Nitric Oxide Production In Macrophages Research Articles

Biological Actions, Electrical Conductance and Silicon-Containing Microparticles of Arsenicum Album Prepared in Plastic and Glass Vials.

According to the "silica hypothesis" formulated to explain homeopathy, the information of starting materials would be transferred to cells by silica nanoparticles detached from the glassware walls by serial dilution and agitation through epitaxy. We compared the biological activity, electrical current and silicon microparticle content (by means of scanning electron microscopy/energy-dispersive X-ray spectroscopy) of high dilutions (HDs) of arsenic prepared in plastic and glass vials to investigate the role of silica in their biological effects in vitro. Co-cultures of macrophages and yeast (Saccharomyces cerevisiae) were treated with different HDs of arsenic prepared in plastic and glass vials. Macrophage morphology, phagocytosis index, nitric oxide (NO), and cytokine production were evaluated. Measurable amounts of silicon microparticles were detected only in the HDs prepared in glass vials, but ultra-centrifugation eliminated them. Specific and non-specific results were observed. Non-specific pro-inflammatory effects were seen in all dilutions prepared in plastic vials, including elevation of pro-inflammatory cytokines, NO and macrophage phagocytic index. Only the 200th centesimal dilution of arsenic produced specific decrease in interleukin-6 production in macrophages, and it was independent of the vial type or the presence of microparticles of silica in the medicine samples. The nature of the vials had an impact on the electric flow in the respective fluids. The non-specific, pro-inflammatory effects might be attributed to organic residuals detached from the vials' plastic walls during manipulation. Instead, specific silica-independent effects of the homeopathic medicine can be attributed to the decrease of interleukin-6 after treatment with the 200th centesimal dilution of arsenic.

Effect of Aloe Vera Herbal Extract on Innate Immunity in Balb/c Mice Against Candida Albicans

Background: Based on the reports, Aloe vera extract, a herbal medicine, has antimicrobial and anti-inflammatory effects. Also, this medicinal plant has considerable effects on innate immunity through cytokine secretion which resist fungal infections such as Candida albicans. Moreover, Aloe vera is considered as immunomodulatory agent and regulate inflammatory responses. Materials and Methods: In this study, twenty Balb/c mice were divided into five groups: four groups of infected mice with Candida albicans treated with different doses of Aloe vera extract and one healthy control group. Then, macrophage vital activity was measured by NBT assay. Nitric Oxide (NO) production as representative of macrophage activity was assayed by MTT test. Tumor Necrosis Factor alpha (TNF-α) and Interleukin-12 (IL-12) cytokines were measured by ELISA assay.Results: Based on our study results, macrophage vital activity and NO production decreased significantly. However, TNF-α and IL-12 cytokines increased significantly in mice model infected by Candida albicans. Conclusion: Aloe vera extract could enhance macrophage activity through increase in TNF-α and IL-12 cytokines in Candida albicans infections. Therefore, Aloe vera could stimulate innate immune responses to eliminate Candida albicans.

Sodium bicarbonate regulates nitric oxide production in mouse macrophage cell lines stimulated with lipopolysaccharide and interferon γ

Macrophages are known to play pivotal roles in host-defense through inflammation via both innate and acquired immune systems, and so on. In an earlier paper, we showed the influence of the type of culture medium, Ham's F-12 or DMEM, on activated macrophage phenotypes induced by LPS and IFNγ. The production of nitric oxide (NO), pro-inflammatory cytokines such as TNFα and IL-1β, as well as the induction of superoxide-generating activity of J774.1/JA-4 cells was different depending on the type of culture medium. In this present study, we showed that sodium bicarbonate concentrations in these culture media, 14 mM in Ham's F-12 and 44 mM in DMEM, were crucial to explaining the differences in the induction of activated macrophage phenotypes, especially in that of iNOS. A concentration-dependent change in pH did not result in any remarkable difference in iNOS expression or NO production. Moreover, high sodium bicarbonate in culture medium increased not only NO production but also TNFα production in the activated macrophages. These results suggest that sodium bicarbonate would be a regulatory factor of NO and TNFα production in macrophages and that its concentration has a crucial role in macrophage activation.

