IL-12 suppression,enhanced endocytosis and up-regulation of MHC-II and CD80 in dendritic cells during experimental endotoxin tolerance

Aim:

The aim of this study was to investigate endocytosis, MHC-II expression and co-stimulatory molecule expression, as well as interleukin-12 (IL-12) production, in bone marrow dendritic cells (DCs) derived from endotoxin tolerant mice.

Methods:

Endotoxin tolerance was induced in C57BL/10J mice through four consecutive daily intraperitoneal injections of 1.0 mg/kg of 055:B5 Escherichia coli lipopolysaccharide (LPS). Bone marrow DCs were isolated in the presence of GM-CSF and IL-4 and purified by anti-CD11c Micro beads. FITC–dextran uptake by DCs was tested by flow cytometric analysis and the percentage of dextran-containing cells was calculated using a fluorescence microscope. The expression of surface MHC-II, CD40, CD80, and CD86 was also detected by flow cytometric analysis. An ELISA was used for the measurement of IL-12 production by DCs with or without LPS stimulation.

Results:

Endotoxin tolerance was successfully induced in C57BL/10J mice, evidenced by an attenuated elevation of systemic TNF-α. DCs from endotoxin tolerant mice possessed enhanced dextran endocytosis ability. The expression of surface MHC-II and CD80 was higher in DCs from endotoxin tolerant mice than in DCs from control mice, whereas the expression of CD40 and CD86 was not altered. Compared with DCs from normal control mice, DCs from endotoxin tolerant mice produced less IL-12 after subsequent in vitro stimulation with LPS.

Conclusion:

These data suggest enhanced endocytosis, selective up-regulation of MHC-II and CD80 and IL-12 suppression in DCs during in vivo induction of endotoxin tolerance.     

9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol, from Eulophia ochreata, inhibits inflammatory signalling mediated by Toll-like receptors

Background and purpose:

9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol (RSCL-0520) is a phenanthrene isolated from Eulophia ochreata, one of the Orchidaceae family, known by local tradition to exhibit medicinal properties. However, no anti-inflammatory activity or any molecular mechanisms involved have been reported or elucidated. Here, for the first time, we evaluate the anti-inflammatory properties of RSCL-0520 on responses induced by lipopolysaccharide (LPS) and mediated via Toll-like receptors (TLRs).

Experimental approach:

The in vitro anti-inflammatory activities of RSCL-0520 were investigated in LPS-stimulated monocytic cells, measuring activation of cytokine and inflammatory genes regulated by nuclear factor-κB (NF-κB). Tumour necrosis factor (TNF)-α levels in serum following LPS stimulation in mice and carrageenan-induced paw oedema in rats were used as in vivo models.

Key results:

Pretreatment with RSCL-0520 effectively inhibited LPS-induced, TLR4-mediated, NF-κB-activated inflammatory genes in vitro, and reduced both LPS-induced TNF-α release and carrageenan-induced paw oedema in rats. Treatment with RSCL-0520 reduced LPS-stimulated mRNA expression of TNF-α, COX-2, intercellular adhesion molecule-1, interleukin (IL)-8 and IL-1β, all regulated through NF-κB activation. RSCL-0520, however, did not interfere with any cellular processes in the absence of LPS.

Conclusions and implications:

RSCL-0520 blocked signals generated by TLR4 activation, as shown by down-regulation of NF-κB-regulated inflammatory cytokines. The inhibitory effect involved both MyD88-dependent and -independent signalling cascades. Our data elucidated the molecular mechanisms involved, and support the search for plant-derived TLR antagonists, as potential anti inflammatory agents.     

Exogenous carbon monoxide suppresses Escherichia coli vitality and improves survival in an Escherichia coli-induced murine sepsis model

Aim:

Endogenous carbon monoxide (CO) has been shown to modulate inflammation and inhibit cytokine production both in vivo and in vitro. The aim of this study was to examine whether exogenous carbon monoxide could suppress the vitality of Escherichia coli (E coli) and improve the survival rate in an E coli-induced murine sepsis model.

