Tumor Necrosis Factor-alpha Synthesis Research Articles

12 Impact of progesterone signaling on tumor necrosis factor-alpha synthesis by luteal macrophages

Abstract Understanding the mechanism in which luteolysis occurs is an important step in improving the reproduction of livestock in the industry. The corpus luteum (CL) is a transient endocrine organ found on the ovary responsible for progesterone secretion. It is well known that prostaglandin F2-alpha (PGF) is responsible for luteal regression, however, the process by which luteal cells (LC) become sensitive to the luteolytic effects of PGF remain unclear. Therefore, the objective of this study was to determine if suppression of progesterone (P4) synthesis or blocking P4 signaling activates tumor necrosis factor (TNF)-alpha production by the luteal macrophages. Ovaries were collected from an abattoir and transported to the laboratory on ice. Ovary status (i.e. approximate days post ovulation), was determined by phenotypic description of ovarian structures. Only ovaries (n = 5) containing midcycle (d 12/13 post ovulation) CL were used in these experiments. Luteal tissue was isolated and dissociated to collect LC and and luteal macrophages (MAC). Isolated cells were incubated independently at 37°C at 5% CO2 for 24 h in 24-well plates. Luteal cells and MACs were then harvested and placed in the following conditions: 1) LC + MAC + DMSO (control), 2) LC + MAC + trilostane (TRIL 1.0mg/mL), 3) LC + MAC + TRIL 1.5 mg/mL, 4) MACS only, 5) MACS +LPS. Supernatant was harvested at 24 h whereas both cells and supernatant were harvested after 48 h, in vitro. Supernatant was immediately frozen for subsequent P4 analysis, via RIA. Data results were analyzed using a MIXED procedure of SAS and evaluated the effects of cell-type, treatment, and culture time. Statistical significance was determined at P < 0.05 and a tendency at 0.05 < P < 0.10. Overall, TRIL treatment reduced supernatant P4 concentration (8.3 and 6.8 ng/mL to 1.0 and 1.5 mg/mL TRIL, respectively) compared with controls (22.8 ng/mL; P = 0.0012); however there was not a difference when comparing the TRIL treated cultures. Progesterone concentrations tended to decrease over time (P = 0.0661). At 24 h in vitro, P4 concentrations were suppressed in 1.0 mg/mL TRIL and 1.5 mg/mL TRIL treated wells (5.3 and 4.2 ng/mL, respectively), compared with controls (16.4 ng/mL; P = 0.0014). Whereas P4 concentrations were reduced in the 1.5 mg/mL TRIL treated wells (1.6 ng/mL) compared with controls (9.95 ng/mL; P = 0.0419) and tended to differ in the1.0 mg/mL TRIL treated wells (2.4 ng/mL; P = 0.0634), at 48 h in vitro. Progesterone concentrations were undetectable in wells containing MAC only. Collectively, TRIL treatment effectively reduced P4 synthesis in LC cultures. Therefore, the greater concentration of TRIL is needed for P4 suppression in longer term culture systems. Additional analyses will be conducted to determine the effect of P4 suppression on macrophage function.

Nutrients in Pharmaceutical

Nutraceutical is any substance that might be categorized as food or a component of food that are beneficial in the prevention and treatment of diseases. Categories of nutraceuticals are dietary fiber, prebiotics, probiotics, fatty acids, polyphenols, antioxidants etc. Dietary fiber is made up of lignins and non-digestible carbohydrates that are intrinsic in plants. Probiotics are beneficial live bacteria and yeasts for our digestive system. Prebiotics are substances that stimulate the activity or growth of microorganisms that improve the health of their host. Omega-3 polyunsaturated fatty acids have a significant anti-inflammatory function. The synthesis of proinflammatory cytokines such prostaglandins, leukotrienes, and tumor necrosis factor-alpha, interferon-gamma, and interleukins-6 and 12 is inhibited by alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid. The anti-inflammatory characteristics of curcumin are demonstrated by its capacity to inhibit pro-inflammatory cytokines. Epigallocatechin Gallate is a tea polyphenols decrease DNA damage caused by UV radiation, prevent lipid peroxidation, and lessen the production of ROS and free radicals in the skin. Antioxidants are also essential for maintenance of health and wellness since they act as our body's first line of defense against free radical damage. Antioxidants like N-acetyl cysteine, lycopene, beta-carotene, lutein and zeaxanthin, vitamin C and vitamin E are used as a nutraceuticals. For the prevention and treatment of CVD, it is also advised to combine physical activity with nutraceuticals such as antioxidants, dietary fiber, omega-3 PUFA, vitamins, and minerals. Nutraceuticals like flavonoids, resveratrol, saponins, are used to cure cancer.

