Regulation Of iNOS Expression Research Articles

Studies of the Impact of the Bifidobacterium Species on Inducible Nitric Oxide Synthase Expression and Nitric Oxide Production in Murine Macrophages of the BMDM Cell Line

Bifidobacterium species are one of the most important probiotic microorganisms which are present in both, infants and adults. Nowadays, growing data describing their healthy properties arise, indicating they could act at the cellular and molecular level. However, still little is known about the specific mechanisms promoting their beneficial effects. Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), is involved in the protective mechanisms in the gastrointestinal tract, where it can be provided by epithelial cells, macrophages, or bacteria. The present study explored whether induction of iNOS-dependent NO synthesis in macrophages stems from the cellular action of Bifidobacterium species. The ability of ten Bifidobacterium strains belonging to 3 different species (Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium animalis) to activate MAP kinases, NF-κB factor, and iNOS expression in a murine bone-marrow-derived macrophages cell line was determined by Western blotting. Changes in NO production were determined by the Griess reaction. It was performed that the Bifidobacterium strains were able to induce NF-қB-dependent iNOS expression and NO production; however, the efficacy depends on the strain. The highest stimulatory activity was observed for Bifidobacterium animalis subsp. animals CCDM 366, whereas the lowest was noted for strains Bifidobacterium adolescentis CCDM 371 and Bifidobacterium longum subsp. longum CCDM 372. Both TLR2 and TLR4 receptors are involved in Bifidobacterium-induced macrophage activation and NO production. We showed that the impact of Bifidobacterium on the regulation of iNOS expression is determined by MAPK kinase activity. Using pharmaceutical inhibitors of ERK 1/2 and JNK, we confirmed that Bifidobacterium strains can activate these kinases to control iNOS mRNA expression. Concluding, the induction of iNOS and NO production may be involved in the protective mechanism of action observed for Bifidobacterium in the intestine, and the efficacy is strain-dependent.

The ERK-p38MAPK-STAT3 Signalling Axis Regulates iNOS Expression and Salmonella Infection in Senescent Cells.

The cellular changes occurring due to senescence like proliferation arrest, increase in free radical levels, and secretion of pro-inflammatory cytokines have been well studied, but its associated alteration in intracellular signalling networks has been scarcely explored. In this study, we examine the roles of three major kinases viz. p38 MAPK, ERK, and STAT3 in regulating iNOS expression and thereby the levels of the free radical Nitric oxide in senescent cells. Our study revealed that these kinases could differentially regulate iNOS in senescent cells compared to non-senescent cells. Further, we tested the physiological relevance of these alterations with Salmonella infection assays and established an inter-regulatory network between these kinases unique to infected senescent cells. Overall, our findings show how key signalling networks may be rewired in senescent cells rendering them phenotypically different.

Autonomous regulation of inducible nitric oxide synthase and cytochrome P450 2E1-mediated oxidative stress in maneb- and paraquat-treated rat polymorphs

Maneb (MB)- and paraquat (PQ)-induced oxidative stress in rat polymorphonuclear leukocytes (PMNs) is regulated in parallel by cytochrome P450 2E1 (CYP2E1) and inducible nitric oxide synthase (iNOS). However, mechanism underlying their regulation is not yet understood. The study investigated the role of nuclear factor- kappa B (NF-κB) and mitogen-activated protein kinase/extracellular signal regulated kinase/protein kinase C (MEK/ERK/PKC) pathway in the regulation of iNOS- and CYP2E1-induced oxidative stress in PMNs. MB + PQ-induced changes in nitrite content, lipid peroxidation (LPO), iNOS expression/activity and inflammatory mediators were alleviated by aminoguanidine (AG), an iNOS inhibitor, without any change in CYP2E1. Alternatively, diallyl sulphide (DAS), a CYP2E1 inhibitor, rescued from MB + PQ-induced changes in CYP2E1 activity/expression, free radical generation, superoxide dismutase (SOD) activity, LPO and pro-inflammatory cytokines without any alterations in nitrite content and iNOS activity/expression. Pyrrolidine dithiocarbamate (PDTC), NF-κB inhibitor, did not alter CYP2E1 but mitigated free radical generation, SOD activity, LPO, nitrite content, iNOS activity/expression and levels of pro-inflammatory cytokines (tumor necrosis factor-α, interleukine-1β and interleukine-4). Ex-vivo treatment with MEK inhibitor (PD98059), ERK1/2 inhibitor (AG126) or PKC inhibitor (rottlerin) ameliorated MB + PQ-induced increase in free radical generation and CYP2E1 activity/expression in PMNs. While PD98059 and AG126 abated MB + PQ-induced increase in ERK1/2, PKC-α/δ and CYP2E1 levels, rottlerin restored PKC-α/δ and CYP2E1 towards normalcy without affecting ERK1/2 level in MB + PQ-treated group. The results suggest that iNOS and CYP2E1 contributing to MB + PQ-induced oxidative stress in rat PMNs exhibit differential regulatory mechanisms. The inflammatory mediators regulate iNOS expression while CYP2E1 expression is triggered via MEK-ERK1/2-PKC pathway.

