Higher Inducible Nitric Oxide Synthase Research Articles

Macrophages of different tissue origin exhibit distinct inflammatory responses to mycobacterial infection.

Macrophages display marked plasticity with functions in both inflammation and tissue repair. Evidence demonstrates that this spectrum of macrophage phenotypes is influenced by their local microenvironment and tissue origin. However, invitro macrophage experiments often do not or cannot readily use macrophages from the most relevant tissue of origin. This study investigated if the origin of two C57BL/6 mouse macrophage cell lines of alveolar (AMJ2-C11) and peritoneal (IC-21) origin may influence their response to mycobacterial infection. Both cell lines equally controlled the growth of Mycobacterium bovis BCG and Mycobacterium tuberculosis, although the expression of all proinflammatory cytokines and chemokines measured (TNF, IL-6, MCP-1, MIP-1α, MIP-1β, and RANTES) was significantly higher in AMJ2-C11 cells than in IC-21 cells. During M. tuberculosis infection, IL-6, MCP-1, and RANTES expression increased 5-fold, and MIP-1β expression increased 30-fold. Additionally, AMJ2-C11 cells exhibited significantly higher inducible nitric oxide synthase activity than IC-21 cells, indicative of a more polarized M1 response. The expression of multiple surface markers was also assessed by flow cytometry. CD80 and CD86 were significantly upregulated in AMJ2-C11 cells and downregulated in IC-21 cells during M. tuberculosis infection. The results support the notion that the origin of tissue-resident macrophages influences their phenotype and antimicrobial response and demonstrate hereto unrecognized potential for these cell lines in invitro studies.

Angelica dahurica and Rheum officinale Facilitated Diabetic Wound Healing by Elevating Vascular Endothelial Growth Factor.

Traditional Chinese medicine (TCM) provides alternative treatment choices for diabetic wounds. The aim of this study was to evaluate the effects of Angelica dahurica and Rheum officinale (ARE) on diabetic wounds and its underlying action mechanism. Atotal of 36 healthy male Sprague-Dawley rats were randomly divided into three groups: diabetes mellitus (DM) rats treated with ARE (DM-ARE), DM rats treated with 0.9% saline (DM-NS), and non-DM rats treated with 0.9% saline (NDM-NS). DM was induced by intraperitoneal administration of 40 mg/kg of streptozotocin after a 2-week high-fat diet feeding. After excisional skin wounds and treatments, the remaining wound area (RWA) in each group was measured. The RWA in the DM-NS group (69.60% ± 2.35%) was greater than that in the DM-ARE (55.70% ± 1.85%) and NDM-NS groups (52.50% ± 2.77%) on day 6. Besides, the DM-ARE group showed higher vascular endothelial growth factor (VEGF), higher inducible nitric oxide synthase (iNOs), higher [Formula: see text]-smooth muscle actin ([Formula: see text]-SMA), and lower nuclear factor kappa-light-chain-enhancer of activated B cell (NF-[Formula: see text]B) expression in the wound skin tissue. These results showed that treatment with ARE shifted the recovery pattern of diabetic rats to the pattern of nondiabetic rats, indicating that ARE may improve wound healing in diabetic conditions.

The impact of l-arginine supplementation on the enteral phaseof experimental Trichinella spiralis infection in treated and untreated mice.

The role of nitric oxide (NO) in the immunopathological response during Trichinella spiralis (T. spiralis) infection remains controversial. The amino acid, l-arginine is a NO precursor commonly used by athletes and bodybuilders as a protein supplement. As to our knowledge, there are no published studies which have tested the effect of l-arginine on the intestinal phase of experimental trichinellosis. The present work aims to investigate the effect of l-arginine on the enteral phase of experimental T. spiralis infection in albendazole-treated and untreated mice. Forty BALB/C mice infected orally with T. spiralis larvae were divided into 4 groups as follows: Group A were infected and untreated (control) mice, Group B received albendazole alone, Group C received l-arginine alone, and Group D received both l-arginine and albendazole. Compared to the control group, l-arginine supplementation showed; a significant increase in the intestinal adult worm burden, a significantly high inducible NO synthase (iNOS) expression, elevated immune markers; tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), and enhanced apoptosis. Albendazole treated-group had a significant reduction in the adult worm number (90.9%), while combined albendazole-arginine regimen showed a lower percentage of worm reduction (72.7%). During the enteral phase of T. spiralis infection, l-arginine supplementation should be taken cautiously, as it may modulate the proinflammatory immune response and subsequently affect the outcome of the infection and/or treatment.

