Induction of distinct immune responses by cell death and NLRP3 signaling

Abstract

While adjuvants have been used in vaccines in order to stimulate an adaptive immune response against specific pathogens for almost a century, the mechanism by which they do so is still unclear. Recent studies suggested that prototypical adjuvants, such as alum, mimic the ability of cytopathic bacteria to activate the adaptive immune response by inducing necrotic cell death. Specifically, these studies suggested that alum stimulates the Nod-like receptor, Nlrp3, which is able to respond to specific danger signals. The goal of this thesis was to understand the role of cell death and Nlrp3 signaling in adjuvant-mediated immune responses.;In our initial study aimed at analyzing the cellular and systemic responses to NLR signaling, we found that overstimulation of NLRs and caspase-1 could actually have detrimental effects on the host. We demonstrated that anthrax lethal toxin activates the closely related NLR, Nlrpl/b, resulting in a caspase-l-mediated cytokine storm and rapid septic shock in susceptible rodents.;We then tested the role of Nlrp3 as it has been implicated in adjuvant-mediated immune responses. Nlrp3 is the best-characterized member of the Nod-like receptor (NLR) family, and has been shown to activate the adaptive immune response following challenge with specific microbial pathogens. Activation of the Nlrp3 inflammasome results in two proinflammatory events: (i) caspase- 1-mediated processing of specific cytokines (IL-lbeta and IL-18), and (ii) caspase-1 dependent necrosis (pyroptosis). It has been shown by several groups that the adjuvant alum is able to activate Nlrp3, presumably by way of destabilization of lysosomes. We further assessed the role of Nlrp3 in adjuvant-mediated immune responses. Surprisingly, we found that lysosome rupture mediated by alum and by the lysosome-destabilizing agent, Leu-Leu-OMe (LLOMe), resulted in the degradation of proinflammatory proteins, including components of the Nlrp3 inflammasome. It is therefore not surprising that alum and LLOMe treatment of macrophages resulted in only minimal NIrp3 signaling, insufficient for inducing caspase-1-mediated pyroptosis, and Nlrp3-mediated immune responses. We analyzed the alum and LLOMe-mediated protein degradation in detail, and found that this process is dependent on lysosomal cathepsins and low pH, but independent of the inflammasome, proteasome, and autophagic cell death. In summary, we believe that our findings could explain some of the inconsistencies that exist in the adjuvant field. These findings could explain why alum activates Nlrp3 (yet with limited efficiency), but fails to activate an Nlrp3-dependent immune response.;We next addressed the second hypothesis that suggested involvement of necrotic cell death in alum-induced immune responses. According to this theory alum-induced cell death results in the release of proinflammatory cellular compounds that trigger the adaptive immune response. Initially, we postulated that if cell death could trigger the adaptive immune response, then cytopathic agents should have adjuvant potential. To test this hypothesis, we used the cytopathic agent LLOMe, which shares with alum the ability to destabilize lysosomes, and to specifically kill antigen-presenting cells. In fact, LLOMe triggered a strong Th2-biased adaptive immune response. To determine whether cell death was critical for the Th2-baised immune response, we used genetics to identify proteins critical for adjuvant-mediated necrotic cell death. We found that cathepsin C was critical for LLOMe-mediated lysosome rupture and the ensuing necrotic cell death while being dispensable for alum mediated cell death. Intriguingly, cathepsin C-deficiency not only prevented LLOMe-mediated cytopathic effects, but also impaired the LLOMe-induced Th2 response. In contrast to LLOMe, cathepsins B and S were critical for alum-induced lysosome rupture and necrotic cell death, indicating that these processes were inducer and cathepsin-specific. Taken together, these studies strongly support the notion that necrotic cell death triggers a Th2 response. (Abstract shortened by UMI.).