Global society is aging.1 Accordingly, elderly asthma (EA) involving asthma in people aged 65 years and over is expected to increase. The estimated prevalence of EA in developed countries is 6%-10%.2 Previous reports suggest that EA is phenotypically different from non-elderly asthma (NEA).345 However, the pathogenesis underlying EA has not been clearly elucidated.

Induced sputum is a reliable non-invasive assessment of bronchial inflammation in asthma.6 The evaluation of gene expression profiles of sputum cells has been successfully applied to understand asthma pathogenesis.78 The present study searched biological pathways related to EA using genome-wide gene expression profiles of sputum cells from EA. We selected genes showing significantly differential expressions between EA and healthy elderly controls and identified 2 distinct clusters by hierarchical clustering of these genes. We performed gene set enrichment analysis (GSEA) to gain further biological insights of each cluster. Finally, we confirmed that our findings were replicated in a dependent gene expression profile of sputum cells obtained from Gene Expression Omnibus (GEO), a publicly available database of gene expression profiles. To the best of our knowledge, this is the first study to identify peculiar biological pathways contributing to the EA pathogenesis based on sputum gene expression profiles.

Fifty-five patients with EA were enrolled. Based on the differential gene expression patterns of sputum cells, 2 distinct clusters were identified (Fig. 1A and Supplementary Fig. S1). Cluster 1 consisted of 35 patients with EA. Cluster 1 featured a significantly lower proportion of eosinophils in the sputum and less severe airway obstruction as measured by the post-BD ratio of the forced expiratory volume in 1 second and forced vital capacity (FEV1/FVC) compared to cluster 2. Detailed characteristics of the 2 clusters are provided in Table 1.

GSEA results (Table 2A and Fig. 2) revealed the significant enrichment of 5 gene sets in cluster 1 (OXIDATIVE_PHOSPHORYLATION [OXPHOS], UNFOLDED_PROTEIN_RESPONSE [UPR], MYC_TARGETS_V1, DNA_REPAIR, and ADIPOGENESIS) and 3 gene sets in cluster 2 (EPITHELIAL_MESENCHYMAL_TRANSITION [EMT], MYOGENESIS, and KRAS_SIGNALING_DN). In the replication analysis, hierarchical clustering of gene expression patterns also revealed 2 clusters (cluster 1' with 8 EA patients and cluster 2' with 7 EA patients, Fig. 1B). Clinical characteristics of each cluster are shown in Supplementary Table S1. GSEA identified 8 gene sets (OXPHOS, ESTROGEN_RESPONSE_EARLY, E2F_TARGETS, MYC_TARGETS_V1, FATTY_ACID_METABOLISM, MYC_TARGETS_V2, ESTROGEN_RESPONSE_LATE, and DNA_REPAIR) were significantly enriched in cluster 1' and 3 gene sets (TNFA_SIGNALING_VIA_NFKB, INFLAMMATORY_RESPONSE, and EMT) in cluster 2' (Table 2B and Supplementary Fig. S2). The OXPHOS gene set was significantly enriched in clusters 1 and 1' and the EMT gene set in both clusters 2 and 2'. To assess the potential functional relevance of the replicated gene sets, the leading edge genes of gene sets enriched in both discovery and replication datasets (Supplementary Table S2) were summarized into a single metagene metric using a principal component (PC) analysis. Then, the correlations between PC1, and serum uric acid levels and post-BD FEV1/FVC values were measured in each cluster. Circulating uric acid is a major antioxidant that might help protect against oxidative stress.13 The post-BD FEV1/FVC value is an indirect indicator of airway remodeling.14 Levels of serum uric acid were lower in cluster 1 with a borderline significance (P = 0.083) compared to cluster 2, whereas post-BD FEV1/FVC values were significantly higher in cluster 1 (P = 0.008) (Supplementary Fig. S3). PC1 of leading edge genes in the OXPHOS gene set showed a negative correlation with serum uric acid levels (P = 0.075) only in cluster 1 (Fig. 3). Meanwhile, PC1 of leading edge genes in the EMT gene set showed a significantly negative correlation with post-BD FEV1/FVC values (P = 0.005) only in cluster 2 (Fig. 3). For individual gene, we found 4 genes (MRPS11, HSPA9, NDUFB4, and ACAT1) in the leading edge genes of the OXPHOS gene set and 2 genes (SNTB1 and FUCA1) in leading edge genes of the EMT gene set belonged to genes which showed more than 1.5 log 2-fold expression difference between clusters 1 and 2 with P values less than 0.01 (Fig. 4).

Using gene expression profiles on sputum cells, we could identify 2 molecular clusters. Cluster 1 showed a significantly lower proportion of eosinophils in sputum and less severe airway obstruction compared to cluster 2. GSEA revealed that different biologic pathways were enriched in each cluster, implying that peculiar and discriminative pathogenesis mechanisms might exist. In replication analysis, we also identified 2 distinct molecular clusters. In addition, similar to the discovery dataset, the OXPHOS gene set was significantly enriched in one cluster and the EMT gene set was significantly enriched in the other cluster.

