1'-Acetoxychavicol acetate inhibits NLRP3-dependent inflammasome activation via mitochondrial ROS suppression.

The nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing (NLRP) 3 inflammasome is a multiprotein complex that triggers Caspase-1-mediated IL-1ß production and pyroptosis, and its dysregulation is associated with the pathogenesis of inflammatory diseases. 1'-Acetoxychavicol acetate (ACA) is a natural compound in the rhizome of tropical ginger Alpinia species with anti-microbial, anti-allergic and anti-cancer properties. In this study, we found that ACA suppressed NLRP3 inflammasome activation in mouse bone marrow-derived macrophages and human THP-1 monocytes. ACA inhibited Caspase-1 activation and IL-1ß production by NLRP3 agonists such as nigericin, monosodium urate (MSU) crystals, and ATP. Moreover, it suppressed oligomerization of the adapter molecule, apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1-mediated cleavage of pyroptosis executor Gasdermin D. Mechanistically, ACA inhibited generation of mitochondrial reactive oxygen species (ROS) and prevented release of oxidized mitochondrial DNA, which trigger NLRP3 inflammasome activation. ACA also prevented NLRP3 inflammasome activation in vivo, as evidenced in the MSU crystal-induced peritonitis and dextran sodium sulfate-induced colitis mouse models accompanied by decreased Caspase-1 activation. Thus, ACA is a potent inhibitor of the NLRP3 inflammasome for prevention of NLRP3-associated inflammatory diseases.

The Ca2+-dependent pathway contributes to changes in the subcellular localization and extracellular release of interleukin-33.

Interleukin-33 (IL-33) is a member of the IL-1 cytokine family and plays critical roles in facilitating type-2 immune responses. IL-33 is localized in the nucleus and released to the extracellular milieu during cell death, although the precise mechanisms underlying IL-33 mobilization remain unclear. Here, we found that nigericin, a toxin derived from Streptomyces hygroscopicus, promoted IL-33 translocation from the nucleus to the cytosol before extracellular release. This translocation was inhibited by chelating Ca2+ with EGTA or membrane protection by glycine treatment. Ca2+ ionophore A23187 stimulation caused IL-33 translocation to the cytoplasm but was not sufficient for extracellular release. However, IL-33 release was induced by detergent treatment, which indicates that membrane rupture is required for IL-33 release. The pore-forming pyroptosis executor gasdermin D was cleaved following nigericin stimulation, and overexpression of the cleaved gasdermin D-N-terminal fragment that forms the membrane pore sufficiently induced IL-33 release, which was blocked by EGTA and glycine. Together, these findings suggest that Ca2+-dependent signals and gasdermin D pore formation are required for robust IL-33 production.

IL-33 Is Essential for Adjuvant Effect of Hydroxypropyl-ß-Cyclodexrin on the Protective Intranasal Influenza Vaccination.

Vaccine adjuvants are traditionally used to augment and modulate the immunogenicity of vaccines, although in many cases it is unclear which specific molecules contribute to their stimulatory activity. We previously reported that both subcutaneous and intranasal administration of hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a pharmaceutical excipient widely used to improve solubility, can act as an effective adjuvant for an influenza vaccine. However, the mechanisms by which mucosal immune pathway is critical for the intranasal adjuvant activity of HP-ß-CD have not been fully delineated. Here, we show that intranasally administered HP-ß-CD elicits a temporary release of IL-33 from alveolar epithelial type 2 cells in the lung; notably, IL-33 expression in these cells is not stimulated following the use of other vaccine adjuvants. The experiments using gene deficient mice suggested that IL-33/ST2 signaling is solely responsible for the adjuvant effect of HP-ß-CD when it is administered intranasally. In contrast, the subcutaneous injection of HP-ß-CD and the intranasal administration of alum, as a damage-associated molecular patterns (DAMPs)-inducing adjuvant, or cholera toxin, as a mucosal adjuvant, enhanced humoral immunity in an IL-33-independent manner, suggesting that the IL-33/ST2 pathway is unique to the adjuvanticity of intranasally administered HP-ß-CD. Furthermore, the release of IL-33 was involved in the protective immunity against influenza virus infection which is induced by the intranasal administration of HP-ß-CD-adjuvanted influenza split vaccine. In conclusion, our results suggest that an understanding of administration route- and tissue-specific immune responses is crucial for the design of unique vaccine adjuvants.

