Macrophage Nitric Oxide Release Research Articles

Anti-inflammatory effects of Lonomia obliqua caterpillar protein rlosac on macrophages and chondrocytes

Purpose: Ostheoarthritis (OA) is a disease characterized by the degeneration of articular cartilage. The role of activated macrophages in the synovial inflammation has been reported to contribute with the knee OA. Activated macrophages increase inflammatory mediators in the synovial fluid, such as IL-1β, TNF-α and nitric oxide (NO), acting on chondrocytes which in turn produce an excess of matrix metalloproteinases (MMPs) and aggrecanases ), which result in the degradation of extracellular matrix (ECM) components such as collagen II (Col-2) and aggrecan. Chondrocytes respond to these stimuli by secreting cytokines like IL-1β, TNFα, IL-6 and IL-8, and altering gene expression leading to a feedback activation of inflammation. Studies using the bristle extract of the Lonomia obliqua caterpillar (LOCBE) allowed the identification of the protein LOSAC, a cytoprotective molecule with effect on serum-deprived cells including HUVECs, fibroblasts, and neurons. Moreover, rLOSAC was reported to promote wound healing (in vivo) by increasing expression of ECM involved in tissue remodeling (as collagen), preservation of substrate attachment and ECM integrity in fibroblast, and prevention of ROS generation, suggesting an anti-oxidative activity. The aim of this work was to evaluate the cytotoxic effects of rLOSAC in chondrocytes and macrophages and to establish if this protein has anti-inflammatory activity on these types of cells. Methods: For this study, rLOSAC was obtained in bacteria system as previously reported. Normal Human Articular Primary human chondrocytes cells (NHAC-Kn) were obtained from LONZA and used until passage 6 for all the assays. Expansion and subculture of chondrocytes were performed according the supplier. Peritoneal macrophage were obtained from male Wistar rats (I. Butantan Ethical Committee CEUA n° 9139050517. For chondrocytes and macrophages inflammation was induced by treatment with 1 ng/mL IL-1β or 1 μg/mL LPS, respectively. Under these conditions, rLOSAC was added and the cytoprotective effect assessed by the MTT assay. Expression of metalloprotease-1 (MMP-1) and NF-kB-p65 translocation were analyzed by the ImageXpress Micro-Confocal High-Content Screening (HCS) with specific antibodies. Cytokines present in the supernatants, after 24h of treatment, were assayed using a Milliplex MAP Cytokine/Chemokine Magnetic Bead Panel with detection and analysis using the Multiplex system (Millipore). The kit was performed according to the manufacturer’s specification. Range of detection was in pg/mL and designed for 3 analytes for chondrocytes (IL-6, IL-8 and TNF-α), and 10 cytokines for macrophages (IL-1α, MIP-1α, IL-4, IL-6, IL-10, IL-17, GRO/KC/CINC-1, LIX, TNF-α, Rantes). Results: To verify the toxic effect of this protein, several concentrations of rLOSAC, in the presence or absence of LPS (macrophages) or IL-1β (chondrocytes) were added to the cells. None concentrations assayed were toxic to those cells. Therefore, rLOSAC showed cytoprotective effect at the nanomolar concentration, while under inflammatory conditions (in the presence of 1ng/mL IL-1β), rLOSAC was cytoprotective at the concentrations of 10 and 20 nM. Regarding chondrocytes, the expression of MMP-1 and NF-kB translocation were analyzed by HCS using IL-1β at 1ng/ml and 24h of incubation for cytokines and MMP-1 release, and 30 min for NF-κB-p65, as well as, the nuclei count. rLOSAC was able to reduce the release of IL-6, IL-8 and TNF-α in a statistically significant manner. However, rLOSAC was no able to affect the LPS-induced NO release in macrophages. rLOSAC did not induced directly NF-κB-p65 translocation. However, in the presence of 1ng/mL IL-1β, some concentrations of rLOSAC and LOCBE increased the translocation of NF-κB in chondrocytes. MMP-1 release was reduced in the presence of rLOSAC in chondrocytes. In macrophages, rLOSAC was also no toxic to the cells. Ten cytokines/chemokines were analyzed in LPS-treated or not-treated macrophages supernatants in the presence or absence of rLOSAC. rLOSAC alone was able to reduce the release of IL-4, IL-1α and TNF-α. Both, in the presence or absence of LPS, rLOSAC was able to induce the release of IL-10, a potent anti-inflammatory cytokine. Conclusions: The data presented in this report showed that the rLOSAC shows a cytoprotective and anti-inflammatory activity. Further studies will be performed with co-culture of chondrocytes and activated macrophages in order to confirm the anti-inflammatory effect of rLOSAC. This makes it clear that rLOSAC is a potential molecule to be explored in order to seek new molecular targets that may be related to osteoarthritis. The combination of HCS and cytokines release analysis had shown to be effective for the screening of new compounds or molecular targets/pathways for OA. Supported by Grant 2015/50040-4, São Paulo Research Foundation (FAPESP), GlaxoSmithKline and Instituto Butantan; FAPESP Grant 2018/04467-4, FAPESP Grant 2013/07467-1 (CETICs).

