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.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).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 (Methods, Table E1, Fig.E1 ).
At baseline, five expression clusters were identified (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
(PTGS2 and PTGES ). Cluster
2 included PTGER2 encoding
the EP2 receptor for PGE2. Absence of genetic
co-expression for PGE2 biosynthesis and the type-2
receptor indicated their transcriptional independence. The best grouped
Cluster 3 consisted of lipoxygenases ALOX5 , ALOX12 ,ALOX15, HPGDS encoding hematopoietic prostaglandins D synthase
and its inactivating hydroxyprostaglandin dehydrogenase HPGD .
Interestingly, we recently reported that high sputum HPGDexpression can predict better response to a high-dose aspirin therapy in
N-ERD.2 Cluster 4 included genes encoding receptors
for eicosanoids PTGDR2, CYSLTR1, CYSLTR2 , and PTER4 . The
fifth cluster included COX-1 mRNA co-expressed with LTC4S andALOX15 transcripts (Fig.2 ).
After 52 weeks high-dose aspirin therapy, only two clusters were
identified: cluster 1 containing, LTC4S and ALOX15 ; and
cluster 2 containing the remaining 13 genes of AA-related group
(Fig.2 , Supplementary material Fig.E3-E4 ).
Grouping LTC4S andALOX15 together as co-regulated was associated with clinical
improvement.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.4 Moreover,
high sputum HPGD expression may predict benefits from subsequent
aspirin therapy.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.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.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.