L-arginine Catabolism Research Articles

Host–pathogen interaction between macrophage co-cultures with Staphylococcus aureus biofilms

The ability of Staphylococcus aureus to form biofilms is an important virulence factor. During the infectious process, the interaction between biofilms and immune cells is determinant; however, the properties that make biofilms resistant to the immune system are not well characterized. In order to better understand this, we evaluated the in vitro interaction of macrophages during the early stages of S. aureus biofilm formation. Biofilm formation was evaluated by crystal violet staining, light microscopy, and confocal scanning laser microscopy. Furthermore, different activation on L-arginine pathways such as nitric oxide (NO•) release and the arginase, the production of reactive oxygen species (ROS), the total oxidative stress response (OSR), and levels of cytokine liberation, were determined. Our findings show that the interaction between biofilms and macrophages results in stimuli for catabolism of L-arginine via arginase, but not for NO•, an increase of ROS production, and activation of the non-enzymatic OSR. We also observed the production of IL-6, but not of TNFα o IL-10 in these co-cultures. These results contribute to a better understanding of host-pathogen interactions and suggest that biofilms increase resistance against immune cell mechanisms, a phenomenon that could contribute to the ability of S. aureus biofilms to establish mature biofilms.

Abstract 5537: Anti-PD-1 reduces the immuno-suppressive phenotype of myeloid subsets in the murine Hepa 1-6 tumor

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer related deaths and is refractory to standard chemo and radiation therapies. Clinical trials are tackling unresectable HCC through combination approaches using immunotherapies and have shown evidence of improved survival but additional treatment options are needed. Hepa 1-6 is a murine model for HCC characterized by suppressive M2 tumor-associated macrophages (TAM) and reduced costimulatory molecule expression in dendritic cells (DC), which impairs T cell activity. To expose new options for therapeutic intervention, we performed immunophenotypic analysis in Hepa 1-6 tumor-bearing mice treated with checkpoint blockade (anti-PD-1) or isotype control antibodies. Flow cytometry and FluoroSpot were used to measure tumor infiltrates and provide a deep dive into the biology of the immune response. Our results demonstrated Hepa 1-6 tumors were responsive to anti-PD-1 treatment with an increased time to progression of 60%. The response coincided with several functional changes in the lymphocyte compartment consistent with enhanced anti-tumor activity. Anti-PD-1 increased tumor infiltrating CD8 T cells, upregulated ICOS expression, and increased the frequency of polyfunctional T cells producing IFNγ, TNFα, and IL-2. Granzyme B and IFNγ responses were also increased in tumor-derived NK and NKT cells. FluoroSpot revealed that white blood cells from anti-PD-1 treated mice were more responsive to stimulation with Hepa 1-6 cells suggesting anti-PD-1 increased systemic anti-tumor specific activity. Examination of the myeloid subsets indicated anti-PD-1 reduced immunosuppressive pressure in the tumor as the number of tumor-infiltrating granulocytic myeloid-derived suppressor cells (G-MDSC) decreased in treated mice. We also measured Arg1 and iNOS expression. In TAMs, iNOS activity is associated with enhanced anti-tumor efficacy. Although the increase in M1 TAMs triggered by anti-PD-1 did not reach significance (p>0.05), iNOS expression increased significantly by 34% in total TAMs. In tumor-derived MDSCs, Arg1 and iNOS increased with treatment. Notably, Arg1 and iNOS mediate MDSC immunosuppressive function via catabolism of L-arginine, suggesting some pro-tumor activity can also be triggered by checkpoint blockade. Finally, DCs play a key role in T cell activation. Analysis revealed the dominant B7 co-stimulatory molecule CD86 was upregulated in the DC1 subset following anti-PD-1 treatment. Together, these data demonstrate that anti-PD-1 enhances lymphocyte activity and inhibits Hepa 1-6 tumor growth. This can potentially be attributed to reduced G-MDSC recruitment, reprograming of TAMs from an M2 to an M1 phenotype, and enhanced DC-mediated T cell activation. Further investigation into combination therapies to enhance the response to checkpoint inhibition in the hepa 1-6 model can inform new strategies to treat HCC. Citation Format: David W. Draper, Philip Lapinski, Brogan Yarzabek, Hillary Evens, Scott Wise, Maryland Franklin. Anti-PD-1 reduces the immuno-suppressive phenotype of myeloid subsets in the murine Hepa 1-6 tumor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5537.

