Role of Interleukin 5 (IL-5) and IL-5R Signaling in Non-eosinophil Upper Airway Cells in Chronic Rhinosinusitis With Nasal Polyps
Conditions: Nasal Polyps, Aspirin-exacerbated Respiratory Disease
Sponsor: Brigham and Women's Hospital

 
Study title: Role of IL-5R Signaling in Non-eosinophil Upper Airway Cells in 
Chronic Rhinosinusitis with Nasal Polyposis 
 
NCT number: NCT05672030 
 
Document date: December 18, 2023 
 
 
 
Study Protocol and Statistical Analysis Plan on following pages. 
 
 

 
 
Study Protocol and Statistical Analysis Plan  
 
Research Plan/Approach: 
 
Subject selection and recruitment.  
These observational clinical studies will be conducted on individuals with AERD and 
CRSwNP who are referred to the BWH Allergy or Otolaryngology Clinics. For the surgically 
excised sinus tissue, patients undergoing sinus surgery will be screened to determine if they 
meet criteria: a diagnosis of CRSwNP, no history of cystic fibrosis or primary ciliary dyskinesia, 
and no unilateral nasal polyposis. Patients for this study will also be excluded if they are on any 
targeted respiratory biologic therapy, or on chronic oral steroids or other immunosuppressive 
agents. Those who meet criteria and want to participate will sign informed consent as approved 
by the Mass General Brigham IRB [the research proposed here is already covered under our 
existing IRBs, with #2019P001829 (Collection and Analysis of Healthy Sinus Mucosa and 
#2003P002088 (Analysis of Cells and Mediators from Aspirin-Tolerant and Aspirin-Intolerant 
Donors)].  
Subjects will be between the ages of 18-65 years old, and we will aim to have equal 
numbers of male and female participants for each of the research approaches proposed below. 
A full physical exam, along with pertinent medical history, concomitant medications, prior history 
of response to medications, and prior surgical history will be collected for all participants.  
For some portions of the proposal, tissue and cell samples have already been collected 
and are available for analysis in our tissue repository of human surgical samples (Aim 1a), and 
for others (Aim 1b and 2) all samples will be prospectively collected. 
 
SPECIFIC AIM 1: Characterize the plasma cell subsets, and the functional 
significance of their IL-5Rα, in nasal polyps from patients with CRSwNP and/or 
AERD. 
Overview. Nasal polyp tissue from patients with AERD contains a distinct population of 
plasma cells when compared to CRSwNP, marked by increased IL5RA expression. In this Aim 
we will test the hypothesis that a unique subset of IL-5-responsive nasal polyp plasma 
cells drives the proliferation of sinus tissue plasma cells and the production of 
pathogenic antibodies in the respiratory tract of patients with CRSwNP and AERD.  
To identify transcriptional, cytofluorographic, and functional characteristics of plasma cells from 
nasal polyps in AERD and CRSwNP, we will employ two approaches, (1) RNA sequencing and 
(2) functional studies. We will then use qPCR and flow cytometry to verify the results on a set of 
markers selected based on pathobiologic interest, including IL-5Rα.  
Sub-Aim 1a. Use RNA sequencing to characterize the transcriptional profile of nasal 
polyp plasma cells in nasal polyps from patients with AERD and aspirin-tolerant 
CRSwNP. 
Methods.  Sinus tissue from 6 subjects with AERD and 6 subjects with aspirin-tolerant 
CRSwNP has been obtained. Subjects were be matched for age, gender, and intranasal 
corticosteroid use. Freshly excised tissue was digested into a single cell suspension. Using a 