PKC δ gene can induce macrophages to release inflammatory factors against Mycobacterium tuberculosis infection.

To investigate the effect of PKC δ gene on the anti-tuberculosis activity of macrophages and the mechanism. Bone marrow cells of PKC δ knockout mice and wild-type mice were cultured and L929 cells were induced to differentiate into macrophages. Lipopolysaccharide (LPS) and trehalose 6,6'-dimycolate (TDM) were used to stimulate macrophages respectively. After 24 and 96 hours, cells and the supernatant were collected to evaluate the inflammatory cytokines produced by macrophages using ELISA method. Real-time PCR was performed to detect the expression of macrophage mRNA level and nitric oxide (NO) production of macrophages was measured by NO assay. The results showed that, after TDB stimulation, IL-1β, IL-6, and other cytokines, as well as NO produced by macrophages of PKC δ knockout mice, were significantly decreased (p < 0.01) compared with the wild-type mice. In PKC δ knockout macrophages, the above protein-coding genes were also decreased significantly at the transcriptional level (p < 0.01). PKC δ can enhance the anti-tuberculosis capacity of macrophages by inducing to the release of inflammatory factors by macrophages.

Protective effects of total alkaloids from Dendrobium crepidatum against LPS-induced acute lung injury in mice and its chemical components

Dendrobium crepidatum was one of the sources of Herba Dendrobii, a famous and precious traditional Chinese medicine. Indolizine-type alkaloids are the main characteristic ingredients of D. crepidatum, which possesses a variety of changeable skeletons. In the present study, we found that the total alkaloids of D. crepidatum (TAD) can inhibit the production of nitric oxide (NO) in lipopolysaccharide (LPS)-activated macrophages and showed protective effects against LPS-induced acute lung injury (ALI) in mice through downregulating the TLR4-mediated MyD88/MAPK signaling pathway. Further phytochemical study showed that six previously undescribed indolizine-type compounds, including a racemic mixture (dendrocrepidine A-E) were isolated from TAD. Meanwhile, dendrocrepidine F was separated into a pair of enantiomers by a chiral chromatography, and their absolute configurations were assigned by single-crystal X-ray diffraction analysis. The isomer (−)-dendrocrepidine F showed higher anti-inflammatory effects by inhibiting NO production in LPS-treated macrophages with an IC50 value of 13.3 μM. Taken together, indolizine-type alkaloids are the active components of D. crepidatum through downregulating the TLR4-mediated pathway, indicating some kind of therapy of TAD for ALI treatment.

Differential effects of experimental hyperthyroidism on declined immunity of broiler chicken.

Thyroid hormones (THs) are involved in the development of lymphoid organs and regulation of immune function in birds. However, their role as an immune-modulator in the hyperthyroid state is still debatable. To explore the interrelationship of thyroxine (T4 ) and the immune system, chicks were divided into three groups. Group I was comprised of control birds, who received the basal diet while group II and III were given diets supplemented with 5μg and 10μg thyroxine/kg feed, respectively, from 15 to 28days of age. Cell-mediated immune response was evaluated through in vitro abdominal macrophage phagocytosis assay, macrophage nitric oxide (NO) production, heterophil-to-lymphocyte (H:L) ratio and delayed-type hypersensitivity response against phytohemagglutinin (PHA). Humoural immune response was assessed through serum IgG and IgM antibody production against sheep red blood cells (SRBCs) and antibody production against infectious bronchitis virus (IBV). Sampling was carried out at 7, 14 and 21days of treatment. Results have shown higher levels (p<.001) of circulating T4 in both treatment groups compared to the control group. There was a lower (p<.05) macrophage engulfment percentage, an increase in H:L ratio (p<.001) in treated birds, while their NO production remained higher (p<.05) in thyroxine supplemented groups after bacterial lipopolysaccharide stimulation. The humoural immune response revealed a significant decline (p<.001) in IgG, IgM antibody production against SRBCs but IBV circulating antibodies increased with age. In conclusion, hyperthyroidism has a strong co-relation with decreased immune performance of birds.

