ARG2 Research Articles

Injecting Immunosuppressive M2 Macrophages Alleviates the Symptoms of Periodontitis in Mice.

Periodontitis is the second most common oral disease affecting tooth-supporting structures. The tissue damage is mainly initiated by the excessive secretion of proinflammatory cytokines by immune cells. Macrophages are a type of antigen-presenting cells that influence the adaptive immunity function. We used a unique set of cytokines, i.e., a combination of IL-4, IL-13, and IL-10, to stimulate macrophages into a subset of M2 polarization cells that express much higher levels of ARG-1, CD206, and PDL-2 genes. The cells’ anti-inflammatory potential was tested with mixed-lymphocyte reaction assay, which showed that this subset of macrophages could increase IL-2 secretion and suppress IL-17, IL-6, and TNF-α secretion by splenocytes. The gram-negative bacterial species Porphyromonas gingivalis was used to initiate an inflammatory process in murine periodontal tissues. In the meantime, cell injection therapy was used to dampen the excessive immune reaction and suppress osteoclast differentiation during periodontitis. Maxilla was collected and analyzed for osteoclast formation. The results indicated that mice in the cell injection group exhibited less osteoclast activity within the periodontal ligament region than in the periodontitis group. Moreover, the injection of M2 macrophages sustained the regulatory population ratio. Therefore, the M2 macrophages induced under the stimulation of IL-4, IL-13, and IL-10 combined had tremendous immune modulation ability. Injecting these cells into local periodontal tissue could effectively alleviate the symptom of periodontitis.

Metabolic and genes expression analyses involved in proline metabolism of two rose species under drought stress

An investigation was conducted to assess proline anabolism and catabolism pathway genes under drought stress. Treatments were irrigation in three levels (25, 50 and 100% field capacity) at 1, 3, 6 and 12 d and two rose species (Rosa canina L. and Rosa damascene Mill.). The results showed that the potential for proline accumulation in R. damascena was higher than R. canina under drought. Simultaneous with proline accumulation, expression of P5CS and P5CR genes increased from 1 to 12 d under 50% FC whereas their expression had an increasing trends from 1 to 6 d at 25% FC and expression of both genes decreased at 12 d in both species. The highest accumulation of proline was observed under 25% FC at 12 d, but expression of genes involved in proline synthesis main pathway decreased on this day. Furthermore, expression of genes (PDH and P5CDH) involved in proline catabolism pathway decreased in 50% FC from 1 to 12 d while their expression remarkably decreased from 1 to 6 d and increased at 12 d under 25% FC. These findings showed that under conditions of 50 and 25% FC, arginine accumulation resulted in the increased expression of the ARG gene, which led to ornithine production. Furthermore, ornithine accumulation increased OAT expression. Therefore, it seems that OAT-induced P5C is transported from the mitochondria to the cytosol and reduced to proline by the P5CR.

The role of polymorphic variants of arginase genes (<i>ARG1, ARG2</i>) involved in beta-2-agonist metabolism in the development and course of asthma

Asthma is a common severe disease of the respiratory tract, it leads to a significant impairment in the quality of a patient’s life unless effectively treated. Uncontrolled asthma symptoms are a cause of disease progression and development, they lead to an increase in the patient’s disability. The sensitivity to asthma therapy largely depends on the interaction of genetic and epigenetic factors, which account for about 50–60 % of variability of therapeutic response. Beta-2-agonists are some of the major class of bronchodilators used for asthma management. According to published data, allelic variants of the arginase ARG1 and ARG2 genes are associated with a risk of asthma development, spirometry measures and efficacy of bronchodilator therapy. High arginase activity results in a low level of plasma L-arginine and in a decrease in nitric oxide, and, as a result, in an increase in airway inflammation and remodeling. Arginase genetic polymorphisms (rs2781667 of the ARG1 gene, rs17249437, rs3742879, rs7140310 of the ARG2 gene) were studied in 236 children with asthma and 194 unrelated healthy individuals of Russian, Tatar and Bashkir ethnicity from the Republic of Bashkortostan. Association analysis of the studied polymorphisms with asthma development and course, the sensitivity to therapy in patients was carried out. It was found that the rs2781667*C allele of the ARG1 gene is a marker of an increased risk of asthma in Tatars. In Russians, the association of rs17249437*TT and rs3742879*GG genotypes of the ARG2 gene with a decrease in spirometry measures (FEV1, MEF25) was established. In Russians and Tatars receiving glucocorticoid monotherapy or combination therapy, the association of the rs17249437*T allele and rs17249437*TT genotype of the ARG2 gene with a partially controlled and uncontrolled course of asthma was shown.

