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Objective and Methods IL-33 is present in endothelial, epithelial, and fibroblast-like cells and released upon cell injury. IL-33 reportedly induces mast-cell degranulation and is involved in various diseases, including allergic diseases. So, IL-33-related diseases seem to overlap with histamine-related diseases. In addition to the release from mast cells, histamine is newly formed by the induction of histidine decarboxylase (HDC). Some inflammatory and/or hematopoietic cytokines (IL-1, IL-3, etc.) are known to induce HDC, and the histamine produced by HDC induction is released without storage. We examined the involvement of HDC and histamine in the effects of IL-33. Results A single intraperitoneal injection of IL-33 into mice induced HDC directly and/or via other cytokines (including IL-5) within a few hours in various tissues, particularly strongly in hematopoietic organs. The major cells exhibiting HDC-induction were mast cells and c-kit+cells in bone marrow. HDC was also induced in non-mast cells in non-hematopoietic organs. HDC, histamine, and histamine H4 receptors (H4Rs) contributed to suppression of IL-33-induced eosinophilia. Conclusion IL-33 directly and indirectly (via IL-5) induces HDC in various cells, particularly potently in c-kit+cells and mature mast cells, and the newly formed histamine contributes to negative regulation of IL-33-induced eosinophilia via H4Rs.

Among the bisphosphonates (BPs), nitrogen-containing BPs (N-BPs) have much stronger anti-bone-resorptive actions than non-N-BPs. However, N-BPs have various side effects such as acute influenza-like reactions after their initial administration and osteonecrosis of the jawbones after repeated administration. The mechanisms underlying such effects remain unclear. To overcome these problems, it is important to profile the inflammatory nature of N-BPs. Here, we analyzed the inflammatory reactions induced in mouse ear pinnae by the N-BPs alendronate (Ale) and zoledronate (Zol). We found the following: (i) Ale and Zol each induced two phases of inflammation (early weak and late strong ear swelling); (ii) both phases were augmented by lipopolysaccharides (LPSs; cell-surface constituent of gram-negative bacteria, including oral bacteria), but prevented by inhibitors of the phosphate transporters of solute carrier 20/34 (SLC20/SLC34); (iii) macrophages and neutrophils were involved in both phases of Ale+LPS-induced ear-swelling; (iv) Ale increased or tended to increase various cytokines, and LPS augmented these effects, especially that on interleukin 1β (IL-1β); (v) adenosine triphosphate (ATP) was involved in both phases, and Ale alone or Ale+LPS increased ATP in ear pinnae; (vi) the augmented late-phase swelling induced by Ale+LPS depended on both IL-1 and neutrophil extracellular traps (NETs; neutrophil-derived net-like complexes); (vii) neutrophils, together with macrophages and dendritic cells, also functioned as IL-1β-producing cells, and upon stimulation with IL-1β, neutrophils produced NETs; (viii) stimulation of the purinergic 2X7 (P2X7) receptors by ATP induced IL-1β in ear pinnae; (ix) NET formation by Ale+LPS was confirmed in gingiva, too. These results suggest that (i) N-BPs induce both early-phase and late-phase inflammation via ATP-production and P2X7 receptor stimulation; (ii) N-BPs and LPS induce mutually augmenting responses both early and late phases via ATP-mediated IL-1β production by neutrophils, macrophages, and/or dendritic cells; and (iii) NET production by IL-1β-stimulated neutrophils may mediate the late phase, leading to prolonged inflammation. These results are discussed in relation to the side effects seen in patients treated with N-BPs. © 2021 American Society for Bone and Mineral Research (ASBMR).

Nickel (Ni) is the most frequent metal allergen and induces Th1-dependent type-IV allergies. In local skin, epidermal Langerhans cells (LCs) and/or dermal dendritic cells (DCs) uptake antigens and migrate to draining lymph nodes (LNs). However, the subsets of antigen-presenting cells that contribute to Ni presentation have not yet been identified. In this study, we analyzed the Ni-binding capabilities of murine DCs using fluorescent metal indicator Newport Green. Elicitation of Ni allergy was assessed after intradermal (i.d.) injection of Ni-treated DCs into ear pinnae of Ni-sensitized mice. The Ni-binding capabilities of MHC class IIhi CD11cint migratory DCs were significantly stronger than those of MHC class IIint CD11chi resident DCs and CD11cint PDCA1+ MHC class IIint B220+ plasmacytoid DCs. Migratory DCs in skin-draining and mandibular LNs showed significantly stronger Ni-binding capabilities than those in mesenteric and medial iliac LNs. An i.d. injection of IL-1β induced the activation of LCs and dermal DCs with strong Ni-binding capabilities. Ni-binding LCs were detected in draining LNs after i.d. challenge with IL-1β and Ni. Moreover, an i.d. injection of Ni-treated DCs purified from skin-draining LNs elicited Ni-allergic inflammation. These results demonstrated that migratory DCs in skin-draining LNs have strong Ni-binding capabilities and elicit Ni allergy.

Nitrogen-containing bisphosphonates (NBPs, anti-bone-resorptive agents) have inflammatory side-effects. Alendronate (Ale, an NBP) intradermally injected into mouse ear-pinnae together with LPS (bacterial cell-wall component) induces augmented ear-swelling that depends on IL-1 and neutrophils. Using this model, we examined histamine's involvement in Ale + LPS-induced inflammation.Ale increased histamine in ear-pinnae by inducing histidine decarboxylase (HDC). This induction was augmented by LPS. In HDC-deficient mice, such augmented ear-swelling was not induced. At peak-swelling, 74.5% of HDC-expressing cells were neutrophils and only 0.2% were mast cells (MCs). The augmented swelling was markedly reduced by a histamine H4-receptor (H4R) antagonist, but not by an H1R antagonist. In MC-deficient mice, unexpectedly, Ale + LPS induced prolonged ear-swelling that was augmented and more persistent than in normal mice. MCs highly expressed H4Rs and produced MCP-1(inflammatory cytokine that recruits macrophages) and IL-10 (anti-inflammatory cytokine) in response to an H4R agonist.Histamine produced by HDC-induction mainly in infiltrated neutrophils stimulates H4Rs, leading to augmented Ale + LPS-induced ear-swelling via MCP-1 production by MCs. Since MCP-1 is produced by other cells, too, the contribution of MCs and their H4Rs to augmented ear-swelling is partial. In the later phase of the swelling, MCs may be anti-inflammatory via IL-10 production.