Pectolinarigenin Suppresses LPS-Induced Inflammatory Response in Macrophages and Attenuates DSS-Induced Colitis by Modulating the NF-κB/Nrf2 Signaling Pathway.

Pectolinarigenin (PEC), a natural flavonoid present in cirsium chanroenicum and citrus fruits, has possess the distinct pharmacological activities. However, its molecular mechanisms and pharmacological effects on intestinal illness have not been elucidated. In the present study, we investigated the potential beneficial effects of pectolinarigenin (PEC) on lipopolysaccharide (LPS)-induced macrophage cells and the dextran sulfate sodium (DSS)-induced colitis model. Our findings showed that PEC pretreatment inhibits the LPS-induced nuclear factor-kappa B (NF-κB) activation by interfering with the degradation of IκB-α. Further, increased Nrf2 protein expression was reported on PEC treated RAW 264.7 and THP1 cell lines. In addition, we revealed that PEC mediated the NF-κB/Nrf2 pathway regulation, which in turn inhibits the synthesis of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), interleukin-1beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) on RAW 264.7 and THP1 cells. Furthermore, PEC dose-dependently reduced the DSS-induced inflammation in the colon by regulating NF-κB/Nrf2 signaling pathway and enhancing the myeloperoxidase (MPO) activity and redox regulators such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and lipid peroxidation byproduct malondialdehyde (MDA) in DSS-induced inflamed colon. Similarly, we reported the minimal pathological damages in the PEC-treated mice colon, as well as increase goblet cell population and mucin-2 production. In conclusion, our findings demonstrate that PEC reduces the DSS-induced colitis in mice by regulating the NF-κB/Nrf2 pathway. Thus, PEC might be a promising therapeutic agent for the treatment of inflammatory bowel disease.

Biogenic silver nanoparticles reduce Toxoplasma gondii infection and proliferation in RAW 264.7 macrophages by inducing tumor necrosis factor-alpha and reactive oxygen species production in the cells

Owing to the serious adverse effects caused by pyrimethamine and sulfadiazine, the drugs commonly used to treat toxoplasmosis, there is a need for treatment alternatives for this disease. Nanotechnology has enabled significant advances toward this goal. This study was conducted to evaluate the activity of biogenic silver nanoparticles (AgNp-Bio) in RAW 264.7 murine macrophages infected with the RH strain of Toxoplasma gondii. The macrophages were infected with T. gondii tachyzoites and then treated with various concentrations of AgNp-Bio. The cells were evaluated by microscopy, and culture supernatants were collected for ELISA determination of their cytokine concentration. Treatment with 6 μM AgNp-Bio reduced the infection and parasite load in infected RAW 264.7 macrophages without being toxic to the cells. The treatment also induced the synthesis of reactive oxygen species and tumor necrosis factor-alpha (both pro-inflammatory mediators), which resulted in ultrastructural changes in the tachyzoites and their intramacrophagic destruction. Our findings suggest that AgNp-Bio affect T. gondii tachyzoites by activating microbicidal and pro-inflammatory mechanisms and may be a potential alternative treatment for toxoplasmosis.

Perspective on the relationship between microbiota dysbiosis and neuroinflammation: probiotics as a treatment option.

Perspective on the relationship between microbiota dysbiosis and neuroinflammation: probiotics as a treatment option.

Association between nuclear factor of activated T cells C2 polymorphisms and treatment response in rheumatoid arthritis patients receiving tumor necrosis factor-alpha inhibitors.

