Array of sorted lung conventional dendritic cells (cDCs) from cell-specific MyD88 KO at 0h and 6h 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) to define the contribution of Myd88 expression in each of these cell types. To examine crosstalk from ECs to conventional (c)DCs in vivo, we examined transcriptional profiles from lung cDCs sorted at baseline or 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 specific transcriptional changes in cDCs that were cell-intrinsic as well as resulting from in vivo crosstalk from ECs. We also observed 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, sorted alveolar macrophage RNA, and sorted cDC by Nanostring nCounter Immunology Codeset Analysis (additional linked files). Overall design: Lung cDCs from all genotypes were flow cytometry sorted at baseline or 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 2 timepoints (0h baseline, 6 h OVA/stFLA) = 8 conditions. 8 conditions x 2 replicates per genotype-timepoint = 16 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 Affymetrix Mouse Transcriptome 1.0 arrays (Affymetrix, Santa Clara, CA) on 2 biological replicates for each genotype and timepoint.