BD FACSAria Fusion Cell Sorter, plasma cells were purified and collected in TCL buffer for 
storage. Going forward, 1000 cells from each patient phenotype will be used for low-input RNA 
sequencing, and the remainder set aside for qRT-PCR analysis. RNA will be sequenced with 
Smart-seq2 using the Broad Institute Technology Labs facilities. Unbiased differential gene 
expression will be characterized using the Kallisto program to map reads to the genome, taking 
into account uncertainty in read mapping. To identify gene sets that may relate to biologically 
relevant differences in cellular function we will utilize linear mixed models. To confirm 
expression results from our RNA sequencing, we will validate the gene quantification of the top 
5 differentially expressed genes within the plasma cell populations with qRT-PCR, using 
commercially available primers.  
Anticipated Results.  We expect that the transcriptional signature of the plasma cells 
from patients with AERD will show increased cell survival and activation. We expect that these 
cells will show upregulation of transcripts involved in activation of the cell cycle (CCND2), 
antiapoptotic genes (BCL2, CFLAIR), and those encoding surface receptors and adhesion 
molecules (IL5RA, ICAM1, CD9), along with downregulation of negative regulators of the cell 
cycle (KLF2, KLF6). We predict that the plasma cells in nasal polyps from patients with AERD 
will have higher IL5RA expression overall, and will express more IGHG4 and IGHE transcripts, 
suggesting that the overproduction of nasal IgE in inflamed tissues may be driven in part by IL-5 
signaling.  
Sub-Aim1b.  Determine the functional significance of IL-5Rα expression and IL-5 
signaling on the plasma cells in nasal polyp tissue. 
Overview.  To exert effects, IL-5 induces phosphorylation and activation of several JAK 
and STAT proteins, and may be involved in regulation of transcripts involved in B-cell 
maturation, differentiation, and survival. While IL-5 supports murine plasma cell survival, IL-5-
driven survival of human plasma cells has not been demonstrated. Our preliminary data has 
confirmed that IL-5Rα+ plasma cells are enriched in nasal polyps, suggesting a disease-specific 
mechanism that may sustain antibody production. Moreover, our studies suggest that IL-5 can 
upregulate expression of transcripts involved in proliferation, survival, migration and class switch 
in these cells. In this Sub-Aim, we will test the hypothesis that IL-5 stimulation of nasal polyp 
plasma cells induces signaling (A) and regulates transcriptional activity relevant to 
pathogenetic function (B). 
Methods. (A) To determine the signaling capability of IL-5Rα on nasal polyp plasma 
cells, we will measure the phosphorylation status of their JAK1/JAK2 and STAT1/STAT5, which 
are rapidly activated upon IL-5 binding to IL-5Rα and can be surrogates of IL-5Rα signaling in 
vivo. We will prospectively collect surgical samples and digest nasal polyp tissue from 5 AERD 
patients and 5 CRSwNP patients into a single cell suspension as above, and sort plasma cells 
as CD38HI/CD27HI/CD138+/CD20-. These will be evaluated using intracellular flow cytometry for 
phosphorylated JAK1, JAK2, STAT1, and STAT5. We will first measure the phosphorylation 
levels on freshly isolated cells, to determine differences in their baseline in vivo status. We will 
also measure their phosphorylation levels following a 6h and 24h stimulation with or without IL-5 
(1, 10 and 100 ng/mL), as described previously, to confirm their IL-5Rα signaling capacity. We 
will use BD Phosflow™ to optimize fixation and permeabilization for flow cytometric analysis of 
intracellular phosphoproteins. (B) To verify the transcriptional consequence of IL-5 signaling on 
plasma cells, we have already collected and sorted nasal polyp plasma cells from 5 patients 
with AERD and 5 CRSwNP and stimulated them for 6h with or without IL-5 (1, 10 and 100 
ng/mL). The stimulated/unstimulated pairs of plasma cells have been collected in TCL buffer, 

