Angiotensin II type-1 receptor antagonist attenuates LPS-induced acute lung injury

Angiotensin II is able to trigger inflammatory responses through an angiotensin II type 1 (AT1) receptor. The role of AT1 receptor in acute lung injury (ALI) is poorly understood. Mice were randomly divided into three groups (n = 40 each groups): NS group; LPS group (2 mg/kg LPS intratracheally); and LPS + ZD 7155 group, 10 mg/kg ZD 7155 (an AT1 receptor antagonist) intraperitoneally 30 min prior to LPS exposure. Samples from the lung were isolated and assayed for histopathology analyses or proinflammatory gene expressions, angiotensin II receptors expressions and nuclear factors activities. LPS exposure resulted in severe ALI, elevated levels of TNF-α and IL-1β mRNA expressions, and increased activities of NF-κB and activated protein (AP)-1. Upregulation of AT1 receptor and down-regulation of AT2 receptor were also observed after LPS challenge. Pretreatment with ZD 7155 significantly inhibited the increase of AT1 receptor expression and upregulated AT2 receptor expression. ZD 7155 also reduced the mRNA expression of TNF-α and IL-1β, inhibited the activation of NF-κB and AP-1, and improved lung histopathology. These findings suggest that antagonism of AT1 receptor inhibits the activation of NF-κB and AP-1 in the lung, which may mediate the release of TNF-α and IL-1β and contribute to LPS-induced ALI.     

Burn-induced alterations in toll-like receptor-mediated responses by bronchoalveolar lavage cells

Burn is associated with profound inflammation and activation of the innate immune system in multiple organ beds, including the lung. Similarly, toll-like receptors (TLR) are associated with innate immune activation. Nonetheless, it is unclear what impact burn has on TLR-induced inflammatory responses in the lung.MethodsMale C57BL/6 mice were subjected to burn (3rd degree, 25% TBSA) or sham procedure and 1, 3 or 7 days thereafter, bronchoalveolar lavage (BAL) fluid was collected and cells were isolated and cultured in vitro with specific TLR agonists as follows: Zymosan (TLR-2), LPS (TLR-4) and CpG-ODN (TLR-9). Supernatants were collected 48 h later and assayed for inflammatory cytokine levels (IL-1β, IL-6, IL-10, IL-17, TNF-α, KC, MCP-1, MIP-1α, MIP-1β and RANTES) by Bioplex.ResultsBAL fluid from sham and burn mice did not contain detectable cytokine levels. BAL cells, irrespective of injury, were responsive to TLR-2 and TLR-4 activation. Seven days after burn, TLR-2 and TLR-4 mediated responses by BAL cells were enhanced as evidenced by increased production of IL-6, IL-17, TNF-α, MCP-1, MIP-1β and RANTES.ConclusionsBurn-induced changes in TLR-2 and TLR-4 reactivity may contribute to the development of post-burn complications, such as acute lung injury (ALI) and adult respiratory distress syndrome (ARDS).     

Elevated level of pro inflammatory cytokine and chemokine expression in chicken bone marrow and monocyte derived dendritic cells following LPS induced maturation

The study was designed to characterize and compare chicken bone marrow and peripheral blood monocyte derived dendritic cells (chBM-DC and chMoDC) and to evaluate inflammatory cytokine and chemokine alterations in response upon LPS stimulation. Typical morphology was observed in DCs from 48 h of culture using recombinant chicken GM-CSF and IL-4. Maturation of DCs with LPS (1 μg/ml) showed significant up regulation of mRNA of surface markers (CD40, CD80, CD83, CD86, MHC-II and DC-LAMP (CD208)), pro-inflammatory cytokines (IL-1β, IL-6, TNF-α (LITAF)), iNOS, chemokine CXCli2 and TLRs4 and 15. Basal level of TLR1 mRNA expression was higher followed by TLR15 in both DCs irrespective of their origin. Expression of iNOS and CXCLi2 mRNA in mature DCs of both origins were higher than other surface molecules and cytokines studied. Hence, its level of expression can also be used as an additional maturation marker for LPS induced chicken dendritic cell maturation along with CD83 and CD40. LPS matured DCs of both origins upregulated IL-12 and IFN-γ. Based on CD40 and CD83 mRNA expression, it was observed that LPS induced the maturation in both DCs, but chMoDCs responded better in expression of surface markers and inflammatory mediator genes.     