Nuclear factor of activated T cells C2 (NFATC2) is known as a member of the transcription family and enhances tumor necrosis factor-alpha (TNF-α) synthesis in human T cells at the gene transcription level. Although NFATC2 has a potential role in rheumatoid arthritis (RA) progression and treatment, no study has investigated the association between NFATC2 gene polymorphisms and response status in RA patients receiving TNF-α inhibitors. This study aimed to examine the effects of polymorphisms in NFATC2, a TNF-α transcription factor, on response to TNF-α inhibitors. This prospective observational study was performed in two centers. Seven single nucleotide polymorphisms (SNPs) were investigated. Good responders were defined as patients with disease activity score (DAS)28 ≤3.2 after 6 months of treatment. Logistic regression analyses were used to investigate the association between genetic polymorphisms and response to the treatment. To test the model's goodness of fit, a Hosmer-Lemeshow test was performed. This study included 98 patients, among whom 46 showed favorable responses to the treatment. Patients with hypertension revealed an approximately three-fold lower response to TNF-α inhibitors compared to those without hypertension (23.5 vs. 76.5%; P = 0.049). After adjusting for covariates, C allele carriers of NFATC2 rs3787186 exhibited approximately three-fold lower rates of treatment response compared to those with TT genotype (P = 0.037). The Hosmer-Lemeshow test showed that the fitness of the multivariable analysis model was satisfactory (χ2 = 9.745; 8 degrees of freedom; P = 0.283). This study suggested an association between the C allele of rs3787186 and treatment response in RA patients receiving TNF-α inhibitors.

Functions of interleukin-6 in ovulation of female climbing perch, Anabas testudineus

In mammals, interleukin 6 (IL-6) has an important function during ovulation, however, the functions of IL-6 in fish have not been elucidated. In the present study, there was quantification of de novo synthesis of ovarian IL-6 and tumor necrosis factor-alpha (TNFα) in control and hCG-treated fish and results were compared with those from an in vitro study where there was evaluation of the regulatory functions of gonadotropins and TNFα of IL-6 secretions. Relatively greater concentrations of ovarian IL-6 at the post-GVBD (post-germinal vesicle breakdown) stage indicates IL-6 modulates ovulatory processes. The hCG-induced increase in relative abundance of IL-6 (in vitro) mRNA transcript and secretion from the ovary were attenuated when there was administration of the inhibitor of TNFα secreting enzyme, TAPI-I, which indicates TNFα modulates IL-6 secretion. Treatments with IL-6 induced a marked increase in ovulation rate in vitro when there was induction of activating matrix metalloproteinase (MMP). Furthermore, treatment with IL-6 resulted in production of prostaglandin as indicated by the IL-6 induced increase in the abundance of ptgs2 mRNA transcript in the ovary of Anabas testudineus. Furthermore, results indicate the source of IL-6 in the ovary is the granulosa cells with secretion of IL-6 being induced by the additions of hCG and TNFα in the medium. There was also an IL-6-induced increase in abundance of receptors (IL-6 Rα and gp130) to which it binds indicating IL-6 autoregulates this population of receptors. Results from this study, for the first time, elucidate the reproductive functions of IL-6 in a teleost fish.

Anti-Inflammatory Effects of Aurantio-Obtusin from Seed of Cassia obtusifolia L. through Modulation of the NF-κB Pathway.

Aurantio-obtusin, an anthraquinone compound, isolated from dried seeds of Cassia obtusifolia L. (syn. Senna obtusifolia; Fabaceae) and Cassia tora L. (syn. Senna tora). Although the biological activities of Semen Cassiae have been reported, the anti-inflammatory mechanism of aurantio-obtusin, its main compound, on RAW264.7 cells, remained unknown. We investigated the anti-inflammatory effect of aurantio-obtusin on lipopolysaccharide- (LPS)-induced RAW264.7 cells in vitro and elucidated the possible underlying molecular mechanisms. Nitric oxide production (NO) and prostaglandin E2 (PGE2) were measured by the Griess colorimetric method and enzyme-linked immunosorbent assay (ELISA), respectively. Protein expression levels of cyclooxygenase 2 (COX-2) were monitored by cell-based ELISA. Interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) synthesis were analyzed using ELISA. The mRNA expression of nitric oxide synthase (iNOS), COX-2, and the critical pro-inflammatory cytokines (IL-6 and TNF-α) were detected by quantitative real-time PCR. Aurantio-obtusin significantly decreased the production of NO, PGE2, and inhibited the protein expression of COX-2, TNF-α and IL-6, which were similar to those gene expression of iNOS, COX-2, TNF-α and IL-6 (p < 0.01). Consistent with the pro-inflammatory gene expression, the Aurantio-obtusin efficiently reduced the LPS-induced activation of nuclear factor-κB in RAW264.7 cells. These results suggested that aurantio-obtusin may function as a therapeutic agent and can be considered in the further development of treatments for a variety of inflammatory diseases. Further studies may provide scientific evidence for the use of aurantio-obstusin as a new therapeutic agent for inflammation-related diseases.

