Vitamin D-independent benefits of safe sunlight exposure.

This review examines the beneficial effects of ultraviolet radiation on systemic autoimmune diseases, including multiple sclerosis and type I diabetes, where the epidemiological evidence for the vitamin D-independent effects of sunlight is most apparent. Ultraviolet radiation, in addition to its role in the synthesis of vitamin D, stimulates anti-inflammatory pathways, alters the composition of dendritic cells, T cells, and T regulatory cells, and induces nitric oxide synthase and heme oxygenase metabolic pathways, which may directly or indirectly mitigate disease progression and susceptibility. Recent work has also explored how the immune-modulating functions of ultraviolet radiation affect type II diabetes, cancer, and the current global pandemic caused by SARS-CoV-2. These diseases are particularly important amidst global changes in lifestyle that result in unhealthy eating, increased sedentary habits, and alcohol and tobacco consumption. Compelling epidemiological data shows increased ultraviolet radiation associated with reduced rates of certain cancers, such as colorectal cancer, breast cancer, non-Hodgkins lymphoma, and ultraviolet radiation exposure correlated with susceptibility and mortality rates of COVID-19. Therefore, understanding the effects of ultraviolet radiation on both vitamin D-dependent and -independent pathways is necessary to understand how they influence the course of many human diseases.

Quantitative analysis of enteric neurons containing choline acetyltransferase and nitric oxide synthase immunoreactivities in the submucosal and myenteric plexuses of the porcine colon.

The enteric nervous system (ENS) controls gastrointestinal functions. In large mammals' intestine, it comprises an inner (ISP) and outer (OSP) submucous plexus and a myenteric plexus (MP). This study quantifies enteric neurons in the ISP, OSP, and MP of the pig ascending (AC) and descending colon (DC) using the HuC/D, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) neuronal markers in whole mount preparations with multiple labeling immunofluorescence. We established that the ISP contains the highest number of HuC/D neurons/mm2, which were more abundant in AC vs. DC, followed by OSP and MP with similar density in AC and DC. In the ISP, the density of ChAT immunoreactive (IR) neurons was very similar in AC and DC (31% and 35%), nNOS-IR neurons were less abundant in AC than DC (15% vs. 42%, P < 0.001), and ChAT/nNOS-IR neurons were 5% and 10%, respectively. In the OSP, 39-44% of neurons were ChAT-IR in AC and DC, while 45% and 38% were nNOS-IR and 10-12% were ChAT/nNOS-IR (AC vs. DC P < 0.05). In the MP, ChAT-IR neurons were 44% in AC and 54% in DC (P < 0.05), nNOS-IR neurons were 50% in both, and ChAT/nNOS-IR neurons were 12 and 18%, respectively. The ENS architecture with multilayered submucosal plexuses and the distribution of functionally distinct groups of neurons in the pig colon are similar to humans, supporting the suitability of the pig as a model and providing the platform for investigating the mechanisms underlying human colonic diseases.

Cellular and Biochemical Analysis of Bronchoalveolar Lavage Fluid from Murine Lungs.

Bronchoalveolar lavage (BAL) is a technique used to collect the contents of the airways. The fluid recovered, called BAL fluid (BALF), serves as a dynamic tool to identify various disease pathologies ranging from asthma to infectious diseases to cancer in the lungs. A wide array of tests can be performed with BALF, including total and differential leukocyte counts (DLC), enzyme-linked immunosorbent assays (ELISA) or flow-cytometric quantitation of inflammatory mediators, such as cytokines, chemokines and adhesion molecules, and assessment of nitrate and nitrite content for estimation of nitric oxide synthase (NOS) activity. Here, we describe a detailed procedure for the collection of BALF for a variety of downstream usages, including DLC by cytological and flow-cytometry-based methods, multiplex cytokine analysis by flow cytometry, and NOS activity analysis by determining nitrate and nitrite levels.

Propacin, a coumarinolignoid isolated from durian, inhibits the lipopolysaccharide-induced inflammatory response in macrophages through the MAPK and NF-κB pathways.

