Nanostring of sorted lung alveolar macrophages (AM) from cell-specific MyD88 KO at 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 from ECs to AMs in vivo, we examined transcriptional profiles from lung AMs sorted following 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 AMs that were cell-intrinsic as well as resulting from in vivo crosstalk from ECs. 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 whole lung RNA, sorted EC RNA, and sorted cDC RNA by Nanostring nCounter Immunology Codeset Analysis (additional linked files). Overall design: Alveolar macrophages from all genotypes were flow cytometry sorted following 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 1 timepoint (6 h OVA/stFLA) = 4 conditions. 4 conditions x 2 replicates per genotype-timepoint = 8 total samples. 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 2 biological replicates for each genotype.