Role of Polyamines in Immune Cell Functions.

The immune system is remarkably responsive to a myriad of invading microorganisms and provides continuous surveillance against tissue damage and developing tumor cells. To achieve these diverse functions, multiple soluble and cellular components must react in an orchestrated cascade of events to control the specificity, magnitude and persistence of the immune response. Numerous catabolic and anabolic processes are involved in this process, and prominent roles for l-arginine and l-glutamine catabolism have been described, as these amino acids serve as precursors of nitric oxide, creatine, agmatine, tricarboxylic acid cycle intermediates, nucleotides and other amino acids, as well as for ornithine, which is used to synthesize putrescine and the polyamines spermidine and spermine. Polyamines have several purported roles and high levels of polyamines are manifest in tumor cells as well in autoreactive B- and T-cells in autoimmune diseases. In the tumor microenvironment, l-arginine catabolism by both tumor cells and suppressive myeloid cells is known to dampen cytotoxic T-cell functions suggesting there might be links between polyamines and T-cell suppression. Here, we review studies suggesting roles of polyamines in normal immune cell function and highlight their connections to autoimmunity and anti-tumor immune cell function.

Biochemical and biophysical insights into the metal binding spectrum and bioactivity of arginase of Entamoeba histolytica.

The human protozoan pathogens possess the essential metalloenzyme arginase (Arg) which catalyses the catabolism of l-arginine to l-ornithine and urea. This being the first committed step in polyamine biosynthesis is a potential drug target for protozoan diseases. In pathogenic organisms, arginase plays a crucial role in depleting host l-arginine, a substrate for nitric oxide synthase (NOS) that participates in protective immunity, thereby evading host immune response. In this study, the metal binding spectrum of EhArg has been determined. This study focuses on the biochemical and biophysical characterization of arginase from Entamoeba histolytica (EhArg), majorly characterizing the bivalent metal selectivity and metal binding kinetics of purified EhArg using Surface Plasmon Resonance and inductively coupled plasma mass spectroscopy. Investigation of the active site chemistry and total metal content using molecular docking and ICP-MS unraveled the fact that two Mn2+ ions are required for the enzyme to be fully functional. However, chelating loosely bound Mn2+ and replacing it with a variety of bivalent metal ions including Mg2+, Zn2+, Ni2+, Hg2+, Cu2+, Co2+, Ca2+ and Cd2+ retains its enzymatic activity. Further, the role of nine bivalent ions in the activation of EhArg was studied thermodynamically and biochemically. Phylogenetic and sequence analysis and oligomerization studies of EhArg show that unlike other eukaryotic arginases, EhArg exists in monomeric and dimeric form in solution and shows the highest similarity with bacterial arginase. This study unveiled interesting facts about EhArg that the enzyme has evolved to utilize available metal ion cofactors and survive the inhospitable environment within the host.

Arginase activity in pathogenic and non-pathogenic species of Leishmania parasites.