Microbial cell components induced tolerance to flagellin-stimulated inflammation through Toll-like receptor pathways in intestinal epithelial cells

In the intestine, bacterial components activate innate responses that protect the host. We hypothesize that bacterial components reduce Interleukin-8 (IL-8) production in intestinal epithelial cells stimulated by flagellin via the Toll-like receptor (TLR) signaling pathway. Caco-2 cells were pretreated with various doses of lipopolysaccharide (LPS), lipoteichoic acid (LTA), or low-dose flagellin (LDFL) for 24 h. Cells were then treated with flagellin (FL) 500 ng/ml (HDFL) for another 48 h. IL-8 production was measured in the cell culture medium by ELISA. Eighty-four genes in the TLR pathway were evaluated by RT Profiler PCR Array. Pathway Studio 8.0 software was used for altered pathway analysis. HDFL induced IL-8 production by 19-fold (p < 0.01). Pretreatment with LDFL at 20, 10 or 1 ng/ml reduced HDFL-induced IL-8 production by 61%, 52% and 40%, respectively (p < 0.05). LPS at 50 μg/ml decreased HDFL–induced IL-8 production by 38% (p < 0.05). HDFL up-regulated CXCL10, IL1B, IL-8, IRAK2, NF-κB1 and I-κB (all p < 0.05). Pathway Studio analysis showed that HDFL induced cell processes including inflammation, cell death and apoptosis. Pretreatment with LDFL at 10 ng/ml down-regulated FADD, FOS, MAP4K4, MyD88, TLR2, TLR3 and TNFERSF1A compared to HDFL (all p < 0.05). These down-regulated genes are integral for numerous cell functions including inflammatory response, cell death, apoptosis and infection. These results demonstrate that LPS and LDFL provoke tolerance to HDFL-induced IL-8 production. This tolerance effect was accompanied by a complex interaction of multiple genes related to inflammatory as well as other responses in the TLR pathway rather than a single gene alteration.     

Vasoactive intestinal peptide (VIP) differentially affects inflammatory immune responses in human monocytes infected with viable Salmonella or stimulated with LPS

We compared the effect of VIP on human blood monocytes infected with Salmonella typhimurium 4/74 or stimulated with LPS. VIP (10⁻⁷ M) increased monocyte viability by 24% and 9% when cultured for 24 h with 4/74 or Salmonella LPS (100 ng/ml), respectively. Significantly increased (P < 0.05) numbers of 4/74 were also recovered from monocytes co-cultured with VIP after 6 h post-infection (pi) and this remained high after 24 h pi. Both 4/74 and LPS increased (P < 0.05) the concentration of TNF-α, IL-1β and IL-6 measured in monocyte supernatants. However, LPS induced this effect more rapidly while, with the exception of IL-6, 4/74 induced higher concentrations (P < 0.05). VIP significantly decreased (P < 0.05) TNF-α and IL-1β production by 4/74-infected monocytes after 6 pi, but only after 24 h in LPS-cultured monocytes. This trend was reversed for IL-6 production. However, TNF-α and IL-1β production by 4/74-infected monocytes, cultured with VIP, still remained higher (P < 0.05) than concentrations measured in supernatants cultured only with LPS. VIP also increased (P < 0.05) production of anti-inflammatory IL-10 in both 4/74 and LPS cultures after 24 h. We also show a differential effect of VIP on the expression of TNFα and IL-6 receptors, since VIP was only able to decreased expression in LPS-stimulated monocytes but not in 4/74-infected monocytes.In conclusion, we show a differential effect of VIP on human monocytes infected with virulent Salmonella or stimulated with LPS. Our study suggests that the use of VIP in bacteraemia and/or sepsis may be limited to an adjunctive therapy to antibiotic treatment.     

