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Peer reviewed: True, Funder: Trinity College Dublin; FundRef: http://dx.doi.org/10.13039/501100001637, Macrophages are a highly adaptive population of innate immune cells. Polarization with IFNγ and LPS into the 'classically activated' M1 macrophage enhances pro-inflammatory and microbicidal responses, important for eradicating bacteria such as Mycobacterium tuberculosis. By contrast, 'alternatively activated' M2 macrophages, polarized with IL-4, oppose bactericidal mechanisms and allow mycobacterial growth. These activation states are accompanied by distinct metabolic profiles, where M1 macrophages favor near exclusive use of glycolysis, whereas M2 macrophages up-regulate oxidative phosphorylation (OXPHOS). Here, we demonstrate that activation with IL-4 and IL-13 counterintuitively induces protective innate memory against mycobacterial challenge. In human and murine models, prior activation with IL-4/13 enhances pro-inflammatory cytokine secretion in response to a secondary stimulation with mycobacterial ligands. In our murine model, enhanced killing capacity is also demonstrated. Despite this switch in phenotype, IL-4/13 trained murine macrophages do not demonstrate M1-typical metabolism, instead retaining heightened use of OXPHOS. Moreover, inhibition of OXPHOS with oligomycin, 2-deoxy glucose or BPTES all impeded heightened pro-inflammatory cytokine responses from IL-4/13 trained macrophages. Lastly, this work identifies that IL-10 attenuates protective IL-4/13 training, impeding pro-inflammatory and bactericidal mechanisms. In summary, this work provides new and unexpected insight into alternative macrophage activation states in the context of mycobacterial infection.

Summary: Increased glycolytic metabolism recently emerged as an essential process driving host defense against Mycobacterium tuberculosis (Mtb), but little is known about how this process is regulated during infection. Here, we observe repression of host glycolysis in Mtb-infected macrophages, which is dependent on sustained upregulation of anti-inflammatory microRNA-21 (miR-21) by proliferating mycobacteria. The dampening of glycolysis by miR-21 is mediated through targeting of phosphofructokinase muscle (PFK-M) isoform at the committed step of glycolysis, which facilitates bacterial growth by limiting pro-inflammatory mediators, chiefly interleukin-1β (IL-1β). Unlike other glycolytic genes, PFK-M expression and activity is repressed during Mtb infection through miR-21-mediated regulation, while other less-active isoenzymes dominate. Notably, interferon-γ (IFN-γ), which drives Mtb host defense, inhibits miR-21 expression, forcing an isoenzyme switch in the PFK complex, augmenting PFK-M expression and macrophage glycolysis. These findings place the targeting of PFK-M by miR-21 as a key node controlling macrophage immunometabolic function. : Hackett et al. identify a role for the anti-inflammatory miR-21 in limiting host glycolysis during tuberculosis (TB) infection to favor bacterial replication. This occurs by targeting a pro-glycolytic isoform at the rate-limiting step in glycolysis, PFK-M, a process antagonized by the host Th1-cytokine IFN-γ, to promote full macrophage activation and antimicrobial function. Keywords: macrophage, metabolic reprogramming, tuberculosis, mycobacterium tuberculosis, glycolysis, microRNA, miR-21, interleukin-1b, phosphofructokinase, interferon gamma

Despite being in the midst of a global pandemic of infections caused by the pathogen Chlamydia trachomatis, a vaccine capable of inducing protective immunity remains elusive. Given the C. trachomatis mucosal port of entry, a formulation compatible with mucosal administration and capable of eliciting potent genital tract immunity is highly desirable. While subunit vaccines are considered safer and better tolerated, these are typically poorly immunogenic and require co-formulation with immune-potentiating adjuvants. However, of the adjuvants licensed for use in humans, very few drive robust cellular responses, a pre-requisite for protection against C. trachomatis infection. Recently, the cationic adjuvant formulations (CAF) have been shown to induce robust humoral and cellular immunity in pre-clinical models of chlamydia, malaria and tuberculosis (TB). Here, we demonstrate that CAF01 induces potent immune responses when combined with the major outer membrane protein (MOMP) of C. trachomatis following parenteral immunisation and also as part of a heterologous prime/boost regime. We show that a subcutaneous prime with CAF01-adjuvanted recombinant MOMP licenses antigen-specific immunity at distant mucosal sites which can be activated following oral antigen re-encounter in the absence of concomitant adjuvant stimulation. Finally, we shed light on the mechanism(s) through which CAF01 elicits robust antigen-specific immunity to co-formulated MOMP via type I interferon (IFN) signalling.

Graphene-based materials are of increasing interest for their potential use in biomedical applications. However, there is a need to gain a deeper understanding of how graphene modulates biological responses before moving towards clinical application. Innate immune training is a recently described phenomenon whereby cells of the innate immune system are capable of being programmed to generate an increased non-specific response upon subsequent challenge. This has been well established in the case of certain microbes and microbial products. However, little is known about the capacity of particulate materials, such as pristine graphene (pGr), to promote innate immune training. Here we report for the first time that while stimulation with pGr alone does not directly induce cytokine secretion by bone-marrow derived macrophages (BMDMs), it programs them for enhanced secretion of proinflammatory cytokines (IL-6, TNF-α) and a concomitant decrease in production of the regulatory cytokine, IL-10 after Toll-like receptor (TLR) ligand stimulation. This capacity of pGr to program cells for enhanced inflammatory responses could be overcome if the nanomaterial is incorporated in a collagen matrix. Our findings thus demonstrate the potential of graphene to modulate innate immunity over long timescales and have implications for the design and biomedical use of pGr-based materials.

Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae (V. cholerae) that results in 3–4 million cases globally with 100,000–150,000 deaths reported annually. Mostly confined to developing nations, current strategies to control the spread of cholera include the provision of safe drinking water and improved sanitation and hygiene, ideally in conjunction with oral vaccination. However, difficulties associated with the costs and logistics of these strategies have hampered their widespread implementation. Specific challenges pertaining to oral cholera vaccines (OCVs) include a lack of safe and effective adjuvants to further enhance gut immune responses, the complex and costly multicomponent vaccine manufacturing, limitations of conventional liquid formulation and the lack of an integrated delivery platform. Herein we describe the use of the orally active adjuvant α-Galactosylceramide (α-GalCer) to strongly enhance intestinal bacterium- and toxin-specific IgA responses to the OCV, Dukoral® in C57BL/6 and BALB/c mice. We further demonstrate the mucosal immunogenicity of a novel multi-antigen, single-component whole-cell killed V. cholerae strain and the enhancement of its immunogenicity by adding α-GalCer. Finally, we report that combining these components and recombinant cholera toxin B subunit in the SmPill® minisphere delivery system induced strong intestinal and systemic antigen-specific antibody responses.

