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Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA
Published on: November 9, 2019
Human primary neutrophil mRNA does not contaminate human resolving macrophage mRNA after efferocytosis
M Maraux1, A Gaillardet1, A Gally1
1Univ. Bourgogne Franche-Comté, INSERM, EFS BFC, UMR1098, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et Génique, Fédération Hospitalo-Universitaire INCREASE, LabEx LipSTIC, F-25000 Besançon, France.
Abstract:
The ingestion of apoptotic corpses by macrophages, a process called efferocytosis, is a crucial step in inflammation resolution, since it alters macrophage phenotype toward a pro-resolving profile to foil inflammation and to favor tissue repair. Up to now, the resolving macrophages remain poorly characterized, especially in humans. Global investigations, like RNA sequencing, would be very helpful to unravel some features of these elusive cells. Nonetheless, these inquiries may be challenging in a single-species model, since the fate of ingested mRNA remains unknown and may hinder any subsequent mRNA investigations in the phagocyte. A full human model consisting of primary human neutrophil and primary human monocyte-derived macrophage co-culture was set up several decades ago to mimic in vitro the efferocytosis process. However, to our knowledge, this model has not been characterized as a suitable model to perform global mRNA investigations. Indeed, the extent of ingested neutrophil mRNA contamination has not been assessed in resolving macrophages. This work answers to this crucial question. Indeed, based on the protocols presented in this article, we demonstrate that neutrophil mRNA is severely degraded and is not able to cross-contaminate resolving macrophage mRNA, contrary to apoptotic human peripheral blood derived mononuclear cell (PBMC) or apoptotic leukemic Jurkat cell mRNA. Moreover, this allogenic co-culture system does not favor neither neutrophil activation nor macrophage pro-inflammatory cytokine release. Collectively, we highlight that this model of primary human neutrophil and primary human monocyte-derived macrophage co-culture is the best model for mRNA investigations in human resolving macrophages to help improving our knowledge on these crucial cells.
Insights
This study validates a human cell co-culture model for studying resolving macrophages. It confirms that neutrophil mRNA does not contaminate macrophage mRNA, making it ideal for RNA sequencing in inflammation resolution research.
Area of Science:
- Immunology
- Cell Biology
Background:
- Efferocytosis, the engulfment of apoptotic cells by macrophages, is vital for resolving inflammation and promoting tissue repair.
- Resolving macrophages, key players in this process, are poorly understood, particularly in human systems.
- Global gene expression studies like RNA sequencing are needed to characterize these cells, but ingested mRNA contamination is a concern.
Purpose of the Study:
- To assess the suitability of a primary human neutrophil and monocyte-derived macrophage co-culture model for investigating resolving macrophages.
- To determine if ingested neutrophil mRNA contaminates macrophage mRNA in this co-culture system, impacting downstream RNA sequencing.
- To evaluate if this co-culture model induces unwanted neutrophil activation or macrophage pro-inflammatory responses.
Main Methods:
- Established a co-culture system using primary human neutrophils and primary human monocyte-derived macrophages.
- Developed and applied protocols to assess mRNA degradation and cross-contamination within the co-culture.
- Analyzed neutrophil activation markers and macrophage pro-inflammatory cytokine release.
Main Results:
- Demonstrated severe degradation of ingested neutrophil mRNA, preventing cross-contamination of resolving macrophage mRNA.
- Showed that this degradation is specific, as apoptotic peripheral blood mononuclear cells (PBMCs) or Jurkat cells did cause contamination.
- Confirmed that the co-culture system does not induce significant neutrophil activation or pro-inflammatory cytokine release from macrophages.
Conclusions:
- The primary human neutrophil and monocyte-derived macrophage co-culture model is validated as a suitable system for mRNA investigations in human resolving macrophages.
- This model effectively minimizes confounding factors like mRNA cross-contamination, enabling accurate characterization of resolving macrophages.
- The findings support the use of this model to advance our understanding of inflammation resolution and tissue repair mechanisms.
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