Vocal fold fibroblasts immunoregulate activated macrophage phenotype

Recent evidence suggests that fibroblasts play a critical role in regulating inflammation during wound healing because they express several inflammatory mediators in response to bacteria. The objective of this study was to analyze the effects of lipopolysaccharide (LPS) on the immunomodulatory properties of vocal fold fibroblasts (VFFs) derived from polyps, scar and normal tissue co-cultured with macrophages, to provide insight into their interactions during the inflammatory process. Fibroblasts were co-cultured with CD14+ monocytes and after 7 days, wells were treated with LPS for 24 and 72 h. Culture supernatants were collected and concentrations of TNF-α, IL-6, IL-8, IL-10, IL-12, IL-1β and MCP-1 were quantified by ELISA. Normal VFF and CD14+ monocultures were used as controls. Twenty-four hours after LPS activation, macrophages co-cultured with polyp VFF had significantly increased expression of TNF-α, IL-1β, IL-12 and IL-10 compared to controls (p < 0.0001). In contrast, macrophages co-cultured with scar VFF had significantly lower expression of TNF-α, IL-1β and IL-12 with significantly higher IL-10 compared to control (p < 0.0001). After 72 h, macrophages co-cultured with polyp VFF increased expression of TNF-α, IL-1β, IL-10, IL-6, IL-8, MCP-1 and TGF-β (p < 0.01) and macrophages co-cultured with scar VFF significantly decreased their expression of IL-1β and IL-12 compared to control (p < 0.0001). Scar VFF at both time points produced significantly lower levels of IL-8, MCP-1, IL-6 and TGF-β compared to controls (p < 0.05). Based on our findings, VFF and macrophages secrete several inflammatory mediators that modify their diverse functions. Polyp and scar VFF may play a role in regulating abnormal inflammatory responses, which could result in excessive ECM deposition that disrupts the function of the vocal folds.     

Antithrombin III,but not C1 esterase inhibitor reduces inflammatory response in lipopolysaccharide-stimulated human monocytes in an ex-vivo whole blood setting

In order to examine the immunomodulatory effects of antithrombin III (AT-III) and C1 esterase inhibitor (C1-INH) in human monocytes, we investigated the intracellular expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in an ex-vivo laboratory study in a whole blood setting.Heparinized whole blood samples from 23 healthy male and female volunteers (mean age: 27 ± 7 years) were pre-incubated with clinically relevant concentrations of AT-III (n = 11) and C1-INH (n = 12), then stimulated with 0.2 ng/mL lipopolysaccharide (LPS) for 3 h. After phenotyping CD14⁺ monocytes, intracellular expression of IL-6, IL-8, and TNF-α was assessed using flow cytometry. In addition, 12 whole blood samples (AT-III and C1-INH, n = 6 each) were examined using hirudin for anticoagulation; all samples were processed in the same way. To exclude cytotoxicity effects, 7-amino-actinomycin D and Nonidet P40 staining were used to investigate probes.This study is the first to demonstrate the influence of C1-INH and AT-III on the monocytic inflammatory response in a whole blood setting, which mimics the optimal physiological setting. Cells treated with AT-III exhibited significant downregulation of the proportion of gated CD14⁺ monocytes for IL-6 and IL-8, in a dose-dependent manner; downregulation for TNF-α did not reach statistical significance. There were no significant effects on mean fluorescence intensity (MFI). In contrast, C1-INH did not significantly reduce the proportion of gated CD14⁺ monocytes or the MFI regarding IL-6, TNF-α, and IL-8. When using hirudin for anticoagulation, no difference in the anti-inflammatory properties of AT-III and C1-INH in monocytes occurs.Taken together, in contrast to TNF-α, IL-6 and IL-8 were significantly downregulated in monocytes in an ex-vivo setting of human whole blood when treated with AT-III. This finding implicates monocytes as an important point of action regarding the anti-inflammatory properties of AT-III in sepsis. C1-INH was unable to attenuate the monocytic response, which supports the hypothesis that other cellular components in whole blood (e.g., neutrophils) might be responsible for the known effects of C1-INH in inflammation.     