Proliferation of Leishmania (L.) parasites depends on polyamine availability, which can be generated by the L-arginine catabolism and the enzymatic activity of arginase (ARG) of the parasites and of the mammalian hosts. In the present study, we characterized and compared the arginase (arg) genes from pathogenic L. major and L. tropica and from non-pathogenic L. tarentolae. We quantified the level of the ARG activity in promastigotes and macrophages infected with pathogenic L. major and L. tropica and non-pathogenic L. tarentolae amastigotes. The ARG's amino acid sequences of the pathogenic and non-pathogenic Leishmania demonstrated virtually 98.6% and 88% identities with the reference L. major Friedlin ARG. Higher ARG activity was observed in all pathogenic promastigotes as compared to non-pathogenic L. tarentolae. In vitro infection of human macrophage cell line (THP1) with pathogenic and non-pathogenic Leishmania spp. resulted in increased ARG activities in the infected macrophages. The ARG activities present in vivo were assessed in susceptible BALB/c and resistant C57BL/6 mice infected with L. major, L. tropica and L. tarentolae. We demonstrated that during the development of the infection, ARG is induced in both strains of mice infected with pathogenic Leishmania. However, in L. major infected BALB/c mice, the induction of ARG and parasite load increased simultaneously according to the time course of infection, whereas in C57BL/6 mice, the enzyme is upregulated solely during the period of footpad swelling. In L. tropica infected mice, the footpads' swellings were slow to develop and demonstrated minimal cutaneous pathology and ARG activity. In contrast, ARG activity was undetectable in mice inoculated with the non-pathogenic L. tarentolae. Our data suggest that infection by Leishmania parasites can increase ARG activity of the host and provides essential polyamines for parasite salvage and its replication. Moreover, the ARG of Leishmania is vital for parasite proliferation and required for infection in mice. ARG activity can be used as one of the main marker of the disease severity.

Platelet Supernatant Suppresses LPS-Induced Nitric Oxide Production from Macrophages Accompanied by Inhibition of NF-κB Signaling and Increased Arginase-1 Expression

We previously reported that mouse bone marrow-derived macrophages (BMDMs) that had been co-cultured with platelets exhibited lower susceptibility to bacterial lipopolysaccharide (LPS) and produced lower levels of nitric oxide (NO) and inflammatory cytokines including TNF-α and IL-6. The suppression of macrophage responses was mediated, at least in part, by platelet supernatant. In the present study, we assessed phenotypic changes of BMDMs induced by incubation with the supernatant from thrombin-activated platelets (PLT-sup) and found that BMDMs cultured with PLT-sup (PLT-BMDMs) expressed a lower level of inducible NO synthase (iNOS) and a higher level of arginase-1, both of which are involved in the L-arginine metabolism, upon stimulation with LPS or zymosan. We also examined possible modulation of the NF-κB signaling pathway and observed suppression of IκBα phosphorylation and a decrease of NF-κB p65 expression in LPS-stimulated PLT-BMDMs. These results suggest that PLT-sup suppresses inflammatory responses of BMDMs via negative regulation of NF-κB signaling leading to lowered expression of iNOS and enhanced L-arginine catabolism by arginase-1.

Catabolism and safety of supplemental L-arginine in animals.

L-arginine (Arg) is utilized via multiple pathways to synthesize protein and low-molecular-weight bioactive substances (e.g., nitric oxide, creatine, and polyamines) with enormous physiological importance. Furthermore, Arg regulates cell signaling pathways and gene expression to improve cardiovascular function, augment insulin sensitivity, enhance lean tissue mass, and reduce obesity in humans. Despite its versatile roles, the use of Arg as a dietary supplement is limited due to the lack of data to address concerns over its safety in humans. Data from animal studies are reviewed to assess arginine catabolism and the safety of long-term Arg supplementation. The arginase pathway was responsible for catabolism of 76-85 and 81-96% Arg in extraintestinal tissues of pigs and rats, respectively. Dietary supplementation with Arg-HCl or the Arg base [315- and 630-mg Arg/(kg BWd) for 91d] had no adverse effects on male or female pigs. Similarly, no safety issues were observed for male or female rats receiving supplementation with 1.8- and 3.6-g Arg/(kg BWd) for at least 91d. Intravenous administration of Arg-HCl to gestating sheep at 81 and 180mg Arg/(kg BWd) is safe for at least 82 and 40d, respectively. Animals fed conventional diets can well tolerate large amounts of supplemental Arg [up to 630-mg Arg/(kg BWd) in pigs or 3.6-g Arg/(kg BWd) in rats] for 91d, which are equivalent to 573-mg Arg/(kg BWd) for humans. Collectively, these results can help guide studies to determine the safety of long-term oral administration of Arg in humans.