Dietary citrulline improves survival of rainbow trout Oncorhynchus mykiss juveniles challenged with Vibrio anguillarum

Arginine is a versatile amino acid that plays important roles in various physiological functions. However, supplementation with arginine can also increase urea excretion of the fish. Thus, given than ornithine and citrulline can potentially be used to increase arginine availability or improve fish immunity, this study aimed to compare the effects of dietary supplementation of arginine, ornithine, and citrulline on the survival of rainbow trout Oncorhynchus mykiss against Vibrio anguillarum. Dietary treatment consisted of a control diet (CTRL) and three treatment diets comprising CTRL supplemented with 2% L-arginine (+ARG), L-ornithine (+ORN), or L-citrulline (+CIT). Protein and lipid content of the diets were confirmed at 48.9 and 18.9%, 53.1 and 18.7%, 51.7 and 18.1% and 52.9 and 18.9% for the CTRL, +ARG, +ORN & + CIT diets respectively. Postprandial plasma amino acid levels were measured at 6, 15, and 30 h after feeding. A feeding trial was conducted for 30 days, and at the end of the trial, the rainbow trout were challenged with V. anguillarum. At 24 h post-injection, plasma amino acid and renal gene expression levels were examined. Fish exhibited better weight gain in +ARG treatment compared to +CIT treatment. Similar postprandial levels of plasma arginine were observed in +ARG and + CIT, and the levels in both treatments were significantly higher than those in CTRL. A survival analysis revealed that fish in +CIT (69.2%) were more resistant than those in CTRL (25.0%). There was significantly higher inducible nitric oxide synthase (iNOS) expression in the kidneys of fish in +CIT than those in CTRL. A significant difference in interleukin-1-beta expression was found between +ORN and CTRL treatments, while there was no significant difference in arginase II expression among the treatments. These result shows that citrulline supplementation can be used to increase arginine availability and better at enhancing the immune performance of rainbow trout compared to arginine supplementation when challenged with V. anguillarum.

Structure and immunostimulating activity of a galactofuranose-rich polysaccharide from the bamboo parasite medicinal fungus Shiraia bambusicola

Ethnopharmacological relevanceShiraia bambusicola is a parasitic fungus on the twigs of bamboos. Its relatively large stroma has high medicinal value and can treat a variety of diseases such as rheumatoid arthritis, cold stomach pain, sciatica, injuries, chronic bronchitis, and infantile. It is widely distributed in many provinces in Southern China and also is also found in Japan. Aim of the studyMedicinal fungi were important resources for bioactive polysaccharides. To explore bioactive polysaccharides from Shiraia bambusicola, a heteropolysaccharide SB2-1 was purified and obtained from S. bambusicola and its immunostimulating activity was researched. Materials and methodsThe polysaccharide from S. bambusicola was extracted and purified using enzyme assisted extraction, ethanol precipitation, anion-exchange and size-exclusion chromatography. Molecular weight of polysaccharide was estimated by high performance gel permeation chromatography. Monosaccharide compositions were determined by high performance liquid chromatography after pre-column derivatization and UV detection. Structure information was elucidated by IR spectrum, GC-MS analysis after methylation and gradual acid hydrolysis of the polysaccharide. The RAW264.7 cells were used to study the immunostimulating activity in vitro. ResultsPhysicochemical and structural analyses showed that SB2-1 was a neutral heteropolysaccharide with molecular weight at 22.2 kDa and consisted of glucose, galactose and mannose at a ratio of 2.0:1.5:1.0. The structure of SB2-1 was a branched polysaccharides composed of a mannan core and side chains consisted of glucose and galactose. The mannan core was composed of (1→2)-Manp as the main chain. Glucose with (1→4)-D-Glcp, (1→2)-D-Glcp and (1→6)-D-Glcp at different degrees of polymerization were linked at C-6 and C-3 of the (1→2)-Manp as the side chains. The galactose with the linages of (1→6)-D-Galf, →2)-D-Galf(1→ and terminal D-Galf(1→ also existed in the side chain. The study on the immunostimulating activities of SB2-1 and its core structure P-2 were investigated on RAW264.7 macrophages. The results showed that SB2-1 could activate RAW264.7 macrophage and significantly improve its phagocytic ability by neutral red uptake experiment. Meanwhile, SB2-1 increased significantly higher inducible nitric oxide synthase (iNOS) production and the productions of IL-1, IL-6, IL-12 and TNF-α. The effect of SB2-1 was better than its core structure P-2 produced by gradual acid hydrolysis, which meant the side chains played an important role in the immunostimulating activities. ConclusionsThe investigation demonstrated that the galactofuranose-containing mannogalactoglucan was characteristic polysaccharides in S. bambusicola and could enhance the activation of macrophages.

