Training vs. Tolerance: The Yin/Yang of the Innate Immune System.

For almost nearly a century, memory functions have been attributed only to acquired immune cells. Lately, this paradigm has been challenged by an increasing number of studies revealing that innate immune cells are capable of exhibiting memory-like features resulting in increased responsiveness to subsequent challenges, a process known as trained immunity (known also as innate memory). In contrast, the refractory state of endotoxin tolerance has been defined as an immunosuppressive state of myeloid cells portrayed by a significant reduction in the inflammatory capacity. Both training as well tolerance as adaptive features are reported to be accompanied by epigenetic and metabolic alterations occurring in cells. While training conveys proper protection against secondary infections, the induction of endotoxin tolerance promotes repairing mechanisms in the cells. Consequently, the inappropriate induction of these adaptive cues may trigger maladaptive effects, promoting an increased susceptibility to secondary infections-tolerance, or contribute to the progression of the inflammatory disorder-trained immunity. This review aims at the discussion of these opposing manners of innate immune and non-immune cells, describing the molecular, metabolic and epigenetic mechanisms involved and interpreting the clinical implications in various inflammatory pathologies.

Vitamin D status can affect COVID-19 outcomes also in pediatric population.

Background: vitamin D influences the immune system and the inflammatory response. It is known that vitamin D supplementation reduces the risk of acute respiratory tract infection. In the last two years, many researchers have investigated vitamin D's role in the pathophysiology of COVID-19 disease. Results: the findings obtained from clinical trials and systematic reviews highlight that most patients with COVID-19 have decreased vitamin D levels and low levels of vitamin D increase the risk of severe disease. This evidence seems to be also confirmed in the pediatric population. Conclusions: further studies (systematic review and meta-analysis) conducted on children are needed to confirm that vitamin D affects COVID-19 outcomes and to determine the effectiveness of supplementation and the appropriate dose, duration and mode of administration.

Targeting whole body metabolism and mitochondrial bioenergetics in the drug development for Alzheimer's disease.

Aging is by far the most prominent risk factor for Alzheimer's disease (AD), and both aging and AD are associated with apparent metabolic alterations. As developing effective therapeutic interventions to treat AD is clearly in urgent need, the impact of modulating whole-body and intracellular metabolism in preclinical models and in human patients, on disease pathogenesis, have been explored. There is also an increasing awareness of differential risk and potential targeting strategies related to biological sex, microbiome, and circadian regulation. As a major part of intracellular metabolism, mitochondrial bioenergetics, mitochondrial quality-control mechanisms, and mitochondria-linked inflammatory responses have been considered for AD therapeutic interventions. This review summarizes and highlights these efforts.

The Yin and Yang of Immunity in Stem Cell Decision Guidance in Tissue Ecologies: An Infection Independent Perspective.

The impact of immune system and inflammation on organ homeostasis and tissue stem cell niches in the absence of pathogen invasion has long remained a conundrum in the field of regenerative medicine. The paradoxical role of immune components in promoting tissue injury as well as resolving tissue damage has complicated therapeutic targeting of inflammation as a means to attain tissue homeostasis in degenerative disease contexts. This confound could be resolved by an integrated intricate assessment of cross-talk between inflammatory components and micro- and macro-environmental factors existing in tissues during health and disease. Prudent fate choice decisions of stem cells and their differentiated progeny are key to maintain tissue integrity and function. Stem cells have to exercise this fate choice in consultation with other tissue components. With this respect tissue immune components, danger/damage sensing molecules driving sterile inflammatory signaling cascades and barrier cells having immune-surveillance functions play pivotal roles in supervising stem cell decisions in their niches. Stem cells learn from their previous damage encounters, either endogenous or exogenous, or adapt to persistent micro-environmental changes to orchestrate their decisions. Thus understanding the communication networks between stem cells and immune system components is essential to comprehend stem cell decisions in endogenous tissue niches. Further the systemic interactions between tissue niches integrated through immune networks serve as patrolling systems to establish communication links and orchestrate micro-immune ecologies to better organismal response to injury and promote regeneration. Understanding these communication links is key to devise immune-centric regenerative therapies. Thus the present review is an integrated attempt to provide a unified purview of how inflammation and immune cells provide guidance to stem cells for tissue sculpting during development, organismal aging and tissue crisis based on the current knowledge in the field.

