Macrophage Nitric Oxide Synthase Research Articles

Immunomodulatory Properties of Masticadienonic Acid and 3α-Hydroxy Masticadienoic Acid in Dendritic Cells.

Dendritic cells are antigen-presenting cells, which identify and process pathogens to subsequently activate specific T lymphocytes. To regulate the immune responses, DCs have to mature by the recognition of TLR ligands, TNFα or IFNγ. These ligands have been used as adjuvants to activate DCs in situ or in vitro, with toxic effects. It has been shown that some molecules affect the immune system, e.g., Masticadienonic acid (MDA) and 3α-hydroxy masticadienoic acid (3α-OH MDA) triterpenes naturally occurring in several medicinal plants, since they activate the nitric oxide synthase in macrophages and induce T lymphocyte proliferation. The DCs maturation induced by MDA or 3a-OH MDA was determined by incubating these cells with MDA or 3α-OH MDA, and their phenotype was afterwards analyzed. The results showed that only 3α-OH MDA was able to induce DCs maturation. When mice with melanoma were inoculated with DCs/3α-OH MDA, a decreased tumor growth rate was observed along with an extended cell death area within tumors compared to mice treated with DCs incubated with MDA. In conclusion, it is proposed that 3α-OH MDA may be an immunostimulant molecule. Conversely, it is proposed that MDA may be a molecule with anti-inflammatory properties.

Boswellia serrata Extract Containing 30% 3-Acetyl-11-Keto-Boswellic Acid Attenuates Inflammatory Mediators and Preserves Extracellular Matrix in Collagen-Induced Arthritis.

Boswellia serrata extracts have been traditionally employed for the treatment of inflammatory diseases. In the present study, we have evaluated the mechanism of activity of Boswellin Super® FJ (BSE), a standardized extract of B. serrata containing not less than 30% 3-acetyl-11-keto-β-boswellic acid along with other β-boswellic acids. The in vitro anti-inflammatory activities were carried out in RAW 264.7 macrophages or human peripheral blood mononuclear cells stimulated with bacterial lipopolysaccharides (LPS) and treated with 1.25-5μg/ml BSE. The anti-arthritic activity of the extract was evaluated in a rat model of collagen-induced arthritis. BSE at 40 and 80mg/kg and celecoxib 10mg/kg were orally dosed for 21days. BSE showed significant (p<0.05) inhibition of inflammation (TNF-α, IL-6, nitric oxide, and COX-2 secretion) and downregulates the mRNA levels of TNF-α, IL-6, IL1-β, and inducible nitric oxide synthase in macrophages. BSE treatment reduced the levels of phosphorylated-NF-κB (P65), suggesting an anti-inflammatory activity mediated by blocking this key signal transduction pathway. In addition, BSE showed inhibition (p<0.05) of collagenase, elastase, hyaluronidase enzymes, and a reduction in reactive oxygen species and matrix-degrading proteins in RAW 264.7 macrophages stimulated with LPS. BSE treatment significantly (p<0.05) reduced the arthritic index, paw volume, and joint inflammation comparable to celecoxib in collagen-induced arthritis (CIA) in rats. The circulating anti-collagen antibodies were reduced in BSE and celecoxib-treated animals as compared to the CIA. In confirmation with in vitro data, BSE showed a significant (p<0.05) dose-dependent effect on C-reactive protein, prostaglandin E2, and erythrocyte sedimentation rate, which is widely used as a blood marker of inflammation. Further, BSE treatment suppressed the cartilage oligomeric matrix protein and significantly enhanced the hyaluronan levels in synovial fluid. As observed by collagen staining in joints, the loss of matrix proteins was lower in BSE-treated animals, suggesting that BSE could preserve the extracellular matrix in RA. The extract showed inhibition of collagenase enzyme activity in vitro, further strengthening this hypothesis. BSE treatment was found to be safe, and rats displayed no abnormal behavior or activities. The results suggest that Boswellin Super® mediates its activity by preserving matrix proteins, reducing pro-inflammatory mediators, and oxidative stress.

Severity of Experimental Autoimmune Uveitis Is Reduced by Pretreatment with Live Probiotic Escherichia coli Nissle 1917.

