Arg1 expression defines immunosuppressive subsets of tumor-associated macrophages.

Tumor-associated macrophages (TAM) have attracted attention as they can modulate key cancer-related activities, yet TAM represent a heterogenous group of cells that remain incompletely characterized. In growing tumors, TAM are often referred to as M2-like macrophages, which are cells that display immunosuppressive and tumorigenic functions and express the enzyme arginase 1 (Arg1). Methods: Here we combined high resolution intravital imaging with single cell RNA seq to uncover the topography and molecular profiles of immunosuppressive macrophages in mice. We further assessed how immunotherapeutic interventions impact these cells directly in vivo. Results: We show that: i) Arg1+ macrophages are more abundant in tumors compared to other organs; ii) there exist two morphologically distinct subsets of Arg1 TAM defined by previously unknown markers (Gbp2b, Bst1, Sgk1, Pmepa1, Ms4a7); iii) anti-Programmed Cell Death-1 (aPD-1) therapy decreases the number of Arg1+ TAM while increasing Arg1- TAM; iv) accordingly, pharmacological inhibition of arginase 1 does not synergize with aPD-1 therapy. Conclusion: Overall, this research shows how powerful complementary single cell analytical approaches can be used to improve our understanding of drug action in vivo.

Techniques to harvest diseased human peripheral arteries and measure endothelial function in an ex vivo model.

OBJECTIVE: Endothelial dysfunction has been studied in animal models. However, direct evidence of endothelial function from human vessels is limited. Our objectives were to optimize methods in harvesting human arteries from amputation specimens, determine endothelial function, and measure responsiveness to l-arginine, a nitric oxide precursor. METHODS: Fresh amputation specimens were transferred expeditiously from the operating room to the bench laboratory for dissection and arterial harvest in an Investigational Review Board-approved protocol. Popliteal and tibial vessels were examined in pilot experiments leading to the use of the anterior tibial artery in consecutive experiments. Human lower extremity anterior tibial artery segments were harvested from 14 amputation specimens. Specimens were rapidly collected and divided for endothelial-dependent relaxation (EDR) studies in a tissue bath apparatus, immunohistochemistry, and intravascular ultrasound-derived virtual histology. A total of 47 ring segments were studied. The data were compared with two-way analysis of variance. RESULTS: Human lower extremity arteries exhibited low responsiveness to acetylcholine (EDR, 24.9%; acetylcholine, 10(-4)). L-arginine supplementation enhanced EDR by 38.5% (P < .0001). N-nitro-L-arginine methyl ester abrogated EDR (P < .0001) in vessels exposed to L-arginine. Arterial responsiveness was intact in all vessels (endothelial independent relaxation to sodium nitroprusside, 113.2% ± 28.1%). Histology and immunohistochemistry confirmed intact endothelium by morphometric analysis, cluster of differentiation 31, endothelial nitric oxide synthase, and arginase II staining. Intravascular ultrasound-derived virtual histology indicated atheroma burden was 11.9 ± 4.7 mm(3)/cm, and plaque stratification indicated fibrous morphology was predominant (59.9%; necrotic core, 16.9%; calcium, 11.2%). Variations in plaque morphology did not correlate with endothelial function or responsiveness to L-arginine. CONCLUSIONS: Human lower extremity arteries demonstrate low baseline endothelial function in patients requiring amputation. Endothelial dysfunction is improved by L-arginine supplementation in an ex vivo model. These results support strategies to increase local levels of nitric oxide in human vessels.

Areca nut extracts enhance the development of CD11b(+) Gr-1(+) cells with the characteristics of myeloid-derived suppressor cells in antigen-stimulated mice.

BACKGROUND: Areca quid chewing is an etiological factor contributing to the development of oral cancer and pre-cancers, whose pathophysiology has been linked to inflammation and immune deterioration. Myeloid-derived suppressor cells (MDSC) play a key role in the regulation of immunity under certain pathological conditions, such as inflammation and cancer. As areca nut extracts (ANE) have been reported to induce a proinflammatory effect in antigen-stimulated mice, we hypothesized that ANE might enhance the development of MDSC. METHODS: Ovalbumin (OVA)-sensitized BALB/c mice were daily administered with ANE (5-50 mg/kg), polyphenol-enriched ANE (PANE; 25 mg/kg) or arecoline (5 mg/kg) by intraperitoneal injection for 10 doses. The mouse footpads were then subcutaneously challenged with OVA to induce local inflammatory responses. RESULTS: ANE and PANE treatment significantly increased the spleen index and the population of CD11b(+) Gr-1(+) cells in the spleen and peripheral blood, whereas arecoline was inactive. In addition, ANE and PANE treatment enhanced the expression of cytokines and enzymes associated with the immunosuppressive function of MDSC, including IL-10, arginase-I and iNOS in splenic CD11b(+) cells. Concordantly, ANE and PANE treatment augmented the infiltration of Gr-1(+) IL-10(+) cells in the footpads challenged with OVA. CONCLUSIONS: Our results suggested that areca nut constituents, in particular, polyphenols enhanced the development of myeloid-derived suppressor cells in vivo, which may be a critical mechanism linking inflammation and the compromised immunity reported to be associated with the pathophysiology of areca-related oral diseases.

Arginase activity in mitochondria--An interfering factor in nitric oxide synthase activity assays.

Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT). Of those techniques, the NOS-catalyzed [(14)C]-L-arginine to [(14)C]-L-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent. Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography. Results obtained from these samples showed no radioactive band for L-citrulline. However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples. In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [(14)C]-L-arginine to [(14)C]-urea. The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays.

Role of arginine in superficial wound healing in man.