DNA methylation in the promoters of PD-L1, MMP9, ARG1, galectin-9, TIM-3, VISTA and TGF-β genes in HLA-DR– myeloid cells, compared with HLA-DR+ antigen-presenting cells

ABSTRACT Myeloid cells, including antigen-presenting cells (APCs) and myeloid-derived suppressor cells (MDSCs) play opposing roles to orchestrate innate and adaptive immune responses during physiological and pathological conditions. We investigated the role of DNA methylation in regulating the transcription of inhibitory/suppressive molecules in myeloid suppressive cells (identified as CD33+HLA-DR–) in comparison to APCs. We selected a number of immune checkpoints (ICs), IC ligands, and immunosuppressive molecules that have been implicated in MDSC function, including PD-L1, TIM-3, VISTA, galectin-9, TGF-β, ARG1 and MMP9. We examined their mRNA expression levels, and investigated whether DNA methylation regulates their transcription in sorted myeloid cell subpopulations. We found that mRNA levels of PD-L1, TIM-3, TGF-β, ARG1 and MMP9 in CD33+HLA-DR– cells were higher than APCs. However, VISTA and galectin-9 mRNA levels were relatively similar in both myeloid subpopulations. CpG islands in the promoter regions of TGF-β1, TIM-3 and ARG1 were highly unmethylated in CD33+HLA-DR–cells, compared with APCs, suggesting that DNA methylation is one of the key mechanisms, which regulate their expression. However, we did not find differences in the methylation status of PD-L1 and MMP9 between CD33+HLA-DR– and APCs, suggesting that their transcription could be regulated via other genetic and epigenetic mechanisms. The promoter methylation status of VISTA was relatively similar in both myeloid subpopulations. This study provides novel insights into the epigenetic mechanisms, which control the expression of inhibitory/suppressive molecules in circulating CD33+HLA-DR– cells in a steady-state condition, possibly to maintain immune tolerance and haemostasis.

Flux Balance Analysis for Media Optimization and Genetic Targets to Improve Heterologous Siderophore Production.

SummarySiderophores are small molecule metal chelators secreted in sparse quantities by their native microbial hosts but can be engineered for enhanced production from heterologous hosts like Escherichia coli. These molecules have been proved to be capable of binding heavy metals of commercial and/or environmental interest. In this work, we incorporated, as needed, the appropriate pathways required to produce several siderophores (anguibactin, vibriobactin, bacillibactin, pyoverdine, and enterobactin) into the base E. coli K-12 MG1655 metabolic network model to computationally predict, via flux balance analysis methodologies, gene knockout targets, gene over-expression targets, and media modifications capable of improving siderophore reaction flux. E. coli metabolism proved supportive for the underlying production mechanisms of various siderophores. Within such a framework, the gene deletion and over-expression targets identified, coupled with complementary insights from medium optimization predictions, portend experimental implementation to both enable and improve heterologous siderophore production. Successful production of siderophores would then spur novel metal-binding applications.

Клинико-генетические ассоциации полиморфизмов генов NO-синтаз и аргиназы и межгенные взаимодействия при бронхиальной астме у детей

