Possible role of arginase 1 positive microglia on depressive/anxiety-like behaviors in atopic dermatitis mouse model.

Atopic dermatitis (AD) and mood disorder comorbidities are typical, but the exact mechanism underlying their interplay has not been clarified. In this study, we aimed to identify the possible mechanisms of anxiety/depressive-like behaviors observed in AD, focusing on microglia. AD was induced by Dermatophagoides farinae body extract (Dfb) in NC/Nga mice and anxiety/depressive-like behaviors were analyzed by behavioral assessments such as open field test (OFT), tail suspension test (TST), sucrose preference test (SPT), and social interaction. As clinical symptoms of AD induced, anxiety/depressive-like behaviors were increased in the OFT and TST and serum glucocorticoid was elevated. AD mice showed an increased mRNA expression of interleukin-4 (IL-4) in lymph nodes but decreased arginase 1 (Arg1) mRNA expression without a change of IL-4 in the hippocampus. In addition, AD mice showed microglia with a shortened branch of de-ramified form and astrocytes with longer processes and decreased branching in the hippocampus, especially in the dentate gyrus (DG). The immunofluorescence study of the DG confirmed that Arg1 reduction was associated with microglia, but not astrocytes. Furthermore, glucocorticoid receptor reduction, increased 5-HT1AR, reduced phosphorylated cAMP response element-binding protein (pCREB), and brain-derived neurotrophic factor (BDNF) expression were identified in the hippocampus of AD mice. Notably, an immunofluorescence study confirmed that pCREB was decreased in the DG of AD mice. Collectively, our data suggest that the reduced Arg1 positive microglia might contribute to anxiety/depressive-like behaviors via pCREB/BDNF reduction in AD.

Granulocytic Myeloid-Derived Suppressor Cells in Cystic Fibrosis.

Cystic Fibrosis (CF) is a genetic disease that causes chronic and severe lung inflammation and infection associated with high rates of mortality. In CF, disrupted ion exchange in the epithelium results in excessive mucus production and reduced mucociliary clearance, leading to immune system exacerbation and chronic infections with pathogens such as P. aeruginosa and S. aureus. Constant immune stimulation leads to altered immune responses including T cell impairment and neutrophil dysfunction. Specifically, CF is considered a Th17-mediated disease, and it has been proposed that both P. aeruginosa and a subset of neutrophils known as granulocytic myeloid suppressor cells (gMDSCs) play a role in T cell suppression. The exact mechanisms behind these interactions are yet to be determined, but recent works demonstrate a role for arginase-1. It is also believed that P. aeruginosa drives gMDSC function as a means of immune evasion, leading to chronic infection. Herein, we review the current literature regarding immune suppression in CF by gMDSCs with an emphasis on T cell impairment and the role of P. aeruginosa in this dynamic interaction.

Breast cancer-derived GM-CSF regulates arginase 1 in myeloid cells to promote an immunosuppressive microenvironment.

Tumor-infiltrating myeloid cells contribute to the development of the immunosuppressive tumor microenvironment. Myeloid cell expression of arginase 1 (ARG1) promotes a protumor phenotype by inhibiting T cell function and depleting extracellular l-arginine, but the mechanism underlying this expression, especially in breast cancer, is poorly understood. In breast cancer clinical samples and in our mouse models, we identified tumor-derived GM-CSF as the primary regulator of myeloid cell ARG1 expression and local immune suppression through a gene-KO screen of breast tumor cell-produced factors. The induction of myeloid cell ARG1 required GM-CSF and a low pH environment. GM-CSF signaling through STAT3 and p38 MAPK and acid signaling through cAMP were required to activate myeloid cell ARG1 expression in a STAT6-independent manner. Importantly, breast tumor cell-derived GM-CSF promoted tumor progression by inhibiting host antitumor immunity, driving a significant accumulation of ARG1-expressing myeloid cells compared with lung and melanoma tumors with minimal GM-CSF expression. Blockade of tumoral GM-CSF enhanced the efficacy of tumor-specific adoptive T cell therapy and immune checkpoint blockade. Taken together, we show that breast tumor cell-derived GM-CSF contributes to the development of the immunosuppressive breast cancer microenvironment by regulating myeloid cell ARG1 expression and can be targeted to enhance breast cancer immunotherapy.