Non-infectious uveitis is considered an autoimmune disease responsible for a significant burden of blindness in developed countries and recent studies have linked its pathogenesis to dysregulation of the gut microbiota. We tested the immunomodulatory properties of two probiotics, Escherichia coli Nissle 1917 (EcN) and E. coli O83:K24:H31 (EcO), in a model of experimental autoimmune uveitis (EAU). To determine the importance of bacterial viability and treatment timing, mice were orally treated with live or autoclaved bacteria in both preventive and therapeutic schedules. Disease severity was assessed by ophthalmoscopy and histology, immune phenotypes in mesenteric and cervical lymph nodes were analyzed by flow cytometry and the gut immune environment was analyzed by RT-PCR and/or gut tissue culture. EcN, but not EcO, protected against EAU but only as a live organism and only when administered before or at the time of disease induction. Successful prevention of EAU was accompanied by a decrease in IRBP-specific T cell response in the lymph nodes draining the site of immunization as early as 7 days after the immunization and eye-draining cervical lymph nodes when the eye inflammation became apparent. Furthermore, EcN promoted an anti-inflammatory response in Peyer’s patches, increased gut antimicrobial peptide expression and decreased production of inducible nitric oxide synthase in macrophages. In summary, we show here that EcN controls inflammation in EAU and suggest that probiotics may have a role in regulating the gut–eye axis.

Regulatory Role of Heme Oxygenase and Nitric Oxide Synthase in Macrophages

Regulatory Role of Heme Oxygenase and Nitric Oxide Synthase in Macrophages

Glyceraldehyde-3-phosphate dehydrogenase is a chaperone that allocates labile heme in cells

Cellular heme is thought to be distributed between a pool of sequestered heme that is tightly bound within hemeproteins and a labile heme pool required for signaling and transfer into proteins. A heme chaperone that can hold and allocate labile heme within cells has long been proposed but never been identified. Here, we show that the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills this role by acting as an essential repository and allocator of bioavailable heme to downstream protein targets. We identified a conserved histidine in GAPDH that is needed for its robust heme binding both in vitro and in mammalian cells. Substitution of this histidine, and the consequent decreases in GAPDH heme binding, antagonized heme delivery to both cytosolic and nuclear hemeprotein targets, including inducible nitric-oxide synthase (iNOS) in murine macrophages and the nuclear transcription factor Hap1 in yeast, even though this GAPDH variant caused cellular levels of labile heme to rise dramatically. We conclude that by virtue of its heme-binding property, GAPDH binds and chaperones labile heme to create a heme pool that is bioavailable to downstream proteins. Our finding solves a fundamental question in cell biology and provides a new foundation for exploring heme homeostasis in health and disease.

Arginase expression modulates nitric oxide production in Leishmania (Leishmania) amazonensis.

BackgroundArginase is an enzyme that converts L-arginine to urea and L-ornithine, an essential substrate for the polyamine pathway supporting Leishmania (Leishmania) amazonensis replication and its survival in the mammalian host. L-arginine is also the substrate of macrophage nitric oxide synthase 2 (NOS2) to produce nitric oxide (NO) that kills the parasite. This competition can define the fate of Leishmania infection.Methodology/Principal findingsThe transcriptomic profiling identified a family of oxidoreductases in L. (L.) amazonensis wild-type (La-WT) and L. (L.) amazonensis arginase knockout (La-arg-) promastigotes and axenic amastigotes. We highlighted the identification of an oxidoreductase that could act as nitric oxide synthase-like (NOS-like), due to the following evidences: conserved domain composition, the participation of NO production during the time course of promastigotes growth and during the axenic amastigotes differentiation, regulation dependence on arginase activity, as well as reduction of NO amount through the NOS activity inhibition. NO quantification was measured by DAF-FM labeling analysis in a flow cytometry.Conclusions/SignificanceWe described an arginase-dependent NOS-like activity in L. (L.) amazonensis and its role in the parasite growth. The increased detection of NO production in the mid-stationary and late-stationary growth phases of La-WT promastigotes could suggest that this production is an important factor to metacyclogenesis triggering. On the other hand, La-arg- showed an earlier increase in NO production compared to La-WT, suggesting that NO production can be arginase-dependent. Interestingly, La-WT and La-arg- axenic amastigotes produced higher levels of NO than those observed in promastigotes. As a conclusion, our work suggested that NOS-like is expressed in Leishmania in the stationary growth phase promastigotes and amastigotes, and could be correlated to metacyclogenesis and amastigotes growth in a dependent way to the internal pool of L-arginine and arginase activity.