Objective. To analyze the association between NOS1, NOS2, NOS3, ARG1, and ARG2 gene polymorphisms and clinical, laboratory, and functional characteristics of bronchial asthma (BA) in children by evaluating gene-gene interactions. Patients and methods. We have evaluated the associations between the NOSs and ARG genes and gene-gene interactions in 107 children with BA. We performed comparative genetic analysis of associations between polymorphic variants of candidate genes in 107 BA patients and clinical manifestations of the disease, laboratory, and functional parameters. Results. We have found an association between the short (S) allele and SS genotype of the NOS1 (AAT)n gene and early disease onset (<5 years), severe BA, and antiinflammatory targeted therapy (with omalizumab and combination inhaled corticosteroids with long-acting β2-agonists). We have also revealed an association between the short (S) allele and SS genotype of the NOS1 (AAT)n gene, as well as long (L) alleles and LL genotypes of the NOS2A (CCTTT)n gene in BA patients and elevated FeNO levels in the exhaled air. The following association have also been identified: between alleles and genotypes containing long (L) tandem repeats in the NOS2A (CCTTT)n gene and reduced FEV1; between AG genotype of the ARG2 gene (rs3742879), alleles and genotypes containing long tandem repeats (L) of the NOS2А (CCTTT)n gene and reduced FEV1/ FVC; between alleles and genotypes containing long tandem repeats (L), combinations of genotypes SL+LL of the NOS2А (CCTTT)n and impaired patency at the level of the middle bronchi FEF50; between allele G, heterozygous genotype AG of the ARG2 (rs3742879) gene with impaired patency at the level of peripheral bronchi FEF75. Conclusion. Our analysis of gene-gene interactions has demonstrated phenotypic characteristics of BA: early BA onset, severe BA, decreased FEV1 and FEF50, increased NO concentration in exhaled air, and presence of fungal and epidermal sensitization. Key words: children, bronchial asthma, NOS and ARG gene polymorphisms, gene-gene interactions

Pan-cancer analysis of the metabolic reaction network

Metabolic reprogramming is considered a hallmark of malignant transformation. However, it is not clear whether the network of metabolic reactions expressed by cancers of different origin differ from each other or from normal human tissues. In this study, we reconstructed functional and connected genome-scale metabolic models for 917 primary tumor samples across 13 types based on the probability of expression for 3765 reference metabolic genes in the sample. This network-centric approach revealed that tumor metabolic networks are largely similar in terms of accounted reactions, despite diversity in the expression of the associated genes. On average, each network contained 4721 reactions, of which 74% were core reactions (present in >95% of all models). Whilst 99.3% of the core reactions were classified as housekeeping also in normal tissues, we identified reactions catalyzed by ARG2, RHAG, SLC6 and SLC16 family gene members, and PTGS1 and PTGS2 as core exclusively in cancer. These findings were subsequently replicated in an independent validation set of 3388 genome-scale metabolic models. The remaining 26% of the reactions were contextual reactions. Their inclusion was dependent in one case (GLS2) on the absence of TP53 mutations and in 94.6% of cases on differences in cancer types. This dependency largely resembled differences in expression patterns in the corresponding normal tissues, with some exceptions like the presence of the NANP-encoded reaction in tumors not from the female reproductive system or of the SLC5A9-encoded reaction in kidney-pancreatic-colorectal tumors. In conclusion, tumors expressed a metabolic network virtually overlapping the matched normal tissues, raising the possibility that metabolic reprogramming simply reflects cancer cell plasticity to adapt to varying conditions thanks to redundancy and complexity of the underlying metabolic networks. At the same time, the here uncovered exceptions represent a resource to identify selective liabilities of tumor metabolism.

Enhanced acid-stress tolerance in Lactococcus lactis NZ9000 by overexpression of ABC transporters

