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Career situation of first and presenting author Student for a master or a PhD. Introduction The activator protein (AP)-1 transcription factor family, especially its subfamily of FOS proteins (cFos, FosB, Fra-1 and Fra-2) are associated to the regulatory network of macrophage responses. Moreover, it is well known that macrophages are central player during rheumatoid arthritis (RA). Objectives This study aims to delineate the role of Fra-1 in macrophages during the acute destructive inflammatory phase of RA. Methods Therefore, we applied the serum-induced arthritis (K/BxN) model to Fra-1 deficient mice controlled by the Mx1 promoter (Fra-1ΔMx) or the LysM promoter (Fra-1ΔLysM). Moreover, we performed in vitro analyses of macrophage polarization in wildtype and Fra-1 deficient macrophages, as well as micro-arrays and ChIP sequencing analyses to delineate Fra-1 targets in activated macrophages. We completed our analysis by studying Fra-1 expression in RA patients’ synovium. Results Fra-1 mutant mice had decreased arthritis severity compared to their littermate wildtype mice. The alleviated arthritis was accompanied to increased arginase-1 (Arg1) expression and activity in the joints, suggesting that its anti-inflammatory features milder RA inflammation. Sorting of immune cell populations revealed macrophages as the major source of Arg1, which was increased in Fra-1 mutant mice. Mechanistically, Fra-1 transcriptionally inhibited Arg1 expression in macrophages. Moreover, inhibition of Arginase in Fra-1 mutant mice restored a full blunt inflammatory RA response and the supplementation of mice with l-arginine, leading to increased arginase activity in the joint, is sufficient to milder arthritis. Synovium histological sections from RA patients showed a correlation between Arg1, Fra-1 and the DAS28 score, confirming that increased Arg1 activity is of benefit also for human inflammatory joint disease. Conclusions Our data show for the first time that Fra-1 is a pivot between pro- and anti-inflammatory macrophage. By inhibiting Arg1 activity, Fra-1 exacerbates RA inflammation and joint destruction. Disclosure of Interest None declared.

Background Tumor orchestrated metabolic changes in the microenvironment limit generation of anti-tumor immune responses. Availability of arginine, a semi-essential amino acid, is critical for lymphocyte proliferation and function. Levels of arginine are regulated by the enzymes arginase 1,2 and nitric oxide synthase (NOS). However, the role of arginase activity in lung tumor maintenance has not been investigated in clinically relevant orthotopic tumor models. Methods RNA sequencing (RNA-seq) of sorted cell populations from mouse lung adenocarcinomas derived from immunocompetent genetically engineered mouse models (GEMM)s was performed. To complement mouse studies, a patient tissue microarray consisting of 150 lung adenocarcinomas, 103 squamous tumors, and 54 matched normal tissue were stained for arginase, CD3, and CD66b by multiplex immunohistochemistry. Efficacy of a novel arginase inhibitor compound 9 in reversing arginase mediated T cell suppression was determined in splenocyte ex vivo assays. Additionally, the anti-tumor activity of this compound was determined in vitro and in an autochthonous immunocompetent KrasG12D GEMM of lung adenocarcinoma model. Results Analysis of RNA-seq of sorted myeloid cells suggested that arginase expression is elevated in myeloid cells in the tumor as compared to the normal lung tissue. Accordingly, in the patient samples arginase 1 expression was mainly localized in the granulocytic myeloid cells and significantly elevated in both lung adenocarcinoma and squamous tumors as compared to the controls. Our ex vivo analysis demonstrated that myeloid derived suppressor cell (MDSC)s cause T cell suppression by arginine depletion, and suppression of arginase activity by a novel ARG1/2 inhibitor, compound 9, led to restoration of T cell function by increasing arginine. Treatment of KrasG12D GEMM of lung cancer model with compound 9 led to a significant tumor regression associated with increased T cell numbers and function, while it had no activity across several murine and human non-small cell (NSCLC) lung cancer lines in vitro. Conclusions We show that arginase expression is elevated in mouse and patient lung tumors. In a KRASG12D GEMM arginase inhibition diminished growth of established tumors. Our data suggest arginase as an immunomodulatory target that should further be investigated in lung tumors with high arginase activity. Electronic supplementary material The online version of this article (10.1186/s40425-019-0504-5) contains supplementary material, which is available to authorized users.

Immune checkpoints arise from physiological changes during tumorigenesis that reprogramme inflammatory, immunological and metabolic processes in malignant lesions and local lymphoid tissues, which constitute the immunological tumour microenvironment (TME). Improving clinical responses to immune checkpoint blockade will require deeper understanding of factors that impact local immune balance in the TME. Elevated catabolism of the amino acids tryptophan (Trp) and arginine (Arg) is a common TME hallmark at clinical presentation of cancer. Cells catabolizing Trp and Arg suppress effector T cells and stabilize regulatory T cells to suppress immunity in chronic inflammatory diseases of clinical importance, including cancers. Processes that induce Trp and Arg catabolism in the TME remain incompletely defined. Indoleamine 2,3 dioxygenase (IDO) and arginase 1 (ARG1), which catabolize Trp and Arg, respectively, respond to inflammatory cues including interferons and transforming growth factor-β (TGFβ) cytokines. Dying cells generate inflammatory signals including DNA, which is sensed to stimulate the production of type I interferons via the stimulator of interferon genes (STING) adaptor. Thus, dying cells help establish local conditions that suppress antitumour immunity to promote tumorigenesis. Here, we review evidence that Trp and Arg catabolism contributes to inflammatory processes that promote tumorigenesis, impede immune responses to therapy and might promote neurological comorbidities associated with cancer. This Review discusses how increased catabolism of the amino acids tryptophan and arginine driven by inflammatory processes in the tumour microenvironment contributes to tumorigenesis, suppression of antitumour immunity and potentially neurological comorbidities associated with cancer.

The cytokine interleukin IL‐35 is known to exert strong immunosuppressive functions. Indoleamine 2,3‐dioxygenase 1 (IDO1) and Arginase 1 (Arg1) are metabolic enzymes that, expressed by dendritic cells (DCs), contribute to immunoregulation. Here, we explored any possible link between IL‐35 and the activity of those enzymes. We transfected a single chain IL‐35Ig gene construct in murine splenic DCs (DC 35) and assessed any IDO1 and Arg1 activities as resulting from ectopic IL‐35Ig expression, both in vitro and in vivo. Unlike Ido1, Arg1 expression was induced in vitro in DC 35, and it conferred an immunosuppressive phenotype on those cells, as revealed by a delayed‐type hypersensitivity assay. Moreover, the in vivo onset of a tolerogenic phenotype in DC 35 was associated with the detection of CD25+ CD39+, rather than Foxp3+, regulatory T cells. Therefore, Arg1, but not Ido1, expression in DC 35 appears to be an early event in IL‐35Ig–mediated immunosuppression.

ARG1, which encodes Arginase1, is expressed in the liver cytoplasm and plays a major role in the hepatic urea cycle. The past research works shed light on the fact that ARG1 participates in anti-inflammation, tumor immunity, and immunosuppression-related diseases. Nevertheless, the concrete role and clinical significance of ARG1 in the progression of hepatocellular carcinoma (HCC) remain unclear. Herein, we aimed at examining the expression and clinicopathological significance of ARG1 in HCC, together with determining the effect of ARG1 on the progression and metastasis of HCC. In the current study, evaluation of the expression of ARG1 and clinicopathological significance of ARG1 was carried out in the human HCC tissues microarray, and the ARG1 overexpression vector and shRNA-ARG1 plasmids were constructed for the assessment of the concrete effect of ARG1 on cellular behaviors of Huh7 cells. As our data revealed, ARG1 was significantly downregulated in HCC, and the higher expression of ARG1 was positively correlated with more aggressive tumor growth, size, ALT, and GGT level. Significantly, we found that the high expression of ARG1 was correlated with poor DFS of HCC patients. Besides, in vitro study revealed that overexpression of ARG1 could enhance arginase activity, cell viability, migration, and invasion of Huh7 cells, and loss-of-function of ARG1 by shRNA interference could inhibit these cellular behaviors. Additionally, overexpression of ARG1 led to a significant increase in the expression of Vimentin, N-cadherin, and