ZBP1 governs the inflammasome-independent IL-1α and neutrophil inflammation that play a dual role in anti-influenza virus immunity.

Influenza A virus (IAV) triggers the infected lung to produce IL-1 and recruit neutrophils. Unlike IL-1ß, however, little is known about IL-1α in terms of its mechanism of induction, action and physiological relevance to the host immunity against IAV infection. In particular, whether Z-DNA-binding protein 1 (ZBP1), a key molecule for IAV-induced cell death, is involved in the IL-1α induction, neutrophil infiltration and the physiological outcome has not been elucidated. Here, we show in a murine model that the IAV-induced IL-1α is mediated solely by ZBP1, in an NLRP3-inflammasome-independent manner, and is required for the optimal IL-1ß production followed by the formation of neutrophil extracellular traps (NETs). During IAV infection, ZBP1 displays a dual role in anti-IAV immune responses mediated by neutrophils, resulting in either protective or pathological outcomes in vivo. Thus, ZBP1-mediated IL-1α production is the key initial step of IAV-infected NETs, regulating the duality of the consequent lung inflammation.

Heparin induces neutrophil elastase-dependent vital and lytic NET formation.

Heparin is used extensively as an anticoagulant in a broad range of diseases and procedures; however, its biological effects are not limited to coagulation and remain incompletely understood. Heparin usage can lead to the life-threatening complication known as heparin-induced thrombocytopenia (HIT), caused by the development of antibodies against heparin/PF4 complexes. Here, we demonstrate the ability of heparin to induce neutrophil extracellular traps (NETs). NETs occurred with cell lysis and death, but live neutrophils releasing extracellular DNA strands, known as vital NETs, also occurred abundantly. Formation of NETs was time and dose dependent, and required reactive oxygen species and neutrophil elastase. Other compounds related to heparin such as low molecular weight heparin, fondaparinux and heparan sulfate either failed to induce NETs, or did so to a much lesser extent. Our findings suggest the ability of heparin to directly induce NET formation should be considered in the context of heparin treatment and HIT pathogenesis.

Cyclic GMP-AMP Triggers Asthma in an IL-33-Dependent Manner That Is Blocked by Amlexanox, a TBK1 Inhibitor.

Extracellular host-derived DNA, as one of damage associated molecular patterns (DAMPs), is associated with allergic type 2 immune responses. Immune recognition of such DNA generates the second messenger cyclic GMP-AMP (cGAMP) and induces type-2 immune responses; however, its role in allergic diseases, such as asthma, has not been fully elucidated. This study aimed to determine whether cGAMP could induce asthma when used as an adjuvant. We intranasally sensitized mice with cGAMP together with house dust mite antigen (HDM), followed by airway challenge with HDM. We then assessed the levels of eosinophils in the broncho-alveolar lavage fluid (BALF) and serum HDM-specific antibodies. cGAMP promoted HDM specific allergic asthma, characterized by significantly increased HDM specific IgG1 and total IgE in the serum and infiltration of eosinophils in the BALF. cGAMP stimulated lung fibroblast cells to produce IL-33 in vitro, and mice deficient for IL-33 or IL-33 receptor (ST2) failed to develop asthma enhancement by cGAMP. Not only Il-33-/- mice, but also Sting-/-, Tbk1-/-, and Irf3-/-Irf7-/- mice which lack the cGAMP-mediated innate immune activation failed to increase eosinophils in the BALF than that from wild type mice. Consistently, intranasal and oral administration of amlexanox, a TBK1 inhibitor, decreased cGAMP-induced lung allergic inflammation. Thus, cGAMP functions as a type 2 adjuvant in the lung and can promote allergic asthma in manners that dependent on the intracellular STING/TBK1/IRF3/7 signaling pathway and the resultant intercellular signaling pathway via IL-33 and ST2 might be a novel therapeutic target for allergic asthma.

