Identifying mediators of cytosolic myddosome assembly

However, not all cells respond, macrophage populations consistently contain bystanders whose frequency tracks with ligand concentration and activated TLR. We established a flow cytometry–based assay to separate responder and bystander cells via SSC changes (SSClo vs SSChi) reflecting cytosolic myddosomes in immortalized bone marrow-derived macrophages (iBMDM) with endogenously-tagged MyD88 (MyD88-AGF).

We performed bulk RNA-Seq to compare responder and bystander cells to untreated cells and LPS-treated but unsorted pools. Clustering and individual comparisons confirmed high similarity between responders and the LPS pool and a distinct bystander signature. Comparing bystanders to responders and untreated to LPS-treated pools showed reduced induction of TLR primary and secondary response genes (PRGs/SRGs) in bystanders, while interferon-stimulated genes (ISGs) induced via paracrine signaling remained intact.