Durian, known as the king of fruits, is rich in nutrients and bioactive phytochemicals. Propacin is a bioactive coumarinolignoid isolated from durian. In this study, we demonstrated its anti-inflammatory effect on lipopolysaccharide (LPS)-induced RAW264.7 cells and explored the underlying mechanism. Propacin suppressed nitric oxide (NO) and prostaglandin E2 (PGE2) production in LPS-stimulated macrophages significantly by downregulating the mRNA and protein expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, propacin decreased the overexpression of the LPS-induced reactive oxygen species (ROS) and maintained the mitochondrial integrity in active macrophages. Furthermore, propacin inhibited the translocation of the nuclear factor-κB (NF-κB) p65 subunit into the nucleus and the phosphorylation of mitogen-activated protein kinases (MAPKs), especially JNK and ERK. Collectively, these data indicated that propacin may have the potential to be developed as a novel therapeutic agent for inflammatory-related diseases.

Alterations in nitric oxide homeostasis during traumatic brain injury.

Changes in nitric oxide (NO) levels have been often associated with various forms of trauma, including secondary damage after traumatic brain injury (TBI). Several studies demonstrate the upregulation of NO synthase (NOS) enzymes, and concomitant increases in brain NO levels, which contribute to the TBI-associated glutamate cytotoxicity, including the pathogenesis of mitochondrial dysfunction. TBI is also associated with elevated NO levels in remote organs, indicating that TBI can induce systemic changes in NO regulation, which can be either beneficial or detrimental. Here we review the possible mechanisms responsible for changes in NO metabolism during TBI. Better understanding of the changes in NO homeostasis in TBI will be necessary to design rational therapeutic approaches for TBI. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.

Chemical Coding of Sensory Neurons Supplying the Hip Joint Capsule in the Sheep.

Immunohistochemical properties of nerve fibres supplying the joint capsule were previously described in many mammalian species, but the localization of sensory neurons supplying this structure was studied only in laboratory animals, the rat and rabbit. However, there is no comprehensive data on the chemical coding of sensory neurons projecting to the hip joint capsule (HJC). The aim of this study was to establish immunohistochemical properties of sensory neurons supplying HJC in the sheep. The study was carried out on 10 sheep, weighing about 30-40 kg. The animals were injected with a retrograde neural tracer Fast Blue (FB) into HJC. Sections of the spinal ganglia (SpG) with FB-positive (FB+) neurons were stained using antibodies against calcitonin gene-related peptide (CGRP) substance P (SP), pituitary adenylate cyclase-activating peptide (PACAP), nitric oxide synthase (n-NOS), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), Leu-5-enkephalin (Leu-Enk), galanin (GAL) and vesicular acetylcholine transporter (VACHT). The vast majority of FB+ neurons supplying HJC was found in the ganglia from the 5th lumbar to the 2nd sacral. Immunohistochemistry revealed that most of these neurons were immunoreactive to CGRP or SP (80.7 ± 8.0% or 56.4 ± 4.8%, respectively) and many of them stained for PACAP or GAL (52.9 ± 2.9% or 50.6 ± 19.7%, respectively). Other populations of FB+ neurons were those immunoreactive to n-NOS (37.8 ± 9.7%), NPY (34.6 ± 6.7%), VIP (28.7 ± 4.8%), Leu-Enk (27.1 ± 14.6) and VACHT (16.7 ± 9.6).

Activity of Zearalenone in the Porcine Intestinal Tract.

This study demonstrates that low doses (somewhat above the No Observed Adverse Effect Level, NOAEL) of the mycoestrogen zearalenone (ZEN) and its metabolites display multispecificity towards various biological targets in gilts. The observed responses in gilts were surprising. The presence of ZEN and zearalenols (ZELs) did not evoke a response in the porcine gastrointestinal tract, which was attributed to dietary tolerance. Lymphocyte proliferation was intensified in jejunal mesenteric lymph nodes, and lymphocyte counts increased in the jejunal epithelium with time of exposure. In the distal digestive tract, fecal bacterial counts decreased, the activity of fecal bacterial enzymes and lactic acid bacteria increased, and cecal water was characterized by higher genotoxicity. The accompanying hyperestrogenism led to changes in mRNA activity of selected enzymes (cytochrome P450, hydroxysteroid dehydrogenases, nitric oxide synthases) and receptors (estrogen and progesterone receptors), and it stimulated post-translational modifications which play an important role in non-genomic mechanisms of signal transmission. Hyperestrogenism influences the regulation of the host's steroid hormones (estron, estradiol and progesteron), it affects the virulence of bacterial genes encoding bacterial hydroxysteroid dehydrogenases (HSDs), and it participates in detoxification processes by slowing down intestinal activity, provoking energy deficits and promoting antiporter activity at the level of enterocytes. In most cases, hyperestrogenism fulfils all of the above roles. The results of this study indicate that low doses of ZEN alleviate inflammatory processes in the digestive system, in particular in the proximal and distal intestinal tract, and increase body weight gains in gilts.

