis during JP2 infection, enhancing bacterial clearance but potentially contributing to inflammation.
- Macrophage depletion in mice reduced bacterial clearance and neutrophil CD47 reduction, mirroring in vitro findings.
Conclusions:
- Reduced neutrophil efferocytosis during JP2 infection may promote bacterial clearance.
- This process could contribute to inflammatory-mediated periodontal tissue damage, highlighting a critical aspect of MIPP pathogenesis.
More Related Videos
09:22High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization
Published on: November 27, 2014
07:45Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis
Published on: November 28, 2015
Related Concept Videos
01:25Immune Surveillance by NK Cells and Phagocytes
Natural Killer Cells: The Fast Responders
NK cells are large granular lymphocytes found in the blood and lymphatic system. These...
01:17Phagocytosis of Apoptotic Cells
Normal cells contain receptors that prevent them from being recognized...
01:38Inflammation