Methods:

ICR mice were infected with E coli, and immediately injected intravenously with carbon monoxide releasing molecule-2 (CORM-2, 8 mg/kg) or inactive CORM-2 (8 mg/kg). The survival rate was monitored 6 times daily for up to 36 h. The blood samples, liver and lung tissues were collected at 6 h after the infection. Bacteria in peritoneal lavage fluid, blood and tissues were enumerated following culture. Tissue iNOS mRNA expression was detected using RT-PCR. NF-κB expression was detected with Western blotting.

Results:

Addition of CORM-2 (200 and 400 μmol/L) into culture medium concentration-dependently suppressed the growth of E coli and decreased the colony numbers, but inactive CORM-2 had no effect. Treatment of the infected mice with CORM-2 significantly increased the survival rate to 55%, while all the infected mice treated with inactive CORM-2 died within 36 h. E coli infection caused severe pathological changes in liver and lungs, and significantly increased serum transaminases, lipopolysaccharide, TNF-α and IL-1β levels, as well as myeloperoxidase activity, TNF-α and IL-1β levels in the major organs. Meanwhile, E coli infection significantly increased the number of colonies and the expression of iNOS mRNA and NF-κB in the major organs. All these abnormalities were significantly attenuated by CORM-2 treatment, while inactive CORM-2 was ineffective.

Conclusion:

In addition directly suppressing E coli, CORM-2 protects the liver and lungs against E coli-induced sepsis in mice, thus improving their survival.     

BAY 41-2272, a soluble guanylate cyclase agonist, activates human mononuclear phagocytes

BACKGROUND AND PURPOSE

Phagocyte function is critical for host defense against infections. Defects in phagocytic function lead to several primary immunodeficiencies characterized by early onset of recurrent and severe infections. In this work, we further investigated the effects of BAY 41-2272, a soluble guanylate cyclase (sGC) agonist, on the activation of human peripheral blood monocytes (PBM) and THP-1 cells.

EXPERIMENTAL APPROACH

THP-1 cells and PBM viability was evaluated by methylthiazoletetrazolium assay; reactive oxygen species production by lucigenin chemiluminescence; gene and protein expression of NAPDH oxidase components by qRT-PCR and Western blot analysis, respectively; phagocytosis and microbicidal activity by co-incubation, respectively, with zymosan and Escherichia coli; and cytokine release by elisa.

KEY RESULTS

BAY 41-2272, compared with the untreated group, increased spreading of monocytes by at least 35%, superoxide production by at least 50%, and gp91^PHOX and p67^PHOX gene expression 20 to 40 times, in both PBM and THP-1 cells. BAY 41-2272 also augmented phagocytosis of zymosan particles threefold compared with control, doubled microbicidal activity against E. coli and enhanced the release of TNF-α and IL-12p70 by both PBM and THP-1 cells. Finally, by inhibiting sGC with ODQ, we showed that BAY 41-2272-induced superoxide production and phagocytosis is not dependent exclusively on sGC activation.

CONCLUSIONS AND IMPLICATIONS

In addition to its ability to induce vasorelaxation and its potential application for therapy of vascular diseases, BAY 41-2272 was shown to activate human mononuclear phagocytes. Hence, it is a novel pro-inflammatory drug that may be useful for controlling infections in the immunocompromised host.     

Apolipoprotein A-I inhibits LPS-induced atherosclerosis in ApoE?/? mice possibly via activated STAT3-mediated upregulation of tristetraprolin

Aim:

To investigate the effects of the major component of high-density lipoprotein apolipoprotein A-I (apoA-I) on the development of atherosclerosis in LPS-challenged ApoE^−/− mice and the underlying mechanisms.

Methods:

Male ApoE-KO mice were daily injected with LPS (25 μg, sc) or PBS for 4 weeks. The LPS-challenged mice were intravenously injected with rAAV-apoA-I-GFP or rAAV-GFP. After the animals were killed, blood, livers and aortas were collected for biochemical and histological analyses. For ex vivo experiments, the abdominal cavity macrophages were harvested from each treatment group of mice, and cultured with autologous serum, then treated with LPS.