Hypertension is a chronic-low grade inflammatory disease, which is known to be associated with increased bone loss. Excessive activity of the local renin-angiotensin system (RAS) in bone leads to increased bone resorption. As inflammatory cytokines may activate RAS components, we hypothesized that the elevated proinflammatory cytokine levels in hypertension activate bone RAS and thus lead to increased bone resorption. To investigate whether salt-sensitive hypertension (SSHTN) induces osteoclastogenesis and bone resorption, we generated a model of SSHTN in C57BL/6J mice by post

Gland macrophages are primed for gland development and functions through interactions within their niche. However, the phenotype, ontogeny, and function of steady-state salivary gland (SG) macrophages remain unclear. We herein identified CD11c+ and CD11c− subsets among CD64+ macrophages in steady-state murine SGs. CD11c− macrophages were predominant in the SGs of embryonic and newborn mice and decreased with advancing age. CD11c+ macrophages were rarely detected in the embryonic period, but rapidly expanded after birth. CD11c+, but not CD11c−, macrophage numbers decreased in mice treated with a CCR2 antagonist, suggesting that CD11c+ macrophages accumulate from bone marrow-derived progenitors in a CCR2-dependent manner, whereas CD11c− macrophages were derived from embryonic progenitors in SGs. CD11c+ and CD11c− macrophages strongly expressed colony-stimulating factor (CSF)-1 receptor, the injection of an anti-CSF-1 receptor blocking antibody markedly reduced both subsets, and SGs strongly expressed CSF-1, indicating the dependency of SG resident macrophage development on CSF-1. The phagocytic activity of SG macrophages was extremely weak; however, the gene expression profile of SG macrophages indicated that SG macrophages regulate gland development and functions in SGs. These results suggest that SG CD11c+ and CD11c− macrophages are developed and instructed to perform SG-specific functions in steady-state SGs.

Gland macrophages are primed for gland development and functions through interactions within their niche. However, the phenotype, ontogeny, and function of steady-state salivary gland (SG) macrophages remain unclear. We herein identified CD11c+and CD11c−subsets among CD64+macrophages in steady-state murine SGs. CD11c−macrophages were predominant in the SGs of embryonic and newborn mice and decreased with advancing age. CD11c+macrophages were rarely detected in the embryonic period, but rapidly expanded after birth. CD11c+, but not CD11c−, macrophage numbers decreased in mice treated with a CCR2 antagonist, suggesting that CD11c+macrophages accumulate from bone marrow-derived progenitors in a CCR2-dependent manner, whereas CD11c−macrophages were derived from embryonic progenitors in SGs. CD11c+and CD11c−macrophages strongly expressed colony-stimulating factor (CSF)-1 receptor, the injection of an anti-CSF-1 receptor blocking antibody markedly reduced both subsets, and SGs strongly expressed CSF-1, indicating the dependency of SG resident macrophage development on CSF-1. The phagocytic activity of SG macrophages was extremely weak; however, the gene expression profile of SG macrophages indicated that SG macrophages regulate gland development and functions in SGs. These results suggest that SG CD11c+and CD11c−macrophages are developed and instructed to perform SG-specific functions in steady-state SGs.

Background Sublingual immunotherapy (SLIT) is used for the treatment of type 1 allergies, such as allergic rhinitis. SLIT leads to tolerance against allergens possibly via the redirection of allergen-specific T helper 2 cells to T helper 1 cells and the generation of peripheral regulatory T (Treg) cells. However, the detailed mechanisms remain unclear. Systemic tolerance to orally administered antigens (oral tolerance) has been extensively investigated. Recent studies have recognized the central role of Treg cells and classical dendritic cells (cDCs) in oral tolerance development. Highlight This review focuses on recent advances in the understanding of the underlying mechanisms of SLIT compared with those of oral tolerance. The sublingual administration of soluble protein antigens has been reported to induce antigen-specific Treg cells in oral mucosa-draining submandibular lymph nodes in mice. The generation of Treg cells is critical for SLIT efficacy because the transfer of SLIT-induced Treg cells confers tolerance against the antigens. A large number of oral cDCs with the CD103−CD11b+ phenotype exert retinoic acid-producing activity and convert naive CD4+ T cells into Foxp3+ Treg cells in vitro in a transforming growth factor-β-dependent and retinoic acid-dependent manner. Oral CD103−CD11b+ cDCs transport sublingual antigens to submandibular lymph nodes and induce antigen-specific Treg cells. Sublingual antigens enter the mucosa most likely by crossing the sublingual ductal epithelium and are captured by oral antigen-presenting cells, especially macrophages. Conclusion Oral CD103−CD11b+ cDCs are specialized for the induction of Treg cells in mice; thus, targeting their human counterpart may enhance the therapeutic effects of SLIT.

Interleukin-33 (IL-33) is a pleiotropic cytokine that can promote type 2 inflammation but also drives immunoregulation through Foxp3+Treg expansion. How IL-33 is exported from cells to serve this dual role in immunosuppression and inflammation remains unclear. Here, we demonstrate that the biological consequences of IL-33 activity are dictated by its cellular source. Whereas IL-33 derived from epithelial cells stimulates group 2 innate lymphoid cell (ILC2)-driven type 2 immunity and parasite clearance, we report that IL-33 derived from myeloid antigen-presenting cells (APCs) suppresses host-protective inflammatory responses. Conditional deletion of IL-33 in CD11c-expressing cells resulted in lowered numbers of intestinal Foxp3+Treg cells that express the transcription factor GATA3 and the IL-33 receptor ST2, causing elevated IL-5 and IL-13 production and accelerated anti-helminth immunity. We demonstrate that cell-intrinsic IL-33 promoted mouse dendritic cells (DCs) to express the pore-forming protein perforin-2, which may function as a conduit on the plasma membrane facilitating IL-33 export. Lack of perforin-2 in DCs blocked the proliferative expansion of the ST2+Foxp3+Treg subset. We propose that perforin-2 can provide a plasma membrane conduit in DCs that promotes the export of IL-33, contributing to mucosal immunoregulation under steady-state and infectious conditions.

