Inflammation & Immunology Research Unit, Pfizer, Cambridge, MA 02139, United States; Department of Pharmacology & Experimental Therapeutics, Boston University School of Medicine, Boston, MA 02118, United States.
Inflammation & Immunology Research Unit, Pfizer, Cambridge, MA 02139, United States.
J Immunol Methods. 2019 Apr;467:19-28. doi: 10.1016/j.jim.2019.02.002. Epub 2019 Feb 5.
The NLRP3 inflammasome is a complex multimeric signaling apparatus that regulates production of the pro-inflammatory cytokine IL-1β. To overcome both the variability among primary immune cells and the limitations of genetic manipulation of differentiated human or murine macrophages, we developed a simplified, reliable and relevant cell-based model for studying the NLRP3 inflammasome using the undifferentiated human myelomonocytic cell line THP1. Undifferentiated THP1 cells constitutively express NLRP3, and NLRP3 inflammasome activation occurred in response to canonical NLRP3 activation stimuli including nigericin, ATP, and urea crystals, culminating in pro-IL-1β cleavage, extracellular release of mature IL-1β, and pyroptosis. We used this THP1 cell system to investigate potential targets of the potent, NLRP3 inflammasome selective inhibitor CP-456,773. We optimized a viral shRNA transduction method for gene expression knockdown (KD), and the KD of NLRP3 itself eliminated inflammasome activation and IL-1β production. NLRP3 inflammasome activation and CP-453,773 pharmacology were not altered in ABCb7- or ABCb10-deficient THP1 cells, eliminating these gene products as candidate pharmacological targets of CP-453,773. For ABCb10, we confirmed our results using CRISPR/CAS9-mediated ABCb10 knockout (KO) THP1 sub-lines. In summary, undifferentiated THP1 cells are fully competent for activation of the NLRP3 inflammasome and production of IL-1β, without differentiation into macrophages, and we describe optimized KD and KO methodologies to manipulate gene expression in these cells. As an example of the utility of undifferentiated THP1 cells for investigations into the biology of the NLRP3 inflammasome, we have used this cell system to rule out ABCb7 and ABCb10 as potential targets of the NLRP3 inflammasome inhibitor CP-453,773.
NLRP3 炎性小体是一种复杂的多聚体信号装置，调节前炎症细胞因子 IL-1β 的产生。为了克服原代免疫细胞的变异性和分化的人类或鼠源巨噬细胞遗传操作的局限性，我们使用未分化的人髓样单核细胞系 THP1 开发了一种简化、可靠且相关的基于细胞的 NLRP3 炎性小体模型。未分化的 THP1 细胞持续表达 NLRP3，并且 NLRP3 炎性小体的激活发生在对包括 Nigericin、ATP 和尿素晶体等经典 NLRP3 激活刺激物的反应中，最终导致前 IL-1β 的切割、成熟的 IL-1β 的细胞外释放和细胞焦亡。我们使用这种 THP1 细胞系统来研究强效、选择性 NLRP3 炎性小体抑制剂 CP-456,773 的潜在靶标。我们优化了病毒 shRNA 转导方法用于基因表达敲低（KD），并且 NLRP3 本身的 KD 消除了炎性小体的激活和 IL-1β 的产生。在 ABCb7 或 ABCb10 缺陷的 THP1 细胞中，NLRP3 炎性小体的激活和 CP-453,773 药理学没有改变，消除了这些基因产物作为 CP-453,773 的候选药理学靶标。对于 ABCb10，我们使用 CRISPR/CAS9 介导的 ABCb10 敲除（KO）THP1 亚系证实了我们的结果。总之，未分化的 THP1 细胞在不分化为巨噬细胞的情况下完全有能力激活 NLRP3 炎性小体并产生 IL-1β，我们描述了优化的 KD 和 KO 方法来操纵这些细胞中的基因表达。作为未分化的 THP1 细胞在 NLRP3 炎性小体生物学研究中的应用实例，我们已经使用该细胞系统排除了 ABCb7 和 ABCb10 作为 NLRP3 炎性小体抑制剂 CP-453,773 的潜在靶标。