Results:

Chronic administration of LPS in ApoE^−/− mice significantly increased the expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1), increased infiltration of inflammatory cells, and enhanced the development of atherosclerosis. In LPS-challenged mice injected with rAAV-apoA-I-GFP, viral particles and human apoA-I were detected in the livers, total plasma human apoA-I levels were grammatically increased; HDL-cholesterol level was significantly increased, TG and TC were slightly increased. Furthermore, overexpression of apoA-I significantly suppressed the expression of proinflammatory cytokines, reduced the infiltration of inflammatory cells, and decreased the extent of atherosclerotic lesions. Moreover, overexpression of apoA-I significantly increased the expression of the cytokine mRNA-destabilizing protein tristetraprolin (TTP), and phosphorylation of JAK2 and STAT3 in aortas. In ex vivo mouse macrophages, the serum from mice overexpressing apoA-I significantly increased the expression of TTP, accompanied by accelerated decay of mRNAs of the inflammatory cytokines.

Conclusion:

ApoA-I potently suppresses LPS-induced atherosclerosis by inhibiting the inflammatory response possibly via activation of STAT3 and upregulation of TTP.     

Kukoamine B, a novel dual inhibitor of LPS and CpG DNA, is a potential candidate for sepsis treatment

BACKGROUND AND PURPOSE

Lipopolysaccharides (LPS) and oligodeoxynucleotides containing CpG motifs (CpG DNA) are important pathogenic molecules for the induction of sepsis, and thus are drug targets for sepsis treatment. The present drugs for treating sepsis act only against either LPS or CpG DNA. Hence, they are not particularly efficient at combating sepsis as the latter two molecules usually cooperate during sepsis. In this study, a natural alkaloid compound kukoamine B (KB) is presented as a potent dual inhibitor for both LPS and CpG DNA.

EXPERIMENTAL APPROACH

The affinities of KB for LPS and CpG DNA were assessed using biosensor technology. Direct interaction of KB with LPS and CpG DNA were evaluated using neutralization assays. Selective inhibitory activities of KB on pro-inflammatory signal transduction and cytokine expression induced by LPS and CpG DNA were analysed by cellular assays. Protective effects of KB in a sepsis model in mice were elucidated by determining survival and circulatory LPS and tumour necrosis factor-alpha (TNF-α) concentrations.

KEY RESULTS

KB had high affinities for LPS and CpG DNA. It neutralized LPS and CpG DNA and prevented them from interacting with mouse macrophages. KB selectively inhibited LPS- and CpG DNA-induced signal transduction and expression of pro-inflammatory mediators without interfering with signal pathways or cell viability in macrophages. KB protected mice challenged with heat-killed Escherichia coli, and reduced the circulatory levels of LPS and TNF-α.

CONCLUSIONS AND IMPLICATIONS

This is the first report of a novel dual inhibitor of LPS and CpG DNA. KB is worthy of further investigation as a potential candidate to treat sepsis.     

Selenium nanoparticle-enriched Lactobacillus brevis causes more efficient immune responses in vivo and reduces the liver metastasis in metastatic form of mouse breast cancer

Background and the purpose of the study

Selenium enriched Lactobacillus has been reported as an immunostimulatory agent which can be used to increase the life span of cancer bearing animals. Lactic acid bacteria can reduce selenium ions to elemental selenium nanoparticles (SeNPs) and deposit them in intracellular spaces. In this strategy two known immunostimulators, lactic acid bacteria (LAB) and SeNPs, are concomitantly administered for enhancing of immune responses in cancer bearing mice.

Methods

Forty five female inbred BALB/c mice were divided into three groups of tests and control, each containing 15 mice. Test mice were orally administered with SeNP-enriched Lactobacillus brevis or Lactobacillus brevis alone for 3 weeks before tumor induction. After that the administration was followed in three cycles of seven days on/three days off. Control group received phosphate buffer saline (PBS) at same condition. During the study the tumor growth was monitored using caliper method. At the end of study the spleen cell culture was carried out for both NK cytotoxicity assay and cytokines measurement. Delayed type hypersensitivity (DTH) responses were also assayed after 72h of tumor antigen recall. Serum lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) levels were measured, the livers of mice were removed and prepared for histopathological analysis.