Pinostrobin and Cajanus lactone isolated from Cajanus cajan (L.) leaves inhibits TNF-α and IL-1β production: In vitro and in vivo experimentation

The tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) inhibitory activities of Cajanus cajan (leaves) crude methanolic extract, its fractions and its phytochemical constituents were evaluated in lipopolysaccharide (LPS) stimulated RAW 264.7 and J774A.1 cells. Phytochemical investigation of the active ethyl acetate (CCE) and n-butanol (CCB) fractions of C. cajan L. leaves yielded 14 compounds. It was observed that both pinostrobin (9) and cajanus lactone (4) were found to be most active in inhibiting TNF-α (IC₅₀ < 22 μM) and IL-1β (IC₅₀ < 40 μM) whereas compounds 2, 3, 5–8, 10 and 14 showed moderate and mild effects (IC₅₀ = 35.50–81.22 μM for TNF-α and 38.23–89.10 μM for IL-1β) in both the cell lines. Furthermore, at dose of 20 mg/kg, both pinostrobin (9) and cajanus lactone (4) were found to reduce LPS-induced TNF-α levels by 48.6% and 55.0% respectively and IL-1β levels by 53.1% and 41.8% respectively in Sprague Dawley (SD) rats. These findings suggest that C. cajan L. leaves can be developed as an effective herbal remedy for the treatment and prevention of inflammation or associated ailments.     

Dynorphin 1-17 and Its N-Terminal Biotransformation Fragments Modulate Lipopolysaccharide-Stimulated Nuclear Factor-kappa B Nuclear Translocation,Interleukin-1beta and Tumor Necrosis Factor-alpha in Differentiated THP-1 Cells

Dynorphin 1–17, (DYN 1–17) opioid peptide produces antinociception following binding to the kappa-opioid peptide (KOP) receptor. Upon synthesis and release in inflamed tissues by immune cells, DYN 1–17 undergoes rapid biotransformation and yields a unique set of opioid and non-opioid fragments. Some of these major fragments possess a role in immunomodulation, suggesting that opioid-targeted therapeutics may be effective in diminishing the severity of inflammatory disorders. This study aimed to examine the immunomodulatory effects of DYN 1–17 and major N-terminal fragments found in the inflammatory environment on nuclear factor-kappaB/p65 (NF-κB/p65) nuclear translocation and the release of interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) from lipopolysaccharide (LPS)-stimulated, differentiated THP-1 cells. The results demonstrate that NF-κB/p65 nuclear translocation was significantly attenuated following treatment with DYN 1–17 and a specific range of fragments, with the greatest reduction observed with DYN 1–7 at a low concentration (10 nM). Antagonism with a selective KOP receptor antagonist, ML-190, significantly reversed the inhibitory effects of DYN 1–17, DYN 1–6, DYN 1–7 and DYN 1–9, but not other DYN 1–17 N-terminal fragments (DYN 1–10 and 1–11) on NF-κB/p65 nuclear translocation. DYN 1–17 and selected fragments demonstrated differential modulation on the release of IL-1β and TNF-α with significant inhibition observed with DYN 1–7 at low concentrations (1 nM and 10 pM). These effects were blocked by ML-190, suggesting a KOP receptor-mediated pathway. The results demonstrate that DYN 1–17 and certain N-terminal fragments, produced in an inflamed environment, play an anti-inflammatory role by inhibiting NF-κB/p65 translocation and the subsequent cytokine release through KOP receptor-dependent and independent pathways.     

