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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.

Mucosal vaccines offer the potential to trigger robust protective immune responses at the predominant sites of pathogen infection. In principle, the induction of adaptive immunity at mucosal sites, involving secretory antibody responses and tissue-resident T cells, has the capacity to prevent an infection from becoming established in the first place, rather than only curtailing infection and protecting against the development of disease symptoms. Although numerous effective mucosal vaccines are in use, the major advances seen with injectable vaccines (including adjuvanted subunit antigens, RNA and DNA vaccines) have not yet been translated into licensed mucosal vaccines, which currently comprise solely live attenuated and inactivated whole-cell preparations. The identification of safe and effective mucosal adjuvants allied to innovative antigen discovery and delivery strategies is key to advancing mucosal vaccines. Significant progress has been made in resolving the mechanisms that regulate innate and adaptive mucosal immunity and in understanding the crosstalk between mucosal sites, and this provides valuable pointers to inform mucosal adjuvant design. In particular, increased knowledge on mucosal antigen-presenting cells, innate lymphoid cell populations and resident memory cells at mucosal sites highlights attractive targets for vaccine design. Exploiting these insights will allow new vaccine technologies to be leveraged to facilitate rational mucosal vaccine design for pathogens including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for cancer., Here, Ed Lavelle and Ross Ward discuss the unique aspects of mucosal immunity that must be considered when developing effective mucosal vaccines. The authors highlight the key immune cell populations that are targeted by mucosal vaccination strategies and explain how innovative adjuvant and delivery approaches should lead to new vaccines for infectious diseases and cancers.

Adjuvants can be incorporated into vaccines to enhance the magnitude and functionality of adaptive immune responses. In this issue of Immunity, Alameh et al. (2021) reveal that lipid nanoparticles, which are key components of effective SARS-CoV-2 mRNA vaccines, have broad adjuvant function, enhancing B cell responses and protective efficacy of protein-based subunit in addition to mRNA antigens.

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

Publsher's version (útgefin grein), Therapeutic vaccination offers great promise as an intervention for a diversity of infectious and non-infectious conditions. Given that most chronic health conditions are thought to have an immune component, vaccination can at least in principle be proposed as a therapeutic strategy. Understanding the nature of protective immunity is of vital importance, and the progress made in recent years in defining the nature of pathological and protective immunity for a range of diseases has provided an impetus to devise strategies to promote such responses in a targeted manner. However, in many cases, limited progress has been made in clinical adoption of such approaches. This in part results from a lack of safe and effective vaccine adjuvants that can be used to promote protective immunity and/or reduce deleterious immune responses. Although somewhat simplistic, it is possible to divide therapeutic vaccine approaches into those targeting conditions where antibody responses can mediate protection and those where the principal focus is the promotion of effector and memory cellular immunity or the reduction of damaging cellular immune responses as in the case of autoimmune diseases. Clearly, in all cases of antigen-specific immunotherapy, the identification of protective antigens is a vital first step. There are many challenges to developing therapeutic vaccines beyond those associated with prophylactic diseases including the ongoing immune responses in patients, patient heterogeneity, and diversity in the type and stage of disease. If reproducible biomarkers can be defined, these could allow earlier diagnosis and intervention and likely increase therapeutic vaccine efficacy. Current immunomodulatory approaches related to adoptive cell transfers or passive antibody therapy are showing great promise, but these are outside the scope of this review which will focus on the potential for adjuvanted therapeutic active vaccination strategies., This article/publication is based upon work from COST Action CA16231 ENOVA (European Network of Vaccine Adjuvants), supported by COST (European Cooperation in Science and Technology—www.cost.eu).

