A proteolytic cascade controls lysosome rupture and necrotic cell death mediated by lysosome-destabilizing adjuvants

Abstract

Recent studies have linked necrotic cell death and proteolysis of inflammatory proteins to the adaptive immune response mediated by the lysosome-destabilizing adjuvants, alum and Leu-Leu-OMe (LLOMe). However, the mechanism by which lysosome-destabilizing agents trigger necrosis and proteolysis of inflammatory proteins is poorly understood. The proteasome is a cellular complex that has been shown to regulate both necrotic cell death and proteolysis of inflammatory proteins. We found that the peptide aldehyde proteasome inhibitors, MG115 and MG132, block lysosome rupture, degradation of inflammatory proteins and necrotic cell death mediated by the lysosome-destabilizing peptide LLOMe. However, non-aldehyde proteasome inhibitors failed to prevent LLOMe-induced cell death suggesting that aldehyde proteasome inhibitors triggered a pleotropic effect. We have previously shown that cathepsin C controls lysosome rupture, necrotic cell death and the adaptive immune response mediated by LLOMe. Using recombinant cathepsin C, we found that aldehyde proteasome inhibitors directly block cathepsin C, which presumably prevents LLOMe toxicity. The cathepsin B inhibitor CA-074-Me also blocks lysosome rupture and necrotic cell death mediated by a wide range of necrosis inducers, including LLOMe. Using cathepsin-deficient cells and recombinant cathepsins, we demonstrate that the cathepsins B and C are not required for the CA-074-Me block of necrotic cell death. Taken together, our findings demonstrate that lysosome-destabilizing adjuvants trigger an early proteolytic cascade, involving cathepsin C and a CA-074-Me-dependent protease. Identification of these early events leading to lysosome rupture will be crucial in our understanding of processes controlling necrotic cell death and immune responses mediated by lysosome-destabilizing adjuvants.

Figure 1. The proteasome inhibitor MG115 blocks LLOMe-mediated cell death and protein degradation.

(A) BALB/c-derived macrophages were primed with 250 ng/ml LPS for 2 hours, and then treated with 2.5 mM LLOMe, anthrax lethal toxin (LT: 500 ng/ml PA and 250 ng/ml LF), alum (150 µg/ml), or 10 µM nigericin (LN) in the absence or presence of 100 µM MG115 or 100 µM CA-074-Me. Cell death was measured after 2 hours (except for alum, which was exposed for 8 hours) by propidium iodide exclusion. Relative levels are blotted with inducer alone adjusted to 100% cell death. (B) BALB/c-derived macrophages were exposed to 2.5 mM LLOMe (LL) or anthrax lethal toxin (500 ng/ml PA and 250 ng/ml LF) in the absence or presence of 100 µM MG115 (MG). Cell death was measured by propidium iodide exclusion. (C) BALB/c macrophages were exposed to 2.5 mM LLOMe in the absence or presence of 100 µM MG115. Cellular lysates were isolated at different time points after LLOMe exposure, and were subjected to immunoblotting and probed with anti-caspase-1 and actin antibodies.

Figure 2. Effect of proteasome inhibitors on cell death mediated by LLOMe and anthrax lethal toxin (LT).

BALB/c-derived macrophages were exposed to 2.5 mM LLOMe (A) or anthrax lethal toxin (500 ng/ml PA and 250 ng/ml LF) (B) in the presence of increasing concentrations of the aldehyde proteasome inhibitors, MG115 and MG132, and the non-aldehyde proteasome inhibitor bortezomib. Cell death was measured by PI exclusion two hours after LLOMe/LT exposure. The above data is a representative experiment performed in triplicate.

Figure 3. MG115 inhibits cathepsin C activity.

(A) The cathepsin C assay was performed in the presence of 1 ng recombinant cathepsin C, the cathepsin C substrate Gly-Arg-AMC, and increasing concentrations of MG115 or the cathepsin C inhibitor Gly-Phe-DMK (GF-DMK). Cathepsin C activity was measured by analyzing Gly-Arg-AMC cleavage at 460 nm. (B) C57BL/6-derived macrophages were exposed to 2.5 mM LLOMe in the presence of increasing concentrations of MG115 or GF-DMK, and cell death was measured by PI exclusion two hours after LLOMe exposure.

Figure 4. The proteasome inhibitor MG115 blocks LLOMe-mediated lysosome rupture.

(A) Flow cytometry analysis of LLOMe-, alum- and LT-treated cells. BALB/c-derived macrophages were exposed to 2.5 mM LLOMe, LT (500 ng/ml PA and 250 ng/ml LF), or alum (150 µg/ml) in the absence and presence of 100 µM MG115 and 50 µM bortezomib. Lysosome and membrane integrity were measured using LysoTracker and PI by flow cytometry 2 hours post LLOMe and LT challenge, and 6 hours post alum challenge. The flow cytometry plots are representative images of two experiments each performed in triplicate. (B) C57BL/6-derived macrophages were exposed to 2.5 mM LLOMe in the absence and presence of 100 µM MG115. Analysis of lysosome integrity was determined by acridine orange (AO) staining 2 hours post LLOMe exposure. The above data is representative of three experiments.

Figure 5. CA-074-Me and Cathepsin C deficiency blocks cell death and protein degradation mediated by LLOMe.

(A) J774A.1 macrophages, and wild type, cathepsin B- or C-deficient C57BL/6-derived macrophages were treated with 2.5 mM LLOMe, and cell death was measured by PI exclusion two hours after LLOMe exposure. Corresponding lysates were subjected to immunoblotting and were probed with anti-caspase-1 and actin antibodies (lower panel). (B) The in vitro assays were performed in the presence of recombinant cathepsin B or cathepsin C, the corresponding cathepsin B and C substrates, and increasing concentrations (0.01, 0.1, 1 and 10 µM) of MG132, the cathepsin C inhibitor GF-DMK or the cathepsin B inhibitor CA-074-Me. Cathepsin B and C activity was measured by analyzing Gly-Arg-AMC and Arg-Arg-AMC cleavage at 460 nm, respectively.

Figure 6. The cathepsin B inhibitor blocks LLOMe-induced necrotic cell death.

(A) C57BL/6-derived BMDCs were exposed 2.5 mM LLOMe and analyzed by electron microscopy 30 and 60 min post-LLOMe exposure, respectively. Bars correspond to 1 µm. (B) C57BL/6-derived macrophages were treated with 2.5 mM LLOMe in the presence of increasing amounts of CA-074-Me (in µM). At different time points, cell death was measured by propidium iodide exclusion, and lysates were probed for IL-1β and actin by immunoblotting. All measurements were performed in triplicate.