Inhaled Fine Particles Induce Alveolar Macrophage Death and Interleukin-1α Release to Promote Inducible Bronchus-Associated Lymphoid Tissue Formation.

Particulate pollution is thought to function as an adjuvant that can induce allergic responses. However, the exact cell types and immunological factors that initiate the lung-specific immune responses are unclear. We found that upon intratracheal instillation, particulates such as aluminum salts and silica killed alveolar macrophages (AMs), which then released interleukin-1α (IL-1α) and caused inducible bronchus-associated lymphoid tissue (iBALT) formation in the lung. IL-1α release continued for up to 2 weeks after particulate exposure, and type-2 allergic immune responses were induced by the inhalation of antigen during IL-1α release and iBALT formation, even long after particulate instillation. Recombinant IL-1α was sufficient to induce iBALTs, which coincided with subsequent immunoglobulin E responses, and IL-1-receptor-deficient mice failed to induce iBALT formation. Therefore, the AM-IL-1α-iBALT axis might be a therapeutic target for particulate-induced allergic inflammation.

RAGE in circulating immune cells is fundamental for hippocampal inflammation and cognitive decline in a mouse model of latent chronic inflammation.

BACKGROUND: Latent chronic inflammation has been proposed as a key mediator of multiple derangements in metabolic syndrome (MetS), which are increasingly becoming recognized as risk factors for age-related cognitive decline. However, the question remains whether latent chronic inflammation indeed induces brain inflammation and cognitive decline. METHODS: A mouse model of latent chronic inflammation was constructed by a chronic subcutaneous infusion of low dose lipopolysaccharide (LPS) for four weeks. A receptor for advanced glycation end products (RAGE) knockout mouse, a chimeric myeloid cell specific RAGE-deficient mouse established by bone marrow transplantation and a human endogenous secretory RAGE (esRAGE) overexpressing adenovirus system were utilized to examine the role of RAGE in vivo. The cognitive function was examined by a Y-maze test, and the expression level of genes was determined by quantitative RT-PCR, western blot, immunohistochemical staining, or ELISA assays. RESULTS: Latent chronic inflammation induced MetS features in C57BL/6J mice, which were associated with cognitive decline and brain inflammation characterized by microgliosis, monocyte infiltration and endothelial inflammation, without significant changes in circulating cytokines including TNF-α and IL-1ß. These changes as well as cognitive impairment were rescued in RAGE knockout mice or chimeric mice lacking RAGE in bone marrow cells. P-selectin glycoprotein ligand-1 (PSGL-1), a critical adhesion molecule, was induced in circulating mononuclear cells in latent chronic inflammation in wild-type but not RAGE knockout mice. These inflammatory changes and cognitive decline induced in the wild-type mice were ameliorated by an adenoviral increase in circulating esRAGE. Meanwhile, chimeric RAGE knockout mice possessing RAGE in myeloid cells were still resistant to cognitive decline and brain inflammation. CONCLUSIONS: These findings indicate that RAGE in inflammatory cells is necessary to mediate stimuli of latent chronic inflammation that cause brain inflammation and cognitive decline, potentially by orchestrating monocyte activation via regulation of PSGL-1 expression. Our results also suggest esRAGE-mediated inflammatory regulation as a potential therapeutic option for cognitive dysfunction in MetS with latent chronic inflammation.

Anti-tumor immunity by transcriptional synergy between TLR9 and STING activation.