Neohesperidin dihydrochalcone down-regulates MyD88-dependent and -independent signaling by inhibiting endotoxin-induced trafficking of TLR4 to lipid rafts

Fulminant hepatic failure (FHF) is a lethal clinical syndrome characterized by the activation of macrophages and the increased production of inflammatory mediators. The purpose of this study was to investigate the effects of neohesperidin dihydrochalcone (NHDC), a widely-used low caloric artificial sweetener against FHF. An FHF experimental model was established in mice by intraperitoneal injection of D-galactosamine (d-GalN) (400 mg/kg)/lipopolysaccharides (LPS) (10 μg/kg). Mice were orally administered NHDC for 6 continuous days and at 1 h before d-GalN/LPS administration. RAW264.7 macrophages were used as an in vitro model. Cells were pre-treated with NHDC for 1 h before stimulation with LPS (10 μg/ml) for 6 h. d-GalN/LPS markedly increased the serum transaminase activities and levels of oxidative and inflammatory markers, which were significantly attenuated by NHDC. Mechanistic analysis indicated that NHDC inhibited LPS-induced myeloid differentiation factor 88 (MyD88) and TIR-containing adapter molecule (TRIF)-dependent signaling. Transient transfection of TLR4 or MyD88 siRNA inhibited the downstream inflammatory signaling. This effect could also be achieved by the pretreatment with NHDC. The fluorescence microscopy and flow cytometry results suggested that NHDC potently inhibited the binding of LPS to TLR4 in RAW264.7 macrophages. In addition, the inhibitory effect of NHDC on LPS-induced translocation of TLR4 into lipid raft domains played an important role in the amelioration of production of downstream pro-inflammatory molecules. Furthermore, the activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) by NHDC inhibited TLR4 signaling. In conclusion, our results suggest that NHDC attenuates d-GalN/LPS-induced FHF by inhibiting the TLR4-mediated inflammatory pathway, demonstrating a new application of NHDC as a hepatoprotective agent.     

Effects of pro-inflammatory cytokines,lipopolysaccharide and COX-2 mediators on human colonic neuromuscular function and epithelial permeability

Chronic colitis is associated with decreased colonic muscle contraction and loss of mucosal barrier function. Pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) are important in the generation and maintenance of inflammation. While colitis is associated with upregulated COX-2 -derived prostanoids and nitric oxide (NO), the direct activity of pro-inflammatory cytokines on human colonic neuromuscular function is less clear. This study investigated the effects of IBD-associated pro-inflammatory cytokines IL-17, TNF-α, IL-1β and LPS on human colonic muscle strip contractility, alone and following inhibition of COX-2 or nitric oxide production. In addition, human colonic epithelial Caco-2 cell monolayers were treated with LPS or COX-2 mediators including prostaglandins (PGE₂, PGF_2α) or their corresponding ethanolamides (PGE₂-EA or PGF_2α-EA) over 48 h and trans-epithelial electrical resistance used to record permeability changes. Longitudinal muscle strips were obtained from healthy colonic resection margins and mounted in organ baths following IL-17, TNF-α, IL-1β and bacterial LPS incubations in an explant setting over 20 h. Contraction in response to acetylcholine (ACh) was then measured, before and after either COX-2 inhibition (nimesulide; 10⁻⁵ M) or nitric oxide synthase (NOS) inhibition (l-NNA; 10⁻⁴ M). None of the cytokine or LPS explant incubations affected the potency or maximum cholinergic contraction in vitro, and subsequent COX-2 blockade with nimesulide revealed a significant but similar decrease in potency of ACh-evoked contraction in control, LPS and cytokine-incubated muscle strips. Pre-treatment with l-NNA provided no functional differences in the potency or maximum contractile responses to ACh in cytokine or LPS-incubated colonic longitudinal smooth muscle. Only PGE₂ transiently increased Caco-2 monolayer permeability at 24 h, while LPS (10 μg/ml) increased permeability over 24–48 h.These findings indicate that cholinergic contractility in the human colon can be decreased by the blockade of COX-2 generated excitatory prostanoids, but major pro-inflammatory cytokines or LPS do not alter the sensitivity or amplitude of this contraction ex vivo. While PGE₂ transiently increase epithelial permeability, LPS generates a significant and sustained increase in permeability indicative of an important role on barrier function at the mucosal interface.     