An iso-α-acid-rich extract from hops (Humulus lupulus) attenuates acute alcohol-induced liver steatosis in mice.

Results of invitro and invivo studies suggest that consumption of beer is less harmful for the liver than consumption of spirits. It also has been suggested that secondary plant compounds derived from hops such as xanthohumol or iso-α-acids may have beneficial effects on the development of liver diseases of various etiologies. The aim of this study was to determine whether iso-α-acids consumed in doses achieved by "normal" beer consumption have beneficial effects on health. Female C57 Bl/6J mice, pretreated for 4d with an iso-α-acid-rich extract (∼30% iso-α-acids from hops, 0.75mg/kg body weight), were fed one bolus of ethanol (6g/kg body weight intragastric) or an iso-caloric maltodextrin solution. Markers of liver damage, toll-like receptor-4 signaling, and lipid peroxidation were determined. Furthermore, the effect of isohumulone on the lipopolysaccharide-dependent activation of J774 A.1 macrophages, used as a model of Kupffer cells, was determined. In the liver, acute ethanol administration led to a significant accumulation of fat (∼10-fold), which was accompanied by significantly higher inducible nitric oxide synthase protein level, elevated nitric oxide production, and increased plasminogen activator inhibitor 1 protein concentration when compared to controls. In mice pretreated with iso-α-acids, these effects of alcohol were markedly attenuated. Pretreatment of J774 A.1 macrophages with isohumulone significantly attenuated lipopolysaccharide-induced mRNA expression of inducible nitric oxide synthase and interleukin-6 as well as the release of nitric oxide. Taken together, iso-α-acids markedly attenuated the development of acute alcohol-induced damage in mice.

Macrophage Cyclooxygenase-2 Protects Against Development of Diabetic Nephropathy.

Diabetic nephropathy (DN) is characterized by increased macrophage infiltration, and proinflammatory M1 macrophages contribute to development of DN. Previous studies by us and others have reported that macrophage cyclooxygenase-2 (COX-2) plays a role in polarization and maintenance of a macrophage tissue-reparative M2 phenotype. We examined the effects of macrophage COX-2 on development of DN in type 1 diabetes. Cultured macrophages with COX-2 deletion exhibited an M1 phenotype, as demonstrated by higher inducible nitric oxide synthase and nuclear factor-κB levels but lower interleukin-4 receptor-α levels. Compared with corresponding wild-type diabetic mice, mice with COX-2 deletion in hematopoietic cells (COX-2 knockout bone marrow transplantation) or macrophages (CD11b-Cre COX2f/f) developed severe DN, as indicated by increased albuminuria, fibrosis, and renal infiltration of T cells, neutrophils, and macrophages. Although diabetic kidneys with macrophage COX-2 deletion had more macrophage infiltration, they had fewer renal M2 macrophages. Diabetic kidneys with macrophage COX-2 deletion also had increased endoplasmic reticulum stress and decreased number of podocytes. Similar results were found in diabetic mice with macrophage PGE2 receptor subtype 4 deletion. In summary, these studies have demonstrated an important but unexpected role for macrophage COX-2/prostaglandin E2/PGE2 receptor subtype 4 signaling to lessen progression of diabetic kidney disease, unlike the pathogenic effects of increased COX-2 expression in intrinsic renal cells.

Macrophage-mediated inflammatory response decreases mycobacterial survival in mouse MSCs by augmenting NO production.