Macrophages play a pivotal role in the development of emphysema and depending on the microenvironment stimuli can be polarized into M1- or M2-like macrophage phenotypes. We compared macrophage polarizations in cigarette smoke (CS)- and porcine pancreatic elastase (PPE)-induced emphysema models. C57BL/6 mice were subdivided into four experimental groups. In the PPE group, animals received an intranasal instillation of PPE (0.677 IU); in the saline group, animals received an intranasal instillation of saline (0.9%). Animals from both groups were euthanized on day 28. In the CS group, animals were exposed to CS for 30 min, twice a day, 5 days per week for 12 weeks. In the control group, animals received filtered air. We observed an increase in total macrophages for both experimental models. For M1-like macrophage markers, we observed an increase in TNF-α+ and IFN-γ+ cells, Cxcl-9 and Cxcl-10 expressions in PPE and CS groups. Only in the CS group, we detected an increased expression of IL-12b. For M2-like macrophages markers we observed a down regulation in IL-10, IL-4, IL-13, Arg1 and Fizz1 and an increase of TGF-β+ cells in the PPE group, while for the CS group there was an increase in TGF-β+ cells and IL-10 expression. All exposure groups were compared to their respective controls. In summary, we demonstrated that CS- and PPE-induced models resulted in different microenvironmental stimuli. CS exposure induced an environmental stimulus related to M1- and M2-like macrophage phenotypes similar to previous results described in COPD patients, whereas the elastase-induced model provided an environmental stimulus related only to the M1 phenotype., Summary: The CS-induced model showed M1 and M2-related genes and chemokines, whereas the PPE-induced model showed a downregulation of M2- and an increase in M1-related genes and chemokines.

Over the last decade, remarkable progress has been made in the areas of targeted therapy and immunotherapy for the treatment of cancer patients. However, durable clinical benefit has occurred only in a subset of responding patients. In our field of melanoma, we believe that the arginine metabolism leads to nitric oxide (NO) production, resulting in marker and functional changes in both melanoma and the microenvironmental cells, including potential effector T lymphocytes, concurrently suppressing the local immune response. Arginine metabolism is dependent on the activity of the families of nitric oxide synthase (NOS) and arginase (ARG) enzymes. NOS oxidizes arginine to citrulline and NO, and ARG hydrolyzes arginine into ornithine and urea. NO can react with oxides to form peroxynitrite (NO3−) that rapidly forms two posttranslational modifications (PTMs) on proteins, nitrosylation of thiols (SNO) and nitration of tyrosines (NT). We and others have shown the expression of the inducible isoform of NOS enzyme (iNOS) in many human tumors, including melanoma. ARG1 activity has also been shown to be associated with the M2-polarized, protumoral tumor-associated macrophages (TAMs). Collectively, ARG and NOS enzymes metabolize arginine and are crucial components of immune suppression pathways, and the metabolic products of these enzymes are important moderators of T-cell function in cancer.

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., Graphical abstract fx1, Highlights • HYCO-3 is a novel hybrid between an Nrf2 activator and a CO-releasing molecule. • HYCO-3 induces Nrf2 and simultaneously delivers CO in vitro and in vivo. • Oral administration of HYCO-3 reduces inflammation in mice challenged with LPS. • In Nrf2-/- mice, the anti-inflammatory action of HYCO-3 is organ specific.

Cerebral ischemia induces neuroinflammation and microglial activation, in which activated microglia upregulate their proliferative activity and change their metabolic states. In activated microglia, l-arginine is metabolized competitively by inducible nitric oxide synthase (iNOS) and arginase (Arg), which then synthesize NO or polyamines, respectively. Our previous study demonstrated that Sema4D deficiency inhibits iNOS expression and promotes proliferation of ionized calcium-binding adaptor molecule 1 (Iba1)-positive (Iba1+) microglia in the ischemic cortex, although the underlying mechanisms were unclear. Using middle cerebral artery occlusion, we tested the hypothesis that Sema4D deficiency alters the balance of l-arginine metabolism between iNOS and Arg, leading to an increase in the production of polyamines, which are an essential factor for cell proliferation. In the peri-ischemic cortex, almost all iNOS+ and/or Arg1+ cells were Iba1+ microglia. In the peri-ischemic cortex of Sema4D-deficient (Sema4D-/-) mice, the number of iNOS+ Arg1- Iba1+ microglia was smaller and that of iNOS- Arg1+ Iba1+ microglia was greater than those of wild-type (WT) mice. In addition, urea and polyamine levels in the ischemic cortex of Sema4D-/- mice were higher than those of WT mice; furthermore, the presence of Sema4D inhibited polyamine production in primary microglia obtained from Sema4D-/- mice. Finally, microglia cultured under polyamine putrescine-supplemented conditions demonstrated increased proliferation rates over non-supplemented controls. These findings indicate that Sema4D regulates microglial proliferation at least in part by regulating the competitive balance of l-arginine metabolism.

Arginase 1 (Arg1), which converts l-arginine into ornithine and urea, exerts pleiotropic immunoregulatory effects. However, the function of Arg1 in inflammatory bowel disease (IBD) remains poorly characterized. Here, we found that Arg1 expression correlated with the degree of inflammation in intestinal tissues from IBD patients. In mice, Arg1 was upregulated in an IL-4/IL-13(–) and intestinal microbiota–dependent manner. Tie2-Cre Arg1(fl/fl) mice lacking Arg1 in hematopoietic and endothelial cells recovered faster from colitis than Arg1-expressing (Arg1(fl/fl)) littermates. This correlated with decreased vessel density, compositional changes in intestinal microbiota, diminished infiltration by myeloid cells, and an accumulation of intraluminal polyamines that promote epithelial healing. The proresolving effect of Arg1 deletion was reduced by an l-arginine–free diet, but rescued by simultaneous deletion of other l-arginine–metabolizing enzymes, such as Arg2 or Nos2, demonstrating that protection from colitis requires l-arginine. Fecal microbiota transfers from Tie2-Cre Arg1(fl/fl) mice into WT recipients ameliorated intestinal inflammation, while transfers from WT littermates into Arg1-deficient mice prevented an advanced recovery from colitis. Thus, an increased availability of l-arginine as well as altered intestinal microbiota and metabolic products accounts for the accelerated resolution from colitis in the absence of Arg1. Consequently, l-arginine metabolism may serve as a target for clinical intervention in IBD patients.

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.

Background: The coronavirus disease 2019 (COVID-19) outbreak, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been declared a global pandemic. It is well-established that SARS-CoV-2 infection can lead to dysregulated immune responses. Arginase-1 (Arg1), which has a pivotal role in immune cells, can be expressed in most of the myeloid cells, e.g., neutrophils and macrophages. Arg1 has been associated with the suppression of antiviral immune responses. Methods: Whole blood was taken from 21 COVID-19 patients and 21 healthy individuals, and after RNA extraction and complementary DNA (cDNA) synthesis, gene expression of Arg1 was measured by real-time PCR. Results: The qPCR results showed that the expression of Arg1 was significantly increased in COVID-19 patients compared to healthy individuals (p &lt, 0.01). The relative expression analysis demonstrated there were approximately 2.3 times increased Arg1 expression in the whole blood of COVID-19 patients. Furthermore, the receiver operating characteristic (ROC) analysis showed a considerable diagnostic value for Arg1 expression in COVID-19 (p = 0.0002 and AUC = 0.8401). Conclusion: Arg1 might be a promising marker in the pathogenesis of the disease, and it could be a valuable diagnostic tool.