Requirement for memory B-cell activation in protection from heterologous influenza virus reinfection.

While two memory compartments, memory B cells and long-lived plasma cells, are thought to contribute to the successful establishment of memory recall responses, the unique roles of each cellular compartment are still unclear. Herein, by tracing influenza anti-hemagglutinin (HA)-specific antibodies in mice, we demonstrate that pre-existing antibodies secreted by long-lived plasma cells are essential for protection from reinfection with the same influenza virus, whereas protection from secondary infection with an antigenically distinct influenza virus requires memory B-cell activation. These activated memory B cells were largely specific for the conserved HA stem region, and generated sufficient levels of antibodies for protection from heterologous reinfection. Given that the anti-stem plasmablasts derived from the memory B cells were higher affinity than those from naive B cells, our results suggest that maturation of anti-stem memory B cells during primary influenza infection and their subsequent activation are required for protection from reinfection by mutant viruses.

Rapid Quantification of NETs In Vitro and in Whole Blood Samples by Imaging Flow Cytometry.

Neutrophil extracellular trap (NET) formation involves the release of DNA outside the cell to neutralize pathogens. Techniques such as live microscopy, flow cytometry, and intravital imaging allow the characterization of NETs, but these either cannot be applied in vivo, lack specificity or require invasive procedures. We developed an automated analysis method to rapidly acquire and characterize cells as NETs or NET precursors, as opposed to cells undergoing other forms of cell death, using imaging flow cytometry. NETs were maintained in solution using a novel three-dimensional cell culture system in which cells are suspended at the interface of two liquids of different density. Critically, we identify NETs using an image analysis algorithm based on morphological data showing the extrusion of DNA beyond the cell boundaries. In vitro, we used this technique to demonstrate different requirements for NET formation in human and mouse neutrophils. We also measured NETs in whole blood during infection of mice with the malaria parasite Plasmodium yoelii. We expect this technique will provide a valuable approach to better understand the process of NET formation and its importance in disease. © 2019 International Society for Advancement of Cytometry.

A unique nanoparticulate TLR9 agonist enables a HA split vaccine to confer FcγR-mediated protection against heterologous lethal influenza virus infection.

The development of a universal influenza vaccine that can provide a robust and long-lasting protection against a broader range of influenza virus strains is a global public health priority. One approach to improve vaccine efficacy is to use an adjuvant to boost immune responses to the target antigens; nevertheless, the role of adjuvants in the context of influenza vaccines is not fully understood. We have previously developed the K3-schizophyllan (SPG) adjuvant, which is composed of nanoparticulated oligodeoxynucleotides K3, a TLR9 agonist, with SPG, a non-agonistic ß-glucan ligand of Dectin-1. In this study, K3-SPG given with conventional influenza hemagglutinin (HA) split vaccine (K3-SPG HA) conferred protection against antigenically mismatched heterologous virus challenge. While K3-SPG HA elicited robust cross-reactive HA-specific IgG2c and CD8 T-cell responses, CD8 T-cell depletion had no impact on this cross-protection. In contrast, K3-SPG HA was not able to confer protection against heterologous virus challenge in FcRγ-deficient mice. Our results indicated that FcγR-mediated antibody responses induced by the HA antigen and K3-SPG adjuvant were important for potent protection against antigenically mismatched influenza virus infection. Thus, we demonstrated that the K3-SPG-adjuvanted vaccine strategy broadens protective immunity against influenza and provides a basis for the development of next-generation influenza vaccines.

DAMP-Inducing Adjuvant and PAMP Adjuvants Parallelly Enhance Protective Type-2 and Type-1 Immune Responses to Influenza Split Vaccination.

