IRAK1 attenuates TLR signaling and activates the inflammasome in response to dual-TLR stimulation

Abstract Macrophages encounter many kinds of pathogen-associated molecules and a coherent immune response involves crosstalk among various immune pathways. How can a cogent response form in the face of crosstalk between various pathways? We address this in the context of combinatorial stimulation of multiple TLRs to assess TLR pathway crosstalk. We find that IRAK1 forms cytoplasmic clusters that sequester downstream signaling proteins upon dual TLR stimulation. This could explain the less-than additive gene response on dual TLR activation. Accordingly, IRAK1 deficient IMMs and BMDMs have higher phosphoprotein, cytokine and transcriptional responses to dual TLR ligands than WT cells, but their single TLR ligand responses are comparable to WT cells. This suggests that IRAK1-dependent sequestration of key TLR signaling proteins might be a mechanism of modulating the innate immune response to high pathogen load. We also find that IRAK1 cluster-enriched cells exhibited higher ASC nucleation and increased IL1α/β secretion, which are hallmarks of an active inflammasome. The colocalization of ASC suggests a possible role for dual TLR-induced IRAK1 clustering in diversifying signaling to multiple innate immune signaling pathways. We conclude that IRAK1 aids in tuning the TLR arm of signaling in response to multi-TLR ligands, allowing the engagement of other PRR pathways like the inflammasome. Thus, the rerouting of immune signals by IRAK1 clusters allows the cell to diversify the immune response to complex microbial signals through activation of multiple host response pathways, and improve the quality of the immune response during an infection. This work was supported by the Intramural Research Program of NIAID, NIH.