The NLRP3 inflammasome is a critical component of sterile inflammation, which is involved in many diseases. However, there is currently no known proximal biomarker for measuring NLRP3 activation in pathological conditions. Protein kinase D (PKD) has emerged as an important NLRP3 kinase that catalyzes the release of a phosphorylated NLRP3 species that is competent for inflammasome complex assembly. To explore the potential for PKD activation to serve as a selective biomarker of the NLRP3 pathway, we tested various stimulatory conditions in THP-1 and U937 cell lines, probing the inflammasome space beyond NLRP3. We analyzed the correlation between PKD activation (monitored by its auto-phosphorylation) and functional inflammasome readouts. PKD activation/auto-phosphorylation always preceded cleavage of caspase-1 and gasdermin D, and treatment with the PKD inhibitor CRT0066101 could block NLRP3 inflammasome assembly and interleukin-1β production. Conversely, blocking NLRP3 either genetically or using the MCC950 inhibitor prevented PKD auto-phosphorylation, indicating a bidirectional functional crosstalk between NLRP3 and PKD. Further assessments of the pyrin and NLRC4 pathways, however, revealed that PKD auto-phosphorylation can be triggered by a broad range of stimuli unrelated to NLRP3 inflammasome assembly. Thus, although PKD and NLRP3 become functionally interconnected during NLRP3 activation, the promiscuous reactivity of PKD challenges its potential use for tracing the NLRP3 inflammasome pathway.

中文翻译：

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评价蛋白激酶D自磷酸化作为NLRP3炎性体激活的生物标志物

NLRP3炎性小体是无菌炎症的重要组成部分，它与许多疾病有关。然而，目前尚无用于在病理状况下测量NLRP3活化的已知近端生物标志物。蛋白激酶D（PKD）已作为一种重要的NLRP3激酶出现，该酶催化释放能够完成炎性体复合物组装的磷酸化NLRP3物种。为了探索PKD激活作为NLRP3途径的选择性生物标志物的潜力，我们测试了THP-1和U937细胞系中的各种刺激条件，探测了NLRP3以外的炎性体空间。我们分析了PKD激活（通过其自身磷酸化水平进行监测）与功能性炎症小体读数之间的相关性。PKD激活/自磷酸化总是先于caspase-1和gasdermin D的裂解，PKD抑制剂CRT0066101的治疗可能会阻断NLRP3炎症小体的组装和白介素1β的产生。相反，通过基因或使用MCC950抑制剂阻断NLRP3可以防止PKD自磷酸化，这表明NLRP3与PKD之间存在双向功能性串扰。然而，对吡啶和NLRC4途径的进一步评估表明，与NLRP3炎症小体组装无关的广泛刺激可触发PKD自磷酸化。因此，尽管在NLRP3激活过程中PKD和NLRP3在功能上互连，但是PKD的混杂反应性挑战了其追踪NLRP3炎性体途径的潜在用途。通过遗传或使用MCC950抑制剂阻断NLRP3可以防止PKD自磷酸化，这表明NLRP3和PKD之间存在双向功能性串扰。然而，对吡啶和NLRC4途径的进一步评估表明，与NLRP3炎症小体组装无关的广泛刺激可触发PKD自磷酸化。因此，尽管在NLRP3激活过程中PKD和NLRP3在功能上互连，但是PKD的混杂反应性挑战了其追踪NLRP3炎性体途径的潜在用途。通过遗传或使用MCC950抑制剂阻断NLRP3可以防止PKD自磷酸化，这表明NLRP3和PKD之间存在双向功能性串扰。然而，对吡啶和NLRC4途径的进一步评估表明，与NLRP3炎症小体组装无关的广泛刺激可触发PKD自磷酸化。因此，尽管在NLRP3激活过程中PKD和NLRP3在功能上互连，但是PKD的混杂反应性挑战了其追踪NLRP3炎性体途径的潜在用途。