IL-1β is an important inflammatory mediator of type 2 diabetes (T2D). Here we show that oligomers of islet amyloid polypeptide (IAPP), a protein that forms amyloid deposits in the pancreas during T2D, trigger the Nlrp3 inflammasome and generate mature interleukin (IL)-1β. A T2D therapy, glyburide, suppresses IAPP-mediated IL-1β production in vitro. Processing of IL-1β initiated by IAPP first requires priming, a process that involves glucose metabolism and can be facilitated by minimally oxidized low density lipoprotein. Finally, mice transgenic for human IAPP have increased IL-1β in pancreatic islets, which colocalizes with amyloid and macrophages. Our findings reveal novel mechanisms in the pathogenesis of T2D and treatment of pathology caused by IAPP.

Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1␤ secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1␤ in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1␤ was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1␤ and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1␤ by antigen-presenting cells in an NLRP3-and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1␤ in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1␤ through at least two separate mechanisms: by targeting pro-IL-1␤ for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.IL-1␤ is an important proinflammatory cytokine, released at sites of infection or injury, that regulates diverse physiological responses, including cellular recruitment, appetite, sleep, and body temperature (1). IL-1␤ is first produced as a proform in response to inflammatory stimuli, such as TLR ligands. This inactive precursor is cleaved into the bioactive (p17) molecule by caspase 1, following the activation of an inflammasome (2).Inflammasomes are molecular scaffolds that trigger the activation of caspase 1 and subsequent maturation of IL-1␤ and IL-18. Typically, inflammasomes are formed from at least one member of the cytosolic innate immune sensor family, the NOD-like receptors (NLRs), including NLRP1, NLRP3, and NLRC4, coupled with the adaptor apoptosis-associated specklike protein containing a caspase recruitment domain (ASC or PYCARD) and caspase 1 (2). The NLRP3 inflammasome is the best characterized to date and is activated by a number of endogenous and exogenous signals.Most studies in vitro employ TLR ligands, particularly LPS, to induce pro-IL-1␤ formation, but in many cases, this is not enough to stimulate inflammasome activation and secretion of the mature cytokine. Instead, a second signal is commonly required, and this can come from a number of endogenous and exogenous sources, including ATP and particulates, including uric acid crystals, amyloid-␤, silica, asbestos, synthetic microparticles, and alum (3-8). Extracellular ATP triggers the P2X7 ATP-gated ion channel, leading to K ϩ efflux and induces recruitment of the pannexin-1 membrane pore (9). This may then allow extracellular NLRP3 agonists to enter the cell and activate inflammasome assembly (9). Particulates have been proposed to act through one of two mechanisms. Uptake of particulates by phagocytes may lead to lysosomal damage and release of lysosomal products into the cytosol, which a...

Many currently used and candidate vaccine adjuvants are particulate in nature, but their mechanism of action is not well understood. Here, we show that particulate adjuvants, including biodegradable poly(lactide-co-glycolide) (PLG) and polystyrene microparticles, dramatically enhance secretion of interleukin-1␤ (IL-1␤) by dendritic cells (DCs). The ability of particulates to promote IL-1␤ secretion and caspase 1 activation required particle uptake by DCs and NALP3. Uptake of microparticles induced lysosomal damage, whereas particle-mediated enhancement of IL-1␤ secretion required phagosomal acidification and the lysosomal cysteine protease cathepsin B, suggesting a role for lysosomal damage in inflammasome activation. Although the presence of a Toll-like receptor (TLR) agonist was required to induce IL-1␤ production in vitro, injection of the adjuvants in the absence of TLR agonists induced IL-1␤ production at the injection site, indicating that endogenous factors can synergize with particulates to promote inflammasome activation. The enhancement of antigenspecific antibody production by PLG microparticles was independent of NALP3. However, the ability of PLG microparticles to promote antigen-specific IL-6 production by T cells and the recruitment and activation of a population of CD11b ؉ Gr1 ؊ cells required NALP3. Our data demonstrate that uptake of microparticulate adjuvants by DCs activates the NALP3 inflammasome, and this contributes to their enhancing effects on innate and antigenspecific cellular immunity.Caspase-1 ͉ IL-1 ͉ microparticle

At mammalian body temperature, the plague bacillus Yersinia pestis synthesizes lipopolysaccharide (LPS)-lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity. To address the effect of weak TLR4 stimulation on virulence, we modified Y. pestis to produce a potent TLR4-stimulating LPS. Modified Y. pestis was completely avirulent after subcutaneous infection even at high challenge doses. Resistance to disease required TLR4, the adaptor protein MyD88 and coreceptor MD-2 and was considerably enhanced by CD14 and the adaptor Mal. Both innate and adaptive responses were required for sterilizing immunity against the modified strain, and convalescent mice were protected from both subcutaneous and respiratory challenge with wild-type Y. pestis. Despite the presence of other established immune evasion mechanisms, the modified Y. pestis was unable to cause systemic disease, demonstrating that the ability to evade the LPS-induced inflammatory response is critical for Y. pestis virulence. Evading TLR4 activation by lipid A alteration may contribute to the virulence of various Gram-negative bacteria.