A role of aryl hydrocarbon receptor (AhR) in mesenchymal stem cells

Abstract Aryl hydrocarbon receptor (AhR), a ligand-induced transcription factor, is known to be involved in the balance between inflammatory and suppressive/regulatory T cell activity. Mesenchymal stem cells (MSCs) possess immunomodulatory activities, including suppression of T- and B-cell activation. MSC-mediated immunosuppression occurs via nitric oxide (NO) in mice. Here, we isolated MSCs from Ahr−/− bone marrow in mice and found less immunosuppressive function compared to the wild type (WT)-MSCs. When MSCs are co-cultured with T cells in the presence of anti-CD3 and anti-CD28 antibodies, the AhR-deficient MSCs less inhibited T-cell proliferation, CD25 and CD122 expression and cytokine productions such as IFN-γ, IL-17A compared with wild type MSCs. In addition, we found AhR-deficient MSCs produce less inducible nitric oxide synthase (iNOS) and NO compared with wild type MSCs, when MSCs were stimulated with IFN-γ, TNF-α, and IL-1β. Since MSCs has been used for the treatment of graft-vs-host disease (GvHD), we injected the WT and AhR-deficient MSCs into the GvHD mice. The AhR-deficient MSCs showed less therapeutic efficacy compared to the WT MSCs. We think that the AhR is involved in the production of NO via regulating iNOS expression which is required for the therapeutic efficacy of MSCs.

HDAC6 Mediates Macrophage iNOS Expression and Excessive Nitric Oxide Production in the Blood During Endotoxemia.

Excessive nitric oxide (NO) production and NO-mediated nitrative stress contribute to vascular dysfunction, inflammation, and tissue injury in septic shock. New therapeutic targets are urgently needed to provide better control of NO level during septic shock. In the present study, we investigated the role of HDAC6 in the regulation of NO production and nitrative stress in a mouse model of endotoxin-induced septic shock. HDAC6 deficient mice and a specific HDAC6 inhibitor were utilized in our studies. Our data clearly indicate that HDAC6 is an important mediator of NO production in macrophages. HDAC6 mediates NO production through the regulation of iNOS expression in macrophages. HDAC6 up-regulates iNOS expression in macrophages by modulating STAT1 activation and IRF-1 expression. HDAC6 inhibition potently blocked endotoxin-induced STAT1 activation and iNOS expression in macrophages. Furthermore, HDAC6 contributes to excessive NO production and nitrotyrosine level in the blood and promotes iNOS expression in the lung tissues during septic shock. Our data reveal a novel HDAC6/STAT1/iNOS pathway that mediates excessive NO production and nitrative stress in septic shock.

Imperatorin suppresses IL-1β-induced iNOS expression via inhibiting ERK-MAPK/AP1 signaling in primary human OA chondrocytes