Regulatory properties of macrophages associated with alternative activation serve to limit the exaggerated inflammatory response during pneumonia caused by Pseudomonas aeruginosa infection. Arginase-1 is an important effector of these macrophages believed to play an essential role in decreasing injury and promoting repair. We investigated the role of arginase-1 in the control of inflammatory immune responses to P. aeruginosa pneumonia in mice that exhibit different immunologic phenotypes. C57BL/6 mice with conditional knockout of the arginase-1 (Arg1) gene from myeloid cells (Arg1(ΔM)) or BALB/c mice treated with small molecule inhibitors of arginase were infected intratracheally with P. aeruginosa. Weight loss, mortality, bacterial clearance, and lung injury were assessed and compared, as were the characterization of immune cell populations over time post-infection. Myeloid arginase-1 deletion resulted in greater morbidity along with more severe inflammatory responses compared to littermate control mice. Arg1(ΔM) mice had greater numbers of neutrophils, macrophages, and lymphocytes in their airways and lymph nodes compared to littermate controls. Additionally, Arg1(ΔM) mice recovered from inflammatory lung injury at a significantly slower rate. Conversely, treatment of BALB/c mice with the arginase inhibitor S-(2-boronoethyl)-L-cysteine hydrochloride (BEC) did not change morbidity as defined by weight loss, but mice at day 10 post-infection treated with BEC had gained significantly more weight back than controls. Neutrophil and macrophage infiltration were similar between groups in the lung parenchyma, and neutrophil migration into the airways was reduced by BEC treatment. Differences seem to lie in the impact on T cell subset disposition. Arg1(ΔM) mice had increased total CD4+ T cell expansion in the lymph nodes, and increased T cell activation, IFNγ production, and IL-17 production in the lymph nodes, lung interstitium, and airways, while treatment with BEC had no impact on T cell activation or IL-17 production, but reduced the number of T cells producing IFNγ in the lungs. Lung injury scores were increased in the Arg1(ΔM) mice, but no differences were observed in the mice treated with pharmacologic arginase inhibitors. Overall, myeloid arginase production was demonstrated to be essential for control of damaging inflammatory responses associated with P. aeruginosa pneumonia in C57BL/6 mice, in contrast to a protective effect in the Th2-dominant BALB/c mice when arginase activity is globally inhibited.

The biological features of porcine alveolar macrophages (PAMs) and interstitial macrophages (IMs) were investigated, including morphology, nitric oxide (NO) secretion, cell viability and porcine reproductive and respiratory syndrome virus (PRRSV) mRNA expression post-inoculation with TJ-F10 or TJM-F92. Viability and NO secretion of PAMs and IMs were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Griess's assay, respectively. mRNA expression of PRRSV, inducible nitric oxide synthase (iNOS) and Arginase1 (Arg1) in PAMs and IMs were detected by quantitative real-time polymerase chain reaction technique. Our results show that PAMs were bigger and more granular than IMs and the Arg1/iNOS value was much higher in PAMs than in IMs. In addition, the vaccine strain TJM-F92 evoked higher NO production in PAMs and IMs compared with the wild type strain TJ-F10. In conclusion, our results indicate that the PAMs and IMs are heterogeneous in morphology, NO production and susce...

The small intestine is the exclusive site of arginine synthesis in neonates. Low levels of circulating arginine have been associated with the occurrence of necrotizing enterocolitis (NEC) but the mechanism of arginine dysregulation has not been fully elucidated. We aimed to investigate (i) expressional changes of arginine synthesizing and catabolic enzymes in human intestinal tissues of NEC, spontaneous intestinal perforation (SIP) and noninflammatory surgical conditions (Surg-CTL) and to investigate the (ii) mechanisms of arginine dysregulation and enterocyte proliferation upon stimulation by bacterial components, arginine depletion, ARG1 overexpression and nitric oxide (NO) supplementation. Our results showed that expressions of arginine synthesizing enzymes ALDH18A1, ASL, ASS1, CPS1, GLS, OAT and PRODH were significantly decreased in NEC compared with Surg-CTL or SIP tissues. Catabolic enzyme ARG1 was increased (>100-fold) in NEC tissues and histologically demonstrated to be expressed by infiltrating neutrophils. No change in arginine metabolic enzymes was observed between SIP and Surg-CTL tissues. In CaCO2 cells, arginine metabolic enzymes were differentially dysregulated by lipopolysaccharide or lipoteichoic acid. Depletion of arginine reduced cell proliferation and this phenomenon could be partially rescued by NO. Overexpression of ARG1 also reduced enterocyte proliferation. We provided the first expressional profile of arginine metabolic enzymes at the tissue level of NEC. Our findings suggested that arginine homeostasis was severely disturbed and could be triggered by inflammatory responses of enterocytes and infiltrating neutrophils as well as bacterial components. Such reactions could reduce arginine and NO, resulting in mucosal damage. The benefit of arginine supplementation for NEC prophylaxis merits further clinical evaluation.

Nitric oxide (NO) is derived from multiple isoforms of the Nitric Oxide Synthases (NOSs) within the lung for a variety of functions; however, NOS2-derived nitrogen oxides seem to play an important role in inflammatory regulation. In this study, we investigate the role of NOS2 in pulmonary inflammation/fibrosis in response to intratracheal bleomycin instillation (ITB) and to determine if these effects are related to macrophage phenotype. Systemic NOS2 inhibition was achieved by administration of 1400 W, a specific and potent NOS2 inhibitor, via osmotic pump starting six days prior to ITB. 1400 W administration attenuated lung inflammation, decreased chemotactic activity of the broncheoalveolar lavage (BAL), and reduced BAL cell count and nitrogen oxide production. S-nitrosylated SP-D (SNO-SP-D), which has a pro-inflammatory function, was formed in response to ITB; but this formation, as well as structural disruption of SP-D, was inhibited by 1400 W. mRNA levels of IL-1β, CCL2 and Ptgs2 were decreased by 1400 W treatment. In contrast, expression of genes associated with alternate macrophage activation and fibrosis Fizz1, TGF-β and Ym-1 was not changed by 1400 W. Similar to the effects of 1400 W, NOS2−/− mice displayed an attenuated inflammatory response to ITB (day 3 and day 8 post-instillation). The DNA-binding activity of NF-κB was attenuated in NOS2−/− mice; in addition, expression of alternate activation genes (Fizz1, Ym-1, Gal3, Arg1) was increased. This shift towards an increase in alternate activation was confirmed by western blot for Fizz-1 and Gal-3 that show persistent up-regulation 15 days after ITB. In contrast arginase, which is increased in expression at 8 days post ITB in NOS2−/−, resolves by day 15. These data suggest that NOS2, while critical to the development of the acute inflammatory response to injury, is also necessary to control the late phase response to ITB.

The concept of macrophage polarization toward different phenotypes after CNS injury has been increasingly discussed. Here, we propose that CD200 treatment may help shift pro-inflammatory macrophages to an arginase 1 (Arg1)-, transglutaminase 2 (TGM2)-, and transforming growth factor beta 1 (TGF-β)-positive phenotype. Rat macrophages were stimulated by interferon γ and lipopolysaccharide (LPS) to induce pro-inflammatory phenotypes. Treatment with human CD200-Fc up-regulated expression levels of alternatively activated M2-like markers such as Arg1 and TGM2 but suppressed pro-inflammatory M1-like markers such as toll-like receptor 4, interleukin 1 beta (IL-1β), IL-6, and GM-CSF. Concomitantly, CD200-Fc enhanced (CCAAT/enhancer-binding protein) C/EBP-beta promoter activity, whereas NF-κB activity was suppressed. Treatment with CD200-Fc also up-regulated potentially beneficial TGF-β expression in macrophages. When C/EBP-beta signaling was suppressed with siRNA, the effect of CD200-Fc on Arg1, TGM2 and TGF-β up-regulation was canceled. Taken together, these data provide proof-of-principle that targeting CD200 signaling may be a novel therapeutic approach to shift macrophages toward M2-like polarization via modulating cAMP-response element binding protein-C/EBP-beta transcriptional activity. We showed that CD200 treatment decreased pro-inflammatory cytokines (IL-1β, IL-6, and GM-CSF) along with suppressed inflammatory NF-κB activity in pro-inflammatory Mφ. On the other hand, CD200 increased Arg1, TGM2, and TGF-β production through CREB-C/EBPβ signaling. We think that these findings provide proof-of-concept that CD200 signaling may play a key role in regulating macrophage polarization toward anti-inflammatory phenotypes.