Myeloid-Derived Suppressor Cells: Possible Link Between Chronic Obstructive Pulmonary Disease and Lung Cancer

Chronic obstructive pulmonary disease (COPD) and lung cancer (LC) are prevalent diseases and a leading cause of morbidity and mortality worldwide. There is strong evidence to show that COPD is an independent risk factor for LC. Chronic inflammation plays a significant pathogenic role in COPD comorbidities, particularly in LC. On the one hand, cellular and molecular inflammatory mediators promote carcinogenesis and, on the other, chronic inflammation impairs the capacity of the immune system to identify and destroy pre-malignant and malignant cells, a process known as tumor immune surveillance. This altered antitumor immunity is due in part to the expansion of myeloid-derived suppressor cells (MDSC), which are characterized by an ability to suppress the antitumor activity of T-cells by down-regulation of the T-cell receptor ζ chain (TCR ζ) through the catabolism of l-arginine. COPD and LC patients share a common pattern of expansion and activation of circulating MDSC associated with TCR ζ downregulation and impaired peripheral T-cell function. The objectives of this study were to review the evidence on the association between COPD and LC and to analyze how MDSC accumulation may alter tumor immune surveillance in COPD, and therefore, promote LC development.

Células mieloides supresoras: potencial vínculo entre la enfermedad pulmonar obstructiva crónica y el cáncer de pulmón

Chronic obstructive pulmonary disease (COPD) and lung cancer (LC) are prevalent diseases and are a leading cause of morbidity and mortality worldwide. There is strong evidence to show that COPD is an independent risk factor for LC. Chronic inflammation plays a significant pathogenic role in COPD comorbidities, particularly in LC. On the one hand, cellular and molecular inflammatory mediators promote carcinogenesis and, on the other, chronic inflammation impairs the capacity of the immune system to identify and destroy pre-malignant and malignant cells, a process known as tumor immune surveillance. This altered antitumor immunity is due in part to the expansion of myeloid-derived suppressor cells (MDSC), which are characterized by an ability to suppress the antitumor activity of T-cells by down-regulation of the T-cell receptor ζ chain (TCRζ) through the catabolism of L-arginine. COPD and LC patients share a common pattern of expansion and activation of circulating MDSC associated with TCRζ downregulation and impaired peripheral T-cell function. The objectives of this study were to review the evidence on the association between COPD and LC and to analyze how MDSC accumulation may alter tumor immune surveillance in COPD, and therefore, promote LC development.

L-arginine catabolism is driven mainly towards nitric oxide synthesis in the erythrocytes of patients with type 2 diabetes at first clinical onset

We investigated the l-arginine (l-Arg)-nitric oxide (NO) metabolic pathway in the erythrocytes (RBCs) and plasma of subjects with type 2 diabetes at first clinical onset. RBCs and plasma were collected from 26 patients with type 2 diabetes at first clinical onset and 19 age-matched non-diabetes subjects as controls. l-Arg content was assayed by capillary electrophoresis. We measured arginase activity and nitrate/nitrite concentrations by spectrophotometry, and glycosylated haemoglobin (HbA1c) by standardized immunoturbidimetry. We found that, when compared with controls, l-Arg content was similar in RBCs while decreased in the plasma of patients with type 2 diabetes. Interestingly, arginase activity was lower in RBCs and increased in plasma of patients with diabetes. NO production was higher in RBCs in patients with type 2 diabetes, while no difference was found in the plasma of our subjects. l-Arg catabolism is driven mainly towards NO synthesis in RBCs of patients with type 2 diabetes at first clinical onset. The decreased RBC arginase activity could be considered a potential mechanism of increased RBC NO production in early diabetes. Therefore, the RBC pool would represent a potentially compensatory intravascular compartment for endothelial dysfunction in diabetes.