A Novel Genistein Prodrug: Design, Synthesis and Bioactivity on Mouse RAW264.7 Macrophages

Genistein, a naturally occurring isoflavone, possesses many beneficial health effects. To improve the bioactivity of the natural compound, we designed and synthesized the genistein prodrug FEHH6-1. In the present study, we evaluated the biological effects of FEHH6-I on mouse RAW264.7 macrophages and compared them with those obtained with the parent drug genistein. The characteristics of FEHH6-1 were determined by melting point, nuclear magnetic resonance spectroscopy (NMR), and mass spectrometric analysis. The effects of FEHH6-I on cell proliferation, apoptosis, and pro-inflammatory cytokine expression were monitored by XTT-assay, Annexin-V/7-AAD staining, Western blotting, and ELISA. FEHH6-1 showed NMR spectra and relative molecular mass in agreement with the designed structure. In mouse RAW264.7 macrophages, FEHH6-1 inhibited proliferation, induced apoptotic cell death and blocked interleukin 6 and tumor necrosis factor alpha synthesis. At low concentrations, FEHH6-1 induced phosphorylation of AKT1, a kinase involved in cell proliferation and survival. Our data demonstrate that the genistein prodrug FEHH6-1 is a bioactive molecule but its solubility and therefore also its efficacy was significantly lower compared with genistein.

Comparative studies of paeoniflorin and albiflorin from Paeonia lactiflora on anti-inflammatory activities

Context: Paeonia lactiflora Pall. (Ranunculaceae) has been used for more than 1000 years in traditional Chinese medicine for the treatment of gynecological problems, cramp, pain, giddiness, and congestion. Paeoniflorin, monoterpene glycosides isolated from P. lactiflora, possesses a variety of pharmacological activities. However, the pharmacological activity of the pharmacological activity of albiflorin, another main monoterpene glycoside, has not been well studied.Objectives: The present study investigated the anti-inflammatory activities of paeoniflorin and albiflorin using models of lipopolysaccharides (LPS) induced RAW 264.7 cells.Materials and methods: Production of nitric oxide (NO) was measured by the Griess colorimetric method. In addition, prostaglandin E2 (PGE2), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) synthesis were analyzed using an enzyme-linked immunosorbent assay (ELISA). The protein expression of cyclooxygenase-2 (COX-2) was detected by a cell-based ELISA. The gene expression levels of inducible nitric oxide synthase (iNOS), COX-2, TNF-α, and IL-6 were detected by quantitative real-time reverse-transcription polymerase chain reaction (real-time RT-PCR).Results: Compared with the LPS-induced group, the inhibition rates of NO, PGE2, TNF-α, and IL-6 production were 17.61, 27.56, 20.57, and 29.01% by paeoniflorin and 17.35, 12.94, 15.29, and 10.78% by albiflorin. The IC50 values of paeoniflorin and albiflorin on NO production were 2.2 × 10−4 mol/L and 1.3 × 10−2 mol/L, respectively. The protein expression of COX-2 was reduced by 50.98% with paeoniflorin and 17.21% with albiflorin. The inhibition rates of gene expression of iNOS, COX-2, IL-6, and TNF-α were 35.65, 38.08, 19.72, and 45.19% by paeoniflorin and 58.36, 47.64, 50.70, and 12.43% by albiflorin, respectively.Conclusion: These results show that albiflorin has similar anti-inflammatory effects to paeoniflorin, which provides new evidence that albiflorin can serve as a new chemical marker for the quality control of Paeoniae Radix and the Chinese Pharmacopoeia can be updated.

