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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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

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, intraperitoneally inject female or male mice with 50 µg each of emulsified OVA.
  3. On days 21, 22, 23, 24, and 25, intranasally instill mice with 50 µg of OVA dissolved in 30 µL of sterile PBS (15 µL per nostril) under inhalation anesthesia (2-3% isoflurane with an Oxygen flow rate or 0.5 L/min).
  4. Euthanize the mice 24 h after the last challenge (day 26).

2. Isolation of mononuclear cells (MNCs) from the nasal mucosa

  1. Euthanize the mice by cervical dislocation under deep anesthesia. Soak the mice head up in 75% ethanol for 5 min and avoid ethanol entering the external nostril. Place the abdomen downward on the operating table.
  2. Cut off the fore-teeth. At the midline of the head, make an incision, and cut open the skin using scissors.
  3. Remove the lower jaw, cut off the entire nose along the end of the palate, and place the tissue into a 60 mm Petri dish containing 5 mL of ice-cold PBS. Using scissors and forceps, remove the flesh and muscles adhering to the bones.
  4. Transfer the mouse nose to a new 60 mm Petri dish containing 5 mL of ice-cold PBS. Wash the bones twice with 5 mL of ice-cold PBS.
  5. Transfer the nose into a 1.5 mL microcentrifuge tube.
  6. Sufficiently smash and transfer the nose to a 15 mL tube containing 2 mL of prewarmed digest buffer (RPMI 1640 medium supplemented with 1 mg/mL of collagenase IV and 10 U/mL DNase I).
  7. Fasten the lid and place the tube vertically in an orbital shaker at 37 °C, with continuous agitation at 120-150 rpm for 40 min.
    NOTE: Pre-warm the digest buffer to 37 °C to achieve the highest enzyme activity.
  8. Add 5 mL of ice-cold RPMI 1640 medium containing 10% fetal bovine serum (FBS) to stop the digestion process.
  9. Filter through a 70 µm cell strainer to remove solid fragments.
  10. Centrifuge at 500 x g for 5 min, and then gently discard the supernatant.
  11. Resuspend the pellet in ice-cold RPMI 1640 medium and centrifuge at 500 x g for 5 min. Gently discard the supernatant.
  12. Resuspend the cell pellet in 4 mL of 40% density gradient media (160 µL of 10x PBS + 1.44 mL of density gradient media stock solution + 2.4 mL of RPMI 1640 medium).
  13. Gently insert a Pasteur pipette to the bottom of the tube and slowly add 2.5 mL of 80% density gradient media (200 µL of 10x PBS + 1.8 mL of density gradient media stock solution + 0.5 mL of RPMI 1640 medium).
  14. Set the acceleration and deceleration rate of the centrifuge lower than the third gear and centrifuge at 400 x g for 15 min at room temperature (RT).
  15. Remove the top layer of impurities before draining the cells at the interface to avoid potential contamination. Carefully drain the mononuclear cells (MNCs) layer at the 40%/80% density gradient media interface into a 15 mL tube containing 2 mL of ice-cold PBS using a pipette. Wash the cells with ice-cold PBS twice.

3. Surface staining for FCM analysis

  1. Harvest the cells and centrifuge at 500 x g for 5 min at 4 °C. Resuspend the cell pellet in staining buffer (PBS supplemented with 2% FBS and 0.1% NaN3) and centrifuge at 500 x g for 5 min at 4 °C. Discard the supernatant.
    CAUTION: Be cautious when preparing the staining buffer. NaN3 is very toxic and may cause damage to organs if swallowed, inhaled, or in contact with skin. Also, avoid release to the environment.
  2. Resuspend the cells in 80 µL blocking solution (anti-CD16/32 antibody (1:100) diluted in staining buffer) per tube. Incubate for 30 min in the dark at 4 °C.
  3. Without washing, add 20 µL of the appropriate dilution of the surface-staining antibody cocktail (Table 1).
  4. Add 1 µL (per test) of fixable viability dye 520 (FVD) just before adding the antibody cocktail to the samples. Incubate in the dark at 4 °C for 30 min. Set matching isotype antibodies and fluorescence minus one (FMO) as the negative controls.
  5. Wash the cells in 500 µL of staining buffer by centrifuging at 500 x g for 5 min at 4 °C.
  6. Resuspend the cell pellet in 200 µL of staining buffer. Add 50 µL of vortexed absolute counting beads to the stained cells and agitate. Subject them to a flow cytometry (FCM) analysis.
    NOTE: FCM data were obtained using a spectral cell analyzer and analyzed with flow cytometry (FCM) analysis software.

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 modifications28,29,30,31. 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...

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

NameCompanyCatalog NumberComments
Aluminum hydroxideMeilun biological Technology21645-51-2
CD11beBioscience11-0112-82Used in antibody coctail
CD11cBioLegend117306Used in antibody coctail
CD16/32BioLegend101302Clone: 93; Dilution 1:100
CD226BioLegend128812Used in antibody coctail
CD3eBioLegend100306Used in antibody coctail
CD45BioLegend103128Used in antibody coctail
CD45ReBioscience11-0452-82Used in antibody coctail
CD90.2BD Pharmingen553014Used in antibody coctail
Collagenase IVDIYIBioDY40128
CountBright absolute counting beadsInvitrogenC36950absolute counting beads
Dnase figure-materials-1052BeyotimeD7076
Fetal Bovine Serumgibco10270-106
Fixable Viability Dye eFluor 520 (FITC)eBioscience65-0867-14FVD
HBSS, calcium, magnesiumServicebioG4204-500
KLRG1eBioscience17-5893-81Used in antibody coctail
NaN3SIGMAS2002
NovoExpress softwareAgilentTechnologiesVersion 1.5.0flow cytometry (FCM) analysis software
OVASIGMA9006-59-1
PBS, 1xServicebioG4202-500
PBS, 10xServicebioG4207-500
PercollYeasen40501ES60density gradient media
RPMI 1640 culture mediaCorning10-040-CVRV
Spectral cell analyzerSONYSA3800

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Group 2 Innate Lymphoid CellsNasal MucosaCD226 ExpressionIsolation TechniqueFlow CytometryAllergic Rhinitis ModelOvalbumin InjectionC57BL 6 MiceSterile PBSDigestion BufferTissue ProcessingRPMI 1640 Medium

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