Salidroside Exerts Anti-Inflammatory Effect by Reducing Nuclear Factor κB and Nitric Oxide Production in Macrophages

Salidroside Exerts Anti-Inflammatory Effect by Reducing Nuclear Factor κB and Nitric Oxide Production in Macrophages

Salidroside Exerts Anti-Inflammatory Effect by Reducing Nuclear Factor κB and Nitric Oxide Production in Macrophages

Salidroside Exerts Anti-Inflammatory Effect by Reducing Nuclear Factor κB and Nitric Oxide Production in Macrophages

Emu Oil Reduces LPS-Induced Production of Nitric Oxide and TNF-α but not Phagocytosis in RAW 264 Macrophages.

Emu is the second-largest extant bird native to Australia. Emu oil, obtained from the emu's fat deposits, is used as an ingredient in cosmetic skincare products. Emu oil has been reported to improve several inflammatory symptoms; however, the mechanisms of these anti-inflammatory effects are largely unknown. This study investigated the effects of emu oil on the inflammatory macrophage response in vitro. A murine macrophage cell line, RAW 264, was incubated in culture media supplemented with or without emu oil and stimulated with lipopolysaccharide (LPS). We determined phagocytic activity by measuring the number of fluorescent microspheres taken up by the cells. The phagocytic activity of RAW 264 cells in the presence of LPS was unaffected by emu oil. We also determined production of nitric oxide (NO) and tumor necrosis factor (TNF)-α in the culture medium using the Griess reaction and an enzyme-linked immunosorbent assay, respectively, and the protein expression of inducible NO synthase (iNOS) using western blotting. The results indicated that emu oil reduced the LPS-induced production of NO, TNF-α, and iNOS expression in a dose-dependent manner. The results suggested that emu oil does not reduce the phagocytic clearance rate of inflammatory matter; however, it does reduce the production of NO and TNF-α in macrophages. These latter products enhance the inflammatory response and emu oil thereby demonstrated anti-inflammatory properties.

Cathepsin B-Mediated NLRP3 Inflammasome Formation and Activation in Angiotensin II -Induced Hypertensive Mice: Role of Macrophage Digestion Dysfunction

Background/Aims: Angiotensin II (Ang II) is an octapeptide hormone that plays a significant role in mediating hypertension. Although hypertension is considered a chronic inflammatory disease, the molecular basis of the sterile inflammatory response involved in hypertension remains unclear. Methods: We investigated the role of macrophage NLRP3 inflammasomes in engulfing and digesting microbes, a key macrophage function, and in early onset of hypertension-associated macrophage injury using biochemical analyses, gene silencing, molecular biotechnology, immunofluorescence, and microbiology. Results: Ang II stimulation decreased nitric oxide (NO) release and macrophage digestion in cultured THP-1 cells and markedly increased NLRP3 inflammasome formation and activation. NO release and macrophage digestion were restored by NLRP3 inflammasome inhibition with isoliquiritigenin and gene silencing. This Ang II-induced upregulation of NLRP3 inflammasomes in macrophages was attributed to lysosomal damage and release of cathepsin B. Mechanistically, losartan, a nonpeptide Ang II receptor antagonist, decreased Ang II-induced NLRP3 inflammasome activation, lysosomal membrane permeability, lysosomal cathepsin B release, and macrophage digestion dysfunction. Similarly, Ang II-induced macrophage microbe digestion and NO production, which were blocked by ATI gene silencing. In addition, in vivo experiments showed that the bacteria scavenging function was clearly decreased in macrophages from Ang II-induced hypertensive mice. Conclusion: Angiotensin II enhances lysosomal membrane permeabilization and the consequent release of lysosomal cathepsin B, resulting in activation of the macrophage NLRP3 inflammasome. This may contribute to NO mediation of dysfunction in digesting microbes.

