Cathepsin-mediated necrosis controls the adaptive immune response by Th2 (T helper type 2)-associated adjuvants.

Immunologic adjuvants are critical components of vaccines, but it remains unclear how prototypical adjuvants enhance the adaptive immune response. Recent studies have shown that necrotic cells could trigger an immune response. Although most adjuvants have been shown to be cytotoxic, this activity has traditionally been considered a side effect. We set out to test the role of adjuvant-mediated cell death in immunity and found that alum, the most commonly used adjuvant worldwide, triggers a novel form of cell death in myeloid leukocytes characterized by cathepsin-dependent lysosome-disruption. We demonstrated that direct lysosome-permeabilization with a soluble peptide, Leu-Leu-OMe, mimics the alum-like form of necrotic cell death in terms of cathepsin dependence and cell-type specificity. Using a combination of a haploid genetic screen and cathepsin-deficient cells, we identified specific cathepsins that control lysosome-mediated necrosis. We identified cathepsin C as critical for Leu-Leu-OMe-induced cell death, whereas cathepsins B and S were required for alum-mediated necrosis. Consistent with a role of necrotic cell death in adjuvant effects, Leu-Leu-OMe replicated an alum-like immune response in vivo, characterized by dendritic cell activation, granulocyte recruitment, and production of Th2-associated antibodies. Strikingly, cathepsin C deficiency not only blocked Leu-Leu-OMe-mediated necrosis but also impaired Leu-Leu-OMe-enhanced immunity. Together our findings suggest that necrotic cell death is a powerful mediator of a Th2-associated immune response.

Proteasome inhibitors prevent caspase-1-mediated disease in rodents challenged with anthrax lethal toxin.

NOD-like receptors (NLRs) and caspase-1 are critical components of innate immunity, yet their over-activation has been linked to a long list of microbial and inflammatory diseases, including anthrax. The Bacillus anthracis lethal toxin (LT) has been shown to activate the NLR Nalp1b and caspase-1 and to induce many symptoms of the anthrax disease in susceptible murine strains. In this study we tested whether it is possible to prevent LT-mediated disease by pharmacological inhibition of caspase-1. We found that caspase-1 and proteasome inhibitors blocked LT-mediated caspase-1 activation and cytolysis of LT-sensitive (Fischer and Brown-Norway) rat macrophages. The proteasome inhibitor NPI-0052 also prevented disease progression and death in susceptible Fischer rats and increased survival in BALB/c mice after LT challenge. In addition, NPI-0052 blocked rapid disease progression and death in susceptible Fischer rats and BALB/c mice challenged with LT. In contrast, Lewis rats, which harbor LT-resistant macrophages, showed no signs of caspase-1 activation after LT injection and did not exhibit rapid disease progression. Taken together, our findings indicate that caspase-1 activation is critical for rapid disease progression in rodents challenged with LT. Our studies indicate that pharmacological inhibition of NLR signaling and caspase-1 can be used to treat inflammatory diseases.

Distinct cathepsins control necrotic cell death mediated by pyroptosis inducers and lysosome-destabilizing agents.

Necrotic cell death triggers a range of biological responses including a strong adaptive immune response, yet we know little about the cellular pathways that control necrotic cell death. Inhibitor studies suggest that proteases, and in particular cathepsins, drive necrotic cell death. The cathepsin B-selective inhibitor CA-074-Me blocks all forms of programmed necrosis by an unknown mechanism. We found that cathepsin B deficiency does not prevent induction of pyroptosis and lysosome-mediated necrosis suggesting that CA-074-Me blocks necrotic cell death by targeting cathepsins other than cathepsin B. A single cathepsin, cathepsin C, drives necrotic cell death mediated by the lysosome-destabilizing agent Leu-Leu-OMe (LLOMe). Here we present evidence that cathepsin C-deficiency and CA-074-Me block LLOMe killing in a distinct and cell type-specific fashion. Cathepsin C-deficiency and CA-074-Me block LLOMe killing of all myeloid cells, except for neutrophils. Cathepsin C-deficiency, but not CA-074-Me, blocks LLOMe killing of neutrophils suggesting that CA-074-Me does not target cathepsin C directly, consistent with inhibitor studies using recombinant cathepsin C. Unlike other cathepsins, cathepsin C lacks endoproteolytic activity, and requires activation by other lysosomal proteases, such as cathepsin D. Consistent with this theory, we found that lysosomotropic agents and cathepsin D downregulation by siRNA block LLOMe-mediated necrosis. Our findings indicate that a proteolytic cascade, involving cathepsins C and D, controls LLOMe-mediated necrosis. In contrast, cathepsins C and D were not required for pyroptotic cell death suggesting that distinct cathepsins control pyroptosis and lysosome-mediated necrosis.

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

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.

