Extracellular cathepsin S and intracellular caspase 1 activation are surrogate biomarkers of particulate-induced lysosomal disruption in macrophages

Abstract

BackgroundParticulate matter has been shown to stimulate the innate immune system and induce acute inflammation. Therefore, while nanotechnology has the potential to provide therapeutic formulations with improved efficacy, there are concerns such pharmaceutical preparations could induce unwanted inflammatory side effects. Accordingly, we aim to examine the utility of using the proteolytic activity signatures of cysteine proteases, caspase 1 and cathepsin S (CTSS), as biomarkers to assess particulate-induced inflammation.MethodsPrimary peritoneal macrophages and bone marrow-derived macrophages from C57BL/6 mice and ctss −/− mice were exposed to micro- and nanoparticulates and also the lysosomotropic agent, L-leucyl-L-leucine methyl ester (LLOME). ELISA and immunoblot analyses were used to measure the IL-1β response in cells, generated by lysosomal rupture. Affinity-binding probes (ABPs), which irreversibly bind to the active site thiol of cysteine proteases, were then used to detect active caspase 1 and CTSS following lysosomal rupture. Reporter substrates were also used to quantify the proteolytic activity of these enzymes, as measured by substrate turnover.ResultsWe demonstrate that exposure to silica, alum and polystyrene particulates induces IL-1β release from macrophages, through lysosomal destabilization. IL-1β secretion positively correlated with an increase in the proteolytic activity signatures of intracellular caspase 1 and extracellular CTSS, which were detected using ABPs and reporter substrates. Interestingly IL-1β release was significantly reduced in primary macrophages from ctss −/− mice.ConclusionsThis study supports the emerging significance of CTSS as a regulator of the innate immune response, highlighting its role in regulating IL-1β release. Crucially, the results demonstrate the utility of intracellular caspase 1 and extracellular CTSS proteolytic activities as surrogate biomarkers of lysosomal rupture and acute inflammation. In the future, activity-based detection of these enzymes may prove useful for the real-time assessment of particle-induced inflammation and toxicity assessment during the development of nanotherapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-016-0129-5) contains supplementary material, which is available to authorized users.

Phagolysosome acidification is required for silica and engineered nanoparticle-induced lysosome membrane permeabilization and resultant NLRP3 inflammasome activity

“…Elevated extracellular cathepsin activity can be a consequence of lysosome disruption and associated cell death (Hughes et al , 2016). We observed greater extracellular cathepsins (L, B, and/or V) activity in the lavage fluid of silica-exposed mice at 7 d (Figure 4A).…”

Multi-hierarchical Profiling the Structure-Activity Relationships of Engineered Nanomaterials at Nano-Bio Interfaces

“…, proactivator polypeptide (PSAP), cathepsin S (CTSS), and cation-independent mannose-6-phosphate receptor (IGF2R). These proteins have effects on phagocytosis and lysosomal dysfunction 31-33 , implying a lysosome-involved mechanism. We further investigated this by detecting the pro-inflammatory cytokine release in THP-1 cells.…”

Engineered nanomaterial-induced lysosomal membrane permeabilization and anti-cathepsin agents

“…Several cathepsins have been associated with inflammatory diseases, while links to LMP were correlated with cathepsins B, C, D, and S (Hughes et al 2016; Jacobson et al 2013; Hornung et al 2008). Cathepsin B has been associated with ischemic cell death resulting from LMP in cerebral ischemia/reperfusion injured rats, an effect that is attenuated by CA074-me, a cathepsin B inhibitor that protects against lysosomal rupture (Xu et al 2016).…”

“…Extracellular cathepsin S and intracellular Caspase 1 were suggested to be regulators of the innate immune response, resulting in release of IL-1β. In particular, their proteolytic activities were associated with LMP following particle exposures (Hughes et al 2016). Cathepsin S was also correlated with autoimmune responses as well as acute and chronic inflammation.…”