Anti-allergic principles of Rhinacanthus nasutus leaves

Three naphthoquinone derivatives, rhinacanthin-C (1), -D (2) and -N (3) were isolated from the extract of Rhinacanthus nasutus Kurz leaves and were tested for anti-allergic effect. The result indicated that all three compounds possessed very potent anti-allergic activity against antigen-induced β-hexosaminidase release as a marker of degranulation in RBL-2H3 cells with IC₅₀ values of 6.9, 8.9 and 6.4 μM, respectively. In addition, the effects of rhinacanthin-C, -D and -N on antigen-induced release of TNF-α and IL-4 were also examined. It was found that rhinacanthin-C showed the most potent on antigen-induced TNF-α release with an IC₅₀ value of 0.7 μM, followed by rhinacanthin-D (IC₅₀=3.8 μM) and rhinacanthin-N (IC₅₀=10.3 μM), whereas those for IL-4 were rhinacanthin-D (IC₅₀=5.4 μM), rhinacanthin-C (IC₅₀=7.0 μM) and rhinacanthin-N (IC₅₀=12.0 μM), respectively. The mechanisms in the late phase reaction of rhinacanthin-C, -D and -N were found to inhibit TNF-α and IL-4 gene expression in antigen-induced TNF-α and IL-4 releases on from RBL-2H3 cells as dose-dependent manners.The structure-activity trends of rhinacanthin-C,-D and-N on the inhibition of TNF-α release are as follow; substitution with octadienoic acid (rhinacanthin-C) conferred much higher activity than that of benzodioxo carboxylic acid ester (rhinacanthin-D) as well as naphthalene carboxylic acid ester (rhinacanthin-N). For the inhibition of IL-4 release, the substitution with octadienoic acid (rhinacanthin-C) and benzodioxo carboxylic acid ester (rhinacanthin-D) possessed the effect two-fold higher than that of naphthalene carboxylic acid ester (rhinacanthin-N).As regards active constituents for anti-allergic activity of R. nasutus, rhinacanthin-C, -D and -N are responsible for anti-allergic effect of this plant on both the early phase and late phase reactions. The finding may support the traditional use of R. nasutus leaves for treatment of allergy and allergy-related diseases.     

Cytokine profiles in acute myeloid leukemia patients at diagnosis: Survival is inversely correlated with IL-6 and directly correlated with IL-10 levels

BackgroundSeveral evidences support the existence of cytokine deregulation in acute myeloid leukemia (AML) patients that may be associated with pathogenesis, disease progression and patient survival.MethodsIn the present study, we analyzed plasma levels of pro- and anti-inflammatory cytokines in AML patients and age-matched healthy donors. TNF-α, IL-6, IL-1β, IL-2, IFN-γ, IL-17A, IL-12p70, IL-8, IL-10, IL-4 and IL-5 were analyzed using fluorescent bead-based technology and TGF-β by ELISA technique. Because age-associated differences in cytokine profiles have been described, patients and healthy individuals were divided into two age groups: up to 65 years and over 65 years.ResultsOur results showed that plasma TNF-α, IL-6 and IL-10 levels were higher in AML patients from both groups of age. IL-8 was increased in AML patients less than 65 years while the plasma concentrations of IL-4, IL-5 and IL-12p70 were significantly higher only in elderly AML patients compared with aged-matched healthy controls. Moreover, plasma levels of IL-6 and IL-10 were associated with patient survival and event-free survival.ConclusionsAn aberrant production of the pro-inflammatory cytokines IL-6 and TNF-α and the anti-inflammatory cytokine IL-10 is observed in AML patients. Low levels of IL-6 and high levels of IL-10 represent favorable prognostic factors for survival in AML patients. These results support the idea that cytokine deregulation may be useful as a marker for predicting clinical evolution in AML patients.     

