Associate Professor University of Massachusetts Amherst Amherst, Massachusetts, United States
Introduction:: The Inflammasome, an intracellular multimeric protein complex that induces metabolic inflammation has been implicated in the onset and progression of the majority of chronic inflammatory diseases. This innate immune response involves the activation of the caspase-1 enzyme responsible for cleaving inactive inflammatory cytokines and other downstream proteins to their respective active forms that eventually lead to pyroptosis and enhanced immune response. Current techniques provide a limited understanding of real-time inflammasome kinetics during a disease progression and the mechanisms of inflammasome activation during disease progression remain poorly understood due to lack of proper tools and techniques. Moreover, one of the shortcomings of the existing platforms include short circulating half-life and reduced bioavailability which makes them inefficient for clinical use. These aspects can be addressed by the use of nanoparticles which not only increase the bioavailability of the probe in inflamed sites but also increases their retention via a sustained release. Here, we propose to design a nanoreporter encapsulated with a caspase-1 cleavable probe, that could enable real-time inflammasome monitoring in-vivo, during an inflammatory disease progression and image the efficacy of inflammasome-targeted agents in real-time.
Materials and Methods:: We first synthesized nanoreporters by encapsulating with a caspase-1 activatable probe (containing a dye and a quencher, conjugated via caspase substrate) in a lipid nanoparticle. Next, these nanoreporters were characterized for size, zeta potential and stability using Dynamic Light Scattering (DLS). Caspase-1 free probes were also tested and validated using recombinant enzymes before proceeding toward cell-based assays. For in-vitro activity studies, immortalized bone marrow-derived macrophages (iBMDMs) were used. We designed a high-content microscopy imaging analysis assay to quantitatively analyze inflammasome activation in iBMDMs after treatment with nanoreporters and monitor the kinetics at different conditions including concentrations and time points. We also performed nanoreporter uptake analysis, lactate dehydrogenase (LDH) assay (to measure membrane integrity), western blot analysis to measure activated caspase-1 levels, and ELISA assay to measure IL1β and IL18 cytokine levels. Finally, we evaluated the efficacy of nanoreporter in monitoring inflammasome activation in the DSS-induced colitis model.
Results, Conclusions, and Discussions:: Results and Discussion: Inflammasome nanoreporters showed a loading of 80% caspase-1 probe. Stability studies indicated that nanoreporters are stable for a month in PBS and atleast 24hours in serum. DLS studies indicated the nanoreporters’ average hydrodynamic size to be around 100-150nm with a neutral zeta potential. Furthermore, probe testing and validation studies displayed increased fluorescence intensity over time by active recombinant caspase-1 but no signal by the active caspase-3 enzyme when incubated with a reporter probe, indicating that that the probe is specifically responsive to caspase-1 enzyme activity. The internalization and colocalization studies specified 4 hours of treatment as the optimum time point for the future set of experiments. In-vitro nanoreporter activity testing displayed a significantly higher signal at 45minutes of nigericin treatment as compared to 0minute treatment when imaged using confocal. We demonstrated that the nanoreporter enables time-dependent and selective imaging of inflammasome activation which correlates with the ASC speck formation, cytokines release and pyroptosis. Altogether, our system exhibits an efficient monitoring of inflammasome activation in-vitro. Studies in the DSS-induced colitis model demonstrated robust inflammasome-specific monitoring and treatment efficacy (Figure 1).
Conclusions: We have successfully demonstrated efficient in vitro imaging of active caspase-1 (an important inflammasome marker) in inflammasome-activated bone-marrow-derived macrophages. We have further translated this into animal models, specifically inflammatory models. This system overall will help in a better understanding of the kinetics of active caspase-1 and inflammasome activation in vivo during a disease progression. It also helps in detecting inflammation early-on and identifying targeted treatment efficacy during disease progression.