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\begin{document}

\title{Disrupted epithelial barrier in nasal polyps characterizes aspirin
exacerbated respiratory disease}



\author[1]{Anand Kumar Andiappan}%
\author[2]{Mohammad Asad}%
\author[3]{Celine Chua}%
\author[2]{Esha Sehanobish}%
\author[4]{Zhen Ren}%
\author[1]{Xue Ying Chan}%
\author[1]{Josephine Lum}%
\author[1]{Nicholas Ang}%
\author[1]{Kaibo Duan}%
\author[2]{Adam Gersten}%
\author[5]{Waleed M. Abuzeid}%
\author[2]{Nadeem Akbar}%
\author[2]{Marc Gibber}%
\author[1]{Shanshan Howland}%
\author[1]{Bernett Lee}%
\author[1]{Olaf Rotzschke}%
\author[2]{Steven A. Porcelli}%
\author[2]{Elina Jerschow}%
\affil[1]{Singapore Immunology Network}%
\affil[2]{Montefiore Medical Center}%
\affil[3]{National University of Singapore Department of Biological Sciences}%
\affil[4]{Washington University in St Louis John T Milliken Department of Medicine}%
\affil[5]{University of Washington Department of Otolaryngology Head and Neck Surgery}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\textbf{Disrupted epithelial barrier in nasal polyps characterizes
aspirin exacerbated respiratory disease}

Anand Kumar Andiappan\textsuperscript{1*}, Mohammad
Asad\textsuperscript{2}, Celine Chua\textsuperscript{3}, Esha
Sehanobish\textsuperscript{2} , Zhen Ren\textsuperscript{4}, Xue Ying
Chan\textsuperscript{1}, Josephine Lum\textsuperscript{1}, Nicholas
Ang\textsuperscript{1}, Duan Kaibo\textsuperscript{1}, Adam
Gersten\textsuperscript{2}, Waleed M. Abuzeid\textsuperscript{5}, Nadeem
Akbar\textsuperscript{2}, Marc Gibber\textsuperscript{2}, Shanshan
Howland\textsuperscript{1}, Bernett Lee\textsuperscript{1}, Olaf
Rotzschke\textsuperscript{1}, Steven A. Porcelli\textsuperscript{2},
Elina Jerschow\textsuperscript{2*}

\textsuperscript{1} Singapore Immunology Network, Agency for Science,
Technology and Research

\textsuperscript{2} Albert Einstein College of Medicine/Montefiore
Medical Center, Bronx, NY, USA

\textsuperscript{3} Department of Biological Science, National
University of Singapore

\textsuperscript{4} Division of Allergy and Immunology, Department of
Medicine, Washington University Schoolf of Medicine, St Louis, MO, USA

\textsuperscript{5}Rhinology and Endoscopic Skull Base Surgery,
Department of Otolaryngology -- Head and Neck Surgery, University of
Washington, Seattle, WA, USA

*Correspondence: EJERSCHO@montefiore.org;
anand\_andiappan@immunol.a-star.edu.sg

\textbf{Conflict of interest:}

\textbf{:}

For Elina Jerschow: Advisory Board - GlaxoSmithKline, Sanofi-Regeneron,
Novartis/Genentech.Consultant --GlaxoSmithKline, none of these are
relevant to this work.

Other authors declare no conflict of interest

\textbf{Funding:}

This work was supported by CTSA grant number 5KL2TR001071from the
National Center for Advancing Translational Sciences (NCATS), a
component of the National Institutes of Health (NIH). Portions of this
work were funded by~R21AI146804 and~the Stony Wold-Herbert Fund research
grant (to EJ). Its contents are solely the responsibility of the authors
and do not represent the official views of the NIH. A.K. Andiappan was
supported by an NMRC (National Medical Research Council) YIRG (Young
Individual Research Grant) grant (OFYIRG17nov065), an Agency for
Science, Technology and Research CDA (Career Development Award) grant
(202D800012),