Arginase 1 (ARG1) deficiency is a rare urea cycle disorder (UCD). This hypothesis-generating study explored clinical phenotypes, metabolic profiles, molecular genetics, and treatment approaches in a cohort of children and adults with ARG1 deficiency to add to our understanding of the underlying pathophysiology.Clinical data were retrieved retrospectively from physicians using a questionnaire survey. Plasma aminoacids, guanidinoacetate (GAA), parameters indicating oxidative stress and nitric oxide (NO) synthesis as well as asymmetric dimethylarginine (ADMA) were measured at a single study site.Nineteen individuals with ARG1 deficiency and 19 matched controls were included in the study. In patients, paraparesis, cognitive impairment, and seizures were significantly associated suggesting a shared underlying pathophysiology. In patients plasma GAA exceeded normal ranges and plasma ADMA was significantly elevated. Compared to controls, nitrate was significantly higher, and the nitrite:nitrate ratio significantly lower in subjects with ARG1 deficiency suggesting an advantage for NO synthesis by inducible NO synthase (iNOS) over endothelial NOS (eNOS). Logistic regression revealed no significant impact of any of the biochemical parameters (including arginine, nitrates, ADMA, GAA, oxidative stress) or protein restriction on long-term outcome.Three main hypotheses which must be evaluated in a hypothesis driven confirmatory study are delineated from this study: 1) clinical manifestations in ARG1 deficiency are not correlated with arginine, protein intake, ADMA, nitrates or oxidative stress. 2) GAA is elevated and may be a marker or an active part of the pathophysiology of ARG1 deficiency. 3) Perturbations of NO metabolism merit future attention in ARG1 deficiency.

In the adipose tissue (AT), an increase in the M1 macrophage (M1Ø)/M2 macrophage (M2Ø) polarization ratio can be a risk factor enhancing the inflammatory response during aging, as well as increasing the risk of chronic disease, thereby reducing lifespan, or at least reducing "healthy" lifespan. The purpose of this study was to analyze and compare the AT M1Ø/M2Ø polarization ratio at the final lifespan stage in aged and control animals performing lifelong spontaneous wheel running. Based on flow cytometric analysis, the AT ratio of macrophages revealed M2Ø polarization following lifelong spontaneous exercise (LSE) regardless of age. However, for Icam1 and Tnf, the qPCR analysis showed no difference in gene expressions in young mice; Arg1 expression was higher in Young-EXE (exercising) than in Young-CON (control) mice (p .0001). In Old-EXE, Icam1 (p .0001) and Tnf (p .0001) expression were lower than in Old-CON; for Arg1, gene expression in Old-EXE was higher than in Old-CON (p .0001). LSE prevents deterioration of physical fitness owing to aging, maintaining high M2Ø polarization levels in the AT. Additionally, LSE does not downregulate Icam1 and Tnf in the AT but appears to suppress the increased M1Ø polarization ratio attributed to aging by upregulating Arg1.

To uncover mechanisms and explore novel biomarkers of obesity, type 2 diabetes (T2DM) and nonalcoholic steatohepatitis (NASH)-associated hepatocarcinogenesis, cellular and molecular alterations in the liver, and hepatocellular carcinomas (HCCs) were investigated in NASH model 60-week-old Tsumura, Suzuki, Obese Diabetic (TSOD) mice and NASH HCC patients. Markedly elevated lipid deposition, inflammation, fibrosis, and peroxisome proliferation in the liver, preneoplastic lesions, and HCCs of TSOD mice were accompanied by accumulation of polysaccharides in the cellular cytoplasm and nuclei and increase of oxidative DNA damage marker, 8-hydroxydeoxyguanosine (8-OHdG) formation in the liver and altered foci. Metabolomics of TSOD mice HCCs demonstrated significant elevation of the concentration of amino acid L-arginine, phosphocreatine, S-adenosylmethionine/S-adenosylhomocysteine ratio, adenylate, and guanylate energy charges in coordination with tremendous rise of glucose metabolites, mostly fructose 1,6-diphosphate. L-arginine accumulation in HCCs was associated with significant under-expression of arginase 1 (ARG1), suppression of the urea cycle, methionine and putrescine degradation pathways, activation of Ser and Thr kinase Akt AKT, phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinase 1/2 (ERK1/2) kinases, &beta, catenin, mammalian target of rapamycin (mTOR), and cell proliferation. Furthermore, clinicopathological analysis in 20 metabolic syndrome/NASH and 80 HCV-positive HCC patients demonstrated significant correlation of negative ARG1 expression with poor tumor differentiation, higher pathological stage, and significant decrease of survival in metabolic syndrome/NASH-associated HCC patients, thus indicating that ARG1 could become a potential marker for NASH HCC. From these results, formation of oxidative stress and 8-OHdG in the DNA and elevation of glucose metabolites and L-arginine due to ARG1 suppression in mice liver cells are the important characteristics of T2DM/NASH-associated hepatocarcinogenesis, which may take part in activating oxidative stress resistance, synthesis of phosphocreatine, cell signaling, methylation, and proliferation.

Introduction: Arginineemia, also known as arginase deficiency, is a rare autosomal recessive metabolic disease. The diagnosis sometimes may be delayed due to atypical clinical manifestations. Confirmation of arginineemia depends on genetic testing. Patient concerns: We reported a Chinese male child presenting with hyperargininemia and progressive spastic diplegia, who has a novel compound heterozygous mutation in the arginase-1 (ARG1) gene (c.263-266delAGAA, p.K88Rfs∗45;c.674T>C,p.L216P), respectively, coming from his mother and father. Diagnosis: The patient was diagnosed with argininemia with a novel compound homozygous mutation of the ARG1 gene at the age of 12 years. Interventions: The patient had a low-protein diet (0.8 g/kg/day). Baclofen, eperisone hydrochloride, botulinum toxin, and rehabilitation training were used to improve his spastic diplegia symptoms for 3 months. Outcomes: The patient's blood arginine was still high after 3 months’ low-protein diet. His spastic diplegia symptoms had not aggravated after 3 months’ treatment. Conclusions: Argininemia should be considered in a patient with slowly progressive neurologic manifestations, especially spastic diplegia. This case also suggests that tandem mass spectrometry should be used as an effective tool in the validity of neonatal screening for early diagnosis.

Arginase is a widely known enzyme of the urea cycle that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. The action of arginase goes beyond the boundaries of hepatic ureogenic function, being widespread through most tissues. Two arginase isoforms coexist, the type I (Arg1) predominantly expressed in the liver and the type II (Arg2) expressed throughout extrahepatic tissues. By producing L-ornithine while competing with nitric oxide synthase (NOS) for the same substrate (L-arginine), arginase can influence the endogenous levels of polyamines, proline, and NO•. Several pathophysiological processes may deregulate arginase/NOS balance, disturbing the homeostasis and functionality of the organism. Upregulated arginase expression is associated with several pathological processes that can range from cardiovascular, immune-mediated, and tumorigenic conditions to neurodegenerative disorders. Thus, arginase is a potential biomarker of disease progression and severity and has recently been the subject of research studies regarding the therapeutic efficacy of arginase inhibitors. This review gives a comprehensive overview of the pathophysiological role of arginase and the current state of development of arginase inhibitors, discussing the potential of arginase as a molecular imaging biomarker and stimulating the development of novel specific and high-affinity arginase imaging probes.

Psoriasis is a chronic inflammatory disease whose etiology is multifactorial. The contributions of cellular metabolism to psoriasis are unclear. Here, we report that interleukin-17 (IL-17) downregulated Protein Phosphatase 6 (PP6) in psoriatic keratinocytes, causing phosphorylation and activation of the transcription factor C/EBP-β and subsequent generation of arginase-1. Mice lacking Pp6 in keratinocytes were predisposed to psoriasis-like skin inflammation. Accumulation of arginase-1 in Pp6-deficient keratinocytes drove polyamine production from the urea cycle. Polyamines protected self-RNA released by psoriatic keratinocytes from degradation and facilitated the endocytosis of self-RNA by myeloid dendritic cells to promote toll-like receptor-7 (TLR7)-dependent RNA sensing and IL-6 production. An arginase inhibitor improved skin inflammation in murine and non-human primate models of psoriasis. Our findings suggest that urea cycle hyperreactivity and excessive polyamine generation in psoriatic keratinocytes promote self-RNA sensation and PP6 deregulation in keratinocytes is a pivotal event that amplifies the inflammatory circuits in psoriasis.