Extracellular DNA as an elicitor of broad-spectrum resistance in Arabidopsis thaliana.

Like in mammals, the plant immune system has evolved to perceive damage. Damaged-associated molecular patterns (DAMPs) are endogenous signals generated in wounded or infected tissue after pathogen or insect attack. Although extracellular DNA (eDNA) is a DAMP signal that induces immune responses, plant responses after eDNA perception remain largely unknown. Here, we report that signaling defenses but not direct defense responses are induced after eDNA applications enhancing broad-range plant protection. A screening of defense signaling and hormone biosynthesis marker genes revealed that OXI1, CML37 and MPK3 are relevant eDNA-Induced Resistance markers (eDNA-IR). Additionally, we observed that eDNA from several Arabidopsis ecotypes and other phylogenetically distant plants such as citrus, bean and, more surprisingly, a monocotyledonous plant such as maize upregulates eDNA-IR marker genes. Using 3,3'-Diaminobenzidine (DAB) and aniline blue staining methods, we observed that H2O2 but not callose was strongly accumulated following self-eDNA treatments. Finally, eDNA resulted in effective induced resistance in Arabidopsis against the pathogens Hyaloperonospora arabidopsidis, Pseudomonas syringae, and Botrytis cinerea and against aphid infestation, reducing the number of nymphs and moving forms. Hence, the unspecificity of DNA origin and the wide range of insects to which eDNA can protect opens many questions about the mechanisms behind eDNA-IR.

Targeting Toll-like Receptor (TLR) Pathways in Inflammatory Arthritis: Two Better Than One?

Inflammatory arthritis is a cluster of diseases caused by unregulated activity of the immune system. The lost homeostasis is followed by the immune attack of one's self, what damages healthy cells and tissues and leads to chronic inflammation of various tissues and organs (e.g., joints, lungs, heart, eyes). Different medications to control the excessive immune response are in use, however, drug resistances, flare-reactions and adverse effects to the current therapies are common in the affected patients. Thus, it is essential to broaden the spectrum of alternative treatments and to develop disease-modifying drugs. In the last 20 years, the involvement of the innate immune receptors TLRs in inflammatory arthritis has been widely investigated and targeting either the receptor itself or the proteins in the downstream signalling cascades has emerged as a promising therapeutic strategy. Yet, concerns about the use of pharmacological agents that inhibit TLR activity and may leave the host unprotected against invading pathogens and toxicity issues amid inhibition of downstream kinases crucial in various cellular functions have arisen. This review summarizes the existing knowledge on the role of TLRs in inflammatory arthritis; in addition, the likely druggable related targets and the developed inhibitors, and discusses the pros and cons of their potential clinical use.

Gasdermin D in pyroptosis.

Pyroptosis is the process of inflammatory cell death. The primary function of pyroptosis is to induce strong inflammatory responses that defend the host against microbe infection. Excessive pyroptosis, however, leads to several inflammatory diseases, including sepsis and autoimmune disorders. Pyroptosis can be canonical or noncanonical. Upon microbe infection, the canonical pathway responds to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), while the noncanonical pathway responds to intracellular lipopolysaccharides (LPS) of Gram-negative bacteria. The last step of pyroptosis requires the cleavage of gasdermin D (GsdmD) at D275 (numbering after human GSDMD) into N- and C-termini by caspase 1 in the canonical pathway and caspase 4/5/11 (caspase 4/5 in humans, caspase 11 in mice) in the noncanonical pathway. Upon cleavage, the N-terminus of GsdmD (GsdmD-N) forms a transmembrane pore that releases cytokines such as IL-1ß and IL-18 and disturbs the regulation of ions and water, eventually resulting in strong inflammation and cell death. Since GsdmD is the effector of pyroptosis, promising inhibitors of GsdmD have been developed for inflammatory diseases. This review will focus on the roles of GsdmD during pyroptosis and in diseases.