Mycobacterium tuberculosis (MTB) is a hard-to-eradicate intracellular microbe, which escapes host immune attack during latent infection. Recent studies reveal that mesenchymal stem cells (MSCs) provide a protective niche for MTB to maintain latency. However, the regulation of mycobacterial residency in MSCs in the infectious microenvironment remains largely unknown. Here, we found that macrophage-mediated inflammatory response during MTB infection facilitated the clearance of bacilli residing in mouse MSCs. Higher inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production were observed in mouse MSCs under macrophage-mediated inflammatory circumstance. Blocking NO production in MSCs increased the survival of intracellular mycobacteria, indicating NO-mediated antimycobacterial activity. Moreover, both nuclear factor κB (NF-κB) and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways were involved in iNOS expression and NO production in inflammatory microenvironment. Furthermore, pro-inflammatory cytokine interleukin-1β could trigger NO production in MSCs and exert anti-mycobacterial activity via NF-κB signaling pathway. Neutralization of interleukin-1β in macrophage-mediated inflammatory microenvironment dampened the ability of mouse MSCs to produce NO. Together, our findings demonstrated that macrophage-mediated inflammatory response during mycobacterial infection promotes the clearance of bacilli in mouse MSCs by increasing NO production, which may provide a better understanding of latent MTB infection.

MiR-155 Suppresses Bacterial Clearance in Pseudomonas aeruginosa–Induced Keratitis by Targeting Rheb

miR-155 (microRNA-155) is an important noncoding RNA in regulating host inflammatory responses. However, its regulatory role in ocular infection remains unclear. Our study first explored the function of miR-155 in Pseudomonas aeruginosa-induced keratitis, one of the most common sight-threatening ocular diseases. We found that miR-155 expression was enhanced in human and mouse corneas after P. aeruginosa infection and was mainly expressed in macrophages but not neutrophils. In vivo studies demonstrated that miR-155 knockout mice displayed more resistance to P. aeruginosa keratitis, with a higher inducible nitric oxide synthase level and a lower bacterial burden. More importantly, in vitro data indicated that miR-155 suppressed the macrophage-mediated bacterial phagocytosis and intracellular killing of P. aeruginosa by targeting Rheb (Ras homolog enriched in brain). To the best of our knowledge, this is the first study to explore the role of miR-155 in bacterial keratitis, which may provide a promising target for clinical treatment of P. aeruginosa keratitis and other infectious diseases.

Caveolin-1 knockout mice exhibit airway hyperreactivity

Caveolae are flask-shaped plasma membrane invaginations expressing the scaffolding caveolin proteins. Although caveolins have been found in endothelium and epithelium (where they regulate nitric oxide synthase activity), their role in smooth muscle is still under investigation. We and others have previously shown that caveolae of human airway smooth muscle (ASM), which express caveolin-1, contain Ca(2+) and force regulatory proteins and are involved in mediating the effects of inflammatory cytokines such as TNF-α on intracellular Ca(2+) concentration responses to agonist. Accordingly, we tested the hypothesis that in vivo, absence of caveolin-1 leads to reduced airway hyperresponsiveness, using a knockout (KO) (Cav1 KO) mouse and an ovalbumin-sensitized/challenged (OVA) model of allergic airway hyperresponsiveness. Surprisingly, airway responsiveness to methacholine, tested by use of a FlexiVent system, was increased in Cav1 KO control (CTL) as well as KO OVA mice, which could not be explained by a blunted immune response to OVA. In ASM of wild-type (WT) OVA mice, expression of caveolin-1, the caveolar adapter proteins cavins 1-3, and caveolae-associated Ca(2+) and force regulatory proteins such as Orai1 and RhoA were all increased, effects absent in Cav1 KO CTL and OVA mice. However, as with WT OVA, both CTL and OVA Cav1 KO airways showed signs of enhanced remodeling, with high expression of proliferation markers and increased collagen. Separately, epithelial cells from airways of all three groups displayed lower endothelial but higher inducible nitric oxide synthase and arginase expression. Arginase activity was also increased in these three groups, and the inhibitor nor-NOHA (N-omega-nor-l-arginine) enhanced sensitivity of isolated tracheal rings to ACh, especially in Cav1 KO mice. On the basis of these data disproving our original hypothesis, we conclude that caveolin-1 has complex effects on ASM vs. epithelium, resulting in airway hyperreactivity in vivo mediated by altered airway remodeling and bronchodilation.

