Immunology

Adipose tissue macrophages (ATMs) critically influence obesity-induced inflammation and metabolic dysfunction. Recent studies identified distinct ATM subsets characterized by markers such as CD11c, CD9, and Trem2, associated with pro-inflammatory and metabolically activated states. This protocol outlines a detailed, reproducible methodology for isolating, characterizing, and sorting these ATM subsets from murine epididymal white adipose tissue (eWAT) using multicolor flow cytometry. Key steps include stromal vascular fraction (SVF) isolation, immunophenotyping, sequential gating strategies, and fluorescence-activated cell sorting (FACS) for downstream gene expression analysis. The protocol was validated in diet-induced obese (DIO) mice treated with the IRE1 RNase inhibitor STF-083010, demonstrating its utility for studying ATMs in the context of obesity and metabolic disease.

The growing demand for advanced analytical techniques to explore complex cellular targets of nanotherapeutics has driven the development of innovative methodologies. This protocol presents a refined approach for fluorescent labeling and flow cytometric analysis of colonic cells following oral lipid nanoparticle (LNP) treatment, focusing on LNP uptake in colonic cell subpopulations in a DSS-induced colitis mouse model. By integrating optimized fluorochrome selection and gating strategies with advanced t-distributed stochastic neighbor embedding (t-SNE) analysis, this method enables precise identification and multidimensional visualization of LNP-targeted epithelial and macrophage populations under the complex conditions of inflamed colon tissue. Building on our previous studies demonstrating the effectiveness of nanoparticles in targeted drug delivery, this approach highlights the utility of flow cytometry for assessing uptake efficiency and cellular targeting. Unlike conventional protocols, it incorporates t-SNE for enhanced multidimensional analysis, allowing for the detection of subtle cellular patterns and the delineation of intricate clusters. By addressing gaps in traditional methodologies, this protocol provides a robust and reproducible framework for investigating in vivo cellular targets and optimizing drug delivery strategies for nanomedicines.

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.

Macrophages are a heterogeneous class of innate immune cells that offer a primary line of defense to the body by phagocytizing pathogens, digesting them, and presenting the antigens to T and B cells to initiate adaptive immunity. Through specialized pro-inflammatory or anti-inflammatory activities, macrophages also directly contribute to the clearance of infections and the repair of tissue injury. Macrophages are distributed throughout the body and largely carry out tissue-specific functions. In skeletal muscle, macrophages regulate tissue repair and regeneration; however, the characteristics of these macrophages are not yet fully understood, and their involvement in skeletal muscle aging remains to be elucidated. To investigate these functions, it is critical to efficiently isolate macrophages from skeletal muscle with sufficient purity and yield for various downstream analyses. However, methods to prepare enriched skeletal muscle macrophages are scarce. Here, we describe in detail an optimized method to isolate skeletal muscle macrophages from mice. This method has allowed the isolation of CD45⁺/CD11b⁺ macrophage-enriched cells from young and old mice, which can be further used for flow cytometric analysis, fluorescence-activated cell sorting (FACS), and single-cell RNA sequencing.

Phlebotomine vectors, sand flies of the order Diptera, are known to transmit Leishmania parasites as well as RNA viruses (arboviruses) to humans. The arbovirus, Icoaraci Phlebovirus (BeAN 24262 - ICOV), used in this study was isolated from Nectomys rodents, a mammalian species that is the same natural sylvatic reservoir of Leishmania (Leishmania) amazonensis. This Leishmania species is distributed in primary and secondary forests in Brazil and other countries in America and causes localized and diffuse anergic skin lesions. In our recent studies, we observed an aggravation of the protozoan infection by ICOV through the modulation of cytokine expression, such as IL-10 and IFN-β, enhancing the parasite load and possibly the pathogenesis. Efficient viral production and quantitation had to be developed and standardized to ensure that immuno-molecular assays provide consistent and reproducible viral infection results. The standardization of these procedures becomes a particularly useful tool in research, with several applications in understanding the interaction between the host cell and Phlebovirus, as well as co-infections, allowing the study of intracellular signaling pathways. Here, we detail a protocol that allows the production and quantitation of the Icoaraci Phlebovirus using BHK-21 cells (baby hamster kidney cells) and subsequent infection of peritoneal macrophages from C57BL/6 mice.