Selective inhibition of arginase-2 in endothelial cells but not proximal tubules reduces renal fibrosis.

Fibrosis is the final common pathway in the pathophysiology of most forms of chronic kidney disease (CKD). As treatment of renal fibrosis still remains largely supportive, a refined understanding of the cellular and molecular mechanisms of kidney fibrosis and the development of novel compounds are urgently needed. Whether arginases play a role in the development of fibrosis in CKD is unclear. We hypothesized that endothelial arginase-2 (Arg2) promotes the development of kidney fibrosis induced by unilateral ureteral obstruction (UUO). Arg2 expression and arginase activity significantly increased following renal fibrosis. Pharmacologic blockade or genetic deficiency of Arg2 conferred kidney protection following renal fibrosis, as reflected by a reduction in kidney interstitial fibrosis and fibrotic markers. Selective deletion of Arg2 in endothelial cells (Tie2Cre/Arg2fl/fl) reduced the level of fibrosis after UUO. In contrast, selective deletion of Arg2 specifically in proximal tubular cells (Ggt1Cre/Arg2fl/fl) failed to reduce renal fibrosis after UUO. Furthermore, arginase inhibition restored kidney nitric oxide (NO) levels, oxidative stress, and mitochondrial function following UUO. These findings indicate that endothelial Arg2 plays a major role in renal fibrosis via its action on NO and mitochondrial function. Blocking Arg2 activity or expression could be a novel therapeutic approach for prevention of CKD.

Role of Arginase in Selective Impairment of Endothelium-Dependent Nitric Oxide Synthase-Mediated Dilation of Retinal Arterioles during Early Diabetes.

Purpose: Retinal vasomotor activity can be regulated by two major endothelial enzymes, nitric oxide synthase (NOS) and cyclooxygenase (COX). The vascular arginase also consumes a NOS substrate and thus impedes NOS-mediated vasodilation. Diabetes mellitus exhibits vascular complications in the retina with elevated oxidative stress and compromised NOS-mediated vasodilation. However, the underlying molecular mechanisms remain unclear, and the effect of diabetes on COX-mediated vasodilation is unknown. Herein, we examined the relative impact of diabetes on retinal arteriolar dilations to COX and NOS activation and the roles of arginase and superoxide in diabetes-induced vasomotor dysfunction. Methods: Retinal arterioles were isolated from streptozocin-induced diabetic pigs (2 weeks of hyperglycemia, 433 ± 27 mg/dL) or age-matched control pigs (97 ± 4 mg/dL). The vasodilations to bradykinin (NOS activator) and histamine (NOS/COX activator) were examined in vitro. Results: Retinal arteriolar dilations to histamine and bradykinin were significantly reduced after 2 weeks of diabetes. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) attenuated the dilations of control vessels, but not diabetic vessels, to histamine. In the presence of L-NAME and COX inhibitor indomethacin, histamine-induced dilations of control and diabetic vessels were reduced similarly. Treatment of diabetic vessels with arginase inhibitor nor-NOHA, but not superoxide dismutase mimetic TEMPOL, preserved both histamine- and bradykinin-induced dilations in an L-NAME-sensitive manner. Conclusions: Arginase, rather than superoxide, impairs endothelium-dependent NOS-mediated dilation of retinal arterioles during diabetes, whereas vasodilation mediated by COX remains intact. Blockade of vascular arginase may improve endothelial function of retinal arterioles during early onset of diabetes.

Genetic regulation, biochemical properties and physiological importance of arginase from Sinorhizobium meliloti.