Localization and Expression of Inducible Nitric Oxide Synthase in Biopsies From Patients With Interstitial Cystitis

Interstitial cystitis is a chronic inflammatory disease of the bladder and luminal nitric oxide has been shown to be increased in the bladder in patients with interstitial cystitis. We analyzed endogenous nitric oxide formation and inducible nitric oxide synthase gene expression in the bladder of patients with interstitial cystitis to obtain further knowledge of the localization of inducible nitric oxide synthase in the bladder mucosa. Six patients with interstitial cystitis and 8 controls were studied. In these 2 groups endogenous nitric oxide formation was measured and inducible nitric oxide synthase expression in bladder biopsies was analyzed at the transcriptional and protein levels by real-time polymerase chain reaction and Western blot, respectively. Immunohistochemistry for inducible nitric oxide synthase was also performed. Patients with interstitial cystitis had higher inducible nitric oxide synthase mRNA expression and nitric oxide formation than controls (p <0.01 and <0.001, respectively). Inducible nitric oxide synthase protein expression was up-regulated in the interstitial cystitis group. Immunohistochemistry showed that inducible nitric oxide synthase was predominantly localized to the urothelium in patients with interstitial cystitis but inducible nitric oxide synthase-like immunoreactivity was also found in macrophages in the bladder mucosa. The increased levels of endogenously formed nitric oxide in patients with interstitial cystitis correspond to increased inducible nitric oxide synthase mRNA expression and protein levels in these patients. Furthermore, inducible nitric oxide synthase was found to be localized to the urothelium but it was also found in macrophages in the bladder mucosa. Whether high levels of endogenously formed nitric oxide are a part of the pathogenesis in interstitial cystitis and whether it has a protective or damaging role remain to be elucidated.

Inducible nitric oxide synthase gene promoter polymorphism is associated with increased gastric mRNA expression of inducible nitric oxide synthase and increased risk of gastric carcinoma

Stimulation of inducible nitric oxide synthase gene expression by Helicobacter pylori, with subsequent overproduction of nitric oxide, has been implicated in gastric carcinogenesis. We investigated whether inducible nitric oxide synthase promoter gene polymorphisms are associated with (a) inducible nitric oxide synthase mRNA expression in the gastric mucosa, and (b) the risk of gastric carcinoma. The relationship between gastric inducible nitric oxide synthase mRNA expression and inducible nitric oxide synthase promoter polymorphisms (CCTTT repeat polymorphism and -2445 C-->G SNP) was examined in 74 H. pylori-infected patients with gastric cancer, peptic ulcer, or functional dyspepsia. In a case-control study the prevalence of the polymorphisms was examined in H. pylori-infected gastric carcinomas (n=77) and noncancerous controls (n=154). Inducible nitric oxide synthase mRNA levels were significantly higher in long CCTTT repeat (either allele>11) carriers than in short ones (P=0.015). Multivariate regression analysis showed that inducible nitric oxide synthase mRNA expression was significantly linked to long CCTTT repeat and gastric cancer (P=0.026), but not to -2445 C-->G SNP and other parameters. The case-control study showed that long CCTTT repeat carriers had an increased risk of gastric cancer with an odds ratio of 2.0 (P=0.021). -2445 C-->G SNP was not associated with the risk. Helicobacter pylori induces higher inducible nitric oxide synthase mRNA expression in carriers of long CCTTT repeats of inducible nitric oxide synthase promoter, and this polymorphism is associated with an increased risk of gastric carcinoma.