Agonists for TLR9 and stimulator of IFN genes (STING) offer therapeutic applications as both anti-tumor agents and vaccine adjuvants, though their clinical applications are limited; the clinically available TLR9 agonist is a weak IFN inducer and STING agonists induce undesired type 2 immunity. Yet, combining TLR9 and STING agonists overcame these limitations by synergistically inducing innate and adaptive IFNγ to become an advantageous type 1 adjuvant, suppressing type 2 immunity, in addition to exerting robust anti-tumor activities when used as a monotherapeutic agent for cancer immunotherapy. Here, we sought to decipher the immunological mechanisms behind the synergism mediated by TLR9 and STING agonists and found that their potent anti-tumor immunity in a Pan02 peritoneal dissemination model of pancreatic cancer was achieved only when agonists for TLR9 and STING were administered locally, and was via mechanisms involving CD4 and CD8 T cells as well as the co-operative action of IL-12 and type I IFNs. Rechallenge studies of long-term cancer survivors suggested that the elicitation of Pan02-specific memory responses provides protection against the secondary tumor challenge. Mechanistically, we found that TLR9 and STING agonists synergistically induce IL-12 and type I IFN production in murine APCs. The synergistic effect of the TLR9 and STING agonists on IL-12p40 was at protein, mRNA and promoter activation levels, and transcriptional regulation was mediated by a 200 bp region situated 983 bp upstream of the IL-12p40 transcription initiation site. Such intracellular transcriptional synergy may hold a key in successful cancer immunotherapy and provide further insights into dual agonism of innate immune sensors during host homeostasis and diseases.

CpG ODN (K3)-toll-like receptor 9 agonist-induces Th1-type immune response and enhances cytotoxic activity in advanced lung cancer patients: a phase I study.

BACKGROUND: Cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) (K3)-a novel synthetic single-stranded DNA immune adjuvant for cancer immunotherapy-induces a potential Th1-type immune response against cancer cells. We conducted a phase I study of CpG ODN (K3) in patients with lung cancer to assess its safety and patients' immune responses. METHODS: The primary endpoint was the proportion of dose-limiting toxicities (DLTs) at each dose level. Secondary endpoints included safety profile, an immune response, including dynamic changes in immune cell and cytokine production, and progression-free survival (PFS). In a 3 + 3 dose-escalation design, the dosage levels for CpG ODN (K3) were 5 or 10 mg/body via subcutaneous injection and 0.2 mg/kg via intravenous administration on days 1, 8, 15, and 29. RESULTS: Nine patients (eight non-small-cell lung cancer; one small-cell lung cancer) were enrolled. We found no DLTs at any dose level and observed no serious treatment-related adverse events. The median observation period after registration was 55 days (range: 46-181 days). Serum IFN-α2 levels, but not inflammatory cytokines, increased in six patients after the third administration of CpG ODN (K3) (mean value: from 2.67 pg/mL to 3.61 pg/mL after 24 hours). Serum IFN-γ (mean value, from 9.07 pg/mL to 12.7 pg/m after 24 hours) and CXCL10 levels (mean value, from 351 pg/mL to 676 pg/mL after 24 hours) also increased in eight patients after the third administration. During the treatment course, the percentage of T-bet-expressing CD8+ T cells gradually increased (mean, 49.8% at baseline and 59.1% at day 29, p = 0.0273). Interestingly, both T-bet-expressing effector memory (mean, 52.7% at baseline and 63.7% at day 29, p = 0.0195) and terminally differentiated effector memory (mean, 82.3% at baseline and 90.0% at day 29, p = 0.0039) CD8+ T cells significantly increased. The median PFS was 398 days. CONCLUSIONS: This is the first clinical study showing that CpG ODN (K3) activated innate immunity and elicited Th1-type adaptive immune response and cytotoxic activity in cancer patients. CpG ODN (K3) was well tolerated at the dose settings tested, although the maximum tolerated dose was not determined. TRIAL REGISTRATION: UMIN-CTR number 000023276. Registered 1 September 2016, https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000026649.

Thymic stromal lymphopoietin contributes to protection of mice from Strongyloides venezuelensis infection by CD4+ T cell-dependent and -independent pathways.