Roots drive oligogalacturonide-induced systemic immunity in tomato.

Oligogalacturonides (OGs) are fragments of pectin released from the plant cell wall during insect or pathogen attack. They can be perceived by the plant as damage signals, triggering local and systemic defence responses. Here, we analyse the dynamics of local and systemic responses to OG perception in tomato roots or shoots, exploring their impact across the plant and their relevance in pathogen resistance. Targeted and untargeted metabolomics and gene expression analysis in plants treated with purified OGs revealed that local responses were transient, while distal responses were stronger and more sustained. Remarkably, changes were more conspicuous in roots, even upon foliar application of the OGs. The treatments differentially activated the synthesis of defence-related hormones and secondary metabolites including flavonoids, alkaloids and lignans, some of them exclusively synthetized in roots. Finally, the biological relevance of the systemic defence responses activated upon OG perception was confirmed, as the treatment induced systemic resistance to Botrytis cinerea. Overall, this study shows the differential regulation of tomato defences upon OGs perception in roots and shoots and reveals the key role of roots in the coordination of the plant responses to damage sensing.

Enhancing innate immunity against virus in times of COVID-19: Trying to untangle facts from fictions.

INTRODUCTION: In light of the current COVID-19 pandemic, during which the world is confronted with a new, highly contagious virus that suppresses innate immunity as one of its initial virulence mechanisms, thus escaping from first-line human defense mechanisms, enhancing innate immunity seems a good preventive strategy. METHODS: Without the intention to write an official systematic review, but more to give an overview of possible strategies, in this review article we discuss several interventions that might stimulate innate immunity and thus our defense against (viral) respiratory tract infections. Some of these interventions can also stimulate the adaptive T- and B-cell responses, but our main focus is on the innate part of immunity. We divide the reviewed interventions into: 1) lifestyle related (exercise, >7 h sleep, forest walking, meditation/mindfulness, vitamin supplementation); 2) Non-specific immune stimulants (letting fever advance, bacterial vaccines, probiotics, dialyzable leukocyte extract, pidotimod), and 3) specific vaccines with heterologous effect (BCG vaccine, mumps-measles-rubeola vaccine, etc). RESULTS: For each of these interventions we briefly comment on their definition, possible mechanisms and evidence of clinical efficacy or lack of it, especially focusing on respiratory tract infections, viral infections, and eventually a reduced mortality in severe respiratory infections in the intensive care unit. At the end, a summary table demonstrates the best trials supporting (or not) clinical evidence. CONCLUSION: Several interventions have some degree of evidence for enhancing the innate immune response and thus conveying possible benefit, but specific trials in COVID-19 should be conducted to support solid recommendations.

Role of Damage-Associated Molecular Patterns in Light of Modern Environmental Research: A Tautological Approach.

Two prominent models emerged as a result of intense interdisciplinary discussions on the environmental health paradigm, called the "exposome" concept and the "adverse outcome pathway" (AOP) concept that links a molecular initiating event to the adverse outcome via key events. Here, evidence is discussed, suggesting that environmental stress/injury-induced damage-associated molecular patterns (DAMPs) may operate as an essential integrating element of both environmental health research paradigms. DAMP-promoted controlled/uncontrolled innate/adaptive immune responses reflect the key events of the AOP concept. The whole process starting from exposure to a distinct environmental stress/injury-associated with the presence/emission of DAMPs-up to the manifestation of a disease may be regarded as an exposome. Clinical examples of such a scenario are briefly sketched, in particular, a model in relation to the emerging COVID-19 pandemic, where the interaction of noninfectious environmental factors (e.g., particulate matter) and infectious factors (SARS CoV-2) may promote SARS case fatality via superimposition of both exogenous and endogenous DAMPs.