Potential of Inducible Nitric Oxide Synthase as a Therapeutic Target for Allergen-Induced Airway Hyperresponsiveness: A Critical Connection to Nitric Oxide Levels and PARP Activity.

Although expression of inducible NO synthase (iNOS) in the lungs of asthmatics and associated nitrosative damage are established, iNOS failed as a therapeutic target for blocking airway hyperresponsiveness (AHR) and inflammation in asthmatics. This dichotomy calls for better strategies with which the enzyme is adequately targeted. Here, we confirm iNOS expression in the asthmatic lung with concomitant protein nitration and poly(ADP-ribose) polymerase (PARP) activation. We show, for the first time, that iNOS is highly expressed in peripheral blood mononuclear cells (PBMCs) of asthmatics with uncontrolled disease, which did not correspond to protein nitration. Selective iNOS inhibition with L-NIL protected against AHR upon acute, but not chronic, exposure to ovalbumin or house dust mite (HDM) in mice. Supplementation of NO by nitrite administration significantly blocked AHR in chronically HDM-exposed mice that were treated with L-NIL. Protection against chronic HDM exposure-induced AHR by olaparib-mediated PARP inhibition may be associated with the partial but not the complete blockade of iNOS expression. Indeed, L-NIL administration prevented olaparib-mediated protection against AHR in chronically HDM-exposed mice. Our study suggests that the amount of iNOS and NO are critical determinants in the modulation of AHR by selective iNOS inhibitors and renews the potential of iNOS as a therapeutic target for asthma.

Transcriptional activation and translocation of ancient NOS during immune response.

NOS is the key component of the NO system, which plays an indispensable role in many physiologic and immunologic processes; however, the process that underlies the activation of ancient NOSs and their functions remains unclear. Expression of Crassostrea gigas NOS (CgNOS) mRNA in hemocytes was examined after stimulating oysters with LPS and TNF-α. Expression level of CgNOS mRNA was increased significantly, by 2.61-fold (P < 0.05), at 24 h poststimulation. A positive CgNOS signal was detected via immunoprecipitation, and only one protein was detected in oyster hemocytes. Shifting and supershifting bands were observed in EMSAs between the CgNOS promoter and the transcription factors CgNF-κB1 and Cg-signal transducer and activator of transcription (STAT). CgNF-κB1 was detected in the nucleus only at 12 h, whereas CgSTAT was observed in the cytoplasm and nucleus at 12 and 24 h. Expression levels of tyrosine-protein kinase receptor Tie-1, phosphatidylinositide phosphatase SAC2, phosphatidylinositol-4-phosphate 5-kinase type-1α, diacylglycerol kinase θ, LPS-induced TNF-α factor-like protein, cAMP-dependent transcription factor-2, NF-κB1, and STAT6 were significantly elevated in a transcriptome analysis after 12 h of LPS and TNF-α stimulation. An immunoreactive CgNOS signal was observed in both the cell membrane and cytoplasm at 12 h, whereas it was mainly localized to the cytoplasm at 24 h post-LPS and -TNF-α stimulation. These findings revealed that CgNOS could be transcriptionally activated by CgNF-κB1 and CgSTAT via the PI3K-Akt pathway, similar to what occurs for iNOS, but CgNOS translocated to the cytoplasm, similar to neuronal NOS, to modulate downstream signals during an immune defense. These results collectively provide crucial knowledge about the evolution of NOS structure and function.-Jiang, Q., Liu, Z., Zhou, Z., Wang, L., Wang, L., Yue, F., Wang, J., Wang, H., Song, L. Transcriptional activation and translocation of ancient NOS during immune response.

Bacterial Exposure at the Larval Stage Induced Sexual Immune Dimorphism and Priming in Adult Aedes aegypti Mosquitoes.