Joint inflammation is a key player in the pathogenesis of osteoarthritis (OA). Imperatorin, a plant-derived small molecule has been reported to have anti-inflammatory properties; however, its effect on chondrocytes is not known. Here, we investigated the effects of Imperatorin on interleukin-1β (IL-1β) induced expression of inducible nitric oxide synthase (iNOS) and nitric oxide production in primary human OA chondrocytes and cartilage explants culture under pathological conditions and explored the associated signaling pathways. We pretreated chondrocytes or explants with Imperatorin (50 μM) followed by IL-1β (1 ng/ml), and the culture supernatant was used to determine the levels of nitrite production by Griess assay and chondrocytes were harvested to prepare cell lysate or RNA for gene expression analysis of iNOS by Western blot or qPCR and in explants by immunohistochemistry (IHC). Pretreatment of primary chondrocytes and cartilage explants with Imperatorin suppressed IL-1β induced expression of iNOS and NO production. Imperatorin blocked the IL-1β-induced phosphorylation of ERK-MAPK/AP1 signaling pathway to suppress iNOS expression. The role of ERK in the regulation of iNOS expression was verified by using ERK inhibitor. Interestingly, we also found that Imperatorin binds to iNOS protein and inhibits its activity in vitro. Our data demonstrated that Imperatorin possess strong anti-inflammatory activity and may be developed as a therapeutic agent for the management of OA.

The Potential Role of iNOS in Ovarian Cancer Progression and Chemoresistance.

Inducible nitric oxide synthase (iNOS), the enzyme responsible for nitric oxide (NO) production, is not present in most cells under normal conditions. The expression of its mRNA, as well as its protein synthesis and full enzymatic activity, undergoes multilevel regulation including transcriptional and posttranscriptional mechanisms, the availability of iNOS substrate and cofactors and oxygen tension. However, in various malignant diseases, such as ovarian cancer, the intracellular mechanisms controlling iNOS are dysregulated, resulting in the permanent induction of iNOS expression and activation. The present review summarizes the multistaged processes occurring in normal cells that promote NO synthesis and focuses on factors regulating iNOS expression in ovarian cancer. The possible involvement of iNOS in the chemoresistance of ovarian cancer and its potential as a prognostic/predictive factor in the course of disease development are also reviewed. According to the available yet limited data, it is difficult to draw unequivocal conclusions on the pros and cons of iNOS in ovarian cancer. Most clinical data support the hypothesis that high levels of iNOS expression in ovarian tumors are associated with a greater risk of disease relapse and patient death. However, in vitro studies with various ovarian cancer cell lines indicate a correlation between a high level of iNOS expression and sensitivity to cisplatin.

Crosstalk between TAp73 and TGF-β in fibroblast regulates iNOS expression and Nrf2-dependent gene transcription

Inducible nitric oxide synthase (iNOS) activity produces anti-tumor and anti-microbial effects but also promotes carcinogenesis through mutagenic, immunosuppressive and pro-angiogenic mechanisms. The tumor suppressor p53 contributes to iNOS downregulation by repressing induction of the NOS2 gene encoding iNOS, thereby limiting NO-mediated DNA damages. This study focuses on the role of the p53 homologue TAp73 in the regulation of iNOS expression. Induction of iNOS by immunological stimuli was upregulated in immortalized MEFs from TAp73−/− mice, compared to TAp73+/+ fibroblasts. This overexpression resulted both from increased levels of NOS2 transcripts, and from an increased stability of the protein. Limitation of iNOS expression by TAp73 in wild-type cells is alleviated by TGF-β receptor I inhibitors, suggesting a cooperation between TAp73 and TGF-β in suppression of iNOS expression. Accordingly, downregulation of iNOS expression by exogenous TGF-β1 was impaired in TAp73−/− fibroblasts. Increased NO production in these cells resulted in a stronger, NO-dependent induction of Nrf2 target genes, indicating that the Nrf2-dependent adaptive response to nitrosative stress in fibroblasts is proportional to iNOS activity. NO-dependent induction of two HIF-1 target genes was also stronger in TAp73-deficient cells. Finally, the antimicrobial action of NO against Trypanosoma musculi parasites was enhanced in TAp73−/− fibroblasts. Our data indicate that tumor suppressive TAp73 isoforms cooperate with TGF-β to control iNOS expression, NO-dependent adaptive responses to stress, and pathogen proliferation.

Interaction of mTOR and iNOS pathways in protection against Ischemia/Reperfusion injury.

