A Proteolytic Cascade Controls Lysosome Rupture and Necrotic Cell Death Mediated by Lysosome-Destabilizing Adjuvants

<div><p>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.</p></div

MG115 inhibits cathepsin C activity.

<p>(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.</p

The proteasome inhibitor MG115 blocks LLOMe-mediated lysosome rupture.

<p>(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.</p

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

<p>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.</p

The proteasome inhibitor MG115 blocks LLOMe-mediated lysosome rupture.

<p>(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.</p

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

<p>(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 <i>in vitro</i> 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.</p

Enhanced germination rate of B. cereus spores germinated in conditioned supernatants.

<p>Wild-type <i>B. cereus</i> spores were germinated with a 0.2 mM inosine solution (•) or in conditioned supernatants containing 0.2 mM inosine (▪). <i>B. cereus</i> spores were also germinated with 0.2 mM inosine and 20 µM alanine (X).</p

<i>B. cereus</i> spore germination at low spore concentrations.

<p>(A) <i>B. cereus</i> spores were diluted in 10 or 4000 ml (OD₅₈₀ of 1 or 0.0025, respectively) of germination buffer supplemented with 0.2 mM inosine and 0 or 40 µM alanine. Thirty min post-inosine exposure, spores were collected by centrifugation, and pellets were treated with malachite green to stain resting spores and safranin-O to stain germinated cells. Samples were placed under a microscope and a field selected at random. (B) <i>B. cereus</i> spores were diluted in germination buffer (OD₅₈₀ of 1 to 0.0025) in the presence of 0.2 mM inosine. Stained samples were placed under a light microscope and the amount of resting spores and germinated cells were counted on three different fields selected at random. The percentage of germinated spores was plotted against the initial spore optical density.</p

7-AMC adducts detected in <i>wt B. cereus</i> conditioned supernatants.

<p>Wild type <i>B. cereus</i> 569 spores were resuspended in 200 µl TMB buffer to OD₅₈₀ = 1. Spores were treated with 0.2 mM inosine and supernatants were collected 30 min post-inosine addition. Collected supernatants were labeled with 7-AMC. 7-AMC adducts sere separated by RP-HPLC and identified by mass spectrometry. Concentrations were calculated by fluorescence spectroscopy.</p

<i>B. cereus</i> spore germination in the presence of Ca-DPA.

<p>(A) Wild-type <i>B. cereus</i> spores were germinated in the presence of 0.2 mM inosine (□). <i>B. cereus</i> spores were also germinated in the presence of conditioned supernatants containing 0.2 mM inosine (+). Spores were also germinated with 0.2 mM inosine supplemented with 0.18 mM Ca-DPA (▪).</p