Loneliness Promotes Inflammation During Acute Stress

Although evidence suggests that loneliness may increase risk for health problems, the mechanisms responsible are not well understood. Immune dysregulation is one potential pathway: Elevated proinflammatory cytokines such as interleukin-6 (IL-6) increase risk for health problems. In our first study (N = 134), lonelier healthy adults exposed to acute stress exhibited greater synthesis of tumor necrosis factor-alpha (TNF-α) and IL-6 by peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharide (LPS) than their less lonely counterparts. Similarly, in the second study (N = 144), lonelier posttreatment breast-cancer survivors exposed to acute stress exhibited greater synthesis of IL-6 and interleukin-1 beta (IL-1β) by LPS-stimulated PBMCs than their counterparts who felt more socially connected. However, loneliness was unrelated to TNF-α in Study 2, although the result was in the expected direction. Thus, two different populations demonstrated that lonelier participants had more stimulated cytokine production in response to stress than less lonely participants, which reflects a proinflammatory phenotype. These data provide a glimpse into the pathways through which loneliness may affect health.

Serum adipocyte fatty acid-binding protein in the critically ill

Sepsis due to unabated inflammation is common. Increased production of pro-inflammatory cytokines, free radicals, and eicosanoids has been detected in sepsis and other critical illnesses but could also be due to decreased synthesis and release of anti-inflammatory molecules. Increased serum adipose-fatty acid-binding protein (A-FABP) levels can cause insulin resistance and have been reported in the critically ill, serve as a marker of prognosis, and thus link metabolic homeostasis and inflammation. A-FABP can be linked to the expression of Toll-like receptors, macrophage activation, synthesis and release of pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha, activation of cyclooxygenase 2 (COX-2) expression, and eicosanoid synthesis, events that can cause insulin resistance and initiation and progression of inflammation and sepsis. Unsaturated fatty acids and their anti-inflammatory products, such as lipoxins, resolvins, and protectins, may suppress A-FABP expression, inhibit macrophage and COX-2 activation, and decrease production of pro-inflammatory cytokines and ultimately could lead to a decrease in insulin resistance and resolution of inflammation and recovery from sepsis. Serial measurement of these pro- and anti-inflammatory molecules and correlation of their levels to the progression to or recovery from (or both) sepsis and other inflammatory processes may form a new approach to predict prognosis in inflammatory conditions and eventually could lead to the development of new therapeutic strategies.

Oxidation of HMGB1 During Liver Ischemia and Reperfusion Causes Attenuation of Its Pro-Inflammatory Activity

High mobility group box 1 (HMGB1) is a nuclear transcription factor. Once HMGB1 is released by damaged cells or activated immune cells, it acts as danger molecule and triggers the inflammatory signaling cascade. Currently, evidence is accumulating, that posttranslational modifications such as oxidation may modulate the pro-inflammatory potential of danger signals. We hypothesized that: (1) effluent HMGB1 from cold preserved liver grafts may act as pro-inflammatory cytokine; (2) oxidation of HMGB1 may reduce its pro-inflammatory potential; and (3) oxidation of HMGB1 occurs during prolonged ischemia and upon reperfusion. Liver grafts were cold preserved for 24 h and flushed with saline in hourly intervals to collect the effluent. Liver grafts, cold-preserved for 6 h, were transplanted into syngeneic recipients to obtain serum and liver samples 24 h after initiation of reperfusion. Addition of the effluent to a macrophage culture induced the synthesis of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6. The stimulatory activity of graft effluent was reduced after depletion of HMGB1 via immunoprecipitation. Oxidation of the effluent HMGB1 using H2O2 attenuated its stimulatory activity as well. Liver transplantation of cold preserved grafts caused HMGB1 translocation and release as determined by immunohistochemistry and ELISA-assay, respectively. Using Western blot with non-reducing conditions revealed the presence of oxidized HMGB1 in liver samples obtained after 4 h and in effluent samples after 16 h of cold preservation as well as in liver and serum samples obtained 24 h after reperfusion. These observations confirm that post-translational oxidation of HMGB1 attenuates its pro-inflammatory activity. Our findings also call for future studies to investigate the mechanism of the inhibitory effect of oxidized HMGB1 on the pro-inflammatory potential.

