Identification of Group 2 Innate Lymphoid Cells in Mouse Lung, Liver, Small Intestine, Bone Marrow, and Mediastinal and Mesenteric Lymph Nodes

Abstract

Innate lymphoid cells (ILCs) are a heterogeneous family of lymphocytes that populate barrier and non‐barrier tissues. ILCs regulate immune responses to pathogens and commensals but also sustain metabolic homeostasis, tissue remodeling after injury and establish dialogue with the nervous system. ILCs rapidly become activated in the absence of adaptive antigen receptors by responding to signaling molecules provided by hematopoietic or non‐hematopoietic cells. Here we provide protocols designed for processing the lung, liver, small intestine, bone marrow, mediastinal and mesenteric lymph nodes in order to obtain a purified leukocyte fraction of cells, in which ILC2 enrichment is optimized. In addition, we describe in detail the methodologies used to activate ILC2s and the assays necessary for the detection of their effector cytokines. We highlight the differences in ILC2 characterization within distinct tissues that we have recently identified. © 2019 by John Wiley & Sons, Inc.

Single-cell analysis of RORα tracer mouse lung reveals ILC progenitors and effector ILC2 subsets

“…This was followed by RBC lysis by ammonium chloride. Liver tissues were processed as indicated previously (Romera-Hernández et al, 2019).…”

Tissue-Resident Type 2 Innate Lymphoid Cells Arrest Alveolarization in Bronchopulmonary Dysplasia

“…After red cell lysis using ACK (Leagene, China), the cell suspension obtained from a single mouse was washed with phosphate-buffered saline (PBS), and the cells counted. For FACS analysis, the single-cell suspensions were stained with antibodies to CD11b-FITC (101206), F4/80-APC/Cyanine7 (123118), CD86-PE (E-AB-F0994D), CD206-PE/Cyanine7 (E-AB-F1135D), CD90.2-PE (E-AB-F10940) [ 3 , 22 ], and Sca-1-APC (108112), as well as antibodies to Lineage-FITC (133302) [ 4 ] (all purchased from eBioscience or Biolegend, USA). Cells were analyzed on a flow cytometer (Beckman CytoFLEX, USA).…”

“…ILC2s are the most abundant in pulmonary mucosa; although, they have also been detected in the liver, small intestine, bone marrow, and mediastinal and mesenteric lymph nodes [ 4 ]. Mirroring Th2 cells, ILC2s are activated by epithelial-derived cytokines, IL-25, IL-33, thymic stromal lymphopoietin (TSLP) through their cognate receptors, IL17RB, suppression of tumorigenicity 2 (ST2), and TSLPR, respectively.…”

Interrogating the Small Intestine Tuft Cell–ILC2 Circuit Using In Vivo Manipulations

“…Protocols for the isolation of lamina propria cells from intestine typically involve sequential steps of mucus depletion using reducing agents such as DTT, removal of epithelial cells with EDTA, and enzymatic digestion of the lamina propria (Sheridan & Lefrancois, 2012). Procedures for the isolation and detection of mouse intestinal ILCs were recently summarized in detail (Burrows, Chiaranunt, Ngai, & Mortha, 2020; Romera‐Hernández, Mathä, Steer, Ghaedi, & Takei, 2019). However, the fast and extensive cell death that occurs when isolating intestinal lamina propria cells from tissues with strong type 2 immune activation, including after helminth infection, has greatly limited the study of cellular processes in the small intestinal tissue through subsequent downstream applications such as multiparameter flow cytometry and scRNA‐seq, which rely on tissue dissociation and generation of single‐cell suspensions.…”