Recently, it was reported that 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CyD), a common pharmaceutical additive, can act as a vaccine adjuvant to enhance protective type-2 immunogenicity to co-administered seasonal influenza split vaccine by inducing host-derived damage-associated molecular patterns (DAMPs). However, like most other DAMP-inducing adjuvants such as aluminum hydroxide (Alum), HP-ß-CyD may not be sufficient for the induction of protective type-1 (cellular) immune responses, thereby leaving room for improvement. Here, we demonstrate that a combination of HP-ß-CyD with a humanized TLR9 agonist, K3 CpG-ODN, a potent pathogen-associated molecular pattern (PAMP), enhanced the protective efficacy of the co-administered influenza split vaccine by inducing antigen-specific type-2 and type-1 immune responses, respectively. Moreover, substantial antigen-specific IgE induction by HP-ß-CyD, which can cause an allergic response to immunized antigen was completely suppressed by the addition of K3 CpG-ODN. Furthermore, HP-ß-CyD- and K3 CpG-ODN-adjuvanted influenza split vaccination protected the mice against lethal challenge with high doses of heterologous influenza virus, which could not be protected against by single adjuvant vaccines. Further experiments using gene deficient mice revealed the unique immunological mechanism of action in vivo, where type-2 and type-1 immune responses enhanced by the combined adjuvants were dependent on TBK1 and TLR9, respectively, indicating their parallel signaling pathways. Finally, the analysis of immune responses in the draining lymph node suggested that HP-ß-CyD promotes the uptake of K3 CpG-ODN by plasmacytoid dendritic cells and B cells, which may contributes to the activation of these cells and enhanced production of IgG2c. Taken together, the results above may offer potential clinical applications for the combination of DAMP-inducing adjuvant and PAMP adjuvant to improve vaccine immunogenicity and efficacy by enhancing both type-2 and type-1 immune responses in a parallel manner.

Recombinant production and characterization of human anti-influenza virus monoclonal antibodies identified from hybridomas fused with human lymphocytes.

In previous studies, hybridomas producing human immunoglobulin G, the antibodies 5E4 and 5A7 against influenza A and B virus were established using a novel human lymphocyte fusion partner, SPYMEG. In the present study, we succeeded in achieving the recombinant production and secretion of 5E4 and 5A7 in Chinese hamster ovary cells. Our N-glycan analysis by intact-mass detection and liquid chromatography mass spectrometry showed that recombinant 5E4 and 5A7 have one N-glycan and the typical mammalian-type N-glycan structures similar to those in hybridomas. However, the glycan distribution was slightly different among these antibodies. The amount of high-mannose-type structures was under 10% of the total N-glycans of recombinant 5E4 and 5A7, compared to 20% of the 5E4 and 5A7 produced in hybridomas. The amount of galactosylated N-glycans was increased in recombinants. Approximately 80% of the N-glycans of all antibodies was fucosylated, and no sialylated N-glycan was found. Recombinant 5E4 and 5A7 neutralized pandemic influenza A virus specifically, and influenza B virus broadly, quite similar to the 5E4 and 5A7 produced in hybridomas, respectively. Here we demonstrated that recombinants of antibodies identified from hybridomas fused with SPYMEG have normal N-glycans and that their neutralizing activities bear comparison with those of the original antibodies.

RNA is an Adjuvanticity Mediator for the Lipid-Based Mucosal Adjuvant, Endocine.

Nasal vaccination has the potential to elicit systemic and mucosal immunity against pathogens. However, split and subunit vaccines lack potency at stimulating mucosal immunity, and an adjuvant is indispensable for eliciting potent mucosal immune response to nasal vaccines. Endocine, a lipid-based mucosal adjuvant, potentiates both systemic and mucosal immune responses. Although Endocine has shown efficacy and tolerability in animal and clinical studies, its mechanism of action remains unknown. It has been reported recently that endogenous danger signals are essential for the effects of some adjuvants such as alum or MF59. However, the contribution of danger signals to the adjuvanticity of Endocine has not been explored. Here, we show that RNA is likely to be an important mediator for the adjuvanticity of Endocine. Administration of Endocine generated nucleic acids release, and activated dendritic cells (DCs) in draining lymph nodes in vivo. These results suggest the possibility that Endocine indirectly activates DCs via damage-associated molecular patterns. Moreover, the adjuvanticity of Endocine disappeared in mice lacking TANK-binding kinase 1 (Tbk1), which is a downstream molecule of nucleic acid sensing signal pathway. Furthermore, co-administration of RNase A reduced the adjuvanticity of Endocine. These data suggest that RNA is important for the adjuvanticity of Endocine.