When animals are infected with helminthic parasites, resistant hosts mount type II helper T (Th2) immune responses to expel worms. Recent studies have clearly shown that epithelial cell-derived cytokines contribute to the induction of Th2 immune responses. Here we demonstrate the role of endogenous thymic stromal lymphopoietin (TSLP) for protection against Strongyloides venezuelensis (S. venezuelensis) infection, utilizing TSLP receptor-deficient Crlf2-/- mice. The number of eggs per gram of feces (EPG) and worm burden were significantly higher in Crlf2-/- mice than in wild type (WT) mice. S. venezuelensis infection induced Tslp mRNA expression in the skin, lung, and intestine and also facilitated the accumulation of mast cells in the intestine in a TSLP-dependent manner. Furthermore, CD4+ T cells from S. venezuelensis-infected Crlf2-/- mice showed diminished capacity to produce Th2 cytokines in the early stage of infection. Finally, CD4+ cell-depleted Crlf2-/- mice still showed higher EPG counts and worm burden than CD4+ cell-depleted WT mice, indicating that TSLP contributes to protecting mice against S. venezuelensis infection in both CD4+ T cell-dependent and -independent manners.

Murine Cross-Reactive Nonneutralizing Polyclonal IgG1 Antibodies Induced by Influenza Vaccine Inhibit the Cross-Protective Effect of IgG2 against Heterologous Virus in Mice.

Annual vaccination against influenza viruses is the most reliable and efficient way to prevent and control annual epidemics and protect from severe influenza disease. However, current split influenza vaccines are generally not effective against antigenically mismatched (heterologous) strains. To broaden the protective spectrum of influenza vaccines, adjuvants that can induce cross-reactive antibodies with cross-protection via Fc-mediated effector functions are urgently sought. Although IgG2 antibodies are generally more efficient than IgG1 antibodies in Fc-mediated effector functions, it is not yet clear which IgG isotypes show superior cross-protection against heterologous strains. It also remains unclear whether these IgG isotypes interfere with each other's protective effects. Here, we found that influenza split vaccine adjuvanted with aluminum salts, which predominantly induce cross-reactive IgG1, did not confer cross-protection against heterologous virus challenge in mice. In contrast, split vaccine adjuvanted with CpG oligodeoxynucleotides, which predominantly induce cross-reactive IgG2, showed cross-protection through the interaction of cross-reactive nonneutralizing IgG2 and alveolar macrophages, indicating the importance of cross-reactive nonneutralizing IgG2 for cross-protection. Furthermore, by using serum samples from immunized mice and isolated polyclonal antibodies, we show that vaccine-induced cross-reactive nonneutralizing IgG1 suppress the cross-protective effects of IgG2 by competitively inhibiting the binding of IgG2 to virus. Thus, we demonstrate the new concept that cross-reactive IgG1 may interfere with the potential for cross-protection of influenza vaccine. We propose that adjuvants that selectively induce virus-specific IgG2 in mice, such as CpG oligodeoxynucleotides, are optimal for heterologous protection.IMPORTANCE Current influenza vaccines are generally effective against highly similar virus strains by inducing neutralizing antibodies. However, these antibodies fail to neutralize antigenically mismatched (heterologous) strains and therefore provide limited protection against them. Efforts are being made to develop vaccines with cross-protective ability that would protect broadly against heterologous strains, because the mismatch between predicted and epidemic strains cannot always be avoided, resulting in low vaccine efficacy. Here, we show that nonneutralizing IgG2 antibodies induced by an optimal adjuvant play a crucial role in cross-protection against heterologous virus challenge in mice. Furthermore, nonneutralizing polyclonal IgG1 suppressed the cross-protective effects of nonneutralizing polyclonal IgG2 by competitively blocking the binding of IgG2 to its antigen. These data shed new light on the importance of IgG isotypes and the selection of appropriate adjuvants for the development of universal influenza vaccines. Furthermore, our findings are applicable to the rational design of vaccines against other pathogens.

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.

Lung fibroblasts produce IL-33 in response to stimulation with retinoblastoma-binding protein 9 via production of prostaglandin E2.