BackgroundMicrobial cell factories are widely used in the production of acidic products such as organic acids and amino acids. However, the metabolic activity of microbial cells and their production efficiency are severely inhibited with the accumulation of intracellular acidic metabolites. Therefore, it remains a key issue to enhance the acid tolerance of microbial cells. In this study, we investigated the effects of four ATP-binding cassette (ABC) transporters on acid stress tolerance in Lactococcus lactis.ResultsOverexpressing the rbsA, rbsB, msmK, and dppA genes exhibited 5.8-, 12.2-, 213.7-, and 5.2-fold higher survival rates than the control strain, respectively, after acid shock for 3 h at pH 4.0. Subsequently, transcriptional profile alterations in recombinant strains were analyzed during acid stress. The differentially expressed genes associated with cold-shock proteins (csp), fatty acid biosynthesis (fabH), and coenzyme A biosynthesis (coaD) were up-regulated in the four recombinant strains during acid stress. Additionally, some genes were differentially expressed in specific recombinant strains. For example, in L. lactis (RbsB), genes involved in the pyrimidine biosynthetic pathway (pyrCBDEK) and glycine or betaine transport process (busAA and busAB) were up-regulated during acid stress, and the argG genes showed up-regulations in L. lactis (MsmK). Finally, we found that overexpression of the ABC transporters RbsB and MsmK increased intracellular ATP concentrations to protect cells against acidic damage in the initial stage of acid stress. Furthermore, L. lactis (MsmK) consistently maintained elevated ATP concentrations under acid stress.ConclusionsThis study elucidates the common and specific mechanisms underlying improved acid tolerance by manipulating ABC transporters and provides a further understanding of the role of ABC transporters in acid-stress tolerance.

Procyanidin B2 Activates PPARγ to Induce M2 Polarization in Mouse Macrophages.

Procyanidins, a subclass of flavonoids found in commonly consumed foods, possess potential anti-inflammatory activity. Manipulation of M1/M2 macrophage homeostasis is an effective strategy for the treatment of metabolic inflammatory diseases. The objective of this study was to determine the effect of procyanidins on macrophage polarization. Procyanidin B2 (PCB2), the most widely distributed natural procyanidins, enhanced the expressions of M2 macrophage markers (Arg1, Ym1, and Fizz1). PCB2 activated peroxisome proliferator-activated receptor γ (PPARγ) activity and increased the expressions of PPARγ target genes (CD36 and ABCG1) in macrophages. Inhibition of PPARγ using siRNA or antagonist GW9662 attenuated the PCB2-induced expressions of M2 macrophage markers. In addition, we identified cognate PPAR-responsive elements (PPREs) within the 5'-flanking regions of the mouse Arg1, Ym1, and Fizz1 genes. Furthermore, macrophages isolated from db/db diabetic mice showed lower expressions of M2 markers. PCB2 effectively restored the Arg1, Ym1, and Fizz1 expressions in a PPARγ-dependent manner. These findings support the notion that PCB2 regulated macrophage M2 polarization via the activation of PPARγ. Our results provide a new mechanism by which procyanidins exert their beneficial anti-inflammatory effects.

Abstract 527: Cardiac Fibroblast Ablation in Chronic Fibrosis

Cardiac fibrosis is a common feature of heart failure independent of etiology. Although much is known about initiation of fibrosis in acute injury, factors leading to the persistence of fibrosis are poorly understood. We sought to determine if cardiac fibroblasts are necessary for the persistence of fibrosis by ablating resident cardiac fibroblasts in a mouse model of cardiac fibrosis. Methods: We generated mice with inducible ablation of cardiac fibroblasts (fb) by crossing the tcf21-cre gene into a MerCreMer-DTA mouse. These mice were then crossed into SR-uPA transgene which promotes diffuse interstitial fibrosis by 12 weeks of age. Control tcf21-DTA and tcf-21-DTA-SRuPA mice were treated with tamoxifen for 1-4 weeks to activate DTA and ablate cardiac fibroblasts. Echocardiography, histology and qPCR of isolated fb and macrophages (mac) was performed. Results: Treatment of wildtype tcf21-DTA mice with tamoxifen for 1-4 weeks resulted in a decrease in alpha-SMA positive cells in the heart but no change in left ventricular size or function. There was no excess mortality. Hearts showed no evidence of increased inflammation or fibrosis. Tcf21-DTA-SRuPA mice were viable but exhibited increased mortality starting at 10 days following initiation of tamoxifen. There were no changes in echocardiographic parameters, histologic inflammation or fibrosis. Fb had no change in expression of Col1a1 normalized to GAPDH. However, mac from Tcf21-DTA-SRuPA mice had a significant decrease in expression of M2 genes Arg1 and YM1. Conclusion: Ablation of fibroblasts was toxic to mice with established fibrosis, without changes in cardiac structure or function. Mac phenotype shifted from pro-fibrotic/M2 to pro-inflammatory/M1 with fibroblast ablation. These data point to important compensatory mechanisms that may maintain fibrosis in the heart.