Results

High level of IFN-γ and IL-17 besides the significant raised in NK cytotoxicity and DTH responses were observed in SeNP-enriched L. brevis administered mice and the extended life span and decrease in the tumor metastasis to liver were also recorded in this group compared to the control mice or L.brevis alone administered mice.

Conclusion

Our results suggested that the better prognosis could be achieved by oral administration of SeNP-enriched L. brevis in highly metastatic breast cancer mice model.     

Tanshinone IIA therapeutically reduces LPS-induced acute lung injury by inhibiting inflammation and apoptosis in mice

Aim:

To study the effects of tanshinone IIA (TIIA) on lipopolysaccharide (LPS)-induced acute lung injury in mice and the underlying mechanisms.

Methods:

Mice were injected with LPS (10 mg/kg, ip), then treated with TIIA (10 mg/kg, ip). Seven hours after LPS injection, the lungs were collected for histological study. Protein, LDH, TNF-α and IL-1β levels in bronchoalveolar lavage fluid (BALF) and myeloperoxidase (MPO) activity in lungs were measured. Cell apoptosis and Bcl-2, caspase-3, NF-κB and HIF-1α expression in lungs were assayed.

Results:

LPS caused marked histological changes in lungs, accompanied by significantly increased lung W/D ratio, protein content and LDH level in BALF, and Evans blue leakage. LPS markedly increased neutrophil infiltration in lungs and inflammatory cytokines in BALF. Furthermore, LPS induced cell apoptosis in lungs, as evidenced by increased TUNEL-positive cells, decreased Bcl-2 content and increased cleaved caspase-3 content. Moreover, LPS significantly increased the expression of NF-κB and HIF-1α in lungs. Treatment of LPS-injected mice with TIIA significantly alleviated these pathological changes in lungs.

Conclusion:

TIIA alleviates LPS-induced acute lung injury in mice by suppressing inflammatory responses and apoptosis, which is mediated via inhibition of the NF-κB and HIF-1α pathways.     

PPARα mediates the anti-inflammatory effect of simvastatin in an experimental model of zymosan-induced multiple organ failure

BACKGROUND AND PURPOSE

Zymosan-induced non-septic shock is a multi-factorial pathology that involves several organs including the kidneys, liver and lungs. Its complexity and diversity presents a continuing therapeutic challenge. Given their pleiotropic effect, statins could be beneficial in non-septic shock. One of the molecular mechanisms underlying the anti-inflammatory effect of statins involves the peroxisome proliferator-activated receptor (PPAR) α. We used a zymosan-induced non-septic shock experimental model to investigate the role of PPARα in the anti-inflammatory effects of simvastatin.

EXPERIMENTAL APPROACH

Effects of simvastatin (5 or 10 mg·kg⁻¹ i.p.) were analysed in PPARα knock-out (KO) and PPARα wild type (WT) mice after zymosan or vehicle administration. Organ injury in lung, liver, kidney and intestine was evaluated by immunohistology. PPARα mRNA expression and nuclear factor-κB activation were evaluated in all experimental groups, 18 h after study onset. Cytokine levels were measured in plasma, and nitrite/nitrate in plasma and peritoneal exudate. Nitric oxide synthase, nitrotyrosine and poly ADP-ribose were localized by immunohistochemical methods.

KEY RESULTS

Simvastatin significantly and dose-dependently increased the zymosan-induced expression of PPARα levels in all tissues analysed. It also dose-dependently reduced systemic inflammation and the organ injury induced by zymosan in lung, liver, intestine and kidney. These effects were observed in PPARαWT mice and in PPARαKO mice.

CONCLUSIONS AND IMPLICATIONS

Simvastatin protected against the molecular and cellular damage caused by systemic inflammation in our experimental model. Our results also provide new information regarding the role of PPARα in the anti-inflammatory effects of statins.     