Intestinal nematode infection induces pulmonary eosinophilia via IL-33, although the mechanism of pulmonary IL-33 induction remains unclear. Because nematode migration damages lungs, we speculated that lung-derived damage-associated molecular patterns (DAMPs) possess an IL-33-inducing activity (IL33ia). Indeed, intra-nasal administration of a lung extract induced IL-33 production in lungs. Additionally, lung extracts increased Il33 mRNA expression in primary lung fibroblasts. Proteomic analysis identified retinoblastoma-binding protein 9 (RBBP9) as a major DAMP with IL33ia. RBBP9 was originally discovered as a protein that provides cells with resistance to the growth inhibitory effect of transforming growth factor (TGF)-ß1. Here, we found that stimulation by RBBP9 induced primary fibroblasts to produce prostaglandin E2 (PGE2) that, in turn, induced fibroblasts to produce IL-33. RBBP9-activated fibroblasts expressed mRNAs of cyclooxygenase-2 (COX-2) and PGE2 synthase-1 that convert arachidonic acid to PGE2. Furthermore, they expressed PGE2 receptors E-prostanoid (EP) 2 and EP4. Thus, treatment with a COX-2 inhibitor or EP2 and/or EP4 receptor antagonists inhibited RBBP9-induced IL-33 production. Nematode infection induced pulmonary Il33 mRNA expression, which was inhibited by the COX-2 inhibitor or EP2 and EP4 antagonists, suggesting that nematode infection induced pulmonary Il33 mRNA via PGE2. RBBP9 was expressed constitutively in the lung in the steady state, which did not increase after nematode infection. Finally, we found that Rbbp9-deficient mice had a significantly diminished capacity to increase pulmonary Il33 mRNA expression following nematode infection. Thus, the PGE2-EP2/EP4 pathway activated by RBBP9 released from damaged lungs is important for pulmonary IL-33 production in nematode-infected animals.

B cell-intrinsic MyD88 signaling controls IFN-γ-mediated early IgG2c class switching in mice in response to a particulate adjuvant.

Adjuvants improve the potency of vaccines, but the modes of action (MOAs) of most adjuvants are largely unknown. TLR-dependent and -independent innate immune signaling through the adaptor molecule MyD88 has been shown to be pivotal to the effects of most adjuvants; however, MyD88's involvement in the TLR-independent MOAs of adjuvants is poorly understood. Here, using the T-dependent antigen NIPOVA and a unique particulate adjuvant called synthetic hemozoin (sHZ), we show that MyD88 is required for early GC formation and enhanced antibody class-switch recombination (CSR) in mice. Using cell-type-specific MyD88 KO mice, we found that IgG2c class switching, but not IgG1 class switching, was controlled by B cell-intrinsic MyD88 signaling. Notably, IFN-γ produced by various cells including T cells, NK cells, and dendritic cells was the primary cytokine for IgG2c CSR and B-cell intrinsic MyD88 is required for IFN-γ production. Moreover, IFN-γ receptor (IFNγR) deficiency abolished sHZ-induced IgG2c production, while recombinant IFN-γ administration successfully rescued IgG2c CSR impairment in mice lacking B-cell intrinsic MyD88. Together, our results show that B cell-intrinsic MyD88 signaling is involved in the MOA of certain particulate adjuvants and this may enhance our specific understanding of how adjuvants and vaccines work.

Cholera toxin B induces interleukin-1ß production from resident peritoneal macrophages through the pyrin inflammasome as well as the NLRP3 inflammasome.

