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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.

Abstract 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.

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 naïve 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 . The latter was more pronounced and attributed to reduced responsiveness to IL-2. Acute deletion of PRMT5 revealed that not only naïve 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 . 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.

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.

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 II hi CD11c int migratory DCs were significantly stronger than those of MHC class II int CD11c hi resident DCs and CD11c int PDCA1 + MHC class II int 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.

Objective and methodsIL-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.ResultsA 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 the bone marrow. HDC was also induced in non-mast cells in non-hematopoietic organs. HDC, histamine, and histamine H4 receptors (H4Rs) contributed to the suppression of IL-33-induced eosinophilia.ConclusionIL-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 the negative regulation of IL-33-induced eosinophilia via H4Rs.

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.

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.

Background Chronic neuropathic pain, particularly in the orofacial region, markedly reduces quality of life. Peripheral trigeminal nerve injury activates satellite glial cells (SGCs) in the trigeminal ganglion (TG), which contributes to orofacial neuropathic pain. However, the upstream signal responsible for SGC activation remains unclear. This study investigated the role and cellular sources of interferon gamma (IFN-γ) signaling in the TG following infraorbital nerve injury (IONI) in rats. Methods Mechanical sensitivity of the whisker pad skin was assessed after IONI. Changes in IFN-γ levels, IFN-γ receptor expression, and glial fibrillary acidic protein (GFAP; a marker of SGC activation) were examined in the TG by immunohistochemistry. The effects of intra-TG administration of IFN-γ, an IFN-γ receptor antagonist, and isolated CD8⁺ T cells on mechanical hypersensitivity were evaluated. GFAP expression after intra-TG administration of IFN-γ or the receptor antagonist was also quantified. Flow cytometry and immunohistochemistry were used to identify IFN-γ–producing cells. In primary SGC cultures, IFN-γ–induced interleukin-1β (IL-1β) release was measured, and the impact of IL-1 receptor (IL-1R1) antagonism on mechanical hypersensitivity was tested. IL-1R1 localization and expression in TG neurons was further evaluated after IONI. Results IONI induced persistent mechanical hypersensitivity and upregulated IFN-γ, IFN-γ receptor, and GFAP expression in the TG. CD8⁺ T cells were the primary source of IFN-γ after IONI, and intra-TG transfer of isolated CD8⁺ T cells transiently induced mechanical hypersensitivity. IFN-γ receptors were localized to SGCs, with expression levels increasing after IONI. Intra-TG IFN-γ administration triggered mechanical hypersensitivity and SGC activation, and its receptor antagonism attenuated the hypersensitivity. IFN-γ stimulation of cultured SGCs enhanced IL-1β release. Co-administration of an IL-1R1 antagonist prevented IFN-γ–induced mechanical hypersensitivity. IL-1R1 was localized on TG neurons and were upregulated following IONI. Conclusions CD8⁺ T cell–derived IFN-γ activates SGCs in the TG, leading to IL-1β release that promotes neuronal hyperactivity and orofacial neuropathic pain following IONI. Targeting the IFN-γ–SGC–IL-1β signaling axis may represent a novel therapeutic strategy for orofacial neuropathic pain. Graphical Abstract