Influence of surgery on in-vitro cytokine production by human monocytes

Surgery leads to significant modulation of the immune system, in which cytokines play a major role. Circulating interleukin 6 (IL-6) and IL-1 have been reported following surgery whereas tumor necrosis factor alpha (TNF-alpha) is only found in gut ischemia-associated surgery. We have investigated the consequences of surgery on in-vitro cytokine production by human monocytes stimulated by lipopolysaccharide (LPS) and staphylococcal toxic shock syndrome toxin-1 (TSST-1). Comparisons were made between the responsiveness of cells obtained the day before (D-1), during (D0) and after (D1, D2, D3) surgery. Patients undergoing abdominal aortic surgery (N = 9), carotid surgery (N = 4) and spinal surgery (N = 4) have been studied. A significant decrease of TNF-alpha, IL-1 beta and IL-1 alpha production by monocytes prepared from blood samples taken during the surgery was noticed, whereas IL-6 production was not significantly modified. On D2 a significant increase of monocyte responsiveness was observed and levels of cytokine productions rose back to initial values by the end of the follow up. The diminished in-vitro cytokine production observed during surgery might be the consequence of the effects of anaesthetic drugs, whereas the enhancement observed on D2 might reflect the surgical stress, leading to in-vivo priming of circulating monocytes.     

Silibinin attenuates cardiac hypertrophy and fibrosis through blocking EGFR‐dependent signaling

Cardiac hypertrophy is a major determinant of heart failure. The epidermal growth factor receptor (EGFR) plays an important role in cardiac hypertrophy. Since silibinin suppresses EGFR in vitro and in vivo, we hypothesized that silibinin would attenuate cardiac hypertrophy through disrupting EGFR signaling. In this study, we examined this hypothesis using neonatal cardiac myocytes and fibroblasts induced by angiotensin II (Ang II) and animal model by aortic banding (AB) mice. Our data revealed that silibinin obviously blocked cardiac hypertrophic responses induced by pressure overload. Meanwhile, silibinin markedly reduced the increased generation of EGFR. Moreover, these beneficial effects were associated with attenuation of the EGFR‐dependent ERK1/2, PI3K/Akt signaling cascade. We further demonstrated silibinin decreased inflammation and fibrosis by blocking the activation of NF‐κB and TGF‐β1/Smad signaling pathways in vitro and in vivo. Our results indicate that silibinin has the potential to protect against cardiac hypertrophy, inflammation, and fibrosis through blocking EGFR activity and EGFR‐dependent different intracellular signaling pathways. J. Cell. Biochem. 110: 1111–1122, 2010. Published 2010 Wiley‐Liss, Inc.     

Diacyltrehalose of Mycobacterium tuberculosis inhibits lipopolysaccharide- and mycobacteria-induced proinflammatory cytokine production in human monocytic cells

The lipids located in the outer layer of Mycobacterium tuberculosis, which include sulfolipid, phthiocerol dimycocerosate (PDIM), diacyltrehalose, and polyacyltrehalose, may play a role in host-pathogen interactions. These lipids were purified using thin-layer chromatography, and their ability to induce proinflammatory cytokines in human monocytes and in a human acute monocytic leukemia cell line (THP-1) was examined. None of the lipids tested induced significant interleukin (IL)-12p40 or tumor necrosis factor (TNF)-alpha production in monocytic cells. Diacyltrehalose significantly inhibited lipopolysaccharide- and M. tuberculosis-induced IL-12p40, TNF-alpha, and IL-6 productions in human monocytes, whereas other lipids had no effect. However, diacyltrehalose was unable to inhibit peptidoglycan-induced IL-12p40 production. These results suggest that diacyltrehalose is a mycobacterial factor capable of modulating host immune responses.     