In bacteria, l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine to l-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiont Sinorhizobium meliloti 1021 has two genes annotated as arginases, argI1 (smc03091) and argI2 (sma1711). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021 argI1 null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in the argI1 mutant genetically complemented with a genomically integrated argI1 gene. In the wild-type, arginase activity and argI1 transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highest in vitro enzymatic activity at pH 7.5 with Ni2+ as cofactor. The enzyme was also active with Mn2+ and Co2+, both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator (smc03092) divergently transcribed from argI1 was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to the argI1 promoter in a region preceding the predicted argI1 transcriptional start. Our results indicate that ArgI1 is the sole arginase in S. meliloti, that it contributes substantially to arginine catabolism in vivo and that argI1 induction by arginine is dependent on ArgIR.

Renal tubular arginase-2 participates in the formation of the corticomedullary urea gradient and attenuates kidney damage in ischemia-reperfusion injury in mice.

AIM: Arginase 2 (ARG2) is a mitochondrial enzyme that catalyses hydrolysis of l-arginine into urea and l-ornithine. In the kidney, ARG2 is localized to the S3 segment of the proximal tubule. It has been shown that expression and activity of this enzyme are upregulated in a variety of renal pathologies, including ischemia-reperfusion (IR) injury. However, the (patho)physiological role of ARG2 in the renal tubule remains largely unknown. METHODS: We addressed this question in mice with conditional knockout of Arg2 in renal tubular cells (Arg2lox/lox /Pax8-rtTA/LC1 or, cKO mice). RESULTS: We demonstrate that cKO mice exhibit impaired urea concentration and osmolality gradients along the corticomedullary axis. In a model of unilateral ischemia-reperfusion injury (UIRI) with an intact contralateral kidney, ischemia followed by 24 hours of reperfusion resulted in significantly more pronounced histological damage in ischemic kidneys from cKO mice compared to control and sham-operated mice. In parallel, UIRI-subjected cKO mice exhibited a broad range of renal functional abnormalities, including albuminuria and aminoaciduria. Fourteen days after UIRI, the cKO mice exhibited complex phenotype characterized by significantly lower body weight, increased plasma levels of early predictive markers of kidney disease progression (asymmetric dimethylarginine and symmetric dimethylarginine), impaired mitochondrial function in the ischemic kidney but no difference in kidney fibrosis as compared to control mice. CONCLUSION: Collectively, these results establish the role of ARG2 in the formation of corticomedullary urea and osmolality gradients and suggest that this enzyme attenuates kidney damage in ischemia-reperfusion injury.

Endothelin-1 increases expression and activity of arginase 2 via ETB receptors and is co-expressed with arginase 2 in human atherosclerotic plaques.

BACKGROUND AND AIMS: Endothelin-1 (ET-1) and arginase are both suggested to be involved in the inflammatory processes and development of endothelial dysfunction in atherosclerosis. However, information regarding the roles of ET-1 and arginase, as well as the interactions between the two in human atherosclerosis, is scarce. We investigated the expression of ET-1 and its receptors, ETA and ETB, as well as arginase in human carotid atherosclerotic plaques and determined the functional interactions between ET-1 and arginase in endothelial cells and THP-1-derived macrophages. METHODS: Carotid plaques and blood samples were retreived from patients undergoing surgery for symptomatic or asymptomatic carotid stenosis. Plaque gene and protein expression was determined and related to clinical characteristics. Functional interactions between ET-1 and arginase were investigated in endothelial cells and THP-1 cells. RESULTS: Expression of ET-1 and ETB receptors was increased in plaques from patients with symptomatic carotid artery disease. ET-1 was co-localized with arginase 1 and arginase 2 in the necrotic core, together with macrophage markers CD163 and CD68. Arginase 2, ET-1 and ETB receptors were expressed in endothelial cells as well as in smooth muscle cells in the fibrous cap. ET-1 increased arginase 2 mRNA expression and arginase activity in endothelial cells and arginase activity in macrophages. Moreover, ET-1 stimulated formation of reactive oxygen species (ROS) in THP-1-derived macrophages via an arginase-dependent mechanism. CONCLUSIONS: This is the first study that demonstrates co-localization of ET-1 and arginase 2 in human atherosclerotic plaques. ET-1 stimulated arginase 2 expression and activity in endothelial cells, as well as arginase activity and ROS formation in macrophages via an arginase-dependent mechanism. These results indicate an important interaction between the ET pathway and arginase in human atherosclerotic plaques.

