Translating the Biology of IL-9 into the Pathogenesis of Chronic Rhinosinusitis
Abstract
Background: Chronic rhinosinusitis (CRS) is an inflammatory disease of the nasal and paranasal sinus mucosa that can have profound effects on patient quality of life and US healthcare costs. Increased IL-9 expression has been identified in CRS patients, particularly those with nasal polyposis (CRSwNP) or eosinophilic CRS (ECRS). Il-9 gene expression has been found to be uniquely regulated by super enhancer RNA (eRNA).
Aim 1: Perform an in-depth literature review to summarize the current understanding of IL-9 biology, with an emphasis on IL-9’s contribution to CRS pathology.
Aim 2: Use antisense treatment of mouse Th9 cells in vitro to suppress IL-9 as initial validation of a possible immunotherapeutic treatment strategy for CRS patients.
Methods: CD4+ T-cells harvested from mouse lymph nodes and spleen were cultured under Th9 polarizing conditions, confirming Th9 differentiation with flow cytometry. Best strategies for robust baseline IL-9 and super enhancer RNA expression were determined by comparing qPCR values with various IL-9 stimulating conditions and RNA extraction methods. Antisense oligonucleotides (ASOs) targeting the Il-9 gene and super enhancer eRNA were designed using the UCSC In-Silico PCR tool and IDT’s OligoAnalyzer tool and were introduced to Th9 cell cultures using electroporation with the Lonza Nucelofector 2b. The fold decrease in gene expression in ASO vs. control conditions were determined using qPCR relative quantification.
Results: Baseline super enhancer expression was 8.9, 7.6, and 3.8 times higher for 3 different super enhancer targets using organic extraction compared to a spin column method for RNA extraction, and 6.8, 7.5, and 6.5 times higher in culture conditions utilizing anti-GITR stimulation compared to OX40L stimulation. Preliminary results of unmodified ASO knockdown studies showed ~2-fold decrease in IL-9 expression in ASO conditions, but this was not consistent across all conditions.
Conclusions: The addition of anti-GITR to Th9 cultures lead to the greatest baseline super enhancer expression and super enhancer RNA was best isolated using organic extraction methods. Though preliminary ASO studies are promising for IL-9 suppression, additional testing is necessary to determine the ideal concentrations of ASOs and timing of ASO introduction to cell cultures, as well as replicate experiments with modified ASOs.
Subject
Chronic RhinosinusitisInterleukin-9
super enhancers
enhancer RNA
antisense oligonucleotides
cytokine profiling
CRS endotyping
Citation
Newstrom, Emily Carole (2022). Translating the Biology of IL-9 into the Pathogenesis of Chronic Rhinosinusitis. Master's thesis, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /197140.