The Emerging Immunological Role of Post-Translational Modifications by Reactive Nitrogen Species in Cancer Microenvironment

Under many inflammatory contexts, such as tumor progression, systemic and peripheral immune response is tailored by reactive nitrogen species (RNS)-dependent post-translational modifications, suggesting a biological function for these chemical alterations. RNS modify both soluble factors and receptors essential to induce and maintain a tumor-specific immune response, creating a “chemical barrier” that impairs effector T cell infiltration and functionality in tumor microenvironment and supports the escape phase of cancer. RNS generation during tumor growth mainly depends on nitric oxide production by both tumor cells and tumor-infiltrating myeloid cells that constitutively activate essential metabolic pathways of l-arginine catabolism. This review provides an overview of the potential immunological and biological role of RNS-induced modifications and addresses new approaches targeting RNS either in search of novel biomarkers or to improve anti-cancer treatment.

Inhibition of Arginase Activity Ameliorates L-Arginine-Induced Acute Pancreatitis in Rats

Intraperitoneal (IP) injection of 3.5 g/kg L-arginine (known to induce acute pancreatitis) in rats will result in much greater increases in serum ornithine versus citrulline concentration (Crit Care Med. 2008;36:2117-2127). These data indicate a major role of arginase in the catabolism of L-arginine. Therefore, we tested the effects of the irreversible arginase inhibitor (+)-S-2-amino-6-iodoacetamidohexanoic acid (AIHA) on L-arginine-induced acute pancreatitis. The inhibitory effect of AIHA on arginase activity was tested on rat liver homogenate and purified bovine arginase. Male Wistar rats were administered 15 mg/kg AIHA or its vehicle IP 1 hour before the injection of physiological saline or 3.5 g/kg L-arginine IP. Laboratory and histological parameters of pancreatitis were determined 24 hours after the last injection. Sixty micromolars of AIHA (equimolar to an in vivo dose of 15 mg/kg) significantly inhibited arginase activity by about 25%. Pretreatment with AIHA significantly ameliorated pancreatic damage caused by L-arginine administration. It decreased pancreatic weight/body weight ratio, pancreatic glutathione peroxidase and myeloperoxidase activities, and histological damage. Administration of AIHA in itself significantly increased levels of pancreatic heat shock proteins. Pretreatment with AIHA reduces the severity of L-arginine-induced pancreatitis most likely by inhibiting arginase activity.

Plasticity of the mitoproteome to nitrogen sources (nitrate and ammonium) in Chlamydomonas reinhardtii: The logic of Aox1 gene localization

Nitrate and ammonium constitute primary inorganic nitrogen sources that can be incorporated into carbon skeletons in photosynthetic eukaryotes. In Chlamydomonas, previous studies and the present one showed that the mitochondrial AOX is up-regulated in nitrate-grown cells in comparison with ammonium-grown cells. In this work, we have performed a comparative proteomic analysis of the soluble mitochondrial proteome of Chlamydomonas cells growth either on nitrate or ammonium. Our results highlight important proteomics modifications mostly related to primary metabolism in cells grown on nitrate. We could note an up-regulation of some TCA cycle enzymes and a down-regulation of cytochrome c1 together with an up-regulation of l-arginine and purine catabolism enzymes and of ROS scavenging systems. Hence, in nitrate-grown cells, AOX may play a dual role: (1) lowering the ubiquinone pool reduction level and (2) permitting the export of mitochondrial reducing power under the form of malate for nitrate and nitrite reduction. This role of AOX in the mitochondrial plasticity makes logical the localization of Aox1 in a nitrate assimilation gene cluster.

