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PIWIL4 knockdown alters gene expression in RSV-infected immortalized small airway epithelial cells

ABSTRACT: Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infection in young children, the elderly, and immunocompromised hosts, and no effective vaccine or therapy exists for infants. PIWIL4, a member of the PIWI protein family known to interact with PIWI-interacting RNAs (piRNAs), was found to be induced by RSV infection in airway epithelial cells. To investigate the role of PIWIL4 in the transcriptional response to RSV, immortalized small airway epithelial (iSAE) cells were transfected with either a non-targeting or a PIWIL4-targeting siRNA and then left uninfected or infected with RSV for 15 hours. Gene expression was profiled using the Agilent Whole Human Genome Oligo Microarray (4x44K) to identify genes differentially expressed upon PIWIL4 knockdown under basal and RSV-infected conditions. 4 samples, one array per condition (no biological replicate arrays; RNA from three pooled technical replicate wells per condition was hybridized to a single array). 2x2 factorial design: siRNA treatment (non-targeting control vs. PIWIL4-targeting) x infection status (uninfected control vs. RSV Long strain infection, MOI 5, 15 hours post-infection) in immortalized human small airway epithelial (iSAE) cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE341202 | GEO | 2026/07/28

REPOSITORIES: GEO

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Project description:Lower respiratory tract infections (LRTI) from human Respiratory Syncytial Virus (RSV) are a significant cause of morbidity in children. A component of LRTI pathogenesis is due to signals generated by infected lower airway cells that alter lymphocyte populations and trigger airway remodeling. To obtain insights into this process, we applied an unbiased quantitative proteomics analysis of the RSV-induced epithelial secretory response in cells representative of the trachea (hBECs) vs small airway bronchiolar cells (hSAECs). A workflow was standardized initially using telomerase immortalized human epithelial cells that showed high reproducibility and cell-type differences in proteomic signatures of both secreted proteins and isolated nanoparticles (exosomes). Over half of secretome proteins were contained within exosomal, with the remainder originating from lysosomal and vaculolar compartments. We applied this workflow to three independently derived primary human cultures. 577 differentially expressed proteins from control supernatants and 966 differentially expressed proteins from RSV-infected cell supernatants were identified at a 1% false discovery rate (FDR). 15 proteins were unique to RSV-infected hBECs regulated by epithelial-specific ets homology factor (EHF). 106 proteins were unique to RSV-infected hSAECs enriched in proteins regulated by the NFB transcription factor. In this latter group, we independently validated the differential expression of Chemokine (C-C Motif) Ligand 20 (CCL20)/macrophage inducible protein (MIP)3, Thymic Stromal Lymphopoietin (TSLP) and chemokine (CC) ligand 3-like 1(CCL3-L1). CCL20/MIP3 is the most active mucin inducing factor in the RSV infected hSAEC secretome, and is differentially expressed in smaller airways in a mouse model of RSV infection. These studies provide insight into role of exosomal production in innate responses and regional differences in epithelial secretomes that participate in pathogenesis of RSV LRTI-induced airway remodeling.

Project description:Lower respiratory tract infections (LRTI) from human Respiratory Syncytial Virus (RSV) are a significant cause of morbidity in children. A component of LRTI pathogenesis is due to signals generated by infected lower airway cells that alter lymphocyte populations and trigger airway remodeling. To obtain insights into this process, we applied an unbiased quantitative proteomics analysis of the RSV-induced epithelial secretory response in cells representative of the trachea (hBECs) vs small airway bronchiolar cells (hSAECs). A workflow was standardized initially using telomerase immortalized human epithelial cells that showed high reproducibility and cell-type differences in proteomic signatures of both secreted proteins and isolated nanoparticles (exosomes). Over half of secretome proteins were contained within exosomal, with the remainder originating from lysosomal and vaculolar compartments. We applied this workflow to three independently derived primary human cultures. 577 differentially expressed proteins from control supernatants and 966 differentially expressed proteins from RSV-infected cell supernatants were identified at a 1% false discovery rate (FDR). 15 proteins were unique to RSV-infected hBECs regulated by epithelial-specific ets homology factor (EHF). 106 proteins were unique to RSV-infected hSAECs enriched in proteins regulated by the NF?B transcription factor. In this latter group, we independently validated the differential expression of Chemokine (C-C Motif) Ligand 20 (CCL20)/macrophage inducible protein (MIP)3?, Thymic Stromal Lymphopoietin (TSLP) and chemokine (CC) ligand 3-like 1(CCL3-L1). CCL20/MIP3? is the most active mucin inducing factor in the RSV infected hSAEC secretome, and is differentially expressed in smaller airways in a mouse model of RSV infection. These studies provide insight into role of exosomal production in innate responses and regional differences in epithelial secretomes that participate in pathogenesis of RSV LRTI-induced airway remodeling.

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PIWIL4 knockdown alters gene expression in RSV-infected immortalized small airway epithelial cells

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