TM
Thomas Marichal
  • Faculty, University of Liege Liege
Differentiation of Bone Marrow Monocytes into Alveolar Macrophages-like Cells through Co-culture with Lung Epithelial Cells and Group 2 Innate Lymphoid Cells
Authors:  Pauline Loos, Thomas Marichal, Bénédicte Machiels and Laurent Gillet, date: 09/20/2023, view: 807, Q&A: 0

During life, the embryonic alveolar macrophage (AM) population undergoes successive waves of depletion and replenishment in response to infectious and inflammatory episodes. While resident AMs are traditionally described as from embryonic origin, their ontogeny following inflammation or infection is much more complex. Indeed, it appears that the contribution of monocytes (MOs) to the AM pool is variable and depends on the type of inflammation, its severity, and the signals released in the microenvironment of the pulmonary niche (peripheral imprinting) and/or in the bone marrow (central imprinting). Deciphering the cellular and molecular mechanisms regulating the differentiation of MOs into AMs remains an area of intense investigation, as this could potentially explain part of the inter-individual susceptibility to respiratory immunopathologies. Here, we detail a relevant ex vivo co-culture model to investigate how lung epithelial cells (ECs) and group 2 lung innate lymphoid cells (ILC2s) contribute to the differentiation of recruited MOs into AMs. Interestingly, the presence of lung ILC2s and ECs provides the necessary niche signals to ensure the differentiation of bone marrow MOs into AMs, thus establishing an accessible model to study the underlying mechanisms following different infection or inflammation processes.


Key features

• Ex vivo co-culture model of the alveolar niche.

• Deciphering the particular niche signals underlying the differentiation of MO into AMs and their functional polarization.


Graphical overview
This protocol described the isolation of bone marrow monocytes (MOs), lung epithelial cells (ECs), and lung group 2 lung innate lymphoid cells (ILC2s) and the ex vivo co-culture of these cells to drive the differentiation of bone marrow MOs into alveolar macrophages (AMs).




This co-culture experiment is composed of three steps (Graphical overview):
1. Identification and FACS-sorting of ECs and MOs isolated from the lung and the bone marrow of naive mice, respectively.
2. Culture of these ECs and bone marrow MOs for three days.
3. Addition of ILC2s isolated from the lung of naïve mice or mice subjected to a treatment/infection of interest.

Identification and Quantitation of Neutrophil Extracellular Traps in Human Tissue Sections
Authors:  Coraline Radermecker, Alexandre Hego, Philippe Delvenne and Thomas Marichal, date: 09/20/2021, view: 3536, Q&A: 0

Neutrophils are one of the first innate immune cells recruited to tissues during inflammation. An important function of neutrophils relies on their ability to release extracellular structures, known as Neutrophil Extracellular Traps or NETs, into their environment. Detecting such NETs in humans has often proven challenging for both biological fluids and tissues; however, this can be achieved by quantitating NET components (e.g., DNA or granule/histone proteins) or by directly visualizing them by microscopy, respectively. Direct visualization by confocal microscopy is preferably performed on formalin-fixed paraffin-embedded (FFPE) tissue sections stained with a fluorescent DNA dye and antibodies directed against myeloperoxidase (MPO) and citrullinated histone 3 (Cit-H3), two components of NETs, following paraffin removal, antigen retrieval, and permeabilization. NETs are defined as extracellular structures that stain double-positive for MPO and Cit-H3. Here, we propose a novel software-based objective method for NET volume quantitation in tissue sections based on the measurement of the volume of structures exhibiting co-localization of Cit-H3 and MPO outside the cell. Such a technique not only allows the unambiguous identification of NETs in tissue sections but also their quantitation and relationship with surrounding tissues.


Graphic abstract:



Graphical representation of the methodology used to stain and quantitate NETs in human lung tissue.


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