Local Immunomodulation Using an Adhesive Hydrogel Loaded with miRNA-Laden Nanoparticles Promotes Wound Healing.

Chronic wounds are characterized by impaired healing and uncontrolled inflammation, which compromise the protective role of the immune system and may lead to bacterial infection. Upregulation of miR-223 microRNAs (miRNAs) shows driving of the polarization of macrophages toward the anti-inflammatory (M2) phenotype, which could aid in the acceleration of wound healing. However, local-targeted delivery of microRNAs is still challenging, due to their low stability. Here, adhesive hydrogels containing miR-223 5p mimic (miR-223*) loaded hyaluronic acid nanoparticles are developed to control tissue macrophages polarization during wound healing processes. In vitro upregulation of miR-223* in J774A.1 macrophages demonstrates increased expression of the anti-inflammatory gene Arg-1 and a decrease in proinflammatory markers, including TNF-α, IL-1β, and IL-6. The therapeutic potential of miR-223* loaded adhesive hydrogels is also evaluated in vivo. The adhesive hydrogels could adhere to and cover the wounds during the healing process in an acute excisional wound model. Histological evaluation and quantitative polymerase chain reaction (qPCR) analysis show that local delivery of miR-223* efficiently promotes the formation of uniform vascularized skin at the wound site, which is mainly due to the polarization of macrophages to the M2 phenotype. Overall, this study demonstrates the potential of nanoparticle-laden hydrogels conveying miRNA-223* to accelerate wound healing.

The effects of fine particulate matter constituents on exhaled nitric oxide and DNA methylation in the arginase–nitric oxide synthase pathway

BackgroundFine particulate matter (PM2.5) has been widely associated with airway inflammation represented by increased fractional concentration of exhaled nitric oxide (FeNO). However, it remains unclear whether various PM2.5 constituents have different impacts on FeNO and its production process from the arginase (ARG)–nitric oxide synthase (NOS) pathway. ObjectivesTo investigate the acute effects of PM2.5 constituents on FeNO and DNA methylation of genes involved. MethodsWe conducted a longitudinal panel study among 43 young adults in Shanghai, China from May to October in 2016. We monitored the concentrations of 25 constituents of PM2.5. We applied the linear mixed-effect model to evaluate the associations of PM2.5 constituents with FeNO and DNA methylation of the ARG2 and NOS2A genes. ResultsFollowing PM2.5 exposure, NOS2A methylation decreased and ARG2 methylation increased only on the concurrent day, whereas FeNO increased most prominently on the second day. Nine constituents (OC, EC, K, Fe, Zn, Ba, Cr, Se, and Pb) showed consistent associations with elevated FeNO and decreased NOS2A methylation or increased ARG2 methylation in single-constituent models and models adjusting for PM2.5 total mass and collinearity. An interquartile range increase of these constituents was associated with respective decrements of 0.27–1.20 in NOS2A methylation (%5mC); increments of 0.48–1.56 in ARG2 methylation (%5mC); and increments of 7.12%–17.54% in FeNO. ConclusionsOur results suggested that OC, EC, and some metallic elements may be mainly responsible for the development and epigenetic regulation of airway inflammatory response induced by short-term PM2.5 exposure.

Heterologous Expression of Argininosuccinate Synthase From Oenococcus oeni Enhances the Acid Resistance of Lactobacillus plantarum.

