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Summary: Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1β (IL-1β), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarify the roles of inositol-requiring enzyme 1 alpha (IRE1α), an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1β production in RPMs. In RPMs, CTB is incorporated into the ER and induces ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1α-deficient RPMs show a significant impairment of CT- or CTB-induced IL-1β production, indicating that IRE1α is required for CT- or CTB-induced IL-1β production in RPMs. This study demonstrates the critical roles of IRE1α in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages.

5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a mouse-selective stimulator of interferon gene (STING) agonist exerting STING-dependent anti-tumor activity. Although DMXAA cannot fully activate human STING, DMXAA reached phase III in lung cancer clinical trials. How DMXAA is effective against human lung cancer is completely unknown. Here, we show that DMXAA is a partial STING agonist interfering with agonistic STING activation, which may explain its partial anti-tumor effect observed in humans, as STING was reported to be pro-tumorigenic for lung cancer cells with low antigenicity. Furthermore, we developed a DMXAA derivative—3-hydroxy-5-(4-hydroxybenzyl)-4-methyl-9H-xanthen-9-one (HHMX)—that can potently antagonize STING-mediated immune responses both in humans and mice. Notably, HHMX suppressed aberrant responses induced by STING gain-of-function mutations causing STING-associated vasculopathy with onset in infancy (SAVI) in in vitro experiments. Furthermore, HHMX treatment suppressed aberrant STING pathway activity in peripheral blood mononuclear cells from SAVI patients. Lastly, HHMX showed a potent therapeutic effect in SAVI mouse model by mitigating disease progression. Thus, HHMX offers therapeutic potential for STING-associated autoinflammatory diseases.

Abstract 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

Summary: Efficient delivery of subunit vaccines to dendritic cells (DCs) is necessary to improve vaccine efficacy, because the vaccine antigen alone cannot induce sufficient protective immunity. Here, we identified DC-targeting peptides using a phage display system and demonstrated the potential of these peptides as antigen-delivery carriers to improve subunit vaccine effectiveness in mice. The fusion of antigen proteins and peptides with DC-targeting peptides induced strong antigen-specific IgG responses, even in the absence of adjuvants. In addition, the DC-targeting peptide improved the distribution of antigens to DCs and antigen presentation by DCs. The combined use of an adjuvant with a DC-targeting peptide improved the effectiveness of the vaccine. Furthermore, nucleolin, located on the DC surface, was identified as the receptor for DC-targeting peptide, and nucleolin was indispensable for the vaccine effect of the DC-targeting peptide. Overall, the findings of this study could be useful for developing subunit vaccines against infectious diseases.

Adjuvants are important vaccine components, composed of a variety of chemical and biological materials that enhance the vaccine antigen-specific immune responses by stimulating the innate immune cells in both direct and indirect manners to produce a variety cytokines, chemokines, and growth factors. It has been developed by empirical methods for decades and considered difficult to choose a single screening method for an ideal vaccine adjuvant, due to their diverse biochemical characteristics, complex mechanisms of, and species specificity for their adjuvanticity. We therefore established a robust adjuvant screening strategy by combining multiparametric analysis of adjuvanticity in vivo and immunological profiles in vitro (such as cytokines, chemokines, and growth factor secretion) of various library compounds derived from hot-water extracts of herbal medicines, together with their diverse distribution of nano-sized physical particle properties with a machine learning algorithm. By combining multiparametric analysis with a machine learning algorithm such as rCCA, sparse-PLS, and DIABLO, we identified that human G-CSF and mouse RANTES, produced upon adjuvant stimulation in vitro, are the most robust biological parameters that can predict the adjuvanticity of various library compounds. Notably, we revealed a certain nano-sized particle population that functioned as an independent negative parameter to adjuvanticity. Finally, we proved that the two-step strategy pairing the negative and positive parameters significantly improved the efficacy of screening and a screening strategy applying principal component analysis using the identified parameters. These novel parameters we identified for adjuvant screening by machine learning with multiple biological and physical parameters may provide new insights into the future development of effective and safe adjuvants for human use.