Myeloid Cell Crosstalk Regulates the Efficacy of the DNA/ALVAC/gp120 HIV Vaccine Candidate.

Vaccination with DNA-SIV + ALVAC-SIV + gp120 alum results in inflammasome activation, high levels of IL-1ß production, emergency myelopoiesis, and the egress of CXCR4+ CD14+ pre-monocytes from bone marrow. Previously we have shown that this vaccine-induced innate monocyte memory is associated with decreased risk of SIVmac251 acquisition. Because IL-1ß also promotes the propagation of monocyte-derived suppressor (M-MDSC)-like cells, here we extended our analysis to this negative regulator subset, characterizing its levels and functions in macaques. Interestingly, we found that DNA prime engages M-MDSC-like cells and their levels are positively associated with the frequency of CD14+ classical monocytes, and negatively with the levels of CD16+ monocytes, correlates of decreased and increased risk of SIV acquisition, respectively. Accordingly, M-MDSC frequency, arginase activity, and NO were all associated with decrease of CD8 T cells responses and worse vaccination outcome. DNA vaccination thus induces innate immunity by engaging three subsets of myeloid cells, M-MDSCs, CD14+ innate monocyte memory, and CD16+ monocytes all playing different role in protection. The full characterization of the immunological space created by myeloid cell crosstalk will likely provide clues to improve the efficacy of HIV vaccine candidates.

Critical role for arginase II in osteoarthritis pathogenesis.

OBJECTIVE: Osteoarthritis (OA) appears to be associated with various metabolic disorders, but the potential contribution of amino acid metabolism to OA pathogenesis has not been clearly elucidated. Here, we explored whether alterations in the amino acid metabolism of chondrocytes could regulate OA pathogenesis. METHODS: Expression profiles of amino acid metabolism-regulating genes in primary-culture passage 0 mouse chondrocytes were examined by microarray analysis, and selected genes were further characterised in mouse OA chondrocytes and OA cartilage of human and mouse models. Experimental OA in mice was induced by destabilisation of the medial meniscus (DMM) or intra-articular (IA) injection of adenoviruses expressing catabolic regulators. The functional consequences of arginase II (Arg-II) were examined in Arg2-/- mice and those subjected to IA injection of an adenovirus encoding Arg-II (Ad-Arg-II). RESULTS: The gene encoding Arg-II, an arginine-metabolising enzyme, was specifically upregulated in chondrocytes under various pathological conditions and in OA cartilage from human patients with OA and various mouse models. Adenovirus-mediated overexpression of Arg-II in mouse joint tissues caused OA pathogenesis, whereas genetic ablation of Arg2 in mice (Arg2-/-) abolished all manifestations of DMM-induced OA. Mechanistically, Arg-II appears to cause OA cartilage destruction at least partly by upregulating the expression of matrix-degrading enzymes (matrix metalloproteinase 3 [MMP3] and MMP13) in chondrocytes via the nuclear factor (NF)-κB pathway. CONCLUSIONS: Our results indicate that Arg-II is a crucial regulator of OA pathogenesis in mice. Although chondrocytes of human and mouse do not identically, but similarly, respond to Arg-II, our results suggest that Arg-II could be a therapeutic target of OA pathogenesis.

Hyperglycemia-impaired aortic vasorelaxation mediated through arginase elevation: Role of stress kinase pathways.