Disturbed Yin-Yang balance: stress increases the susceptibility to primary and recurrent infections of herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1), a neurotropic herpes virus, is able to establish a lifelong latent infection in the human host. Following primary replication in mucosal epithelial cells, the virus can enter sensory neurons innervating peripheral tissues via nerve termini. The viral genome is then transported to the nucleus where it can be maintained without producing infectious progeny, and thus latency is established in the cell. Yin-Yang balance is an essential concept in traditional Chinese medicine (TCM) theory. Yin represents stable and inhibitory factors, and Yang represents the active and aggressive factors. When the organism is exposed to stress, especially psychological stress caused by emotional stimulation, the Yin-Yang balance is disturbed and the virus can re-engage in productive replication, resulting in recurrent diseases. Therefore, a better understanding of the stress-induced susceptibility to HSV-1 primary infection and reactivation is needed and will provide helpful insights into the effective control and treatment of HSV-1. Here we reviewed the recent advances in the studies of HSV-1 susceptibility, latency and reactivation. We included mechanisms involved in primary infection and the regulation of latency and described how stress-induced changes increase the susceptibility to primary and recurrent infections.

A dose-response study of a novel method of selective tissue modification of cellular structures in the skin with nanosecond pulsed electric fields.

BACKGROUND AND OBJECTIVES: This study describes the effects of nanosecond pulsed electric fields (nsPEF) on the epidermis and dermis of normal skin scheduled for excision in a subsequent abdominoplasty. NsPEF therapy applies nanosecond pulses of electrical energy to induce regulated cell death (RCD) in cellular structures, with negligible thermal effects. Prior pre-clinical studies using nsPEF technology have demonstrated the ability to stimulate a lasting immune response in animal tumor models, including melanoma. This first-in-human-use of nsPEF treatment in a controlled study to evaluate the dose-response effects on normal skin and subcutaneous structures is intended to establish a safe dose range of energies prior to use in clinical applications using nsPEF for non-thermal tissue modification. STUDY DESIGN/MATERIALS AND METHODS: Seven subjects with healthy tissue planned for abdominoplasty excision were enrolled. Five subjects were evaluated in a longitudinal, 60-day study of effects with doses of six nsPEF energy levels. A total of 30 squares of spot sizes 25mm2 or less within the planned excision area were treated and then evaluated at 1 day, 5 days, 15 days, 30 days, and 60 days prior to surgery. Photographs were taken over time of each treated area and assessed by three independent and blinded dermatologists for erythema, flaking and crusting using a 5-point scale (0 = low, 4 = high). Punch biopsies of surgically removed tissue were processed and evaluated for tissue changes using hematoxylin and eosin, trichome, caspase-3, microphthalmia transcription factor, and elastin stains and evaluated by a dermatopathologist. The skin of two subjects received additional treatments at 2 and 4 hours post-nsPEF and was evaluated in a similar manner. RESULTS: Most energy settings exhibited delayed epidermal loss followed by re-epithelization by day 15 and a normal course of healing. Histologic analysis identified the appearance of activated caspase-3 at two and four hours after nsPEF treatment, but not at later time points. At the 1-day time point, a nucleolysis effect was observed in epidermal cells, as evidenced by the lack of nuclear staining while the epidermal plasma membranes were still intact. Cellular structures within the treatment zone such as melanocytes, sebaceous glands, and hair follicles were damaged while acellular structures such as elastic fibers and collagen were largely unaffected except for TL6 which showed signs of dermal damage. Melanocytes reappeared at levels comparable with untreated controls within 1 month of nsPEF treatment. CONCLUSIONS: The selective effect of nsPEF treatment on cellular structures in the epidermal and dermal layers suggests that this non-thermal mechanism for targeting cellular structures does not affect the integrity of dermal tissue within a range of energy levels. The specificity of effects and a favorable healing response makes nsPEF ideal for treating cellular targets in the epidermal or dermal layers of the skin, including treatment of benign and malignant lesions. NsPEF skin treatments provide a promising, non-thermal method for treating skin conditions and removing epidermal lesions. © 2019 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

Cell wall traits that influence plant development, immunity, and bioconversion.