Neurorestorative Therapy of Stroke in Type 2 Diabetes Mellitus Rats Treated With Human Umbilical Cord Blood Cells

Diabetes mellitus is a high-risk factor for ischemic stroke. Diabetic stroke patients suffer worse outcomes, poor long-term recovery, risk of recurrent strokes, and extensive vascular damage. We investigated the neurorestorative effects and the underlying mechanisms of stroke treatment with human umbilical cord blood cells (HUCBCs) in type 2 diabetes mellitus (T2DM) rats. Adult male T2DM rats were subjected to 2 hours of middle cerebral artery occlusion (MCAo). Three days after MCAo, rats were treated via tail-vein injection with (1) PBS and (2) HUCBCs (5×10(6)), n=10 per group. HUCBC stroke treatment initiated 3 days after MCAo in T2DM rats did not significantly decrease blood-brain barrier leakage (P=0.1) and lesion volume (P=0.078), but significantly improved long-term functional outcome and decreased brain hemorrhage (P<0.05) when compared with the PBS-treated T2DM MCAo control group. HUCBC treatment significantly promoted white matter remodeling as indicated by increased expression of Bielschowsky silver (axons marker), Luxol fast blue (myelin marker), SMI-31 (neurofilament), and Synaptophysin in the ischemic border zone. HUCBC promoted vascular remodeling and significantly increased arterial and vascular density. HUCBC treatment of stroke in T2DM rats significantly increased M2 macrophage polarization (increased M2 macrophage, CD163and CD 206; decreased M1 macrophage, ED1 and inducible nitric oxide synthase expression) in the ischemic brain compared with PBS-treated T2DM MCAo controls (P<0.05). HUCBC also significantly decreased proinflammatory factors, that is, matrix metalloproteinase 9, receptor for advanced glycation end products and toll-like receptor 4 expression in the ischemic brain. HUCBC treatment initiated 3 days after stroke significantly increased white matter and vascular remodeling in the ischemic brain as well as decreased neuroinflammatory factor expression in the ischemic brain in T2DM rats and promoted M2 macrophage polarization. HUCBC reduction of neuroinflammation and increased vascular and white matter axonal remodeling may contribute to the HUCBC-induced beneficial effects in T2DM stroke rats.

Facile and Quantitative Synthesis of a Poly(ethylene glycol)-b-Poly(l-arginine) Block Copolymer and Its Use for the Preparation of Polyion Complex Micelles with Polyanions for Biomedical Applications.

Though l-arginine-containing polymers show versatile biological functions, a precisely controlled synthesis of poly(ethylene glycol)-b-poly(l-arginine) (PEG-b-PArg) block copolymers has not been reported. Here, an effective method for the synthesis of PEG-b-PArg block copolymers is developed. In order to obtain PEG-b-PArg, a two-step reaction, i.e., synthesis of PEG-b-poly(l-ornithine) is employed, followed by guanidinylation with N,N'-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine. This procedure quantitatively converts amino groups to guanidium groups at the side chains of peptide segments under mild conditions. Polyion complex (PIC) micelles are prepared by mixing the positively charged PEG-b-PArg with negatively charged homo-polyelectrolytes such as hyaluronic acid (HA) or chondroitin sulfate C (CS). PIC micelles prepared with CS show a higher stability than those prepared with HA, probably due to strong interactions between guanidium cations in PEG-b-PArg and carboxylate/sulfate in CS. Thus, PIC micelles containing PArg are a potentially effective arginine carrier for the development of in vivo therapeutic applications for various diseases related to nitric oxide, which is generated from inducible nitric oxide synthase in macrophages using l-arginine as a substrate.

Plasma gelsolin improves lung host defense against pneumonia by enhancing macrophage NOS3 function.