Diabetes-induced vascular endothelial dysfunction has been reported to involve hyperglycemia-induced increases in arginase activity. However, upstream mediators of this effect are not clear. Here, we have tested involvement of Rho kinase, ERK1/2 and p38 MAPK pathways in this process. Studies were performed with aortas isolated from wild type or hemizygous arginase 1 knockout (Arg1+/-) mice and bovine aortic endothelial cells exposed to high glucose (HG, 25 mmol/l) or normal glucose (NG, 5.5 mmol/l) conditions for different times. Effects of inhibitors of arginase, p38 MAPK, ERK1/2 or ROCK and ex vivo adenoviral delivery of active Arg1 and inactive (D128-Arg1) cDNA were also determined. Exposure in wild type aorta or endothelial cells to HG significantly increased arginase activity and Arg1 expression and impaired aortic relaxation. Transduction of wild type aorta with active Arg1 cDNA impaired vascular relaxation, whereas inactive Arg1 had no effect. The HG-induced vascular endothelial dysfunction was associated with increased phosphorylation (activation) of ERK1/2 and p38 MAPK. Pretreatment with inhibitors of ERK1/2, p38 MAPK, ROCK or arginase blocked HG-induced elevation of arginase activity and Arg1 expression and prevented the vascular dysfunction. Inhibition of ROCK blunted the HG-induced activation of ERK1/2 and p38 MAPK. In summary, activated ROCK and subsequent activation of ERK1/2 or p38 MAPK elevates arginase activity and Arg1 expression in hyperglycemic states. Targeting this pathway may provide an effective means for preventing diabetes/hyperglycemia-induced vascular endothelial dysfunction.

Arginase Inhibition Improves Endothelial Function in an Age-Dependent Manner in Healthy Elderly Humans.

Reduced bioavailability of nitric oxide (NO) is accompanied by endothelial dysfunction, which precedes clinical signs of atherosclerosis. The metalloenzyme arginase reciprocally inhibits the formation of NO and available data demonstrate that arginase contributes to reduced bioavailability of NO and increases the formation of reactive oxygen species. Emerging evidence suggests that arginase thereby plays a key role in the pathophysiology of age-associated vascular complications in animal models. However, the role of arginase in elderly human subjects in vivo with regard to macrovascular endothelial function is unclear. We hypothesized that arginase inhibition improves endothelial function in an age-dependent manner in elderly healthy subjects. Twenty-one subjects ranging from 48 to 75 years of age were included for evaluation of endothelial function using forearm venous occlusion plethysmography. Endothelium-dependent vasodilatation (EDV) and endothelium-independent vasodilatation (EIDV) were evaluated before and after 2-hour intra-arterial infusion of the arginase inhibitor Nω-hydroxy-nor-L-arginine (nor-NOHA). Baseline EDV, but not EIDV, was inversely correlated with age (p < 0.05, R = -0.48 and p = 0.62, R = -0.14, respectively). The magnitude of the improvement in EDV induced by arginase inhibition was significantly correlated with age (p < 0.05, R = 0.56), whereas EIDV was not (p = 0.25, R = 0.26). Arginase inhibition improves endothelial function in an age-dependent manner in elderly healthy subjects. The results suggest that arginase is a key factor contributing to endothelial dysfunction and may serve as a future pharmacological target for improving endothelial function in elderly subjects.

Mechanisms of cell death induced by arginase and asparaginase in precursor B-cell lymphoblasts.

Arginase has therapeutic potential as a cytotoxic agent in some cancers, but this is unclear for precursor B acute lymphoblastic leukaemia (pre-B ALL), the commonest form of childhood leukaemia. We compared arginase cytotoxicity with asparaginase, currently used in pre-B ALL treatment, and characterised the forms of cell death induced in a pre-B ALL cell line 697. Arginase and asparaginase both efficiently killed 697 cells and mature B lymphoma cell line Ramos, but neither enzyme killed normal lymphocytes. Arginase depleted cellular arginine, and arginase-treated media induced cell death, blocked by addition of arginine or arginine-precursor citrulline. Asparaginase depleted both asparagine and glutamine, and asparaginase-treated media induced cell death, blocked by asparagine, but not glutamine. Both enzymes induced caspase cleavage and activation, chromatin condensation and phosphatidylserine exposure, indicating apoptosis. Both arginase- and asparaginase-induced death were blocked by caspase inhibitors, but with different sensitivities. BCL-2 overexpression inhibited arginase- and asparaginase-induced cell death, but did not prevent arginase-induced cytostasis, indicating a different mechanism of growth arrest. An autophagy inhibitor, chloroquine, had no effect on the cell death induced by arginase, but doubled the cell death induced by asparaginase. In conclusion, arginase causes death of lymphoblasts by arginine-depletion induced apoptosis, via mechanism distinct from asparaginase. Therapeutic implications for childhood ALL include: arginase might be used as treatment (but antagonised by dietary arginine and citrulline), chloroquine may enhance efficacy of asparaginase treatment, and partial resistance to arginase and asparaginase may develop by BCL-2 expression. Arginase or asparaginase might potentially be used to treat Burkitt lymphoma.