Clinical translation of cell therapies requires strategies that can manufacture cells efficiently and economically. One promising way to reproducibly expand T cells for cancer therapy is by attaching the stimuli for T cells onto artificial substrates with high surface area. Here, we show that a carbon nanotube-polymer composite can act as an artificial antigen-presenting cell to efficiently expand the number of T cells isolated from mice. We attach antigens onto bundled carbon nanotubes and combined this complex with polymer nanoparticles containing magnetite and the T-cell growth factor interleukin-2 (IL-2). The number of T cells obtained was comparable to clinical standards using a thousand-fold less soluble IL-2. T cells obtained from this expansion were able to delay tumour growth in a murine model for melanoma. Our results show that this composite is a useful platform for generating large numbers of cytotoxic T cells for cancer immunotherapy.

Vaccine development has progressed significantly and has moved from whole microorganisms to subunit vaccines that contain only their antigenic proteins. Subunit vaccines are often less immunogenic than whole pathogens; therefore, adjuvants must amplify the immune response, ideally establishing both innate and adaptive immunity. Incorporation of antigens into biomaterials, such as liposomes and polymers, can achieve a desired vaccine response. The physical properties of these platforms can be easily manipulated, thus allowing for controlled delivery of immunostimulatory factors and presentation of pathogen-associated molecular patterns (PAMPs) that are targeted to specific immune cells. Targeting antigen to immune cells via PAMP-modified biomaterials is a new strategy to control the subsequent development of immunity and, in turn, effective vaccination. Here, we review the recent advances in both immunology and biomaterial engineering that have brought particulate-based vaccines to reality.

Alum is the principal vaccine adjuvant for clinical applications but it is a poor inducer of cellular immunity and is not an optimal adjuvant for vaccines where Th1 responses are required for protection. The mechanism underlying the inefficiency of alum in promoting Th1 responses is not fully understood. We show that aluminium hydroxide, aluminium phosphate, and calcium phosphate adjuvants inhibit the secretion of the Th1 polarizing cytokine, IL-12 by dendritic cells (DCs). Alum selectively inhibited DC expression of the IL-12p35 subunit and the inhibitory effect results from adjuvantinduced PI3 kinase signaling. To develop a more effective adjuvant for promoting cellmediated immunity, we investigated alternative particulates and found that in contrast to alum, the cationic polysaccharide chitosan did not inhibit IL-12 secretion. A combination of chitosan and the TLR9 agonist CpG activated the NLRP3 inflammasome and enhanced secretion of IL-12 and the other key Th1 and Th17-cell polarizing cytokines. When used as an adjuvant, CpG-chitosan induced NLRP3-dependent antigen-specific Th1 and Th17 responses. A combination of alum and CpG also enhanced Th1 and Th17 responses but was less effective than CpG-chitosan. Therefore, chitosan is an attractive alternative to alum in adjuvants for vaccines where potent cell-mediated immunity is required. Keywords IntroductionThere is a pressing need for novel vaccine adjuvants that are effective in safely promoting cellular immunity for diseases including Correspondence: Dr. Ed C. Lavelle e-mail: lavellee@tcd.ie tuberculosis (TB), malaria, and HIV [1]. Alum is the most widely used adjuvant for clinical applications and has been used in multiple vaccines for the past 80 years. However, while alum is a very effective adjuvant for promoting humoral immunity and Th2 type * These authors contributed equally to this work. The inefficiency of alum and related adjuvants as activators of Th1 responses suggests that additional signals are required for particulates to effectively promote cell-mediated immunity. Indeed, it has been shown that combining alum with IL-12 enhanced IgG2a antibody and Th1 responses to an HIV antigen compared with alum alone [19]. Combinations of emulsions, alum, or microparticles with PAMPs, particularly TLR4 agonists, are being used to amplify adaptive immunity [20][21][22] and are under active investigation for inclusion in a new generation of vaccines. To advance the development of this new generation of vaccine adjuvants with greater potency, it is essential to optimize both the particulate and immunostimulatory adjuvants to produce systems that act in synergy to drive appropriate T-cell responses. A recent report suggests that co-administration of CpG with alum results in a suppression of Th1-dependent antigen-specific IgG2a responses compared with administration of CpG alone [23]. We demonstrate that alum strongly inhibits the secretion of TLR agonistinduced IL-12 by DCs and these inhibitory effects are dependent on PI3 kinase signaling. In contrast to a...

The effectiveness of many vaccines licensed for clinical use relates to the induction of neutralising antibodies, facilitated by the inclusion of vaccine adjuvants, particularly alum. However, the ability of alum to preferentially promote humoral rather than cellular, particularly Th1-type responses, is not well understood. We demonstrate that alum activates immunosuppressive mechanisms following vaccination, which limit its capacity to induce Th1 responses. One of the key cytokines limiting excessive immune responses is IL-10. Injection of alum primed draining lymph node cells for enhanced IL-10 secretion ex vivo. Moreover, at the site of injection, macrophages and dendritic cells were key sources of IL-10 expression. Alum strongly enhanced the transcription and secretion of IL-10 by macrophages and dendritic cells. The absence of IL-10 signalling did not compromise alum-induced cell infiltration into the site of injection, but resulted in enhanced antigen-specific Th1 responses after vaccination. In contrast to its decisive regulatory role in regulating Th1 responses, there was no significant change in antigen-specific IgG1 antibody production following vaccination with alum in IL-10-deficient mice. Overall, these findings indicate that injection of alum promotes IL-10, which can block Th1 responses and may explain the poor efficacy of alum as an adjuvant for inducing protective Th1 immunity.