\textbf{Keywords} : asthma, chronic rhinosinusitis, nasal polyps,
barrier dysfunction, AERD

\textbf{To the Editor}

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a debilitating
inflammatory disease of the sinonasal
cavities.\textsuperscript{1}Concomitant CRSwNP and asthma, together with
hypersensitivity reactions to cyclooxygenase-1 (COX-1) inhibitors, is a
particular phenotype known as aspirin exacerbated respiratory disease
(AERD), which is associated with greater disease
severity.\textsuperscript{2}

In this study, we attempted to characterize clinical, laboratory, and
transcriptomic differences between patients with AERD (N=13) and CRSwNP
patients without AERD (N=13).

We compared patient demographics and clinical characteristics
(\textbf{Table 1} ). A more severe clinical phenotype with asthma
presence and a higher number of sinus surgeries was noticed in AERD
patients. LM scores in AERD were significantly higher compared to CRSwNP
patients (20 (IQR 16-23) vs. 14 (IQR 14-18), respectively, P =
0.0065,\textbf{Figure 1A} ). Peripheral blood eosinophil counts were not
significantly different between the groups (\textbf{Figure E1A} ).
However, blood eosinophil counts had a positive correlation with LM
scores in CRSwNP patients (R = 0.65, P = 0.018, \textbf{Figure E1C} ),
while this correlation was absent in AERD (R = -0.33, P =
0.26,\textbf{Figure E1B)} . Neutrophil counts were higher in the polyp
tissues of AERD patients (8.5 cells per hpf (IQR 3-25) vs. 2.5 cells
(IQR 2-5) in CRSwNP (P = 0.026, \textbf{Figure 1B} ). Analysis of the
inflammatory cell infiltrates on histologic slides showed that both
groups had abundant but similar eosinophil counts (\textbf{Figure 1C} ).

We then conducted the whole-transcriptomic sequencing, followed by
pairwise comparisons of gene expression between the AERD and CRSwNP
polyps. A stringent analysis of the transcriptomic data using a false
discovery correction (FDR) narrowed down the differentially expressed
genes (DEGs) to 10 protein-coding genes significantly downregulated in
AERD compared to CRSwNP (\textbf{Figure 1D} , \textbf{Table E1).} In
AERD, there was a downregulation of the genes coding for epidermal
differentiation marker (\emph{CALM3, CRNN),} keratinsation (\emph{CNFN}
,\emph{KRT13} , \emph{SPINK5} )\emph{,} secreted mucin protein
\emph{MUC2,}and epidermal barrier marker genes (\emph{SPINK5, CRNN,
A2ML1)} . Functional analysis of significant DEGs using Ingenuity
Pathway Analysis highlighted biological pathways enriched with AERD
status such as organismal injury (N = 264, P = 1.66 x
10\textsuperscript{-6}), inflammatory responses (N = 147, p = 5.95 x
10\textsuperscript{-20}), humoral immune response (N = 34, p =5.95 x
10\textsuperscript{-20}), and cell morphology (N = 73, p = 4.45 x
10\textsuperscript{-14};\textbf{Figure 1E-G} ).

Taken together, these findings underline the key differences in patients
with CRSwNP, with and without AERD. Compared to CRSwNP patients, we
observed that AERD patients were likely to have a more severe clinical
disease. This was associated with a downregulation of genes responsible
for the epithelial barrier function. AERD has a strong type-2
inflammation, but a simultaneous increase in the presence of neutrophils
suggests that AERD is characterized by mixed Th17 along with Th1 and Th2
immune responses assocaited with eosinophils\textsuperscript{3} While
blood eosinophils were not sufficiently predictive of type 2
inflammation in patients with chronic rhinosinusitis without nasal
polyps,\textsuperscript{4} here we show that peripheral blood
eosinophilia was significantly associated with LM scores in CRSwNP
patients. This finding was not relevant to AERD patients, indicating
that other cells, perhaps neutrophils, contribute to the severity of the
polyp burden in AERD. The presence of a mixed eosinophilic and
neutrophilic inflammation has recently been reported in a more severe
phenotype of CRSwNP.\textsuperscript{5} Such phenotype is likely due to
the presence of Charcot-Leyden crystals that attract neutrophils and
lead to a greater barrier dysfunction and bacterial
colonization.\textsuperscript{5,6} Our observations also suggest that
the clinical characteristics of AERD, as a more severe phenotype of
CRSwNP, are linked to a distinct dysregulation of epithelial barrier
function in AERD polyps, offering a basis for further mechanistic
studies on epithelial remodeling and inflammation of the upper and lower
airways.