ABSTRACT Mycoplasma pneumoniae (Mp) residing extracellularly in the respiratory tract is the primary cause of bacterial community-acquired pneumonia in humans. However, the detailed pathological mechanism of Mp infection, especially inflammation in the lung, remains unclear. This study examined the role of the neutrophils in the inflammation of Mp-induced pneumonia in mice and the mechanism of neutrophil infiltration into the lungs in the Mp-induced pneumonia. We observed massive infiltration of neutrophils in the bronchoalveolar lavage fluid (BALF) and lung injury after the Mp challenge. The neutrophils were shown to contribute to lung injury in Mp pneumonia but were not involved in eliminating Mp, suggesting that neutrophils are detrimental to the host in Mp pneumonia. Mp also induced the production of inflammatory cytokines and chemokines in the BALF in a toll-like receptor 2 (TLR2)-dependent manner. Particularly, both interleukin (IL)-1α and IL-12 p40 played a crucial role in neutrophil infiltration into the BALF in a coordinated manner. Both IL-1α and IL-12 p40 were released from the alveolar macrophages depending on the TLR2 and reactive oxygen species. In addition, the community-acquired respiratory distress syndrome (CARDS) toxin from Mp were found to induce neutrophil infiltration into BALF in a TLR2-independent and IL-1α-dependent manner. Collectively, the TLR2-dependent production of both IL-1α and IL-12 p40, and CARDS toxin have been elucidated to play an important role in neutrophil infiltration into the lungs subsequently leading to the lung injury upon Mp infection in mice. These data will aid in the development of therapeutics and vaccines for Mp pneumonia. IMPORTANCE Although Mp-induced pneumonia is usually a self-limiting disease, refractory life-threatening pneumonia is often induced. In addition, the development of alternative therapeutic strategies for Mp is expected because of the emergence of antibiotic-resistant Mp. However, the lack of knowledge regarding the pathogenesis of Mp-induced pneumonia, especially inflammation upon the Mp infection, makes it tedious to design novel therapeutics and vaccines. For example, although neutrophil infiltration is widely recognized as one of the characteristics of Mp-induced pneumonia, the precise role of neutrophils in the aggravation of Mp pneumonia remains unclear. This study showed that the infiltration of neutrophils in the lungs is detrimental to the host in Mp-induced pneumonia in mice. Furthermore, the TLR2-dependent IL-1α and IL-12 p40 production, and CARDS toxin play important roles in neutrophil infiltration into the lung, following lung injury. Our findings apply to the rational design of novel therapeutics and vaccines against Mp.

We previously generated a transgenic mouse line expressing skin-specific IL-33 (IL33tg mice) and showed that IL-33 elicits group 2 innate lymphoid cell (ILC2)–dependent atopic dermatitis–like skin inflammation. ILC2s are believed to be tissue-resident cells under steady-state conditions, but the dynamics of ILC2 migration are not fully understood. We sorted ILC2s from the skin and draining lymph nodes of IL33tg mice and analyzed their transcriptomes using the single-cell RNA sequencing technique, which revealed that the skin ILC2s had split into two clusters: circulating ILC2 and skin-resident ILC2. The circulating ILC2s expressed H2-related major histocompatibility complex class II genes. Conversely, the skin-resident ILC2s demonstrated increased mRNA expression of the ICOS, IL-5, and IL-13. Next, we tracked ILC2 migration using IL33tg–Kikume Green-Red mice. Exposing the IL33tg–Kikume Green-Red mice’s inflamed skin to violet light allowed us to label the circulating ILC2s in their skin and track the ILC2 migration from the skin to the draining lymph nodes. Cutaneous local innate responses could transition to systemic type 2 responses by migrating the activated ILC2s from the skin into the draining lymph node. Conversely, the skin-resident ILC2s produced a large number of cytokines. Thus, the skin ILC2s turned out to be a heterogeneous cell population.

