Systemic NO production during (septic) shock depends on parenchymal and not on hematopoietic cells: in vivo iNOS expression pattern in (septic) shock

Objective. To investigate the immunomodulatory activity of a traditional Sri Lankan concoction of Coriandrum sativum L. and Coscinium fenestratum (Gaertn.) Colebr., which is a Sri Lankan traditional medicine used to relieve inflammation and cold. Methods. In vivo anti-inflammatory activity was tested using carrageenan-induced rat paw-edema model. Mechanism of anti-inflammatory activity was assessed by investigating the production of nitric oxide (NO), expression of iNOS enzyme, and reactive oxygen species (ROS) by rat peritoneal cells. The membrane stabilizing activity was also tested. The antibody response was determined by assessing the specific haemagglutination antibodies raised against sheep red blood cells. Results. The three doses of freeze-dried concoction used ((human equivalent dose (HED)—183 mg/kg) 2 × HED and 1/2HED; n = 6 rats/group) showed significant inhibition of paw edema compared to water control at 3rd–5th hours (p<0.05). Both HED and 1/2HED exhibited marked anti-inflammatory activity (72–83% inhibition at 4th-5th hours; p<0.05). The HED of the concoction showed significant inhibition of NO (77.5 ± 0.73%, p<0.001) and ROS production (26.9 ± 2.55%; p<0.01) by rat peritoneal cells. Inhibition of NO production in the concoction treated rat peritoneal cells was confirmed by the lack of iNOS expression. The concoction also exhibited significant membrane stabilizing activity (IC50 = 0.0006 μg/ml; p=0.001). HED resulted in a significantly high induction of specific antibody production against SRBC antigens as detected by SRBC haemagglutination assay (mean day 14 titers 253.3 compared to control: 66.7) (p<0.01). Conclusions. The traditional Sri Lankan concoction of C. sativum and C. fenestratum demonstrated potent in vivo anti-inflammatory activity, significant reduction of ROS, and NO production by rat peritoneal cells and the lack of iNOS expression confirmed the low NO production. The increased membrane stability also supports the anti-inflammatory activity of the concoction. Further, this concoction induced a significantly high antibody response reflecting its immunostimulatory activity. Together these results scientifically validate the therapeutic use of the concoction of C. sativum and C. fenestratum in Sri Lankan traditional medicinal system for immunomodulatory effects.

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microRNA-146a promotes mycobacterial survival in macrophages through suppressing nitric oxide production

Scientific Reports ◽

10.1038/srep23351 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 31

Author(s):

Miao Li ◽

Jinli Wang ◽

Yimin Fang ◽

Sitang Gong ◽

Meiyu Li ◽

...

Keyword(s):

Nitric Oxide ◽

Mycobacterial Infection ◽

Nuclear Factor Κb ◽

Inos Expression ◽

Transcriptional Level ◽

No Production ◽

Promising Therapeutic Target ◽

Mitogen Activated Protein

Abstract Macrophages play a crucial role in host innate anti-mycobacterial defense, which is tightly regulated by multiple factors, including microRNAs. Our previous study showed that a panel of microRNAs was markedly up-regulated in macrophages upon mycobacterial infection. Here, we investigated the biological function of miR-146a during mycobacterial infection. miR-146a expression was induced both in vitro and in vivo after Mycobacterium bovis BCG infection. The inducible miR-146a could suppress the inducible nitric oxide (NO) synthase (iNOS) expression and NO generation, thus promoting mycobacterial survival in macrophages. Inhibition of endogenous miR-146a increased NO production and mycobacterial clearance. Moreover, miR-146a attenuated the activation of nuclear factor κB and mitogen-activated protein kinases signaling pathways during BCG infection, which in turn repressed iNOS expression. Mechanistically, miR-146a directly targeted tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) at post-transcriptional level. Silencing TRAF6 decreased iNOS expression and NO production in BCG-infected macrophages, while overexpression of TRAF6 reversed miR-146a-mediated inhibition of NO production and clearance of mycobacteria. Therefore, we demonstrated a novel role of miR-146a in the modulation of host defense against mycobacterial infection by repressing NO production via targeting TRAF6, which may provide a promising therapeutic target for tuberculosis.

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Intra-Articular Route for the System of Molecules 14G1862 from Centella asiatica: Pain Relieving and Protective Effects in a Rat Model of Osteoarthritis

Nutrients ◽

10.3390/nu12061618 ◽

2020 ◽

Vol 12 (6) ◽

pp. 1618

Author(s):

Laura Micheli ◽

Lorenzo Di Cesare Mannelli ◽

Luisa Mattoli ◽

Sara Tamimi ◽

Enrico Flamini ◽

...

