Name: noninvasive sampling of mucosal lining fluid for the quantification of in vivo upper airway immune-mediator levels
Uploaded: 2017-08-07T12:30:00
Description: Watch this Scientific Journal Video about Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels at JoVE.com

We express our gratitude to the children and families of the COPSAC2010 cohort study for all their support and commitment. We acknowledge and appreciate the unique efforts of the COPSAC research team and the technical help from Technician Lisbeth Buus Rosholm, Technical University of Denmark, for the measurement of cytokines and chemokines.

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
<ol>
	<li>Use sheets of filter paper (fibrous hydroxylated-polyester sheets, see the Table of Materials...

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Response+to+A(H1N1)pnd09+Vaccination+in+Pregnant+Women:+A+Single+Blinded+Randomized+Controlled+Trial.">Altered Response to A(H1N1)pnd09 Vaccination in Pregnant Women: A Single Blinded Randomized Controlled Trial.</a> PloS One. 8 (4), 56700 (2013).</li><li>Wolsk, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picornavirus-Induced+Airway+Mucosa+Immune+Profile+in+Asymptomatic+Neonates.">Picornavirus-Induced Airway Mucosa Immune Profile in Asymptomatic Neonates.</a> J Infect Dis. 213 (8), 1262-1270 (2016).</li><li>Wolsk, H. M., Chawes, B. L., F&#248;lsgaard, N. V., Rasmussen, M. 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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 picornaviruses7,11, as well as other pre&#160;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...

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.
Here, we describe the implementation of a method to sample undisturbed mucosal lining fluid from the nasal cavity using a filter paper-based, noninvasive technique, as well as a subsequent extraction procedure, used to elute protein-based analytes from the filter papers prior to their quantification. This technique can, for example, be used to obtain in vivo immune signatures of both healthy individuals and individuals with various respiratory diseases. Furthermore, it is possible to examine exposures of importance for a specific immune signature and to evaluate if it is a predictor or mediator of later disease development.
Mucosal lining fluid has previously been obtained by nasal lavage2, which is often preceded by a nasal challenge test, where an allergen is introduced in high levels to stimulate an inflammatory response3,4. However, the nasal lavage technique is not feasible in young children and introduces an unknown dilution factor, which confounds the outcomes, as the diluted mediator levels can fall below the detection limit of the assay5. Moreover, due to the unknown dilution factor, the measured analyte responses from the nasal challenge tests are not comparable between individuals, thereby limiting the usefulness of the nasal lavage technique in a cohort setting. Finally, allergen challenge is only applicable in sensitized subjects, and other challenges, such as the histamine challenge, are not physiologically relevant, potentially causing a ceiling effect on mediator release. These problems are circumvented in the presented filter paper-based technique for mucosal lining fluid collection, where the individual secretion of fluids and analyte levels are the only factors that influence inter-individual variance.
During the extraction procedure, analytes are eluted from the filter papers after the addition of identical volumes of buffer to all samples. This favors similar ex vivo dilution of all samples. An extraction buffer of albumin-based isotonic salt solution is used for the extraction step; it enables the extraction of protein-based mediators and stabilizes proteins to limit denaturation during the subsequent freezing of eluted proteins prior to quantification. To avoid protein degradation during the extraction phase, a cocktail of protease inhibitors is added to the extraction buffer.
The implementation of techniques that allow for the quantification of undisturbed, in vivo-generated immune mediators at mucosal sites is of the utmost importance. First, the mucosal site makes up the largest immunological organ in the body. Second, the nasal location is the primary site of airborne exposure and is tightly connected to the respiratory immunological compartment of the lungs1. Third, the possibility of surveying this important organ with a noninvasive technique opens the possibility to provide a plethora of information on the important microbe-immune interaction axis in relation to health and disease in the airways. Fourth, there are many other possible applications of this technique, such as studying local immunological alterations in randomized, controlled trials of drugs and micronutrients.
We initially implemented the technique in the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort, where we determined the immune profile of the mucosal lining fluid in 7-year-old children with allergic rhinitis versus healthy controls13. Subsequently, we successfully applied this technique to the longitudinal COPSAC2010 cohort and assessed airway immune profiles at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. Results from the 1-month-old neonates have demonstrated important associations between the immune signature and early-life environmental exposure7,8,9,10,11,12.