and banked for low-input RNA sequencing as above. Going forward, we will conduct differential 
expression analysis on IL-5-stimulated and unstimulated plasma cells using sample pairing, with 
a linear model framework. Our criterion for transcripts induced or repressed by IL-5 is that 
genes are more than twofold changed and obtain a false discovery rate of <1%.  
Anticipated Results. (A) Plasma cell populations with higher IL-5Rα expression should 
exhibit increased phosphorylation of JAK1, JAK2, STAT1, and STAT5 and should demonstrate 
further increases upon stimulation with IL-5. (B) We expect to confirm that IL-5 stimulation of 
nasal polyp plasma cells induces expression of CCND2, JAK1, IKZF1, ZBTB44, OTG, RAP1B, 
and RAC2, as well as signatures reflecting activation and anti-apoptotic signals (BCL6, AID, 
BLIMP-1).  
Statistical Analyses. With a sample size of 6 AERD subjects and 6 CRSwNP subjects, 
we will be powered at >90%, at a 0.05 level of significance, to detect a difference in antibody-
secreting cells that express IL-5Rα+ between subjects with AERD and CRSwNP. The data will 
be analyzed as described in Aim 2.    
Pitfalls and Alternative Approaches.  Although the proposed work is largely 
hypothesis based and furthers recent preliminary observations made by our group, low-input 
RNA sequencing allows for unbiased analyses. Thus, while we focus on the role of IL-5Rα+ 
plasma cells in CRSwNP and AERD pathogenesis, our approach also allows us to identify a 
range of differentially expressed genes and to broadly phenotype nasal polyp plasma cells.  
 
SPECIFIC AIM 2: Define the IL-5-elicted molecular features of upper airway nasal 
epithelial tissue from subjects with healthy sinus mucosa, CRSwNP, and AERD. 
 
Overview. Bronchial epithelial cells and human sinonasal tissue epithelial cells 
express a functional IL-5Rα. Further, in vivo inhibition of IL-5 leads to transcriptional 
modification of upper respiratory tract epithelial cells. In this aim, we will test the hypothesis 
that IL-5 actions on the human upper airway epithelium to reduce cell-to-cell adhesions 
by downregulating tight junction pathways and impacting transcripts related to cell-to-
cell adhesion.  
 
Methods. We have already established a protocol that for the first time, shows the 
development of IL-5Rα on upper airway (nasal) epithelial cells. Sinonasal epithelial cells 
expanded to an air liquid interface culture begin to express detectable levels of IL5RA by week 
2 and appear to reach maximal IL5RA transcript expression after 4 weeks in culture, which 
parallels their development of cilia and their expression of FOXJ1, a transcript that directly 
regulates genes involved in ciliary structure assembly and motility (Figure 5). 
Using this culture system, we will now explore the function of IL-5 signaling on these cells. 
Sinonasal epithelial cells will be harvested from 10 donors with CRSwNP, 10 donors with 
AERD, and 10 healthy donors undergoing correction of upper airway anatomic abnormalities. 
Epithelial cells will be expanded to passage 3 (week 4-5) in an air liquid interface culture and will 
be stimulated with IL-5 at 3 increasing doses for 24–48 hours. Additionally, to assess any 
possible chronic effects of IL-5, a portion of the air liquid interface cultures will be grown in low-
dose IL-5 added into their media from week 1 (in order to more closely approximate the 
environment of respiratory inflammation with the constant presence of IL-5 that exists in the 
sinuses of patients with CRSwNP).These sets of cultured and chronically and/or acutely 
stimulated or unstimulated epithelial cells will then be stored for RNA and protein 
measurements. They will be assessed by qPCR for expression of IL5RA, CDH1 (E-cadherin), 
CAV1 (caveolin-1), CAV2 (caveolin-2), EGFR (epidermal growth factor receptor), CXCLA, 

CXCL1, IL10RA, IL1RL1 (ST2), and IL6R, as these transcripts are known to be impacted by IL-
5 stimulation in the lower airway. We will also measure IL-5Rα protein levels by Western-blot in 
these epithelial cells. To more broadly assess the effects of IL-5 signaling on human nasal polyp 
epithelial cells and to assess for yet-unidentified impacts of IL-5/IL-5Rα signaling on these cells, 
we will collect IL-5 stimulated/unstimulated sinus tissue epithelial cell pairs for bulk RNA-
sequencing with Smart-seq2.  
 
