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.