Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

Summary

Group 2 innate lymphoid cells (ILC2s), implicated in type 2 inflammation, mainly participate in response to helminth infection, allergic diseases, metabolic homeostasis, and tissue repair. In this study, a procedure to isolate ILC2s from murine nasal mucosa and detect the expression of CD226 is demonstrated.

Abstract

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various pathological processes, including anti-helminth immunity, tissue repair, thermogenesis, and autoimmune diseases such as asthma and allergic rhinitis (AR). ILC2s permanently reside in peripheral tissues such as the skin, gut, lungs, and nasal cavity; however, there is limited information about their exact functions in nasal mucosal immunity. CD226 is an activating costimulatory molecule, mainly expressed on natural killer (NK) cells, T cells, and inflammatory monocytes. However, whether ILC2s express CD226 or play a role in the pathogenesis of ILC2s-related diseases remains unknown. Here, we established a method to isolate and identify ILC2s from the nasal mucosa and detected CD226 expression on ILC2s obtained from healthy and AR mice. Herein, we describe this protocol for the isolation and identification of ILC2s from mouse nasal mucosa, which will help explore the internal pathological mechanism of immunological disorders in nasal mucosal diseases.

Introduction

Group 2 innate lymphoid cells (ILC2s) were first discovered in the peritoneal cavity tissues of mice and were subsequently demonstrated to be present in the blood and other peripheral tissues such as the lungs, skin, and nasal cavity^1,2,3. As tissue-resident cells, ILC2s are mainly maintained and proliferated locally and function as the first guards responding to exogenous harmful stimuli through producing numerous type 2 cytokines and inducing type 2 immunity^4,5,6. ILC2s can also ex....

Protocol

All experiments were performed in accordance with the Care and Use of Laboratory Animals Guidelines. All procedures and protocols were approved by the Scientific Research Ethics Committee of the Fourth Military Medical University (No. 20211008).

1. Murine AR model establishment

1. House male and female wild-type (WT) C57BL/6 mice aged 8-12 weeks under specific pathogen-free conditions and provide standard laboratory chow and water.
2. Emulsify 50 µg of ovalbumin (OVA) in 0.2 mL of sterile PBS containing 2 mg of aluminum hydroxide on a clean bench to maintain sterility. On days 0, 7, and 14, intraperiton....

Results

An OVA-induced murine model was developed to explore the role of ILC2s in AR. The construction of AR murine model was based on previous studies with slight modifications^28,29,30,31. A 10 min video was captured to measure the frequency of sneezing and nasal scratching after the last nasal challenge. Allergic symptoms of the OVA-induced-AR mice were presented in Figure 1

Discussion

ILC2s are closely associated with type 2 inflammation and inflammatory disorders, as demonstrated by an increasing number of studies. Both mouse models and human observation contribute to a better understanding of its function in the upper airway. In asthma pathophysiology, ILC2s are activated through thymic stromal lymphopoietin, IL-25, and IL-33, which are mostly produced by epithelial cells. Then mirroring Th2 cells, ILC2s produce IL-4, IL-5, and IL-13 to aggravate type 2 inflammation³². Furthe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Aluminum hydroxide	Meilun biological Technology	21645-51-2
CD11b	eBioscience	11-0112-82	Used in antibody coctail
CD11c	BioLegend	117306	Used in antibody coctail
CD16/32	BioLegend	101302	Clone: 93; Dilution 1:100
CD226	BioLegend	128812	Used in antibody coctail
CD3e	BioLegend	100306	Used in antibody coctail
CD45	BioLegend	103128	Used in antibody coctail
CD45R	eBioscience	11-0452-82	Used in antibody coctail
CD90.2	BD Pharmingen	553014	Used in antibody coctail
Collagenase IV	DIYIBio	DY40128
CountBright absolute counting beads	Invitrogen	C36950	absolute counting beads
Dnase	Beyotime	D7076
Fetal Bovine Serum	gibco	10270-106
Fixable Viability Dye eFluor 520 (FITC)	eBioscience	65-0867-14	FVD
HBSS, calcium, magnesium	Servicebio	G4204-500
KLRG1	eBioscience	17-5893-81	Used in antibody coctail
NaN3	SIGMA	S2002
NovoExpress software	AgilentTechnologies	Version 1.5.0	flow cytometry (FCM) analysis software
OVA	SIGMA	9006-59-1
PBS, 1x	Servicebio	G4202-500
PBS, 10x	Servicebio	G4207-500
Percoll	Yeasen	40501ES60	density gradient media
RPMI 1640 culture media	Corning	10-040-CVRV
Spectral cell analyzer	SONY	SA3800