Modulation of cytokine production by monocytes and developing-dendritic cells under the influence of leukemia and lymphoma cell products

Cytokines and other soluble factors released by tumor cells play an important role in modulating immune cells to favor tumor development. Monocyte differentiation into macrophages or dendritic cells (DCs) with specific phenotypes is deeply affected by tumor signals and understanding this context is paramount to prevent and propose new therapeutic possibilities. Hence, we developed a study to better describe the modulatory effects of leukemia and lymphoma cell products on human monocytes and monocyte-derived DCs secretion of cytokines such as interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, and IL-12. Except with the promyelocytic leukemia cell supernatants (HL-60), the other two tumor supernatants (chronic myeloid leukemia, K562 and Burkitt lymphoma, DAUDI) increased both TNF-α and IL-1β production by monocytes and monocytes undergoing differentiation. This effect was neither explained by alterations of cell number in culture nor by the high amount of vascular endothelial growth factor (VEGF) present in the tumor supernatants. Moreover, all supernatants used were able to induce drastic reduction of IL-12 secretion by cells induced to activation, suggesting a negative interference with Th1 antitumoral responses that should be a huge advantage for tumor progression.     

The ATP binding cassette transporter A1 contributes to the secretion of interleukin 1beta from macrophages but not from monocytes

Deficiency of ABCA1 causes high density lipoprotein deficiency and macrophage foam cell formation in Tangier disease. ABCA1 was also postulated to mediate the secretion of IL-1beta from monocytes and macrophages. We investigated the contribution of ABCA1 to IL-1beta secretion from human monocytes and macrophages of normal donors and Tangier disease patients. Neither an anti-ABCA1 antisense oligonucleotide nor ABCA1 deficiency interfered with LPS-induced secretion of IL-1beta from full blood or freshly isolated monocytes. By contrast, anti-ABCA1 antisense oligonucleotides decreased the LPS-induced secretion of IL-beta from macrophages by 30-50%. The secretion of the precursor pro-IL-1beta and TNFalpha was not inhibited. Compared to normal macrophages, LPS-stimulated Tangier disease macrophages secreted less IL-1beta relative to TNFalpha. Also the spontaneous secretion of IL-1beta by Tangier macrophages was lower than by control cells. We conclude that IL-1beta is secreted from monocytes by an ABCA1-independent pathway and from macrophages by ABCA1-dependent and -independent pathways.     

Normothermic machine perfusion attenuates hepatic ischaemia-reperfusion injury by inhibiting CIRP-mediated oxidative stress and mitochondrial fission

Extracellular cold-inducible RNA-binding protein (CIRP) is a proinflammatory mediator that aggravates ischaemia-reperfusion injury (IRI). Normothermic machine perfusion (NMP) could effectively alleviate the IRI of the liver, but the underlying mechanism remains to be explored. We show that human DCD livers secreted a large amount of CIRP during static cold storage (CS), which is released into the circulation after reperfusion. The expression of CIRP was related to postoperative IL-6 levels and liver function. In a rat model, the CIRP expression was upregulated during warm ischaemia and cold storage. Then, rat DCD livers were preserved using CS, hypothermic oxygenated machine perfusion (HOPE) and NMP. C23, a CIRP inhibitor, was administrated in the HOPE group. Compared with CS, NMP significantly inhibited CIRP expression and decreased oxidative stress by downregulating NADPH oxidase and upregulating UCP2. NMP markedly inhibited the mitochondrial fission-related proteins Drp-1 and Fis-1. Further, NMP increased the mitochondrial biogenesis-related protein, TFAM. NMP significantly reduced inflammatory reactions and apoptosis after reperfusion, and NMP-preserved liver tissue had higher bile secretion and ICG metabolism compared to the CS group. Moreover, C23 administration attenuated IRI in the HOPE group. Additionally, HL-7702 cells were stimulated with rhCIRP and C23. High rhCIRP levels increased oxidative stress and apoptosis. In summary, NMP attenuates the IRI of DCD liver by inhibiting CIRP-mediated oxidative stress and mitochondrial fission.     