The architecture of the plant cell wall is highly dynamic, being substantially re-modeled during growth and development. Cell walls determine the size and shape of cells and contribute to the functional specialization of tissues and organs. Beyond the physiological dynamics, the wall structure undergoes changes upon biotic or abiotic stresses. In this review several cell wall traits, mainly related to pectin, one of the major matrix components, will be discussed in relation to plant development, immunity and industrial bioconversion of biomass, especially for energy production. Plant cell walls are a source of oligosaccharide fragments with a signaling function for both development and immunity. Sensing cell wall damage, sometimes through the perception of released damage-associated molecular patterns (DAMPs), is crucial for some developmental and immunity responses. Methodological advances that are expected to deepen our knowledge of cell wall (CW) biology will also be presented.

Structural insights into pharmacophore-assisted in silico identification of protein-protein interaction inhibitors for inhibition of human toll-like receptor 4 - myeloid differentiation factor-2 (hTLR4-MD-2) complex.

Toll-like receptor 4 (TLR4) is a member of Toll-Like Receptors (TLRs) family that serves as a receptor for bacterial lipopolysaccharide (LPS). TLR4 alone cannot recognize LPS without aid of co-receptor myeloid differentiation factor-2 (MD-2). Binding of LPS with TLR4 forms a LPS-TLR4-MD-2 complex and directs downstream signaling for activation of immune response, inflammation and NF-κB activation. Activation of TLR4 signaling is associated with various pathophysiological consequences. Therefore, targeting protein-protein interaction (PPI) in TLR4-MD-2 complex formation could be an attractive therapeutic approach for targeting inflammatory disorders. The aim of present study was directed to identify small molecule PPI inhibitors (SMPPIIs) using pharmacophore mapping-based approach of computational drug discovery. Here, we had retrieved the information about the hot spot residues and their pharmacophoric features at both primary (TLR4-MD-2) and dimerization (MD-2-TLR4*) protein-protein interaction interfaces in TLR4-MD-2 homo-dimer complex using in silico methods. Promising candidates were identified after virtual screening, which may restrict TLR4-MD-2 protein-protein interaction. In silico off-target profiling over the virtually screened compounds revealed other possible molecular targets. Two of the virtually screened compounds (C11 and C15) were predicted to have an inhibitory concentration in µM range after HYDE assessment. Molecular dynamics simulation study performed for these two compounds in complex with target protein confirms the stability of the complex. After virtual high throughput screening we found selective hTLR4-MD-2 inhibitors, which may have therapeutic potential to target chronic inflammatory diseases.

Anti-inflammatory and preventive activity of white mulberry root bark extract in an experimental model of pancreatitis.

