Identification of Potential Novel Biomarkers and Signaling Pathways Related to Otitis Media Induced by Diesel Exhaust Particles Using Transcriptomic Analysis in an In Vivo System

Abstract

Introduction: Air pollutants are associated with inflammatory diseases such as otitis media (OM). Significantly higher incidence rates of OM are reported in regions with air pollution. Diesel exhaust particles (DEPs) comprise a major class of contaminants among numerous air pollutants, and they are characterized by a carbonic mixture of polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, and small amounts of sulfate, nitrate, metals and other trace elements. DEP exposure is a risk factor for inflammatory diseases. Our previous study identified potential biomarkers using gene expression microarray and pathway analyses in an in vitro system. Although in vitro investigations have been conducted to elucidate plausible biomarkers and molecular mechanisms related to DEP exposure, in vivo studies are necessary to identify the exact biological relevance regarding the incidence of OM caused by DEP exposure. In this study, we identified potential molecular biomarkers and pathways triggered by DEP exposure in a rodent model.

Methods: Transcriptomic analysis was employed to identify novel potential biomarkers in the middle ear of DEP-exposed mice.

Results: A total of 697 genes were differentially expressed in the DEP-exposed mice; 424 genes were upregulated and 273 downregulated. In addition, signaling pathways among the differentially expressed genes mediated by DEP exposure were predicted. Several key molecular biomarkers were identified including cholinergic receptor muscarinic 1 (CHRM1), erythropoietin (EPO), son of sevenless homolog 1 (SOS1), estrogen receptor 1 (ESR1), cluster of differentiation 4 (CD4) and interferon alpha-1 (IFNA1).

Conclusions: Our results shed light on the related cell processes and gene signaling pathways affected by DEP exposure. The identified biomarkers might be potential candidates for determining early diagnoses and effective treatment strategies for DEP-mediated disorders.

Fig 1. Direct signaling pathways among the genes upregulated in response to diesel exhaust particles (DEPs) in an in vivo system.

The genes in the green box (C-reactive protein [CRP] and solute carrying family 1 member 2 [SLC1A2]) are involved in immunological pathways, whereas the genes in the blue box (erythropoietin [EPO], potassium inwardly rectifying channel, subfamily J, member 3 [KCNJ3], cholinergic receptor, muscarinic 1 [CHRM1], and Ca-channel, voltage-dependent, N type, alpha 1B subunit [CACNA1B]) have numerous interactions.

Fig 2. Direct signaling pathways among the genes downregulated in response to DEPs in an in vivo system.

The genes in the green box (cluster of differentiation 4 [CD4], estrogen receptor 1 [ESR1], interferon alpha-1 [IFNA1], son of sevenless homolog 1 [SOS1], and forkhead box P3 [FOXP3]) are involved in immunological pathways, and the gene in the blue box (Wilms’ tumor 1 [WT1]) has numerous interactions.

Fig 3. Otitis media (OM)-related signaling pathways among the upregulated genes.

The genes upregulated by DEPs, determined by microarray analysis, are highlighted in red. OM-related diseases such as ear disease, hearing loss, infection and neoplasm are associated with the genes upregulated by DEP exposure.

Fig 4. OM-related signaling pathways among the downregulated genes.

The genes downregulated by DEPs, determined by microarray analysis, are highlighted in blue. OM-related cell processes such as those involved in the immune responses against diseases and inflammation are associated with the genes downregulated by DEP exposure.

Fig 5. DEP-induced upregulated and downregulated genes validated by quantitative real-time polymerase chain reaction (qRT-PCR).

Among the upregulated genes, CHRM1 was overexpressed 4-fold, and EPO was overexpressed 1.5-fold. All of the downregulated genes (SOS1, CD4, IFNA1 and ESR1) showed 2-fold decreased expression.