Infections with parasites, especially those involving nematodes that invade tissues, induce a strong Th2-type immune response, which increases immunoglobulin E and eosinophil levels in the blood and tissues. Eosinophils are not effective against all possible helminth infections, but are known to be effective against nematode larvae. In particular, when a host is re-infected by a species of nematode that it previously encountered, the activation of acquired immunity causes robust accumulation and activation of eosinophils that damages the nematode larvae. Eosinophil production and activation processes are mainly induced by interleukin (IL)-5, which is produced by Th2 cells and group 2 innate lymphoid cells, and eosinophils have been shown to generally participate in host defense, inflammation, and immunomodulation. Recently, several papers have reported host defense by non-antigen-specific immune activation, in which group 2 innate lymphoid cells and Th2 cells produce interleukin (IL)-5 and IL-13 in response to IL-33 stimulation. This immune activation is produced by migrating larvae of a species that differs from the species previously encountered. Eosinophils also play an important role in the eradication of migrating larvae. Thus, eosinophils contribute to host defense in both antigen-specific and non-antigen-specific manners.

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-β-cyclodextrin (HP-β-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-β-CD have not been fully delineated. Here, we show that intranasally administered HP-β-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-β-CD when it is administered intranasally. In contrast, the subcutaneous injection of HP-β-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-β-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-β-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.

Photocatalytic filters installed in air purifiers have been used to purify spaces by decomposing allergenic substances. However, we have not found any reports that evaluate the effectiveness of photocatalytic filters in suppressing allergic reactions in living organisms. In this study, we intratracheally instilled ovalbumin (OVA) into OVA-sensitized mice after the OVA was photocatalyzed by a titanium dioxide (TiO2) filter, and verified the experimental model for evaluating the allergy-suppressing effect of photocatalysts. Mice were sensitized to OVA (10 µg/mouse) four times, and were intratracheally instilled with OVA (10 µg/mouse) after photocatalysis three times. Non-sensitized animals were instilled with normal saline following the same exposure schedule. The mice were dissected 24 h after final exposure. The OVA after photocatalysis significantly decreased the number of eosinophils in bronchoalveolar lavage fluid, and the concentration of OVA-specific IgE and IgG1 in serum, which were elevated in untreated OVA. Moreover, our experimental model showed the suppression of allergic reactions in mice, along with the decomposition of OVA after photocatalysis using the photocatalytic filter. Taken together, our experimental model for evaluating allergic reactions in the respiratory tract suggested that the allergy-suppressing effect of the photocatalytic filter can be evaluated.

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.

Recently, it was reported that 2-hydroxypropyl-β-cyclodextrin (HP-β-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-β-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-β-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-β-CyD, which can cause an allergic response to immunized antigen was completely suppressed by the addition of K3 CpG-ODN. Furthermore, HP-β-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-β-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.

Vaccine adjuvants that can induce not only antigen-specific antibody responses but also Th1-type immune responses and CD8+ cytotoxic T lymphocyte responses are needed for the development of vaccines against infectious diseases and cancer. Of many available adjuvants, oligodeoxynucleotides (ODNs) with unmethylated cytosine-phosphate-guanine (CpG) motifs are the most promising for inducing the necessary immune responses, and these adjuvants are currently under clinical trials in humans. However, the development of novel delivery vehicles that enhance the adjuvant effects of CpG ODNs, subsequently increasing the production of cytokines such as type-I interferons (IFNs), is highly desirable. In this study, we demonstrate the potential of pH-responsive biodegradable carbonate apatite (CA) nanoparticles as CpG ODN delivery vehicles that can enhance the production of type-I IFNs (such as IFN-α) relative to that induced by CpG ODNs and can augment the adjuvant effects of CpG ODNs in vivo. In contrast to CpG ODNs, CA nanoparticles containing CpG ODNs (designated CA-CpG) induced significant IFN-α production by mouse dendritic cells and human peripheral blood mononuclear cells in vitro; and production of interleukin-12, and IFN-γ was higher in CA-CpG-treated groups than in CpG ODNs groups. In addition, treatment with CA-CpG resulted in higher cytokine production in draining lymph nodes than did treatment with CpG ODNs in vivo. Furthermore, vaccination with CA-CpG plus an antigen, such as ovalbumin or influenza virus hemagglutinin, resulted in higher antigen-specific antibody responses and CD8+ cytotoxic T lymphocyte responses in vivo, in an interleukin-12- and type-I IFN-dependent manner, than did vaccination with the antigen plus CpG ODNs; in addition, the efficacy of the vaccine against influenza virus was higher with CA-CpG as the adjuvant than with CpG ODNs as the adjuvant. These data show the potential of CA nanoparticles to serve as CpG ODN delivery vehicles that increase the production of cytokines, especially IFN-α, induced by CpG ODNs and thus augment the efficacy of CpG ODNs as adjuvants. We expect that the strategy reported herein will facilitate the design and development of novel adjuvant delivery vehicles for vaccines.