Keyword(s):

Rat Model ◽

Centella Asiatica ◽

Inos Expression ◽

Histological Evaluation ◽

Spontaneous Pain ◽

No Production ◽

Protective Effects ◽

Reference Drug

Current pharmacological therapies for the management of chronic articular diseases are far from being satisfactory, so new strategies need to be investigated. We tested the intra-articular pain relieving properties of a system of molecules from a characterized Centella asiatica extract (14G1862) in a rat model of osteoarthritis induced by monoiodoacetate (MIA). 14G1862 (0.2–2 mg mL−1) was intra-articularly (i.a.) injected 7 days after MIA, behavioural and histological evaluations were performed 14, 30 and 60 days after treatments. Moreover, the effect of 14G1862 on nitrate production and iNOS expression in RAW 264.7 macrophages stimulated with LPS was assessed. In vitro, 14G1862 treatment attenuated LPS-induced NO production and iNOS expression in a comparable manner to celecoxib. In vivo, 14G1862 significantly reduced mechanical allodynia and hyperalgesia, spontaneous pain and motor alterations starting on day 14 up to day 60. The efficacy was higher or comparable to that evoked by triamcinolone acetonide (100 μg i.a.) used as reference drug. Histological evaluation highlighted the improvement of several morphological parameters in MIA + 14G1862-treated animals with particularly benefic effects on joint space and fibrin deposition. In conclusion, i.a. treatment with Centella asiatica is a candidate to be a novel effective approach for osteoarthritis therapy.

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Inducible nitric oxide synthase in the lung and exhaled nitric oxide after hyperoxia

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.277.3.l636 ◽

1999 ◽

Vol 277 (3) ◽

pp. L636-L644 ◽

Cited By ~ 8

Author(s):

Giovanni Cucchiaro ◽

Arthur H. Tatum ◽

Michael C. Brown ◽

Enrico M. Camporesi ◽

John W. Daucher ◽

...

Keyword(s):

Nitric Oxide ◽

Septic Shock ◽

Nitric Oxide Synthase ◽

Inducible Nitric Oxide Synthase ◽

Inos Expression ◽

Control Group ◽

No Production ◽

Exhaled Air ◽

Oxide Synthase ◽

Inducible Nitric Oxide

The effect of hyperoxia on nitric oxide (NO) production in intact animals is unknown. We described the effects of hyperoxia on inducible nitric oxide synthase (iNOS) expression and NO production in the lungs of rats exposed to high concentrations of oxygen. Animals were placed in sealed Plexiglas chambers and were exposed to either 85% oxygen (hyperoxic group) or 21% oxygen (negative control group). Animals were anesthetized after 24 and 72 h of exposure and were ventilated via a tracheotomy. We measured NO production in exhaled air (ENO) by chemiluminescence. The lungs were then harvested and processed for detection of iNOS by immunohistochemistry and Western blotting analysis. The same experiments were repeated in animals exposed to hyperoxia for 72 h after they were infused with l-arginine. We used rats that were injected intraperitoneally with Escherichia coli lipopolysaccharide to induce septic shock as a positive control group. Hyperoxia and septic shock induced expression of iNOS in the lung. However, ENO was elevated only in septic shock rats but was normal in the hyperoxic group. Exogenous infusion of l-arginine after hyperoxia did not increase ENO. To exclude the possibility that in the hyperoxic group NO was scavenged by oxygen radicals to form peroxynitrite, lungs were studied by immunohistochemistry for the detection of nitrotyrosine. Nitrotyrosine was found in septic shock animals but not in the hyperoxic group, further suggesting that NO is not synthesized in rats exposed to hyperoxia. We conclude that hyperoxia induces iNOS expression in the lung without an increase in NO concentration in the exhaled air.

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MKP-1 switches arginine metabolism from nitric oxide synthase to arginase following endotoxin challenge

AJP Cell Physiology ◽

10.1152/ajpcell.00137.2006 ◽

2007 ◽

Vol 293 (2) ◽

pp. C632-C640 ◽

Cited By ~ 42

Author(s):

Leif D. Nelin ◽

Xianxi Wang ◽

Qun Zhao ◽

Louis G. Chicoine ◽

Tamara L. Young ◽

...