Human hepatocyte transplantation corrects the inherited metabolic liver disorder arginase deficiency in mice.

The transplantation, engraftment, and expansion of primary hepatocytes have the potential to be an effective therapy for metabolic disorders of the liver including those of nitrogen metabolism. To date, such methods for the treatment of urea cycle disorders in murine models has only been minimally explored. Arginase deficiency, an inherited disorder of nitrogen metabolism that presents in the first two years of life, has the potential to be treated by such methods. To explore the potential of this approach, we mated the conditional arginase deficient mouse with a mouse model deficient in fumarylacetoacetate hydrolase (FAH) and with Rag2 and IL2-Rγ mutations to give a selective advantage to transplanted (normal) human hepatocytes. On day -1, a uroplasminogen-expressing adenoviral vector was administered intravenously followed the next day with the transplantation of 1 × 106 human hepatocytes (or vehicle alone) by intrasplenic injection. As the initial number of administered hepatocytes would be too low to prevent hepatotoxicity-induced mortality, NTBC cycling was performed to allow for hepatocyte expansion and repopulation. While all control mice died, all except one human hepatocyte transplanted mice survived. Four months after hepatocyte transplantation, 2 × 1011 genome copies of AAV-TBG-Cre recombinase was administered IV to disrupt endogenous hepatic arginase expression. While all control mice died within the first month, human hepatocyte transplanted mice did well. Ammonia and amino acids, analyzed in both groups before and after disruption of endogenous arginase expression, while well-controlled in the transplanted group, were markedly abnormal in the controls. Ammonium challenging further demonstrated the durability and functionality of the human repopulated liver. In conclusion, these studies demonstrate that human hepatocyte repopulation in the murine liver can result in effective treatment of arginase deficiency.

Arginase-1 Expression in Myeloid Cells Regulates Staphylococcus aureus Planktonic but Not Biofilm Infection.

Staphylococcus aureus is a leading cause of device-associated biofilm infections, which represent a serious health care concern based on their chronicity and antibiotic resistance. We previously reported that S. aureus biofilms preferentially recruit myeloid-derived suppressor cells (MDSCs), which promote monocyte and macrophage anti-inflammatory properties. This is associated with increased myeloid arginase-1 (Arg-1) expression, which has been linked to anti-inflammatory and profibrotic activities that are observed during S. aureus biofilm infections. To determine whether MDSCs and macrophages utilize Arg-1 to promote biofilm infection, Arg-1 was deleted in myeloid cells by use of Tie-2Cre mice. Despite Arg-1 expression in biofilm-associated myeloid cells, bacterial burdens and leukocyte infiltrates were similar between wild-type (WT) and Arg-1fl/fl;Tie-2Cre conditional knockout (KO) mice from days 3 to 14 postinfection in both orthopedic implant and catheter-associated biofilm models. However, inducible nitric oxide synthase (iNOS) expression was dramatically elevated in biofilm-associated MDSCs from Arg-1fl/fl;Tie-2Cre animals, suggesting a potential Arg-1-independent compensatory mechanism for MDSC-mediated immunomodulation. Treatment of Arg-1fl/fl;Tie-2Cre mice with the iNOS inhibitor N6-(1-iminoethyl)-l-lysine (l-NIL) had no effect on biofilm burdens or immune infiltrates, whereas treatment of WT mice with the Arg-1/ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) increased bacterial titers, but only in the surrounding soft tissues, which possess attributes of a planktonic environment. A role for myeloid-derived Arg-1 in regulating planktonic infection was confirmed using a subcutaneous abscess model, in which S. aureus burdens were significantly increased in Arg-1fl/fl;Tie-2Cre mice compared to those in WT mice. Collectively, these results indicate that the effects of myeloid Arg-1 are context dependent and are manifest during planktonic but not biofilm infection.