We delineated the expression of DAP12 (DNAX-Activating Protein) and its associated receptors, TREM-1, TREM-2 and MDL-1 in pig alveolar monocyte/macrophages (AMM) that have attained M1 or M2 phenotypes. Pig AMM stimulated in vitro with IFN-γ and IL-4 induced the expression of M1 (TNFα and iNOS) and M2 (ARG1 and no MMR) phenotypic markers, respectively. In influenza virus infected pigs at seven days post-infection, in addition to substantial modulations in the M1 and M2 markers expression, DAP12, TREM-1 and MDL-1 were downregulated in AMM. Thus, DAP12 signaling promoted the anti-inflammatory pathway in AMM of influenza virus infected pigs.

Summary As current therapies benefit only a minority of cancer patients, additional therapeutic targets are needed. Tumor-associated macrophages (TAMs) have attracted attention for improving therapeutic responses, yet regulatory strategies remain elusive. Here, we show that the protein kinase A catalytic subunit (PKA-C) acts as a molecular switch, inducing a pro-tumoral immunosuppressive macrophage phenotype within tumors. In human and murine breast cancer, overactivated PKA in TAMs creates a detrimental microenvironment for cancer progression by inducing vascular endothelial growth factor A (VEGFA), interleukin-10 (IL-10), and macrophage-derived arginase 1 (ARG1) expression. Macrophages with genetic deletion of PKA-C are prone to be pro-inflammatory, suggesting a possible immunotherapeutic target. Delivery of liposomal PKA inhibitor facilitates tumor regression and abrogates pro-tumoral TAM functions in mice. The therapeutic effect of targeting PKA is pronounced when combined with αCTLA-4 antibody, increasing cluster of differentiation 8 (CD8)+GranzymeB+ T cells by about 60-fold. Our findings demonstrate critical roles of TAM PKA-C in tumor progression and suggest that targeting PKA-C efficiently augments cancer treatment responses.

Acute respiratory distress syndrome (ARDS) is characterized as a neutrophil-dominant disorder without effective pharmacological interventions. Knowledge of neutrophils in ARDS patients at the transcriptome level is still limited. We aimed to identify the hub genes and key pathways in neutrophils of patients with ARDS. The transcriptional profiles of neutrophils from ARDS patients and healthy volunteers were obtained from the GSE76293 dataset. The differentially expressed genes (DEGs) between ARDS and healthy samples were screened using the limma R package. Subsequently, functional and pathway enrichment analyses were performed based on the database for annotation, visualization, and integrated discovery (DAVID). The construction of a protein–protein interaction network was carried out using the search tool for the retrieval of interacting genes (STRING) database and the network was visualized by Cytoscape software. The Cytoscape plugins cytoHubba and MCODE were used to identify hub genes and significant modules. Finally, 136 upregulated genes and 95 downregulated genes were identified. Gene ontology analyses revealed MHC class II plays a major role in functional annotations. SLC11A1, ARG1, CHI3L1, HP, LCN2, and MMP8 were identified as hub genes, and they were all involved in the neutrophil degranulation pathway. The MAPK and neutrophil degranulation pathways in neutrophils were considered as key pathways in the pathogenesis of ARDS. This study improves our understanding of the biological characteristics of neutrophils and the mechanisms underlying ARDS, and key pathways and hub genes identified in this work can serve as targets for novel ARDS treatment strategies.

Continual efferocytic clearance of apoptotic cells (ACs) by macrophages prevents necrosis and promotes injury resolution. How continual efferocytosis is promoted is not clear. Here, we show that the process is optimized by linking the metabolism of engulfed cargo from initial efferocytic events to subsequent rounds. We found that continual efferocytosis is enhanced by the metabolism of AC-derived arginine and ornithine to putrescine by macrophage arginase 1 (Arg1) and ornithine decarboxylase (ODC). Putrescine augments HuR-mediated stabilization of the mRNA encoding the GTP-exchange factor Dbl, which activates actin-regulating Rac1 to facilitate subsequent rounds of AC internalization. Inhibition of any step along this pathway after first-AC uptake suppresses second-AC internalization, whereas putrescine addition rescues this defect. Mice lacking myeloid Arg1 or ODC have defects in efferocytosis in vivo and in atherosclerosis regression, while treatment with putrescine promotes atherosclerosis resolution. Thus, macrophage metabolism of AC-derived metabolites allows for optimal continual efferocytosis and resolution of injury.

Iron overload is an undesired effect of frequent blood transfusions or genetic diseases. Myelodysplastic syndrome (MDS) patients become transfusion dependent, but due to the combination of ineffective haematopoiesis and repeated blood transfusions they are often subject to iron overload. In this study, we demonstrate that iron-overload mimicking condition alters bone marrow progenitor differentiation towards dendritic cells (DCs). Cells cultured in iron-enriched culture medium for seven days fail to differentiate into conventional CD11c+MHCIIhi DCs and fail to efficiently respond to LPS (Lipopolysaccharides). Cells appear smaller than control DCs but vital and able to perform FITC-dextran (Fluorescein isothiocyanate-dextran) endocytosis. At molecular level, cells cultured in iron-enriched conditions show increased ARG1 and PU.1, and decreased IRF8 expression.

TanshinoneIIA (TanIIA) has been demonstrated to possess numerous biological effects. However, the specific effect of TanIIA on macrophage polarization has not been reported. In this study, it was revealed that TanIIA might play a pivotal role in macrophage polarization. As our results indicated, cell morphology was changed in RAW264.7 cells which were treated with LPS or LPS/TanIIA (0.1 μM, 1 μM, 10 μM). Subsequently, pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 were measured by ELISA kits. Furthermore, TanIIA enhanced the expression of M2 macrophage markers (Arg1 and FIZZ1) and decreased the expression of markers associated with M1 macrophage polarization (iNOS and IL-1β). Increased expression of CD206 was also detected by flow cytometry in TanIIA-treated groups. Mechanistically, it was revealed that TanIIA modulated macrophage polarization by ameliorating mitochondrial function and regulating TLR4-HMGB1/CEBP-β pathway. In addition, increased expression of miR-155 was observed in RAW264.7 cells incubated with LPS and were effectively inhibited by TanIIA. Taken together, it was suggested that TanIIA inhibits inflammatory response and promotes macrophage polarization toward an M2 phenotype, which provides new evidence for the anti-inflammation activity of TanIIA.

ARG1, which encodes Arginase1, is expressed in the liver cytoplasm and plays a major role in the hepatic urea cycle. The past research works shed light on the fact that ARG1 participates in anti-inflammation, tumor immunity, and immunosuppression-related diseases. Nevertheless, the concrete role and clinical significance of ARG1 in the progression of hepatocellular carcinoma (HCC) remain unclear. Herein, we aimed at examining the expression and clinicopathological significance of ARG1 in HCC, together with determining the effect of ARG1 on the progression and metastasis of HCC. In the current study, evaluation of the expression of ARG1 and clinicopathological significance of ARG1 was carried out in the human HCC tissues microarray, and the ARG1 overexpression vector and shRNA-ARG1 plasmids were constructed for the assessment of the concrete effect of ARG1 on cellular behaviors of Huh7 cells. As our data revealed, ARG1 was significantly downregulated in HCC, and the higher expression of ARG1 was positively correlated with more aggressive tumor growth, size, ALT, and GGT level. Significantly, we found that the high expression of ARG1 was correlated with poor DFS of HCC patients. Besides, in vitro study revealed that overexpression of ARG1 could enhance arginase activity, cell viability, migration, and invasion of Huh7 cells, and loss-of-function of ARG1 by shRNA interference could inhibit these cellular behaviors. Additionally, overexpression of ARG1 led to a significant increase in the expression of Vimentin, N-cadherin, andβ-catenin both at protein and mRNA levels, which promotes the EMT process. On the other hand, these proteins' expression was significantly downregulated in ARG1 silenced Huh7 cells. Besides, the level of E-cadherin protein was upregulated in ARG1 knocked down cells. In conclusion, ARG1 might play a pivotal role as an oncogene in the progression of HCC through promoting the EMT process.