Adjuvants are a miscellaneous range of molecules and materials that can enhance the magnitude, functionality, breadth and durability of immune responses. Despite the multiplicity of compounds with adjuvant properties, less than a dozen are in clinical use in vaccines against infectious diseases. While many factors have contributed to their slow development, among the major challenges are the high safety and efficacy standards set by current adjuvants in human vaccines and our limited understanding of how adjuvants mediate their effects. This review outlines why it is so difficult to elucidate their mechanism of action, highlights areas that require in-depth research and discusses recent advancements that are revitalising adjuvant development. It is hoped that a fuller understanding of adjuvant sensing, signalling and function will facilitate the design of vaccines that promote sustained protective immunity against challenging bacterial and viral pathogens.

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.

Oral vaccination has the potential to offer a safer and more efficacious approach for protection against enteric pathogens than injection-based approaches, especially in developing countries. One key advantage is the potential to induce intestinal immune responses in addition to systemic immunity. In general, antigen delivery via the oral route triggers weak immune responses or immunological tolerance. The effectiveness of oral vaccination can be improved by co-administering adjuvants. However, a major challenge is the absence of potent and safe oral adjuvants for clinical application. Here, the Type II NKT cell activator sulfatide is shown for the first time to be an effective oral adjuvant for Vibrio cholerae vaccine antigens in a mouse model. Specifically, administration of sulfatide with the oral cholera vaccine Dukoral® resulted in enhancement of intestinal antigen-specific IgA in addition to Th1 and Th17 immune responses. In summary, sulfatide is a promising adjuvant for inclusion in an oral cholera vaccine and our data further support the potential of adjuvants targeting NKT cells in new vaccine strategies.

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.

Chitosan is a cationic polysaccharide that has been evaluated as an adjuvant due to its biocompatible and biodegradable nature. The polysaccharide can enhance antibody responses and cell-mediated immunity following vaccination by injection or mucosal routes. However, the optimal polymer characteristics for activation of dendritic cells (DCs) and induction of antigen-specific cellular immune responses have not been resolved. Here, we demonstrate that only chitin-derived polymers with a high degree of deacetylation (DDA) enhance generation of mitochondrial reactive oxygen species (mtROS), leading to cGAS-STING mediated induction of type I IFN. Additionally, the capacity of the polymers to activate the NLRP3 inflammasome was strictly dependent on the degree and pattern of deacetylation and mtROS generation. Polymers with a DDA below 80% are poor adjuvants while a fully deacetylated polyglucosamine polymer is most effective as a vaccine adjuvant. Furthermore, this polyglucosamine polymer enhanced antigen-specific Th1 responses in a NLRP3 and STING-type I IFN-dependent manner. Overall these results indicate that the degree of chitin deacetylation, the acetylation pattern and its regulation of mitochondrial ROS are the key determinants of its immune enhancing effects.

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.

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.

The recent outbreak of coronavirus disease 2019 (COVID‐19), triggered by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) poses an enormous threat to global public health and economies. Human coronaviruses normally cause no or mild respiratory disease but in the past two decades, potentially fatal coronavirus infections have emerged, causing respiratory tract illnesses such as pneumonia and bronchitis. These include severe acute respiratory syndrome coronavirus (SARS‐CoV), followed by the Middle East respiratory syndrome coronavirus (MERS‐CoV), and recently the SARS‐CoV‐2 coronavirus outbreak which emerged in Wuhan, China, in December 2019. Currently, most COVID‐19 patients receive traditional supportive care including breathing assistance. To halt the ongoing spread of the pandemic SARS‐CoV‐2 coronavirus and rescue individual patients, established drugs and new therapies are under evaluation. Since it will be some time until a safe and effective vaccine will be available, the immediate priority is to harness innate immunity to accelerate early antiviral immune responses. Secondly since excessive inflammation is a major cause of pathology, targeted anti‐inflammatory responses are being evaluated to reduce inflammation‐induced damage to the respiratory tract and cytokine storms. Here, we highlight prominent immunotherapies at various stages of development which aim for augmented anti‐coronavirus immunity and reduction of pathological inflammation. This article is protected by copyright. All rights reserved

