β-defensin-1 regulates influenza virus infection in human bronchial epithelial cells through the STAT3 signaling pathway
Updated June 8, 2023
January 2023
NIOSH Dataset RD-1053-2023-0
Introduction
Understanding the host response to influenza A virus (IAV) infection is vital for developing intervention strategies. The primary barriers for invading respiratory pathogens are the airway epithelial cells of the respiratory tract and antimicrobial peptides produced by these cells. The antimicrobial peptide, β-defensin-1, has antiviral activity against both enveloped and non-enveloped viruses. Significant downregulation of β-defensin1 gene (DEFB1) expression was observed when human bronchial epithelial cells (HBEpCs) were exposed to IAV. HBEpCs overexpressing DEFB1 caused a significant reduction in IAV, that was confirmed by IAV matrix gene analysis and confocal microscopy. DEFB1expression after transfection with hsa-miR-186-5p and hsa-miR-340-5p provided evidence that DEFB1 expression could be modulated by these two miRNAs and hsa-miR-186-5p had a higher binding efficiency with DEFB1. Overexpression of DEFB1 in IAV infected HBEpCs led to increased NF-kB expression. In a Polymerase Chain Reaction (PCR) array analysis of 84 transcription factors, either overexpressing DEFB1 or siRNA silencing of DEFB1 expression significantly modulated the expression of STAT3. In addition, Ingenuity Pathway Analysis (IPA) integrated with PCR array data showed that the JAK1/STAT3 pathway was significantly altered by cells overexpressing DEFB1, suggesting that this may be one of the pathways by which defensin regulates IAV replication in HBEpCs. In conclusion, the reduction in IAV copy number in DEFB1 overexpressing cells suggests that β-defensin-1 plays a key role in regulating IAV survival through STAT3 and is a potential target for antiviral drug development.
- Data-Ago-IP [XLS – 311 B]
- Data-BCL-XL [XLS – 349 B]
- Data-copy numjber [XLS – 852 B]
- Data-Ct NFkB1 [XLS – 1 KB]
- Data-DEFB1 time course [XLS – 1 KB]
- Data-DEFB1-3 strains [XLS – 369 B]
- Data-Mimic inhibitors [XLS – 815 B]
- Data-Transcrption factor array [XLS – 6 KB]
- Data-Wblot quantitation [XLS – 424 B]
- Plaque assay [XLS – 442 B]
- Data dictionary -defensin [PDF – 135 KB]
- Methods collection- defensin B1 [PDF – 175 KB]
Data Collection Methods
- Cell culture
- Primary human bronchial epithelial cells HBEp cells. exposed to influenza virus H1N1, H9N1 (1P10) and H9N1 (1WF10)
- Madin Darby Canine Kidney (MDCK) cells were cultured in Eagle’s Minimum Essential Medium (ATCC, Manassas, VA) with 10% fetal bovine serum, and appropriate antibiotics. MDCK cells were used for the propagation of influenza virus H1N1 (A/WSN/33), H9N1 (1P10), and H9N1 (1WF10).
- Transfection of HBEp cells with DEFB1 plasmid and siRNAs specific for DEFB1
- MiRNA analysis
- Quality of RNA was assessed by Agilent 2100 Bioanalyzer
- Co-immunoprecipitation of Argonaute proteins with associated RNAs.
- HBEpC were transfected with miRNAs inhibitor oligonucleotide or mimic oligonucleotide (Thermo Fisher Scientific, Carlsbad, CA) using the lipid-based Lipofectamine 2000.
- Western immunoblotting
- Protein collected from cells infected or uninfected using RIPA lysis buffer. Proteins were quantified by BCA method. Proteins were resolved using a 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
- Viral Detection in cells
- Real-time PCR (Applied Biosystems 7500 RT-PCR System) using matrix gene specific forward and reverse primers, used a standard curve method for copy number calculations.
- Transcription factor assay
- Real-time PCR (Applied Biosystems 7500 RT-PCR System). 84 pathway specific genes investigated; list of genes studied provided in data dictionary with abbreviation.
- Confocal microscopy
- HBEpCs overexpressing DEFB1 were grown on chamber slides overnight (Chamber slide™, Lab-TekII, Thermo Fisher Scientific, Rochester, NY, USA). Cells were then exposed to H1N1 for 24 h. Immunofluorescent staining was done with an antibody that recognizes the influenza virus (Millipore, Billerica, MA, USA), anti-hemagglutinin, anti-influenza PB1, anti-hBD1 antibodies (Abcam, Waltham, MA, USA) and appropriate secondary antibodies (Alexa 488 and Alexa-555; Thermo Fisher Scientific). Photomicrographs were produced using a Zeiss LSM510 (Carl Zeiss, Obertochen, AG Germany).
Publications Based on Dataset
Othumpangat, S.; Noti, J.D. Defensin-1 Regulates Influenza Virus Infection in Human Bronchial Epithelial Cells through the STAT3 Signaling Pathway. Pathogens 2023, 12, 123. https://doi.org/10.3390/ pathogens12010123.
Acknowledgements
This project was supported by the National Institute for Occupational Safety and Health (NIOSH). When a publication makes use of this dataset, acknowledgement of the development of the dataset should be attributed to the NIOSH Health Effects Laboratory Division.
We would also like to recognize the work of Sreekumar Othumpangat and John D. Noti.
Contact
NIOSH/Health Effects Laboratory Division
Allergy and Clinical Immunology Branch
(304) 285-6024