Plasma gelsolin (pGSN) functions as part of the "extracellular actin-scavenging system," but its potential to improve host defense against infection has not been studied. In a mouse model of primary pneumococcal pneumonia, recombinant human pGSN (rhu-pGSN) caused enhanced bacterial clearance, reduced acute inflammation, and improved survival. In vitro, rhu-pGSN rapidly improved lung macrophage uptake and killing of bacteria (Streptococcus pneumoniae, Escherichia coli, and Francisella tularensis). pGSN triggers activating phosphorylation (Ser(1177)) of macrophage nitric oxide synthase type III (NOS3), an enzyme with important bactericidal functions in lung macrophages. rhu-pGSN failed to enhance bacterial killing by NOS3(-/-) macrophages in vitro or bacterial clearance in NOS3(-/-) mice in vivo. Prophylaxis with immunomodulators may be especially relevant for patients at risk for secondary bacterial pneumonia, e.g., after influenza. Treatment of mice with pGSN challenged with pneumococci on postinfluenza day 7 (the peak of enhanced susceptibility to secondary infection) caused a ∼15-fold improvement in bacterial clearance, reduced acute neutrophilic inflammation, and markedly improved survival, even without antibiotic therapy. pGSN is a potential immunomodulator for improving lung host defense against primary and secondary bacterial pneumonia.

γ-Irradiated cancer cells promote tumor growth by activation of Toll-like receptor 1-mediated inducible nitric oxide synthase in macrophages

RT is commonly used to treat malignant tumors. However, tumor regrowth is a major limitation to RT as an antitumor treatment. In the present study, we investigated the tumor-promoting effects of high-dose (or ablative) RT treatments on tumor-bearing mice. We focused on the role of macrophages that interact with IR-CCs in the TME, which cause tumor regrowth. We observed that CT26(H-2(d)) tumor growth was enhanced by i.v. injection of IR-CT26 cells compared with NR control CT26 cells. The levels of iNOS gene expression and NO production from RAW264.7 macrophages (H-2(d)) in response to the interaction with IR-CT26 cells were higher than with NR-CT26 cells. When CT26 tumor-bearing mice were treated i.v. with L-NMMA, a NOS inhibitor, the reduction in in vivo tumor growth was higher in the IR-CT26-injected group compared with the NR-CT26-injected control group. In vivo CT26 tumor growth was decreased after transplanting PEM extracted from L-NMMA-treated, tumor-bearing mice. Although iNOS activity was reduced by inhibiting TLR1 expression with TLR1-siRNA, it was enhanced by TLR1 overexpression. Transcriptional activation and protein expression levels of iNOS were also decreased in the presence of TLR1-siRNA but increased as a result of TLR1 overexpression. These results demonstrate that postradiotherapeutic tumor regrowth may be caused by interaction of IR-CCs with macrophages that induce TLR1-mediated iNOS expression and NO production. Our data suggest that iNOS in macrophages could be a useful target to regulate postradiotherapeutic responses in hosts and subsequently limit tumor regrowth.

Extracellular Signal-regulated Kinase Mediates Expression of Arginase II but Not Inducible Nitric-oxide Synthase in Lipopolysaccharide-stimulated Macrophages

The mitogen-activated protein kinases (MAPK) have been shown to participate in iNOS induction following lipopolysaccharide (LPS) stimulation, while the role of MAPKs in the regulation of arginase remains unclear. We hypothesized that different MAPK family members are involved in iNOS and arginase expression following LPS stimulation. LPS-stimulated RAW 264.7 cells exhibited increased protein and mRNA levels for iNOS, arginase I, and arginase II; although the induction of arginase II was more robust than that for arginase I. A p38 inhibitor completely prevented iNOS expression while it only attenuated arginase II induction. In contrast, a MEK1/2 inhibitor (ERK pathway) completely abolished arginase II expression while actually enhancing iNOS induction in LPS-stimulated cells. Arginase II promoter activity was increased by ∼4-fold following LPS-stimulation, which was prevented by the ERK pathway inhibitor. Arginase II promoter activity was unaffected by a p38 inhibitor or JNK pathway interference. Transfection with a construct expressing a constitutively active RAS mutant increased LPS-induced arginase II promoter activity, while transfection with a vector expressing a dominant negative ERK2 mutant or a vector expressing MKP-3 inhibited the arginase II promoter activity. LPS-stimulated nitric oxide (NO) production was increased following siRNA-mediated knockdown of arginase II and decreased when arginase II was overexpressed. Our results demonstrate that while both the ERK and p38 pathways regulate arginase II induction in LPS-stimulated macrophages, iNOS induction by LPS is dependent on p38 activation. These results suggest that differential inhibition of the MAPK pathway may be a potential therapeutic strategy to regulate macrophage phenotype.