CREB and C/EBP β signaling pathways are modulated during inflammation and also targeted by Bacillus anthracis edema toxin (ET), but how these factors individually and jointly contribute to changes in immune cell function is poorly understood. Using CRISPR/Cas9 gene editing, macrophage cell lines lacking CREB and isoforms of C/EBP β were generated and analyzed for changes in responses to LPS, ET, and IL-4. Macrophages lacking C/EBP β suppressed induction of IL-10 and Arg1, while IL-6 was increased in these cells following exposure to LPS. Examination of C/EBP β isoforms indicated the 38 kDa isoform was necessary for the expression of IL-10 and Arg1. ChIP-Seq analysis of CREB and C/EBP β binding to targets on the chromosome of human PBMC identified several regions where both factors overlapped in their binding, suggesting similar gene targeting or cooperative effects. Based on the ChIP-Seq data, a panel of previously unknown targets of CREB and C/EBP β was identified and includes genes such as VNN2, GINS4, CTNNBL1, and SULF2. Isoforms of a transcriptional corepressor, transducin-like enhancer of Split (TLE), were also found to have CREB and C/EBP β binding their promoter and were up regulated by ET. Finally, we explore a possible layer of C/EBP β regulation by a protein complex consisting of adenomatous polyposis coli (APC) and PKA. Collectively, these data provide new insights into the role of CREB and C/EBP β as immunosignaling regulators and targets of an important bacterial virulence factor.

Arginase I (ARG1) deficiency is an autosomal recessive urea cycle disorder, caused by deficiency of the enzyme Arginase I, resulting in accumulation of arginine in blood. Current Standard of Care (SOC) for ARG1 deficiency in patients or those having detrimental mutations of ARG1 gene is diet control. Despite diet and drug therapy with nitrogen scavengers, ~25% of patients suffer from severe mental deficits and loss of ambulation. 75% of patients whose symptoms can be managed through diet therapy continue to suffer neuro-cognitive deficits. In our research, we demonstrate in vitro and in vivo that administration of ARG1 mRNA increased ARG1 protein expression and specific activity in relevant cell types, including ARG1-deficient patient cell lines, as well as in wild type mice for up to 4 days. These studies demonstrate that ARG1 mRNA treatment led to increased functional protein expression of ARG1 and subsequently an increase in urea. Hence, ARG1 mRNA therapy could be a potential treatment option to develop for patients.

This study aims to explore the effect of Corilagin (Cor) on post-parasiticide schistosome egg-induced hepatic fibrosis through the Stat6 signalling pathway in vitro and in vivo. Cellular and animal models were established and treated by Corilagin. The inhibitory effect of Corilagin was also confirmed in RAW264.7 cells in which Stat6 was overexpressed based on the GV367-Stat6-EGFP lentiviral vector system and in which Stat6 was knock-downed by gene specific siRNAs. As a result, Corilagin prevented increases in the protein level of Phospho-Stat6 (P-Stat6). Both the mRNA and protein levels of the downstream mediators SOCS1, KLF4, and PPARγ/δ were markedly suppressed after Corilagin treatment. Expression of ARG1 and FIZZ1/Retnla, Ym1, TGF-β and PDGF in serum were also inhibited by Corilagin. The pathological changes, area of granulomas of liver sections, and degree of hepatic fibrosis were significantly alleviated in the Corilagin group. The areas of CD68- and CD206-positive cells stained by immunofluorescence were significantly decreased by Corilagin. In conclusion, Corilagin can suppress post-parasiticide schistosome egg-induced hepatic fibrosis by inhibiting the Stat6 signalling pathway and provide a new therapeutic strategy for schistosomiasis liver fibrosis.

Neurodegenerative diseases are a set of disorders characterized by progressive neuronal death and are associated with microglia-mediated neuroinflammation. Recently, neuroinflammation is proposed as a promising therapeutic target for many neurodegenerative diseases. Alpha-1 antitrypsin (AAT) is recognized as a novel immunomodulatory agent in autoimmune diseases and transplantation, however, its impact on neuroinflammation and neurodegeneration remains unknown. This study aims to explore the effects of AAT on microglia-mediated neuroinflammation and retinal degeneration in rd1 mouse model. We found reduced expression of AAT in rd1 retina, and AAT supplement exhibited certain protective effect on retinal degeneration, presenting with increased amount of photoreceptor nuclei, and amplified wave amplitudes in electroretinogram analysis. Of note, AAT shifted microglia phenotype from pro-inflammatory M1 (CD16/CD32+, iNOS+) to anti-inflammatory M2 (CD206+, Arg1+) both in vivo and in vitro, underscoring the concept of immunomodulation on microglia polarization by AAT during neurodegeneration. Furthermore, AAT suppressed the activation of STAT1, promoted the expression of IRF4 while inhibited IRF8 expression, indicating the involvement of these signaling pathways in AAT immunomodulation. Collectively, our data provided evidence for a novel protective role of AAT through immunomodulation on microglia polarization. Attenuating neuroinflammation by AAT may be beneficial to retard neurodegeneration in rd1 mice.

Aim: Arginase catalyzes the hydrolysis of arginine to ornithine and urea, being the final enzyme of the urea cycle, which is a ubiquitous pathway to excrete toxic ammonia from organisms. Lately, it also has emerged as a key regulator of nitric oxide by competing with NO synthase for the same substrate. Therefore, arginase overexpression has been associated with a series of pathogenic processes that can go from cardiovascular, immune-mediated or inflammatory conditions to tumor cell metabolism.(1) Several research groups have been involved in the development of small-molecule arginase inhibitors reaching to data holding great promise. However, only recently the drug development industry is attaining clinical trials.(2)Since the association to PET labeling techniques has great relevance not only for the evaluation and characterization of some of this molecules, but also to increase the library of radiotracers available, our goal with this work was to synthesize and evaluate the [18F]fluorinated equivalent of a late-generation arginase inhibitor.(3) Methods: An arylboronic acid pinacol ester-derived precursor was synthesized in view of a Cu-mediated nucleophilic [18F]fluorination. Radiochemical yield for the conversion of the intermediate species was assessed by TLC-SG and/or radioHPLC. Deprotection of the amino acid moiety was achieved by hydrolysis and the final radiotracer was purified by semi-preparative HPLC and obtained, after reformulation, as a physiological and injectable solution. Further stability and distribution-coefficient studies were performed. Results: In summary, radiochemical yield of the conversion of the arylboronic ester-derived precursor reached ~80% when using 60 to 15 μmol (lower amounts brought significant losses in yield). Final [18F]fluorinated compound was obtained with radiochemical purity ≥95% in an overall yield of 11% (d.c., non-automated synthesis). The radiotracer showed stability in solution up to 4 h and an experimental log D of -0.67±0.05. Conclusion: The [18F]fluorinated arginase inhibitor was efficiently labeled with fluorine-18 in good yield. Preliminary in vitro studies using R22v1 cell lines, which express Arg2, revealed a cellular uptake of the radiotracer susceptible of being blocked after treatment with arginase inhibitors. Further studies are currently being performed in different cell lines which either overexpress Arg1 or Arg2 to evaluate the potential of the developed radiotracer towards arginase mapping. References 1. Caldwell RB, Toque HA, Narayanan SP, Caldwell RW, [2015], Trends Pharmacol Sci, 36(6):395-405 2. Pudlo M, Demougeot C, Girard-Thernier C, [2017], Med Res Rev, 37(3):475-513; (3) Van Zandt M, Golebiowski A, Ji MK, Whitehouse D, Ryder T, Beckett P, [2011], WO Patent 2011/133653A1.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunomodulatory function. To study the mechanism by which MDSC affect antimicrobial immunity, we infected mice with two M. avium strains of differential virulence, highly virulent Mycobacterium avium subsp. avium strain 25291 (MAA) and low virulent Mycobacterium avium subsp. hominissuis strain 104 (MAH). Intraperitoneal infection with MAA, but not MAH, caused severe disease and massive splenic infiltration of monocytic MDSC (M-MDSC; Gr-1intCD11bhiCD11cint) expressing inducible NO synthase (Nos2) and bearing high numbers of mycobacteria. Depletion experiments demonstrated that M-MDSC were essential for disease progression. NO production by M-MDSC influenced antigen-uptake and processing by dendritic cells and proliferation of CD4+ T cells. M-MDSC were also induced in MAA-infected mice lacking Nos2. In these mice CD4+ T cell expansion and control of infection were restored. However, T cell inhibition was only partially relieved and arginase (Arg) 1-expressing M-MDSC were accumulated. Likewise, inhibition of Arg1 also partially rescued T cell proliferation. Thus, mycobacterial virulence results in the induction of M-MDSC that block the T cell response in a Nos2- and Arg1-dependent manner.