Correlation between maternal plasma homocysteine and zinc levels in preeclamptic women

Women with preeclampsia have been shown to have elevated blood levels of the metabolite homocysteine, and alterations in blood levels of zinc and copper have also been reported. This study measured plasma levels of zinc, copper, and homocysteine in women with preeclampsia and in women with healthy, normotensive pregnancies. For the patients with preeclampsia compared with controls, significantly higher mean plasma levels were found of homocysteine (16.39 vs 9.45 nmol/mL; p≤0.001), zinc (15.53 vs 11.93 μg/g protein; p < 0.05), and copper (47.90 vs 31.60 μg/g protein; p=0.001). The ratio of plasma Cu/Zn levels tended to be higher in preeclamptic women and could be taken as an index of inflammatory reaction, but the difference was not significant. Homocysteine concentrations correlated positively with plasma zinc concentrations in women with preeclampsia (r=0.588, p=0.003) but not in women with healthy pregnancies. No correlations were observed between plasma levels of homocysteine and copper. Thus, the present study found evidence that preeclampsia might be associated with hyperhomocysteinemia and elevated blood levels of zinc and copper. Furthermore, elevated blood levels of zinc were significantly associated with hyperhomocysteinemia in preeclampsia. More studies are warranted to investigate further any relationship between altered homocysteine metabolism and levels of zinc and copper in preeclampsia.     

Autophagy alleviates neurodegeneration caused by mild impairment of oxidative metabolism

Thiamine deficiency (TD) causes mild impairment of oxidative metabolism and region‐selective neuronal loss in the brain, which may be mediated by neuronal oxidative stress, endoplasmic reticulum (ER) stress, and neuroinflammation. TD‐induced brain damage is used to model neurodegenerative disorders, and the mechanism for the neuronal death is still unclear. We hypothesized that autophagy might be activated in the TD brain and play a protective role in TD‐induced neuronal death. Our results demonstrated that TD induced the accumulation of autophagosomes in thalamic neurons measured by transmission electron microscopy, and the up‐regulation of autophagic markers LC3‐II, Atg5, and Beclin1 as measured with western blotting. TD also increased the expression of autophagic markers and induced LC3 puncta in SH‐SY5Y neuroblastoma cells. TD‐induced expression of autophagic markers was reversed once thiamine was re‐administered. Both inhibition of autophagy by wortmannin and Beclin1 siRNA potentiated TD‐induced death of SH‐SY5Y cells. In contrast, activation of autophagy by rapamycin alleviated cell death induced by TD. Intraperitoneal injection of rapamycin stimulated neuronal autophagy and attenuated TD‐induced neuronal death and microglia activation in the submedial thalamus nucleus (SmTN). TD inhibited the phosphorylation of p70S6 kinase, suggesting mTOR/p70S6 kinase pathway was involved in the TD‐induced autophagy. These results suggest that autophagy is neuroprotective in response to TD‐induced neuronal death in the central nervous system. This opens a potential therapeutic avenue for neurodegenerative diseases caused by mild impairment of oxidative metabolism.

     

Decreased Bioenergetic Health Index in monocytes isolated from the pericardial fluid and blood of post-operative cardiac surgery patients

Monitoring the bioenergetics of leucocytes is now emerging as an important approach in translational research to detect mitochondrial dysfunction in blood or other patient samples. Using the mitochondrial stress test, which involves the sequential addition of mitochondrial inhibitors to adherent leucocytes, we have calculated a single value, the Bioenergetic Health Index (BHI), which represents the mitochondrial function in cells isolated from patients. In the present report, we assess the BHI of monocytes isolated from the post-operative blood and post-operative pericardial fluid (PO-PCF) from patients undergoing cardiac surgery. Analysis of the bioenergetics of monocytes isolated from patients’ PO-PCF revealed a profound decrease in mitochondrial function compared with monocytes isolated from their blood or from healthy controls. Further, patient blood monocytes showed no significant difference in the individual energetic parameters from the mitochondrial stress test but, when integrated into the BHI evaluation, there was a significant decrease in BHI compared with healthy control monocytes. These data support the utility of BHI measurements in integrating the individual parameters from the mitochondrial stress test into a single value. Supporting our previous finding that the PO-PCF is pro-oxidant, we found that exposure of rat cardiomyocytes to PO-PCF caused a significant loss of mitochondrial membrane potential and increased reactive oxygen species (ROS). These findings support the hypothesis that integrated measures of bioenergetic health could have prognostic and diagnostic value in translational bioenergetics.     