Objectives The cationic biopolymer chitosan (CH) has emerged as a promising candidate adjuvant due to its safety profile and immunostimulatory properties. The presence of endotoxin contamination in biomaterials is generally underappreciated and can generate misleading results. It is important to establish a convenient methodology to obtain large amounts of high quality chitosan nanoparticles for biomedical applications. Methods We developed an easy method to generate endotoxin-free chitosan and assessed its purity using the Limulus amebocyte lysate assay and by measuring dendritic cell activation. Key findings Purified chitosan-based formulations alone failed to induce production of the proinflammatory cytokines tumour necrosis factor alpha (TNF-α) and interleukin (IL)-6 in bone marrow-derived dendritic cells (BMDCs) generated from C57BL/6 mice, while maintaining its ability to promote IL-1β secretion in combination with the Toll-like receptor (TLR)-9 agonist, CpG. Moreover, BMDCs from C3H/HeN and TLR4-deficient mice, C3H/HeJ were stimulated with endotoxin-free chitosan-based formulations and no differences were observed in IL-6 and IL-1β secretion, excluding the involvement of TLR-4 in the immunomodulatory effects of chitosan. Conclusions The developed method provides simple guidelines for the production of endotoxin-free chitosan, ideal for biomedical applications.

The cytokine IL-33 is a well-established inducer of Th2 responses. However, roles for IL-33 in promoting CD8, Th1, and T regulatory cell responses have also emerged. In this study, the role of IL-33 as a regulator of particulate vaccine adjuvant-induced Ag-specific cellular immunity was investigated. We found that polymeric nanoparticles surpassed alum in their ability to enhance Ag-specific CD8 and Th1 responses. IL-33 was a potent negative regulator of both CD8+ T cell and Th1 responses following i.m. vaccination with Ag and nanoparticles, whereas the cytokine was required for the nanoparticle enhancement in Ag-specific IL-10. In contrast to the effect on cellular immunity, Ab responses were comparable between vaccinated wild-type and IL-33–deficient mice. IL-33 did not compromise alum-induced adaptive cellular immunity after i.m. vaccination. These data suggest that IL-33 attenuates the induction of cellular immune responses by nanoparticulate adjuvants and should be considered in the rational design of vaccines targeting enhanced CD8 and Th1 responses.

Type I interferons (IFN‐I) are the principal antiviral molecules of the innate immune system and can be made by most cell types, including central nervous system cells. IFN‐I has been implicated in neuroinflammation during neurodegeneration, but its mechanism of induction and its consequences remain unclear. In the current study, we assessed expression of IFN‐I in murine prion disease (ME7) and examined the contribution of the IFN‐I receptor IFNAR1 to disease progression. The data indicate a robust IFNβ response, specifically in microglia, with evidence of IFN‐dependent genes in both microglia and astrocytes. This IFN‐I response was absent in stimulator of interferon genes (STING−/−) mice. Microglia showed increased numbers and activated morphology independent of genotype, but transcriptional signatures indicated an IFNAR1‐dependent neuroinflammatory phenotype. Isolation of microglia and astrocytes demonstrated disease‐associated microglial induction of Tnfα, Tgfb1, and of phagolysosomal system transcripts including those for cathepsins, Cd68, C1qa, C3, and Trem2, which were diminished in IFNAR1 and STING deficient mice. Microglial increases in activated cathepsin D, and CD68 were significantly reduced in IFNAR1−/− mice, particularly in white matter, and increases in COX‐1 expression, and prostaglandin synthesis were significantly mitigated. Disease progressed more slowly in IFNAR1−/− mice, with diminished synaptic and neuronal loss and delayed onset of neurological signs and death but without effect on proteinase K‐resistant PrP levels. Therefore, STING‐dependent IFN‐I influences microglial phenotype and influences neurodegenerative progression despite occurring secondary to initial degenerative changes. These data expand our mechanistic understanding of IFN‐I induction and its impact on microglial function during chronic neurodegeneration.

It remains unresolved how retinal pigment epithelial cell metabolism is regulated following immune activation to maintain retinal homeostasis and retinal function. We exposed retinal pigment epithelium (RPE) to several stress signals, particularly Toll-like receptor stimulation, and uncovered an ability of RPE to adapt their metabolic preference on aerobic glycolysis or oxidative glucose metabolism in response to different immune stimuli. We have identified interleukin-33 (IL-33) as a key metabolic checkpoint that antagonizes the Warburg effect to ensure the functional stability of the RPE. The identification of IL-33 as a key regulator of mitochondrial metabolism suggests roles for the cytokine that go beyond its extracellular "alarmin" activities. IL-33 exerts control over mitochondrial respiration in RPE by facilitating oxidative pyruvate catabolism. We have also revealed that in the absence of IL-33, mitochondrial function declined and resultant bioenergetic switching was aligned with altered mitochondrial morphology. Our data not only shed new light on the molecular pathway of activation of mitochondrial respiration in RPE in response to immune stressors but also uncover a potentially novel role of nuclear intrinsic IL-33 as a metabolic checkpoint regulator.

Mitochondria are important regulators of macrophage polarisation. Here, we show that arginase-2 (Arg2) is a microRNA-155 (miR-155) and interleukin-10 (IL-10) regulated protein localized at the mitochondria in inflammatory macrophages, and is critical for IL-10-induced modulation of mitochondrial dynamics and oxidative respiration. Mechanistically, the catalytic activity and presence of Arg2 at the mitochondria is crucial for oxidative phosphorylation. We further show that Arg2 mediates this process by increasing the activity of complex II (succinate dehydrogenase). Moreover, Arg2 is essential for IL-10-mediated downregulation of the inflammatory mediators succinate, hypoxia inducible factor 1α (HIF-1α) and IL-1β in vitro. Accordingly, HIF-1α and IL-1β are highly expressed in an LPS-induced in vivo model of acute inflammation using Arg2−/− mice. These findings shed light on a new arm of IL-10-mediated metabolic regulation, working to resolve the inflammatory status of the cell., IL-10 can limit inflammation in part by inhibiting miR-155. Here the authors show how this axis induces mitochondrial arginase-2 to alter the mitochondrial dynamics and bioenergetics of macrophages and make these cells less pro-inflammatory.

Macrophages are key immune cells for combatting

Activated caspase-1 and caspase-11 induce inflammatory cell death in a process termed pyroptosis. Here we show that Prostaglandin E2 (PGE2) inhibits caspase-11-dependent pyroptosis in murine and human macrophages. PGE2 suppreses caspase-11 expression in murine and human macrophages and in the airways of mice with allergic inflammation. Remarkably, caspase-11-deficient mice are strongly resistant to developing experimental allergic airway inflammation, where PGE2 is known to be protective. Expression of caspase-11 is elevated in the lung of wild type mice with allergic airway inflammation. Blocking PGE2 production with indomethacin enhances, whereas the prostaglandin E1 analog misoprostol inhibits lung caspase-11 expression. Finally, alveolar macrophages from asthma patients exhibit increased expression of caspase-4, a human homologue of caspase-11. Our findings identify PGE2 as a negative regulator of caspase-11-driven pyroptosis and implicate caspase-4/11 as a critical contributor to allergic airway inflammation, with implications for pathophysiology of asthma., Caspase 11 activation involves transcriptional upregulation and proteolytic cleavage. Here the authors show that prostaglandin E2 prevents caspase-11-mediated pyroptosis, blocking caspase-11 mRNA and protein upregulation in macrophages and in vivo, and that mice lacking caspase-11 are strongly protected from allergic airway inflammation.