The antimicrobial effects of selenium nanoparticle-enriched probiotics and their fermented broth against Candida albicans

Background

Lactic acid bacteria are considered important probiotics for prevention of some infections. The aim of this work was to investigate the effect of selenium dioxide on the antifungal activity of Lactobacillus plantarum and L. johnsonii against Candida albicans.

Methods

Lactobacillus plantarum and L. johnsonii cells, grown in the presence and absence of selenium dioxide, and their cell-free spent culture media were tested for antifungal activity against C. albicans ATCC 14053 by a hole-plate diffusion method and a time-kill assay.

Results

Both L. plantarum and L. johnsonii reduced selenium dioxide to cell-associated elemental selenium nanoparticles. The cell-free spent culture media, from both Lactobacillus species that had been grown with selenium dioxide for 48 h, showed enhanced antifungal activity against C. albicans. Enhanced antifungal activity of cell biomass against C. albicans was also observed in cultures grown with selenium dioxide.

Conclusions

Selenium dioxide-treated Lactobacillus spp. or their cell-free spent broth inhibited the growth of C. albicans and should be investigated for possible use in anti-Candida probiotic formulations in future.     

Pharmacological inhibition of diacylglycerol acyltransferase 1 reduces body weight gain, hyperlipidemia, and hepatic steatosis in db/db mice

Aim:

To test whether pharmacological inhibition of Diacylglycerol acyltransferase 1 (DGAT1) by a small-molecule inhibitor H128 can improve metabolism disorders in leptin receptor-deficient db/db mice.

Methods:

To investigate the effect of H128 on intestinal fat absorption,db/db mice were acutely given a bolus of corn oil by gavage. The mice were further orally administered H128 (3 and 10 mg/kg) for 5 weeks. Blood glucose, lipids, insulin, ALT, and AST as well as hepatic triglycerides were measured. The insulin tolerance test was performed to evaluate insulin sensitivity. The expression of genes involved in fatty acid oxidation was detected by RT-PCR.

Results:

Oral administration of H128 (10 mg/kg) acutely inhibited intestinal fat absorption following a lipid challenge in db/db mice. Chronic treatment with H128 significantly inhibited body weight gain, decreased food intake, and induced a pronounced reduction of serum triglycerides. In addition, H128 treatment markedly ameliorated hepatic steatosis, characterized by decreased liver weight, lipid droplets, and triglyceride content as well as serum ALT and AST levels. Furthermore, H128 treatment increased the expression of the CPT1 and PPARα genes in liver, suggesting that H128 enhanced fatty acid oxidation in db/db mice. However, neither blood glucose nor insulin tolerance was affected by H128 treatment throughout the 5-week experimental period.

Conclusion:

DGAT1 may be an effective therapeutic target for the treatment of obesity, hyperlipidemia and hepatic steatosis.     

The 5-lipoxygenase inhibitor, zileuton, suppresses prostaglandin biosynthesis by inhibition of arachidonic acid release in macrophages

BACKGROUND AND PURPOSE

Zileuton is the only 5-lipoxygenase (5-LOX) inhibitor marketed as a treatment for asthma, and is often utilized as a selective tool to evaluate the role of 5-LOX and leukotrienes. The aim of this study was to investigate the effect of zileuton on prostaglandin (PG) production in vitro and in vivo.

EXPERIMENTAL APPROACH

Peritoneal macrophages activated with lipopolysaccharide (LPS)/interferon γ (LPS/IFNγ), J774 macrophages and human whole blood stimulated with LPS were used as in vitro models and rat carrageenan-induced pleurisy as an in vivo model.

KEY RESULTS

Zileuton suppressed PG biosynthesis by interference with arachidonic acid (AA) release in macrophages. We found that zileuton significantly reduced PGE2 and 6-keto prostaglandin F1α (PGF1α) levels in activated mouse peritoneal macrophages and in J774 macrophages. This effect was not related to 5-LOX inhibition, because it was also observed in macrophages from 5-LOX knockout mice. Notably, zileuton inhibited PGE2 production in LPS-stimulated human whole blood and suppressed PGE2 and 6-keto PGF1α pleural levels in rat carrageenan-induced pleurisy. Interestingly, zileuton failed to inhibit the activity of microsomal PGE2 synthase1 and of cyclooxygenase (COX)-2 and did not affect COX-2 expression. However, zileuton significantly decreased AA release in macrophages accompanied by inhibition of phospholipase A2 translocation to cellular membranes.

