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\begin{document}

\title{Sputum transcriptome analysis of co-regulated genes related to
arachidonic acid metabolism in N-ERD}



\author[1]{Lucyna Mastalerz}%
\author[1]{Radosław Kacorzyk}%
\author[1]{Bogdan Jakiela}%
\author[2]{Adam Ćmiel}%
\author[1]{Marek Sanak}%
\affil[1]{Uniwersytet Jagiellonski w Krakowie Wydzial Lekarski}%
\affil[2]{Department of Applied Mathematics AGH University of Science and Technology Krakow Poland}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\emph{Title:} \textbf{Sputum transcriptome analysis of co-regulated
genes related to arachidonic acid metabolism in N-ERD}

\emph{Short title:} \textbf{Cluster analysis of sputum transcriptome in
N-ERD}

\textbf{To the Editor}

Nonsteroidal anti-inflammatory drug--exacerbated respiratory disease
(N-ERD) is a disease characterized by alteration of arachidonic acid
(AA) metabolism. The disease has variable pattern of lipid biomarkers
and bronchial inflammation.\textsuperscript{1-3} How this heterogeneity
is reflected by expression of AA-related genes before and after aspirin
therapy remains unknown. We collected induced sputum from 27
participants before aspirin desensitization and after 52-week high-dose
aspirin therapy (650 mg/d).\textsuperscript{2} Abundance of 87 genes
mRNAs, including 15 AA-related ones was assessed in a total RNA from the
sputum cells. By a method of unsupervised cluster analysis the genes
expression was used to find co-regulated transcripts, based on similar
expression patterns before and after aspirin therapy. We hypothesized
that clustering of co-expressed AA-related genes might elucidate the
genetic heterogeneity of N-ERD at baseline. Thereafter, we determined
effect of a high-dose aspirin therapy on the aggregation of co-regulated
AA-related genes into the clusters. Gene expression was assessed using a
quantitative real-time polymerase chain reaction. The cluster analysis
was performed using distances calculated on correlations between the
expression of individual genes. Similar expression patterns were
classified together as co-regulated genes, thus in separate clusters
mRNA expression was independent from one another. The study group
characteristics, data collection, and statistics are described in
Supplementary Material (\textbf{Methods, Table E1, Fig.E1} ).

At baseline, five expression clusters were identified (\textbf{Fig.1,
Supplementary material Fig.E2} ). Each cluster contained genes encoding
AA metabolic enzymes and/or receptors for eicosanoids. The first cluster
included COX-2--pathway co-regulated genes of AA metabolism
(\emph{PTGS2} and \emph{PTGES} ). Cluster 2 included \emph{PTGER2}
encoding the EP2 receptor for PGE\textsubscript{2}. Absence of genetic
co-expression for PGE\textsubscript{2} biosynthesis and the type-2
receptor indicated their transcriptional independence. The best grouped
Cluster 3 consisted of lipoxygenases \emph{ALOX5} , \emph{ALOX12}
,\emph{ALOX15, HPGDS} encoding hematopoietic prostaglandins D synthase
and its inactivating hydroxyprostaglandin dehydrogenase \emph{HPGD} .
Interestingly, we recently reported that high sputum
\emph{HPGD}expression can predict better response to a high-dose aspirin
therapy in N-ERD.\textsuperscript{2} Cluster 4 included genes encoding
receptors for eicosanoids \emph{PTGDR2, CYSLTR1, CYSLTR2} , and
\emph{PTER4} . The fifth cluster included COX-1 mRNA co-expressed with
\emph{LTC4S} and\emph{ALOX15} transcripts (\textbf{Fig.2} ).

After 52 weeks high-dose aspirin therapy, only two clusters were
identified: cluster 1 containing, \emph{LTC4S} and \emph{ALOX15} ; and
cluster 2 containing the remaining 13 genes of AA-related group
(\textbf{Fig.2} , \textbf{Supplementary material Fig.E3-E4} ).

Grouping \emph{LTC4S} and\emph{ALOX15} together as co-regulated was
associated with clinical improvement.\textsuperscript{2} This supports
that cells expressing synthase for cysteinyl leukotrienes and 15-HETE(S)
are mandatory for beneficial response to aspirin therapy in N-ERD. N-ERD
patients with symptom improvement on aspirin had higher plasma 15-HETE
levels at baseline than those with symptom worsening.\textsuperscript{4}
Moreover, high sputum \emph{HPGD} expression may predict benefits from
subsequent aspirin therapy.\textsuperscript{2} Interestingly, HPGD is
required for 15-oxo-eicosatetraenoic acid (15-Oxo-ETE) synthesis, and
15-Oxo-ETE levels were markedly higher in N-ERD. Thus, epithelial and
mast cell interactions leading to 15-Oxo-ETE synthesis could contribute
to AA metabolism alterations via the 15-LOX pathway.\textsuperscript{5}
We suggest that this can be prevented by long-term high-dose aspirin
therapy in patients with N-ERD. In a physiological sputum macrophages
are predominant cells. In N-ERD macrophages seems to be reprogrammed to
produce proinflammatory mediators.\textsuperscript{6} This involves
epigenetic DNA changes leading to enhanced fatty acids metabolism and
release of lipid mediators. Presented clusters after high dose aspirin
therapy may reflect reversal of these alterations. In conclusion, this
is the first study presenting an aggregation of co-regulated genes
related to AA metabolism and signaling in N-ERD patients before and
after aspirin therapy. On aspirin, almost all AA-related genes became
grouped together, indicating aspirin's ability to regulate the
expression of these genes in N-ERD.