Oral vaccines present an attractive alternative to injectable vaccines for enteric diseases due to ease of delivery and the induction of intestinal immunity at the site of infection. However, susceptibility to gastrointestinal proteolysis, limited transepithelial uptake and a lack of clinically acceptable adjuvants present significant challenges. A further challenge to mass vaccination in developing countries is the very expensive requirement to maintain the cold chain. We recently described the effectiveness of a Single Multiple Pill® (SmPill®) adjuvanted capsule approach to enhance the effectiveness of a candidate enterotoxigenic Escherichia coli (ETEC) oral vaccine. Here it was demonstrated that this delivery system maintains the antigenicity of ETEC colonisation factor antigen I (CFA/I) and the immunostimulatory activity of the orally active α-Galactosylceramide (α-GalCer) adjuvant after storage of SmPill® minispheres under room temperature and extreme storage conditions for several months. In addition, the internal structure of the cores of SmPill® minispheres and antigen release features at intestinal pH were found to be preserved under all these conditions. However, changes in the surface morphology of SmPill® minispheres leading to the antigen release at gastric pH were observed after a few weeks of storage under extreme conditions. Those modifications were prevented by the introduction of an Opadry® White film coating layer between the core of SmPill® minispheres and the enteric coating. Under these conditions, protection against antigen release at gastric pH was maintained even under high temperature and humidity conditions. These results support the potential of the SmPill® minisphere approach to maintain the stability of an adjuvanted whole cell killed oral vaccine formulation.

The extent of regeneration following biomaterial implantation is dependent on the microenvironment surrounding the implant. Since implant composition can have a profound effect on inflammation, it is essential to understand this process as a non-resolving inflammatory response can lead to fibrous encapsulation and insufficient integration. Incorporation of particulates into implants confers structural and functional benefits, thus optimizing particulate characteristics to enhance immune mediated efficacy is important. We investigated the relationship between the nature of hydroxyapatite (HA) particles and the innate immune response, focusing on how particle size (0.1 µm, 5 µm, 20 µm, 100 µm) and morphology (needle-shaped/spherical; smooth/rough surface) modulates inflammatory responses. We observed a shape and size-dependent activation of the NLRP3 inflammasome and IL-1β secretion; while needle-shaped and smaller HA particles significantly enhanced cytokine secretion, larger particles did not. Moreover, HA particle characteristics profoundly influenced patterns of innate immune cell recruitment and cytokine production following injection. While small, needle-shaped particles induced a strong inflammatory response, this was not observed with smooth, spherical particles of comparable size or with larger particles. These findings indicate that hydroxyapatite particle characteristics dictate immune cell recruitment and the ensuing inflammatory response, providing an opportunity to tailor HA particle characteristics to regulate immune responses induced after biomaterial implantation.

Mollusk hemocyanins have biomedical uses as carriers/adjuvants and nonspecific immunostimulants with beneficial clinical outcomes by triggering the production of proinflammatory cytokines in antigen-presenting cells (APCs) and driving immune responses toward type 1 T helper (Th1) polarization. Significant structural features of hemocyanins as a model antigen are their glycosylation patterns. Indeed, hemocyanins have a multivalent nature as highly mannosylated antigens. We have previously shown that hemocyanins are internalized by APCs through receptor-mediated endocytosis with proteins that contain C-type lectin domains, such as mannose receptor (MR). However, the contribution of other innate immune receptors to the proinflammatory signaling pathway triggered by hemocyanins is unknown. Thus, we studied the roles of Dectin-1, Dectin-2, and Toll-like receptor 4 (TLR4) in the hemocyanin activation of murine APCs, both in dendritic cells (DCs) and macrophages, using hemocyanins from Megathura crenulata (KLH), Concholepas concholepas (CCH) and Fissurella latimarginata (FLH). The results showed that these hemocyanins bound to chimeric Dectin-1 and Dectin-2 receptors in vitro; which significantly decreased when the glycoproteins were deglycosylated. However, hemocyanin-induced proinflammatory effects in APCs from Dectin-1 knock-out (KO) and Dectin-2 KO mice were independent of both receptors. Moreover, when wild-type APCs were cultured in the presence of hemocyanins, phosphorylation of Syk kinase was not detected. We further showed that KLH and FLH induced ERK1/2 phosphorylation, a key event involved in the TLR signaling pathway. We confirmed a glycan-dependent binding of hemocyanins to chimeric TLR4 in vitro. Moreover, DCs from mice deficient for MyD88-adapter-like (Mal), a downstream adapter molecule of TLR4, were partially activated by FLH, suggesting a role of the TLR pathway in hemocyanin recognition to activate APCs. The participation of TLR4 was confirmed through a decrease in IL-12p40 and IL-6 secretion induced by FLH when a TLR4 blocking antibody was used; a reduction was also observed in DCs from C3H/HeJ mice, a mouse strain with a nonfunctional mutation for this receptor. Moreover, IL-6 secretion induced by FLH was abolished in macrophages deficient for TLR4. Our data showed the involvement of TLR4 in the hemocyanin-mediated proinflammatory response in APCs, which could cooperate with MR in innate immune recognition of these glycoproteins.

The cationic polysaccharide chitosan is an attractive candidate adjuvant capable of driving potent cell-mediated immunity, but the mechanism by which it acts is not clear. We show that chitosan promotes dendritic cell maturation by inducing type I interferons (IFNs) and enhances antigen-specific T helper 1 (Th1) responses in a type I IFN receptor-dependent manner. The induction of type I IFNs, IFN-stimulated genes and dendritic cell maturation by chitosan required the cytoplasmic DNA sensor cGAS and STING, implicating this pathway in dendritic cell activation. Additionally, this process was dependent on mitochondrial reactive oxygen species and the presence of cytoplasmic DNA. Chitosan-mediated enhancement of antigen specific Th1 and immunoglobulin G2c responses following vaccination was dependent on both cGAS and STING. These findings demonstrate that a cationic polymer can engage the STING-cGAS pathway to trigger innate and adaptive immune responses.