Chronic morphine (CM) treatment increases the phosphorylation of the mammalian target of rapamycin (mTOR), which confers neuroprotection against ischemia/reperfusion (I/R) injury. Besides its important regulatory role in the proliferation, metabolism, and survival of cells, the mTOR is critically involved in intracellular signaling events during I/R injury. In the present study, we investigated the interaction between the expressions of the mTOR and inducible nitric oxide synthase (iNOS) and their possible protective effects on hippocampal neurons against I/R injury in morphine-dependent mice. Additive doses of morphine were administered for 5 days to BALB/c mice so as to induce CM preconditioning before I/R injury. Global brain ischemia was induced via the occlusion of bilateral common carotid arteries for 30 min. CM attenuated iNOS expression, NO production, and malondialdehyde activity in the hippocampal tissue. Pretreatment with rapamycin, the inhibitor of mTOR, abolished all the above mentioned effects of CM. These findings suggested that CM acted through the mTOR signaling pathways to regulate iNOS expression and oxidative state in the hippocampal tissue after I/R injury.

Induction of inducible nitric oxide synthase (iNOS) in Porphyromonas gingivalis LPS-treated mouse macrophage cell line (RAW264.7) requires Toll-like receptor 9.

The aim of this study is to investigate the involvement of TLR9 in the regulation of iNOS expression and nitric oxide (NO) production in Porphyromonas gingivalis LPS-treated mouse macrophages. Mouse macrophage cell line (RAW264.7) was transfected with siRNAs against TLR9 and then stimulated with P. gingivalis LPS. At indicated time points, the activated cells were lysed. Gene and protein expression of iNOS were determined by RT-PCR and immunoblotting, respectively. The level of nitric oxide (NO) production in the supernatant of the activated cells was determined by Griess reaction assay. Depletion of TLR9 in mouse macrophages demonstrated the markedly decreased iNOS gene and protein expression by P. gingivalis LPS compared to those of the wild-type or control siRNA transfected cells. In consistent with these results, the level of NO secretion was also significantly diminished in TLR9-depleted cells after challenged with P. gingivalis LPS. These results indicate that TLR9 involves in the regulation of the iNOS expression and the NO secretion in P. gingivalis LPS-treated macrophages.

Tyrosine kinase Fyn regulates iNOS expression in LPS-stimulated astrocytes via modulation of ERK phosphorylation

The high concentrations of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in activated glial cells in response to neuroinflammatory stimuli have neurotoxic effects on the brain. At basal levels, iNOS expression is low, and proinflammatory stimuli induce iNOS expression in astrocytes, microglia, and oligodendrocytes. Fyn, a non-receptor tyrosine kinase, regulates iNOS expression in several types of immune cells. However, its role in stimulated astrocytes is less clear. In this study, we investigated the role of Fyn in the regulation of lipopolysaccharide (LPS)-induced iNOS expression in astrocytes from mice and rats. Intracerebroventricular LPS injections in cortical regions enhanced iNOS mRNA and protein levels, which were increased in Fyn-deficient mice. Accordingly, LPS-induced nitrite production was enhanced in primary astrocytes cultured from Fyn-deficient mice or rats. Similar results were observed in cultured astrocytes after the siRNA-induced knockdown of Fyn expression. Finally, we observed increased LPS-induced extracellular signal-regulated protein kinase (ERK) activation in Fyn-deficient astrocytes. These results suggested that Fyn has a regulatory role in iNOS expression in astrocytes during neuroinflammatory responses.

Cell-specific regulation of iNOS by AMP-activated protein kinase in primary rat hepatocytes

BackgroundAMP-activated protein kinase (AMPK) regulates several metabolic pathways in hepatocytes that are critical to the hepatic response to sepsis and shock. Induction of nitric oxide synthesis is an important response to sepsis, inflammation and shock and many of the stimuli that upregulate inducible nitric oxide synthase (iNOS) also activate AMPK. AMPK inhibits nitric oxide (NO) production in skeletal and cardiac muscle cells, but the role of AMPK in regulating iNOS expression in hepatocytes has not been determined. Materials and methodsPrimary cultured rat hepatocytes were preincubated with an AMPK inhibitor, AMPK activators, or transfected with AMPK siRNA before being treated with the proinflammatory cytokines interleukin-1β (IL-1β) and interferon-γ (IFNγ). The hepatocyte cell lysate and culture supernatants were collected for Western blot analysis and Griess assay. ResultsIL-1β and IFNγ markedly upregulated iNOS expression and AMPK phosphorylation. IL-1β + IFNγ–induced NO production and iNOS expression were significantly decreased in hepatocytes treated with the AMPK inhibitor compound C and AMPK knockdown by AMPK siRNA. Cytokine-induced iNOS expression was increased by AMPK activators 1-oxo-2-(2H-pyrrolium-1-yl)-1H-inden-3-olate, AMPK signaling activator III and AICA-riboside. Compound C upregulated Akt and c-Jun N-terminal kinase phosphorylation but decreased IκBα phosphorylation. AICA-riboside exerted opposite effects on these signaling pathways in hepatocytes. ConclusionsIn contrast to other cell types, AMPK increased IL-1β + IFNγ–induced NO production and iNOS expression through the Akt, c-Jun N-terminal kinase, and NF-κΒ signaling pathways in primary hepatocytes. These data suggest that AMPK-altering medications used clinically may have subsequent effects on iNOS expression and proinflammatory signaling pathways.