<table border="0" cellpadding="0" cellspacing="0" width="1688">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Fibrous hydroxylatedpolyester sheets</td>
			<td style="width:103px;">Accuwik Ultra&#160;</td>
			<td style="width:119px;">SPR0730</td>
			<td style="width:1084px;">Filter paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Milliplex Assay Buffer&#160;</td>
			<td>Millipore</td>
			<td>L-AB</td>
			<td>Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">low-protein binding storage plates&#160;</td>
			<td>Thermo Scientific</td>
			<td>CLS8161</td>
			<td>Plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor</td>
			<td style="width:103px;">Roche</td>
			<td align="right" style="width:119px;">11873580001</td>
			<td>complete EDTA-free Protease Inhibitor Cocktail</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Reader of multi-spot plates</td>
			<td style="width:103px;">Mesoscale</td>
			<td style="width:119px;">NA</td>
			<td>Sector imager 6000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Assays&#160;</td>
			<td style="width:103px;">Mesoscale</td>
			<td style="width:119px;"></td>
			<td>Human 10-plex TH1/TH2 cytokine assay and 9-plex chemokine assay, and singleplex IL-17A, TGF-&#946;1and TSLP. A description can be found online on www.mesoscale.com</td>
		</tr>
	</tbody>
</table>

noninvasive sampling of mucosal lining fluid for the quantification of in vivo upper airway immune-mediator levels

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.&#160;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.

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 sample...

Watch this Scientific Journal Video about Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels at JoVE.com

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

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.

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

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

The overall goal of this mucosal lining fluid sampling method is to study the local immune signature of the airway without the need for stimulation procedures. This method allows you to study key questions in the area of pediatric respiratory diseases such as infections, asthma and allergy. The advantages of the method are that you can measure very low levels of immune mediators in the unstimulated airway mucosa in vivo.Demonstrating the procedure will be done by Dr.Wolsk. For neonate sample collection, first cut at least two three by 15 millimeter strips from fibrous hydroxylated polyester filter sheets. Next, place the neonate on a bed and take note of any symptoms of airway infection.Then, use forceps to insert one piece of filter paper approximately one centimeter into the anterior part of the inferior turbinate of each nostril of the infant and administer sugar water to the neonate for comfort as needed. Take note of any sneezing, persistent crying or epistaxis that occurs during the sampling period. After two minutes of absorption, transfer the filter papers into a labeled tube for immediate storage at negative 80 degrees Celsius.Demonstrating the experimental procedure will be Lisbeth Rosholm, a technician in my laboratory. For airway immune mediator quantification, place up to 10 samples from negative 80 degrees Celsius storage on ice and record their identification numbers. When the samples have thawed, immerse both of the filter papers per subject in 150 microliters of freshly prepared buffer per filter paper, supplemented with one complete protease inhibitor tablet per 25 milliliters of buffer stock solution.Transfer the samples to a plate shaker at 400 rpm for five minutes. Transfer the buffer and filter papers into the cups of individual cellulose acetate tube filters. Centrifuge the samples to collect the filtered nasal extract at the bottom of the microcentrifuge tubes.Then, remove the cups and place the tubes on ice. Next, transfer 150 microliter aliquots of the nasal extract into individual wells of low protein binding storage plates and store the plates at negative 80 degrees Celsius until analysis. Then, measure the concentration of the nasal immune mediators of interest by a high sensitivity electrochemiluminescence-based multiplexed array according to standard assay protocols.In this representative analysis of 620 neonates, most of the immune mediators measured demonstrated a low level of detection. IFN gamma levels were below the lower detection limit in almost half of the samples, while TNF alpha, CCL4, and TGF beta levels were below the lower detection limit in less than 1%of the samples. A strong multicollinearity was evident for the 20 detected mediator levels, as illustrated by heat map.In asymptomatic picornavirus infected neonates, an upregulated profile compromised mainly of type 1 immune mediators was observed suggesting an immune-triggering role of colonizing bacteria and viruses in the earliest part of life. An association between the presence of siblings in the household at birth, and a specific Type 1 Type 17 directed mucosal immune response was also determined with evidence for correlation of this phenomenon with an in utero immune priming effect that is inversely correlated to the time since the last childbirth. In addition, children born to mothers receiving high versus placebo supplements were characterized by an upregulation in specific immune mediators, suggesting that Vitamin D promotes an antibacterial immune response independent of that induced by virus, bacteria, siblings or maternal atopy.This method of microsampling may also be used through a bronchoscope, allowing to study the immune mediators in the lower airways. After watching this video, you should have a good understanding on how to measure mediators in the unstimulated airway mucosa in vivo in neonates and older children.

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