The purpose of this study was to investigate the efficiency of a natural flavonoid, Chrysin (Chr), encapsulated in PLGA-PEG nanoparticles (NPs) for the modulation of macrophage polarity from the pro-inflammatory M1 to anti-inflammatory M2 phenotype. The synthetized NPs were characterized using FTIR, DLS and FE-SEM. MTT assay was used to assess the toxicity of different concentration of Chr-encapsulated NPs on LPS/IFN-γ stimulated peritoneal exudate macrophages. To investigate the repolarization efficiency of Chr-encapsulated NPs, real-time PCR was applied to measure M1 (iNOS and SOCS3) and M2 (Arg1 and Fizz) markers expression. Also, the relative mRNA and protein expression levels of pro-inflammatory cytokines including IL-6, IL-1β and TNF-α were investigated in M1 macrophages treated with Chr-encapsulated NPs. Findings revealed that the Chr-encapsulated NPs with spherical shape and an average diameter of 235 nm were considerably less toxic to the macrophages. Additionally, the nano-formulated Chr efficiently showed a reduction in M1 markers and an increase in M2 markers levels than free Chr. Furthermore, macrophage phenotype switching by PLGA-PEG encapsulated Chr NPs significantly suppressed LPS/IFN-γ induced inflammation by a remarkable reduction in pro-inflammatory cytokine levels, TNF-α, IL-1β, and IL-6. Convincingly, the results revealed that PLGA-PEG encapsulated Chr based drug delivery system might be introduced into biomaterials to fabricate bioactive smart multifunctional nanocomposites with macrophage repolarization activities for regenerative medicine purposes.

In recent years, a large amount of clinical and experimental data has shown that M2-like polarized tumor-associated macrophages (TAMs) play an important role in cancer metastasis. Therefore, TAMs, especially M2-like TAMs is a promising target for anti-tumor metastasis therapy. Here, we found that celastrol dose-dependently suppressed IL-13 induced CD206 expression both in RAW264.7 and in primary macrophages. Consistently, celastrol also inhibited the expression of M2-like specific genes, including MRC1, Arg1, Fizz1, Mgl2 and CD11c. Further, by the employment of 4T1 breast cancer model, we found that celastrol significantly prevented cancer metastasis in vivo. Mechanistically, celastrol completely ameliorated STAT6 phosphorylation, which is the key signal molecule responsible for M2 polarization. Our research puts forward a new application of celastrol in anti-cancer metastasis, by intervening M2-like polarization through inhibiting STAT6.

Ethnopharmacological relevance Huoluo Yinao decoction (HLYND) has been used to ameliorate cognitive impairment induced by chronic cerebral hypoperfusion in clinical for years. However, the exact mechanisms remain unknown. Aim of the study To investigate the effects and mechanisms underlying HLYND-mediated improvement in cognitive deficits associated with chronic cerebral hypoperfusion. Materials and methods Thirty-six Sprague-Dawley rats were randomly allocated to three groups: sham, model, and HLYND. Daily administration of HLYND or volume-matched vehicle by gavage was initiated 1 day after bilateral carotid artery stenosis (BCAS) and continued for 42 days. The Morris water maze (MWM) test was used to assess cognitive functions from days 36–42. Via western blot and immunofluorescent staining, restoration of neuronal plasticity and remyelination of white matter were evaluated by analyzing the expression profiles of MAP-2, synaptophysin and MBP. In addition, macrophage/microglial activation was assessed by quantifying changes in Iba1, and macrophage/microglial polarization was assessed by changes in iNOS and CD16 (M1 markers), as well as Arg1 and CD206 (M2 markers). Results In the MWM test, BCAS rats showed significantly extended escape latency and reduced platform crossing times, while those in the HLYND group had shortened escape latency and increased frequency of platform crossing. In addition, rats in the model group showed decreased levels and abnormal morphological changes of MAP-2, synaptophysin and MBP, whereas HLYND administration reversed these effects. As expected, Iba1 levels were elevated in both the model and HLYND groups but rats in the model group showed increased levels of the M1 markers, iNOS and CD16, and a correspondent decrease in the M2 marker, Arg1. In contrast, in the HLYND group, iNOS and CD16 levels were suppressed, while Arg1 levels were elevated. Conclusions Our findings demonstrate that HLYND mitigates cognitive impairment after chronic cerebral hypoperfusion in rats through mechanisms involving increased neuronal plasticity and white matter remyelination, with a subtile modulation of macrophage/microglial polarization toward the M2 phenotype.

Introduction Osteoclasts are giant, multi-nucleated cells that derive from the monocyte-macrophage linage and are regulators of bone turnover. Availability and catabolism of L-Arginine have been implicated with immune cell biology, skewing inflammatory responses within myeloid cells in a pro- or anti-inflammatory manner. Objectives While the role of L-Arginine within certain myeloid lineages such as macrophages is well appreciated, its role within osteoclasts is relatively unknown. We therefore aim to investigate L-Arginine metabolism in the context of osteoclastogenesis. Methods We analysed osteoclastogenesis of C57BL/6J wildtype bone marrow cells in vitro in the presence and absence of recombinant Arginase 1 (recArg1). This approach was complemented via qPCR analysis of relevant osteoclast marker genes and an extracellular flux assay. We further investigated the effect of recArg1 regarding an in vivo model called serum transfer arthritis, where we treated C57BL/6J wildtype mice with the recombinant enzyme. Disease severity was then assessed using clinical scores and paw histology. Results We observed that ARG1 mRNA expression was downregulated from the progression of a precursor to a mature osteoclast. We incubated day 3 osteoclast precursors with recArg1 and observed that addition of 1000 ng/ml recArg1 abolished osteoclastogenesis. L-Arginine deprivation led to a decrease in the oxygen consumption rate of osteoclast precursor cells, assessed 48 hour after RANKL addition. Using serum transfer arthritis, an established murine in vivo model, recArg1 treated mice showed reduced disease severity combined with a significant decrease in the presence of osteoclasts. Treatment efficiency was evaluated using an L-Arginine ELISA, where the amino acid was found to be absent in the serum of treated mice. Conclusions We propose that the amino acid L-Arginine is critical for the development of osteoclasts from myeloid precursors and hypothesise that its abundance, influenced by recArg1 addition, influences development and severity of osteoclast driven diseases. Disclosure of interest J. Brunner Grant/research support from: Bio Cancer Treatment International Ltd, M. Hofmann Grant/research support from: Bio Cancer Treatment International Ltd, V. Saferding Grant/research support from: Bio Cancer Treatment International Ltd, A. Vogel Grant/research support from: Bio Cancer Treatment International Ltd, A. Lercher: None declared, P. Cheng Shareholder of: Bio Cancer Treatment International Ltd, G. Schabbauer Grant/research support from: Bio Cancer Treatment International Ltd, S. Bluml Grant/research support from: Bio Cancer Treatment International Ltd

