Identifying mediators of cytosolic myddosome assembly

Toll-like receptor (TLR) activation induces the formation of highly dynamic myddosomes in the cytosol of macrophages. 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. Thus, myddosome-containing cells drive the LPS-induced transcriptional program, whereas bystanders only retain IFN responsiveness.