Analysis of Innate Immune Signaling Networks

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Mitogen activated protein kinases (MAPKs) serve as central stress and immune integration hubs during inflammatory responses. While MAPKs are essential for numerous transcriptional events following classical TLR signaling, the role of each major MAPK branch (ERK, p38, and JNK) in post priming inflammasome activation has yet to be clarified. Recent advances in biosensor technologies have established modular MAPK nuclear to cytosol kinase translocation reporters (KTRs) to report intracellular dynamics of each major MAPK signaling branch in live cells. Multiplexing such kinase reporter lines with chemical reporters for reactive oxygen species (ROS) and calcium second messengers required for inflammatory cytokine release, allows correlative, real time associations with MAPK activity. When coupled with inhibitors that can specifically and quickly inactivate distinct intracellular signaling events, this approach has the potential to provide causal insight into intricacies of signal integration accompanying inflammasome activation and pyroptosis that are elusive to traditional genetic approaches. To clarify the requirements and dynamics of MAPK signaling during inflammasome activation, in FY22 we have taken a broad combinatorial approach focusing on multiplex biosensor investigations, acute pharmacological inhibition and screening, and traditional genetic and biochemical approaches to provide an overarching view of MAPK signal cascades during inflammasome activation. By doing so, we have delineated distinct causal MAPK signaling events required for inflammasome activation and pyroptosis through the discovery of a novel GSDMD regulating complex that licenses pre pore oligomerization. Furthermore, we have identified a specific ROS source necessary for cascade activation and validated inhibitors of this complex in both whole animal models of inflammatory colitis and peripheral blood mononuclear cells (PBMCs) from patients suffering from molecularly distinct inflammasomopathies. In additional studies of inflammasome regulation, we are considering not only microbial ligands, but also host-derived triggers associated with inflammatory diseases, such as the biologically abundant saturated fatty acid (SFA) palmitate which is associated with metabolic syndromes. SFAs are known to activate the NLRP3 inflammasome but the intracellular pathways by which this activation occurs are incompletely described. We hypothesized that SFAs activate the NLRP3 inflammasome through different intracellular pathways than classical NLRP3 activators. In FY2022, we found that NLRP3 activation by the SFA palmitic acid (PA) requires not only the canonical inflammasome pathway but also the non-canonical inflammasome components caspase-4/5/11. Disruption of either pathway leads to partial impairments in the inflammasome response to PA, though the canonical pathway generally accounts for more IL-1 secretion while the non-canonical pathway is the primary driver of cell death. PA also has a weaker dependence on reactive oxygen species (ROS) compared to other NLRP3 activators. While classical activators engage multiple ROS sources including both mitochondrial and cytosolic enzymes, PA activation of NLRP3 specifically requires ROS from NADPH oxidases. PA exposure leads to activation of the MAP kinase JNK in the cytosol of macrophages and interference with JNK signaling blocks PA-induced NLRP3 activation. These findings further characterize the mechanisms by which SFAs activate the NLRP3 inflammasome and highlight pathways that may be targeted to delay or reverse the progression of sterile inflammation in many lifestyle diseases.