Oenococcus oeni can survive well in wine (an acid-stress environment) and dominate malolactic fermentation (MLF). To demonstrate a possible role of argininosuccinate synthase gene (argG) in the acid tolerance response of O. oeni, a related argG gene was inserted into a plasmid pMG36e and heterologously expressed in Lactobacillus plantarum SL09, a wine isolate belonging to a species of relevant importance in MLF. The expression levels of the argG gene in L. plantarum were analyzed by RT-qPCR, argininosuccinate synthase (ASS) activity and cell properties (amino acids, pH, H+-ATPase activity, and ATP levels) were determined at pH 3.7 in comparison with that at pH 6.3. Results showed that the recombinant strain L. plantarum SL09 (pMG36eargG) exhibited stronger growth performance compared with the control strain (without argG gene), and the expression levels of hsp1, cfa, atp, the citrate and malate metabolic genes were apparently increased under acid stress. In addition, the recombinant strain exhibited 11.0-, 2.0-, 1.9-fold higher ASS activity, H+-ATPase activity and intracellular ATP level, compared with the corresponding values for control strain during acid-stresses condition, which may take responsible for the acid tolerance enhancement of the recombinant strain. This is the first work report on heterologous expression of argG gene, and the results presented in this study will be beneficial for the research on acid stress response of O. oeni.

C/EBPβ regulates the M2 transcriptome in β-adrenergic-stimulated macrophages

At the M2 terminal of the macrophage activation spectrum, expression of genes is regulated by transcription factors that include STAT6, CREB, and C/EBPβ. Signaling through β-adrenergic receptors drives M2 activation of macrophages, but little is known about the transcription factors involved. In the present study, we found that C/EBPβ regulates the signaling pathway between β-adrenergic stimulation and expression of Arg1 and several other specific genes in the greater M2 transcriptome. β-adrenergic signaling induced Cebpb gene expression relatively early with a peak at 1 h post-stimulation, followed by peak Arg1 gene expression at 8 h. C/EBPβ transcription factor activity was elevated at the enhancer region for Arg 1 at both 4 and 8 h after stimulation but not near the more proximal promoter region. Knockdown of Cebpb suppressed the β-adrenergic-induced peak in Cebpb gene expression as well as subsequent accumulation of C/EBPβ protein in the nucleus, which resulted in suppression of β-adrenergic-induced Arg1 gene expression. Analysis of genome-wide transcriptional profiles identified 20 additional M2 genes that followed the same pattern of regulation by β-adrenergic- and C/EBPβ-signaling. Promoter-based bioinformatic analysis confirmed enrichment of binding motifs for C/EBPβ transcription factor across these M2 genes. These findings pinpoint a mechanism that may be targeted to redirect the deleterious influence of β-adrenergic signaling on macrophage involvement in M2-related diseases such as cancer.

Halobacterium salinarum virus ChaoS9, a Novel Halovirus Related to PhiH1 and PhiCh1.

The unexpected lysis of a large culture of Halobacterium salinarum strain S9 was found to be caused by a novel myovirus, designated ChaoS9. Virus purification from the culture lysate revealed a homogeneous population of caudovirus-like particles. The viral genome is linear, dsDNA that is partially redundant and circularly permuted, has a unit length of 55,145 nt, a G + C% of 65.3, and has 85 predicted coding sequences (CDS) and one tRNA (Arg) gene. The left arm of the genome (0–28 kbp) encodes proteins similar in sequence to those from known caudoviruses and was most similar to myohaloviruses phiCh1 (host: Natrialba magadii) and phiH1 (host: Hbt. salinarum). It carries a tail-fiber gene module similar to the invertible modules present in phiH1 and phiCh1. However, while the tail genes of ChaoS9 were similar to those of phiCh1 and phiH1, the Mcp of ChaoS9 was most similar (36% aa identity) to that of Haloarcula hispanica tailed virus 1 (HHTV-1). Provirus elements related to ChaoS9 showed most similarity to tail/assembly proteins but varied in their similarity with head/assembly proteins. The right arm (29–55 kbp) of ChaoS9 encoded proteins involved in DNA replication (ParA, RepH, and Orc1) but the other proteins showed little similarity to those from phiH1, phiCh1, or provirus elements, and most of them could not be assigned a function. ChaoS9 is probably best classified within the genus Myohalovirus, as it shares many characteristics with phiH1 (and phiCh1), including many similar proteins. However, the head/assembly gene region appears to have undergone a recombination event, and the inferred proteins are different to those of phiH1 and phiCh1, including the major capsid protein. This makes the taxonomic classification of ChaoS9 more ambiguous. We also report a revised genome sequence and annotation of Natrialba virus phiCh1.