Expression of inducible nitric oxide synthase in macrophages inversely correlates with parasitism of lymphoid tissues in dogs with visceral leishmaniasis.

BackgroundThere are only a few studies reporting the role of nitric oxide metabolites for controlling macrophage intracellular parasitism, and these are controversial. Therefore, the present study aimed to evaluate the expression of inducible nitric oxide synthase (iNOS) in the lymph nodes and spleen of dogs affected by visceral leishmaniasis through immunohistochemistry and to determine its correlation with tissue parasite burden and serum interferon (IFN)-γ levels. Twenty-eight dogs were selected and assigned to one of two groups, symptomatic (n = 18) and asymptomatic (n = 10), according to clinical status and laboratory evaluation. A negative control group (n = 6) from a non-endemic region for visceral leishmaniasis was included as well.ResultsParasite density (amastigotes/mm2) was similar between clinical groups in the lymph nodes (P = 0.2401) and spleen (P = 0.8869). The density of iNOS+ cells was higher in infected dogs compared to controls (P < 0.05), without a significant difference in lymph node (P = 0.3257) and spleen (P = 0.5940) densities between symptomatic and asymptomatic dogs. A positive correlation was found between the number of iNOS+ cells in lymph nodes and interferon-γ levels (r = 0.3776; P = 0.0303), and there was a negative correlation between parasites and iNOS+ cell densities both in lymph nodes (r = −0.5341; P = 0.0034) and spleen (r = −0.4669; P = 0.0329).ConclusionThe negative correlation observed between tissue parasitism and the expression of iNOS may be a reflection of NO acting on the control of parasites.

Host Immune Response to Intestinal Amebiasis

Entamoeba histolytica is an invasive enteric protozoan parasite that causes amebiasis. Globally, diarrheal disease is second only to pneumonia as a leading cause of death in children under five, and intestinal amebiasis is one of the top ten causes of severe diarrhea in the developing world. Amebiasis is more common in malnourished children, a state that afflicts approximately one-third of children in the developing world. In the critical first year of life, 11% of Bangladeshi infants living in poverty suffer from E. histolytica diarrhea [1], [2]. There is currently no vaccine for this devastating disease, thus an understanding of the human immune response toward the parasite would greatly enhance the ability to develop effective immunotherapies. The host deploys a series of immune defenses against the parasite as it invades the colon. The ameba, however, has developed complex strategies to evade host defenses and promote its own survival. Here, we summarize the dynamics of the interaction of parasite with host and its importance in the pathogenesis of amebiasis (Figure 1). Figure 1 A. Host Immune Response to Intestinal Amebiasis. Innate Immunity Stomach acid serves as an important first line of defense against enteropathogens through its ability to kill acid-sensitive microorganisms. However, infectious amebic cysts are highly resistant and survive passage through the acidic environment of the stomach. In the intestine, the next layer of innate defense may be the mucus layer, which is thought to act as a protective barrier, preventing E. histolytica from invading intestinal epithelial cells (IECs). Mucin, a major constituent of the intestinal mucus layer, is a glycoprotein secreted by goblet cells and submucosal glands. Mucin glycoproteins bind to and inhibit the Gal/GalNAc adherence lectin of the parasite, preventing in vitro adherence and killing of CHO cells [3]. Trophozoites, however, can disrupt the mucus layer and intestinal barrier by secreting cysteine proteases (CPs) and glycosidases to allow for penetration of the colonic mucosa. Specifically, E. histolytica cysteine protease-A5 (EhCP-A5) degrades mucin-2 (MUC2) and extracellular matrix (ECM) proteins [4]. The importance of cysteine proteases was demonstrated by an ex vivo human intestinal model, where EhCP-A5–silenced parasites failed to penetrate into the colonic lamina propria [5]. IECs exposed to E. histolytica trophozoites secrete potent chemokines, such as IL-8, resulting in immune cell recruitment and infiltration of the lamina propria and intestinal epithelium [6]. Neutrophils are one of the first immune cells to respond to amebic invasion. Neutrophils activated by interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), or lipopolysaccharides (LPS) carry out amebicidal activity in vitro by releasing reactive oxygen species (ROS) [7], [8]. Depletion of neutrophils with anti-Gr-1 antibodies resulted in exacerbated intestinal disease in murine models, supporting the protective role of neutrophils in amebiasis [9]. It should be noted, however, that anti-Gr-1 antibodies can deplete other granulocytes such as eosinophils. Macrophages also play a crucial role in the host response against intestinal amebiasis. Macrophages are amebicidal after stimulation with IFN-γ or TNF-α [10], [11]. Several amebic antigens are known to activate these cells via pattern recognition receptors. Toll-like receptor (TLR)-2 expression in macrophages is upregulated when exposed to the Gal/GalNAc lectin of E. histolytica, triggering pro-inflammatory cytokine production via NF-κB activation [12]. Macrophages that lack TLR-2 and TLR-4 displayed impaired response to E. histolytica lipopeptidophosphoglycan (LPPG), suggesting that pattern recognition is essential to the immune response [13]. Additionally, E. histolytica DNA can activate macrophages through interacting with TLR-9 [14]. Amebicidal activity of macrophages is contributed to by the production of nitric oxide (NO) from L-arginine, which is mediated by macrophage nitric oxide synthase. Inducible nitric oxide synthase (iNOS)–deficient mice were more susceptible to amebic liver abscess and to E. histolytica–induced hepatocytic apoptosis, implicating a critical role for NO in the host defense against amebiasis [15].