Arginine methylation is a post-translational modification that regulates many biological processes. However, the role of arginine methylation in immune cells is not well studied. Here we report an essential role of protein arginine methyltransferase 5 (PRMT5) in T cell homeostasis and activation-induced expansion. Using T cell-specific PRMT5 conditional knockout mice, we found that PRMT5 is required for natural killer T (NKT) cell but not for conventional or regulatory T (Treg) cell development after the double positive (DP) stage in the thymus. In contrast, PRMT5 was required for optimal peripheral T cell maintenance, for the transition of naive T cells to effector/memory phenotype, and for early T cell development before the DP stage in a cell-intrinsic manner. Accordingly, PRMT5-deleted T cells showed impaired IL-7-mediated survival and TCR-induced proliferation in vitro. The latter was more pronounced and attributed to reduced responsiveness to IL-2. Acute deletion of PRMT5 revealed that not only naive but also effector/memory T cells were impaired in TCR-induced proliferation in a development-independent manner. Reduced expression of common γ chain (γc), a shared receptor component for several cytokines including IL-7 and IL-2, on PRMT5-deleted T cells may be in part responsible for the defect. We further showed that PRMT5 was partially required for homeostatic T cell survival but absolutely required for lymphopenic T cell expansion in vivo. Thus, we propose that PRMT5 is required for T cell survival and proliferation by maintaining cytokine signaling, especially during proliferation. The inhibition of PRMT5 may provide a novel strategy for the treatment of diseases where uncontrolled T cell activation has a role, such as autoimmunity.

Since the first report in 2003, bisphosphonate-related osteonecrosis of the jaw (BRONJ) has been increasing, without effective clinical strategies. Osteoporosis is common in elderly women, and bisphosphonates (BPs) are typical and widely used anti-osteoporotic or anti-bone-resorptive drugs. BRONJ is now a serious concern in dentistry. As BPs are pyrophosphate analogues and bind strongly to bone hydroxyapatite, and the P-C-P structure of BPs is non-hydrolysable, they accumulate in bones upon repeated administration. During bone-resorption, BPs are taken into osteoclasts and exhibit cytotoxicity, producing a long-lasting anti-bone-resorptive effect. BPs are divided into nitrogen-containing BPs (N-BPs) and non-nitrogen-containing BPs (non-N-BPs). N-BPs have far stronger anti-bone-resorptive effects than non-N-BPs, and BRONJ is caused by N-BPs. Our murine experiments have revealed the following. N-BPs, but not non-N-BPs, exhibit direct and potent inflammatory/necrotic effects on soft-tissues. These effects are augmented by lipopolysaccharide (the inflammatory component of bacterial cell-walls) and the accumulation of N-BPs in jawbones is augmented by inflammation. N-BPs are taken into soft-tissue cells via phosphate-transporters, while the non-N-BPs etidronate and clodronate inhibit this transportation. Etidronate, but not clodronate, has the effect of expelling N-BPs that have accumulated in bones. Moreover, etidronate and clodronate each have an analgesic effect, while clodronate has an anti-inflammatory effect via inhibition of phosphate-transporters. These findings suggest that BRONJ may be induced by phosphate-transporter-mediated and infection-promoted mechanisms, and that etidronate and clodronate may be useful for preventing and treating BRONJ. Our clinical trials support etidronate being useful for treating BRONJ, although additional clinical trials of etidronate and clodronate are needed.

Ribosome engineering, originally applied to Streptomyces lividans, has been widely utilized for strain improvement, especially for the activation of bacterial secondary metabolism. This study assessed ribosome engineering technology to modulate primary metabolism, taking butanol production as a representative example. The introduction into Clostridium saccharoperbutylacetonicum of mutations conferring resistance to butanol (ButR) and of the str mutation (SmR; a mutation in the rpsL gene encoding ribosomal protein S12), conferring high-level resistance to streptomycin, increased butanol production 1.6-fold, to 16.5 g butanol/L. Real-time qPCR analysis demonstrated that the genes involved in butanol metabolism by C. saccharoperbutylacetonicum were activated at the transcriptional level in the drug-resistant mutants, providing a mechanism for the higher yields of butanol by the mutants. Moreover, the activity of enzymes butyraldehyde dehydrogenase (AdhE) and butanol dehydrogenases (BdhAB), the key enzymes involved in butanol synthesis, was both markedly increased in the ButR SmR mutant, reflecting the significant up-regulation of adhE and bdhA at transcriptional level in this mutant strain. These results demonstrate the efficacy of ribosome engineering for the production of not only secondary metabolites but of industrially important primary metabolites. The possible ways to overcome the reduced growth rate and/or fitness cost caused by the mutation were also discussed.

We established a mouse model of contact hypersensitivity (CHS) to hydroquinone (HQ), a widespread chemical in our environment. HQ was painted onto flanks; then, HQ was challenged by painting onto ear pinnas on days 7 and 14. The CHS after the second challenge was markedly greater than that after the first challenge. Both challenges increased thymic stromal lymphopoietin and T helper type 2 cytokines in ear pinnas, whereas IFN-γ (typical T helper type 1 cytokine) was decreased, despite an increase in IL-18 (typical IFN-γ inducer). In nude mice (T cell-reduced), although a first challenge induced CHS, a second challenge did not augment it. In severe combined immunodeficient, severe combined immunodeficient-beige, and IL-1-deficient mice, CHS was not induced. However, CHS was inducible in severe combined immunodeficient-beige mice after transfer of natural killer cells from HQ-sensitized normal mice. Tretinoin (used for enhancing the skin-whitening effect of HQ) and resin monomers (used to prevent polymerization of HQ) lowered the HQ concentration needed to establish sensitization to HQ. The augmented CHS after a second challenge was reduced by JNJ7777120, dexamethasone, suplatast tosilate (T helper type 2-cytokine inhibitor), and anti-thymic stromal lymphopoietin antibody. These results suggest that (i) thymic stromal lymphopoietin, IL-1, and T and/or natural killer cells are important in establishing and augmenting CHS to HQ and (ii) inflammatory chemicals may promote CHS to HQ as adjuvants.