Intranasal hydroxypropyl-ß-cyclodextrin-adjuvanted influenza vaccine protects against sub-heterologous virus infection.

Intranasal vaccination with inactivated influenza viral antigens is an attractive and valid alternative to currently available influenza (flu) vaccines; many of which seem to need efficient and safe adjuvant, however. In this study, we examined whether hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a widely used pharmaceutical excipient to improve solubility and drug delivery, can act as a mucosal adjuvant for intranasal flu vaccines. We found that intranasal immunization of mice with hemagglutinin split- as well as inactivated whole-virion influenza vaccine with HP-ß-CD resulted in secretion of antigen-specific IgA and IgGs in the airway mucosa and the serum as well. As a result, both HP-ß-CD adjuvanted-flu intranasal vaccine protected mice against lethal challenge with influenza virus, equivalent to those induced by experimental cholera toxin-adjuvanted ones. Of note, intranasal use of HP-ß-CD as an adjuvant induced significantly lower antigen-specific IgE responses than that induced by aluminum salt adjuvant. These results suggest that HP-ß-CD may be a potent mucosal adjuvant for seasonal and pandemic influenza vaccine.

A novel dual luciferase assay for the simultaneous monitoring of HIV infection and cell viability.

Human immunodeficiency virus type 1 (HIV-1) reporter cell lines are critical tools for drug development. However, one disadvantage of HIV-1 reporter cell lines is that reductions in reporter gene activity need to be normalized to cytotoxicity, i.e., live cell numbers. Here, we developed a dual luciferase assay based on a R. reniformis luciferase (hRLuc)-expressing R5-type HIV-1 (NLAD8-hRLuc) and a CEM cell line expressing CCR5 and firefly luciferase (R5CEM-FiLuc). The NLAD8-hRLuc reporter virus was replication competent in peripheral blood mononuclear cells. The level of hRLuc was correlated with p24 antigen levels (p<0.001, R=0.862). The target cell line, R5CEM-FiLuc, stably expressed the firefly luciferase (FiLuc) reporter gene and allowed the simultaneous monitoring of compound cytotoxicity. The dual reporter assay combining a NLAD8-hRLuc virus with R5CEM-FiLuc cells permitted the accurate determination of drug susceptibility for entry, reverse transcriptase, integrase, and protease inhibitors at different multiplicities of infection. This dual reporter assay provides a rapid and direct method for the simultaneous monitoring of HIV infection and cell viability.

Species-dependent role of type I IFNs and IL-12 in the CTL response induced by humanized CpG complexed with ß-glucan.

CpG oligodeoxynucleotide (ODN) is one of promising nucleic acid-based adjuvants. We recently improved its ability to enhance CD8(+) T-cell responses to coadministered protein antigen without conjugation or emulsion, by forming a nanoparticulate complex between CpG ODN (K3) and mushroom-derived ß-glucan schizophyllan (SPG), namely K3-SPG. Here, we sought to elucidate the cellular immunological mechanisms by which K3-SPG induce such potent CD8(+) T-cell responses to coadministered antigen. By focusing on two DC subsets, plasmacytoid DCs and CD8α(+) DCs, as well as the secreted cytokines, IFN-α and IL-12, we found that K3-SPG strongly activates mouse plasmacytoid DCs to secrete IFN-α and CD8α(+) DCs to secrete IL-12, respectively. Although a single cytokine deficiency had no impact on adjuvant effects, the lack of both type I IFN and IL-12 in mice resulted in a significant reduction of Th1 type immune responses and CD8(+) T-cell responses elicited by protein vaccine model. By sharp contrast, type I IFN, but not IL-12, was required for the production of IFN-γ by human PBMCs as well as antigen-specific CD8(+) T-cell proliferation. Taken together, K3-SPG may overcome the species barrier for CpG ODN to enhance antigen-specific CD8(+) T-cell responses despite the differential role of IL-12 between human and mice.