IP3R attenuates oxidative stress and inflammation damage in smoking-induced COPD by promoting autophagy

Tobacco smoking is one of the most important risk factors for chronic obstructive pulmonary disease (COPD). However, the most critical genes and proteins remain poorly understood. Therefore, we aimed to investigate these hub genes and proteins in tobacco smoke-induced COPD, together with the potential mechanism(s). Differentially expressed genes (DEGs) were analysed between smokers and patients with COPD. mRNA expression and protein expression of IP₃R were confirmed in patients with COPD and extracted smoke solution (ESS)-treated human bronchial epithelial (HBE) cells. Moreover, expression of oxidative stress, inflammatory cytokines and/or autophagy-related protein was tested when IP₃R was silenced or overexpressed in ESS-treated and/or 3-MA-treated cells. A total of 30 DEGs were obtained between patients with COPD and smoker samples. IP₃R was identified as one of the key targets in tobacco smoke-induced COPD. In addition, IP₃R was significantly decreased in patients with COPD and ESS-treated cells. Loss of IP₃R statistically increased expression of oxidative stress and inflammatory cytokines in ESS-treated HBE cells, and overexpression of IP₃R reversed the above functions. Furthermore, the autophagy-related proteins (Atg5, LC3 and Beclin1) were statistically decreased, and p62 was increased by silencing of IP₃R cells, while overexpression of IP₃R showed contrary results. Additionally, we detected that administration of 3-MA significantly reversed the protective effects of IP₃R overexpression on ESS-induced oxidative stress and inflammatory injury. Our results suggest that IP₃R might exert a protective role against ESS-induced oxidative stress and inflammation damage in HBE cells. These protective effects might be associated with promoting autophagy.     

Thioredoxin-interacting protein promotes activation and inflammation of monocytes with DNA demethylation in coronary artery disease

Numerous studies have demonstrated that thioredoxin-interacting protein (TXNIP) expression of peripheral blood leucocytes is increased in coronary artery disease (CAD). However, the molecular mechanism of this phenomenon remained unclear. DNA methylation plays important roles in the regulation of gene expression. Therefore, we speculated there might be a close association between the expression of TXNIP and methylation. In this study, we found that compared with controls, DNA methylation at cg19693031 was decreased in CAD, while mRNA expressions of TXNIP and inflammatory factors, NLRP3, IL-1β, IL-18, were increased. Methylation at cg19693031 was negatively associated with TXNIP expression in the cohort, THP-1 and macrophages/foam cells. Furthermore, Transwell assay and co-cultured adhesion assay were performed to investigate functions of TXNIP on the migration of THP-1 or the adhesion of THP-1 on the surface of endothelial cells, respectively. Notably, overexpressed TXNIP promoted the migration and adhesion of THP-1 cells and expressions of NLRP3, IL-18 and IL-1β. Oppositely, knock-down TXNIP inhibited the migration and adhesion of THP-1 and expressions of NLRP3, IL-18. In conclusion, increased TXNIP expression, related to cg19693031 demethylation orientates monocytes towards an inflammatory status through the NLRP3 inflammasome pathway involved in the development of CAD.     

