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Detecting Anastasis In Vivo by CaspaseTracker Biosensor
Published on: February 1, 2018
CASPorter: A Novel Inducible Human CASP1/NALP3/ASC Inflammasome Biosensor
Chan Zou1,2, Jordan A Beard1, Guoping Yang2,3,4
1Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, TN, USA.
Background:
Following our 2015 elucidation of the CASP1/NALP3 inflammasome mechanism of glucocorticoid (GC)-resistance in pediatric acute lymphoblastic leukemia (ALL) patients, we engineered a cell-based CASP1/NALP3 reporter system suitable for high-throughput screening (HTS) of small molecule libraries, with the purpose of identifying compounds capable of inhibiting the CASP1/NALP3 inflammasome and synergizing with GC drugs for the treatment of GC-resistant ALL patients and various autoinflammatory diseases.
Methods:
A Dox-controlled system was utilized to induce the expression of the ASC transgene in HEK293 cells while simultaneously overexpressing NLRP3 and CASP1. ASC/CASP1/NALP3 inflammasome complex formation was confirmed by co-immunoprecipitation (co-IP) experiments. Next, a LV fluorescence-based biosensor (CASPorter) was transduced in the HEK293-iASC-NLRP3/CASP1 cell line to monitor the real-time activation of CASP1/NALP3 inflammasome in live cells. The applicability and effectiveness of the CASPorter cell line were tested by co-treatment with Dox and four known CASP1/NLRP3 inhibitors (MCC950, Glyburide, VX-765 and VRT-043198). Inflammasome activation and inhibitions were assessed by Western blotting, fluorescence microscopy and flow cytometry (FC) methods.
Results:
Dox treatment significantly induced ASC expression and increased levels of cleaved and catalytically active CASP1, co-IPs further demonstrated that CASP1 was pulled-down with NLRP3 in HEK293-iASC-NLRP3/CASP1 cells after induction of ASC by Dox treatment. In HEK293-iASC-NLRP3/CASP1-CASPorter cell system, cleavage of the CASP1 consensus site (YVAD) in the CASPorter protein after Dox treatment causing excitation/emission of green fluorescence and the 71% GFP+ cell population increase quantified by FC (78.1% vs 6.90%). Dox-induced activation of the NLRP3 inflammasome was dose-dependently inhibited by Dox co-treatment with four known CASP1/NLRP3 inhibitors.
Conclusion:
We have established a cell-based CASP1/NLRP3 inflammasome model, utilizing a fluorescence biosensor as readout for qualitatively observing and quantitatively determining the activation of caspase 1 and NLRP3 inflammasomes in living cells and easily define the inhibitory effect of inhibitors with high efficacy.
Insights
We developed a novel cell-based reporter system to screen for compounds that inhibit the CASP1/NALP3 inflammasome, offering new therapeutic avenues for glucocorticoid-resistant acute lymphoblastic leukemia and autoinflammatory diseases.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Established the CASP1/NALP3 inflammasome mechanism in glucocorticoid (GC)-resistant pediatric acute lymphoblastic leukemia (ALL) in 2015.
- Identified the need for high-throughput screening (HTS) to find inhibitors of this pathway.
Purpose of the Study:
- Engineer a cell-based CASP1/NALP3 reporter system for HTS.
- Identify compounds that inhibit the CASP1/NALP3 inflammasome.
- Discover synergistic drug combinations with GC for GC-resistant ALL and autoinflammatory diseases.
Main Methods:
- Utilized a Dox-controlled system to induce ASC, NLRP3, and CASP1 expression in HEK293 cells.
- Confirmed inflammasome complex formation via co-immunoprecipitation (co-IP).
- Developed and employed a fluorescence biosensor (CASPorter) for real-time monitoring of inflammasome activation using flow cytometry (FC) and fluorescence microscopy.
Main Results:
- Dox treatment successfully induced ASC expression and active CASP1 cleavage.
- CASPorter system demonstrated a significant increase in GFP+ cells (71%) upon inflammasome activation.
- Known CASP1/NALP3 inhibitors dose-dependently inhibited Dox-induced inflammasome activation.
Conclusions:
- Successfully established a cell-based CASP1/NALP3 inflammasome model.
- The fluorescence biosensor provides a reliable readout for inflammasome activation and inhibitor efficacy.
- This system facilitates the identification of potent inhibitors for therapeutic applications.