Artemin transiently increases iNOS expression in primary cultured trigeminal ganglion neurons.

Artemin, a member of the glial cell line-derived neurotrophic factor family, is an important cytokine and a critical participant in trigeminal pain disorders such as tongue pain and migraine. However, the mechanisms underlying artemin's activity are largely unknown. In the present study, we used primary cultured trigeminal ganglion neurons (TGNs) to determine the effect of artemin on the expression of the inducible form of nitric oxide synthase (iNOS), which is released in response to painful and inflammatory stimuli. Following artemin treatment, western blot analysis showed that the protein level of iNOS was transiently elevated after artemin treatment for 15min (p<0.05). Immunofluorescence revealed that both the expressions of iNOS and GFRα3 were significantly up-regulated after artemin treatment for 15min. In addition, iNOS expression induced by artemin was co-localized with GFRα3 and TUJ-1 in primary cultured TGNs, respectively. Our results indicate a previously unknown role of artemin in regulating iNOS expression in primary cultured TGNs, and regulation of iNOS might be involved in the mechanism through which artemin participates in the trigeminal pain pathway.

Influence of a High-Fat Diet on Cardiac iNOS in Female Rats

Overexpression of inducible nitric oxide synthase (iNOS) is a key link between high-fat (HF) diet induced obesity and cardiovascular disease. Oestradiol has cardioprotective effects that may be mediated through reduction of iNOS activity/expression. In the present study, female Wistar rats were fed a standard diet or a HF diet (42% fat) for 10 weeks. iNOS gene and protein expressions were measured in heart tissue. HF-fed rats exhibited a significant increase in cardiac iNOS mRNA by 695% (p<.05), iNOS protein level by 248% (p<0.01), without changes in nitrate/nitrite levels. Expression of CD36 protein in plasma membranes was increased by 37% (p<0.05), while the concentration of free fatty acids (FFA) was reduced by 25% (p<0.01) in HF-fed rats. Expression of the p50 subunit of nuclear factor-kB (NFkB-p50) in heart was increased by 77% (p<0.01) in HF-fed rats. Expression of protein kinase B (Akt) and extracellular signalregulated kinases 1/2 (ERK1/2) were unchanged between the groups. There was a significant increase in the ratio of phospho-Akt/total Akt but not for phospho-ERK1/2/total ERK1/2 in HF-fed rats. Estrogen receptor-α levels (by 50%; p<0.05) and serum oestradiol concentrations (by 35%; p<0.05) were shown to be significantly reduced in HF-fed rats. Our results revealed that a HF diet led to increased iNOS expression, most likely via a mechanism involving Akt and NFκB-p50 proteins. Decreased levels of oestradiol and ERα protein in the HF-fed group, in combination with increased iNOS levels are consistent with the hypothesis that oestradiol has a cardioprotective effect through its ability to regulate iNOS expression.

TLR2 signal influences the iNOS/NO responses and worm development in C57BL/6J mice infected with Clonorchis sinensis