Sorting of Leishmania-bearing dendritic cells reveals subtle parasite-induced modulation of host-cell gene expression

Once in the mouse skin, Leishmania (L) amazonensis amastigotes are hosted by professional mononuclear phagocytes such as dendritic cells (DCs). When monitored after parasite inoculation, the frequency of amastigote-hosting DCs is very low (<1%) in both the skin and skin-draining lymph nodes. Therefore, we designed and validated an efficient procedure to purify live amastigotes-hosting DCs with the objective to facilitate quantitative and qualitative analysis of such rare cells. To this end, a L. amazonensis transgenic parasite expressing DsRed2 fluorescent protein was generated and added to mouse bone marrow-derived DC cultures. Then, a high speed sorting procedure, performed in BSL-2 containment, was setup to pick out only DCs hosting live amastigotes. This study reveals, for the first time, a unique transcript pattern from sorted live amastigotes-hosting DCs that would have been undetectable in unsorted samples. It was indeed possible to highlight a significant and coordinated up-regulation of l-arginine transporter and arginase2 transcripts in Leishmania-hosting DCs compared to un-parasitized DCs. These results indicate that arginine catabolism for polyamine generation is dominating over L-arginine catabolism for NO generation. In conclusion, this approach provides a powerful method for further characterisation, of amastigote-hosting DCs in the skin and the skin-draining lymph nodes.

Proteophosophoglycans Regurgitated by Leishmania-Infected Sand Flies Target the L-Arginine Metabolism of Host Macrophages to Promote Parasite Survival

All natural Leishmania infections start in the skin; however, little is known of the contribution made by the sand fly vector to the earliest events in mammalian infection, especially in inflamed skin that can rapidly kill invading parasites. During transmission sand flies regurgitate a proteophosphoglycan gel synthesized by the parasites inside the fly midgut, termed promastigote secretory gel (PSG). Regurgitated PSG can exacerbate cutaneous leishmaniasis. Here, we show that the amount of Leishmania mexicana PSG regurgitated by Lutzomyia longipalpis sand flies is proportional to the size of its original midgut infection and the number of parasites transmitted. Furthermore, PSG could exacerbate cutaneous L. mexicana infection for a wide range of doses (10–10,000 parasites) and enhance infection by as early as 48 hours in inflamed dermal air pouches. This early exacerbation was attributed to two fundamental properties of PSG: Firstly, PSG powerfully recruited macrophages to the dermal site of infection within 24 hours. Secondly, PSG enhanced alternative activation and arginase activity of host macrophages, thereby increasing L-arginine catabolism and the synthesis of polyamines essential for intracellular parasite growth. The increase in arginase activity promoted the intracellular growth of L. mexicana within classically activated macrophages, and inhibition of macrophage arginase completely ablated the early exacerbatory properties of PSG in vitro and in vivo. Thus, PSG is an essential component of the infectious sand fly bite for the early establishment of Leishmania in skin, which should be considered when designing and screening therapies against leishmaniasis.

Inhibition of arginase activity ameliorates L-arginine-induced acute pancreatitis in rats

Introduction: Intraperitoneal (i.p.) injection of 3.5g/kg L-arginine (which induces experimental acute necrotizing pancreatitis in rats) will result in much greater increases in serum ornithine vs. citrulline level. This suggests that arginase is the key enzyme in the catabolism of L-arginine. Furthermore, equimolar L-ornithine induces a more severe acute pancreatitis compared to L-arginine. Aim: To test the effects of the irreversible arginase inhibitor (+)-S-2-amino-6-iodoacetamidohexanoic acid (AIHA) on L-arginine-induced acute pancreatitis.