SummaryBackground Nickel (Ni) is the most frequent metal allergen and induces a TH1-dependent type-IV allergy. Although Ni2+ is considered to bind to endogenous proteins, it currently remains unclear whether these Ni-binding proteins are involved in Ni allergy in vivo. We previously reported the adjuvant effects of lipopolysaccharide (LPS) in a Ni allergy mouse model. As LPS induces a number of inflammatory mediators, we hypothesized that Ni-binding protein(s) are also induced by LPS. Objective The objective of this study was to purify and identify Ni-binding protein(s) from serum taken from LPS-injected mice (referred as LPS serum) and examined the augmenting effects of these Ni-binding protein(s) on Ni allergy in an in vivo model. Methods BALB/cA mice were sensitized with an i.p. injection of NiCl2 and LPS. Ten days after sensitization, mice were challenged with NiCl2 by an i.d. injection into ear pinnae. Ni-binding protein(s) were purified by Ni-affinity column chromatography and gel filtration. Results Lipopolysaccharide serum, but not serum taken from saline-injected mice, augmented ear swelling induced by Ni-allergic inflammation. Ni-binding, but not non-binding fraction, purified from LPS serum augmented Ni-allergic inflammation. Mass spectrometry and Western blotting detected CXCL4 in the active fraction. A batch analysis with Ni-sepharose and a surface plasmon resonance analysis revealed direct binding between CXCL4 and Ni2+. Recombinant CXCL4 augmented Ni-allergic inflammation and exerted adjuvant effects at the sensitization phase. Conclusions These results indicate that CXCL4 is a novel Ni-binding protein that augments Ni allergy at the elicitation and sensitization phases. This is the first study to demonstrate that the Ni-binding protein augments Ni allergy in vivo.

Although transcriptional activation of pathwayspecific positive regulatory genes and/or biosynthetic genes is primarily important for enhancing secondary metabolite production, reinforcement of substrate supply, as represented by primary metabolites, is also effective. For example, partial inhibition of fatty acid synthesis with ARC2 (an analog of triclosan) was found to enhance polyketide antibiotic production. Here, we demonstrate that this approach is effective even for industrial high-producing strains, for example enhancing salinomycin production by 40%, reaching 30.4 g/l of salinomycin in an industrial Streptomyces albus strain. We also hypothesized that a similar approach would be applicable to another important antibiotic group, nonribosomal peptide (NRP) antibiotics. We therefore attempted to partially inhibit protein synthesis by using ribosome-targeting drugs at subinhibitory concentrations (1/50∼1/2 of MICs), which may result in the preferential recruitment of intracellular amino acids to the biosynthesis of NRP antibiotics rather than to protein synthesis. Among the ribosome-targeting drugs examined, chloramphenicol at subinhibitory concentrations was most effective at enhancing the production by Streptomyces of NRP antibiotics such as actinomycin, calcium-dependent antibiotic (CDA), and piperidamycin, often resulting in an almost 2-fold increase in antibiotic production. Chloramphenicol activated biosynthetic genes at the transcriptional level and increased amino acid pool sizes 1.5- to 6-fold, enhancing the production of actinomycin and CDA. This "metabolic perturbation" approach using subinhibitory concentrations of ribosome-targeting drugs is a rational method of enhancing NRP antibiotic production, being especially effective in transcriptionally activated (e.g., rpoB mutant) strains. Because this approach does not require prior genetic information, it may be widely applicable for enhancing bacterial production of NRP antibiotics and bioactive peptides.

Sublingual immunotherapy (SLIT) is a safe and efficient treatment for type 1 allergies; however, the underlying immunological mechanisms, particularly the phenotype of oral antigen-presenting cells (APCs) responsible for the induction of regulatory T (Treg) cells, remain unclear. We show here that the sublingual application of ovalbumin (OVA) induced antigen-specific Foxp3+ Treg cells in draining submandibular lymph nodes (ManLNs). Oral APCs were classified into macrophages, classical dendritic cells (cDCs), and Langerhans cells by flow cytometry. A major subset of oral cDCs with the CD103-CD11b+ phenotype showed retinoic acid (RA)-producing activity and converted naive CD4+ T cells to Foxp3+ Treg cells in a transforming growth factor-β- and RA-dependent manner in vitro. In the ManLNs, migratory CD103-CD11b+ cDCs also showed RA-producing activity. After the sublingual application of fluorescent OVA, fluorescence was detected in oral macrophages in tissues, followed by migratory CD103-CD11b+ cDCs in ManLNs and migratory CD103-CD11b+ cDCs were the main APCs responsible for the induction of sublingual antigen-specific Treg cells. The transfer of OVA-SLIT-induced Treg cells suppressed the OVA-induced hypersensitivity response. These results suggest that oral CD103-CD11b+ cDCs transport sublingual antigens to draining ManLNs and induce antigen-specific Foxp3+ Treg cells, and, thus, provide a rationale for developing cDC-based therapeutic approaches in SLIT.

Dendritic cells (DCs) in lymphoid and non-lymphoid tissues are professional antigen-presenting cells that are essential for effective immunity and tolerance. However, the presence and characteristics of DCs in steady-state salivary glands (SGs) currently remain unknown. We herein identified CD64- CD11c+ classical DCs (cDCs) as well as CD64+ macrophages among CD45+ MHC class II+ antigen-presenting cells in steady-state murine SGs. SG cDCs were divided into CD103+ CD11b- and CD103- CD11b+ cDCs. CD103+ CD11b- cDCs expressed XCR1, CLEC9A, and interferon regulatory factor 8, whereas CD103- CD11b+ cDCs strongly expressed CD172a. Both cDC subsets in SGs markedly expanded in response to the Flt3 ligand (Flt3L), were replenished by bone marrow-derived precursors, and differentiated from common DC precursors, but not monocytes. Furthermore, ovalbumin-pulsed SG CD103+ CD11b- cDCs induced the proliferation of naive ovalbumin-specific CD8+ T cells and production of interferon-γ from proliferating T cells. SG CD103+ CD11b- cDCs expanded by Flt3L in vivo exhibited the same properties. These results indicate that bona fide cDCs reside in steady-state murine SGs and cDCs with the CD103+ CD11b- phenotype possess antigen cross-presenting capacity. Moreover, Flt3L enhances protective immunity by expanding cDCs. Taken together, SG cDCs might play an important role in maintaining immune homeostasis in the tissues.

