Diacylglycerol kinase zeta deficiency attenuates papain-induced type 2 airway inflammation.

Allergic airway diseases are caused by inappropriate immune responses directed against inhaled environmental antigens. We previously reported that the inhibition of diacylglycerol (DAG) kinaseζ (DGKζ),an enzyme that terminates DAG-mediated signaling,protects against T cell-mediated allergic airway inflammation by blocking Th2 cell differentiation.In this study, we tested whether DGKζ deficiency also affects allergic airway disease mediated by type 2 innate lymphoid cells (ILC2)s. DGKζ-deficient mice displayed diminished ILC2 function and reduced papain-induced airway inflammation compared to wildtype mice. Unexpectedly, however, mice with hematopoietic cell-specific deletion ofDGKζ displayed intact airway inflammation upon papain challenge. Rather, bone marrow chimera studies revealed thatDGKζ deficiency in the non-hematopoietic compartment was responsible for the reduction in papain-induced airway inflammation. These data suggest that DGK might represent a novel therapeutic target not only for T cell-dependent but also ILC2-dependent allergic airway inflammation by affecting non-hematopoietic cells.

IL-33 induces histidine decarboxylase, especially in c-kit+ cells and mast cells, and roles of histamine include negative regulation of IL-33-induced eosinophilia.

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 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. 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 the negative regulation of IL-33-induced eosinophilia via H4Rs.

Differential expression of CD11c defines two types of tissue-resident macrophages with different origins in steady-state salivary glands.

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.

Nitrogen-containing bisphosphonates and lipopolysaccharide mutually augment inflammation via adenosine triphosphate (ATP)-mediated and interleukin 1ß (IL-1ß)-mediated production of neutrophil extracellular traps (NETs).

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 1ß (IL-1ß); (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-1ß-producing cells, and upon stimulation with IL-1ß, neutrophils produced NETs; (viii) stimulation of the purinergic 2X7 (P2X7) receptors by ATP induced IL-1ß 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-1ß production by neutrophils, macrophages, and/or dendritic cells; and (iii) NET production by IL-1ß-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).

Cellular context of IL-33 expression dictates impact on anti-helminth immunity.

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.

Migratory dendritic cells in skin-draining lymph nodes have nickel-binding capabilities.

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-1ß 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-1ß 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.

PRMT5 Associates With the FOXP3 Homomer and When Disabled Enhances Targeted p185erbB2/neu Tumor Immunotherapy.

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.

Combined Drug Resistance Mutations Substantially Enhance Enzyme Production in Paenibacillus agaridevorans.

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.

Retained Myogenic Potency of Human Satellite Cells from Torn Rotator Cuff Muscles Despite Fatty Infiltration.

Rotator cuff tears (RCTs) are a common shoulder problem in the elderly that can lead to both muscle atrophy and fatty infiltration due to less physical load. Satellite cells, quiescent cells under the basal lamina of skeletal muscle fibers, play a major role in muscle regeneration. However, the myogenic potency of human satellite cells in muscles with fatty infiltration is unclear due to the difficulty in isolating from small samples, and the mechanism of the progression of fatty infiltration has not been elucidated. The purpose of this study was to analyze the population of myogenic and adipogenic cells in disused supraspinatus (SSP) and intact subscapularis (SSC) muscles of the RCTs from the same patients using fluorescence-activated cell sorting. The microstructure of the muscle with fatty infiltration was observed as a whole mount condition under multi-photon microscopy. Myogenic differentiation potential and gene expression were evaluated in satellite cells. The results showed that the SSP muscle with greater fatty infiltration surrounded by collagen fibers compared with the SSC muscle under multi-photon microscopy. A positive correlation was observed between the ratio of muscle volume to fat volume and the ratio of myogenic precursor to adipogenic precursor. Although no difference was observed in the myogenic potential between the two groups in cell culture, satellite cells in the disused SSP muscle showed higher intrinsic myogenic gene expression than those in the intact SSC muscle. Our results indicate that satellite cells from the disused SSP retain sufficient potential of muscle growth despite the fatty infiltration.

Metabolic perturbation to enhance polyketide and nonribosomal peptide antibiotic production using triclosan and ribosome-targeting drugs.

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.

Skin-derived TSLP systemically expands regulatory T cells.

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.

CD103+ CD11b- salivary gland dendritic cells have antigen cross-presenting capacity.

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 naïve 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.

The mthA mutation conferring low-level resistance to streptomycin enhances antibiotic production in Bacillus subtilis by increasing the S-adenosylmethionine pool size.

Certain Str(r) mutations that confer low-level streptomycin resistance result in the overproduction of antibiotics by Bacillus subtilis. Using comparative genome-sequencing analysis, we successfully identified this novel mutation in B. subtilis as being located in the mthA gene, which encodes S-adenosylhomocysteine/methylthioadenosine nucleosidase, an enzyme involved in the S-adenosylmethionine (SAM)-recycling pathways. Transformation experiments showed that this mthA mutation was responsible for the acquisition of low-level streptomycin resistance and overproduction of bacilysin. The mthA mutant had an elevated level of intracellular SAM, apparently acquired by arresting SAM-recycling pathways. This increase in the SAM level was directly responsible for bacilysin overproduction, as confirmed by forced expression of the metK gene encoding SAM synthetase. The mthA mutation fully exerted its effect on antibiotic overproduction in the genetic background of rel(+) but not the rel mutant, as demonstrated using an mthA relA double mutant. Strikingly, the mthA mutation activated, at the transcription level, even the dormant ability to produce another antibiotic, neotrehalosadiamine, at concentrations of 150 to 200 µg/ml, an antibiotic not produced (<1 µg/ml) by the wild-type strain. These findings establish the significance of SAM in initiating bacterial secondary metabolism. They also suggest a feasible methodology to enhance or activate antibiotic production, by introducing either the rsmG mutation to Streptomyces or the mthA mutation to eubacteria, since many eubacteria have mthA homologues.

