Yuhui Ouyang1,2, Ying Li3, Zhaojun Xu4,5, Yusan An3, Luo Zhang6,7,8. 1. Department of Allergy, Beijing Tongren Hospital, Capital University of Medical Science, Beijing, 100730, China. 2. Beijing Key Laboratory of Nasal Diseases, Beijing Institute of Otolaryngology, No. 17, HouGouHuTong, Dongcheng District, Beijing, 100005, China. 3. Department of Otolaryngology Head and Neck, Beijing Tongren Hospital, Capital University of Medical Science, Beijing, 100730, China. 4. Department of Environmental Medicine, Quanzhou Medical College, Quanzhou, 362011, Fujian, China. 5. Department of Biochemistry, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, 409-3898, Japan. 6. Department of Allergy, Beijing Tongren Hospital, Capital University of Medical Science, Beijing, 100730, China. dr.luozhang@139.com. 7. Department of Otolaryngology Head and Neck, Beijing Tongren Hospital, Capital University of Medical Science, Beijing, 100730, China. dr.luozhang@139.com. 8. Beijing Key Laboratory of Nasal Diseases, Beijing Institute of Otolaryngology, No. 17, HouGouHuTong, Dongcheng District, Beijing, 100005, China. dr.luozhang@139.com.
Abstract
BACKGROUND: Air pollution may be associated with increased airway responsiveness to allergens in allergic rhinitis (AR). Ozone-aged environmental black carbon (O3BC) is an important constituent of atmospheric particulate matter (PM), for which the mechanisms underlying its effects have not been fully elucidated in AR. The objective of the present study was to determine the O3BC and pollen-induced alterations in the transcriptome in human nasal epithelial cells (hNECs) in vitro. METHODS: hNECs from nasal epithelial mucosal samples of healthy individuals undergoing nasal surgery (turbinoplasty or septoplasty) were established as air-liquid interface (ALI) cultures and exposed to O3BC, pollen, or a combination of O3BC+ pollen. Changes in cell viability were analyzed by fluorescence and changes in the transcriptome by high-throughput RNA sequencing (RNA-seq). Several differentially expressed genes were verified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Enrichment analysis, based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, was performed to determine major biological functions and pathways involved. RESULTS: Exposure to ≥ 50 μg/ml O3BC or 25 μg/ml O3BC+ 200 μg /ml pollen significantly decreased cell viability of the hNECs compared to control (p < 0.05) or 25 μg/ml O3BC alone (p < 0.05); whereas exposure to pollen alone did not alter cell viability at any concentration investigated. High-throughput RNA sequencing analysis indicated that there was significant difference in gene expression between pollen or O3BC alone and O3BC+ pollen exposed cells. Exposure to 200 μg/ml O3BC was associated with hypoxia stress response GO terms, whereas exposure to 25 μg/ml O3BC+ 200 μg/ml pollen was associated with inflammatory response GO terms; including regulation of neutrophil migration and chemotaxis, macrophage differentiation and chemotaxis, mast cell activation, and phagocytosis. KEGG pathway analysis indicated the top 10 upstream regulators to be IL1B, CSF1, CCL2, TLR2, LPL, IGF8, SPP1, CXCL8, FCER1G and IL1RN; of which expressions of inflammation-related genes IL1B, CSF1 and FCER1G were significantly increased. CONCLUSION: O3BC and pollen allergen combined exposure may induce innate immune and allergic inflammation in hNECs, and therefore potentially exacerbate the symptoms of AR in affected individuals.
BACKGROUND: Air pollution may be associated with increased airway responsiveness to allergens in allergic rhinitis (AR). Ozone-aged environmental black carbon (O3BC) is an important constituent of atmospheric particulate matter (PM), for which the mechanisms underlying its effects have not been fully elucidated in AR. The objective of the present study was to determine the O3BC and pollen-induced alterations in the transcriptome in human nasal epithelial cells (hNECs) in vitro. METHODS: hNECs from nasal epithelial mucosal samples of healthy individuals undergoing nasal surgery (turbinoplasty or septoplasty) were established as air-liquid interface (ALI) cultures and exposed to O3BC, pollen, or a combination of O3BC+ pollen. Changes in cell viability were analyzed by fluorescence and changes in the transcriptome by high-throughput RNA sequencing (RNA-seq). Several differentially expressed genes were verified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Enrichment analysis, based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, was performed to determine major biological functions and pathways involved. RESULTS: Exposure to ≥ 50 μg/ml O3BC or 25 μg/ml O3BC+ 200 μg /ml pollen significantly decreased cell viability of the hNECs compared to control (p < 0.05) or 25 μg/ml O3BC alone (p < 0.05); whereas exposure to pollen alone did not alter cell viability at any concentration investigated. High-throughput RNA sequencing analysis indicated that there was significant difference in gene expression between pollen or O3BC alone and O3BC+ pollen exposed cells. Exposure to 200 μg/ml O3BC was associated with hypoxia stress response GO terms, whereas exposure to 25 μg/ml O3BC+ 200 μg/ml pollen was associated with inflammatory response GO terms; including regulation of neutrophil migration and chemotaxis, macrophage differentiation and chemotaxis, mast cell activation, and phagocytosis. KEGG pathway analysis indicated the top 10 upstream regulators to be IL1B, CSF1, CCL2, TLR2, LPL, IGF8, SPP1, CXCL8, FCER1G and IL1RN; of which expressions of inflammation-related genes IL1B, CSF1 and FCER1G were significantly increased. CONCLUSION:O3BC and pollen allergen combined exposure may induce innate immune and allergic inflammation in hNECs, and therefore potentially exacerbate the symptoms of AR in affected individuals.