Effects of the synthetic psychedelic 2,5-dimethoxy-4-iodoamphetamine (DOI) on ethanol consumption and place conditioning in male mice.

Approximately 20 million adults in the USA have an alcohol use disorder (AUD). There are clinical and preclinical data suggesting that psychedelics may have benefits for AUD. To investigate the effects of the synthetic psychedelic 2,5-dimethoxy-4-iodoamphetamine (DOI) on the behavioral effects of ethanol. The effects of DOI were examined using ethanol-induced place conditioning (1.8g/kg ethanol) and 2-bottle choice ethanol drinking (20% v/v), using a dose of DOI (3mg/kg) that produced the maximal response in the serotonin 2A (5-HT2A) receptor-dependent head-twitch assay. Interactions between DOI and ethanol (3g/kg) were examined using the ethanol-induced loss of righting reflex procedure and blood-ethanol analysis. To examine additional mechanisms by which psychedelics may interact with ethanol, we determined whether DOI reverses ethanol-induced nitric oxide release in macrophages, a marker of inflammation. DOI significantly attenuated ethanol-induced place conditioning and ethanol drinking. DOI-induced suppression of alcohol drinking depended upon 5-HT2A receptors, was selective for alcohol over water, and was selective for high alcohol-preferring subjects. DOI had no apparent pharmacokinetic interactions with ethanol, and DOI reduced ethanol-induced nitric oxide release. Our findings demonstrate that DOI blocks ethanol place conditioning and selectively reduces voluntary ethanol consumption. This may be related to modulation of the effects of ethanol in the reward circuitry of the brain, ethanol-induced neuroinflammation, or a combination of both. Additional studies to elucidate the mechanisms through which psychedelics attenuate the effects of ethanol would inform the pathophysiology of AUD and potentially provide new treatment options.

Lipids From Trypanosoma cruzi Amastigotes of RA and K98 Strains Generate a Pro-inflammatory Response via TLR2/6.

Lipids from microorganisms are ligands of Toll like receptors (TLRs) and modulate the innate immune response. Herein, we analyze in vitro the effect of total lipid extracts from Trypanosoma cruzi amastigotes of RA and K98 strains (with polar biological behavior) on the induction of the inflammatory response and the involvement of TLRs in this process. We demonstrated that total lipid extracts from both strains induced lipid body formation, cyclooxygenase-2 expression and TNF-α and nitric oxide release in macrophages, as well as NF-κB activation and IL-8 release in HEK cells specifically through a TLR2/6 dependent pathway. We also evaluated the inflammatory response induced by total lipid extracts obtained from lysed parasites that were overnight incubated to allow the action of parasite hydrolytic enzymes, such as Phospholipase A1, over endogenous phospholipids. After incubation, these total lipid extracts showed a significantly reduced pro-inflammatory response, which could be attributed to the changes in the content of known bioactive lipid molecules like lysophospholipids and fatty acids, here reported. Moreover, analyses of total fatty acids in each lipid extract were performed by gas chromatography-mass spectrometry. Our results indicate a relevant role of T. cruzi lipids in the induction of a pro-inflammatory response through the TLR2/6 pathway that could contribute to the modulation of the immune response and host survival.