BackgroundAlthough the responses of inducible nitric oxide synthase (iNOS) and associated cytokine after Clonorchis sinensis infection have been studied recently, their mechanisms remain incompletely understood. In this study, we investigated the effects of toll-like receptor 2 (TLR2) signals on iNOS/nitric oxide (NO) responses after C. sinensis infection. We also evaluated the correlations between iNOS responses and worm development, which are possibly regulated by TLR2 signal.MethodsTLR2 wild-type and mutant C57BL/6 J mice were infected with 60 C. sinensis metacercariae, and the samples were collected at 30, 60, 90 and 120 days post-infection (dpi). The total serum NO levels were detected using Griess reagent after nitrate was reduced to nitrite. Hepatic tissue samples from the infected mice were sliced and stained with hematoxylin and eosin (HE) to observe worm development in the intrahepatic bile ducts. The iNOS mRNA transcripts in the splenocytes were examined by real time reverse transcriptase polymerase chain reaction (qRT-PCR), and iNOS expression was detected by immunohistochemistry.ResultsDeveloping C. sinensis juvenile worms were more abundant in the intrahepatic bile ducts of TLR2 mutant mice than those of TLR2 wild-type mice. However, no eggs were found in the faeces of both mice samples. The serum levels of total NO significantly increased in TLR2 mutant mice infected with C. sinensis at 30 (t(5) = 2.595, P = 0.049), 60 (t(5) = 7.838, P = 0.001) and 90 dpi (t(5) = 3.032, P = 0.029). Meanwhile, no changes occurred in TLR2 wild-type mice compared with uninfected controls during the experiment. The iNOS expression in splenocytes showed unexpected higher background levels in TLR2 mutant mice than those in TLR2 wild-type mice. Furthermore, the iNOS mRNA transcripts in splenocytes were significantly increased in the TLR2 wild-type mice infected with C. sinensis at 30 (t(5) = 5.139, P = 0.004), 60 (t(5) = 6.138, P = 0.002) and 90 dpi (t(5) = 6.332, P = 0.001). However, the rising of iNOS transcripts dropped under the uninfected control level in the TLR2 mutant mice at 120 dpi (t(5) = -9.082, P < 0.0001). Both total NO and iNOS transcripts were significantly higher in the TLR2 mutant mice than those in the TLR2 wild-type mice at 30 (t(5) = 3.091/2.933, P = 0.027/0.033) and 60 dpi (t(5) = 2.667/6.331, P = 0.044/0.001), respectively. In addition, the remarkable increase of iNOS expressions was immunohistochemically detected in the splenic serial sections of TLR2 wild-type mice at 30 and 60 dpi. However, the expressions of iNOS were remarkably decreased in the splenocytes of both TLR2 wild-type and mutant mice at 120 dpi.ConclusionsThese results demonstrate that TLR2 signal plays an important role in the regulation of iNOS expression after C. sinensis infection. TLR2 signal is also beneficial to limiting worm growth and development and contributing to the susceptibility to C. sinensis in which the iNOS/NO reactions possibly participate.

Increased intracellular Ca2+ decreases cisplatin resistance by regulating iNOS expression in human ovarian cancer cells

Previous studies have reported that intracellular Ca2+ signals and inducible nitric oxide synthase (iNOS) are involved in cell apoptosis. However, the role of iNOS in cisplatin resistance in ovarian cancer remains unclear. Here, we demonstrate that SKOV3/DDP ovarian cancer cells were more resistant to cisplatin than were SKOV3 ovarian cancer cells. The expression of intracellular Ca2+ and iNOS was more strongly induced by cisplatin in SKOV3 cells than in SKOV3/DDP cells. TAT-conjugated IP3R-derived peptide (TAT-IDPS) increased cisplatin-induced iNOS expression and apoptosis in SKOV3/DDP cells. 2-Aminoethoxydiphenyl borate (2-APB) decreased cisplatin-induced iNOS expression and apoptosis in SKOV3 cells. Thus, iNOS induction may be a valuable strategy for improving the anti-tumor efficacy of cisplatin in ovarian cancer.

A Cytokine Signalling Network for the Regulation of Inducible Nitric Oxide Synthase Expression in Rheumatoid Arthritis

