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 cavity1,2,3. 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 immunity4,5,6. ILC2s can also exert their effects by trafficking toward the infected tissues7,8.
Similar to T-helper 2 (Th2) cells, the complicated regulatory networks of ILC2s ensure their significant involvement in the progression of various type 2 inflammatory diseases, including airway allergic diseases8,9. In asthma, epithelial cell-derived alarmins can activate ILC2s, which further promote pulmonary inflammation through the secretion of interleukin (IL)-4, IL-5, and IL-1310. Clinical studies have also indicated that ILC2 levels were significantly elevated in the sputum and blood of patients with severe asthma, suggesting an association of ILC2s with asthma severity and their function as a predictor of asthma progression11.
Allergic rhinitis (AR) is a common chronic inflammatory disease that affects millions of people annually, and effective treatments for this disease are limited12,13. ILC2s play crucial roles in the pathophysiology of AR, whether in the sensitization phase or symptom generation and inflammation phase14. In patients with AR, the levels of ILC2 in the peripheral blood have been reported to be elevated both locally and systemically15. However, certain effects and the underlying mechanisms of ILC2s on the pathophysiology and progression of AR still require further exploration.
CD226 – a transmembrane glycoprotein that serves as a costimulatory molecule – is primarily expressed on natural killer (NK) cells, T cells, and other inflammatory monocytes16,17. The interaction of CD226 and its ligands (CD155 and/or CD112) or competitor (TIGIT) allows it to participate in the biological functions of various immune cells18. The binding of the ligands on antigen-presenting cells to CD226 on cytotoxic lymphocyte (CTL) promotes the activation of both cells simultaneously, while the activation of CTL can be further suppressed by TIGIT (T cell immunoreceptor with Ig and ITIM domains), the competitor of CD22619,20. A human ex vivo study revealed that CD226 and CD155 on T cells regulate the balance between Th1/Th17 and Th2 through differentially modulating Th subsets21. CD226 can likewise mediate platelet adhesion and NK tumor-killing activity22,23. Meanwhile, CD226 is well-studied in the pathogenesis of various infectious diseases, autoimmune diseases, and tumors18,24,25. At present, CD226 has become a new bright spot for immunotherapy. Studies have found that extracellular vesicles can reverse CD226 expression on NK cells to reinstate their cytotoxic activity and intervene in the progression of lung cancer26. A recent study has revealed a subcluster of fetal intestinal group 3 ILCs characterized with high CD226 expression by single-cell RNA sequencing27, which indicated that CD226 might exert roles in the innate lymphoid cell-mediated immunity.
Our knowledge about ILC2s in airway inflammation is primarily based on studies on asthma; however, little is known about their functions in nasal mucosal immunity. Thus, a protocol was established to isolate and identify ILC2s from the nasal mucosa. The study focuses on the expression of CD226 on ILC2s in nasal tissues and its variation between healthy and AR mice. This may provide novel insights into the underlying mechanisms of ILC2-mediated regulation in the local immunity and serve as a basis for developing new approaches for AR treatment.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
R.Z. was supported by the National Natural Science Foundation of China (No. 81871258) and funds provided by Fourth Military Medical University (No.2020rcfczr). Y.Z. was supported by the Natural Science Basic Research Program of Shaanxi (No. 2021JM-081).
Materials
| 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 |
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