Irradiation by Gallium–Aluminum–Arsenate Diode Laser Enhances the Induction of Nitric Oxide by Porphyromonas gingivalis in RAW 264.7 Cells

Low-level laser irradiation promotes cell viability and wound healing in periodontal tissue. However, its effect on periodontal pathogenic bacteria is unknown. The purpose of this study is to investigate the biologic effect of low-level laser irradiation on Porphyromonas gingivalis. A murine macrophage cell line (RAW 264.7) was cultured and treated with gallium-aluminum-arsenate (GaAlAs) laser-irradiated P. gingivalis with varying levels of energy fluency. Gene expression of monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), interferon-β (IFN-β), and inducible nitric oxide synthase (iNOS) was examined by reverse transcription-polymerase chain reaction. Production of iNOS was determined by Western blot analysis, and nitric oxide (NO) release was assessed using Griess reagent. Flow cytometric analysis was performed to determine the activation of Toll-like receptors (TLRs) in response to P. gingivalis. The laser-irradiated P. gingivalis significantly enhanced messenger RNA and protein levels of iNOS in RAW 264.7. Although the laser irradiation on P. gingivalis did not alter the expression level of MCP-1, IL-6, and IFN-β, it showed a noticeable effect on NO production in RAW 264.7. Furthermore, the laser-irradiated P. gingivalis accelerated TLR2 activation, but not TLR4 activation. This study reveals that GaAlAs laser irradiation on P. gingivalis induced iNOS expression at the transcriptional and translation levels and increased NO release in macrophages. Moreover, it is confirmed that this process was mediated specifically by TLR2 activation. These findings suggest that low-level laser irradiation to periodontal pathogenic bacteria could be detrimental to periodontal treatments.

Ramaline inhibits LPS‐induced NO release in macrophages by regulating MAPK and NF‐κB activities.

Lipopolysaccharide (LPS) induces innate immune responses through TLR4 signaling and activated macrophages produce various inflammatory mediators including Nitric oxide (NO). NO produced by inducible NO synthase (iNOS) mediates a number of pathophysiological processes. Therefore, inhibiting NO production in macrophages is a potential therapeutic target in inflammatory diseases. Herein, we demonstrate that ramalin, a newly discovered antioxidant that is extracted from polar lichen, inhibits production of inflammatory mediators in macrophage‐like Raw 264.7 cell line. RT‐PCR and Western blot analysis demonstrated that ramalin suppressed the level of NO release through down‐regulating both mRNA and protein levels of iNOS. Western blot analysis also revealed that ramalin abrogated LPS‐induced phosphorylation of ERK, p38, and JNK as well as the expression of p65. Attenuated activity of NF‐κB by ramalin was also confirmed by Luciferase promoter assay. In addition, not only the surface level of LPS‐induced TLR4 but also its mRNA and protein levels were decreased by ramalin. Together, these results suggest that ramalin reduces NO release in macrophage through down‐regulation of TLR4 signaling which in turn suppresses MAPK and NF‐κB activities, and it would be a possible therapeutic approach to the treatment of inflammatory diseases.

Mycobacterium tuberculosis 6 kDa early secreted antigenic target stimulates activation of J774 macrophages

The influence of the 6 kDa early-secreted antigenic target (ESAT-6) of Mycobacterium tuberculosis on macrophage activation was investigated using J774 macrophage cell line. While without effect if applied alone, ESAT-6 in a dose-dependent manner enhanced nitric oxide (NO) release by IFN-γ-stimulated J774 cells. However, it completely failed to modulate NO production in J774 cells activated with E. coli lipopolysaccharide. The effect of ESAT-6 on NO synthesis in IFN-γ-activated J774 cells was accompanied by up-regulated expression of inducible NO synthase (iNOS), an enzyme that catalyzes NO formation. ESAT-6-mediated enhancement of macrophage NO release was markedly suppressed by pharmacological agents that selectively inhibit iNOS enzymatic activity or protein tyrosine kinase and mitogen-activated protein kinase activation. Finally, treatment of J774 macrophages with ESAT-6 also enhanced IFN-γ-induced expression of the surface molecules B7.1, MHC-II, and ICAM-1. Collectively, these data suggest that ESAT-6 might contribute to macrophage activation in tuberculosis.

Iron down-regulates macrophage anti-tumour activity by blocking nitric oxide production.

