Analysis of Innate Immune Signaling Networks

丝裂原活化蛋白激酶（MAPK）在炎症反应中充当中枢应激和免疫整合枢纽。虽然MAPK对于经典TLR信号传导后的许多转录事件是必不可少的，但每个主要MAPK分支（ERK、p38和JNK）在引发后炎性小体激活中的作用还有待澄清。生物传感器技术的最新进展已经建立了模块化MAPK核到胞质溶胶激酶易位报告子（KTR），以报告活细胞中每个主要MAPK信号分支的细胞内动力学。将这种激酶报告细胞系与炎性细胞因子释放所需的活性氧（ROS）和钙第二信使的化学报告细胞复合，允许与MAPK活性相关的、真实的时间关联。当与可以特异性地和快速地抑制不同的细胞内信号传导事件的抑制剂结合时，这种方法有可能提供对伴随炎性小体激活和焦亡的信号整合的复杂性的因果洞察，这是传统遗传方法难以实现的。 为了阐明炎性小体活化过程中MAPK信号传导的要求和动力学，在2022财年，我们采取了广泛的组合方法，重点是多重生物传感器研究、急性药理学抑制和筛选以及传统的遗传和生物化学方法，以提供炎性小体活化过程中MAPK信号级联的总体视图。通过这样做，我们通过发现一种新的GSDMD调节复合物，许可前孔寡聚化，描绘了炎性小体激活和细胞凋亡所需的不同的因果MAPK信号传导事件。此外，我们已经确定了级联激活所必需的特定ROS源和该复合物的经验证的抑制剂，在炎症性结肠炎的整个动物模型和患有分子上不同的炎性疾病的患者的外周血单核细胞（PBMC）中。 在炎性小体调节的其他研究中，我们不仅考虑了微生物配体，还考虑了与炎性疾病相关的宿主来源的触发因子，例如与代谢综合征相关的生物丰富的饱和脂肪酸（SFA）棕榈酸酯。已知SFA激活NLRP 3炎性体，但这种激活发生的细胞内途径没有完全描述。我们假设SFA通过与经典NLRP 3激活剂不同的细胞内途径激活NLRP 3炎性体。在FY 2022中，我们发现SFA棕榈酸（PA）激活NLRP 3不仅需要经典炎性体途径，还需要非经典炎性体组分caspase-4/5/11。任一途径的破坏导致对PA的炎性小体反应的部分损伤，尽管经典途径通常导致更多的IL-1分泌，而非经典途径是细胞死亡的主要驱动因素。与其他NLRP 3激活剂相比，PA对活性氧（ROS）的依赖性也较弱。虽然经典活化剂涉及多种ROS来源，包括线粒体和胞质酶，但NLRP 3的PA活化特别需要来自NADPH氧化酶的ROS。PA暴露导致巨噬细胞胞质溶胶中MAP激酶JNK的活化，并且干扰JNK信号传导阻断PA诱导的NLRP 3活化。这些发现进一步表征了SFA激活NLRP 3炎性体的机制，并突出了可能靶向延迟或逆转许多生活方式疾病中无菌炎症进展的途径。

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.