Gender differences in the immune response of insects are driven by natural selection for females and sexual selection for males. These natural forces entail a multitude of extrinsic and intrinsic factors involved in a genotype-environment interaction that results in sex-biased expression of the genes shared by males and females. However, little is known about how an infection at a particular ontogenetic stage may influence later stages, or how it may impact sexual immune dimorphism. Using Aedes aegypti mosquitoes, the aim of the present study was to analyze the effect of a bacterial exposure at the larval stage on adult immunity in males and females. The parameters measured were phenoloxidase activity, nitric oxide production, antimicrobial activity, and the antimicrobial peptide transcript response. As a measure of the immune response success, the persistence of injected bacteria was also evaluated. The results show that males, as well as females, were able to enhance survival in the adult stage as a result of being exposed at the larval stage, which indicates a priming effect. Moreover, there was a differential gender immune response, evidenced by higher PO activity in males as well as higher NO production and greater antimicrobial activity in females. The greater bacterial persistence in females suggests a gender-specific strategy for protection after a previous experience with an elicitor. Hence, this study provides a primary characterization of the complex and gender-specific immune response of male and female adults against a bacterial challenge in mosquitoes primed at an early ontogenetic stage.

Immunolocalization of nitric oxide synthase (NOS) isoforms in ovarian follicles of the catfish, Clarias batrachus and its relation with ovarian activity.

Nitric oxide, a gaseous molecule, is produced during the conversion of arginine to citrulline by the action of NOS isoforms (eNOS, iNOS or nNOS). Role of NO in regulation of mammalian reproduction is well established; however, practically no report is available on fishes. Hence, in the present study, expression of all three isoforms of NOS was worked out in the ovary of Clarias batrachus immunohistochemically during different phases of its reproductive cycle and its relation with ovarian activities. No immunoreactivity of eNOS was observed in the ovary of C. batrachus during the late-quiescence and early-recrudescence phases. While during the recrudescence phase (April and May) it expressed intensely in thecal and granulosa cells of the oocyte-II and III, but immune-intensity decreased in the late-recrudescence and spawning phases (June and July). Similar pattern of immunoprecipitation was also observed in case of iNOS. However, the immunoreactivity pattern of nNOS was quite varied, it expressed moderately only in the nucleus and cytoplasm of perinuclear and oocyte-I stages during late-quiescence phase. While during the early recrudescence phase, the expression of nNOS disappeared completely from the nucleus and cytoplasm, rather it expressed intensely in the thecal and granulosa cells, which declined in the late-recrudescence and spawning phases. Moderate immunoreactivity of iNOS could also be localized in the zona radiata of ovulated oocyte. The intense NOS immunoreactivity in the thecal and granulosa cells coincided with increased levels of ovarian NO and 17ß-estradiol content. They exhibited statistically significant positive correlation amongst themselves, suggesting the involvement of ovarian NOS/NO system in oogenesis and steroidogenesis in the catfish.

Expression of pattern recognition receptors in cholesteatoma.

Although many immunologic mechanisms have been investigated in studies of the pathogenesis of cholesteatoma, the role of pattern recognition receptors (PRRs) has not been fully determined. Therefore, we assessed innate immune responses in patients with cholesteatoma. We prospectively evaluated 21 patients with acquired cholesteatoma between August 2010 and July 2012. Cholesteatoma specimens were obtained during surgery, and skin from the external meatus of each patient was used as a control. RNA was extracted from these tissue samples, followed by real-time PCR to quantitatively assess the relative expression of toll-like receptors (TLRs), NOD-like receptors (NLRs), retinoic acid-inducible gene (RIG)-I, NO synthase (NOS) and cytokines. The levels of TLR-2, -3, -4, -6, -7, and -10, NOD-2, and IL-1 and -8 mRNAs were significantly higher in the cholesteatoma than in the skin specimens (p < .05). The expression levels of TLR-2 and -3, RIG-I, IL-6, and TNF-α mRNAs were significantly higher in cholesteatomas from women than from men. The levels of TLR-8, NOD-2, IL-12, and TNF-α mRNAs were significantly higher in recurrent than in initial cholesteatoma specimens (p < .05). Hearing level did not correlate with the levels of expression of mRNAs encoding TLRs, NLRs, NOS, RIG-I and related cytokines (p > .05). In conclusion, alterations in innate immunity triggered by PRRs are important in the pathophysiology of cholesteatoma. Gender differences and frequency of surgery may affect the expression of PRRs in cholesteatomas.