In rheumatoid arthritis (RA), nitric oxide (NO) is implicated in inflammation, angiogenesis and tissue destruction. The enzyme inducible nitric oxide synthase (iNOS) is responsible for the localised over-production of NO in the synovial joints affected by RA. The pro- and anti-inflammatory cytokines stimulate the synovial macrophages and the fibroblast-like synoviocytes to express iNOS. Therefore, the cytokine signalling network underlying the regulation of iNOS is essential to understand the pathophysiology of the disease. By using information from the literature, we have constructed, for the first time, the cytokine signalling network involved in the regulation of iNOS expression. Using the differential expression patterns obtained by re-analysing the microarray data on the RA synovium and the synovial macrophages available in the Gene Expression Omnibus (GEO) database, we aimed to establish the role played by the network genes towards iNOS regulation in the RA synovium. Our analysis reveals that the network genes belonging to interferon (IFN) and interleukin-10 (IL-10) pathways are always up-regulated in the RA synovium whereas the genes which are part of the anti-inflammatory transforming growth factor-beta (TGF-β) signalling pathway are mostly down-regulated. We observed a consistent up-regulation of the transcription factor signal transducers and activators of transcription 1 (STAT1) in the RA synovium and the macrophages. Interestingly, we found a consistent up-regulation of the iNOS interacting protein ras-related C3 botulinum toxin substrate 2 (RAC2) in the RA synovium as well as the macrophages. Importantly, we have constructed a model to explain the impact of IFN and IL-10 pathways on Rac2-iNOS interaction leading to over-production of NO and thereby causing chronic inflammation in the RA synovium. The interplay between STAT1 and RAC2 in the regulation of NO could have implications for the identification of therapeutic targets for RA.

Abstract LB-265: IRF8 is an essential transcriptional activator of iNOS although MDSCs up-regulate iNOS expression via an IRF8-independent mechanism

Abstract Nitric Oxide Synthase (NOS) is a family of enzymes that catalyzes L-arginine metabolism to generate nitric oxide (NO). There are three main isoforms of NOS, named neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS or NOS2). These three distinct isoforms of NOS have different cellular localization, regulation, and catalytic properties. iNOS is the most extensively studied one among these three NOS isoforms. iNOS was originally identified in myeloid cells, and it is known that its expression is often induced after myeloid cell activation by endotoxins or cytokines to generate NO which then acts as a defense effector to suppress invading microorganisms or neoplastic cells. It is known that IFNγ signaling pathway and NF-κB synergistically regulate iNOS expression, and that IFN regulatory factor 8 (IRF8) enhances interaction between IFNγ signaling and NF-κB in regulation of iNOS expression. However, the molecular mechanism underlying iNOS expression regulation in myeloid cell lineage is still not fully understood. We report here that IRF8 is an essential transcription activator of iNOS in myeloid cells since IRF8-deficient myeloid cells lose iNOS expression. Furthermore, a key phenotype of IRF8 KO mice is the mass accumulation of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs), suggesting that IRF8 is a key suppressor of MDSC differentiation under physiological conditions. On the other hand, accumulation of MDSCs is a hallmark of cancers and iNOS expression is dramatically elevated in tumor-induced MDSCs, despite that tumor-induced MDSCs exhibit diminished IRF8 expression. Furthermore, the IFNγ signaling pathway is impaired and NF-κB subunits are undetectable in tumor-induced MDSCs. Our data therefore indicate that iNOS expression is activated by an IRF8-independent mechanism that is also IFNγ and NF-κB-independent in tumor-induced MDSCs under pathological conditions. At the functional level, we determined that iNOS-KO mice exhibit lower tumor incidence as compared to WT mice. However, the sizes of established tumors are not different between iNOS-KO mice and WT mice. Taken together, our data determine that IRF8 is an essential transcriptional activator of iNOS expression in myeloid cells under physiological conditions, although iNOS expression is activated by an IFNγ-, NF-κB-, and IRF8-independent mechanism in tumor-induced MDSCs under pathological conditions. iNOS acts as a suppressor of tumor cell colonization but not tumor growth. Citation Format: Mohammed M. Ibrahim, Amy V. Paschall, Priscilla S. Redd, Kebin Liu. IRF8 is an essential transcriptional activator of iNOS although MDSCs up-regulate iNOS expression via an IRF8-independent mechanism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-265.