Heat-killed Tsukamurella inchonensis reduces lipopolysaccharide-induced inflammatory responses in activated murine peritoneal macrophages

Tsukamurella inchonensis (T. inchonensis) is an aerobic species of Actinomycetales which has immunomodulatory activities when used as a suspension of killed bacilli. Here, the effects of T. inchonensis on lipopolysaccharide-induced inflammatory responses in mouse peritoneal macrophages have been examined. Peritoneal macrophages were harvested by lavaging with ice cold phosphate-buffered saline. Macrophages acquired from mice treated with different doses of T. inchonensis for seven days were cultured with 20 U/ml interferon-γ and 10 µg/ml lipopolysaccharide for in vivo assays. Nitrite levels were measured by using the diazotization method based on the Griess reaction, an indirect technique to determine nitric oxide (NO) production. T. inchonensis inhibited lipopolysaccharide-stimulated NO production in mouse peritoneal macrophages from mice previously exposed to concentrations of 108 and 5 × 10&lt;sup&gt;7&lt;/sup&gt; CFU per flask. Also, T. inchonensis decreased lipopolysaccharide-induced production of pro-inflammatory cytokines, including interleukin-6 and tumor necrosis factor-α. Thus, it can be concluded that T. inchonensis is a powerful inhibitor of lipopolysaccharide-induced NO production in activated murine macrophages, and T. inchonensis may be useful as a novel agent for chemoprevention in inflammatory diseases.

Antioxidant, antiproliferative, and immunostimulatory effects of cell wall α-d-mannan fractions from Kluyveromyces marxianus

This study evaluated the antioxidant, antiproliferative, and immunostimulatory properties of cell wall α-d-mannan fractions from yeast Kluyveromyces marxianus CCT7735. Filter centrifugation was used to obtain four fractions (KMM-1, KMM-2, KMM-3, and KMM-4) with molecular weight ranging from 7.6 to 75.1kDa. KMM-1 and KMM-2 comprised D-mannose with traces of D-glucose, whereas other fractions contained only D-mannose. Total sugar found in samples ranged from 85.9% to 96.1%, while protein and phenolic contents were 1.21% and 0.41%, respectively. Although only KMM-1 was able to scavenge superoxide radicals, all fractions presented total antioxidant capacity as well as reducing power, hydroxyl-radical scavenging, and copper- and iron-chelating activities. No fraction was cytotoxic to HeLa cells. However, all samples inhibited the proliferation of the tumor cell Hep-G2 and presented minor cytotoxicity against normal 3T3 cells. All fractions showed mitogenic activity in macrophages and all, except KMM-4, induced nitric oxide production in macrophages, suggestive of their immunostimulatory effects.

Anti-inflammatory properties of oolong tea (Camellia sinensis) ethanol extract and epigallocatechin gallate in LPS-induced RAW 264.7 cells