Cholera toxin B (CTB) is a subunit of cholera toxin, a bacterial enterotoxin secreted by Vibrio cholerae and also functions as an immune adjuvant. However, it remains unclear how CTB activates immune cells. We here evaluated whether or how CTB induces production of a pro-inflammatory cytokine, interleukin-1ß (IL-1ß). CTB induced IL-1ß production not only from bone marrow-derived macrophages (BMMs) but also from resident peritoneal macrophages in synergy with O111:B4-derived lipopolysaccharide (LPS O111:B4) that can bind to CTB. Meanwhile, when prestimulated with O55:B5-derived LPS (LPS O55:B5) that fails to bind to CTB, resident peritoneal macrophages, but not BMMs, produced IL-1ß in response to CTB. The CTB-induced IL-1ß production in synergy with LPS in both peritoneal macrophages and BMMs was dependent on ganglioside GM1, which is required for internalization of CTB. Notably, not only the NLRP3 inflammasome but also the pyrin inflammasome were involved in CTB-induced IL-1ß production from resident peritoneal macrophages, while only the NLRP3 inflammasome was involved in that from BMMs. In response to CTB, a Rho family small GTPase, RhoA, which activates pyrin inflammasome upon various kinds of biochemical modification, increased its phosphorylation at serine-188 in a GM1-dependent manner. This phosphorylation as well as CTB-induced IL-1ß productions were dependent on protein kinase A (PKA), indicating critical involvement of PKA-dependent RhoA phosphorylation in CTB-induced IL-1ß production. Taken together, these results suggest that CTB, incorporated through GM1, can activate resident peritoneal macrophages to produce IL-1ß in synergy with LPS through novel mechanisms in which pyrin as well as NLRP3 inflammasomes are involved.

Silica crystals and aluminum salts regulate the production of prostaglandin in macrophages via NALP3 inflammasome-independent mechanisms.

Particulates such as silica crystal (silica) and aluminum salts (alum) activate the inflammasome and induce the secretion of proinflammatory cytokines in macrophages. These particulates also induce the production of immunoglobulin E via a T helper 2 (Th2) cell-associated mechanism. However, the mechanism involved in the induction of type 2 immunity has not been elucidated. Here, we showed that silica and alum induced lipopolysaccharide-primed macrophages to produce the lipid mediator prostaglandin E2 (PGE2) and interleukin-1ß (IL-1ß). Macrophages deficient in the inflammasome components caspase 1, NALP3, and ASC revealed that PGE2 production was independent of the NALP3 inflammasome. PGE2 expression was markedly reduced in PGE synthase-deficient (Ptges⁻/⁻) macrophages, and Ptges⁻/⁻ mice displayed reduced antigen-specific serum IgE concentrations after immunization with alum or silica. Our results indicate that silica and alum regulate the production of PGE2 and that the induction of PGE2 by particulates controls the immune response in vivo.

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.

Immunological association of inducible bronchus-associated lymphoid tissue organogenesis in Ag85B-rHPIV2 vaccine-induced anti-tuberculosis mucosal immune responses in mice.

We previously reported that Ag85B-expressing human parainfluenza type 2 virus (Ag85B-rHPIV2) was effective as a nasal vaccine against tuberculosis in mice; however, the mechanism by which it induces an immune response remains to be investigated. In the present study, we found that organogenesis of inducible bronchus-associated lymphoid tissue (iBALT) played a role in the induction of antigen-specific T cells and IgA antibody responses in the lung of mice intra-nasally administered Ag85B-rHPIV2. We found that expression of Ag85B was dispensable for the development of iBALT, suggesting that HPIV2 acted as an iBALT-inducing vector. When iBALT organogenesis was disrupted in Ag85B-rHPIV2-immunized mice, either by neutralization of the lymphotoxin pathway or depletion of CD11b+ cells, Ag85B-specific immune responses (i.e. IFN γ-producing T cells and IgA antibody) were diminished in the lung. Furthermore, we found that immunization with Ag85B-rHPIV2 induced neutrophil and eosinophil infiltration temporally after the immunization in the lung. Thus, our results show that iBALT organogenesis contributes to the induction of antigen-specific immune responses by Ag85B-rHPIV2 and that Ag85B-rHPIV2 provokes its immune responses without inducing long-lasting inflammation.

CD63-Mediated Antigen Delivery into Extracellular Vesicles via DNA Vaccination Results in Robust CD8+ T Cell Responses.