Oral Angiotensin-(1–7) prevented obesity and hepatic inflammation by inhibition of resistin/TLR4/MAPK/NF-κB in rats fed with high-fat diet

Obesity is characterized by a pro-inflammatory state commonly associated with type 2 diabetes and fat-liver disease. In the last few years, different studies pointed out the role of Angiotensin (Ang)-(1–7) in the metabolic regulation. The aim of the present study was to evaluate the effect of oral-administration of Ang-(1–7) in metabolism and inflammatory state of high-fat feed rats. Twenty-four male Sprague Dawley rats were randomized into three groups: High Fat Diet (HFD); Standard Diet (ST); High Fat Diet + Angiotensin-(1–7) [HFD + Ang-(1–7)]. Glycemic profile was evaluated by glucose tolerance and insulin sensitivity tests, plasmatic glucose and insulin. Cholesterol, HDL and triglycerides analyses presented lipidic profile. RT-PCR evaluated mRNA expression to ACE, ACE2, resistin, TLR4, IL-6, TNF-α and NF-κB genes. The main results showed that oral Ang-(1–7) decreased body weight and abdominal fat-mass. In addition, HFD + Ang-(1–7) treated rats presented enhanced glucose tolerance, insulin-sensitivity and decreased plasma-insulin levels, as well as a significant decrease in circulating lipid levels. These alterations were accompanied by a marked decreased expression of resistin, TLR4, ACE and increased ACE2 expression in liver. Furthermore, Ang-(1–7) decreases phosphorylation of MAPK and increases NF-κB expression. These alterations diminished expression of interleukin-6 and TNF-α, ameliorate inflammatory state in liver. In summary, the present study showed that oral-treatment with Ang-(1–7) in high-fat feed rats improved metabolism down-regulating resistin/TLR4/NF-κB-pathway.     

The −251A>T polymorphism of interleukin-8 is associated with longer mechanical ventilation and hospital staying after coronary surgery

Background: Cardiac surgery is associated with inflammatory responses that are known to affect its outcome. The present study was designed to define whether post-operative release of interleukin (IL)-6, 8 and tumor necrosis factor-alpha (TNF-α) is related to the presence of a certain allele in functional polymorphism and its relationship to clinical outcome after off-pump coronary artery bypass (OPCAB). Methods: One hundred and forty-five patients undergoing first time elective OPCAB were genotyped for IL-6(−174G>C), IL-8(−251A>T) and TNF-α(−308G>A) polymorphisms using polymerase chain reaction (PCR) and gene sequencing. Cytokine levels were measured in plasma samples taken before the operation and 4, 24 and 72 h postoperatively by suspension array system. Results: Levels of IL-6 and IL-8 increased significantly after OPCAB. Patients with IL-6−174GG and IL-8−251AA genotypes had higher post-operative circulating levels of IL-6 and IL-8, respectively. Logistic regression showed that IL-8−251AA genotype was an independent risk factor of ventilation time more than 1 day (OR = 11.80, 95% CI: 1.87–74.48) and hospital staying more than 14 days (OR = 38.00, 95% CI: 4.15–347.87) after surgery. Conclusions: OPCAB results in post-operative inflammatory responses. Genetic backgrounds alter the extent of inflammatory response and might relate to clinical outcome of OPCAB.     

Beta blocker metoprolol protects against contractile dysfunction in rats after coronary microembolization by regulating expression of myocardial inflammatory cytokines

AimsTo examine the effects of metoprolol on expression of myocardial inflammatory cytokines and myocardial function in rats following coronary microembolization (CME).Main methodsMale rats were randomly assigned to receive either sham-operation (control group), CME plus saline (CME group), or CME plus metoprolol (metoprolol group). CME was induced by injecting 3000 polyethylene microspheres (42 μm) into the left ventricle during a 10-second occlusion of the ascending aorta. Metoprolol (2.5 mg/kg) or saline was administered as three intravenous bolus injections after CME. At 3 h, 6 h, 12 h, 24 h and 4 weeks after CME, myocardial function was measured with echocardiography; and the mRNA and protein levels of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10) and interleukin 1-β (IL-1β) were determined.Key findingsInduced CME led to markedly higher mRNA and protein levels of TNF-α, IL-1β and IL-10 at 3, 6, 12, and 24 h, as well as reduced left ventricular function, compared to the control group. Metoprolol administration reduced TNF-α and IL-1β levels, but increased IL-10 levels at 3, 6, 12, and 24 h compared to the CME group. Moreover, metoprolol treatment resulted in significantly improved left ventricular function at 12 h, 24 h and 4 weeks, but afforded no cardiac protection at 3 h and 12 h, compared to the CME group.SignificanceHigher levels of TNF-α and IL-1β in rats following CME are associated with the development of myocardial contractile dysfunction. Metoprolol-conferred protection against progressive contractile dysfunction following CME may be mediated by its anti-inflammation potential.     