Pancreatitis is characterized by highly morbid inflammation in the pancreas. Currently, there is no specific drug available for pancreatitis except supportive medicines. The present study assessed the pancreato-protective effect of Morus alba root bark extract by using alcohol and cerulein-induced model of pancreatitis. The study also investigated the phytochemical profile through GC-MS and HPLC. Methanolic extract of Morus alba root bark extract (MEMARB) was subjected to GC-MS and HPLC studies. Male albino Wistar rats were administered ethanol (0%-36%) and cerulein (20 µg/kg b.wt. i.p.) with or without MEMARB. Serum lipase, amylase, caspase-1, lipid peroxidation products, glutathione and enzymatic antioxidants were determined. Histological changes in the pancreas were assessed. Cudraflavone B in MEMARB was quantified by HPLC. Significant amount of Cudraflavone B was detected by quantitative HPLC. Marked increase in the levels of serum amylase, lipase, caspase-1, IL-18 and IL-1ß were observed in ethanol and cerulein administered rats than in MEMARB co-administered rats. In MEMARB co-administered rats, the antioxidant status was restored to near normal levels. Histological examinations showed that MEMARB significantly reduced the inflammatory and fibrotic changes. The results reveal the potent pancreato-protective effects of Morus alba root bark. The anti-inflammatory effect of Morus alba root bark extract might be due to the presence of various phytonutrients including Cudraflavone B.

Targeting Cytosolic Nucleic Acid-Sensing Pathways for Cancer Immunotherapies.

The innate immune system provides the first line of defense against pathogen infection though also influences pathways involved in cancer immunosurveillance. The innate immune system relies on a limited set of germ line-encoded sensors termed pattern recognition receptors (PRRs), signaling proteins and immune response factors. Cytosolic receptors mediate recognition of danger damage-associated molecular patterns (DAMPs) signals. Once activated, these sensors trigger multiple signaling cascades, converging on the production of type I interferons and proinflammatory cytokines. Recent studies revealed that PRRs respond to nucleic acids (NA) released by dying, damaged, cancer cells, as danger DAMPs signals, and presence of signaling proteins across cancer types suggests that these signaling mechanisms may be involved in cancer biology. DAMPs play important roles in shaping adaptive immune responses through the activation of innate immune cells and immunological response to danger DAMPs signals is crucial for the host response to cancer and tumor rejection. Furthermore, PRRs mediate the response to NA in several vaccination strategies, including DNA immunization. As route of double-strand DNA intracellular entry, DNA immunization leads to expression of key components of cytosolic NA-sensing pathways. The involvement of NA-sensing mechanisms in the antitumor response makes these pathways attractive drug targets. Natural and synthetic agonists of NA-sensing pathways can trigger cell death in malignant cells, recruit immune cells, such as DCs, CD8+ T cells, and NK cells, into the tumor microenvironment and are being explored as promising adjuvants in cancer immunotherapies. In this minireview, we discuss how cGAS-STING and RIG-I-MAVS pathways have been targeted for cancer treatment in preclinical translational researches. In addition, we present a targeted selection of recent clinical trials employing agonists of cytosolic NA-sensing pathways showing how these pathways are currently being targeted for clinical application in oncology.

Laser immunotherapy for cutaneous squamous cell carcinoma with optimal thermal effects to enhance tumour immunogenicity.

BACKGROUND: Laser immunotherapy is a new anti-cancer therapy combining photothermal therapy and immunostimulation. It can eliminate the tumours by damaging tumour cells directly and promoting the release of damage-associated molecular patterns (DAMPs) to enhance tumour immunogenicity. The aim of this study was to investigate the thermal effects of laser immunotherapy and to evaluate the effectiveness and safety of laser immunotherapy for cutaneous squamous cell carcinoma (cSCC). METHODS: The cell viability and the DAMPs productions of heat-treated cSCC A431 cells in different temperatures were investigated. Laser immunotherapy with the optimal thermal effect for DAMPs production was performed on SKH-1 mice bearing ultraviolet-induced cSCC and a patient suffering from a large refractory cSCC. RESULTS: The temperature in the range of 45-50 °C killing half of A431 cells had an optimal thermal effect for the productions of DAMPs. The thermal effect could be further enhanced by local application of imiquimod, an immunoadjuvant. Laser immunotherapy eliminated most tumours and improved the survival rate of the ultraviolet-induced cSCC-bearing SKH-1 mice (p < 0.05). The patient with cSCC treated by laser immunotherapy experienced a significant tumour reduction after laser immunotherapy increased the amounts of infiltrating lymphocytes in the tumour. No obviously adverse effect was observed in the mice experiment or in the clinical application. CONCLUSIONS: Our results strongly indicate that laser immunotherapy with optimal thermal effects is an effective and safe treatment modality for cSCC.