Anti-inflammatory effect of Pueraria tuberosa extracts through improvement in activity of red blood cell anti-oxidant enzymes

Changing life style and over-nutrition causes low-grade inflammation (LGI), with obesity and hyper-lipidemia as basic factors. The physiological state polarizes macrophages to classical type (M1), which is pro-inflammatory and promotes ectopic fat deposition in the body. Both factors induce inflammatory cascade, where free radicals (FRs) play an important role. Thus, pharmacological and non-pharmacological interventions would be effective in the management of LGI and plant products would be used as food supplement or as a drug. Previously, a study has reported the anti-oxidant potential of methanolic extract of tubers of Pueraria tuberosa (PTME) and inhibitory role of tuberosin on lipopolysaccharides-induced expression of inducible nitric oxide synthase in macrophages in an in vitro study model. Here, the effect of PTME has been explored on carrageenan-induced inflammatory changes in rats. The activity of antioxidant enzymes in red blood cell hemolysate has been assessed. PTME was orally given to rats for 9 days and periodical changes (every 3rd day) in the activity/concentration of superoxide dismutase (SOD), catalase, reduced glutathione (GSH), lipid peroxides (LPO), and C-reactive proteins (CRP) were monitored. The PTME significantly prevented carrageenan-induced decline in GSH content, lowering of catalase and SOD activity, and rise in LPO and CRP in rats in a time-dependent, sequential manner. Thus, it could be suggested that the anti-inflammatory role of PTME is primarily mediated through its FR scavenging potential.

Acute traumatic spinal cord injury induces glial activation in the cynomolgus macaque (Macaca fascicularis)

Traumatic spinal cord injury leads to direct myelin and axonal damage and leads to the recruitment of inflammatory cells to site of injury. Although rodent models have provided the greatest insight into the genesis of traumatic spinal cord injury (TSCI), recent studies have attempted to develop an appropriate non-human primate model. We explored TSCI in a cynomolgus macaque model using a balloon catheter to mimic external trauma to further evaluate the underlying mechanisms of acute TSCI. Following 1hour of spinal cord trauma, there were focal areas of hemorrhage and necrosis at the site of trauma. Additionally, there was a marked increased expression of macrophage-related protein 8, MMP9, IBA-1, and inducible nitric oxide synthase in macrophages and microglia at the site of injury. This data indicate that acute TSCI in the cynomolgus macaque is an appropriate model and that the earliest immunohistochemical changes noted are within macrophage and microglia populations.