NPFF2 receptor is involved in the modulatory effects of neuropeptide FF for macrophage cell line.

Neuropeptide FF (NPFF) interacts with specific receptors to regulate diverse biological processes. Its modulatory effect in the immune field, however, has not been fully explored yet. Here, we report that NPFF2 receptors may be functionally expressed in two immune cell models, the primary peritoneal macrophage and RAW 264.7 macrophage. Firstly, the mRNA levels of NPFF2 receptor were up-regulated in macrophages when treated with LPS for 24 to 72 h. Subsequently, our data hinted that NPFF regulates the viability of both kinds of macrophages. After treatment with RF9, a reported antagonist for both NPFF receptors, delayed or inhibited the NPFF-induced macrophages viability augmentation, suggesting the involvement of NPFF2 receptor. Furthermore, down-regulation of nitric oxide (NO) synthases (NOSs) partially significantly inhibited the viability augmentation of macrophages induced by NPFF, implying a nitric oxide synthases- dependent pathway is involved. However, the NOSs are not the only route by which NPFF affects the viability of macrophages. Pharmacological inhibitors of NF-κB signal pathway also blocked the NPFF-induced macrophages growth, suggesting the involvement of the NF-κB signal pathway. The regulation activity of NPFF for macrophages suggests that NPFF could act as a potential hormone in the control of immune system. Collectively, our data provide new evidence about the immune modulatory effect of NPFF, which will be helpful in extending the scope of NPFF functions.

Increased exhaled nitric oxide and its oxidation metabolism in eosinophilic chronic rhinosinusitis.

OBJECTIVE: Monitoring of fractional concentrations of exhaled nitric oxide (FeNO) has become a reliable marker of inflammation in human nose and paranasal sinuses. However, it is still unknown to what extent nasal NO levels contribute to the pathology of chronic rhinosinusitis (CRS). In the present study, we aimed to examine FeNO levels and the underlying mechanism of NO production and metabolism in patients with eosinophilic chronic rhinosinusitis (ECRS) and non-ECRS. METHODS: Thirty-three untreated ECRS patients, 16 non-ECRS patients, and 38 normal subjects were enrolled in this cross-sectional study of FeNO levels. Oral and nasal FeNO levels were measured before treatment using an electrochemical NO analyzer (NObreath(®)) with a nose adaptor. The mRNA expression of three nitric oxide synthase (NOS) isoforms, interleukin-5 (IL-5), and transforming growth factor-beta (TGF-ß) in the ethmoid sinus mucosa and nasal polyps were analyzed by real-time PCR. Immunohistological localization of inducible NOS (iNOS) and nitrotyrosine (NT), a marker for oxidized NO metabolites, was also examined. RESULTS: ECRS patients showed significantly higher oral FeNO levels compared to non-ECRS patients and normal subjects (mean values, 47.6, 13.5, and 15.3ppb, respectively). Nasal FeNO levels of the non-ECRS patients (30.5ppb) were significantly lower than those of the ECRS patients (53.9ppb) and normal subjects (45.5ppb). Positive correlations existed between the blood eosinophil percentage and FeNO levels in ECRS patients. Histologically, ECRS patients showed higher eosinophil accumulation in the ethmoid mucosa than non-ECRS patients (103.1 vs. 16.3cells/HPF). Real-time PCR analysis showed significant upregulation of iNOS and IL-5 mRNA expression in the ethmoid mucosa of the ECRS patients compared to those of non-ECRS patients. Positive iNOS immunoreactivity was observed in ciliated epithelial cells, submucosal glands and associated inflammatory cells in both groups. NT immunoreactivity was detected in the epithelium and around inflammatory cells. Intense NT staining was co-localized with eosinophil accumulation and ECRS patients showed significantly higher rates of NT-positive cells than non-ECRS patients. CONCLUSION: A combination of oral and nasal FeNO measurement is a valid marker for the classification and definition of different CRS subtypes in Japan. Higher levels of oral and nasal FeNO in ECRS patients may reflect the persistence of eosinophilic inflammation in sinus mucosa with concomitant iNOS upregulation and accompanying deposition of oxidized NO metabolites.

Increased ornithine-derived polyamines cause airway hyperresponsiveness in a mouse model of asthma.

