Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

Summary

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Abstract

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood₂₀₀₀ (COPSAC₂₀₀₀) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC₂₀₁₀ birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e. from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Introduction

The mucosal lining fluid of the nose makes up the liquid part of the upper-airway system. It consists of a complex matrix of mediators derived from the interplay between the epithelium and the immune cells that make up the first line of defense against invading microorganisms. The nasal mucosa is easily accessible, and there is a strong functional and immunological relationship between the nose and the bronchi¹. This compartment is of special interest in relation to airway diseases that are common in childhood, such as asthma and allergic rhinitis, but also to a range of other respiratory disorders more prevalent later in life.

Protocol

The studies were conducted in accordance with the guiding principles of the Declaration of Helsinki. Approvals from the Ethics Committee for Copenhagen (KF 01-289/96 for COPSAC₂₀₀₀ and H-B-2008-093 for COPSAC₂₀₁₀) and the Danish Data Protection Agency were obtained, and written informed consent was obtained from both parents of each subject before enrollment.

1. Experimental Setup

1. Use sheets of filter paper (fibrous hydroxylated-polyester sheets, see the Table of Materials)^6,7.
   1. Cut out strips 3 x 15 mm in size for a 1-month-old child an....

Results

Baseline Characteristics of the Airway Immune Profiles:
Complete data on upper airway mucosal immune mediator levels at 1 month of age was obtained in 620 (89%) of the 700 children enrolled in the COPSAC₂₀₁₀ cohort. Ten neonates were enrolled before the technique was established, and 19 did not attend the 1-month visit. Additionally, 47 samples were excluded because they were extracted and measured in another laboratory used in a pilot study, and 4 samples .......

Discussion

With the technique presented here, we were able to determine the in vivo upper-airway mucosal immune profile in children from as early as 1 month of age, which has not been done previously. We observed that presence of specific airway bacteria and picornaviruses^7,11, as well as other pre and perinatal exposures, were mirrored in the airway immune profile of the neonates. Furthermore, we used these airway immune profile data to study the mechanism of.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fibrous hydroxylatedpolyester sheets	Accuwik Ultra	SPR0730	Filter paper
Milliplex Assay Buffer	Millipore	L-AB	Buffer
Low-protein binding storage plates	Thermo Scientific	CLS8161	Plates
Protease Inhibitor	Roche	11873580001	complete EDTA-free Protease Inhibitor Cocktail
Reader of multi-spot plates	Mesoscale	NA	Sector imager 6000
Assays	Mesoscale		Human 10-plex TH1/TH2 cytokine assay and 9-plex chemokine assay, and singleplex IL-17A, TGF-β1and TSLP. A description can be found online on www.mesoscale.com