Plasma gelsolin enhances lung macrophage host defense against bacterial pneumonia in mice

Plasma gelsolin (pGSN) functions as part of the ‘extracellular actin scavenging system’ but its potential to improve host defense against infection has not been studied. Mice (C57BL/6, male) were pre‐treated with recombinant human plasma gelsolin (rhu‐pGSN, 400 mg/kg, s.c.) or vehicle 2 h before infection with Strep. pneumoniae (serotype 3, 150K CFU, i.n.) and evaluated 24 h later. rhu‐pGSN caused enhanced bacterial clearance (% bacterial left alive in lungs, mean±SD: control, 88 ± 38 vs. rhu‐pGSN‐treated, 10 ± 5; n ≥ 13 mice/grp) and reduced acute inflammation (% BAL PMNs: control, 30 ± 11 vs. rhu‐pGSN‐treated, 8 ± 3). Similar results were seen with aerosolized rhu‐pGSN. In vitro, rhu‐pGSN rapidly improved lung macrophage uptake and killing of bacteria. rhu‐pGSN caused an activating phosphorylation (ser1177) of macrophage nitric oxide synthase type III (NOS3), which has important bactericidal functions in the AM. rhu‐pGSN failed to enhance bacterial killing in NOS3−/− macrophages. pGSN binds sphingosine‐1‐phosphate (S1P), and may enhance its delivery to the S1P1 receptor. Both S1P and the S1P mimic, FTY720P, enhanced macrophage uptake and killing of bacteria, and activated macrophage NOS3 as well. Plasma gelsolin enhances innate resistance to pneumococcal pneumonia via lung macrophage NOS3, and is a potential immunomodulator for improving lung host defense against bacterial pneumonia. Support: NIH ES00002 ES11008

Nitric Oxide Synthases of Bacteria - and Other Unicellular Organisms

The defining feature of a nitric oxide synthase (NOS) is a heme and pterin-binding oxygenase domain, and enzymes that possess this domain are found in animals and bacteria. While the roles of animal NOS in such processes as vasodilation, neurotransmission, and host defence are understandably important for multicellular life, they are meaningless in bacteria, which makes bacterial NOSs all the more interesting as objects of study. This review describes the structural similarities and differences between animal and bacterial NOS, and how such differences may influence the extent and duration of bacterial NO production, and, ultimately the roles of NO in microorganisms. Recent progress in defining the functions of bacterially-derived NO, notably in protection from various stresses and as a potential transcriptional regulator are described. Finally, an attempt is made to span the gap between animal and bacterial nitric oxide synthases by commenting on the search for examples of NOS in single-celled eukaryotes, where progress seems imminent. The story of nitric oxide synthase (NOS) is a wonderful example of how an enzyme's function can evolve over time, even as its structure and mechanism remain largely intact. Constitutive NOS isotypes of mammals are used for the transient production of nanomolar amounts of NO for neurotransmission and vasodilation, while at the higher concentrations produced by inducible NOS in macrophages over sustained periods, NO is cytotoxic and is used to kill pathogens. Yet the origin of NOS is ancient; since the turn of the century the number of bacteria known to carry a NOS gene has increased sharply, and the work of defining the roles of bacterial NOS is an active and exciting area of research. But the story of NOS is also the story of an enzyme that has been characterized mainly in these two distinct kingdoms, the metazoans (overwhelmingly of mammals) and the bacteria, with few studies from other branches of life. If the bacteria originated it and we still have it, why is it seemingly so rare elsewhere? If it can be found in other kingdoms, for example among the protists, what might its functions be? This brief review will begin with the features common to NOS and then, gradually, move towards their differences. This includes the biochemical targets of NO, the reaction catalyzed by NOS, common structural features and differences in the enzyme, and the different roles that these enzymes have, with the focus on bacterial NOSs. Having considered these aspects, the prospects of the presence of NOS in single-celled eukaryotes and their possible functions will be discussed, which, because of the paucity of

Aedes aegypti Saliva Alters Leukocyte Recruitment and Cytokine Signaling by Antigen-Presenting Cells during West Nile Virus Infection