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier superfamily that can mediate the transfer of protons into the mitochondrial matrix from the intermembrane space. We have previously reported UCP3 expression in thymocytes, mitochondria of total splenocytes and splenic lymphocytes. Here, we demonstrate that Ucp3 is expressed in peripheral naive CD4+ T cells at the mRNA level before being markedly downregulated following activation. Non-polarized, activated T cells (Th0 cells) from Ucp3-/- mice produced significantly more IL-2, had increased expression of CD25 and CD69 and were more proliferative than Ucp3+/+ Th0 cells. The altered IL-2 expression observed between T cells from Ucp3+/+ and Ucp3-/- mice may be a factor in determining differentiation into Th17 or induced regulatory (iTreg) cells. When compared to Ucp3+/+, CD4+ T cells from Ucp3-/- mice had increased FoxP3 expression under iTreg conditions. Conversely, Ucp3-/- CD4+ T cells produced a significantly lower concentration of IL-17A under Th17 cell-inducing conditions in vitro. These effects were mirrored in antigen-specific T cells from mice immunized with KLH and CT. Interestingly, the altered responses of Ucp3-/- T cells were partially reversed upon neutralisation of IL-2. Together, these data indicate that UCP3 acts to restrict the activation of naive T cells, acting as a rheostat to dampen signals following TCR and CD28 co-receptor ligation, thereby limiting early activation responses. The observation that Ucp3 ablation alters the Th17:Treg cell balance in vivo as well as in vitro suggests that UCP3 is a potential target for the treatment of Th17 cell-mediated autoimmune diseases.

Despite its long record of successful use in human vaccines, the mechanisms underlying the immunomodulatory effects of alum are not fully understood. Alum is a potent inducer of interleukin-1 (IL-1) secretion in vitro in dendritic cells and macrophages via Nucleotide-binding domain and leucine-rich repeat-containing (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome activation. However, the contribution of IL-1 to alum-induced innate and adaptive immune responses is controversial and the role of IL-1α following alum injection has not been addressed. This study shows that IL-1 is dispensable for alum-induced antibody and CD8 T cell responses to ovalbumin. However, IL-1 is essential for neutrophil infiltration into the injection site, while recruitment of inflammatory monocytes and eosinophils is IL-1 independent. Both IL-1α and IL-1β are released at the site of injection and contribute to the neutrophil response. Surprisingly, these effects are NLRP3-inflammasome independent as is the infiltration of other cell populations. However, while NLRP3 and caspase 1 were dispensable, alum-induced IL-1β at the injection site was dependent on the cysteine protease cathepsin S. Overall, these data demonstrate a previously unreported role for cathepsin S in IL-1β secretion, show that inflammasome formation is dispensable for alum-induced innate immunity and reveal that IL-1α and IL-1β are both necessary for alum-induced neutrophil influx in vivo.

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.

Obesity is characterized by chronic inflammation associated with neutrophil and M1 macrophage infiltration into white adipose tissue. However, the mechanisms underlying this process remain largely unknown. Based on the ability of oil-based adjuvants to induce immune responses, we hypothesized that endogenous oils derived from necrotic adipocytes may function as an immunological “danger signal.” Here we show that endogenous oils of human origin are potent adjuvants, enhancing antibody responses to a level comparable to Freund’s incomplete adjuvant. The endogenous oils were capable of promoting interleukin (IL)-1α–dependent recruitment of neutrophils and M1-like macrophages, while simultaneously diminishing M2-like macrophages. We found that endogenous oils from subcutaneous and omental adipocytes, and from healthy and unhealthy obese individuals, promoted comparable inflammatory responses. Furthermore, we also confirmed that white adipocytes in visceral fat of metabolically unhealthy obese (MUO) individuals are significantly larger than those in metabolically healthy obese individuals. Since adipocyte size is positively correlated with adipocyte death, we propose that endogenous oils have a higher propensity to be released from hypertrophied visceral fat in MUO individuals and that this is the key factor in driving inflammation. In summary, this study shows that adipocytes contain a potent oil adjuvant which drives IL-1α–dependent proinflammatory responses in vivo.

Assembly of inflammasomes after infection or injury leads to the release of interleukin-1β (IL-1β) and to pyroptosis. After inflammasome activation, cells either pyroptose or enter a hyperactivated state defined by IL-1β secretion without cell death, but what controls these different outcomes is unknown. Here, we show that removal of the Toll-IL-1R protein SARM from macrophages uncouples inflammasome-dependent cytokine release and pyroptosis, whereby cells displayed increased IL-1β production but reduced pyroptosis. Correspondingly, increasing SARM in cells caused less IL-1β release and more pyroptosis. SARM suppressed IL-1β by directly restraining the NLRP3 inflammasome and, hence, caspase-1 activation. Consistent with a role for SARM in pyroptosis, Sarm1-/- mice were protected from lipopolysaccharide (LPS)-stimulated sepsis. Pyroptosis-inducing, but not hyperactivating, NLRP3 stimulants caused SARM-dependent mitochondrial depolarization. Thus, SARM-dependent mitochondrial depolarization distinguishes NLRP3 activators that cause pyroptosis from those that do not, and SARM modulation represents a cell-intrinsic mechanism to regulate cell fate after inflammasome activation.

Lung DCs induce the expression of gut-homing molecules on T cells, resulting in their migration to the GI tract and protection against Salmonella infection after immunization, Developing efficacious vaccines against enteric diseases is a global challenge that requires a better understanding of cellular recruitment dynamics at the mucosal surfaces. The current paradigm of T cell homing to the gastrointestinal (GI) tract involves the induction of α4β7 and CCR9 by Peyer’s patch and mesenteric lymph node (MLN) dendritic cells (DCs) in a retinoic acid–dependent manner. This paradigm, however, cannot be reconciled with reports of GI T cell responses after intranasal (i.n.) delivery of antigens that do not directly target the GI lymphoid tissue. To explore alternative pathways of cellular migration, we have investigated the ability of DCs from mucosal and nonmucosal tissues to recruit lymphocytes to the GI tract. Unexpectedly, we found that lung DCs, like CD103+ MLN DCs, up-regulate the gut-homing integrin α4β7 in vitro and in vivo, and induce T cell migration to the GI tract in vivo. Consistent with a role for this pathway in generating mucosal immune responses, lung DC targeting by i.n. immunization induced protective immunity against enteric challenge with a highly pathogenic strain of Salmonella. The present report demonstrates novel functional evidence of mucosal cross talk mediated by DCs, which has the potential to inform the design of novel vaccines against mucosal pathogens.

Autophagy controls IL-1β secretion by regulating inflammasome activation and by targeting pro–IL-1β for degradation. In this article, we show that inhibition of autophagy, either with the PI3K inhibitors 3-methyladenine, wortmannin, and LY294002 or with small interfering RNA against autophagy proteins augmented the secretion of IL-23 by human and mouse macrophages and dendritic cells in response to specific TLR agonists. This process occurred at the transcriptional level and was dependent on reactive oxygen species and IL-1R signaling; it was abrogated with an IL-1R antagonist or with IL-1–neutralizing Abs, whereas treatment with either rIL-1α or IL-1β induced IL-23 secretion. Dendritic cells treated with LPS and 3-methyladenine secreted enhanced levels of both IL-1β and IL-23, and supernatants from these cells stimulated the innate secretion of IL-17, IFN-γ, and IL-22 by γδ T cells. These data demonstrate that autophagy has a potentially pivotal role to play in the induction and regulation of inflammatory responses by innate immune cells, largely driven by IL-1 and its consequential effects on IL-23 secretion.