Summary: Neutrophil granule proteases are thought to function as anti-microbial effectors, cooperatively hydrolyzing microorganisms within phagosomes, or upon deployment into the extracellular space. However, evidence also suggests that neutrophil proteases play an important role in the coordination and escalation of inflammatory reactions, but how this is achieved has been obscure. IL-1 family cytokines are important initiators of inflammation and are typically released via necrosis but require proteolytic processing for activation. Here, we show that proteases liberated from activated neutrophils can positively or negatively regulate the activity of six IL-1 family cytokines (IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ) with exquisite sensitivity. In contrast, extracellular neutrophil proteases displayed very poor bactericidal activity, exhibiting 100-fold greater potency toward cytokine processing than bacterial killing. Thus, in addition to their classical role as phagocytes, neutrophils play an important immunoregulatory role through deployment of their granule proteases into the extracellular space to process multiple IL-1 family cytokines. : Here, Clancy et al. show that proteases released by activated neutrophils into the extracellular space exhibited poor antimicrobial activity but were potent modulators of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ activation states. Thus, neutrophils play a key role in modulating inflammatory responses through processing of multiple IL-1 family cytokines. Keywords: IL-1 family, inflammation, IL-33, IL-36, neutrophil, cathepsin G, elastase, cell death, necrosis, microbial killing

The dogma that immunological memory is an exclusive trait of adaptive immunity has been recently challenged by studies showing that priming of innate cells can also result in modified long-term responsiveness to secondary stimuli, once the cells have returned to a non-activated state. This phenomenon is known as 'innate immune memory', 'trained immunity' or 'innate training'. While the main known triggers of trained immunity are microbial-derived molecules such as β-glucan, endogenous particles such as oxidized low-density lipoprotein and monosodium urate crystals can also induce trained phenotypes in innate cells. Whether exogenous particles can induce trained immunity has been overlooked. Our exposure to particulates has dramatically increased in recent decades as a result of the broad medical use of particle-based drug carriers, theragnostics, adjuvants, prosthetics and an increase in environmental pollution. We recently showed that pristine graphene can induce trained immunity in macrophages, enhancing their inflammatory response to TLR agonists, proving that exogenous nanomaterials can affect the long-term response of innate cells. The consequences of trained immunity can be beneficial, for instance, enhancing protection against unrelated pathogens; however, they can also be deleterious if they enhance inflammatory disorders. Therefore, studying the ability of particulates and biomaterials to induce innate trained phenotypes in cells is warranted. Here we analyse the mechanisms whereby particles can induce trained immunity and discuss how physicochemical characteristics of particulates could influence the induction of innate memory. We review the implications of trained immunity in the context of particulate adjuvants, nanocarriers and nanovaccines and their potential applications in medicine. Finally, we reflect on the unanswered questions and the future of the field.

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.

Helicobacter pylori causes chronic gastric infection that can lead to peptic ulcers and is an identified risk factor for gastric cancer development. Although much effort has been put into the development of a Helicobacter pylori vaccine over the last three decades, none has yet reached clinical application. Specific challenges pertaining to effective H. pylori vaccine development include the lack of proven vaccine-effective antigens and safe mucosal adjuvants to enhance local immune responses as well as the lack of accepted correlates of protection. Herein, we demonstrate that prophylactic intragastric immunisation with a whole-cell killed H. pylori antigen administered together with the non-toxic oral adjuvant α-galactosylceramide (α-GalCer) induced effective immune protection against H. pylori infection in mice, which was of similar magnitude as when using the “gold standard” cholera toxin as adjuvant. We further describe that this α-GalCer-adjuvanted vaccine formulation elicited strong intestinal and systemic Th1 responses as well as significant antigen-specific mucosal and systemic antibody responses. Finally, we report that the protective intestinal Th1 responses induced by α-GalCer are dependent on CD1d, IL-1R as well as IL-17R signalling. In summary, our results show that α-GalCer is a promising adjuvant for inclusion in an oral vaccine against H. pylori infection., Helicobacter pylori: prophylactic intragastric immunisation with oral adjuvant Infection by Helicobacter pylori is highly prevalent in humans and can lead to chronic inflammation and gastric cancer, but to date no effective vaccine has been approved for clinical use owing to the lack of appropriate antigens and of safe mucosal adjuvants that can produce protective and durable immunity to the bacterium. Sukanya Raghavan, Ed Lavelle and colleagues now show that prophylactic intragastric administration of an inactivated whole-cell H. pylori preparation, together with the oral adjuvant α-galactosylceramide, reduced H. pylori infection in mice by eliciting a protective mucosal and systemic TH1 response. The immunisation triggered antigen-specific antibodies and interferon-γ that prevented effective colonisation of H. pylori after challenge in a process dependent on the CD1d, IL-1 receptor and IL-17 receptor pathways. The reported enhanced immune response to this orally adjuvanted vaccine formulation paves the way for further studies of its safety and efficacy.