West Nile virus (WNV) is transmitted during mosquito bloodfeeding. Consequently, the first vertebrate cells to contact WNV are cells in the skin, followed by those in the draining lymph node. Macrophages and dendritic cells are critical early responders in host defense against WNV infection, not just because of their role in orchestrating the immune response, but also because of their importance as sites of early peripheral viral replication. Antigen-presenting cell (APC) signals have a profound effect on host antiviral responses and disease severity. During transmission, WNV is intimately associated with mosquito saliva. Due to the ability of mosquito saliva to affect inflammation and immune responses, and the importance of understanding early events in WNV infection, we investigated whether mosquito saliva alters APC signaling during arbovirus infection, and if alterations in cell recruitment occur when WNV infection is initiated with mosquito saliva. Accordingly, experiments were performed with cultured dendritic cells and macrophages, flow cytometry was used to characterize infiltrating cell types in the skin and lymph nodes during early infection, and real-time RT-PCR was employed to evaluate virus and cytokine levels. Our in vitro results suggest that mosquito saliva significantly decreases the expression of interferon-β and inducible nitric oxide synthase in macrophages (by as much as 50 and 70%, respectively), whilst transiently enhancing interleukin-10 (IL-10) expression. In vivo results indicate that the predominate effect of mosquito feeding is to significantly reduce the recruitment of T cells, leading the inoculation site of mice exposed to WNV alone to have up to 2.8 fold more t cells as mice infected in the presence of mosquito saliva. These shifts in cell population are associated with significantly elevated IL-10 and WNV (up to 4.0 and 10 fold, respectively) in the skin and draining lymph nodes. These results suggest that mosquito saliva dysregulates APC antiviral signaling, and reveal a possible mechanism for the observed enhancement of WNV disease mediated by mosquito saliva via a reduction of T lymphocyte and antiviral activity at the inoculation site, an elevated abundance of susceptible cell types, and a concomitant increase in immunoregulatory activity of IL-10.

An ethanol extract of Piper betle Linn. mediates its anti-inflammatory activity via down-regulation of nitric oxide

The leaves of Piper betle (locally known as Paan) have long been in use in the Indian indigenous system of medicine for the relief of pain; however, the underlying molecular mechanisms of this effect have not been elucidated. The anti-inflammatory and immunomodulatory effects of an ethanolic extract of the leaves of P. betle (100 mg kg(-1); PB) were demonstrated in a complete Freund's adjuvant-induced model of arthritis in rats with dexamethasone (0.1 mg kg(-1)) as the positive control. At non-toxic concentrations of PB (5-25 microg mL(-1)), a dose-dependent decrease in extracellular production of nitric oxide in murine peritoneal macrophages was measured by the Griess assay and corroborated by flow cytometry using the nitric oxide specific probe, 4,5-diaminofluorescein-2 diacetate. This decreased generation of reactive nitrogen species was mediated by PB progressively down-regulating transcription of inducible nitric oxide synthase in macrophages, and concomitantly causing a dose-dependent decrease in the expression of interleukin-12 p40, indicating the ability of PB to down-regulate T-helper 1 pro-inflammatory responses. Taken together, the anti-inflammatory and anti-arthrotic activity of PB is attributable to its ability to down-regulate the generation of reactive nitrogen species, thus meriting further pharmacological investigation.

Bone morphogenetic protein‐6 induces the expression of inducible nitric oxide synthase in macrophages

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily. In the present study, we investigated the effect of BMPs on the production of inducible nitric oxide synthase (iNOS) in the murine macrophage cell line, RAW 264.7, and in mouse peritoneal macrophages. Among the BMPs, only BMP-6 induced iNOS expression in a time-dependent and dose-dependent manner in both cell types. Induction of iNOS was inhibited by both cycloheximide and actinomycin D, indicating that the induction of iNOS expression by BMP-6 requires new protein synthesis. Mechanistic studies revealed that the BMP-6-induced iNOS expression requires both Smads and nuclear factor-kappa B (NF-kappaB) signalling pathways. Furthermore, induction of interleukin-1beta (IL-1beta) was necessary for iNOS induction by BMP-6. These observations suggest that BMP-6 stimulates macrophages to produce iNOS through IL-1beta via Smad and NF-kappaB signalling pathways and that BMP-6 may be an important regulator of macrophages.