C70-10 Il-33 Release From Pediatric Bronchial Epithelial Cells Is Synergistically Increased by Neutrophils Following RV16 Infection

Abstract Rationale Neutrophilic inflammation in the airways is associated with steroid-resistant severe asthma in adults and children. While epithelial-derived alarmins (IL-33, TSLP) are recognized drivers of asthma pathogenesis, mechanisms initiating their release in the context of neutrophilic airway inflammation are poorly understood. We investigated the impact of neutrophil co-culture in organotypic ex vivo epithelium on gene expression from BECs and on secretion of IL-33 from co-cultured neutrophils and BECs. Methods Bronchial epithelial cells (BECs) from children with asthma were differentiated to an organotypic epithelium. The apical surface of the epithelium was infected with RV-16 at a multiplicity of infection of 0.5, and 24 hours after infection neutrophils from a healthy donor were added in co-culture to the basolateral chamber of infected and uninfected BEC cultures. RNA from BECs and neutrophils was collected 24 hours later. RNA sequencing was performed on BEC RNA with and without neutrophils co-cultured under both infected and uninfected conditions. Differentially expressed genes were analyzed using WGCNA co-expression modules for pathway enrichment analysis. Viral copy number was quantified using RNA-seq reads aligned to a panel of viral genomes. IL-33 and soluble ST2 were measured in supernatant using Luminex (IL-33) and ELISA (sST2). Results In BECs, neutrophil co-culture altered expression of 2,743 genes that clustered into 11 modules. Genes for cell migration, TNF signaling, and mucus secretion increased expression with neutrophil co-culture. Genes for antigen processing and presentation clustered in a module with IL-33 and decreased expression in BECs co-cultured with neutrophils. Neutrophils had 258 differentially expressed genes when co-cultured with infected BECs that grouped into 4 distinct modules. In contrast to gene expression, IL-33 protein secretion was markedly increased (5-fold) with neutrophil co-culture prior to infection and then further increased after RV-16 infection. Soluble ST2 secretion increased modestly (1.5-fold) with neutrophil co-culture, but not with RV-16 infection. Viral copy number in BECs did not differ with neutrophil co-culture. Conclusions Neutrophils decrease IL-33 gene expression in bronchial epithelia but stimulate IL-33 protein release from epithelia prior to infection and potentiate increased IL-33 release after RV-16 infection. Increased IL-33 secretion coincides with upregulation of cell migration and TNF signaling gene expression by epithelia. These observations suggest that in children with asthma airway neutrophilic inflammation triggers IL-33 release by epithelia and then further augments release in the setting of rhinovirus infection contributing to enhanced airway dysfunction. This abstract is funded by: SRS (WTP), Parker B Francis Fellowship (WTP), NIH K08HL179396 (WTP), NIH R01AI163160 (JSD), NIH K24AI150991 (JSD), NIH U19AI175089 (JSD, MCA, TSH).