D21-09 Asthmatic Airway Epithelial Cells Differentially Regulate Mast Cell Response to IL-33

Abstract Rationale The epithelial compartment of the airway wall is uniquely infiltrated by mast cells (MCs) in individuals with asthma, and the density of intraepithelial MCs (MCIE) is associated with airway dysfunction (Altman JCI 2019) and distinct type-2 (T2) and non-T2 mechanisms of inflammation (Murphy AJRCCM 2023). We have modeled interactions between airway epithelial cells (AECs) and MCs using a coculture system with primary AECs in the apical chamber and MCs in the basolateral compartment, and have found that AECs promote sustained T2 gene expression in MCs that is further enhanced with IL-33 stimulation (Murphy AJRCCM 2023). Here we aimed to characterize the effect of asthmatic AECs on MC responses to IL-33 using the same ex vivo coculture model system. Methods Laboratory of allergic disease-2 (LAD2) MCs were cocultured with primary AECs obtained from pediatric donors (n = 24 total; n = 14 asthma, n = 10 healthy controls), which were fully differentiated at air-liquid interface. RNA was isolated from MCs at baseline and 48 hours following coculture exposure and/or IL-33 stimulation (10 ng/mL). Bulk RNA-sequencing (RNA-seq) analysis was performed, including assessment for differentially expressed genes (DEGs; defined by FDR 0.05) that underwent subsequent functional enrichment analysis. Results Globally, AECs significant altered the MC transcriptional response to IL-33 stimulation relative to MCs cultured alone (1078 DEGs; 614 upregulated and 464 downregulated with AEC exposure). Notable upregulated DEGs included key cell inflammatory cell surface receptors (IL1RL1, IL18R1, CXCR4), cytokines (IL1B, IL13), chemokines (CCL1, CCL2, CCL3, CCL4, CCL18), MC proteases (CMA1), and genes involved in prostaglandin synthesis (PTGS1, PTGS2). Notable downregulated DEGs were TPSAB1 and PTGES2. We also identified unique differences in MC responses to IL-33 stimulation based on AEC donor phenotype. 244 DEGs were uniquely upregulated in MCs exposed to asthmatic AECs, including modulation of the complement cascade (CD55), MC proteases (CMA1, CTSB), TRAIL (TNFSF10), and enriched in “antigen processing and presentation,” “interferon gamma signaling, and “adaptive immune response.” 363 DEGs were uniquely downregulated in MCs exposed to asthmatic AECs, including cell surface receptors involved in MC activation (SUCNR1, TNFRSF14), prostaglandin E2 synthase (PTGES2), and enriched in “cholesterol biosynthesis” and “steroid metabolism.” Conclusions AECs globally promote sustained MC expression of pro-inflammatory genes implicated in asthma, including distinct alterations in MC responses in the presence of asthmatic AECs. These findings suggest that in asthma, external stimuli that promote epithelial IL-33 release (such as viral infection and allergen exposure) act in part through a coordinated response between AEC and MCs. This abstract is funded by: NIH NIAID and NHLBI awards, Parker B. Francis Foundation