Cathepsin-dependent lysosome rupture controls cell death, Nalp3 impairment, and adaptive immunity by the adjuvants alum and Leu-Leu-OMe
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Modern, antigen-based vaccines depend on immunostimulatory compounds, known as adjuvants, to generate strong and protective adaptive immune responses. Despite the ubiquitous importance of adjuvants to modern medicine, few have been developed that are considered safe and effective enough for human use. Development of new adjuvants is unpredictable, because most are identified empirically. Currently, there is no unifying theory governing adjuvant function. Aluminum salts, collectively known as alum, were the first adjuvants discovered, and remain the most widely used adjuvants in human vaccines. Though alum has been used clinically for over 80 years, our understanding of its immunostimulatory mechanism is limited. Alum's long track record of safe and effective clinical use suggest that mimicking its mechanism may result in potent human-grade adjuvants. Recent studies have suggested two distinct mechanisms for alum's immune enhancement. The first suggests that alum stimulates adaptive immunity by inducing necrosis at the site of injection. The second suggests that alum activates the pattern-recognition receptor Nalp3. In this thesis, we address both theories.;While alum is considered among the safest immunologic adjuvants, aluminum salts have been identified with tissue necrosis, granuloma formation, and myofascitis. Until recently, this toxicity was considered an unfortunate side-effect of alum-adjuvanted immunizations. As a result, alum-mediated necrosis is a poorly understood process. However, several recent studies have suggested that alum-mediated necrosis may contribute to its adjuvant effect. To unravel the relationship between adjuvant-induced necrosis and immunostimulation, we began by identifying the mechanism of alum-mediated cell death. We found that that macrophages challenged with alum undergo cathepsin-dependent lysosome rupture preceding cell death. Alum-mediated necrosis is a cell-type specific process, as it induced robust necrosis in myeloid leukocytes, but failed to kill lymphocytes. Strikingly, direct permeabilization of lysosomes with Leu-Leu-OMe (LL) mimicked alum-mediated cell death with respect to cell-specificity and cathepsin-dependence. Using cathepsin-deficient mice, we found that cathepsins B and S were critical for alum-mediated cell death while cathepsin C controlled LL-mediated necrosis. Just as LL was able to mimic alum-mediated cell death, we found that it also generated alum-like immunoglobulin responses when used as an adjuvant. Strikingly, cathepsin C-deficient mice were resistant to alum-adjuvanted immune responses, indicating that cell death controlled the adjuvant effects.;Alum mediated immunity has also been linked to activation of the intracellular surveillance receptor, Nalp3. Nalp3 is an intracellular surveillance receptor that promotes the release of the caspase-1-dependent cytokines, IL-1beta and IL-18. Recent evidence suggests that lysosome disruption, as by alum and LL, is a potent activator of Nalp3. Here we show that alum and LL are poor inflammasome inducers compared to prototypical channel-forming agents. We found that inflammasome signaling by alum and LL is impaired because lysosome rupture releases active proteases into the cytosol, leading to the degradation of caspase-1 and its associated cytokines. Unlike traditional caspase-1 inducers, alum and LL-induced proteolysis of caspase-1 occurred independently of inflammasome components. This protein degradation was prevented by cathepsin inhibitors and in cells deficient in critical cathepsins. Proteolysis was not restricted to the inflammasome, as cytosolic and cytoskeletal proteins were also degraded. Together, these data indicate that alum and LL-mediated cell death induce widespread protein degradation that antagonizes cytokine signaling pathways within macrophages.
Source: Dissertation Abstracts International, Volume: 74-09(E), Section: B.;Advisors: Jurgen Brojutsch; Arturo Casadevall Committee members: Stephen Baum; Kartik Chandran; Fernando Macias; Xingxing Zang.