DNA vaccines are attractive immunogens for priming humoral and cellular immune responses to the encoded Ag. However, their ability to induce Ag-specific CD8+ T cell responses requires improvement. Among the strategies for improving DNA vaccine immunogenicity are booster vaccinations, alternate vaccine formulations, electroporation, and genetic adjuvants, but few, such as extracellular vesicles (EVs), target natural Ag delivery systems. By focusing on CD63, a tetraspanin protein expressed on various cellular membranes, including EVs, we examined whether a DNA vaccine encoding an Ag fused to CD63 delivered into EVs would improve vaccine immunogenicity. In vitro transfection with plasmid DNA encoding an OVA Ag fused to CD63 (pCD63-OVA) produced OVA-carrying EVs. Immunizations with the purified OVA-carrying EVs primed naive mice to induce OVA-specific CD4+ and CD8+ T cells, whereas immunization with EVs purified from cells transfected with control plasmids encoding OVA protein alone or a calnexin-OVA fusion protein delivered into the endoplasmic reticulum failed to do so. Vaccinating mice with pCD63-OVA induced potent Ag-specific T cell responses, particularly those from CD8+ T cells. CD63 delivery into EVs led to better CD8+ T cell responses than calnexin delivery into the endoplasmic reticulum. When we used a mouse tumor implantation model to evaluate pCD63-OVA as a therapeutic vaccine, the EV-delivered DNA vaccination significantly inhibited tumor growth compared with the control DNA vaccinations. These results indicate that EV Ag delivery via DNA vaccination offers a new strategy for eliciting strong CD8+ T cell responses to the encoded Ag, making it a potentially useful cancer vaccine.

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.

Vaccine adjuvants as potential cancer immunotherapeutics.

Accumulated evidence obtained from various clinical trials and animal studies suggested that cancer vaccines need better adjuvants than those that are currently licensed, which include the most commonly used alum and incomplete Freund's adjuvant, because of either a lack of potent anti-tumor immunity or the induction of undesired immunity. Several clinical trials using immunostimulatory adjuvants, particularly agonistic as well as non-agonistic ligands for TLRs, C-type lectin receptors, retinoic acid-inducible gene I-like receptors and stimulator of interferon genes, have revealed their therapeutic potential not only as vaccine adjuvants but also as anti-tumor agents. Recently, combinations of such immunostimulatory or immunomodulatory adjuvants have shown superior efficacy over their singular use, suggesting that seeking optimal combinations of the currently available or well-characterized adjuvants may provide a better chance for the development of novel adjuvants for cancer immunotherapy.

Hydroxypropyl-ß-cyclodextrin spikes local inflammation that induces Th2 cell and T follicular helper cell responses to the coadministered antigen.

Cyclodextrins are commonly used as a safe excipient to enhance the solubility and bioavailability of hydrophobic pharmaceutical agents. Their efficacies and mechanisms as drug-delivery systems have been investigated for decades, but their immunological properties have not been examined. In this study, we reprofiled hydroxypropyl-ß-cyclodextrin (HP-ß-CD) as a vaccine adjuvant and found that it acts as a potent and unique adjuvant. HP-ß-CD triggered the innate immune response at the injection site, was trapped by MARCO(+) macrophages, increased Ag uptake by dendritic cells, and facilitated the generation of T follicular helper cells in the draining lymph nodes. It significantly enhanced Ag-specific Th2 and IgG Ab responses as potently as did the conventional adjuvant, aluminum salt (alum), whereas its ability to induce Ag-specific IgE was less than that of alum. At the injection site, HP-ß-CD induced the temporary release of host dsDNA, a damage-associated molecular pattern. DNase-treated mice, MyD88-deficient mice, and TBK1-deficient mice showed significantly reduced Ab responses after immunization with this adjuvant. Finally, we demonstrated that HP-ß-CD-adjuvanted influenza hemagglutinin split vaccine protected against a lethal challenge with a clinically isolated pandemic H1N1 influenza virus, and the adjuvant effect of HP-ß-CD was demonstrated in cynomolgus macaques. Our results suggest that HP-ß-CD acts as a potent MyD88- and TBK1-dependent T follicular helper cell adjuvant and is readily applicable to various vaccines.

Nonagonistic Dectin-1 ligand transforms CpG into a multitask nanoparticulate TLR9 agonist.