Keyword(s):

Nitric Oxide ◽

Peritoneal Macrophages ◽

Mapk Signaling ◽

Inos Expression ◽

No Production ◽

Wild Type ◽

Endotoxin Challenge ◽

Raw264.7 Macrophages ◽

Urea Production

l-Arginine (l-arg) is metabolized to nitric oxide (NO) by inducible NO synthase (iNOS) or to urea and l-ornithine (l-orn) by arginase. NO is involved in the inflammatory response, whereas arginase is the first step in polyamine and proline synthesis necessary for tissue repair and wound healing. Mitogen-activated protein kinases (MAPK) mediate LPS-induced iNOS expression, and MAPK phosphatase-1 (MKP-1) plays a crucial role in limiting MAPK signaling in macrophages. We hypothesized that MKP-1, by attenuating iNOS expression, acts as a switch changing l-arg metabolism from NO production to l-orn production after endotoxin administration. To test this hypothesis, we performed studies in RAW264.7 macrophages stably transfected with an MKP-1 expression vector in thioglyollate-elicited peritoneal macrophages harvested from wild-type and Mkp-1−/− mice, as well as in vivo in wild-type and Mkp-1−/− mice. We found that overexpression of MKP-1 resulted in lower iNOS expression and NO production but greater urea production in response to LPS. Although deficiency of MKP-1 resulted in greater iNOS expression and NO production and lower urea production in response to LPS, neither the overexpression nor the deficiency of MKP-1 had any substantial effect on the expression of the arginases.

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In Vitro and in Vivo Therapeutics of β-Thujaplicin on Lps-Induced Inflammation in Macrophages and Septic Shock in Mice

International Journal of Immunopathology and Pharmacology ◽

10.1177/039463201202500106 ◽

2012 ◽

Vol 25 (1) ◽

pp. 39-48 ◽

Cited By ~ 34

Author(s):

M-F. Shih ◽

L-Y. Chen ◽

P-J. Tsai ◽

J-Y. Cherng

Keyword(s):

Septic Shock ◽

Mortality Rate ◽

Molecular Mechanisms ◽

No Production ◽

Active Constituent ◽

Tnf Α ◽

Anti Inflammatory ◽

Inflammatory Effect

β-thujaplicin, an active constituent from Chamaecyparis obtusa, has been shown to have acaricidal and antimicrobial effects. Very few studies have focused on the potential of the anti-inflammatory effect of β-thujaplicin. Moreover, its capability of inhibiting inflammatory mediators e.g. TNF-α gene transcription, nitric oxide (NO) and prostaglandin E2, remains unknown. Besides those molecular mechanisms behind the anti-inflammatory effect of β-thujaplicin, solid proof of its effectiveness in vivo has not yet been studied. In our study, in vitro effects of β-thujaplicin were verified on RAW 264.7 macrophages which were stimulated by LPS. Indomethacin was used as a positive control. The inducible NO production after stimulation was measured by Griess reagent. PGE2, IL-6 and TNF-α were measured by ELISA methods. Protein expressions of iNOS, COX2, and NF-κB were evaluated by Western blotting. Septic ICR mice were administered 20 mg/kg of LPS and then the mortality rate was monitored. Within the concentration range which was devoid of cytotoxicty, β-thujaplicin exhibited a clear dose-dependent inhibition on LPS-induced NO production. Furthermore, β-thujaplicin inhibited LPS-induced PGE2, IL-6, and TNF-α production as well as iNOS, COX2, and NF-κB protein expression more substantially potent than indomethacin. In agreement with the in vitro study, β-thujaplicin was shown to be effective in vivo for inhibiting LPS-induced NO and TNF-α production and a significant decrease in mortality rate of mice suffering from septic shock was observed. This study demonstrates the potential of β-thujaplicin in treatment of inflammation and sepsis. These effects occur through an efficient blockage of TNF-α and iNOS production, β-thujaplicin efficacy is comparable to that of indomethacin thus it can be a substitution but bear less depletion of PGE2, making this compound very promising in clinical applications.

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Polyphenols from Cymbopogon citratus inhibit iNOS expression and NO production – a promising source of new anti-inflammatory drugs

Planta Medica ◽

10.1055/s-0030-1264523 ◽

2010 ◽

Vol 76 (12) ◽

Author(s):

V Francisco ◽

A Figueirinha ◽

B Neves ◽

C Garcia-Rodriguez ◽

M Lopes ◽

...