ObjectiveTo evaluate the anti-inflammatory activity of oolong tea ethanol extract (OTEE) and epigallocatechin gallate (EGCG) on lipopolysaccharide-induced murine macrophage cell line (RAW 264.7). MethodsA cytotoxic assay using MTS tetrazolium was conducted to find a nontoxic level of OTEE and EGCG toward RAW 264.7 cells. Interleukins (IL-6, IL-1β), tumor necrosis factor-α (TNF-α), and cyclooxigenase-2 (COX-2) levels were measured by ELISA, and nitric oxide (NO) levels measured by a nitrate/nitrite colorimetric assay to determine the inhibition activity of OTEE and EGCG. ResultsLipopolysaccharide induction increases NO, COX-2, IL-6, IL-1β, and TNF-α levels compared with the untreated cell (negative control). The positive control, lipopolysaccharide-induced RAW 264.7 without treatments showed the highest level of all pro-inflammatory cytokines and modulators tested in this study. The positive control was used as standard to obtain OTEE and EGCG inhibition activity toward NO, COX-2, IL-6, IL-1β, and TNF-α. OTEE had a higher inhibition activity toward NO, COX-2, IL-6, and IL-1β than EGCG; the reverse was seen for TNF-α. However, both OTEE and EGCG suppressed production of NO, COX-2, IL-6, IL-1β, and TNF-α. ConclusionsOTEE and EGCG have the potential for use as anti-inflammatory drugs, which is shown by their ability to reduce the production of NO, COX-2, IL-6, IL-1β, and TNF-α in active macrophages.

Two goitrogenic 1,3-oxazolidine-2-thione derivatives from Brassicales taxa: Challenging identification, occurrence and immunomodulatory effects

1,3-Oxazolidine-2-thione derivatives are glucosinolate-related food constituents known to impart (thyreo)toxic properties to some cruciferous vegetables. In this work, 5,5-dimethyl-1,3-oxazolidine-2-thione and (-)-(R)-5-phenyl-1,3-oxazolidine-2-thione, known goitrogens, were isolated from Draba lasiocarpa Rochel (Brassicaceae) and Reseda luteola L. (Resedaceae), respectively, and were fully spectrally characterized. Subsequently, the occurrence of the two 1,3-oxazolidine-2-thiones was verified in six additional taxa out of in total 78 screened Serbian Brassicales taxa. The stereochemistry of 5-phenyl-1,3-oxazolidine-2-thione was inferred from nuclear magnetic resonance experiments with a chiral lanthanide-shift reagent, employed in this work for the first time for this type of compounds. Unexpectedly, during gas chromatography, 5-phenyl-1,3-oxazolidine-2-thione underwent an unreported thermal core isomerization (1,3-oxazolidine-2-thione to 1,3-thiazolidine-2-one). These goitrogenic volatile glucosinolate products were tested for their effect on rat macrophage viability (three assays) and nitric oxide production. It was shown that the compounds displayed different levels of cytotoxicity. All tested compounds caused a significant lactate dehydrogenase leakage, but only (R)-5-phenyl-1,3-oxazolidine-2-thione statistically significantly reduced macrophage mitochondrial activity, whereas the racemic 5-phenyl-1,3-oxazolidine-2-thione and 5,5-dimethyl-1,3-oxazolidine-2-thione had little or no effect. Again only (R)-5-phenyl-1,3-oxazolidine-2-thione exerted nitric oxide production-inhibiting properties, suggesting the higher immunomodulatory potential of this enantiomer compared with its antipode and racemic mixture.

Characterisation of a novel coumarin-based fluorescent probe for monitoring nitric oxide production in macrophages

Nitric oxide (NO) is an important effector molecule in host defence against bacterial pathogens. The development of fluorescence imaging to monitor NO production in vitro and in vivo will increase our understanding of its biological role. Recently, a novel ‘trappable’ fluorescent blue ‘turn-on’ Cu(II)-complexed coumarin-based probe (CB) has been developed to detect NO. In this study, CB was investigated to evaluate its ability to detect NO in macrophages. Using confocal microscopy, NO was successfully detected in macrophages in the presence of stimuli that induce nitric oxide synthase (iNOS), the enzyme responsible for production of NO. The time dependence and subcellular compartmentalisation of CB in macrophages were evaluated. The probe can be trapped within cells and reacts directly and specifically with NO, rendering it a promising tool for imaging NO in response to pharmacological agents that modulate its level, for example during bacterial infections.