CpG DNA, a ligand for Toll-like receptor 9 (TLR9), has been one of the most promising immunotherapeutic agents. Although there are several types of potent humanized CpG oligodeoxynucleotide (ODN), developing "all-in-one" CpG ODNs activating both B cells and plasmacytoid dendritic cells forming a stable nanoparticle without aggregation has not been successful. In this study, we generated a novel nanoparticulate K CpG ODN (K3) wrapped by the nonagonistic Dectin-1 ligand schizophyllan (SPG), K3-SPG. In sharp contrast to K3 alone, K3-SPG stimulates human peripheral blood mononuclear cells to produce a large amount of both type I and type II IFN, targeting the same endosome where IFN-inducing D CpG ODN resides without losing its K-type activity. K3-SPG thus became a potent adjuvant for induction of both humoral and cellular immune responses, particularly CTL induction, to coadministered protein antigens without conjugation. Such potent adjuvant activity of K3-SPG is attributed to its nature of being a nanoparticle rather than targeting Dectin-1 by SPG, accumulating and activating antigen-bearing macrophages and dendritic cells in the draining lymph node. K3-SPG acting as an influenza vaccine adjuvant was demonstrated in vivo in both murine and nonhuman primate models. Taken together, K3-SPG may be useful for immunotherapeutic applications that require type I and type II IFN as well as CTL induction.

TLR9 and STING agonists synergistically induce innate and adaptive type-II IFN.

Agonists for TLR9 and Stimulator of IFN Gene (STING) act as vaccine adjuvants that induce type-1 immune responses. However, currently available CpG oligodeoxynucleotide (ODN) (K-type) induces IFNs only weakly and STING ligands rather induce type-2 immune responses, limiting their potential therapeutic applications. Here, we show a potent synergism between TLR9 and STING agonists. Together, they make an effective type-1 adjuvant and an anticancer agent. The synergistic effect between CpG ODN (K3) and STING-ligand cyclic GMP-AMP (cGAMP), culminating in NK cell IFN-γ (type-II IFN) production, is due to the concurrent effects of IL-12 and type-I IFNs, which are differentially regulated by IRF3/7, STING, and MyD88. The combination of CpG ODN with cGAMP is a potent type-1 adjuvant, capable of inducing strong Th 1-type responses, as demonstrated by enhanced antigen-specific IgG2c and IFN-γ production, as well as cytotoxic CD8(+) T-cell responses. In our murine tumor models, intratumoral injection of CpG ODN and cGAMP together reduced tumor size significantly compared with the singular treatments, acting as an antigen-free anticancer agent. Thus, the combination of CpG ODN and a STING ligand may offer therapeutic application as a potent type-II IFN inducer.

Evaluation of Pulmonary Toxicity of Zinc Oxide Nanoparticles Following Inhalation and Intratracheal Instillation.

We conducted inhalation and intratracheal instillation studies of zinc oxide (ZnO) nanoparticles in order to examine their pulmonary toxicity. F344 rats were received intratracheal instillation at 0.2 or 1 mg of ZnO nanoparticles with a primary diameter of 35 nm that were well-dispersed in distilled water. Cell analysis and chemokines in bronchoalveolar lavage fluid (BALF) were analyzed at three days, one week, one month, three months, and six months after the instillation. As the inhalation study, rats were exposed to a concentration of inhaled ZnO nanoparticles (2 and 10 mg/m³) for four weeks (6 h/day, 5 days/week). The same endpoints as in the intratracheal instillation study were analyzed at three days, one month, and three months after the end of the exposure. In the intratracheal instillation study, both the 0.2 and the 1.0 mg ZnO groups had a transient increase in the total cell and neutrophil count in the BALF and in the expression of cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2, chemokine for neutrophil, and heme oxygenase-1 (HO-1), an oxidative stress marker, in the BALF. In the inhalation study, transient increases in total cell and neutrophil count, CINC-1,-2 and HO-1 in the BALF were observed in the high concentration groups. Neither of the studies of ZnO nanoparticles showed persistent inflammation in the rat lung, suggesting that well-dispersed ZnO nanoparticles have low toxicity.