The mucosal surfaces of the gastrointestinal tract are continually exposed to an enormous antigenic load of microbial and dietary origin, yet homeostasis is maintained. Pattern recognition molecules (PRMs) have a key role in maintaining the integrity of the epithelial barrier and in promoting maturation of the mucosal immune system. Commensal bacteria modulate the expression of a broad range of genes involved in maintaining epithelial integrity, inflammatory responses, and production of antimicrobial peptides. Mice deficient in PRMs can develop intestinal inflammation, which is dependent on the microbiota, and in humans, PRM polymorphisms are associated with exacerbated inflammatory bowel disease. Innate immune responses and epithelial barrier function are regulated by PRM-induced signaling at multiple levels, from the selective expression of receptors on mucosal cells or compartments to the expression of negative regulators. Here, we describe recent advances in our understanding of innate signaling pathways, particularly by Toll-like receptors and nucleotide-binding domain and leucine-rich repeat containing receptors at mucosal surfaces.

PUBLISHED, PMID: 19570828, IL-17 producing CD4+ T (Th17) cells are pathogenic in many autoimmune diseases. The induction and expansion of Th17 cells is directed by cytokines, including IL-23 and IL-1?, produced by innate immune cells through activation of pathogen recognition receptors (PRRs). The NF?B and interferon regulatory factor (IRF) families of transcriptional factors mediate IL-12 production; however, distinct signalling pathways appear to be required for IL- 23 production. Here we show that inhibition of ERK MAP kinase suppressed IL-23 and IL-1? production by dendritic cells (DC) stimulated with TLR or dectin-1 agonists, but did not affect IL-12p70 production. Furthermore, an ERK inhibitor suppressed the ability of antigen-pulsed TLR-activated DC to induce antigen-specific Th17 cells in vivo, but interestingly also inhibited the induction of Th1 cells. Treatment with an ERK inhibitor attenuated experimental autoimmune encephalomyelitis (EAE), when administered either at the induction phase of acute EAE or during remission in the relapsing-remitting EAE model. This was associated with significant suppression of autoantigen-specific Th17 and Th1 responses. The suppressive effect of the ERK inhibitor on attenuation of EAE was reversed by administration of IL-1? and IL-23. Our findings suggest that ERK MAP kinase plays a critical and hitherto undescribed role in activating innate production of IL-23 and IL-1?, which promote pathogenic T cell responses, and therefore represents an important target for therapeutic intervention against autoimmune diseases., This work was supported by Science Foundation Ireland.

Th17 cells, CD4(+) T cells that secrete interleukin-17 (IL-17), are pathogenic in autoimmune diseases and their development and expansion is driven by the cytokines IL-6, TGF-beta, IL-21, IL-1, and IL-23. However, there are also innate sources of IL-17. Here, we show that gammadelta T cells express IL-23R and the transcription factor RORgammat and produce IL-17, IL-21, and IL-22 in response to IL-1beta and IL-23, without T cell receptor engagement. IL-17-producing gammadelta T cells were found at high frequency in the brain of mice with experimental autoimmune encephalomyelitis (EAE). gammadelta T cells activated by IL-1beta and IL-23 promoted IL-17 production by CD4(+) T cells and increased susceptibility to EAE, suggesting that gammadelta T cells act in an amplification loop for IL-17 production by Th17 cells. Our findings demonstrate that gammadelta T cells activated by IL-1beta and IL-23 are an important source of innate IL-17 and IL-21 and provide an alternative mechanism whereby IL-1 and IL-23 may mediate autoimmune inflammation.

PUBLISHED, It was recently demonstrated that interleukin (IL)-23?driven IL-17?producing (ThIL-17) T cells mediate inflammatory pathology in certain autoimmune diseases. We show that the induction of antigen-specific ThIL-17 cells, but not T helper (Th)1 or Th2 cells, by immunization with antigens and adjuvants is abrogated in IL-1 receptor type I?deficient (IL-1RI?/?) mice. Furthermore, the incidence of experimental autoimmune encephalomyelitis (EAE) was significantly lower in IL-1RI?/? compared with wild-type mice, and this correlated with a failure to induce autoantigen-specific ThIL-17 cells, whereas induction of Th1 and Th2 responses was not substantially different. However, EAE was induced in IL-1RI?/? mice by adoptive transfer of autoantigen-specific cells from wild-type mice with EAE. IL-23 alone did not induce IL-17 production by T cells from IL-1RI?/? mice, and IL-23?induced IL-17 production was substantially enhanced by IL-1? or IL-1?, even in the absence of T cell receptor stimulation. We demonstrate essential roles for phosphatidylinositol 3-kinase, nuclear factor ?B, and novel protein kinase C isoforms in IL-1? and IL-23?mediated IL-17 production. Tumor necrosis factor ? also synergized with IL-23 to enhance IL-17 production, and this was IL-1 dependent. Our findings demonstrate that IL-1 functions upstream of IL-17 to promote pathogenic ThIL-17 cells in EAE., This work was supported by a Science Foundation Ireland Principal Investigator Award (no. 00/PI.I/B045 to K.H.G. Mills)

Intranasal delivery of vaccines provides an attractive alternative to parenteral delivery, but it requires appropriate mucosal adjuvants. Cholera toxin (CT) is a powerful mucosal adjuvant, but it can undergo retrograde transport to the brain via the olfactory system after intranasal delivery. We demonstrate that intranasal delivery of CT increases the expression of interleukin-1 beta , cyclooxygenase-2, and chemokine messenger RNA in the murine hypothalamus, whereas parenterally delivered CT has little effect. Our findings suggest that CT can induce proinflammatory mediators in the brain when it is administered intranasally but not parenterally, and they raise concerns about the use of AB toxins as adjuvants in intranasal vaccines.

PUBLISHED, Adenylate cyclase toxin (CyaA) from Bordetella pertussis can subvert host immune responses allowing bacterial colonization. Here we have examined its adjuvant and immunomodulatory properties and the possible contribution of lipopolysaccharide (LPS), known to be present in purified CyaA preparations. CyaA enhanced antigen-specific interleukin-5 (IL-5) and IL-10 production and immunoglobulin G1 antibodies to coadministered antigen in vivo. Antigen-specific CD4+-T-cell clones generated from mice immunized with antigen and CyaA had cytokine profiles characteristic of Th2 or type 1 regulatory T (Tr1) cells. Since innate immune cells direct the induction of T-cell subtypes, we examined the influence of CyaA on activation of dendritic cells (DC) and macrophages. CyaA significantly augmented LPS-induced IL-6 and IL-10 and inhibited LPS-driven tumor necrosis factor alpha and IL-12p70 production from bone marrow-derived DC and macrophages. CyaA also enhanced cell surface expression of CD80, CD86, and major histocompatibility class II on immature DC. The stimulatory activity of our CyaA preparation for IL-10 production and CD80, CD86, and major histocompatibility complex class II expression was attenuated following the addition of polymyxin B or with the use of DC from Toll-like receptor (TLR) 4-defective mice. However, treatment of DC with LPS alone at the concentration present in the CyaA preparation (0.2 ng/ml) failed to activate DC in vitro. Our findings demonstrate that activation of innate cells in vitro by CyaA is dependent on a second signal through a TLR and that CyaA can promote Th2/Tr1-cell responses by inhibiting IL-12 and promoting IL-10 production by DC and macrophages., This material is based upon works supported by the Science Foundation Ireland under grant 00/PI.I/B045. Padraig J. Ross also receives support from Enterprise Ireland.