In vitro cytotoxicity of zoledronate (nitrogen-containing bisphosphonate: NBP) and/or etidronate (non-NBP) in tumour cells and periodontal cells.

OBJECTIVE: Nitrogen-containing bisphosphonates (NBPs), the first-choice drugs for diseases that cause enhanced bone resorption, may injure jawbones and gastrointestinal tissues. In rodents, NBPs cause necrosis at injection sites. Bisphosphonates accumulate within bones, especially where there is inflammation. We hypothesized that if jawbone-accumulated NBPs are released, they may directly injure cells around the jawbones. To examine this hypothesis, we compared the direct effects of zoledronate (NBP) and/or etidronate (non-NBP) on various cells, including periodontal cells. DESIGN: Various human tumour cells (such as squamous carcinoma cells and prostate adenocarcinoma cells) and periodontal cells (such as gingival fibroblasts and periodontal ligament cells) were incubated with or without zoledronate and/or etidronate. Cell viability and cytotoxicity were determined by tetrazolium dye assay and by FITC-Annexin V/propidium iodide assay, respectively. RESULTS: Zoledronate, at 100µM, was toxic to all types of cells tested, while its toxicity varied among cells at both 1 and 10µM. There was no clear difference between tumour cells and non-tumour cells in sensitivity to the cytotoxicity of zoledronate. In contrast, etidronate was not toxic at 1-100µM in any of the cells tested. Interestingly, etidronate reduced the cytotoxicity of zoledronate in many cell-types, including gingival fibroblasts. CONCLUSIONS: These results, together with those reported by others and those from our previous in vivo experiments, suggest that NBPs, upon release from jawbones (e.g., during dental surgery or bone infection), may directly injure various cells located around the jawbones, and that etidronate may be protective against the cytotoxicity of NBPs in periodontal tissues.

Histamine reduces susceptibility to natural killer cells via down-regulation of NKG2D ligands on human monocytic leukaemia THP-1 cells.

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.

Stimulation of Ly-6G on neutrophils in LPS-primed mice induces platelet-activating factor (PAF)-mediated anaphylaxis-like shock.

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.

Biotin status affects nickel allergy via regulation of interleukin-1beta production in mice.

Biotin, a water-soluble B complex vitamin, is possibly involved in chronic inflammatory diseases, although the detailed mechanisms are unclear. In this study, we investigated the effects of biotin status on nickel (Ni) allergy in mice. Mice were fed a basal or biotin-deficient (BD) diet for 8 wk and sensitized with an intraperitoneal injection of NiCl(2) and lipopolysaccharide. Ten days after sensitization, NiCl(2) was intradermally injected into pinnas and ear swelling was measured. For in vitro analysis, we cultured a murine macrophage cell line, J774.1, under a biotin-sufficient (C, meaning control) or BD condition for 4 wk and analyzed interleukin (IL)-1 production. Significantly higher ear swelling was induced in BD mice than C mice. Adaptive transfer of splenocytes from both C and BD mice induced Ni allergy in unsensitized mice. Regardless of donor mice, ear swelling was significantly higher in BD recipient mice than C recipient mice. Ni allergy was not induced in either C or BD IL-1(-/-) mice. Splenocytes from BD mice produced a significantly higher amount of IL-1beta than those from C mice. Production and mRNA expression of IL-1beta were significantly higher in BD J774.1 cells than in C cells. Biotin supplementation inhibited the augmentation of IL-1beta production in vitro. In vivo supplementation of biotin in drinking water dose-dependently decreased ear swelling in C and BD mice. These results indicate that biotin status affects Ni allergy in the elicitation phase via the upregulation of IL-1beta production in mice, suggesting that biotin supplementation may have therapeutic effects on human metal allergy.

Inactivation of KsgA, a 16S rRNA methyltransferase, causes vigorous emergence of mutants with high-level kasugamycin resistance.

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.

Human parotid saliva contains soluble toll-like receptor (TLR) 2 and modulates TLR2-mediated interleukin-8 production by monocytic cells.

Toll-like receptor (TLR) family members are pattern-recognition receptors and very important molecules in innate immunity. Although TLRs are originally type I transmembrane receptors, soluble forms of TLRs are detected in human plasma and milk. This study showed that soluble TLR2 (sTLR2) is detected in human parotid saliva. Western blotting with anti-TLR2 antibodies (Abs) showed that three polypeptides are detected as sTLR2 with molecular weights of 55, 40 and 27kDa, respectively. Parotid saliva neutralized the binding of anti-TLR2 polyclonal Ab to cell-surface TLR2 on THP-1, a human monocytic cell line. Immunohistochemical analysis revealed that TLR2 is expressed in serous and interlobular ductal cells of human salivary gland. Human salivary gland cell lines, AZA3 and HSY, constitutively expressed TLR2. Parotid saliva augmented IL-8 production of THP-1 cells stimulated with a synthetic TLR2 ligand, Pam(3)Cys-Ser-(Lys)(4) (Pam(3)CSK(4)). Depletion of sCD14 from parotid saliva by immunoprecipitation eliminated the augmentation of IL-8 production, indicating that the augmentable effects depended on sCD14 in parotid saliva. On the other hand, preincubation of Pam(3)CSK(4) with parotid saliva abrogated the augmentation of IL-8 production, indicating that sTLR2 in saliva bound to Pam(3)CSK(4) and neutralized its function. These results suggest that parotid saliva modulates the TLR2-mediated immune responses with binary mechanisms via sTLR2 and sCD14 in the oral cavity.