\textbf{References}

1. Khan A, Vandeplas G, Huynh TMT, Joish VN, Mannent L, Tomassen P, et
al. The Global Allergy and Asthma European Network (GALEN rhinosinusitis
cohort: a large European cross-sectional study of chronic rhinosinusitis
patients with and without nasal polyps. Rhinology. 2019;57(1):32-42.

2. Kim JE, Kountakis SE. The prevalence of Samter's triad in patients
undergoing functional endoscopic sinus surgery. Ear Nose Throat J.
2007;86(7):396-9.

3. Celejewska-W\selectlanguage{ngerman}ójcik N, Wójcik K, Ignacak\selectlanguage{english}-Popiel M, \selectlanguage{polish}Ć\selectlanguage{english}miel A, Tyrak K,
Gielicz A et al.. Subphenotypes of nonsteroidal antiinflammatory
disease-exacerbated respiratory disease identified by latent class
analysis.~Allergy~2020;\textbf{75} :831--840.

4. Delemarre T, Holtappels G, De Ruyck N, et al. Type 2 inflammation in
chronic rhinosinusitis without nasal polyps: Another relevant endotype.
J Allergy Clin Immunol. 2020;146(2):337-343.e6.

5. Delemarre T, Holtappels G, De Ruyck N, Zhang N, Nauwynck H, Bachert
C, et al. A substantial neutrophilic inflammation as regular part of
severe type 2 chronic rhinosinusitis with nasal polyps. J Allergy Clin
Immunol. 2021;147(1):179-88 e2.

6. Delemarre T, Bochner BS, Simon HU, Bachert C. Rethinking neutrophils
and eosinophils in chronic rhinosinusitis. J Allergy Clin Immunol.
2021;148(2):327-35.

\textbf{Figure legends}

\textbf{Figure 1: Characteristics of the study cohort stratified by AERD
status}

Lund-Mackay (LM) score. \textbf{(B)} Peripheral blood eosinophil counts
(cells/\selectlanguage{greek}μ\selectlanguage{english}l). Correlation between peripheral blood eosinophil count
(cells/\selectlanguage{greek}μ\selectlanguage{english}l) to LM score for CRSwNP \textbf{(C)} and AERD\textbf{(D)} .
Count/hpf for (\textbf{E} ) eosinophils and (\textbf{F} ) neutrophils
from histologic analysis of nasal polyp tissue. Ingenuity pathway
analysis (IPA) of differentially expressed genes (DEGs) highlighting
enriched biological pathways under (\textbf{G} ) Diseases and Disorders,
(\textbf{H} ) Molecular and Cell Function and (\textbf{I} )
Physiological system based on a cut off of nominally significant P-value
\textless{} 0.05.

\textbf{Hosted file}

\verb`Figure 1.pptx`  available at \url{https://authorea.com/users/436799/articles/538872-disrupted-epithelial-barrier-in-nasal-polyps-characterizes-aspirin-exacerbated-respiratory-disease}

\textbf{Hosted file}

\verb`Table 1.docx`  available at \url{https://authorea.com/users/436799/articles/538872-disrupted-epithelial-barrier-in-nasal-polyps-characterizes-aspirin-exacerbated-respiratory-disease}

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