It has previously been reported that cholera toxin (CT) is a potent mucosal adjuvant that enhances Th2 or mixed Th1/Th2 type responses to coadministered foreign Ag. Here we demonstrate that CT also promotes the generation of regulatory T (Tr) cells against bystander Ag. Parenteral immunization of mice with Ag in the presence of CT induced T cells that secreted high levels of IL-4 and IL-10 and lower levels of IL-5 and IFN-γ. Ag-specific CD4+ T cell lines and clones generated from these mice had cytokine profiles characteristic of Th2 or type 1 Tr cells, and these T cells suppressed IFN-γ production by Th1 cells. Furthermore, adoptive transfer of bone marrow-derived dendritic cells (DC) incubated with Ag and CT induced T cells that secreted IL-4 and IL-10 and low concentrations of IL-5. It has previously been shown that IL-10 promotes the differentiation or expansion of type 1 Tr cells. Here we found that CT synergized with low doses of LPS to induce IL-10 production by immature DC. CT also enhanced the expression of CD80, CD86, and OX40 (CD134) on DC and induced the secretion of the chemokine, macrophage inflammatory protein-2 (MIP-2), but inhibited LPS-driven induction of CD40 and ICAM-I expression and production of the inflammatory cytokines/chemokines IL-12, TNF-α, MIP-1α, MIP-1β, and monocyte chemoattractant protein-1. Our findings suggest that CT induces maturation of DC, but, by inducing IL-10, inhibiting IL-12, and selectively affecting surface marker expression, suppresses the generation of Th1 cells and promotes the induction of T cells with regulatory activity.

Age-related macular degeneration (AMD) is the most common form of central retinal blindness globally. Distinct processes of the innate immune system, specifically activation of the NLRP3 inflammasome, have been shown to play a central role in the development of both "dry" and neovascular ("wet") forms of the disease. We show that the inflammatory cytokine interleukin-18 (IL-18) can regulate choroidal neovascularization formation in mice. We observed that exogenous administration of mature recombinant IL-18 has no effect on retinal pigment epithelial (RPE) cell viability, but that overexpression of pro-IL-18 or pro-IL-1β alone can cause RPE cell swelling and subsequent atrophy, a process that can be inhibited by the promotion of autophagy. A direct comparison of local and systemic administration of mature recombinant IL-18 with current anti-VEGF (vascular endothelial growth factor)-based therapeutic strategies shows that IL-18 treatment works effectively alone and more effectively in combination with anti-VEGF therapy and represents a novel therapeutic strategy for the treatment of wet AMD.

Porous silicon (pSi) microparticles, in diverse sizes and shapes, can be functionalized to present pathogen-associated molecular patterns that activate dendritic cells. Intraperitoneal injection of MPL-adsorbed pSi microparticles, in contrast to free MPL, resulted in the induction of local inflammation, reflected in the recruitment of neutrophils, eosinophils and proinflammatory monocytes, and the depletion of resident macrophages and mast cells at the injection site. Injection of microparticle-bound MPL resulted in enhanced secretion of the T helper 1 associated cytokines IFN-γ and TNF-α by peritoneal exudate and lymph node cells in response to secondary stimuli while decreasing the anti-inflammatory cytokine IL-10. MPL-pSi microparticles independently exhibited anti-tumor effects and enhanced tumor suppression by low dose doxorubicin nanoliposomes. Intravascular injection of the MPL-bound microparticles increased serum IL-1β levels, which was blocked by the IL-1 receptor antagonist Anakinra. The microparticles also potentiated tumor infiltration by dendritic cells, cytotoxic T lymphocytes, and F4/80+ macrophages, however, a specific reduction was observed in CD204+ macrophages.

Hemocyanins, the huge oxygen-transporting glycoproteins of some mollusks, are used as immunomodulatory proteins with proven anti-cancer properties. The biodiversity of hemocyanins has promoted interest in identifying new anti-cancer candidates with improved immunological properties. Hemocyanins promote Th1 responses without known side effects, which make them ideal for long-term sustained treatment of cancer. In this study, we evaluated a novel hemocyanin from the limpet/gastropod Fissurella latimarginata (FLH). This protein has the typical hollow, cylindrical structure of other known hemocyanins, such as the keyhole limpet hemocyanin (KLH) and the Concholepas hemocyanin (CCH). FLH, like the KLH isoforms, is composed of a single type of polypeptide with exposed N- and O-linked oligosaccharides. However, its immunogenicity was significantly greater than that of KLH and CCH, as FLH induced a stronger humoral immune response and had more potent anti-tumor activity, delaying tumor growth and increasing the survival of mice challenged with B16F10 melanoma cells, in prophylactic and therapeutic settings. Additionally, FLH-treated mice demonstrated increased IFN-γ production and higher numbers of tumor-infiltrating CD4(+) lymphocytes. Furthermore, in vitro assays demonstrated that FLH, but not CCH or KLH, stimulated the rapid production of pro-inflammatory cytokines (IL-6, IL-12, IL-23 and TNF-α) by dendritic cells, triggering a pro-inflammatory milieu that may explain its enhanced immunological activity. Moreover, this effect was abolished when deglycosylated FLH was used, suggesting that carbohydrates play a crucial role in the innate immune recognition of this protein. Altogether, our data demonstrate that FLH possesses increased anti-tumor activity in part because it activates a more potent innate immune response in comparison to other known hemocyanins. In conclusion, FLH is a potential new marine adjuvant for immunization and possible cancer immunotherapy.

In recent years microspheres or microparticles produced from biodegradable polymers such as poly( d,l -lactide) (PLA) and poly( d,l -lactide-co-glycolide) (PLGA) containing encapsulated vaccine antigens have been investigated for administration via parenteral, oral, and intranasal routes. These microparticles allow the controlled release of vaccines with an aim to reduce the number of doses for primary immunisation or to develop single dose vaccines. The polymer materials have been widely regarded as being of minimal toxicity. Evaluation of candidate systems in animal studies have shown antibody levels and cell responses similar to or greater than those observed with adjuvants such as alum. However, there are concerns regarding the integrity and immunogenicity of the antigen during the encapsulation process when the antigen is exposed to organic solvents, high shear stresses and the exposure of antigen to low pH which is caused by polymer degradation. An alternative approach would be to adsorb antigens to the surface of biodegradable polymer particles. Polymeric lamellar substrate particles (PLSP), produced by a simple precipitation of PLA, are suitable for this purpose. The adsorption of antigens onto these particles is a simple procedure. It avoids pH changes due to bulk polymer degradation and the use of solvents and therefore will be less damaging to the vaccine. Moreover, such systems will be much easier to scale up for a clinical study and eventual manufacture. The aim of this article is to discuss the preparation and physical characteristics of PLSP, antigen adsorption, in vivo efficacy of PLSP antigen systems and to consider the potential of PLSP as controlled release adjuvants for protein, peptide or viral vaccines.