Up-regulation of arginase contributes to airways hyperresponsiveness (AHR) in asthma by reducing L-arginine bioavailability for the nitric oxide (NO) synthase isozymes. The product of arginase activity, L-ornithine, can be metabolized into polyamines by ornithine decarboxylase. We tested the hypothesis that increases in L-ornithine-derived polyamines contribute to AHR in mouse models of allergic airways inflammation. After measuring significantly increased polyamine levels in sputum samples from human subjects with asthma after allergen challenge, we used acute and subacute ovalbumin sensitization and challenge mouse models of allergic airways inflammation and naive mice to investigate the relationship of AHR to methacholine and polyamines in the lung. We found that spermine levels were elevated significantly in lungs from the acute model, which exhibits robust AHR, but not in the subacute murine model of asthma, which does not develop AHR. Intratracheal administration of spermine significantly augmented airways responsiveness to methacholine in both naive mice and mice with subacute airways inflammation, and reduced nitrite/nitrate levels in lung homogenates, suggesting that the AHR developed as a consequence of inhibition of constitutive NO production in the airways. Chronic inhibition of polyamine synthesis using an ornithine decarboxylase inhibitor significantly reduced polyamine levels, restored nitrite/nitrate levels to normal, and abrogated the AHR to methacholine in the acute model of allergic airways inflammation. We demonstrate that spermine increases airways responsiveness to methacholine, likely through inhibition of constitutive NO synthesis. Thus, inhibition of polyamine production may represent a new therapeutic target to treat airway obstruction in allergic asthma.

Decreased cardiac mitochondrial tetrahydrobiopterin in a rat model of pressure overload.

Sustained cardiac pressure overload induces mitochondrial dysfunction and apoptosis of cardiomyocytes leading to pathological cardiac hypertrophy and dysfunction. Mitochondrial nitric oxide synthase (NOS) appears to cause uncoupling, which produces reactive oxygen species (ROS) instead of nitric oxide (NO), by a decrease in the cofactor tetrahydrobiopterin (BH4). This study focused on examining the changes in mitochondrial BH4 levels during cardiac pressure overload. Chronic cardiac pressure overload was generated by abdominal aortic banding in rats. Levels of BH4 and its oxidized form were measured in the mitochondria isolated from the left ventricle (LV) and the post-mitochondrial supernatants. Chronic aortic banding increased blood pressure, and induced cardiac hypertrophy and fibrosis. Notably, the BH4 levels were decreased while its oxidized forms were increased in LV mitochondria, but not in the post-mitochondrial supernatants containing the cytosol and microsome. Anti-neuronal NOS antibody-sensitive protein was detected in the cardiac mitochondria. Moreover, continuous administration of BH4 to rats with pressure overload increased mitochondrial BH4 levels and reduced cardiac fibrosis and matrix metallopeptidase activity, but not cardiac hypertrophy. These findings show the possibility that NOS uncoupling by decreased cardiac mitochondrial BH4 levels is implicated, at least in part, in the development of cardiac fibrosis, leading to cardiac dysfunction induced by pressure overload.

Reactive oxygen and nitrogen species in sepsis-induced hepatic microvascular dysfunction.

OBJECTIVE AND DESIGN: Hepatic microvascular dysfunction is a critical event in the development of liver failure during sepsis. Activated blood cells and reactive oxygen and nitrogen species (RONS) have been implicated in the pathogenesis of sepsis. METHODS: Intravital-videomicroscopy was used to determine whether RONS contribute to the recruitment of leukocytes/platelets in the hepatic microvasculature during sepsis. Six hours following cecal-ligation and puncture (CLP), disturbances of the hepatic microvasculature were assessed in WT-mice (C57Bl/6 J; n = 8), in mice lacking gp91(phox)(n = 5), overexpressing superoxide-dismutase (SOD, n = 8), in WT-mice treated with a NOS-inhibitor (L-NAME, n = 5), lacking nNOS, eNOS or iNOS (n = 5 each), treated with the NO-donor DetaNO (n = 5), in WT-mice treated with gadolinium-chloride (GdCl(2), n = 5) and compared to a group of WT-mice following a sham operation (n = 8). Six hours post-CLP, the adhesion of leukocytes and platelets in terminal hepatic venules (THV) and sinusoids was quantified. RESULTS: In WT-mice, CLP elicited increases in the number of adherent leukocytes and platelets. Similar responses to CLP were noted in mice overexpressing SOD or lacking either eNOS or gp91(phox). The blood-cell recruitment was significantly blunted in septic iNOS-knockout mice and this response was reversed by pre-treatment with DetaNO. CONCLUSION: These findings suggest that iNOS-derived NO is a determinant of the pro-inflammatory phenotype assumed by the hepatic microvasculature during sepsis.