Abstract PR005: TGF-beta1 primed myeloid derived suppressor cells decrease tumor growth and lose their ability to inhibit T cell proliferation via iNOS downregulation

Abstract Background: Myeloid derived suppressor cells or MDSC are a heterogeneous population of bone marrow-derived cells that consist of myeloid progenitor and immature. Cancer-induced pro-inflammatory signals recruit MDSC from the bone marrow and maintain them in an undifferentiated state. MDSCs inhibit T cell proliferation via up regulation of iNOS and ROS thereby suppressing antitumor immune responses. MDSC have also been shown to promote tumor growth by stimulating angiogenesis and other mechanisms. TGF-b1 is a pleiotropic cytokine abundantly expressed in the tumor microenvironment with diverse effects on myeloid, lymphoid and tumor cells. The aim of this study is to determine the effect of TGF-b1 in the generation and function of MDSC, including its effects on T cell proliferation and tumor growth. Methods: Ex vivo MDSC generation: Bone marrow progenitor cells were derived from WT C57bl/6 mice and co-cultured with MTEC (mouse tonsil epithelial cells transformed with HPV16 E6+E7 oncogenes and H-Ras ) tumor supernatants in the presence or absence of TGF-β1 for 5 days at 370C. Cells were then harvested, processed into single cell suspensions, and stained for MDSC surface markers, DAF-DA (to determine nitric oxide levels),iNOS and other functional markers analysis was performed by flow cytometry (FACS). MDSC functional assay: A) T cell proliferation assay MDSCs were generated in the presence or absence of TGF-β1 with supernatants from MTEC cells and co-cultured with CFSE labeled T cells activated with anti CD3 and anti CD28 antibodies. T cell proliferation was measured by using CFSE dilution, which was analyzed by flow cytometry. B) Effect of MDSC on tumor growth Control and TGF-β1 conditioned MDSC were co-cultured with MTEC tumor (grown as spheroids) for 72 hrs at the end of which histological sections of the spheroids were prepared and analyzed for tumor proliferation by Ki-67 staining. Sorted MDSCs were also co-cultured with T-hep3 cells grown in a monolayer (human oral cancer line) and tumor growth was determined by flow cytometry. Results: While control MDSC suppressed T cell proliferation in a dose-dependent fashion, we observed that TGF-β1 primed MDSCs lost the ability to inhibit T cell proliferation. Further, TGF-β1 primed MDSC inhibited tumor growth in an ex vivo co-culture system. Histological sections of tumor spheroid / MDSC co-cultures revealed diminshed ki-67 expression in spheroids cultured with TGF-B1 conditioned MDSC compared to control. Upon further examining the cellular mechanism, it was seen that TGF-β1 treated MDSCs down regulate iNOS expression and produced decreased amounts of nitric oxide compared to their control counterparts, without altering the expression of other MDSC functional markers like arginase, PD-1 and PD-L1. Conclusions: We conclude that TGF-β1 reprograms MDSC via an iNOS/NO dependent mechanism to a) T cell suppressive capacity and b) inhibit tumor cell growth. These observations have a direct translational implication wherein the inherent pro-tumor nature of MDSCs could potentially be reprogrammed with TGF-β1 and directed toward the tumor thereby suppressing tumor growth. Citation Format: Padmini Jayaraman, Falguni Parikh, Rosemarie Krupar, Robin Parihar, Indu Varier, Andrew Sikora. TGF-beta1 primed myeloid derived suppressor cells decrease tumor growth and lose their ability to inhibit T cell proliferation via iNOS downregulation. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr PR005.

Hemozoin Regulates iNOS Expression by Modulating the Transcription Factor NF-κB in Macrophages.

Hemozoin (Hz) is released from ruptured erythrocytes during malaria infection caused by Plasmodium sp., in addition the malaria infected individuals are prone to bacterial sepsis. The molecular interactions between Hz, bacterial components and macrophages remains poorly investigated. In this report, we investigated the combinatorial immune-modulatory effects of phagocytosed Hz, Interferon gamma (IFNγ) or lipopolysaccharide (LPS) in macrophages. Macrophages were treated with various concentrations of commercial synthetic Hz, and surprisingly it did not result in inducible nitric oxide synthase (iNOS) expression. However, when macrophages were pretreated with Hz and then challenged with IFNγ or LPS, there was a differential impact on iNOS expression. There was an increase in iNOS expression when macrophages were pre-treated with Hz and subsequently treated with IFNγ when compared to IFNγ alone. Whereas iNOS expression was reduced when Hz phagocytosed macrophages were stimulated with LPS compared to LPS alone. Furthermore, there was an increased activation of NF-κB in Hz phagocytosed macrophages that were challenged with IFNγ. The interaction between Hz and macrophages has an impact on iNOS expression.