CONCLUSIONS AND IMPLICATION

Zileuton inhibited PG production by interfering at the level of AA release. Its mechanism of action, as well as its use as a pharmacological tool, in experimental models of inflammation should be reassessed.     

Kynurenic acid attenuates multiorgan dysfunction in rats after heatstroke

Aim:

To assess whether systemic delivery of kynurenic acid improves the outcomes of heatstroke in rats.

Methods:

Anesthetized rats were divided into 2 major groups and given vehicle solution (isotonic saline 0.3 mL/kg rat weight) or kynurenic acid (30–100 mg in 0.3 mL saline/kg) 4 h before the start of thermal experiments. They were exposed to an ambient temperature of 43 °C for 68 min to induce heatstroke. Another group of rats were exposed to room temperature (26 °C) and used as normothermic controls. Their core temperatures, mean arterial pressures, serum levels of systemic inflammatory response molecules, hypothalamic values of apoptotic cells and neuronal damage scores, and spleen, liver, kidney and lung values of apoptotic cells were determined.

Results:

The survival time values during heatstroke for vehicle-treated rats were decreased from the control values of 475–485 min to new values of 83–95 min. Treatment with KYNA (30–100 mg/kg, iv) 4 h before the start of heat stress significantly and dose-dependently decreased the survival time to new values of 152–356 min (P<0.05). Vehicle-treated heatstroke rats displayed hypotension, hypothalamic neuronal degeneration and apoptosis, increased serum levels of tunor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1), and interleukin-10 (IL-10), and spleen, liver, kidney, and lung apoptosis. KYNA preconditioning protected against hypotension but not hyperthermia and attenuated hypothalamic neuronal degeneration and apoptosis during heatstroke. KYNA preconditioning attenuated spleen, kidney, liver, and lung apoptosis and up-regulated serum IL-10 levels but down-regulated serum TNF-α and ICAM-1 levels during heatstroke.

Conclusion:

Our results suggest that systemic delivery of kynurenic acid may attenuate multiorgan dysfunction in rats after heatstroke.     

Isochaihulactone protects PC12 cell against H(2)O(2) induced oxidative stress and exerts the potent anti-aging effects in D-galactose aging mouse model

Aim:

To investigate the effect of isochaihulactone (also known as K8), a lignan compound of Bupleurum scorzonerifolium, on H₂O₂-induced cytotoxicity in neuronally differentiated PC12 cells (nPC12).

Methods:

Viability of neuronal PC12 cells was measured using MTT assay. Protein expression was determined by Western blot. Apoptotic cells was determined using TUNEL assay. D-galactose aging mice were used as a model system to study the anti-oxidant effects of isochaihulactone in vivo.

Results:

Pretreatment with isochaihulactone (5–10 μmol/L) increased cell viability and decreased membrane damage, generation of reactive oxygen species and degradation of poly (ADP-ribose) polymerase in H₂O₂-treated nPC12 cells and also decreased the expression of cyclooxygenase-2, via downregulation of NF-kappaB, resulting in a decrease in lipid peroxidation. The results suggest that isochaihulactone is a potential antioxidant agent. In a murine aging model, in which chronic systemic exposure to D-galactose (D-gal) causes the acceleration of senescence, administration of isochaihulactone (10 mg·kg^-1·d^-1, sc) for 7 weeks concomitant with D-gal injection significantly increased superoxide dismutase and glutathione peroxidase activities and decreased the MDA level in plasma. Furthermore, H&E staining to quantify cell death within hippocampus showed that percentage of pyknotic nuclei in the D-gal-treated mice were much higher than in control.

Conclusion:

The results suggest that isochaihulactone exerts potent anti-aging effects against D-gal in mice possibly via antioxidative mechanisms.