Epigenetic therapy attenuates oxidative stress in BMSCs during ageing

Oxidative stress, a hallmark of ageing, inhibits the osteogenic differentiation of bone marrow‐derived mesenchymal stem cells in long bone. The dysfunction of the cellular antioxidant defence system is a critical cause of oxidative stress, but the mechanism of the decline of antioxidant defence in senescent stem cells remains elusive. Here, we found that EZH2, an epigenetic regulator of histone methylation, acted as a suppressor of the antioxidative defence system in BMSCs from the femur. The increased EZH2 led to a decrease in the levels of antioxidant enzymes and exaggerated oxidative damage in aged BMSCs, resulting in the defect of bone formation and regeneration. Mechanistically, EZH2 enhanced the modification of H3K27me3 on the promoter of Foxo1 and suppressed its function to activate the downstream genes in antioxidant defence. Moreover, epigenetic therapy targeting EZH2‐mediated H3K27me3 modification largely recovered the antioxidant defence in BMSCs and attenuate oxidative damage, leading to the recovery of the osteogenesis in old BMSCs. Taken together, our findings revealed novel crosstalk between histone epigenetic modification and oxidative stress during stem cell ageing, suggesting a possibility of epigenetic therapy in the recovery of BMSCs senescence and treatment of age‐related bone disease.     

Melatonin attenuates methotrexate‐induced oxidative stress and renal damage in rats

Nephrotoxicity is an adverse side effect of methotrexate (MTX) chemotherapy. The present study verifies whether melatonin, an endogenous antioxidant prevents MTX‐induced renal damage. Adult rats were administered 7 mg/kg body weight MTX intraperitoneally for 3 days. In the melatonin pretreated rats, 40 mg/ kg body weight melatonin was administered daily intraperitoneally 1 h before the administration of MTX. The rats were killed 12 h after the final dose of MTX/vehicle. The kidneys were used for light microscopic and biochemical studies. The markers of oxidative stress were measured along with the activities of the antioxidant enzymes and myeloperoxidase activity in the kidney homogenates. Pretreatment with melatonin reduced MTX induced renal damage both histologically and biochemically as revealed by normal plasma creatinine levels. Melatonin pretreatment reduced MTX induced oxidative stress, alteration in the activity of antioxidant enzymes as well as elevation in myeloperoxidase activity. The results suggest that melatonin has the potential to reduce MTX induced oxidative stress, neutrophil infiltration as well as renal damage. As melatonin is an endogenous antioxidant and is non‐toxic even in high doses it is suggested that melatonin may be beneficial in minimizing MTX induced renal damage in humans. Copyright © 2010 John Wiley & Sons, Ltd.     

P2 purinergic receptor modulation of cytokine production

Cytokines serve important functions in controlling host immunity. Cells involved in the synthesis of these polypeptide mediators have evolved highly regulated processes to ensure that production is carefully balanced. In inflammatory and immune disorders, however, mis-regulation of the production and/or activity of cytokines is recognized as a major contributor to the disease process, and therapeutics that target individual cytokines are providing very effective treatment options in the clinic. Leukocytes are the principle producers of a number of key cytokines, and these cells also express numerous members of the purinergic P2 receptor family. Studies in several cellular systems have provided evidence that P2 receptor modulation can affect cytokine production, and mechanistic features of this regulation have emerged. This review highlights three separate examples corresponding to (1) P2Y₆ receptor mediated impact on interleukin (IL)-8 production, (2) P2Y₁₁ receptor-mediated affects on IL-12/23 output, and (3) P2X₇ receptor mediated IL-1β posttranslational processing. These examples demonstrate important roles of purinergic receptors in the modulation of cytokine production. Extension of these cellular observations to in vivo situations may lead to new therapeutic strategies for treating cytokine-mediated diseases.     

Alteration of calcium homeostasis in primary preeclamptic syncytiotrophoblasts: effect on calcium exchange in placenta