Novel poly(dl-lactide-co-glycolide) microparticles for oral vaccine delivery were formulated using the enteric polymers Eudragit L100-55 and carboxymethylethylcellulose (CMEC) as stabilisers. To serve as a control, microparticles were also produced using the conventional PVA surfactant. In all three cases the antigen, ovalbumin (OVA)-loaded microparticles produced were less than 5 microm in diameter and had a spherical, smooth rounded appearance. The presence of surfactants at the microparticle surface was demonstrated by the surface analysis techniques, XPS and SSIMS. Incubation of microparticles with solutions of pepsin or trypsin led to the removal of a proportion of the antigen associated with all three systems. However, in three CMEC-stabilised microparticle formulations and one of three Eudragit formulations, a high percentage of the associated antigen was protected from removal by a solution of pepsin at pH 1.2 compared with the PVA-stabilised microparticles. In addition, with certain CMEC and Eudragit formulations a degree of protection was also afforded to the associated OVA against removal by trypsin at pH 7.4. Following the incubation of microparticles in simulated gastric fluid a higher percentage of intact antigenic OVA was detected in microparticles stabilised using CMEC than in the PVA- and Eudragit- stabilised formulations. Oral immunisation of mice with OVA-loaded microparticles stabilised using either of the three surfactants led to the induction of specific serum IgG and salivary IgA antibodies. Significantly higher levels of specific salivary IgA antibody to OVA were measured in mice immunised with the CMEC-stabilised microparticles than with the other two formulations. This novel approach in PLG microparticle formulation may have potential in increasing the efficacy of microparticulate systems for the oral administration of vaccines.

Protein-loaded microparticles were produced from blends of poly(ethylene glycol) (PEG) with poly(L-lactide) (PLA) homopolymer or poly(DL-lactide co-glycolide) copolymers (PLG) using a water-in oil-in oil method. The stability of ovalbumin (OVA) associated with microparticles prepared using PEG and 50:50 PLG, 75:25 PLG and PLA, respectively, was analysed by SDS-PAGE and quantified by scanning densitometry following incubation in PBS at 37 degrees C for up to 1 month. Fragmentation and aggregation of OVA was detected with all 3 formulations. The extent of both processes correlated with the degradation rate of the lactide polymer used and decreased in the order PLA < 75:25 PLG < 50:50 PLG. Extensive degradation of the PLG/PEG microparticles also occurred over 4 weeks whereas the use of PLA/PEG blends resulted in a stable microparticle morphology and much reduced fragmentation and aggregation of the associated protein. Following a single sub-cutaneous immunisation, high levels of specific serum IgG antibody were elicited by OVA associated with the PLA/PEG particles. Injection of OVA associated with the 75:25 PLG/PEG microparticles resulted in very low levels of specific antibody. A higher response was induced by the 50:50 PLG/PEG formulation but there was very large inter-animal variation in this group. Antibody levels elicited by all 3 formulations were significantly higher than those elicited by a single injection of soluble OVA. Analysis of antigen specific IgG1 and IgG2a antibody subtype levels also revealed the greater efficacy of the PLA/PEG microparticles as an adjuvant system. The use of PLA/PEG microparticles shows improved protein loading and delivery capacity while maintaining a high level of stability of the associated protein. These results indicate a strong correlation between the stability of microencapsulated antigen and the magnitude of the immune response following sub-cutaneous immunisation.

The effect of different surfactants on the surface characteristics of poly(d,l-lactide-co-glycolide) microparticles prepared by the emulsification/solvent evaporation technique was investigated and the immune response to a protein antigen (OVA) associated with these microparticles was measured. Three surfactants - polyvinyl alcohol (PVA, a conventional stabiliser of PLG microparticles), the non-ionic surfactant, poly(oxyethylene glycerol mono-oleate) [Tagat] and Bile salts (a natural emulsifier) - were used to produce OVA-loaded PLG microparticles. Antigen was detected at the surface of all three types of OVA-loaded microparticles, in amounts in excess of 40% of the total protein load. The levels of specific serum IgG antibody elicited to OVA were significantly higher (P < 0.05) after a single subcutaneous administration of antigen associated with the Bile salts and Tagat formulations compared to the PVA formulation. A strong correlation was revealed between the levels of antibody measured and the magnitude of negative surface charge of the particulate carrier. The pattern of the IgG antibody response to OVA was similar in all three cases, indicating that the degradation rate of the PLG polymer determined the duration of the response. The results demonstrate the potential of using different surfactants to produce PLG microparticles with increased adjuvant activity.

Despite showing promise in preclinical models, anti-Staphylococcus aureus vaccines have failed in clinical trials. To date, approaches have focused on neutralizing/opsonizing antibodies; however, vaccines exclusively inducing cellular immunity have not been studied to formally test whether a cellular-only response can protect against infection. We demonstrate that nasal vaccination with targeted nanoparticles loaded with Staphylococcus aureus antigen protects against acute systemic S. aureus infection in the absence of any antigen-specific antibodies. These findings can help inform future developments in staphylococcal vaccine development and studies into the requirements for protective immunity against S. aureus.

The ability of particulate materials to provoke inflammatory immune responses has been well documented. In the case of endogenous and environmental particulates, these effects can often lead to pathological disorders. In contrast, particulate adjuvants incorporated into vaccines promote immune responses, which in turn provide efficient protection against infectious diseases. In recent years, studies have revealed that the NLRP3 inflammasome plays a key role in particulate-driven inflammation and its associated cytotoxicity. Hence, this chapter covers protocols useful to (1) assess NLRP3 inflammasome activation triggered by particulate adjuvants or materials in mouse bone marrow-derived dendritic cell (BMDCs) differentiated cultures, and (2) measure particle-induced cytotoxicity. More specifically, protocols are described for the preparation and differentiation of BMDCs, their priming and stimulation using particulate NLRP3 agonists such as monosodium urate monohydrate (MSU) and the vaccine adjuvant alum. We then detail protocols to assess particulate-driven cytotoxicity via flow cytometry using annexin V-propidium iodide (PI) and novel dye LIVE/DEAD(®) aqua stain. General considerations are provided that warn against the use of endotoxin-contaminated particles and emphasize the use of experimental controls. Suggestions are also outlined for further assessment of the immunomodulatory effects of particulate materials in vivo using the mouse peritonitis model.

