Necroptotic signaling in adaptive and innate immunity.

The vertebrate immune system is highly dependent on cell death for efficient responsiveness to microbial pathogens and oncogenically transformed cells. Cell death pathways are vital to the function of many immune cell types during innate, humoral and cellular immune responses. In addition, cell death regulation is imperative for proper adaptive immune self-tolerance and homeostasis. While apoptosis has been found to be involved in several of these roles in immunity, recent data demonstrate that alternative cell death pathways are required. Here, we describe the involvement of a programmed form of cellular necrosis called "necroptosis" in immunity. We consider the signaling pathways that promote necroptosis downstream of death receptors, type I transmembrane proteins of the tumor necrosis factor (TNF) receptor family. The involvement of necroptotic signaling through a "RIPoptosome" assembled in response to innate immune stimuli or genotoxic stress is described. We also characterize the induction of necroptosis following antigenic stimulation in T cells lacking caspase-8 or FADD function. While necroptotic signaling remains poorly understood, it is clear that this pathway is an essential component to effective vertebrate immunity.

Selectively targeting the toll-like receptor 9 (TLR9)--IRF 7 signaling pathway by polymer blend particles.

Signaling through toll-like receptor 9 (TLR9) has been exploited for cancer therapy. The stimulation of TLR9 leads to two bifurcating signaling pathways - NF-κB-dependent pro-inflammatory cytokines pathway and IRF-7-dependent type I interferons (IFNs) pathway. In this study, we employ polymer blend particles to present the synthetic ligand, CpG oligonucleotides (CpG ODNs), to TLR9. The polymer blend particles are made from the blend of pH-insensitive and pH-sensitive copolymer. By tailoring the composition of the pH-sensitive polymer, CpG ODNs are presented to TLR9 in a way that only activates the IRF-7 signaling pathway. CpG ODNs have been used for cancer therapy in both preclinical and clinical studies. The selective activation of IRF-7 could potentially enhance the apoptosis of tumor cells and immunological control of tumor progression without inadvertently activating NF-κB-dependent oncogenesis.

Effects of particle size on toll-like receptor 9-mediated cytokine profiles.

Biomaterials interface with toll-like receptor (TLR) 9-mediated innate immunity in a wide range of medical applications, such as tissue implants and drug delivery systems. The stimulation of TLR9 can lead to two different signaling pathways, resulting in the generation of proinflammatory cytokines (i.e. IL-6) and/or type I interferons (IFNs, i.e. IFN-α). These two categories of cytokines differentially influence both innate and adaptive immunity. Although particle size is known to be a critical parameter of biomaterials, its role in TLR9-mediated cytokine profiles is not clear. Here, we examined how the size of biomaterials impacted cytokine profiles by using polystyrene particles of defined sizes as model carriers for TLR9 agonists (CpG oligonucleotides (CpG ODNs)). CpG ODNs bound to nano- to submicro- particles stimulated the production of both IL-6 and IFN-α, while those bound to micro particles resulted in IL-6 secretions only. The differential TLR9-mediated cytokine profiles were attributed to the pH of endosomes that particles trafficked to. The magnitude of IFN-α production was highly sensitive to the change in endosomal pH in comparison to that of IL-6. Our results define two critical design variables, size and the ability to modulate endosomal pH, for the engineering of biomaterials that potentially interface with TLR9-mediated innate immunity. The fine control of these two variables will allow us to fully exploit the beneficial facets of TLR9-mediated innate immunity while minimizing undesirable side effects.

Thermostability and molecular encapsulation within an engineered caged protein scaffold.

Self-assembling biological complexes such as viral capsids have been manipulated to function in innovative nanotechnology applications. The E2 component of pyruvate dehydrogenase from Bacillus stearothermophilus forms a dodecahedral complex and potentially provides another platform for these purposes. In this investigation, we show that this protein assembly exhibits unusual stability and can be modified to encapsulate model drug molecules. To distill the E2 protein down to its structural scaffold core, we synthesized a truncated gene optimized for expression in Escherichia coli. The correct assembly and dodecahedral structure of the resulting scaffold was confirmed with dynamic light scattering and transmission electron microscopy. Using circular dichroism and differential scanning calorimetry, we found the thermostability of the complex to be unusually high, with an onset temperature of unfolding at 81.1 +/- 0.9 degrees C and an apparent midpoint unfolding temperature of 91.4 +/- 1.4 degrees C. To evaluate the potential of this scaffold for encapsulation of guest molecules, we made variants at residues 381 and 239 which altered the physicochemical properties of the hollow internal cavity. These mutants, yielding 60 and 120 mutations within this cavity, assembled into the correct architecture and exhibited high thermostability that was comparable to the wild-type scaffold. To show the applicability of this scaffold, two different fluorescent dye molecules were covalently coupled to the cysteine mutant at site 381. We demonstrate that these mutations can introduce non-native functionality and enable molecular encapsulation within the cavity while still retaining the dodecahedral structure. The unusually robust nature of this scaffold and its amenability to internal changes reveal its potential for nanoscale applications.