Role of Genomic Biomarkers in Increasing Fetal Hemoglobin Levels Upon Hydroxyurea Therapy and in β-Thalassemia Intermedia: A Validation Cohort Study

Hemoglobinopathies exhibit a remarkable phenotypic diversity in terms of disease severity, while individual genetic background plays a key role in differential response to drug treatment. In the last decade, genomic variants in genes located within, as well as outside the human β-globin cluster have been shown to be significantly associated with Hb F increase, in relation to hydroxyurea (HU) therapy in patients with these diseases. Here, we aim to determine the effect of genomic variants located in genes, such as MAP3K5, ASS1, NOS2A, TOX, PDE7B, NOS1, FLT1 and ARG2, previously shown to modulate fetal hemoglobin (Hb F) levels in patients with β type hemoglobinopathies and reflecting disease severity and response to HU therapy in an independent cohort of Greek patients with these diseases. We recruited and genotyped 45 β-thalassemia patients (β-thal), either transfusion-dependent (TDT) or non transfusion-dependent (NTDT), 42 Hb S (HBB: c.20A>T)-β-thal compound heterozygotes, who were treated with HU, as well as 53 healthy individuals, all of Hellenic origin. Our study showed that genomic variants of the MAP3K5, NOS2A and ARG2 gene are associated with HU therapy efficacy in Hb S-β-thal compound heterozygotes. We have also shown that FLT1 and ARG2 genomic variants are associated with the mild phenotype of NTDT patients. Our findings provide evidence that MAP3K5, NOS2A, ARG2 and FLT1 genomic variants could be considered as genomic biomarkers to predict HU therapy efficacy in Hb S-β-thal compound heterozygotes and also to describe disease severity in patients with β type hemoglobinopathies.

Spermidine-Regulated Biosynthesis of Heat-Stable Antifungal Factor (HSAF) in Lysobacter enzymogenes OH11.

Heat-Stable Antifungal Factor (HSAF) and its analogs are antifungal natural products produced by the biocontrol agent Lysobacter enzymogenes. The production of HSAF is greatly influenced by environmental stimuli and nutrients, but the underlying molecular mechanism is mostly unclear. Here, we found that HSAF production in L. enzymogenes OH11 is strictly controlled by spermidine, which is the most prevalent triamine in bacteria. When added into OH11 cultures, spermidine regulated the production of HSAF and analogs in a concentration-dependent manner. To verify the role of spermidine, we deleted LeSDC and LeADC genes, encoding S-adenosylmethionine decarboxylase and arginine decarboxylase, respectively, that are the key enzymes for spermidine biosynthesis. Both deletion mutants produced barely detectable spermidine and HSAF including its analogs, whereas the antifungals production was restored by exogenous spermidine. The results showed that the OH11 cells must maintain a proper spermidine homeostasis for the antifungals production. Indeed, the expression level of the key HSAF biosynthetic genes was significantly impaired in LeSDC and LeADC mutants, and exogenous spermidine restored the gene expression level in the mutants. Ornithine is a key substrate for HSAF biosynthesis, and OH11 genome contains arg1 and arg2 genes, encoding arginases that convert arginine to ornithine. While the expression of arg1 and arg2 was affected slightly upon mutation of LeSDC and LeADC, exogenous spermidine significantly increased the arginase gene expression in LeSDC and LeADC mutants. Together, the data revealed a previously unrecognized mechanism, in which spermidine controls antibiotic production through controlling both the biosynthetic genes and the substrate-production genes.

HYCO-3, a dual CO-releaser/Nrf2 activator, reduces tissue inflammation in mice challenged with lipopolysaccharide