Inflammatory disorders of the gastro-intestinal tract are a major cause of morbidity and significant burden from a health and economic perspective in industrialized countries. While the incidence of such conditions has a strong environmental component, in particular dietary composition, epidemiological studies have identified specific hereditary mutations which result in disequilibrium between pro- and anti-inflammatory factors. The IL-1 super-family of cytokines and receptors is highly pleiotropic and plays a fundamental role in the pathogenesis of several autoimmune and inflammatory conditions including rheumatoid arthritis, multiple sclerosis and psoriasis. However, the role of this super-family in the aetiology of inflammatory bowel diseases remains incompletely resolved despite extensive research. Herein, we highlight the currently accepted paradigms as they pertain to specific IL-1 family members and focus on some recently described non-classical roles for these pathways in the gastrointestinal tract. Finally, we address some of the shortcomings and sources of variance in the field which to date have yielded several conflicting results from similar studies and discuss the potential effect of these factors on data interpretation.

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.

Glioblastoma (GBM) is an aggressive brain tumour, associated with extremely poor prognosis and although there have been therapeutic advances, treatment options remain limited. This review focuses on the use of immunotherapy, harnessing the power of the host's immune system to reject cancer cells. Key challenges in glioma specific immunotherapy as with many other cancers are the limited immunogenicity of the cancer cells and the immunosuppressive environment of the tumour. Although specific antigens have been identified in several cancers; brain tumours, such as GBM, are considered poorly immunogenic. However, as detailed in this review, strategies aimed at circumventing these challenges are showing promise for GBM treatment; including identification of glioma specific antigens and endogenous immune cell activation in an attempt to overcome the immunosuppressive environment which is associated with GBM tumours. An up-to-date summary of current Phase I/II and ongoing Phase III GBM immunotherapy clinical trials is provided in addition to insights into promising preclinical approaches which are focused predominantly on increased induction of Type 1 helper T cell (Th1) immune responses within patients.

Interleukin (IL)-33, a member of the IL-1 family, was originally described in 2005 as a potent initiator of type 2 immunity found during allergic inflammation and parasitic infections. IL-33 has been shown to play important and potent roles bridging innate and adaptive immunity in the regulation of tissue homeostasis, injury, and repair. Recent discoveries have extended the range of functions for IL-33 beyond type 2 conditions and its role as an alarmin at barrier sites, with emerging central roles for IL-33 in T-cell regulation, obesity, viral and tumor immunity. Here, we review the recent advances on how IL-33 activity is regulated, its immunomodulatory properties on innate and adaptive cells, and the newly discovered roles of IL-33 in obesity, intestinal inflammation, and tumorigenesis.

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.

The Hippo pathway responds to environmental factors including nutrient availability, cell density and substrate stiffness to regulate organ size. This pathway is now shown to also regulate antiviral defence by modulating the TBK1-mediated control of interferon production.

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.