Regulatory T cells (Tregs) are a subpopulation of T cells that are specialized in suppressing immune responses. Here we show that the arginine methyl transferase protein PRMT5 can complex with FOXP3 transcription factors in Tregs. Mice with conditional knock out (cKO) of PRMT5 expression in Tregs develop severe scurfy-like autoimmunity. In these PRMT5 cKO mice, the spleen has reduced numbers of Tregs, but normal numbers of Tregs are found in the peripheral lymph nodes. These peripheral Tregs that lack PRMT5, however, display a limited suppressive function. Mass spectrometric analysis showed that FOXP3 can be di-methylated at positions R27, R51, and R146. A point mutation of Arginine (R) 51 to Lysine (K) led to defective suppressive functions in human CD4 T cells. Pharmacological inhibition of PRMT5 by DS-437 also reduced human Treg functions and inhibited the methylation of FOXP3. In addition, DS-437 significantly enhanced the anti-tumor effects of anti-erbB2/neu monoclonal antibody targeted therapy in Balb/c mice bearing CT26Her2 tumors by inhibiting Treg function and induction of tumor immunity. Controlling PRMT5 activity is a promising strategy for cancer therapy in situations where host immunity against tumors is attenuated in a FOXP3 dependent manner.

This study shows that sequential introduction of drug resistance mutations substantially increased enzyme production in Paenibacillus agaridevorans The triple mutant YT478 (rsmG Gln225→stop codon, rpsL K56R, and rpoB R485H), generated by screening for resistance to streptomycin and rifampin, expressed a 1,100-fold-larger amount of the extracellular enzyme cycloisomaltooligosaccharide glucanotransferase (CITase) than the wild-type strain. These mutants were characterized by higher intracellular S-adenosylmethionine concentrations during exponential phase and enhanced protein synthesis activity during stationary phase. Surprisingly, the maximal expression of CITase mRNA was similar in the wild-type and triple mutant strains, but the mutant showed greater CITase mRNA expression throughout the growth curve, resulting in enzyme overproduction. A metabolome analysis showed that the triple mutant YT478 had higher levels of nucleic acids and glycolysis metabolites than the wild type, indicating that YT478 mutant cells were activated. The production of CITase by the triple mutant was further enhanced by introducing a mutation conferring resistance to the rare earth element, scandium. This combined drug resistance mutation method also effectively enhanced the production of amylases, proteases, and agarases by P. agaridevorans and Streptomyces coelicolor This method also activated the silent or weak expression of the P. agaridevorans CITase gene, as shown by comparisons of the CITase gene loci of P. agaridevorans T-3040 and another cycloisomaltooligosaccharide-producing bacterium, Paenibacillus sp. strain 598K. The simplicity and wide applicability of this method should facilitate not only industrial enzyme production but also the identification of dormant enzymes by activating the expression of silent or weakly expressed genes.IMPORTANCE Enzyme use has become more widespread in industry. This study evaluated the molecular basis and effectiveness of ribosome engineering in markedly enhancing enzyme production (>1,000-fold). This method, due to its simplicity, wide applicability, and scalability for large-scale production, should facilitate not only industrial enzyme production but also the identification of novel enzymes, because microorganisms contain many silent or weakly expressed genes which encode novel antibiotics or enzymes. Furthermore, this study provides a new mechanism for strain improvement, with a consistent rather than transient high expression of the key gene(s) involved in enzyme production.

Ribosome engineering has been widely utilized for strain improvement, especially for the activation of bacterial secondary metabolism. This study assessed ribosome engineering technology to modulate primary metabolism, taking vitamin B12 production as a representative example. The introduction into Propionibacterium shermanii of mutations conferring resistance to rifampicin, gentamicin, and erythromycin, respectively, increased per cell production (μg/L/OD600) of vitamin B12 5.2-fold, although net production (μg/L) was unchanged, as the cell mass of the mutants was reduced. Real-time qPCR analysis demonstrated that the genes involved in vitamin B12 fermentation by P. shermanii were activated at the transcriptional level in the drug-resistant mutants, providing a mechanism for the higher yields of vitamin B12 by the mutants. These results demonstrate the efficacy of ribosome engineering for the production of not only secondary metabolites but of industrially important primary metabolites.

Regulatory T cells (Tregs) are a subset of CD4+ T cells with suppressive function and are critical for limiting inappropriate activation of T cells. Hence, the expansion of Tregs is an attractive strategy for the treatment of autoimmune diseases. Here, we demonstrate that the skin possesses the remarkable capacity to systemically expand Treg numbers by producing thymic stromal lymphopoietin (TSLP) in response to vitamin D receptor stimulation. An ∼2-fold increase in the proportion and absolute number of Tregs was observed in mice treated topically but not systemically with the Vitamin D3 analog MC903. This expansion of Tregs was dependent on TSLP receptor signaling but not on VDR signaling in hematopoietic cells. However, TSLP receptor expression by Tregs was not required for their proliferation. Rather, skin-derived TSLP promoted Treg expansion through dendritic cells. Importantly, treatment of skin with MC903 significantly lowered the incidence of autoimmune diabetes in non-obese diabetic mice and attenuated disease score in experimental autoimmune encephalomyelitis. Together, these data demonstrate that the skin has the remarkable potential to control systemic immune responses and that Vitamin D-mediated stimulation of skin could serve as a novel strategy to therapeutically modulate the systemic immune system for the treatment of autoimmunity.

Since bacteria were found to contain genes encoding enzymes that synthesize a plethora of potential secondary metabolites, interest has grown in the activation of these cryptic pathways. Homologous and heterologous expression of these cryptic secondary metabolite-biosynthetic genes, often “silent” under ordinary laboratory fermentation conditions, may lead to the discovery of novel secondary metabolites. We review current progress on this topic, describing concepts for activating silent genes. We especially focus on genetic manipulation of transcription and translation, as well as the utilization of rare earth elements as a novel method to activate the silent genes. The possible roles of silent genes in bacterial physiology are also discussed.