Arginine supplementation has been identified as advantageous in experimental wound healing. However, the mechanisms underlying this beneficial effect in tissue repair remain unresolved. Animal studies suggest that the beneficial role of arginine supplementation is mediated, at least in part through NO. The latter component mediates processes involved in tissue repair, including angiogenesis, epithelialization and collagen formation. This prospective study is performed to investigate arginine metabolism in acute surgical wounds in man. Expression of enzymes, known to be involved in arginine metabolism, was studied in donor sites of skin grafts of 10 hospitalized patients undergoing skin transplantation. Plasma and wound fluid levels of arginine metabolites (ornithine, citrulline, nitrate and nitrite = NOx) were measured using High Performance Liquid Chromatography. Expression of iNOS, eNOS, arginase-1 and arginase-2 was studied by immunohistochemistry in paraffin sections of skin tissue. Arginase-1 concentration was measured in plasma and wound fluid using ELISA. Arginase-2 was determined using Western blot analysis. We observed increased levels of citrulline, ornithine, NOx and arginase-1 in wound fluid when compared with plasma. Arginase-2 was expressed in both plasma and wound fluid and seemed higher in plasma. iNOS was expressed by neutrophils, macrophages, fibroblasts, keratinocytes and endothelial cells upon wounding, whereas eNOS reactivity was observed in endothelial cells and fibroblasts. Arginase-1 was expressed in neutrophils post-wounding, while arginase-2 staining was observed in endothelial cells, keratinocytes, fibroblasts, macrophages and neutrophils. For the first time, human data support previous animal studies suggesting arginine metabolism for an NO- as well as arginase-mediated reparation of injured skin.

[Pathogenetic mechanisms of action of tarry substances and Mycobacterium tuberculosis in guinea pigs and rats]. / Patogeneticheskie mekhanizmy vozdeistviia smolistykh veshchestv i mikobakterii tuberkuleza na morskikh svinok i krys.

Harmful environmental agents [polycyclic aromatic hydrocarbons (PAH)] have been ascertained to greatly stimulate the biosynthesis of arginine and urea and reduce the amount of sulfur-containing metabolites in the liver of experimental animals by increasing the level of sulfur sulfate. Against this background, contamination with Mycobacteria tuberculosis (MBT) inhibits the activity of arginine and drastically decreases its amount by elevating the concentration of sulfur-containing metabolites. The supplementary administration of sodium glutamate to animals receiving PAH and MBT potentiates a decrease in nitrogen-rich metabolites and increases the level of sulfur-containing metabolites guinea pigs, tuberculosis resistance being on the rise. Under the influence of a combined action of PAH and MBT, the mutagenic effect of the former lowered in rats.

Decreased expression of arginase II in the kidneys of Dahl salt-sensitive rats.

Arginase catalyzes the hydrolysis of arginine to urea and ornithine. Urea is not only an important solute for concentrating urine but also inhibits Na-K-2Cl cotransport. To elucidate the roles of arginase in the development of salt-sensitive hypertension, we examined arginase activity and expression in the kidney and other organs of Dahl/Rapp salt-sensitive (SS) and salt-resistant (SR) rats before and after 4 weeks' administration of a 4% NaCl or control diet. At 4 weeks of age, arginase activity in the kidney was lower in SS rats than in SR rats. Kidney arginase activity was lower in SS rats than in SR rats at 8 weeks of age, and salt loading did not alter arginase activity. Arginase II (the dominant isoform in the kidney) mRNA and protein in the kidney of salt-loaded SS rats were also lower than those of salt-loaded SR rats. Arginase activities in the liver and cerebellum did not differ between SS and SR rats. To examine the effect of urea, the product of arginase reaction, on the development of hypertension, SS rats were given a 4% NaCl diet containing 5% kaolin or 5% urea. Six-week urea supplementation attenuated the development of hypertension in SS rats. These findings suggest that decreased arginase expression in the kidney may be at least partially responsible for the salt-sensitive hypertension in SS rats.

Dexamethasone and glucagon cause synergistic increases of urea cycle enzyme activities in livers of normal but not adrenalectomized rats.

Adrenalectomized and intact rats were given constant high-dose infusions of glucagon, 0.3 mg/kg per day for 7 days, with or without low-dose dexamethasone, 0.01 mg/kg daily, to test whether glucocorticoids potentiate glucagon induction of the 5 urea cycle enzymes as they do in cultured rat hepatocytes. Glucagon did not induce any of the urea cycle enzymes in adrenalectomized Sprague-Dawley rats and only induced argininosuccinate lyase (EC 4.3.2.1) in adrenalectomized inbred Wistar-Furth rats. Dexamethasone alone induced arginase in adrenalectomized and in intact Wistar-Furth rats and restored the other enzymes to normal levels in adrenalectomized rats. In intact Wistar-Furth rats, the combination of hormones gave synergistic increases of all 5 enzymes over the responses to each hormone alone, but in adrenalectomized rats the combination was only additive or less than additive compared with the sum of single hormone responses. The lack of synergism between the two hormones in adrenalectomized rats suggest that other factors play a role in glucagon induction of this cycle.

The significance of the arginine and arginase of tears in experimentally-induced herpes simplex corneae.

Experimental corneal herpes is always accompanied by the accumulation of arginine, the substrate of arginase, in tears, ensuring the multiplication of the herpes hominis virus. The main source of the large amount of arginine is the desquamating corneal epithelium, since after the epithelium of the cornea is abraded the arginine content of the tears again equals that of healthy tears. The low arginase content of rabbit tears can be supplemented by arginase applied as eyedrops, and this results in the cure of the herpetic process.