Anticipated results. We suspect that the sinus tissue epithelial cells from diseased 
patients (CRSwNP and AERD) will express higher levels IL-5Rα compared to healthy control 
tissue, and/or that their development of IL5RA expression will happen earlier in the maturation 
process compared to healthy control epithelial cells. We suspect that IL-5 stimulation of sinus 
tissue epithelial cells will identify downregulation of transcripts related to cellular adhesion and 
tight junction pathways and enrichment of a T2 immune signature.  
Statistical Analyses. Differential expression analysis of RNA-seq data will be 
conducted using the DESeq2 package for R taking patient origin into account. Following 
normalization, DESeq2 will be run in a pairwise manner by comparing stimulated/unstimulated 
cell pairs, identifying significantly differentially expressed genes (DEGs). For pathway analyses, 
WebGestalt online tool will be used to identify the distribution of pre-ranked DEGs involved in 
specific signaling pathways in Gene Ontology Biological Processes database.  
Pitfalls and Alternative Approaches.  The molecular analysis of human upper airway 
epithelial cells is based on prior study of the human bronchial epithelial cells. However, if the 
pre-selected qPCR markers do not detect measurable differences between IL-5 stimulated and 
unstimulated cells, RNA-sequencing allows for unbiased analyses and may detect yet-
undescribed changes mediated by IL-5. Further, if the molecular analyses do not identify IL-5-
elicited changes in sinus tissue airway epithelium, we can plan functional phosphorylation 
studies on the sinus tissue airway epithelial cells as described in Aim 1b.   
 
Protection of Data Security and Confidentiality: 
Blinding to reduce bias. All patient-derived samples will be stored and analyzed using 
only a random Subject ID number, and all research will be kept blinded as to patient disease 
state, and all analyses of mechanistic data (flow cytometric measurements, qPCR 
measurements, Western Blots etc.) will be completed prior to unblinding of research 
investigators. 
Data security. All subjects will be assigned a unique numerical identifier, and study data 
will be stored in a password-secured electronic database on a dedicated server. Double-keyed 
entry will be used to enter the data to ensure the highest levels of validity. The two generated 
files are compared using a verification program that checks any discrepancies among double-
keyed entry. Before being added to the database, the data undergo further within-form and 
across-time verifications to assure completeness, accuracy, and participant identification 
number. Any errors in coding or keying are corrected promptly. Questionable data that need 
additional review are sent to a separate file and then resubmitted to the validation program after 
problem resolution. Nightly backups of all data files are made. All database files can be 
imported in SAS software for validation and data analysis. 
Data confidentiality. All data will remain confidential within the investigation site. A 
record of study subjects entered will remain strictly confidential and will be coded only by a 
study identification number assigned to each subject at the time of enrollment. While the study 
is in progress, identifying information will be maintained to enable collection of all follow-up data. 

All subject CRFs will be coded with a unique patient identifier, which will identify that subject 
only at the investigational site staff level. All direct subject contact (informed consent, subject 
screening and study procedures) and relevant confidentiality protections will be the 
responsibility of the PI. There should be no risk to subject confidentiality because all records 
associated with this study will be confidential within the study staff environment within the AERD 
Center. Safety precautions, password protection and encryption of data, will ensure that 
electronic systems will not be a risk to subject confidentiality.  
Confidentiality of genetic data information. Tissue samples will be taken, from which 
cell populations will be isolated for genetics studies. The relevant tissue specimens and isolated 
cell populations for the genetics studies will be labeled with a unique numerical identification 
code. Subject name or other identifiers will not be placed on the genetics specimens. The key to 
the code will connect the subject’s name to the subject’s study information and specimens. The 
key to the code will be kept in a separate electronic database file and subject’s genetic 
specimens will always remain coded and their identity will remain confidential.