Role of lysosome rupture in controlling Nlrp3 signaling and necrotic cell death.

The Nod-like receptor, Nlrp3, has been linked to inflammatory diseases and adjuvant-mediated immune responses. A wide array of structurally diverse agents does not interact directly with Nlrp3, but is thought to activate the Nlrp3 inflammasome by inducing a common upstream signal, such as lysosome rupture. To test the connection between lysosome integrity and Nlrp3 signaling, we analyzed inflammasome activation following stimulation of murine macrophages with lysosome-destabilizing agents and pyroptosis inducers. Here we provide evidence that lysosomal rupture and the corresponding release of lysosomal hydrolases is an early event in macrophages exposed to the lysosome-destabilizing adjuvants LLOMe and alum. Lysosome rupture preceded cell death induction mediated by these agents and was associated with the degradation of low-molecular weight proteins, including the inflammasome component caspase-1. Proteolysis of caspase-1 was controlled by specific cathepsins, but was independent of autocatalytic processes and Nlrp3 signaling. Consistent with these findings, lysosome-disrupting agents triggered only minimal caspase-1 activation and failed to cause caspase-1-dependent cell death (pyroptosis), generally associated with Nlrp3 signaling. In contrast, lysosome rupture was a late event in macrophages exposed to prototypical pyroptosis inducers. These agents triggered extensive Nlrp3 signaling prior to lysosome rupture with only minimal impact on the cellular proteome. Taken together, our findings suggest that lysosome impairment triggers a cascade of events culminating in cell death but is not crucial for Nlrp3 signaling. The significant differences observed between lysosome-disrupting agents and pyroptosis inducers might explain the distinct immunologic responses associated with these compounds.

Distinct defensin profiles in Neisseria gonorrhoeae and Chlamydia trachomatis urethritis reveal novel epithelial cell-neutrophil interactions.

Defensins are key participants in mucosal innate defense. The varied antimicrobial activity and differential distribution of defensins at mucosal sites indicate that peptide repertoires are tailored to site-specific innate defense requirements. Nonetheless, few studies have investigated changes in peptide profiles and function after in vivo pathogen challenge. Here, we determined defensin profiles in urethral secretions of healthy men and men with Chlamydia trachomatis- and Neisseria gonorrhoeae-mediated urethritis by immunoblotting for the epithelial defensins HBD1, HBD2, and HD5 and the neutrophil defensins HNP1 to -3 (HNP1-3). HBD1 was not detectable in secretions, and HBD2 was only induced in a small proportion of the urethritis patients; however, HD5 and HNP1-3 were increased in C. trachomatis infection and significantly elevated in N. gonorrhoeae infection. When HNP1-3 levels were low, HD5 appeared mostly as the propeptide; however, when HNP1-3 levels were >10 microg/ml, HD5 was proteolytically processed, suggesting neutrophil proteases might contribute to HD5 processing. HD5 and HNP1-3 were bactericidal against C. trachomatis and N. gonorrhoeae, but HD5 activity was dependent upon N-terminal processing of the peptide. In vitro proteolysis of proHD5 by neutrophil proteases and analysis of urethral secretions by surface-enhanced laser desorption ionization substantiated that neutrophils contribute the key convertases for proHD5 in the urethra during these infections. This contrasts with the small intestine, where Paneth cells secrete both proHD5 and its processing enzyme, trypsin. In conclusion, we describe a unique defensin expression repertoire in response to inflammatory sexually transmitted infections and a novel host defense mechanism wherein epithelial cells collaborate with neutrophils to establish an antimicrobial barrier during infection.

Reduced Paneth cell alpha-defensins in ileal Crohn's disease.

The pathogenesis of Crohn's disease (CD), an idiopathic inflammatory bowel disease, is attributed, in part, to intestinal bacteria that may initiate and perpetuate mucosal inflammation in genetically susceptible individuals. Paneth cells (PC) are the major source of antimicrobial peptides in the small intestine, including human alpha-defensins HD5 and HD6. We tested the hypothesis that reduced expression of PC alpha-defensins compromises mucosal host defenses and predisposes patients to CD of the ileum. We report that patients with CD of the ileum have reduced antibacterial activity in their intestinal mucosal extracts. These specimens also showed decreased expression of PC alpha-defensins, whereas the expression of eight other PC products either remained unchanged or increased when compared with controls. The specific decrease of alpha-defensins was independent of the degree of inflammation in the specimens and was not observed in either CD of the colon, ulcerative colitis, or pouchitis. The functional consequence of alpha-defensin expression levels was examined by using a transgenic mouse model, where we found changes in HD5 expression levels, comparable to those observed in CD, had a pronounced impact on the luminal microbiota. Thus, the specific deficiency of PC defensins that characterizes ileal CD may compromise innate immune defenses of the ileal mucosa and initiate and/or perpetuate this disease.