CD40 Negatively Regulates ATP-TLR4-Activated Inflammasome in Microglia.

During acute brain injury and/or sterile inflammation, release of danger-associated molecular patterns (DAMPs) activates pattern recognition receptors (PRRs). Microglial toll-like receptor (TLR)-4 activated by DAMPs potentiates neuroinflammation through inflammasome-induced IL-1ß and pathogenic Th17 polarization which critically influences brain injury. TLR4 activation accompanies increased CD40, a cognate costimulatory molecule, involved in microglia-mediated immune responses in the brain. During brain injury, excessive release of extracellular ATP (DAMPs) is involved in promoting the damage. However, the regulatory role of CD40 in microglia during ATP-TLR4-mediated inflammasome activation has never been explored. We report that CD40, in the absence of ATP, synergizes TLR4-induced proinflammatory cytokines but not IL-1ß, suggesting that the response is independent of inflammasome. The presence of ATP during TLR4 activation leads to NLRP3 inflammasome activation and caspase-1-mediated IL-1ß secretion which was inhibited during CD40 activation, accompanied with inhibition of ERK1/2 and reactive oxygen species (ROS), and elevation in p38 MAPK phosphorylation. Experiments using selective inhibitors prove indispensability of ERK 1/2 and ROS for inflammasome activation. The ATP-TLR4-primed macrophages polarize the immune response toward pathogenic Th17 cells, whereas CD40 activation mediates Th1 response. Exogenous supplementation of IFN-γ (a Th1 cytokine and CD40 inducer) results in decreased IL-1ß, suggesting possible feedback loop mechanism of inflammasome inhibition, whereby IFN-γ-mediated increase in CD40 expression and activation suppress neurotoxic inflammasome activation required for Th17 response. Collectively, the findings indicate that CD40 is a novel negative regulator of ATP-TLR4-mediated inflammasome activation in microglia, thus providing a checkpoint to regulate excessive inflammasome activation and Th17 response during DAMP-mediated brain injury.

A liquid crystal of ascorbyl palmitate, used as vaccine platform, provides sustained release of antigen and has intrinsic pro-inflammatory and adjuvant activities which are dependent on MyD88 adaptor protein.

Modern subunit vaccines require the development of new adjuvant strategies. Recently, we showed that CpG-ODN formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate (Coa-ASC16) is an attractive system for promoting an antigen-specific immune response to weak antigens. Here, we showed that after subcutaneous injection of mice with near-infrared fluorescent dye-labeled OVA antigen formulated with Coa-ASC16, the dye-OVA was retained at the injection site for a longer period than when soluble dye-OVA was administered. Coa-ASC16 alone elicited a local inflammation, but how this material triggers this response has not been described yet. Although it is known that some materials used as a platform are not immunologically inert, very few studies have directly focused on this topic. In this study, we explored the underlying mechanisms concerning the interaction between Coa-ASC16 and the immune system and we found that the whole inflammatory response elicited by Coa-ASC16 (leukocyte recruitment and IL-1ß, IL-6 and IL-12 production) was dependent on the MyD88 protein. TLR2, TLR4, TLR7 and NLRP3-inflammasome signaling were not required for induction of this inflammatory response. Coa-ASC16 induced local release of self-DNA, and in TLR9-deficient mice IL-6 production was absent. In addition, Coa-ASC16 revealed an intrinsic adjuvant activity which was affected by MyD88 and IL-6 absence. Taken together these results indicate that Coa-ASC16 used as a vaccine platform is effective due to the combination of the controlled release of antigen and its intrinsic pro-inflammatory activity. Understanding how Coa-ASC16 works might have significant implications for rational vaccine design.