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


METHODS
Primary 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.


RESULTS
We 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.


CONCLUSIONS
This 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.
Molecular Therapeutics, School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK.
Caroline S
CS
Hughes
Centre for Experimental Medicine, Queen's University Belfast, Belfast, BT12 6BA, UK.
Liza M
LM
Colhoun
Molecular Therapeutics, School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK.
Baljinder K
BK
Bains
Innovative Medicines and Early Development, Drug Safety and Metabolism, Regulatory Safety, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK.
Joanne D
JD
Kilgour
Molecular Therapeutics, School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK.
Roberta E
RE
Burden
Molecular Therapeutics, School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK.
James F
JF
Burrows
Adjuvant Research Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, D02 PN40, Ireland.
Ed C
EC
Lavelle
Molecular Therapeutics, School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK.
Brendan F
BF
Gilmore
Molecular Therapeutics, School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK. c.scott@qub.ac.uk.
Christopher J
CJ
Scott
D000534:Alum Compounds / Q000633:toxicity; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D020170:Caspase 1 / Q000378:metabolism*; D002403:Cathepsins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004151:Dipeptides / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D007113:Immunity, Innate / Q000187:drug effects; D007249:Inflammation / Q000139:chemically induced* / Q000201:enzymology / Q000235:genetics / Q000276:immunology / Q000473:pathology; D053583:Interleukin-1beta / Q000378:metabolism; D007700:Kinetics; D008247:Lysosomes / Q000187:drug effects* / Q000201:enzymology / Q000276:immunology / Q000473:pathology; D017737:Macrophages, Peritoneal / Q000187:drug effects* / Q000201:enzymology / Q000276:immunology / Q000473:pathology; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D053758:Nanoparticles; D052638:Particulate Matter / Q000633:toxicity*; D011137:Polystyrenes / Q000633:toxicity; D061251:Primary Cell Culture; D059748:Proteolysis; D012822:Silicon Dioxide / Q000633:toxicity; D013379:Substrate Specificity; D018675:Toxicity Tests / Q000379:methods*
D000534:Alum Compounds; D015415:Biomarkers; D004151:Dipeptides; C587672:IL1B protein, mouse; D053583:Interleukin-1beta; D052638:Particulate Matter; D011137:Polystyrenes; C045139:leucyl-leucine-methyl ester; C041524:aluminum sulfate; D012822:Silicon Dioxide; D002403:Cathepsins; C028217:cathepsin S; D020170:Caspase 1
Affinity-binding probes; Caspase 1; Cathepsin S; IL-1β; Nanoparticles; Proteolytic activity; Toxicity