Bacillus subtilis contains 10 rRNA ( rrn ) operons. We found that rRNA operon-engineered B. subtilis strain RIK543, with only the rrnO operon, is specifically hypersensitive to RNA polymerase inhibitors such as rifamycin SV and rifampin (80-fold and 20-fold, respectively). In pilot screening experiments, we found actinomycete isolates successfully at an incidence of 1.9% (18/945) that produced antibacterials that were detectable only with RIK543 as the test organism. Strain RIK543 may be a feasible test organism for the discovery of novel RNA polymerase inhibitors.

Dendritic cells (DCs) in lymphoid and non-lymphoid tissues are professional antigen-presenting cells that are essential for effective immunity and tolerance. However, the presence and characteristics of DCs in steady-state salivary glands (SGs) currently remain unknown. We herein identified CD64

Natural killer (NK) group 2D (NKG2D) is a key activating receptor expressed on NK cells, whose interaction with ligands on target cells plays an important role in tumorigenesis. However, the effect of histamine on NKG2D ligands on tumour cells is unclear. Here we showed that human monocytic leukaemia THP-1 cells constitutively express MHC class I-related chain A (MICA) and UL16-binding protein 1 on their surface, and incubation with histamine reduced the expression in a dose-dependent and time-dependent manner as assessed by flow cytometry. Interferon-γ augmented the surface expression of the NKG2D ligands, and this augmentation was significantly attenuated by histamine. The histamine H1 receptor (H1R) agonist 2-pyridylethylamine and H2R agonist dimaprit down-regulated the expression of NKG2D ligands, and activation of H1R and H2R signalling by A23187 and forskolin, respectively, had the same effect, indicating that the histamine-induced down-regulation of NKG2D ligands is mediated by H1R and H2R. Quantitative reverse transcription-PCR showed that mRNA levels of the NKG2D ligands and relevant microRNAs were not significantly changed by histamine. Histamine down-regulated the surface expression of endoplasmic reticulum protein 5, and inhibition of matrix metalloproteinases did not impair this down-regulation, indicating that proteolytic shedding was not involved. Instead, pharmacological inhibition of protein transport and proteasome abrogated it, and histamine enhanced ubiquitination of MICA. Furthermore, histamine treatment significantly reduced susceptibility to NK cell-mediated cytotoxicity. These results suggest that histamine down-regulates NKG2D ligands through the activation of an H1R- and H2R-mediated ubiquitin-proteasome pathway and consequently reduces susceptibility to NK cells.

A P-type icosahedral quasicrystal is formed in Au-Al-Yb alloy of which 6-dimensional lattice parameter a6D = 7.448 {\AA}. The composition of the quasicrystal was analyzed to be Au51Al34Yb15. This quasicrystal is formed in as-cast alloys, and is regarded as metastable because of decomposition into other crystalline phases by annealing at 700 \degree C. Among Tsai-type quasicrystals, this quasicrystal is situated just between Zn-Sc group with smaller a6D and larger Cd-Yb group. Intermediate valence of Yb recently observed in this quasicrystal may be due to this unique situation, namely smaller major component Au-Al than in Cd-Yb. The predominant phase in the annealed specimen is a 1/1 cubic approximant with lattice parameter a = 14.500 {\AA} belonging to the space group Im-3 . This phase is stable at the composition Au51Al35Yb14 at 700 \degree C. Rietveld structural analysis indicated that the crystal structure is understood as periodic arrangement of Tsai-type clusters each including four Au-Al atoms at their centers. Chemical ordering of Au and Al is characteristics of this approximant., Comment: 20 pages including 1 table and 7 figures

Genome sequencing projects have revealed many biosynthesis gene clusters for the production of as-yet unknown secondary metabolites, especially in actinomycetes. Here, we report that the rare earth elements, scandium and/or lanthanum, markedly activate, ranging from 2.5- to 12-fold, the expression of nine genes belonging to nine secondary metabolite-biosynthetic gene clusters of Streptomyces coelicolor A3(2) when added to the medium at low concentrations. HPLC analysis of ethyl acetate-extractable metabolites indicated the detectability of several compounds only in the rare earth-treated cultures. This approach should facilitate discovery of new biologically active compounds and the study of secondary metabolite production.

This paper deals with a new PCD named EC-PCD which is made up of boron doped diamond particles and its properties related to EDM machinability. For the purpose of improving various properties of standard PCD including resistance to heat, wear and reactivity, a new PCD (EC-PCD) was manufactured on a trial basis using electrically conductive diamond particle as a basic ingredient. Grain size, resistivity and thermal conductivity of the boron doped diamond used are 10μm, 5~37×10Ω•m and 440~580W/m•K. In this report, machinability of newly developed PCD (EC-PCD) by wire EDM was investigated in comparison with that of standard PCD. In wire cutting of 2 types of PCD in water under the condition of open gap voltage: ue=80V, set peak current: iP=0.8A and pulse condition: te/to=20/20μs, it was found that roughness of the first cut surface of standard PCD was approximately 8μm Rz, while that of EC-PCD was far better such as 3μm. Also in finish cut (7th cut), the latter achieved the value of Rz=1.7μm while the former achieved only the value of Rz=2.7μm. Expecting better performance, EC-PCD was tested also in oil. As a result, the best achieved roughness was improved to Rz=0.4μm with no chipping on the edge. To explore a reason for such a good roughness obtained, the cut samples were observed on the SEM, which revealed that the diamond particles in EC-PCD were flattened by electro discharge.