Approximately 70% of EA enrolled in the present study displayed symptom onset after 65 years old and the mean symptom duration was 5 years. These findings suggest that EA is not merely a prolongation of NEA but rather that aging itself may pose susceptibility to the development of asthma, as discussed elsewhere.15 In addition to anatomical changes, repetitive and long-standing exposures to environmental noxious stimuli cause the pro-inflammatory state in elderly subjects which results in aging-associated diseases in combination with oxidative stress.1617 The OXPHOS system embedded in mitochondria is the final biochemical pathway to produce adenosine triphosphate. A defect in the coupling between oxidation and phosphorylation causes various pathologic conditions including airway disorders.181920 At least 70 of the OXPHOS subunits are encoded by the nuclear genes whose expressions were measured in the present study and some defects of these genes were related with many diseases.21 The OXPHOS system is an important source of reactive oxygen species (ROS) within cells. ROS production contributes to mitochondrial damage in a range of pathologies and is also important in redox from the organelle to the rest of the cell.2223 Oxidative stress has also been linked to endoplasmic reticulum (ER) stress and to the activation of the UPR, leading to the activation of different inflammatory responses and dysregulation of the innate immune functions in the airways.2425 These prior observations match well with our findings that both the OXPHOS and UPR gene sets were significantly enriched in cluster 1. Given that previous suggestions that the OXPHOS and UPR systems have specific roles in the pathogenesis of asthma,2526 age-related changes in the OXPHOS and UPR systems may influence the development of asthma in the elderly. A decrease in serum uric acid levels in cluster 1 was also an interesting finding. As anti-oxidants may scavenge ROS, an imbalance between ROS and anti-oxidant capacity must be one of the important factors determining the effect of oxidative stress. Currently, it is not clear that patients with EA in cluster 1 have an intrinsic defect in anti-oxidant capacity. However, combined with changes in the OXPHOS and UPR systems, reduced anti-oxidant capacity may contribute to the pathogenesis of asthma in the elderly.

Cluster 2 as characterized by the enrichment of EMT gene sets. EMT describes a situation that epithelial and mesenchymal cells can, under certain conditions, switch their phenotypes27 and emerging evidence suggests that EMT is an important mechanism contributing to airway remodeling in asthma,28 which can explain lower post-BD FEV1 and post-BD FEV1/FVC ratios in cluster 2 compared to those in cluster 1. Senescent cells secrete a variety of proteins collectively known as the senescence-associated secretory phenotype (SASP), which can induce cellular plasticity and tissue change in a paracrine manner.29 Recently, it has been reported that SASP might play a causal role in the pathogenesis of chronic obstructive pulmonary disease.3031 The role of SASP in the pathogenesis of asthma in the elderly has been totally unknown. However, cellular senescence is believed to contribute to cancer progression via EMT.3233 Cellular senescence observed in the airway may induce EMT and increase the severity of asthma in the elderly.

Interestingly, we did not find any specific pathway directly related with eosinophil biology, although a significantly higher sputum eosinophil proportion was observed in cluster 2. However, given that a previous report showed that eosinophil promoted EMT of bronchial epithelial cells,34 airway eosinophilia might be complicated in the pathogenesis underlying cluster 2.

We aimed to identify biological pathways explaining a whole feature of EA, and thus we did not consider asthma-COPD overlap (ACO) as a subtype of EA in this study. If we define ACO as 1) post-BD FEV1/FVC < 0.7 and 2) classic symptoms of chronic bronchitis or suggesting signs of emphysema with chest radiography or pulmonary function test,35 the proportion of ACO is higher in cluster 2 (28.6% vs 55.5%). Although the difference showed a borderline significance (P = 0.083), the present study raised new insight in understanding ACO in the elderly. As is known, ACO is a common clinical problem in the elderly.36 Therefore, large scale studies to exam possible associations between biological pathways enriched in cluster 2 and ACO are necessary.

There are a few limitations to generalize our findings. One is the small number of participants. However, our results were replicated in an independent data set. This was a cross-sectional study, and thus we cannot know whether that the pathways identified were the primary cause or the secondary effect of asthma. Secondly, we used the uric acid level as an indirect biomarker to reflect oxidative stress status as it was easily available from routine laboratory examinations. Additional biomarkers need to be measure to confirm our observations. Finally, studies to elucidate mechanisms need to be followed.

Among leading genes identified in this study, NUDFB4 and FUCA1 are of special interest. NUDFB4 is a subunit of NADH dehydrogenase and its expression increases in smoking associated severe asthmatics.3738 Similar to the present study, it has been reported that a considerable portion of elderly asthmatics were former or current smokers.45 FUCA1 codes α-L-fucosidase that can block wound repair of primary airway epithelium.39 As well known, a delay in airway epithelial damage is associated with airway remodeling.40

In this study, we identified 2 distinct molecular clusters using gene expression profiles in sputum cells from patients with EA. The OXOPHOS and UPR gene sets were significantly enriched in one cluster with lower serum levels of uric acid. The EMT gene sets were significantly enriched in one cluster with airway remodeling defined by lower post-BD FEV/FVC ratios. This is the first study to show biologic pathways possibly related to EA pathogenesis. We postulate that oxidative stress and cellular senescence-associated with aging may be important in the development or progress of EA.