Inhaled fine particulates are thought to cause chronic pulmonary inflammation through the deposition of particulates into the lungs. To investigate the effect of fine particulates on the lungs, instillation of suspension of particulates into the lungs is required. This protocol describes direct injection of suspensions of fine particulates into the airway. We also show examples of typical lung immune responses after particulate administration.

Immunostimulatory CpG ODNs have been developed and utilized as TLR9-dependent innate immune activators and vaccine adjuvants. Four different types of immunostimulatory CpG ODNs (A/D, B/K, C, and P type) have been reported. A/D type ODNs are characterized by high IFN-α production but intrinsically form aggregates, hindering its good manufacturing practice grade preparation. In this study, we developed several D35-derived ODNs (a commonly used A/D type ODN), which were modified with the addition of a phosphorothioate polynucleotide tail (such as dAs40), and examined their physical properties, solubility in saline, immunostimulatory activity on human PBMCs, and vaccine adjuvant potential in monkeys. We found that two modified ODNs including D35-dAs40 and D35core-dAs40 were immunostimulatory, similar to original D35 in human PBMCs, resulting in high IFN-α secretion in a dose-dependent manner. Physical property analysis by dynamic light scattering revealed that both D35-dAs40 and D35core-dAs40 did not form aggregates in saline, which is currently impossible for the original D35. Furthermore, D35-dAs40 and D35core-dAs40 worked as better vaccine adjuvant in monkeys. These results suggested that D35-dAs40 and D35core-dAs40 are two promising prototypes of nonaggregating A/D type ODN with advantages of ease of drug preparation for clinical applications as vaccine adjuvants or IFN-α inducing immunomodifiers.

Pulmonary inflammation, especially persistent inflammation, has been found to play a key role in respiratory disorders induced by nanoparticles in animal models. In inhalation studies and instillation studies of nanomaterials, persistent inflammation is composed of neutrophils and alveolar macrophages, and its pathogenesis is related to chemokines such as the cytokine-induced neutrophil chemoattractant (CINC) family and macrophage inflammatory protein-1α and oxidant stress-related genes such as heme oxygenase-1 (HO-1). DNA damages occur chemically or physically by nanomaterials. Chemical and physical damage are associated with point mutation by free radicals and double strand brake, respectively. The failure of DNA repair and accumulation of mutations might occur when inflammation is prolonged, and finally normal cells could become malignant. These free radicals can not only damage cells but also induce signaling molecules containing immunoreaction. Nanoparticles and asbestos also induce the production of free radicals. In allergic responses, nanoparticles act as Th2 adjuvants to activate Th2 immune responses such as activation of eosinophil and induction of IgE. Taken together, the presence of persistent inflammation may contribute to the pathogenesis of a variety of diseases induced by nanomaterials.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a potent type I interferon (IFN) inducer, was evaluated as a chemotherapeutic agent in mouse cancer models and proved to be well tolerated in human cancer clinical trials. Despite its multiple biological functions, DMXAA has not been fully characterized for the potential application as a vaccine adjuvant. In this report, we show that DMXAA does act as an adjuvant due to its unique property as a soluble innate immune activator. Using OVA as a model antigen, DMXAA was demonstrated to improve on the antigen specific immune responses and induce a preferential Th2 (Type-2) response. The adjuvant effect was directly dependent on the IRF3-mediated production of type-I-interferon, but not IL-33. DMXAA could also enhance the immunogenicity of influenza split vaccine which led to significant increase in protective responses against live influenza virus challenge in mice compared to split vaccine alone. We propose that DMXAA can be used as an adjuvant that targets a specific innate immune signaling pathway via IRF3 for potential applications including vaccines against influenza which requires a high safety profile.