The methyltransferases RsmG and KsgA methylate the nucleotides G535 (RsmG) and A1518 and A1519 (KsgA) in 16S rRNA, and inactivation of the proteins by introducing mutations results in acquisition of low-level resistance to streptomycin and kasugamycin, respectively. In a Bacillus subtilis strain harboring a single rrn operon ( rrnO ), we found that spontaneous ksgA mutations conferring a modest level of resistance to kasugamycin occur at a high frequency of 10 −6 . More importantly, we also found that once cells acquire the ksgA mutations, they produce high-level kasugamycin resistance at an extraordinarily high frequency (100-fold greater frequency than that observed in the ksgA + strain), a phenomenon previously reported for rsmG mutants. This was not the case for other antibiotic resistance mutations (Tsp r and Rif r ), indicating that the high frequency of emergence of a mutation for high-level kasugamycin resistance in the genetic background of ksgA is not due simply to increased persistence of the ksgA strain. Comparative genome sequencing showed that a mutation in the speD gene encoding S -adenosylmethionine decarboxylase is responsible for the observed high-level kasugamycin resistance. ksgA speD double mutants showed a markedly reduced level of intracellular spermidine, underlying the mechanism of high-level resistance. A growth competition assay indicated that, unlike rsmG mutation, the ksgA mutation is disadvantageous for overall growth fitness. This study clarified the similarities and differences between ksgA mutation and rsmG mutation, both of which share a common characteristic—failure to methylate the bases of 16S rRNA. Coexistence of the ksgA mutation and the rsmG mutation allowed cell viability. We propose that the ksgA mutation, together with the rsmG mutation, may provide a novel clue to uncover a still-unknown mechanism of mutation and ribosomal function.

Bacillus subtilis strains produce a wide range of antibiotics, including ribosomal and nonribosomal peptide antibiotics, as well as bacilysocin and neotrehalosadiamine. Mutations in B. subtilis strain 168 that conferred resistance to drugs such as streptomycin and rifampin resulted in overproduction of the dipeptide antibiotic bacilysin. Cumulative drug resistance mutations, such as mutations in the mthA and rpsL genes, which confer low- and high-level resistance, respectively, to streptomycin, and mutations in rpoB , which confer resistance to rifampin, resulted in cells that overproduced bacilysin. Transcriptional analysis demonstrated that the enhanced transcription of biosynthesis genes was responsible for the overproduction of bacilysin. This approach was effective also in activating the cryptic genes of Bacillus amyloliquefaciens , leading to actual production of antibiotic(s).

Improved antibiotic production and silent gene activation in Streptomyces diastatochromogenes by ribosome engineering

The observing method that uses ENA (Energetic Neutral Atom), produced by Charge Exchange Collision, is studied and researched by ISAS in order to estimate the density of neutral particles in the upper atmosphere. Its method is called ENA observation. At present, the ENA observation is done by Charge Exchange Collision between neutrals in the upper atmosphere and the ions in the magnetospheric ring current field that is activated by solar wind. But in this method, it is difficult to separate the informations between neutrals and ions from ENA data because the ions in the magnetospheric ring current field are the natural origin and their energies are not constant. In this study, we propose the new method of ENA observation that uses ions exhausted from ion thruster. Ions from ion thruster are the artificial ones and are more suitable for ENA observation because their energies and densities can be controlled. We evaluate the possibility of application of ion thruster’s exhausted ions to ENA observation. Analytical results show the range of altitude and the operating conditions of ion thruster that this method effectively can apply.

Bisphosphonates (BPs) are pyrophosphate analogs. They are widely used against enhanced bone-resorption in various diseases. Nitrogen-containing BPs (N-BPs) exhibit strong anti-bone-resorptive effects but have inflammatory and necrotic side effects. The non-nitrogen-containing BPs (non-N-BPs) etidronate and clodronate lack such side effects, but their anti-bone-resorptive effects are weak. In mice, etidronate and clodronate reduce the inflammatory/necrotic effects of N-BPs, even those of zoledronate, the N-BP with the strongest anti-bone-resorptive effect yet reported and the highest risk of inflammation/necrosis. Here, to explore the mechanisms underlying this protection, we used a mouse model in which a single reagent or a mixture of two reagents was injected subcutaneously into ear-pinnas. These reagents included zoledronate, four non-N-BPs, pyrophosphate, and inhibitors of various organic-anion-transporters. Pyrophosphate and two of the four non-N-BPs (not etidronate or clodronate) had inflammatory/necrotic effects. These effects were reduced by etidronate and clodronate, but not by phosphonoformate, an inhibitor of two of the three known phosphate-transporter families. Phosphonoformate reduced the inflammatory/necrotic effects of zoledronate, but not those of pyrophosphate or of non-N-BPs. Conversely, pyrophosphate, at non-inflammatory/necrotic concentrations, reduced the inflammatory/necrotic effects of non-N-BPs, but not those of zoledronate. The efficacies of the protective effects against the inflammatory/necrotic effects of zoledronate were clodronate > etidronate > phosphonoformate. These findings suggest that (i) the N-BP zoledronate may enter soft-tissue cells via phosphonoformate-inhibitable phosphate-transporters, (ii) other phosphate-transporters may carry pyrophosphate and inflammatory/necrotic non-N-BPs into such cells, and (iii) etidronate and clodronate inhibit all these transporters, and they ameliorate the side effects of zoledronate by inhibiting phosphonoformate-inhibitable phosphate-transporters.

Diseases involving enhanced bone-resorption (e.g., osteoporosis) are widely treated with bisphosphonates (BPs). BPs are of two types: the nitrogen-containing BPs (N-BPs) and the non-nitrogen-containing BPs (non-N-BPs). N-BPs have much stronger anti-bone-resorptive effects than non-N-BPs, and N-BPs can exert inflammatory and necrotic effects, including osteonecrosis of jawbones. Minodronate, an N-BP, was approved in 2009 in Japan for osteoporosis. Its anti-bone-resorptive effect is comparable to that of zoledronate, the N-BP with the strongest anti- bone-resorptive effect and the highest risk of side effects yet reported. Unlike other N-BPs, minodronate has an analgesic effect, and no serious side effects have been documented. Here, to examine whether minodronate lacks inflammatory and/or necrotic effects, we used mice (since the N-BPs tested so far induce such effects in mice with potencies that parallel those reported in humans). To facilitate comparison with previous studies, we gave a single systemic (intraperitoneal) or local (ear pinna) injection of minodronate (or another N-BP). We measured the systemic responses (weight of thoracic exudate, number of inflammatory cells in the peritoneal cavity, and spleen weight) or local responses (area of inflamed skin and incidence of necrosis). Anti-bone-resorptive effects were evaluated by X-ray analysis of tibias following intraperitoneal injection. Minodronate's anti-bone-resorptive effect and its inflammatory and necrotic effects were as great as, or greater than those of zoledronate. Moreover, in cultured human periodontal ligament cells, the cytotoxicity of minodronate was significantly greater than that of zoledronate. These results suggest that caution may be needed with minodronate in clinical use, as with other N-BPs.