Evaluation of the Effect of Mesenchymal Stem Cells pulsed with Caffeine on the Function of the Peritoneal Macrophages of Rat

Introduction: Pervious studies indicated that the mesenchymal stem cells (MSCs) produce adenosine and express adenosine receptors. Moreover, produced adenosine has biological effect on these cells. On the other hand, MSCs have a wide relationship with macrophages in tissues and bone marrow. The aim of the present study was to determine the effects of the MSCs treated with caffeine, as an adenosine antagonist, on the peritoneal macrophages isolated from rats. Materials & methods: After isolation of mesenchymal stem cells from bone marrow of rats, these cells pulsed with different concentration of caffeine (0, 0.1, 0.5 and 1 mM) for 48h. Then, MSCs were co-cultured with macrophages for 1 day. Then, the level of nitric oxide production and respiratory burst of macrophages after challenge with N-formylmethionine-leucyl-phenylalanine (f-MLP) or opsonized yeast were evaluated. Findings: It seems that MSCs pulsed with caffeine or without it could significantly decrease the respiratory burst and nitric oxide production of co-cultured macrophages more prominent than macrophages alone after unspecific challenged with fMLP. Although, pervious treatment of MSCs with caffeine could decrease the respiratory burst and nitric oxide production of macrophages challenged with f-MLP more prominent than MSCs without treatment. However, the potential of nitric oxide production and respiratory burst of various group after challenge with opsonized yeast didnt show any significant differences. Discussion & conclusions: Collectively, these data may offer new insight into the potential mechanisms underlying the immunomodulatory and anti-inflammatory effects of caffeine.

Identification of new CpG oligodeoxynucleotide motifs that induce expression of interleukin-1β and nitric oxide in avian macrophages.

Unmethylated CpG motifs are known to stimulate mammalian toll-like receptor-9 expressing cells such as macrophages. However, the magnitude of immune-stimulation by CpG-motif can be sequence- and host-specific, implying the importance of identifying new immune-stimulatory motifs. This study aimed to determine the frequency distribution of 256 unique hexamers CpG-motifs in the Salmonella genome and to characterize their immune-stimulatory activity in avian host. We synthesized 256 CpG oligodeoxynucleotides (CpG-ODNs) each containing triplicates of a unique hexamer CpG-motif and tested their ability to induce expression of pro-inflammatory cytokine IL-1β in avian macrophages using q-RT PCR in four rounds of screening assays. CpG-ODNs that induced significantly higher IL-1β expression were also subjected to Griess assay to determine their ability to induce nitric oxide (NO) production in avian macrophages. This analysis resulted in identification of 7 CpG-ODNs that consistently induced IL-1β expression and NO production in avian macrophages at a level similar to the expression achieved using commercially available PTO-CpG-ODN 2007 and LPS derived from Salmonella. To the best of our knowledge, this is the first report showing comprehensive screening of all possible unique CpG hexamer (n=256) motifs for their ability to induce IL-1β expression and NO production in avian macrophages. We also show that the newly identified CpG-motifs with high immune-stimulatory activity are widely distributed in Salmonella genome. The CpG-ODNs identified in this study may serve as promising immunoprophylactics to potentiate innate responses in chickens against Salmonella and other infectious agents.

Porphyromonas gulae Activates Unprimed and Gamma Interferon-Primed Macrophages via the Pattern Recognition Receptors Toll-Like Receptor 2 (TLR2), TLR4, and NOD2.