Galectin-1 Reduces Neuroinflammation via Modulation of Nitric Oxide-Arginase Signaling in HIV-1 Transfected Microglia: a Gold Nanoparticle-Galectin-1 "Nanoplex" a Possible Neurotherapeutic?

Galectins are a family of ß-galactoside-binding lectins that are important modulators of homeostasis in the central nervous system (CNS). Galectin-1 is a pivotal regulator of microglia activation that alters the immune balance from neurodegeneration to neuroprotection and could have therapeutic relevance in HIV associated neurocognitive disorders (HAND). We have previously shown that galectin-1 treatment decreased oxidative stress in microglia and hypothesize that the mechanism underlying this phenomenon is the cross regulatory interactions between Nitric oxide (NO) and Arginase I activity in microglia. We induced microglial activation and examined the effect of galectin-1 on the expression of various M1/M2 microglial phenotypic markers. Since, TNF-α is associated with activation of microglial cells involved in pathogenesis of neurodegenerative diseases, we treated HIV transfected human microglial cell cultures (CHME-5/HIV) with TNF-α followed by treatment with galectin-1, to examine the galectin-1 mediated neuro-modulatory response. Our results show that treatment of CHME-5/HIV microglia with galectin-1 reduced TNF-α induced oxidative stress by ~40%, and also significantly reduced iNOS gene expression and NO production while correspondingly increasing arginase-1, cationic amino acid transporter (CAT-1) gene expression and arginase activity. Galectin-1 treatment results in shifting microglia polarization from M1 toward the beneficial M2 phenotype which may prevent neurodegeneration and promote neuroprotection. Thus, our data suggests that galectin-1 treatment reduces neuroinflammation in the CNS microenvironment via the modulation of the NO-arginase network in microglia and thus could play a neuroprotective role in HAND. Further, the therapeutic potential of galectin-1 could be enhanced by conjugation of galectin-1 onto gold nanoparticles (Au-NP), resulting in a nanogold-galectin-1 (Au-Gal-1) multivalent complex that will have more clinical translational efficacy than free galectin-1 by virtue of increasing the payload influx.

Arginase inhibition attenuates arteriogenesis and interferes with M2 macrophage accumulation.

l-Arginine is the common substrate for nitric oxide synthases (NOS) and arginase. Whereas the contribution of NOS to collateral artery growth (arteriogenesis) has been demonstrated, the functional role of arginase remains to be elucidated and was topic of the present study. Arteriogenesis was induced in mice by ligation of the femoral artery. Laser Doppler perfusion measurements demonstrated a significant reduction in arteriogenesis in mice treated with the arginase inhibitor nor-NOHA (N(ω)-hydroxy-nor-arginine). Accompanying in vitro results on murine primary arterial endothelial cells and smooth muscle cells revealed that nor-NOHA treatment interfered with cell proliferation and resulted in increased nitrate/nitrite levels, indicative for increased NO production. Immuno-histological analyses on tissue samples demonstrated that nor-NOHA administration caused a significant reduction in M2 macrophage accumulation around growing collateral arteries. Gene expression studies on isolated growing collaterals evidenced that nor-NOHA treatment abolished the differential expression of Icam1 (intercellular adhesion molecule 1). From our data we conclude that arginase activity is essential for arteriogenesis by promoting perivascular M2 macrophage accumulation as well as arterial cell proliferation.

Topographical modulation of macrophage phenotype by shrink-film multi-scale wrinkles.