Keyword(s):

Inos Expression ◽

No Production ◽

Cymbopogon Citratus ◽

Inflammatory Drugs ◽

Anti Inflammatory ◽

Promising Source

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Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽

10.1182/blood.v89.11.3919 ◽

1997 ◽

Vol 89 (11) ◽

pp. 3919-3924 ◽

Cited By ~ 328

Author(s):

Jean C.Y. Wang ◽

Monica Doedens ◽

John E. Dick

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Allogeneic Transplantation ◽

Functional Assay ◽

Hematopoietic Stem ◽

Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

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BTK Inhibition Reverses MDSC-Mediated Immunosuppression and Enhances Response to Anti-PDL1 Therapy in Neuroblastoma

Cancers ◽

10.3390/cancers13040817 ◽

2021 ◽

Vol 13 (4) ◽

pp. 817

Author(s):

Mehreen Ishfaq ◽

Timothy Pham ◽

Cooper Beaman ◽

Pablo Tamayo ◽

Alice L. Yu ◽

...

Keyword(s):

T Cell ◽

Tumor Growth ◽

Checkpoint Inhibitors ◽

No Production ◽

Free Survival ◽

T Cell Infiltration ◽

Car T ◽

Checkpoint Inhibitor Therapy

MDSCs are immune cells of myeloid lineage that plays a key role in promoting tumor growth. The expansion of MDSCs in tumor-bearing hosts reduces the efficacy of checkpoint inhibitors and CAR-T therapies, and hence strategies that deplete or block the recruitment of MDSCs have shown benefit in improving responses to immunotherapy in various cancers, including NB. Ibrutinib, an irreversible molecular inhibitor of BTK, has been widely studied in B cell malignancies, and recently, this drug is repurposed for the treatment of solid tumors. Herein we report that BTK is highly expressed in both granulocytic and monocytic murine MDSCs isolated from mice bearing NB tumors, and its increased expression correlates with a poor relapse-free survival probability of NB patients. Moreover, in vitro treatment of murine MDSCs with ibrutinib altered NO production, decreased mRNA expression of Ido, Arg, Tgfβ, and displayed defects in T-cell suppression. Consistent with these findings, in vivo inhibition of BTK with ibrutinib resulted in reduced MDSC-mediated immune suppression, increased CD8+ T cell infiltration, decreased tumor growth, and improved response to anti-PDL1 checkpoint inhibitor therapy in a murine model of NB. These results demonstrate that ibrutinib modulates immunosuppressive functions of MDSC and can be used either alone or in combination with immunotherapy for augmenting antitumor immune responses in NB.

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Neuropilin-1 on hematopoietic cells as a source of vascular development

Blood ◽

10.1182/blood-2002-01-0119 ◽

2003 ◽

Vol 101 (5) ◽

pp. 1801-1809 ◽

Cited By ~ 35

Author(s):

Yoshihiro Yamada ◽

Yuichi Oike ◽

Hisao Ogawa ◽

Yasuhiro Ito ◽

Hajime Fujisawa ◽

...

Keyword(s):

Endothelial Cells ◽

Fetal Liver ◽

Vascular Development ◽

Hematopoietic Cells ◽

Vegf Receptor ◽

Knock Out ◽

Neuropilin 1 ◽

Vasculogenesis And Angiogenesis

Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-165 (VEGF165) and acts as a coreceptor that enhances the function of VEGF165 through VEGF receptor-2 (VEGFR-2). Studies using transgenic and knock-out mice of NP-1 indicated that this molecule is important for vascular development as well as neuronal development. We recently reported that clustered soluble NP-1 phosphorylates VEGFR-2 on endothelial cells with a low dose of VEGF165 and rescues the defective vascularity of the NP-1−/− embryo in vitro and in vivo. Here we show that NP-1 is expressed by CD45+ hematopoietic cells in the fetal liver, can bind VEGF165, and phosphorylates VEGFR-2 on endothelial cells. CD45+NP-1+ cells rescued the defective vasculogenesis and angiogenesis in the NP-1−/− P-Sp (para-aortic splanchnopleural mesodermal region) culture, although CD45+NP-1− cells did not. Moreover, CD45+NP-1+ cells together with VEGF165 induced angiogenesis in an in vivo Matrigel assay and cornea neovascularization assay. The extracellular domain of NP-1 consists of “a,” “b,” and “c” domains, and it is known that the “a” and “c” domains are necessary for dimerization of NP-1. We found that both the “a” and “c” domains are essential for such rescue of defective vascularities in the NP-1 mutant. These results suggest that NP-1 enhances vasculogenesis and angiogenesis exogenously and that dimerization of NP-1 is important for enhancing vascular development. In NP-1−/− embryos, vascular sprouting is impaired at the central nervous system (CNS) and pericardium where VEGF is not abundant, indicating that NP-1–expressing cells are required for normal vascular development.

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