Porphyromonas gulae is an anaerobic, Gram-negative coccobacillus that has been associated with periodontal disease in companion animals. The aims of this study were to analyze the ligation of pattern recognition receptors by P. gulae and the subsequent activation of macrophages. Exposure of HEK cells transfected with Toll-like receptors (TLRs) or NOD-like receptors to P. gulae resulted in the ligation of TLR2, TLR4, and NOD2. The effects of this engagement of receptors were investigated by measuring the synthesis of nitric oxide (NO), CD86 expression, and inflammatory cytokine production by wild-type, TLR2-/-, and TLR4-/- macrophages. The addition of P. gulae to unprimed and gamma interferon (IFN-γ)-primed (M1 phenotype) macrophages significantly increased the surface expression of CD86, but only M1 macrophages produced nitric oxide. P. gulae-induced expression of CD86 on unprimed macrophages was dependent on both TLR2 and TLR4, but CD86 expression and NO production in M1 macrophages were only TLR2 dependent. P. gulae induced an increase in secretion of interleukin-1α (IL-1α), IL-1β, IL-6, IL-12p70, IL-13, tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1α (MIP-1α) by M1 macrophages compared to that by unprimed controls. Among these cytokines, secretion of IL-6 and TNF-α by M1 macrophages was dependent on either TLR2 or TLR4. Our data indicate that TLR2 and TLR4 are important for P. gulae activation of unprimed macrophages and that activation and effector functions induced in M1 macrophages by P. gulae are mainly dependent on TLR2. In conclusion, P. gulae induces a strong TLR2-dependent inflammatory M1 macrophage response which may be important in establishing the chronic inflammation associated with periodontal disease in companion animals.

4'-Hydroxywogonin suppresses lipopolysaccharide-induced inflammatory responses in RAW 264.7 macrophages and acute lung injury mice.

4'-Hydroxywogonin (4'-HW), a flavonoid, has been isolated from various plants and shown to inhibit NO production in macrophages. However, the molecular mechanisms and its in vivo activity have not been determined. Our study aimed to investigate the mechanisms underlying the anti-inflammatory effects of 4'-HW in vitro and in vivo. We showed that 4'-HW potently reduced the expression levels of COX-2 and iNOS as well as their products, prostaglandin E2 (PGE2) and nitric oxide (NO) respectively, in LPS-stimulated RAW 264.7 macrophages. 4'-HW also suppressed LPS-induced pro-inflammatory cytokines at mRNA and protein levels. Moreover, 4'-HW blocked the interaction of TAK1 and TAB1 in LPS-stimulated RAW 264.7 macrophages, resulting in an inhibition of the TAK1/IKK/NF-κB signaling pathway. Furthermore, 4'-HW also reduced the phosphorylation of MAPKs and PI3/Akt signaling pathways in LPS-stimulated RAW 264.7 macrophages. 4'-HW was also significantly decreased the intracellular reactive oxygen species (ROS) level. The effect of 4'-HW was confirmed in vivo. 4'-HW exhibited potent protective effect against LPS-induced ALI in mice. These findings indicate that 4'-HW suppresses the LPS-induced response in vitro and in vivo. It is likely that the inhibition of the TAK1/IKK/NF-κB, MAPKs and PI3/AKT signaling pathways contribute to the anti-inflammatory effects of 4'-HW. Our study suggests that 4'-HW may be an important functional constituent in the plants and has the potential value to be developed as a novel anti-inflammatory agent.

Toll-like receptor (TLR)21 signalling-mediated antiviral response against avian influenza virus infection correlates with macrophage recruitment and nitric oxide production.

Cytosine-guanosinedeoxynucleotide (CpG) DNA can be used for the stimulation of the toll-like receptor (TLR)21 signalling pathway in avian species which ultimately leads to up-regulation of gene transcription for pro-inflammatory molecules including nitric oxide and recruitment of innate immune cells. The objective of this study was to determine the antiviral effect of NO, produced in response to in ovo delivery of CpG DNA, against avian influenza virus (AIV) infection. We found that when CpG DNA is delivered at embryo day (ED)18 in ovo and subsequently challenged with H4N6 AIV at ED19 pre-hatch and day 1 post-hatching, CpG DNA reduces H4N6 AIV replication associated with enhanced NO production and macrophage recruitment in lungs. In vitro, we showed that NO originating from macrophages is capable of eliciting an antiviral response against H4N6 AIV infection. This study provides insights into the mechanisms of CpG DNA-mediated antiviral response, particularly against AIV infection in avian species.