Oxidation of HMGB1 Causes Attenuation of Its Pro-Inflammatory Activity and Occurs during Liver Ischemia and Reperfusion

High mobility group box 1 (HMGB1) is a nuclear transcription factor. Once HMGB1 is released by damaged cells or activated immune cells, it acts as danger molecule and triggers the inflammatory signaling cascade. Currently, evidence is accumulating that posttranslational modifications such as oxidation may modulate the pro-inflammatory potential of danger signals. We hypothesized that oxidation of HMGB1 may reduce its pro-inflammatory potential and could take place during prolonged ischemia and upon reperfusion.Liver grafts were cold preserved for 24 h and flushed with saline in hourly intervals to collect the effluent. Liver grafts, cold-preserved for 6 h, were transplanted into syngeneic recipients to obtain serum and liver samples 24 h after initiation of reperfusion. Addition of the effluent to a macrophage culture induced the synthesis of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6. The stimulatory activity of graft effluent was reduced after depletion of HMGB1 via immunoprecipitation. Oxidation of the effluent HMGB1 using H2O2 attenuated its stimulatory activity as well. Liver transplantation of cold preserved grafts caused HMGB1 translocation and release as determined by immunohistochemistry and ELISA-assay, respectively. Using Western blot with non-reducing conditions revealed the presence of oxidized HMGB1 in liver samples obtained after 12 h and in effluent samples after 16 h of cold preservation as well as in liver and serum samples obtained 24 h after reperfusion.These observations confirm that post-translational oxidation of HMGB1 attenuates its pro-inflammatory activity. Oxidation of HMGB1 as induced during prolonged ischemia and by reoxygenation during reperfusion in vivo might also attenuate its pro-inflammatory activity. Our findings also call for future studies to investigate the mechanism of the inhibitory effect of oxidized HMGB1 on the pro-inflammatory potential.

The protective effects of coumestrol against amyloid-beta peptide- and lipopolysaccharide-induced toxicity on mice astrocytes

Objectives: Estrogen replacement therapy can decrease the risk of developing Alzheimer’s disease. Phytoestrogens have been proposed as potential alternatives to estrogen replacement therapy. The purpose of this study was to evaluate the in vitro protective effects of coumestrol on mice astrocytes.Methods: Different concentrations of coumestrol were tested for their protective efficacy against two toxic insults, lipopolysaccharide (LPS) and amyloid-beta peptide, on astrocytes. The mitochondrial activity of astrocytes was determined, and the protective efficacy and pathway were examined by their specific gene expression and protein change.Results: The results showed that coumestrol induced a modest but significant increase in viability of astrocytes, while the viability of astrocytes was reduced following exposure to LPS and amyloid-beta peptide. The addition of coumestrol could reverse the toxic effect induced by LPS and amyloid-beta peptide. Both the LPS and amyloid-beta peptide enhanced interleukin 1, interleukin 6, and tumor necrosis factor-alpha synthesis and these effects were inhibited by 10−9M coumestrol. This effect was more obvious on the LPS-induced inflammation. The estrogen receptor expression was upregulated by coumestrol, while the effect was more obvious on estrogen receptor-beta (ER-beta). These effects can be inhibited by extracellular signal-regulated kinase and c-Jun N-terminal kinase inhibitors but not p38 inhibitor.Discussion: The current data support a possible role for astrocytes in the mediation of neuroprotection by coumestrol. An indirect extracellular signal-regulated kinase/c-Jun N-terminal kinase signaling pathway to downregulate the expression of interleukin 1, interleukin 6, and the tumor necrosis factor-alpha cytotoxic effect may act in concert with the proposed direct ER-beta biosynethsis pathway to achieve a widespread, global protection of ER-beta positive neurons.

North American Porcine Reproductive and Respiratory Syndrome Viruses Inhibit Type I Interferon Production by Plasmacytoid Dendritic Cells