Activation of foal neutrophils at different ages by CpG oligodeoxynucleotides and Rhodococcus equi

Toll-like receptor 9 (TLR9) activation stimulates protective immune responses against intracellular pathogens by phagocytes, including neutrophils. This study examined TLR9-mediated neutrophil activation in neonatal foals. Unmethylated CpGs, ligands for TLR9, were used to stimulate equine neutrophils, either purified or in contact with other peripheral blood leukocytes. Rhodococcus equi was used as another stimulus in parallel. TLR9 mRNA was constitutively expressed at a similar level in purified equine neutrophils across different ages from birth to adulthood, and expression was not affected by either CpG or R. equi. Purified foal neutrophils were directly sensitive to CpG stimulation, reflected by enhanced reactive oxygen species generation following fMLP stimulation, and by expressing significantly (P < 0.05) greater mRNA of IFN-γ, IL-8, IL-12p35, and significantly (P < 0.05) decreased TNF-α mRNA. In comparison, purified foal neutrophils stimulated by R. equi showed significantly (P < 0.05) increased mRNA production of IL-6, IL-8, IL-23p19, and TNF-α. Neutrophils co-cultured with other leukocytes expressed a distinct profile of cytokine mRNA than purified neutrophils in response to CpG stimulation, whereas the profile was very similar following R. equi stimulation irrespective of neutrophil purity. When co-cultured with other leukocytes, foal neutrophils were significantly (P < 0.05) activated at birth by B-class CpGs and produced IL-6, IL-8, IL-12p40, and IL-23p19 at similar magnitudes to those at 2 months of age. In foal neutrophils at birth, R. equi significantly (P < 0.05) induced all cytokines stimulated by CpGs (except IL-12p40), as well as TNF-α. Our results indicate that foal neutrophils were sensitive to CpG or R. equi activation as early as at birth, and that B-class CpGs enhanced foal neutrophil functions in vitro.     

Haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice

AimsTo investigate whether haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice.Methods and ResultsExperiments were performed using irradiated LDL receptor-deficient (LDLR^−/−) mice with marrow from either TLR4-deficient (TLR4^−/−) or age-matched wild-type (WT) mice. After 12 weeks of being fed a high-cholesterol diet, TLR4^−/− → LDLR^−/− mice developed fewer atherosclerotic lesions in the aorta compared to WT → LDLR^−/− mice. This effect was associated with an increase in multilocular lipid droplets and mitochondria in perivascular adipose tissue (PVAT). Immunofluorescence analysis confirmed that there was an increase in capillary density and M2 macrophage infiltration, accompanied by a decrease in tumour necrosis factor (TNF)-α expression in the localized PVAT of TLR4^−/− → LDLR^−/− mice. In vitro studies indicated that bone marrow-derived macrophages (BMDMs) from WT mice demonstrated an M1-like phenotype and expression of inflammatory cytokines induced by palmitate. These effects were attenuated in BMDMs isolated from TLR4^−/− mice. Furthermore, brown adipocytes incubated with conditioned medium (CM) derived from palmitate-treated BMDMs, exhibited larger and more unilocular lipid droplets, and reduced expression of brown adipocyte-specific markers and perilipin-1 compared to those observed in brown adipocytes exposed to CM from palmitate-treated BMDMs of TLR4^−/− mice. This decreased potency was primarily due to TNF-α, as demonstrated by the capacity of the TNF-α neutralizing antibody to reverse these effects.ConclusionsThese results suggest that haematopoietic-specific deletion of TLR4 promotes PVAT homeostasis, which is involved in reducing macrophage-induced TNF-α secretion and increasing mitochondrial biogenesis in brown adipocytes.