Inflammasome-dependent IFN-γ drives pathogenesis in Streptococcus pneumoniae meningitis.

The pathology associated with Streptococcus pneumoniae meningitis results largely from activation of immune-associated pathways. We systematically investigated the production of IFN subtypes, as well as their influence on pathology, in a mouse model of S. pneumoniae meningitis. Despite the occurrence of a mixed IFN type I/II gene signature, no evidence for production or involvement of type I IFNs in disease progression was found. In contrast, type II IFN (IFN-γ) was strongly induced, and IFN-γ(-/-) mice were significantly protected from severe disease. Using intracellular cytokine staining and targeted cell-depletion approaches, NK cells were found to be the dominant source of IFN-γ. Furthermore, production of IFN-γ was found to be dependent upon ASC and IL-18, indicating that an ASC-dependent inflammasome pathway was responsible for mediating IFN-γ induction. The influence of IFN-γ gene deletion on a range of processes known to be involved in bacterial meningitis pathogenesis was examined. Although neutrophil numbers in the brain were similar in infected wild-type and IFN-γ(-/-) mice, both monocyte recruitment and CCL2 production were less in infected IFN-γ(-/-) mice compared with infected wild-type controls. Additionally, gene expression of NO synthase was strongly diminished in infected IFN-γ(-/-) mice compared with infected controls. Finally, bacterial clearance was enhanced in IFN-γ(-/-) mice, although the underlying mechanism remains unclear. Together, these data suggest that inflammasome-dependent IFN-γ contributes via multiple pathways to pathology during S. pneumoniae meningitis.

Suppression of lipopolysaccharide and galactosamine-induced hepatic inflammation by red grape pomace.

Grape pomace is generated in the production process of wine and grape juices and is an industrial waste. This study investigated whether an intake of grape pomace was able to suppress chronic inflammation induced by lipopolysaccharide (LPS) and galactosamine (GalN) in vivo. When Sprague-Dawley rats were orally given methanolic extracts from red and white grape pomace, the extracts inhibited the LPS/GalN-evoked activation of nuclear factor-κB (NF-κB) dose-dependently, and red grape pomace exerted a stronger effect than white grape one. Next, rats were fed an AIN93 M-based diet containing 5% red grape pomace for 7 days, followed by the intraperitoneal injection of LPS and GalN. The intake of the red grape pomace-supplemented diet was found to suppress the LPS/GalN-induced activation of NF-κB and expression of inducible nitric oxide synthase and cyclooxygenase-2 proteins. These results suggest that red grape pomace may contain an abundance of effective compound(s) for anti-inflammatory action.

Functional changes in myeloid-derived suppressor cells (MDSCs) during tumor growth: FKBP51 contributes to the regulation of the immunosuppressive function of MDSCs.

Myeloid-derived suppressor cells (MDSCs) are increased by tumor-derived factors and suppress anti-tumor immunity. MDSCs obtained at a late time point after tumor injection had stronger suppressive activity than MDSCs obtained at an early time point, as measured by T cell proliferation assays. To find factors in MDSCs that change during tumor growth, we analyzed gene expression profiles from MDSCs at different time points after tumor injection. We found that immune response-related genes were downregulated but protumor function-related genes were upregulated in both monocytic MDSCs (Mo-MDSCs) and polymorphonuclear granulocytic MDSCs (PMN-MDSCs) at the late time point. Among differentially expressed genes, FK506 binding protein 51 (FKBP51), which is a member of the immunophilin protein family and plays a role in immunoregulation, was increased in the Mo-MDSCs and PMN-MDSCs isolated from the late time points. Experiments using small interfering RNA and a chemical inhibitor of FKBP51 revealed that FKBP51 contributes to the regulation of the suppressive function of MDSCs by increasing inducible NO synthase, arginase-1, and reactive oxygen species levels and enhancing NF-κB activity. Collectively, our data suggest that FKBP51 is a novel molecule that can be targeted to regulate the immunosuppressive function of MDSCs.