\textbf{References}

\begin{enumerate}
\tightlist
\item
  Celejewska-W\selectlanguage{ngerman}ójcik N, Wójcik K, Ignacak-Popiel M, et al. Subphenotypes
  of nonsteroidal antiinflammatory disease-exacerbated respiratory
  disease identified by latent class analysis.~\emph{Allergy} .
  2020;75(4):831-840. doi:10.1111/all.14141
\item
  Tyrak KE, Pajdzik K, Jakie\selectlanguage{polish}ł\selectlanguage{english}a B, et al. Biomarkers for predicting
  response to aspirin therapy in aspirin-exacerbated respiratory
  disease.~\emph{Clin Exp Allergy} . 2021;51(8):1046-1056.
  doi:10.1111/cea.13886
\item
  Cahill KN, Cui J, Kothari P, et al. Unique effect of aspirin therapy
  on biomarkers in aspirin-exacerbated respiratory disease. A
  Prospective Trial.~\emph{Am J Respir Crit Care Med} .
  2019;200(6):704-711. doi:10.1164/rccm.201809-1755OC
\item
  Jerschow E, Edin ML, Pelletier T, et al. Plasma
  15-Hydroxyeicosatetraenoic Acid Predicts Treatment Outcomes in
  Aspirin-Exacerbated Respiratory Disease.~\emph{J Allergy Clin Immunol
  Pract} . 2017;5(4):998-1007.e2. doi:10.1016/j.jaip.2016.11.021
\item
  Dwyer DF. Eicosanoid relay: Epithelial and mast cell transmetabolism
  in AERD.~\emph{J Allergy Clin Immunol} . 2021;147(2):501-503.
  doi:10.1016/j.jaci.2020.12.627
\item
  Haimerl P, Bernhardt U, Schindela S, et al. Inflammatory macrophage
  memory in nonsteroidal anti-inflammatory drug-exacerbated respiratory
  disease.~\emph{J Allergy Clin Immunol} . 2021;147(2):587-599.
  doi:10.1016/j.jaci.2020.04.064
\end{enumerate}

\emph{Authors} : Lucyna Mastalerz\textsuperscript{a}, Rados\selectlanguage{polish}ł\selectlanguage{english}aw
Kacorzyk\textsuperscript{a}, Bogdan Jakie\selectlanguage{polish}ł\selectlanguage{english}a\textsuperscript{a}, Adam
\selectlanguage{polish}Ć\selectlanguage{english}miel\textsuperscript{b}, Marek Sanak\textsuperscript{a}

\textsuperscript{a}II Department of Internal Medicine, Jagiellonian
University Medical College, Krakow, Poland

\textsuperscript{b}Department of Applied Mathematics, AGH University of
Science and Technology, Krakow, Poland

\textbf{Address for correspondence:} Prof. Marek Sanak

Department of Internal Medicine, Jagiellonian University Medical
College,

ul. Skawi\selectlanguage{polish}ń\selectlanguage{english}ska 8, 31-066 Krakow, Poland

E-mail: marek.sanak@uj.edu.pl

Telephone number: 12~430 52 66

\textbf{Word count (body):} 599

\textbf{Number of tables:} 0

\textbf{Number of figures:} 2

\textbf{Conflict of Interest:} None declared.

\textbf{Funding information:} This work was supported by the National
Science Centre, Poland (grant number UMO-2015/19/B/NZ5/00096; NCN).

\textbf{ORCiD}

Lucyna Mastalerz:\emph{0000-0002-8994-0036}

Marek Sanak:\emph{0000-0001-7635-8103}

\textbf{Author's contribution statement:}

\begin{enumerate}
\tightlist
\item
  Lucyna Mastalerz: \textbf{50\%}
\item
  Designed the study, conceived, and designed the analysis, wrote the
  paper.
\item
  Rados\selectlanguage{polish}ł\selectlanguage{english}aw Kacorzyk: \textbf{10\%}
\item
  Collected the data, prepared graphics.
\item
  Bogdan Jakie\selectlanguage{polish}ł\selectlanguage{english}a: \textbf{10\%}
\item
  Contributed to the interpretation of the results.
\item
  Adam \selectlanguage{polish}Ć\selectlanguage{english}miel: \textbf{10\%}
\item
  Performed statistical analysis, prepared graphics.
\item
  Marek Sanak: \textbf{20\%}
\item
  Contributed to the interpretation of the results, supervised the
  preparation of the manuscript.
\end{enumerate}

All authors provided critical feedback and helped shape the research,
analysis, and manuscript.

\textbf{Figure 1.} Dendrogram showing a hierarchical cluster analysis of
87 genes in sputum cells from patients with NSAID-exacerbated
respiratory disease at baseline. Cluster analysis was performed using
the Ward method on Spearman's correlation. Each color represents
different cluster: blue -- cluster number 1, yellow -- cluster number 2,
green -- cluster number 3, red -- cluster number 4, and violet -- number
5. Genes related to arachidonic acid pathways are in bold.

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/492952/articles/575518-sputum-transcriptome-analysis-of-co-regulated-genes-related-to-arachidonic-acid-metabolism-in-n-erd}

\textbf{Figure 2.} Clusters at baseline and at 52 weeks of high-dose
aspirin therapy\selectlanguage{english}
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