The inflammatory effects of carbon nanoparticles (NPs) are highly disputed. Here it is demonstrated that endotoxin-free preparations of raw carbon nanotubes (CNTs) are very limited in their capacity to promote inflammatory responses in vitro, as well as in vivo. Upon purification and selective oxidation of raw CNTs, a higher dispersibility is achieved in physiological solutions, but this process also enhances their inflammatory activity. In synergy with toll-like receptor (TLR) ligands, CNTs promote NLRP3 inflammasome activation and it is shown for the first time that this property extends to spherical carbon nano-onions (CNOs) of 6 nm in size. In contrast, the benzoic acid functionalization of purified CNTs and CNOs leads to significantly attenuated inflammatory properties. This is evidenced by a reduced secretion of the inflammatory cytokine IL-1β, and a pronounced decrease in the recruitment of neutrophils and monocytes following injection into mice. Collectively, these results reveal that the inflammatory properties of carbon NPs are highly dependent on their physicochemical characteristics and crucially, that chemical surface functionalization allows significant moderation of these properties.

Inhibitor of apoptosis proteins (IAPs) play a major role in determining whether cells undergo apoptosis in response to TNF as well as other stimuli. However, TNF is also highly proinflammatory through its ability to trigger the secretion of multiple inflammatory cytokines and chemokines, which is arguably the most important role of TNF in vivo. Indeed, deregulated production of TNF-induced cytokines is a major driver of inflammation in several autoimmune conditions such as rheumatoid arthritis. Here, we show that IAPs are required for the production of multiple TNF-induced proinflammatory mediators. Ablation or antagonism of IAPs potently suppressed TNF- or RIPK1-induced proinflammatory cytokine and chemokine production. Surprisingly, IAP antagonism also led to spontaneous production of chemokines, particularly RANTES, in vitro and in vivo. Thus, IAPs play a major role in influencing the production of multiple inflammatory mediators, arguing that these proteins are important regulators of inflammation in addition to apoptosis. Furthermore, small molecule IAP antagonists can modulate spontaneous as well as TNF-induced inflammatory responses, which may have implications for use of these agents in therapeutic settings. Background: IAP antagonists sensitize toward apoptosis induced by TNF and other TNFR family ligands. Results: IAP antagonism exerted effects on spontaneous as well as TNF-induced cytokine and chemokine production. Conclusion: IAPs regulate spontaneous as well as TNF-induced cytokine/chemokine production. Significance: IAP antagonists modulate cytokine production as well as apoptosis, which could influence their utility as adjuncts to chemotherapy.

Apoptosis is commonly thought to represent an immunologically silent or even anti-inflammatory mode of cell death, resulting in cell clearance in the absence of explicit activation of the immune system. However, here we show that Fas/CD95-induced apoptosis is associated with the production of an array of cytokines and chemokines, including IL-6, IL-8, CXCL1, MCP-1, and GMCSF. Fas-induced production of MCP-1 and IL-8 promoted chemotaxis of phagocytes toward apoptotic cells, suggesting that these factors serve as “find-me” signals in this context. We also show that RIPK1 and IAPs are required for optimal production of cytokines and chemokines in response to Fas receptor stimulation. Consequently, a synthetic IAP antagonist potently suppressed Fas-dependent expression of multiple proinflammatory mediators and inhibited Fas-induced chemotaxis. Thus, in addition to provoking apoptosis, Fas receptor stimulation can trigger the secretion of chemotactic factors and other immunologically active proteins that can influence immune responsiveness toward dying cells.

The pro-inflammatory cytokines, TNFα, IL-1 and IL-18, amplify cartilage destruction associated with osteoarthritis (OA). Current data suggest that basic calcium phosphate (BCP) crystals are potent drivers of inflammatory mediator and matrix metalloprotease expression in the OA joint. It has previously been demonstrated that synovial macrophages play a role in initiating and driving BCP-induced inflammation. However, the molecular mechanisms by which BCP crystals exert their effects remain unclear. Here we demonstrate that exposure of macrophages to BCP crystals leads to activation of Syk and PI3 kinase. Furthermore, we show that production of pro-inflammatory cytokines and phosphorylation of the downstream kinase, ERK, are suppressed following treatment with Syk and PI3 kinase inhibitors. Finally, we demonstrate that treatment of macrophages with BCP crystals induces the production of the damage-associated molecule, S100A8, in a Syk dependent manner. We therefore identify Syk and PI3 kinase as potential novel targets for the treatment of BCP-related pathologies.

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 adjuvant-induced PI3 kinase signaling. To develop a more effective adjuvant for promoting cell-mediated 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.

The role of vaccines is to induce protec-tive pathogen-specific adaptive immune responses, but this relies on the efficient and appropriate induction of innate immunity, which instructs the adaptive response. Host recognition of invading organisms is a crucial function of innate immune cells and is mediated by the sensing of conserved microbial moieties by pathogen recognition receptors.

Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1β secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1β in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1β was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1β and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1β 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-1β in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1β through at least two separate mechanisms: by targeting pro-IL-1β for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.

Interleukin 1β (IL-1β) is an important inflammatory mediator of type 2 diabetes. Here we show that oligomers of islet amyloid polypeptide (IAPP), a protein that forms amyloid deposits in the pancreas during type 2 diabetes, triggered the NLRP3 inflammasome and generated mature IL-1β. One therapy for type 2 diabetes, glyburide, suppressed IAPP-mediated IL-1β production in vitro. Processing of IL-1β initiated by IAPP first required priming, a process that involved glucose metabolism and was facilitated by minimally oxidized low-density lipoprotein. Finally, mice transgenic for human IAPP had more IL-1β in pancreatic islets, which localized together with amyloid and macrophages. Our findings identify previously unknown mechanisms in the pathogenesis of type 2 diabetes and treatment of pathology caused by IAPP.

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system., Author Summary Streptococcus pneumoniae (pneumococcus) is a pathogen of global significance, causing diseases including pneumonia, meningitis and septicaemia. In order to develop improved pneumococcal vaccines it is essential to understand how the bacterium interacts with the host immune system. Pneumococci produce a range of pathogenicity factors, among which the toxin pneumolysin plays a central role and has potential as a vaccine candidate. Here, we demonstrate that pneumolysin can directly activate innate immune cells and dramatically amplify the production of pro-inflammatory cytokines. These enhancing effects of the toxin do not require Toll-like receptor (TLR)4. In particular, the toxin exerts a potent effect on interleukin (IL)-1, which is an endogenous pyrogen and powerful activator of IL-17A production. This effect results from activation of the NLRP3 inflammasome complex and NLRP3 is required for protection against the pathogen in vivo. To induce protective immunity against pneumococci, IFN-γ and IL-17A are thought to be essential. We show that pneumolysin plays a key role in promoting these cytokines both in vitro and in vivo during respiratory infection. The results add significantly to our understanding of the interactions between pneumococci and the immune system and support investigations into the inclusion of pneumolysin or its derivatives in novel pneumococcal vaccines.

PUBLISHED, PMID: 19139407, 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 antigen-specific 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 antigen-specific cellular immunity., The project was supported by the Irish Research Council for Science, Engineering and Technology.