Nanostring of whole lung RNA from cell-specific MyD88 KO at 0h, 2h and 6h after in vivo sensitization with OVA/standard flagellin. Mus musculus

Allergic asthma is a chronic disease of the airways characterized by eosinophilic and neutrophilic inflammation. MYD88, the adaptor molecule for TLR and IL-1 family member signaling, is required for allergic sensitization through the airway in animal models of allergic asthma. We generated conditionally mutant mice separately lacking Myd88 in airway epithelial cells (ECs) or dendritic cells (DCs) and alveolar macrophages (AMs) to define the contribution of Myd88 expression in each of these cell types. To examine crosstalk between ECs and CD11c-expressing cells in vivo, we examined transcriptional profiles from whole lung RNA at baseline, or following 2h or 6h in vivo lung allergic sensitization through the airways from WT MyD88 fx/fx, SPC cre+ MyD88 fx/fx (EC-MYD88 KO), CD11c cre+ MyD88 fx/fx (DC-MYD88 KO), and full MyD88 KO mice. We observed immune-specific transcriptional changes in whole lung RNA that were altered based on EC- or CD11c-specific deletion of MYD88. We also observed transcriptional (linked data set) and epigenetic changes in chromatin conformation in cDCs by ATAC-seq (linked data set) as well as changes in immune-specific sorted EC RNA, sorted AM RNA, and sorted cDC RNA by Nanostring nCounter Immunology Codeset Analysis (additional linked files). Overall design: Whole lung RNA was isolated from all genotypes were at baseline or following 2h or 6h of in vivo allergic sensitization with 100 micrograms ovalbumin (OVA)/ 1250 ng standard flagellin (stFLA). Samples from all 4 genotypes (WT MyD88 fx, SPC cre+ MyD88 fx, CD11c cre+ MyD88 fx, MyD88 KO) x 3 timepoints (baseline or 2h or 6h OVA/stFLA) = 12 conditions. 12 conditions x 3 replicates per genotype-timepoint = 36 total samples. Analysis of baseline (0h) timepoint is a control for induced gene changes. WT MyD88 fx/fx mice are a positive control for all genes induced following allergen sensitization, while MyD88 KO is a negative control for genes that are not induced in the full body MyD88 KO mouse. SPC cre+ MyD88 fx/fx mice test for genes that require EC-dependent MYD88 signaling, whereas CD11c cre+ MyD88 fx/fx mice test for genes that require CD11c-expressing cell-dependent MYD88 expression. Gene expression analysis was conducted using nCounter Mouse Immunology Codeset (Nanostring, Seattle, WA) on 3 biological replicates for each genotype and timepoint.