The host immune response to foreign materials is a major hurdle for implanted medical devices. To control this response, modulation of macrophage behavior has emerged as a promising strategy, given their prominent role in inflammation and wound healing. Towards this goal, we explore the effect of biomimetic multi-scale wrinkles on macrophage adhesion and expression of phenotype markers. We find that macrophages elongate along the direction of the uniaxial wrinkles made from shape memory polymers, and express more arginase-1 and IL-10, and less TNF-α, suggesting polarization towards an alternatively activated, anti-inflammatory phenotype. Materials were further implanted in the subcutaneous space of mice and tissue surrounding the material evaluated by histology and immunohistochemistry. We found that material surface topography altered the distribution of collagen deposition in the adjacent tissue, with denser collagen tissue observed near flat materials when compared to wrinkled materials. Furthermore, cells surrounding wrinkled materials exhibited higher arginase-1 expression. Together these data suggest that wrinkled material surfaces promote macrophage alternative activation, and may influence the foreign body response to implants.

Myeloid-derived suppressor cells reveal radioprotective properties through arginase-induced l-arginine depletion.

BACKGROUND AND PURPOSE: High arginase-1 (Arg) expression by myeloid-derived suppressor cells (MDSC) is known to inhibit antitumor T-cell responses through depletion of l-arginine. We have previously shown that nitric oxide (NO), an immune mediator produced from l-arginine, is a potent radiosensitizer of hypoxic tumor cells. This study therefore examines whether Arg(+) overexpressing MDSC may confer radioresistance through depleting the substrate for NO synthesis. MATERIAL AND METHODS: MDSC and Arg expression were studied in preclinical mouse CT26 and 4T1 tumor models and further validated in rectal cancer patients in comparison with healthy donors. The radioprotective effect of MDSC was analyzed in hypoxic tumor cells with regard to l-arginine depletion. RESULTS: In both mouse tumors and cancer patients, MDSC expansion was associated with Arg activation causing accelerated l-arginine consumption. l-Arginine depletion in turn profoundly suppressed the capacity of classically activated macrophages to synthesize NO resulting in impaired tumor cell radiosensitivity. In advanced cT3-4 rectal cancer, circulating neutrophils revealed Arg overexpression approaching that in MDSC, therefore mounting a protumor compartment wherein Arg(+) neutrophils increased from 17% to over 90%. CONCLUSIONS: Protumor Arg(+) MDSC reveal a unique ability to radioprotect tumor cells through l-arginine depletion, a common mechanism behind both T-cell and macrophage inhibition.

CD38-Expressing Myeloid-Derived Suppressor Cells Promote Tumor Growth in a Murine Model of Esophageal Cancer.

Myeloid-derived suppressor cells (MDSC) are an immunosuppressive population of immature myeloid cells found in advanced-stage cancer patients and mouse tumor models. Production of inducible nitric oxide synthase (iNOS) and arginase, as well as other suppressive mechanisms, allows MDSCs to suppress T-cell-mediated tumor clearance and foster tumor progression. Using an unbiased global gene expression approach in conditional p120-catenin knockout mice (L2-cre;p120ctn(f/f)), a model of oral-esophageal cancer, we have identified CD38 as playing a vital role in MDSC biology, previously unknown. CD38 belongs to the ADP-ribosyl cyclase family and possesses both ectoenzyme and receptor functions. It has been described to function in lymphoid and early myeloid cell differentiation, cell activation, and neutrophil chemotaxis. We find that CD38 expression in MDSCs is evident in other mouse tumor models of esophageal carcinogenesis, and CD38(high) MDSCs are more immature than MDSCs lacking CD38 expression, suggesting a potential role for CD38 in the maturation halt found in MDSC populations. CD38(high) MDSCs also possess a greater capacity to suppress activated T cells, and promote tumor growth to a greater degree than CD38(low) MDSCs, likely as a result of increased iNOS production. In addition, we have identified novel tumor-derived factors, specifically IL6, IGFBP3, and CXCL16, which induce CD38 expression by MDSCs ex vivo. Finally, we have detected an expansion of CD38(+) MDSCs in peripheral blood of advanced-stage cancer patients and validated targeting CD38 in vivo as a novel approach to cancer therapy.