Urea cycle enzymes may play important yet poorly characterized roles in Alzheimer's disease (AD). Our previous results showed that amyloid-β (Aβ) affects urea cycle enzymes in rat pheochromocytoma (PC12) cells. The aim of the present study was to investigate the changes in arginases, other urea cycle enzymes, and nitric oxide synthases (NOSs) in PC12 cells transfected with AβPP bearing the double 'Swedish' mutation (APPsw, K670M/N671L) and in postmortem sporadic AD brain hippocampus; the mutation intensifies Aβ production and strongly associates with AD neuropathology. mRNA expression was analyzed using real-time PCR in cell cultures and DNA microarrays in hippocampal CA1 area of human AD brains. Arginase activity was measured spectrophotometrically, and arginine, ornithine, and citrulline levels by high-performance liquid chromatography. Our data demonstrated that the expression and activity of arginases (Arg1 and Arg2), as well as the expression of argininosuccinate synthase (Ass) were significantly reduced in APPsw cells compared to control. However, argininosuccinate lyase (Asl) was upregulated in APPsw cells. Real-time PCR analysis revealed significant elevation of neuronal nitric oxide synthase (Nnos) mRNA in APPsw cells, without changes in the endothelial Enos, whereas inducible Inos was undetectable. The changes were found to follow closely those observed in the human hippocampal CA1 region of sporadic AD brains. The changes in enzyme expression were accompanied in APPsw cells by significantly elevated citrulline, ornithine, and arginine. Our findings demonstrate that AβPP/Aβ alters arginine metabolism and induces a shift of cellular homeostasis that may support the oxidative/nitrosative stress observed in AD.

Activated mouse macrophages metabolize arginine via NO synthase (NOS2) to produce NO as an antimicrobial effector. Published gene expression datasets provide little support for the activation of this pathway in human macrophages. Generation of NO requires the coordinated regulation of multiple genes. We have generated RNA-sequencing data from bone marrow–derived macrophages from representative rodent (rat), monogastric (pig and horse), and ruminant (sheep, goat, cattle, and water buffalo) species, and analyzed the expression of genes involved in arginine metabolism in response to stimulation with LPS. In rats, as in mice, LPS strongly induced Nos2, the arginine transporter Slc7a2, arginase 1 (Arg1), GTP cyclohydrolase (Gch1), and argininosuccinate synthase (Ass1). None of these responses was conserved across species. Only cattle and water buffalo showed substantial NOS2 induction. The species studied also differed in expression and regulation of arginase (ARG2, rather than ARG1), and amino acid transporters. Variation between species was associated with rapid promoter evolution. Differential induction of NOS2 and ARG2 between the ruminant species was associated with insertions of the Bov-A2 retrotransposon in the promoter region. Bov-A2 was shown to possess LPS-inducible enhancer activity in transfected RAW264.7 macrophages. Consistent with a function in innate immunity, NO production and arginine metabolism vary greatly between species and differences may contribute to pathogen host restriction.

Hypoxic microenvironment found in pancreatic ductal adenocarcinoma and other solid tumors is central to physiological and metabolic alterations of immune cells that significantly impact tumor growth dynamics. Hypoxic adaptations in the immune cells are primarily mediated by the stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), which regulates cellular metabolism by modulating glycolysis and other interconnected metabolic pathways. HIF-1α plays distinct roles in M1 and M2 macrophage polarization, which, in turn, regulates tumor cell immune escape and growth. In this chapter, we describe a real-time PCR-based assay to monitor the transcript levels of Arg1 and Nos2 to assess the status of tumor-induced macrophage polarization under hypoxic conditions. This method can be effectively utilized to delineate the genes critical for M1/M2 polarization in the hypoxic tumor microenvironment and would provide opportunities to develop immunomodulating therapies to regulate the tumor growth, progression, and metastatic dissemination.

Fatigue, the most common side effect of cancer treatments, is observed to intensify during external-beam radiation therapy (EBRT). The underlying molecular mechanisms remain unclear. This study investigated the differentially expressed genes/proteins and their association with fatigue intensification during EBRT. Fatigue scores measured by FACT-F and peripheral blood were collected prior to treatment (baseline D0), at midpoint (days 19-21, D21) and endpoint (days 38-42, D42) from men (n=30) with non-metastatic prostate cancer undergoing EBRT. RNA extracted from peripheral blood was used for gene expression analysis. Plasma arginase I and arginine were examined using ELISA and liquid chromatography-tandem mass spectrometry. Differences in fatigue scores, gene and protein expression between times points following EBRT were analyzed by one way ANOVA followed by Post Hoc t-test. Fatigue scores decreased significantly from baseline (44.6 ± 8.1) to midpoint (37.3 ± 10.6, p=0.000, low scores indicating high fatigue) and to endpoint (37.4 ± 10.1, p=0.001) during EBRT. ARG1 (encoding arginase type 1) was significantly up regulated from baseline to midpoint of EBRT (fold change =2.41, p 2-fold between the study time points. The changes in gene expression were associated with patient reported fatigue intensity. Moreover, the upregulation of ARG1 was negatively correlated with the absolute lymphocyte count (R2=0.561, p=0.01) only in the high level of fatigue group (n=17) during EBRT. Increased ARG1 expression is known to result in arginine deficiency, which leads to immunosuppression by impairing lymphocyte proliferation and activation. EBRT-induced ARG1 upregulation may play an essential role in fatigue intensification via the arginine deficiency and suppression of T-cell proliferation pathways. These findings may provide novel insights into the molecular-genetic mechanisms underlying the development and intensification of cancer treatment-related fatigue.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunomodulatory function. To study the mechanism by which MDSC affect antimicrobial immunity, we infected mice with two M. avium strains of differential virulence, highly virulent Mycobacterium avium subsp. avium strain 25291 (MAA) and low virulent Mycobacterium avium subsp. hominissuis strain 104 (MAH). Intraperitoneal infection with MAA, but not MAH, caused severe disease and massive splenic infiltration of monocytic MDSC (M-MDSC; Gr-1intCD11bhiCD11cint) expressing inducible NO synthase (Nos2) and bearing high numbers of mycobacteria. Depletion experiments demonstrated that M-MDSC were essential for disease progression. NO production by M-MDSC influenced antigen-uptake and processing by dendritic cells and proliferation of CD4+ T cells. M-MDSC were also induced in MAA-infected mice lacking Nos2. In these mice CD4+ T cell expansion and control of infection were restored. However, T cell inhibition was only partially relieved and arginase (Arg) 1-expressing M-MDSC were accumulated. Likewise, inhibition of Arg1 also partially rescued T cell proliferation. Thus, mycobacterial virulence results in the induction of M-MDSC that block the T cell response in a Nos2- and Arg1-dependent manner.

The Gram-negative bacterium, Helicobacter pylori, causes chronic gastritis, peptic ulcers, and gastric cancer in humans. Although the gastric epithelium is the primary site of H. pylori colonization, H. pylori can gain access to deeper tissues. Concurring with this notion, H. pylori has been found in the vicinity of endothelial cells in gastric submucosa. Endothelial cells play crucial roles in innate immune response, wound healing and tumorigenesis. This study examines the molecular mechanisms by which H. pylori interacts with and triggers inflammatory responses in endothelial cells. We observed that H. pylori infection of primary human endothelial cells stimulated secretion of the key inflammatory cytokines, interleukin-6 (IL-6) and interleukin-8 (IL-8). In particular, IL-8, a potent chemokine and angiogenic factor, was secreted by H. pylori-infected endothelial cells to levels ~10- to 20-fold higher than that typically observed in H. pylori-infected gastric epithelial cells. These inflammatory responses were triggered by the H. pylori type IV secretion system (T4SS) and the T4SS-associated adhesin CagL, but not the translocation substrate CagA. Moreover, in contrast to integrin α5β1 playing an essential role in IL-8 induction by H. pylori upon infection of gastric epithelial cells, both integrin α5β1 and integrin αvβ3 were dispensable for IL-8 induction in H. pylori-infected endothelial cells. However, epidermal growth factor receptor (EGFR) is crucial for mediating the potent H. pylori-induced IL-8 response in endothelial cells. This study reveals a novel mechanism by which the H. pylori T4SS and its adhesin subunit, CagL, may contribute to H. pylori pathogenesis by stimulating the endothelial innate immune responses, while highlighting EGFR as a potential therapeutic target for controlling H. pylori-induced inflammation. Introduction