Although the inhibitory effect of iron on macrophage production of tumoricidal free radical nitric oxide (NO) has been reported, its possible influence on macrophage anti-tumour activity has not been established. In the present study, FeSO4 markedly reduced IFN-gamma + LPS-induced NO synthesis in mouse and rat macrophages. The effect of iron coincided with the loss of macrophage cytotoxic activity against NO-sensitive C6 rat astrocytoma and L929 mouse fibrosarcoma cell lines, as measured by MTT assay for cellular respiration and the crystal violet test for cell viability. Tumour cell survival did not improve further in the presence of FeSO4 if macrophage NO release and cytotoxicity were already blocked by aminoguanidine. In accordance with the results obtained with exogenous iron, cell membrane permeable iron chelator o-phenanthroline enhanced both macrophage NO release and anti-tumour activity. Iron also down-regulated NO production and increased the viability of L929 fibrosarcoma cells stimulated with IFN-gamma + LPS in the absence of macrophages. However, neither NO release nor cell viability was affected by iron addition to cultures of the C6 astrocytoma cell line. Iron was unable to prevent L929 and C6 cell death induced by the NO releasing chemicals SNP and SIN-1, indicating that iron-mediated inhibition of NO synthesis, rather than interference with its cytotoxic action, was responsible for the protection of tumour cells. Collectively, these results indicate that iron might protect tumour cells by reducing both macrophage and tumour cell-derived NO release.

Influence of aminoguanidine on parameters of liver injury and regeneration induced in rats by a necrogenic dose of thioacetamide.

1. When aminoguanidine, a nucleophilic hydrazine compound, was administered to rats (50 mg kg(-1) body wt) 30 min before a necrogenic dose of thioacetamide (500 mg kg(-1) body wt), significant changes related to liver injury and hepatocellular regeneration were observed. 2. The extent of necrosis was noticeably less pronounced, as detected by the peak of serum aspartate aminotransferase activity. Depletion of hepatic glutathione (GSH) and the increase in malondialdehyde concentration as markers of oxidative stress, produced by thioacetamide metabolism, were significantly diminished. However, the activity of microsomal FAD monooxygenase, the system responsible for thioacetamide oxidation, did not show significant alterations. Antioxidant enzyme systems involved in the glutathione redox cycle, such as glutathione reductase and glutathione peroxidase activities, slightly decreased following aminoguanidine pretreatment. 3. Primary cultures of peritoneal macrophages from control rats, when incubated in the presence of serum collected following thioacetamide intoxication, showed a significant decrease in nitric oxide (NO) release at 24 h, that was more pronounced in the group pretreated with aminoguanidine. However, the sharp and progressive increase in macrophage NO release, when incubated in the presence of serum obtained at 48, 72 and 96 h, were increased by aminoguanidine-pretreatment. 4. The cell population involved in DNA synthesis sharply increased in both groups at 48 h of intoxication, although the values at 0, 24, 72 and 96 h were markedly higher in the group pre-treated with aminoguanidine. Polyploidy at 72 and 96 h of intoxication was delayed by the effect of aminoguanidine and a progressive increase in the hypodiploid hepatocyte population, which reached 16% of the total at 96 h, was observed. 5. These results indicate that a single dose of aminoguanidine before thioacetamide administration, markedly diminished the severity of the liver injury by decreasing oxidative stress and lipoperoxidation, but hepatocellular regeneration was apparently unaffected probably due to an enhanced mitogenic activity.

Effect of modified LDL on the release of NO and PGI2 from rat peritoneal macrophages.

Nitric oxide (NO) and prostacyclin (PGI2) have vasodilative and anti-proliferative effects on smooth muscle cells (SMC) and an anti-aggregating action on platelets. The present study was designed to elucidate the influence of modified low density lipoprotein (LDL) on the release on NO and PGI2 from rat peritoneal macrophages. Cholesteryl ester (CE) content in macrophages markedly increased on incubation with acetylated LDL (ac-LDL), while NO release did not change. Although incubation with mildly oxidized LDL (m-ox-LDL) and highly oxidized LDL (h-ox-LDL) increased CE content in macrophages, only incubation with h-ox-LDL reduced NO release. PGI2 release from macrophages was not affected by incubation with ac-LDL, m-ox-LDL or h-ox-LDL. These results indicate that the degree of suppression of NO release in macrophages by modified LDL is related to the extent of oxidative modification of LDL itself, but not to the extent of the accumulation of CE in macrophages. Although the role of NO released from macrophages in atherosclerosis is still unclear, the observation of reduced production of NO from macrophages in response to ox-LDL may provide new insight into the role of ox-LDL in the pathogenesis of atherosclerosis.