5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a mouse-selective stimulator of interferon gene (STING) agonist exerting STING-dependent anti-tumor activity. Although DMXAA cannot fully activate human STING, DMXAA reached phase III in lung cancer clinical trials. How DMXAA is effective against human lung cancer is completely unknown. Here, we show that DMXAA is a partial STING agonist interfering with agonistic STING activation, which may explain its partial anti-tumor effect observed in humans, as STING was reported to be pro-tumorigenic for lung cancer cells with low antigenicity. Furthermore, we developed a DMXAA derivative—3-hydroxy-5-(4-hydroxybenzyl)-4-methyl-9H-xanthen-9-one (HHMX)—that can potently antagonize STING-mediated immune responses both in humans and mice. Notably, HHMX suppressed aberrant responses induced by STING gain-of-function mutations causing STING-associated vasculopathy with onset in infancy (SAVI) in in vitro experiments. Furthermore, HHMX treatment suppressed aberrant STING pathway activity in peripheral blood mononuclear cells from SAVI patients. Lastly, HHMX showed a potent therapeutic effect in SAVI mouse model by mitigating disease progression. Thus, HHMX offers therapeutic potential for STING-associated autoinflammatory diseases. [ABSTRACT FROM AUTHOR]

Although malaria is a life-threatening disease with severe complications, most people develop partial immunity and suffer from mild symptoms. However, incomplete recovery from infection causes chronic illness, and little is known of the potential outcomes of this chronicity. We found that malaria causes bone loss and growth retardation as a result of chronic bone inflammation induced by Plasmodium products. Acute malaria infection severely suppresses bone homeostasis, but sustained accumulation of Plasmodium products in the bone marrow niche induces MyD88-dependent inflammatory responses in osteoclast and osteoblast precursors, leading to increased RANKL expression and overstimulation of osteoclastogenesis, favoring bone resorption. Infection with a mutant parasite with impaired hemoglobin digestion that produces little hemozoin, a major Plasmodium by-product, did not cause bone loss. Supplementation of alfacalcidol, a vitamin D3 analog, could prevent the bone loss. These results highlight the risk of bone loss in malaria-infected patients and the potential benefits of coupling bone therapy with antimalarial treatment. [ABSTRACT FROM AUTHOR]

The application of nanotechnology in the health care setting has many potential benefits; however, our understanding of the interactions between nanoparticles and our immune system remains incomplete. Although many of the biological effects of nanoparticles are negatively correlated with particle size, some are clearly size specific and the mechanisms underlying these size-specific biological effects remain unknown. Here, we examined the pro-inflammatory effects of silica particles in THP-1 cells with respect to particle size; a large overall size range with narrow intervals between particle diameters (particle diameter: 10, 30, 50, 70, 100, 300, and 1,000 nm) was used. Secretion of the pro-inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α induced by exposure to the silica particles had a bell-shaped distribution, where the maximal secretion was induced by silica nanoparticles with a diameter of 50 nm and particles with smaller or larger diameters had progressively less effect. We found that blockade of IL-1β secretion markedly inhibited TNF-α secretion, suggesting that IL-1β is upstream of TNF-α in the inflammatory cascade induced by exposure to silica particles, and that the induction of IL-1β secretion was dependent on both the NLRP3 inflammasome and on uptake of the silica particles into the cells via endocytosis. However, a quantitative analysis of silica particle uptake showed that IL-1β secretion was not correlated with the amount of silica particles taken up by the cells. Further investigation revealed that the induction of IL-1β secretion and uptake of silica nanoparticles with diameters of 50 or 100 nm, but not of 10 or 1,000 nm, was dependent on scavenger receptor (SR) A1. In addition, of the silica particles examined, only those with a diameter of 50 nm induced strong IL-1β secretion via activation of Mer receptor tyrosine kinase, a signal mediator of SR A1. Together, our results suggest that the SR A1-mediated pro-inflammatory response is dependent on ligand size and that both SR A1-mediated endocytosis and receptor-mediated signaling are required to produce the maximal pro-inflammatory response to exposure to silica particles. [ABSTRACT FROM AUTHOR]