Preeclampsia (PE) is characterized by maternal hypertension, proteinuria, oedema and, in 30% of cases, by intrauterine growth retardation. Causes are still unknown; however, epidemiological and clinical studies have suggested alterations in maternal calcium metabolism. We suggested that in PE, calcium transport by the syncytiotrophoblast (ST) is disturbed. From total placental tissues, we studied the expression of: calcium channels (TRPV5, TRPV6 [transient receptor potential vanilloid]), calcium binding proteins (CaBP‐9K, CaBP‐28K), plasma membrane calcium ATPase (PMCA)1,2,3,4 pumps, ATP synthase, genes implicated in Ca²⁺ release [inositol‐1,4,5‐triphosphate receptor (IP3R)1,2,3; Ryanodine receptor (RyR)1,2,3] and replenishment (SERCA1,2,3 [sarcoendoplasmic reticulum Ca²⁺ ATPases]) from endoplasmic reticulum, channels implicated in mitochondrial Ca²⁺ accumulation (VDAC1,2,3 [voltage‐dependent anion channels]) and a marker of oxidative stress (hOGG1 [Human 8‐oxoguanine‐DNA glycosylase 1]), as well as the influence of these variations on calcium transport in primary ST cultures. The mRNA and protein levels were thereby examined by real‐time PCR and Western blot analysis, respectively, in two different groups of pregnant women with similar gestational age: a normal group (n= 16) and a PE group (n= 8), diagnosed by a clinician. Our study showed a significant decrease in calcium transport by the ST cultured from preeclamptic placentas. We found a significant (P < 0.05) decrease in mRNA levels of TRPV5, TRPV6, CaBP‐9K, CaBP‐28K, PMCA1, PMCA4, ATP synthase, IP3R1, IP3R2, RyR1, RyR2 and RyR3 in PE group compared to normal one. We also noted a significant decrease in protein levels of TRPV5, TRPV6, CaBP‐9K, CaBP‐28K and PMCA1/4 in PE group. In contrast, SERCA1, SERCA2, SERCA3, VDAC3 and hOGG1 mRNA expressions were significantly increased in PE placentas. Calcium homeostasis and transport through placenta is compromised in preeclamptic pregnancies and it appears to be affected by a lack of ATP and an excess of oxidative stress.     

虫草发酵菌丝体对胰岛素抵抗大鼠氧化应激的缓解作用

探究虫草发酵菌丝体对胰岛素抵抗(IR)大鼠氧化应激的影响。采用低中高剂量虫草发酵菌丝体(剂量分别为1.65g/kg饲料,3.30g/kg饲料和6.60g/kg饲料)分别干预IR大鼠。口服葡萄糖耐量实验观察不同剂量虫草发酵菌丝体的降糖效果,同时观察血脂、氧化应激水平的变化情况。RT-PCR法测肌肉、肝脏组织氧化应激相关基因Nrf2、HO-1和NQO1mRNA相对表达量,Westernblot法测其蛋白相对表达量。结果发现CM干预能够显著改善IR大鼠空腹血糖、血脂和机体氧化应激水平,肌肉、肝脏组织Nrf2、HO-1和NQO1mRNA相对表达量显著上升,肝脏Nrf2和NQO1蛋白相对表达量显著上调,并且具有剂量依赖性。说明CM具有缓解IR大鼠氧化应激的作用。     

Enhanced production of lutein in heterotrophic Chlorella protothecoides by oxidative stress

The fast growing unicellular green microalgae Chlorella protothecoides has attracted interest as a promising organism for commercial production of a high-value carotenoid, lutein, by heterotrophic fermentation. Effects of two oxidant-forming reactive oxygen species (ROS) on the biomass concen-tration, and yield and content of lutein in batch culture of heterotrophic Chlorella protothecoides were investigated in this study. The addition of 0.1 mmol/L H2O2 and 0.01 mmol/L NaClO plus 0.5 mmol/L Fe2 to the culture led to the generation of ·OH and enhanced the lutein content from 1.75 to 1.90 and 1.95 mg/g, respectively. The lutein content further increased to 1.98 mg/g when 0.01 mmol/L H2O2 and 0.5 mmol/L NaClO were added to generate 1O2. The maximum yield of lutein (28.5, 29.8 and 31.4 mg/L) and a high biomass concentration (15.0, 15.3 and 15.9 g/L) were also achieved through the above treatments. The results indicated that 1O2 could promote lutein formation and enhance lutein production in hetero-trophic Chlorella protothecoides. Moreover, 1O2 produced from the reaction of H2O2 and NaClO was more effective in enhancing lutein production and reducing biomass loss than ·OH from the reaction of H2O2 or NaClO plus Fe2 .