Oxidative stress and inflammation are predominant features of several chronic diseases. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a major arbiter in counteracting these insults via up-regulation of several defensive proteins, including heme oxygenase-1 (HO-1). HO-1-derived carbon monoxide (CO) exhibits anti-inflammatory actions and can be delivered to tissues by CO-releasing agents. In this study we assessed the pharmacological and anti-inflammatory properties of HYCO-3, a dual activity compound obtained by conjugating analogues of the CO-releasing molecule CORM-401 and dimethyl fumarate (DMF), an immunomodulatory drug known to activate Nrf2. HYCO-3 induced Nrf2-dependent genes and delivered CO to cells in vitro and tissues in vivo, confirming that the two expected pharmacological properties of this agent are achieved. In mice challenged with lipopolysaccharide, orally administered HYCO-3 reduced the mRNA levels of pro-inflammatory markers (TNF-α, IL-1β and IL-6) while increasing the expression of the anti-inflammatory genes ARG1 and IL-10 in brain, liver, lung and heart. In contrast, DMF or CORM-401 alone or their combination decreased the expression of pro-inflammatory genes but had limited influence on anti-inflammatory markers. Furthermore, HYCO-3 diminished TNF-α and IL-1β in brain and liver but not in lung and heart of Nrf2-/- mice, indicating that the CO-releasing part of this hybrid contributes to reduction of pro-inflammation and that this effect is organ-specific. These data demonstrate that the dual activity of HYCO-3 results in enhanced efficacy compared to the parent compounds indicating the potential exploitation of hybrid compounds in the development of effective anti-inflammatory therapies.

Peroxisome proliferator–activated receptor γ (PPARγ) induces the gene expression of integrin αVβ5 to promote macrophage M2 polarization

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily and polarizes the macrophages into an anti-inflammatory M2 state. Integrins are transmembrane receptors that drive various cellular functions, including monocyte adhesion and foam cell formation. In this study, we first reported that the expression of integrins αV and β5 was up-regulated by PPARγ activation in RAW264.7 cells and human peripheral blood monocytes. Luciferase reporter and ChIP assay revealed that PPARγ directly bound to the potential PPAR-responsive elements sites in the 5'-flanking regions of both murine and human integrin αV and β5 genes, respectively. In addition, we showed that PPARγ augmented the ligation of integrins αV and β5 Knockdown of integrin αVβ5 by siRNA strategy or treatment with cilengitide, a potent inhibitor of integrin αVβ5, attenuated PPARγ-induced expression of Ym1 (chitinase-like protein 3), Arg1 (Arginase1), Fizz1 (resistin-like molecule RELMα), and other M2 marker genes, suggesting that the heterodimers of integrin αVβ5 were involved in PPARγ-induced M2 polarization. In conclusion, these results provided novel evidence that PPARγ-mediated gene expression and the ensuing ligation of integrins αV and β5 are implicated in macrophage M2 polarization.

Effects of a Single of Bout Exercise on the Macrophage Phenotypic Ratio in the Adipose Tissue of High-Fat Diet-Induced Obese Mice

PURPOSE Exercise in known to inhibit M1 macrophage (M1ф) differentiation, and increase in M2 macrophage (M2ф) and inhibition of M1ф in adipose tissue are known to reduce obesity. However, studies on the effects of exercise are very limited and it has not yet confirmed whether the polarization of M1ф/M2ф actually changes. Therefore, I tried to confirm the M1ф/M2ф polarization in the real change in single bout of exercise. METHODS Male C57BL/6 mice were divided into four groups; normal diet (ND) control (n=7), ND exercise (n=7), high-fat diet (HFD) control (n=7), and HFD exercise (n=7) groups. All exercise mice ran on a treadmill at 15 m/min for 60 minutes for single bout. All animals were sacrificed two hours after single-bout of exercise and adipose tissue was isolated. Adipocytes were extracted from isolated adipose tissue and FACS was performed. RESULTS Single bout of exercise in high-fat diet induced obese mice did not actually increase M2ф polarization in adipose tissue. Rather, the expression of M1ф was significantly higher than that of the other groups. However, expression of M2ф-related genes (Arg1) was highest in HFD-EX compared to ND control and HFD control. CONCLUSIONS Despite the increase of M1ф due to obesity, the expression of M2ф-related gene Arg1 was found to be increased by exercise regardless of the high-fat diet. These results suggest for the first time that a single bout of exercise can independently affect the change in M1ф/M2ф phenotypic ratio regardless of obesity or high-fat diet. 색인어: 비만, 운동, 지방조직, 대식세포 Keywords: Obesity, Exercise, Adipose tissue, Macrophage