Vaccination has been well recognised as a critically important tool in preventing infectious disease, yet incomplete immunisation coverage remains a major obstacle to achieving disease control and eradication. As medical products for global access, vaccines need to be safe, effective and inexpensive. In line with these goals, continuous improvements of vaccine delivery strategies are necessary to achieve the full potential of immunisation. Novel technologies related to vaccine delivery and route of administration, use of advanced adjuvants and controlled antigen release (single-dose immunisation) approaches are expected to contribute to improved coverage and patient compliance. This review discusses the application of micro- and nano-technologies in the alternative routes of vaccine administration (mucosal and cutaneous vaccination), oral vaccine delivery as well as vaccine encapsulation with the aim of controlled antigen release for single-dose vaccination.

The introduction of vaccines is regarded as one of the most successful medical interventions to date. However, there is a clear need for the development of new vaccines for diseases which require the induction of a potent cellular immune response. Advancements in the field of vaccine research have resulted in a move away from the use of whole organisms and towards the use of subunit vaccines which consist of highly purified antigens with an improved safety profile. Adjuvants are immunostimulatory components that are included in subunit vaccine formulations to help direct and amplify adaptive immune responses. Chitosan is a cationic polysaccharide that has been examined in an adjuvant setting due to its biocompatible and biodegradable nature. The polysaccharide has been shown to have the capacity to induce Th1 cell responses following vaccination by injection or mucosal routes, supporting its application as an alternative to alum for vaccines that promote cell-mediated immunity. Here, we provide an overview of the physico-chemical properties of chitosan with a focus on the specific characteristics that dictate the type and scale of the immune responses induced. The potential to finely tailor chitosan polymers in order to direct specific types of immune responses can provide invaluable tools for the design of novel chitosan-based adjuvants and biomaterials.

Growing awareness of the multiplicity of roles for the IL-1 family in immune regulation has prompted research exploring these cytokines in the context of vaccine-induced immunity. While tightly regulated, cytokines of the IL-1 family are normally released in response to cellular stress and in combination with other danger-/damage-associated molecular patterns (DAMPs), triggering potent local and systemic immune responses. In the context of infection or autoimmunity, engagement of IL-1 family receptors links robust innate responses to adaptive immunity. Clinical and experimental evidence has revealed that many vaccine adjuvants induce the release of one or multiple IL-1 family cytokines. The coordinated release of IL-1 family members in response to adjuvant-induced damage or cell death may be a determining factor in the transition from local inflammation to the induction of an adaptive response. Here, we analyse the effects of IL-1 family cytokines on innate and adaptive immunity with a particular emphasis on activation of antigen-presenting cells and induction of T cell-mediated immunity, and we address in detail the contribution of these cytokines to the modes of action of vaccine adjuvants including those currently approved for human use.

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.

While the majority of human pathogens infect the body through mucosal sites, most licensed vaccines are injectable. In fact the only mucosal vaccine that has been widely used globally for infant and childhood vaccination programs is the oral polio vaccine (OPV) developed by Albert Sabin in the 1950s. While oral vaccines against Cholera, rotavirus and Salmonella typhi have also been licensed, the development of additional non-living oral vaccines against these and other enteric pathogens has been slow and challenging. Mucosal vaccines can elicit protective immunity at the gut mucosa, in part via antigen-specific secretory immunoglobulin A (SIgA). However, despite their advantages over the injectable route, oral vaccines face many hurdles. A key challenge lies in design of delivery strategies that can protect antigens from degradation in the stomach and intestine, incorporate appropriate immune-stimulatory adjuvants and control release at the appropriate gastrointestinal site. A number of systems including micro and nanoparticles, lipid-based strategies and enteric capsules have significant potential either alone or in advanced combined formulations to enhance intestinal immune responses. In this review we will outline the opportunities, challenges and potential delivery solutions to facilitate the development of improved oral vaccines for infectious enteric diseases.