Leukocyte-Derived IL-10 Reduces Subepithelial Fibrosis Associated with Chronically Inhaled Endotoxin

Endotoxin (LPS), a Gram-negative cell wall component, has potent proinflammatory properties. Acute LPS exposure causes airway inflammation; chronic exposure causes airway hyperreactivity and remodeling. IL-10 is an important antiinflammatory cytokine, which is decreased in patients with airway disease, such as asthma and cystic fibrosis. To examine the physiologic and therapeutic role of IL-10 in acute and chronic LPS-induced airway disease. Mice were exposed to aerosolized LPS once or daily for 4 wk. Endpoints were airway inflammation, airway reactivity to methacholine, extracellular matrix protein expression, and histologic analysis. IL-10-deficient mice developed significantly enhanced airway cellularity and remodeling when compared with C57BL/6 mice after chronic LPS inhalation. However they demonstrated less airway hyperreactivity associated with higher inducible nitric oxide synthase (iNOS), endothelial NOS (eNOS), and lung lavage fluid nitrite levels. In a bone marrow transplantation model, the IL-10 antiinflammatory effect was dependent on the hematopoietic but not on the parenchymal IL-10 expression. Induced epithelial human IL-10 expression protected from the LPS effects and led to decreased collagen production. IL-10 attenuates chronic LPS-induced airway inflammation and remodeling. Physiologically, the antiinflammatory effect of IL-10 is mediated by hematopoietic cells. Therapeutically, adenovirus-driven expression of human IL-10 in airway epithelia is sufficient for its protective effect on inflammation and remodeling. The role of IL-10 on airway hyperreactivity is complex: IL-10 deficiency protects against LPS-induced hyperreactivity, and is associated with higher eNOS, iNOS, and airway nitrate levels.

Production of nitric oxide and self-nitration of proteins during monocyte differentiation to dendritic cells

Nitric oxide (NO) can stimulate dendritic cells to a more activated state. However, nitric oxide and peroxynitrites production by dendritic cells has been usually associated with pathological situations such as autoimmunity or inflammatory diseases. This study was designed to determine if dendritic cells obtained from healthy volunteers produce nitric oxide and peroxynitrites, which results in protein nitration. The expression of arginase II, but not arginase I, isoform was detected in monocytes and dendritic cells. There was higher inducible nitric oxide synthase (iNOS) protein expression and lower arginase activity both in immature and mature dendritic cells, compared to monocytes. This caused nitric oxide production, and maturation of dendritic cells which provoked a significative increase of nitrites and nitrates compared to immature dendritic cells. There was also peroxynitrites synthesis during monocyte differentiation as shown by the nitration of proteins. Immunoblot revealed a pattern of nitrated proteins in cell extracts obtained from monocytes and dendritic cells, however there were bands that appeared only in human dendritic cells, in particular an intense 90 kDa band. Nitric oxide production and nitrotyrosine formation could affect the antigen presentation and modify the immune response.

Effects of antibiotic class on the macrophage inflammatory response to Streptococcus pneumoniae.

Antibiotic choice can alter host inflammation during invasive bacterial infections. Previous studies of gram-negative organisms concluded that antibiotic-mediated release of bacterial cell wall components amplifies inflammation. Less has been reported about antibiotic effect on gram-positive organisms. This study explored the hypothesis that Streptococcus pneumoniae would induce greater macrophage inflammatory mediator production when killed with cell wall active antibiotics rather than protein synthesis inhibitors. Stimulation of RAW 264.7 murine macrophages with pneumococci and oxacillin led to significantly higher inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) accumulation than did the same concentrations of pneumococci and clindamycin. Neither antibiotic alone or in combination with lipopolysaccharide acted directly on macrophages to modify the immune response. Endotoxin contamination did not confound the results, as preincubation with polymyxin B did not change iNOS or TNF protein levels. Thus, the antimicrobial mechanism of action affects macrophage inflammatory mediator production after stimulation with pneumococci.

Dexamethasone Inhibits the Expression of an Inducible Nitric Oxide Synthase in Infarcted Rabbit Myocardium

Infarcted areas of rabbit myocardium show relatively higher inducible nitric oxide synthase activity, measured by the conversion of L-[14C]arginine to L-[14C]citrulline. The principal finding in this study is that dexamethasone (2 mg/kg) prevents the induction of inducible nitric oxide synthase in heart muscle when given before, or even 3 hr after coronary artery ligation. Additionally cyclic GMP levels remain unchanged following treatment with dexamethasone. It is possible that the enhanced production of nitric oxide by inducible nitric oxide synthase accounts, at least in part, for the depression of myocardial contractility seen in myocardial infarction and in other clinical conditions.