Over the past decade, a vast array of nanomaterials has been created through the development of nanotechnology. With the increasing application of these nanomaterials in various fields, such as foods, cosmetics, and medicines, there has been concern about their safety, that is, nanotoxicity. Therefore, there is an urgent need to collect information about the biological effects of nanomaterials so that we can exploit their potential benefits and design safer nanomaterials, while avoiding nanotoxicity as a result of inhalation or skin exposure. In particular, the immunomodulating effect of nanomaterials is one of most interesting aspects of nanotoxicity. However, the immunomodulating effects of nanomaterials through skin exposure have not been adequately discussed compared with the effects of inhalation exposure, because skin penetration by nanomaterials is thought to be extremely low under normal conditions. On the other hand, the immunomodulatory effects of nanomaterials via skin may cause severe problems for people with impaired skin barrier function, because some nanomaterials could penetrate the deep layers of their allergic or damaged skin. In addition, some studies, including ours, have shown that nanomaterials could exhibit significant immunomodulating effects even if they do not penetrate the skin. In this review, we summarize our current knowledge of the allergic responses induced by nanomaterials upon skin exposure. First, we discuss nanomaterial penetration of the intact or impaired skin barrier. Next, we describe the immunomodulating effects of nanomaterials, focusing on the sensitization potential of nanomaterials and the effects of co-exposure of nanomaterials with substances such as chemical sensitizers or allergens, on the onset of allergy, following skin exposure. Finally, we discuss the potential mechanisms underlying the immunomodulating effects of nanomaterials by describing the involvement of the protein corona in the interaction of nanomaterials with biological components and by presenting recent data about the adjuvant effects of well-characterized particle adjuvant, aluminum salt, as an example of immunomodulatory particulate. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Pellagra is a photosensitivity syndrome characterized by three "D's": diarrhea, dermatitis, and dementia as a result of niacin deficiency. However, the molecular mechanisms of photosensitivity dermatitis, the hallmark abnormality of this syndrome, remain unclear. We prepared niacin deficient mice in order to develop a murine model of pellagra. Niacin deficiency induced photosensitivity and severe diarrhea with weight loss. In addition, niacin deficient mice exhibited elevated expressions of COX-2 and PGE syntheses (Ptges) mRNA. Consistently, photosensitivity was alleviated by a COX inhibitor, deficiency of Ptges, or blockade of EP4 receptor signaling. Moreover, enhanced PGE2 production in niacin deficiency was mediated via ROS production in keratinocytes. In line with the above murine findings, human skin lesions of pellagra patients confirmed the enhanced expression of Ptges. Niacin deficiency-induced photosensitivity was mediated through EP4 signaling in response to increased PGE2 production via induction of ROS formation. [ABSTRACT FROM AUTHOR]

: BACKGROUND The invasion of first trimester extravillous trophoblast (EVT) to decidua is an important event in placentation. Leukemia inhibitory factor (LIF) is an essential factor for mouse implantation, and it is reported that LIF may be involved in human first trimester EVT invasion. Prostaglandin E2 (PGE2) is also known as a critical factor for first trimester EVT invasion. In this study, we investigated the role of LIF in PGE2 production and EVT invasion using a human first trimester EVT cell line, HTR-8/SVneo. : METHODS and RESULTS Co-stimulation with LIF and IL-1β induced higher amounts of PGE2 production and further migration of HTR-8/SVneo cells compared with that by stimulation with LIF or IL-1β alone. Enhanced PGE2 production was most probably due to the enhanced expression of cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1). PGE2 produced by HTR-8/SVneo cells promoted the migration of HTR-8/SVneo cells. A COX-2 inhibitor suppressed PGE2 production and the migration of HTR-8/SVneo cells. Agonists to PGE2 receptors, EP1, EP2 and EP4, promoted the migration of HTR-8/SVneo cells. Moreover, stimulation with LIF up-regulated EP1, EP2 and EP4 expression in HTR-8/SVneo cells. : CONCLUSIONS It is suggested that LIF participates in placentation through EVT invasion by up-regulating PGE2 production and PGE2 receptor expression in first trimester EVT. [ABSTRACT FROM AUTHOR]

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 DNAvaccine 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. [ABSTRACT FROM AUTHOR]