A subset of rifampin resistance ( rpoB ) mutations result in the overproduction of antibiotics in various actinomycetes, including Streptomyces , Saccharopolyspora , and Amycolatopsis , with H437Y and H437R rpoB mutations effective most frequently. Moreover, the rpoB mutations markedly activate (up to 70-fold at the transcriptional level) the cryptic/silent secondary metabolite biosynthetic gene clusters of these actinomycetes, which are not activated under general stressful conditions, with the exception of treatment with rare earth elements. Analysis of the metabolite profile demonstrated that the rpoB mutants produced many metabolites, which were not detected in the wild-type strains. This approach utilizing rifampin resistance mutations is characterized by its feasibility and potential scalability to high-throughput studies and would be useful to activate and to enhance the yields of metabolites for discovery and biochemical characterization.

A quasiperiodic intermediate-valence (IV) system is realized in an icosahedral Au-Al-Yb quasicrystal. X-ray absorption spectroscopy near the Yb ${L}_{3}$ edge indicates that quasiperiodically arranged Yb ions assume a mean valence of 2.61, between a divalent state ($4{f}^{14}$, $J=0$) and a trivalent one ($4{f}^{13}$, $J=7/2$). Magnetization measurements demonstrate that the $4f$ holes in this quasicrystal have a localized character. The magnetic susceptibility shows a Curie-Weiss behavior above $\ensuremath{\sim}100$ K with an effective magnetic moment of 3.81${\ensuremath{\mu}}_{\mathrm{B}}$ per Yb. Moreover, a crystalline approximant to this quasicrystal is an IV compound. We propose a heterogeneous IV model for the quasicrystal, whereas the crystalline approximant is most likely a homogeneous IV system. At temperatures below $\ensuremath{\sim}10$ K, specific heat and magnetization measurements reveal non-Fermi-liquid behavior in both the quasicrystal and its crystalline approximant without either doping, pressure, or field tuning.

Previously, two anti-Ly-6G mAb—RB6-8C5 and 1A8—have been used to deplete neutrophils in mice and to clarify their involvement in immune responses. During the course of experiments on neutrophil depletion, we noticed that i.v. injection of RB6-8C5 or 1A8 induced anaphylaxis-like shock in mice pretreated i.v. with LPS. Signs of shock, such as hypothermia, appeared within a few minutes, and the mice died of shock within 20 min of the antibody injection. In vivo experiments, including depletion of various cell types, indicated that neutrophils and macrophages (but not platelets, basophils, or mast cells) are involved in the shock. Experiments using various drugs and gene-targeted mice demonstrated that PAF is the central mediator of the shock. Optimal LPS priming required at least 1 h, and the priming was associated with neutrophil accumulation within pulmonary and hepatic blood vessels. Consistently, following 1A8 injection into LPS-pretreated mice, the mRNA for LysoPAFAT (a PAF biosynthetic enzyme) was markedly up-regulated in neutrophils accumulated in the lung but not in macrophages. These results suggest that (1) stimulation of Ly-6G on LPS-primed neutrophils induces PAF-mediated anaphylaxis-like shock in mice, (2) neutrophils are primed by LPS during and/or after their accumulation in lung and liver to rapidly induce LysoPAFAT, and (3) macrophages may play a pivotal role in the priming phase and/or in the challenge phase by unknown mechanisms. These findings may be related to adult respiratory distress syndrome, although the natural ligand for Ly-6G remains to be identified.

Deficient antibiotic production in an afsB mutant, BH5, of Streptomyces coelicolor A3(2) was recently shown to be due to a mutation (G243D) in region 1.2 of the primary sigma factor σ HrdB. Here we show that intracellular ppGpp levels during growth, as well as after amino acid depletion, in the mutant BH5 are lower than those of the afsB+ parent strain. The introduction of certain rifampicin resistance (rif) mutations, which bypassed the requirement of ppGpp for transcription of pathway-specific regulatory genes, actII-ORF4 and redD, for actinorhodin and undecylprodigiosin, respectively, completely restored antibiotic production by BH5. Antibiotic production was restored also by introduction of a new class of thiostrepton-resistance (tsp) mutations, which provoked aberrant accumulation of intracellular ppGpp. Abolition of ppGpp synthesis in the afsB tsp mutant Tsp33 again abolished antibiotic production. These results indicate that intracellular ppGpp level is finely tuned for successful triggering of antibiotic production in the wild-type strain, and that this fine tuning was absent from the afsB mutant BH5, resulting in a failure to initiate antibiotic production in this strain.

During the course of experiments on neutrophil depletion, we happened to find that intravenous injection of antineutrophil monoclonal antibodies (RB6-8C5 and 1A8) induces anaphylaxis-like shock in mice pretreated with lipopolysaccharide. The shock reaction depends largely on neutrophils and Toll-like receptor 4, and significantly on macrophages. Complement C5 may be involved in the shock reaction. We expect that elucidation of behavior of neutrophils may contribute to understand the pathology of septic shock.

Unlike other Streptomyces spp., the streptomycin producer Streptomyces griseus IFO13189 shows emergence of a small fraction of rsmG and rpsL mutants among spontaneous low- or high-level streptomycin-resistant mutants. rsmG, but not rpsL, mutants showed greater ability (two- to threefold) to produce streptomycin, accompanied by enhanced transcription of metK and strR, together with streptomycin biosynthetic genes, such as strB1, strD and strF, thus underlying the observed increase in streptomycin production in the rsmG mutants. Moreover, rsmG mutation was effective for activating the 'silent' or poorly expressed secondary metabolite-biosynthetic genes present in S. griseus.

Certain streptomycin resistance mutations (i.e., rpsL and rsmG ) result in the overproduction of antibiotics in various actinomycetes. Moreover, rpsL rsmG double-mutant strains show a further increase in antibiotic production. rpsL but not rsmG mutations result in a marked enhancement of oligomycin production in Streptomyces avermitilis and erythromycin production in Saccharopolyspora erythraea , accompanied by increased transcription of a key developmental regulator gene, bldD , in the latter organism.