Although enveloped viruses typically trigger the prodigious secretion of alpha interferon (IFN-α) by plasmacytoid dendritic cells (pDC), porcine pDC remain quiescent when exposed to porcine reproductive and respiratory syndrome virus (PRRSV). This inactivity is likely due to virus-mediated interference since the typical IFN-α response by either purified or nonsorted porcine pDC to transmissible gastroenteritis virus (TGEV) or the Toll-like receptor 9 agonist, oligodeoxynucleotide (ODN) D19, was markedly reduced in the presence of PRRSV. Suppression occurred independently of virus viability and acidification of pDC early endosomes but correlated with diminished levels of IFN-α mRNA. This change was attributed to an abrogation of transcription resulting from a decrease in the otherwise enhanced amounts of the requisite interferon regulatory factor 7 (IRF-7), whose gene expression in turn was limited as a consequence of a lessened availability of nuclear-localized signal transducer and activator of transcription 1 (STAT1). While PRRSV also inhibited tumor necrosis factor alpha (TNF-α) synthesis by pDC responding to either agent, only the interleukin-2 (IL-2) and IL-6 production instigated by ODN D19 exposure was blocked. Likewise, PRRSV did not impact a specific TGEV-associated enhancement of IL-8 expression. Moreover, an augmented phosphorylation of NF-κB seen in activated pDC was not only unaffected by PRRSV but actually occurred in its presence. Thus, as supported by a demonstrated resilience of pDC to PRRSV infection, this pathogen may interact with a cell surface protein(s) to selectively impede the completion of cascades involved in cytokine production by stimulated pDC.

Effects of compounds from Bi-Qi Capsule on the expression of inflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 macrophages

The Bi-Qi Capsule (Bi-Qi) has been used in clinic as prescribed drug for the treatment of rheumatic arthritis, rheumatoid arthritis and other osteoarticular diseases about 20 years in China. Pharmacological and clinical studies have confirmed the anti-inflammatory and analgesic action of Bi-Qi in vivo. However, its anti-inflammatory molecular mechanism is still unclear. The objective of the study is to reveal the anti-inflammatory molecular mechanism of Bi-Qi which would form an additional proof to the traditional experience of Bi-Qi in clinical administration. The aqueous extract of Bi-Qi was used to evaluate the anti-inflammatory action in murine macrophage cell line RAW 264.7 treated with lipopolysaccharide (LPS). Cell viability was evaluated by MTT assay. Production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) were measured by the Griess colorimetric method and enzyme-linked immunosorbent assay (ELISA), respectively. Protein expression levels of cyclooxygenase 2 (COX-2) were monitored by cell-based ELISA. Proteome profiler array was analyzed to evaluate 40 cytokines at protein level. In addition, interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) synthesis were analyzed using ELISA to confirm the result of the Proteome profiler array. The gene expression levels of inducible nitric oxide synthase (iNOS), COX-2, TNF-α, IL-6, and interleukin 1β (IL-1β) were detected by quantitative real-time reverse-transcription polymerase chain reaction (real-time RT-PCR). Bi-Qi significantly decreased the production of NO, PGE(2), and inhibited the protein expression of COX-2. The Proteome profiler array showed that eight protein cytokines were down-regulated and six protein cytokines were up-regulated by Bi-Qi. Furthermore, the results of TNF-α and IL-6 protein expression analyzed by ELISA were similar to those of Proteome profiler array. The results of real-time RT-PCR demonstrated that iNOS, COX-2, TNF-α, IL-6 and IL-1β gene expression were also significantly reduced by Bi-Qi. These results suggested that the anti-inflammatory molecular mechanism of Bi-Qi might be the results from modulating the LPS-mediated NO-iNOS pathway, COX-2 pathway via inhibition of iNOS, COX-2, TNF-α, IL-6 and IL-1β expression in activated macrophages. In addition, these results provided evidence to understand the therapeutic effects of Bi-Qi on various inflammatory diseases, e.g. rheumatoid arthritis, rheumatic arthritis and other osteoarticular diseases.

Comparison of the Moonlighting Actions of the Two Highly Homologous Chaperonin 60 Proteins of Mycobacterium tuberculosis