The proteome of Salmonella Typhimurium grown under in vivo‐mimicking conditions

To successfully infect a host, it is a prerequisite for enteric pathogens such as Salmonella enterica serovar Typhimurium to adapt to their environment, in casu the gastrointestinal tract. The adoption of an appropriate lifestyle is triggered by environmental signals such as the low oxygen availability and high osmolarity prevalent in the gut. In order to gain more insight in the changes that are induced when S. Typhimurium is adapting to these particular conditions, we used 2-D DIGE technology to investigate the combined effect of low oxygen tension and high osmolarity on the proteome of S. Typhimurium SL1344 compared to standard laboratory conditions. As a validation of the 2-D DIGE technique, preferential protein labeling by the Cy-dyes was assessed and proved to be negligible. The differentially expressed proteins identified reflect very well the applied culture conditions. Furthermore, reported transcriptional changes and observed changes at the translational level show overlap. Among the metabolic processes that are upregulated under in vivo-mimicking conditions are anaerobic fumarate respiration and the utilization of 1,2-propanediol. We also provide evidence that S. Typhimurium expresses an arginine deiminase pathway for the catabolism of L-arginine. The increased activity of this pathway was biochemically validated. Finally, also proteins involved in quorum sensing and virulence are differentially expressed under in vivo-mimicking conditions. These conditions offer possibilities as a simplified model system for the host environment given the high overlap of identifications in our study and reported genuine in vivo studies, respectively.

Age-Related Alteration of Arginase Activity Impacts on Severity of Leishmaniasis

BackgroundThe leishmaniases are a group of vector-borne parasitic diseases that represent a major international public health problem; they belong to the most neglected tropical diseases and have one of the highest rates of morbidity and mortality. The clinical outcome of infection with Leishmania parasites depends on a variety of factors such as parasite species, vector-derived products, genetics, behaviour, and nutrition. The age of the infected individuals also appears to be critical, as a significant proportion of clinical cases occur in children; this age-related higher prevalence of disease is most remarkable in visceral leishmaniasis. The mechanisms resulting in this higher incidence of clinical disease in children are poorly understood. We have recently revealed that sustained arginase activity promotes uncontrolled parasite growth and pathology in vivo. Here, we tested the hypothesis that arginase-mediated L-arginine metabolism differs with age.MethodologyThe age distribution of patients with visceral or cutaneous leishmaniasis was determined in cohorts of patients in our clinics in endemic areas in Ethiopia. To exclude factors that are difficult to control in patients, we assessed the impact of ageing on the manifestations of experimental leishmaniasis. We determined parasite burden, T cell responses, and macrophage effector functions in young and aged mice during the course of infection.ResultsOur results show that younger mice develop exacerbated lesion pathology and higher parasite burdens than aged mice. This aggravated disease development in younger individuals does not correlate with a change in T helper cytokine profile. To address the underlying mechanisms responsible for the more severe infections in younger mice, we investigated macrophage effector functions. Our results show that macrophages from younger mice do not have an impaired capacity to kill parasites; however, they express significantly higher levels of arginase 1 than aged mice and promote parasite growth more efficiently. Thus, our results demonstrate that ageing differentially impacts on L-arginine metabolism and subsequent effector functions of physiologically distinct macrophage subsets.ConclusionsHere, we show that arginase-mediated L-arginine metabolism is modulated with age and affects the capacity of macrophages to express arginase; the increased capacity to upregulate this enzyme in younger individuals results in a more permissive environment for parasite growth, increased disease severity and pathology. These results suggest that the difference in arginase-mediated L-arginine catabolism is likely to be an important factor contributing to the increased incidence of clinical cases in children. Thus, targeting L-arginine metabolism might be a promising therapeutic strategy against leishmaniasis, especially in children and young adults.