TNF promotes a regulated form of necrosis, called necroptosis, upon inhibition of caspase activity in cells expressing RIPK3. Because necrosis is generally more pro-inflammatory than apoptosis, it is widely presumed that TNF-induced necroptosis may be detrimental in vivo due to excessive inflammation. However, because TNF is intrinsically highly pro-inflammatory, due to its ability to trigger the production of multiple cytokines and chemokines, rapid cell death via necroptosis may blunt rather than enhance TNF-induced inflammation. Here we show that TNF-induced necroptosis potently suppressed the production of multiple TNF-induced pro-inflammatory factors due to RIPK3-dependent cell death. Similarly, necroptosis also suppressed LPS-induced pro-inflammatory cytokine production. Consistent with these observations, supernatants from TNF-stimulated cells were more pro-inflammatory than those from TNF-induced necroptotic cells in vivo. Thus necroptosis attenuates TNF- and LPS-driven inflammation, which may benefit intracellular pathogens that evoke this mode of cell death by suppressing host immune responses.

peer-reviewed There has been a move away from vaccines composed of whole or inactivated antigens toward subunit-based vaccines, which although safe, provide less immunological protection. As a result, the use of adjuvants to enhance and direct adaptive immune responses has become the focus of much targeted bovine vaccine research. However, the mechanisms by which adjuvants work to enhance immunological protection in many cases remains unclear, although this knowledge is critical to the rational design of effective next generation vaccines. This study aimed to investigate the mechanisms by which alum, a commonly used adjuvant in bovine vaccines, enhances IL-1β secretion in bovine peripheral blood mononuclear cells (PBMCs). Unlike the case with human PBMCs, alum promoted IL-1β secretion in a subset of bovine PBMCs without priming with a toll-like receptor agonist. This suggests that PBMCs from some cattle are primed to produce this potent inflammatory cytokine and western blotting confirmed the presence of preexisting pro-IL-1β in PBMCs from a subset of 8-month-old cattle. To address the mechanism underlying alum-induced IL-1β secretion, specific inhibitors identified that alum mediates lysosomal disruption which subsequently activates the assembly of an NLRP3, ASC, caspase-1, and potentially caspase-8 containing complex. These components form an inflammasome, which mediates alum-induced IL-1β secretion in bovine PBMCs. Given the demonstrated role of the NLRP3 inflammasome in regulating adaptive immunity in murine systems, these results will inform further targeted research into the potential of inflammasome activation for rational vaccine design in cattle.

The recent surge in graphene research, since its liquid phase monolayer isolation and characterization in 2004, has led to advancements which are accelerating the exploration of alternative 2D materials such as molybdenum disulphide (MoS2), whose unique physico-chemical properties can be exploited in applications ranging from cutting edge electronic devices to nanomedicine. However, to assess any potential impact on human health and the environment, the need to understand the bio-interaction of MoS2 at a cellular and sub-cellular level is critical. Notably, it is important to assess such potential impacts of materials which are produced by large scale production techniques, rather than research grade materials. The aim of this study was to explore cytotoxicity, cellular uptake and inflammatory responses in established cell-lines that mimic different potential exposure routes (inhalation, A549; ingestion, AGS; monocyte, THP-1) following incubation with MoS2 flakes of varying sizes (50 nm, 117 nm and 177 nm), produced by liquid phase exfoliation. Using high content screening (HCS) and live/dead assays, it was established that 1 μg/ml (for the three different MoS2 sizes) did not induce toxic effects on any of the cell-lines. Confocal microscopy images revealed a normal cellular morphology in all cases. Transmission electron microscopy (TEM) confirmed the uptake of all MoS2 nanomaterials in all the cell-lines, the MoS2 ultimately locating in single membrane vesicles. At such sub-lethal doses, inflammatory responses are observed, however, associated, at least partially, with the presence of lipopolysaccharide endotoxin in nanomaterial suspensions and surfactant samples. Therefore, the inflammatory response of the cells to the MoS2 or endotoxin contamination was interrogated using a 10-plex ELISA which illustrates cytokine production. The experiments carried out using wild-type and endotoxin hyporesponsive bone marrow derived dendritic cells confirmed that the inflammatory responses result from a combination of endotoxin contamination, the MoS2 nanomaterials themselves, and the stabilizing surfactant.