Recognition of Cytoplasmic RNA Results in Cathepsin-Dependent Inflammasome Activation and Apoptosis in Human Macrophages

dsRNA is an important pathogen-associated molecular pattern that is primarily recognized by cytosolic pattern-recognition receptors of the innate-immune system during virus infection. This recognition results in the activation of inflammasome-associated caspase-1 and apoptosis of infected cells. In this study, we used high-throughput proteomics to identify secretome, the global pattern of secreted proteins, in human primary macrophages that had been activated through the cytoplasmic dsRNA-recognition pathway. The secretome analysis revealed cytoplasmic dsRNA-recognition pathway-induced secretion of several exosome-associated proteins, as well as basal and dsRNA-activated secretion of lysosomal protease cathepsins and cysteine protease inhibitors (cystatins). Inflammasome activation was almost completely abolished by cathepsin inhibitors in response to dsRNA stimulation, as well as encephalomyocarditis virus and vesicular stomatitis virus infections. Interestingly, Western blot analysis showed that the mature form of cathepsin D, but not cathepsin B, was secreted simultaneously with IL-18 and inflammasome components ASC and caspase-1 in cytoplasmic dsRNA-stimulated cells. Furthermore, small interfering RNA-mediated silencing experiments confirmed that cathepsin D has a role in inflammasome activation. Caspase-1 activation was followed by proteolytic processing of caspase-3, indicating that inflammasome activation precedes apoptosis in macrophages that had recognized cytoplasmic RNA. Like inflammasome activation, apoptosis triggered by dsRNA stimulation and virus infection was effectively blocked by cathepsin inhibition. In conclusion, our results emphasize the importance of cathepsins in the innate immune response to virus infection.