L-Arginine metabolism in dog kidney and isolated nephron segments

The renal basic amino acid metabolism often differs in rodents, strict carnivores, and omnivore species. Given the pivotal role of l-arginine and l-ornithine in several metabolic pathways and the fact that the dog is closely related to humans, being also an omnivore, we tested whether l-arginine metabolism and l-ornithine catabolism take place in the dog kidney. We examined the metabolism of l-arginine in dog cortical tubules to integrate local l-arginine metabolism into a general physiological and metabolic framework. To achieve these goals, we first ascertained the protein expression of relevant enzymes by Western blot. l-Arginine catabolism was studied in suspensions of canine cortical proximal tubules, medullary thick ascending limbs, and papillary collecting ducts either incubated without exogenous l-arginine being added (small endogenous quantities) or incubated with l-arginine being added in supraphysiological amounts (2 mmol/L with or without the presence of alternative metabolic substrates, 2 mmol/L l-glutamine, or lactate). The results revealed that dog kidneys consumed l-citrulline and released l-arginine and l-ornithine. Argininosuccinate synthetase and lyase, arginase II, and ornithine aminotransferase were detected in the renal cortex. Arginase II activity was found in a suspension of proximal tubules by measuring the amounts of urea and l-ornithine produced. A fraction of this l-ornithine was further partially metabolized through the intramitochondrial ornithine aminotransferase pathway, leading to changes in l-glutamate, glucose, l-alanine, and ammonia metabolism without l-proline accumulation. Medullary thick ascending limbs expressed a very low arginase activity, whereas papillary collecting ducts did not. In conclusion, the dog kidney produces l-arginine. Part of this l-arginine is further catabolized by arginase II, suggesting that its physiological role was to produce l-ornithine for the body.

Bioinformatic evaluation of L-arginine catabolic pathways in 24 cyanobacteria and transcriptional analysis of genes encoding enzymes of L-arginine catabolism in the cyanobacterium Synechocystis sp. PCC 6803

BackgroundSo far very limited knowledge exists on L-arginine catabolism in cyanobacteria, although six major L-arginine-degrading pathways have been described for prokaryotes. Thus, we have performed a bioinformatic analysis of possible L-arginine-degrading pathways in cyanobacteria. Further, we chose Synechocystis sp. PCC 6803 for a more detailed bioinformatic analysis and for validation of the bioinformatic predictions on L-arginine catabolism with a transcript analysis.ResultsWe have evaluated 24 cyanobacterial genomes of freshwater or marine strains for the presence of putative L-arginine-degrading enzymes. We identified an L-arginine decarboxylase pathway in all 24 strains. In addition, cyanobacteria have one or two further pathways representing either an arginase pathway or L-arginine deiminase pathway or an L-arginine oxidase/dehydrogenase pathway. An L-arginine amidinotransferase pathway as a major L-arginine-degrading pathway is not likely but can not be entirely excluded. A rather unusual finding was that the cyanobacterial L-arginine deiminases are substantially larger than the enzymes in non-photosynthetic bacteria and that they are membrane-bound. A more detailed bioinformatic analysis of Synechocystis sp. PCC 6803 revealed that three different L-arginine-degrading pathways may in principle be functional in this cyanobacterium. These are (i) an L-arginine decarboxylase pathway, (ii) an L-arginine deiminase pathway, and (iii) an L-arginine oxidase/dehydrogenase pathway. A transcript analysis of cells grown either with nitrate or L-arginine as sole N-source and with an illumination of 50 μmol photons m-2 s-1 showed that the transcripts for the first enzyme(s) of all three pathways were present, but that the transcript levels for the L-arginine deiminase and the L-arginine oxidase/dehydrogenase were substantially higher than that of the three isoenzymes of L-arginine decarboxylase.ConclusionThe evaluation of 24 cyanobacterial genomes revealed that five different L-arginine-degrading pathways are present in the investigated cyanobacterial species. In Synechocystis sp. PCC 6803 an L-arginine deiminase pathway and an L-arginine oxidase/dehydrogenase pathway represent the major pathways, while the L-arginine decarboxylase pathway most likely only functions in polyamine biosynthesis. The transcripts encoding the enzymes of the two major pathways were constitutively expressed with the exception of the transcript for the carbamate kinase, which was substantially up-regulated in cells grown with L-arginine.