Evidence is emerging that the two chaperonin (Cpn) 60 proteins of Mycobacterium tuberculosis, Cpn60.1 and Cpn60.2, have moonlighting actions that may contribute to the pathology of tuberculosis. We studied the release of Cpn60.1 from M. tuberculosis and infected macrophage like cells and compared recombinant Cpn60.1 and Cpn60.2 in a range of cell-based assays to determine how similar the actions of these highly homologous proteins are. We now establish that Cpns are similar as follows: (i) Cpn60.1, as it has been shown for Cpn60.2, is released by M. tuberculosis in culture, and Cpn60.1 is furthermore released when the bacterium is in quiescent, but not activated, macrophage like cells, and (ii) both proteins only showed a partial requirement for MyD88 for the induction of proinflammatory cytokine production compared to lipopolysaccharide. However, we also found major differences in the cellular action of Cpns. (i) Cpn60.2 proved to be a more potent stimulator of whole blood leukocytes than Cpn60.1 and was the only one to induce tumor necrosis factor alpha synthesis. (ii) Cpn60.1 bound to ca. 90% of circulating monocytes compared to Cpn60.2, which bound <50% of these cells. Both chaperonins bound to different cell surface receptors, while monocyte activation by both proteins was completely abrogated in TLR4-/- mice, although Cpn60.2 also showed significant requirement for TLR2. Finally, an isogenic mutant lacking cpn60.1, but containing intact cpn60.2, was severely inhibited in generating multinucleate giant cells in an in vitro human granuloma assay. These results clearly show that, despite significant sequence homology, M. tuberculosis Cpn60 proteins interact in distinct ways with human or murine macrophages.

Glycosylation regulates NK cell-mediated effector function through PI3K pathway

Aberrant glycosylation, which impairs recognition capability of NK cells or modifies recognition pattern of target cells, is associated with cancer. Synthetic glycoconjugates (GCs), which modulate cell glycosylation, increase the sensitivity of tumor cells to therapy or boost anti-cancer immune response. In the current study, we employed N-acetyl-D-glucosamine-calix[4]arene (GN4C) as a modulator of cell glycosylation of NK cells represented by the NK-92 cell line and fresh human NK cells. For the first time, we have demonstrated that calix[4]arene-based GC down-regulated the expression of glycosyltransferases MGAT3 and MGAT5 in NK-92 and fresh NK cells. GN4C increased the susceptibility of tumor cells to cytotoxicity by purified fresh NK cells or NK-92 cells. This functional activation of NK cells and the NK-92 cell line correlated with an increased expression of NKG2D mRNA. In the NK-92 cell line, GN4C induced the synthesis of IL-2, IFN-gamma and tumor necrosis factor-alpha as well. Cellular signaling triggered by GN4C engaged PI3-kinase/ERK but not phospholipase C-gamma/JNK pathways. Simultaneously, in transformed NK-92 cells, GN4C reduced the rate of proliferation and down-regulated the c-MYC, EGF-receptor 1 and REL-A molecules. In conclusion, the modulation of glycosyltransferases MGAT3 and MGAT5 by synthetic GN4C correlated with the improvement of NK cell effector functions and the augmentation of tumor cells sensitivity to NK cell-mediated cytotoxicity.

CB1 and CB2 cannabinoid receptors differentially regulate the production of reactive oxygen species by macrophages

We investigated the mechanism by which cannabinoid receptors-1 (CB1) and -2 (CB2) modulate inflammatory activities of macrophages. Real-time polymerase chain reaction showed the predominant CB2 expression in freshly isolated human monocytes. PMA, a potent inducer of differentiation, upregulated CB1 and increased CB1:CB2 transcript ratio from 1:17.5 to 1:3 in 5 days of culture. Immunohistochemistry showed that CB1 protein was colocalized in CD68- and CD36-positive macrophages in human atheroma. Through selective expression of CB1 or CB2 to thioglycollate-elicited peritoneal macrophages, we proved that CB1 and CB2 mediate opposing influences on the production of reactive oxygen species (ROS). Flow cytometry showed that cannabinoid-induced ROS production by macrophages was CB1-dependent. Immunoblotting assays confirmed that macrophage CB1, not CB2, induced phosphorylation of p38-mitogen-activated protein kinase, which modulated ROS production and the subsequent synthesis of tumour necrosis factor-alpha and monocyte chemoattractant protein-1. Pull-down assays showed that the Ras family small G protein, Rap1 was activated by CB2. Dominant-negative Rap1 profoundly enhanced CB1-dependent ROS production by macrophages, suggesting CB2 Rap1-dependently inhibits CB1-stimulated ROS production. CB1 promotes pro-inflammatory responses of macrophages through ROS production, which is negatively regulated by CB2 through Rap1 activation. Blocking CB1 together with selective activation of CB2 may suppress pro-inflammatory responses of macrophages.