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Abstract Objective Tristetraprolin (TTP/ZFP36) family proteins exhibit antiinflammatory effects by destabilizing proinflammatory mRNAs. Previous studies showed that bacterial endotoxin lipopolysaccharides (LPS) stimulated TTP and tumor necrosis factor (TNF) gene expression, but less was known about LPS effects on TTP homologues and other proinflammatory gene expression in macrophages. The objective was to investigate LPS regulation of TTP family gene and TTP-targeted gene expression in mouse RAW264.7 macrophages using much higher concentrations of LPS and much longer treatment time than previous studies. Results MTT assay showed that LPS was not toxic to the cells under LPS treatment up to 1000 ng/mL for 2–24 h. LPS mildly affected the soluble protein content in the cells. qPCR assay showed that LPS stimulated TTP mRNA rapidly but not sustainably with 40, 10, and 3 fold of the DMSO control after 2, 8 and 24 h treatment, respectively. Immunoblotting confirmed qPCR results on LPS stimulation of TTP gene expression in the mouse macrophages. LPS exhibited minimal effects on ZFP36L1, ZFP36L2 and ZFP36L3 mRNA levels. LPS increased mRNA levels of TNF, COX2, GM-CSF, INFγ and IL12b up to 311, 418, 11, 9 and 4 fold, respectively. This study demonstrated that LPS did not affect macrophage viability, dramatically increased antiinflammatory TTP gene expression as well as proinflammatory TNF and COX2 gene expression but had only mild effects on TTP homologues and other proinflammatory cytokine gene expression in the mouse macrophages.

Here, we determined the immunostimulatory effects of black radish (Raphanus sativus ver niger) hot water extract (BRHE) on a mouse macrophage cell line (RAW 264.7) and mouse peritoneal macrophages. We found that BRHE treatment increased cell proliferation, phagocytic activity, nitric oxide (NO) levels, cytokine production, and reactive oxygen species synthesis. Moreover, BRHE increased the expression of the following immunomodulators in RAW 264.7 cells and peritoneal macrophages: pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), iNOS, and COX-2. BRHE treatment significantly up-regulated the phosphorylation of components of the mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), Akt, and STAT3 signaling pathways. Further, the effects of BRHE on macrophages were significantly diminished after the cells were treated with the TLR2 antagonist C29 or the TLR4 antagonist TAK-242. Therefore, BRHE-induced immunostimulatory phenotypes in mouse macrophages were reversed by multiple inhibitors, such as TLR antagonist, MAPK inhibitor, and Akt inhibitor indicating that BRHE induced macrophage activation through the TLR2/4–MAPK–NFκB–Akt–STAT3 signaling pathway. These results indicate that BRHE may serve as a potential immunomodulatory factor or functional food and provide the scientific basis for the comprehensive utilization and evaluation of black radish in future applications. [ABSTRACT FROM AUTHOR]

Cystatin, a cysteine protease inhibitor found in many parasites, plays important roles in immune evasion. This study analyzed the molecular characteristics of a cystatin from Fasciola hepatica (FhCystatin) and expressed recombinant FhCystatin (rFhcystatin) to investigate the immune modulatory effects on lipopolysaccharide-induced proliferation, migration, cytokine secretion, nitric oxide (NO) production, and apoptosis in mouse macrophages. The FhCystatin gene encoded 116 amino acids and contained a conserved cystatin-like domain. rFhCystatin significantly inhibited the activity of cathepsin B. rFhCystatin bound to the surface of mouse RAW264.7 cells, significantly inhibited cell proliferation and promoted apoptosis. Moreover, rFhCystatin inhibited the expression of cellular nitric oxide, interleukin-6, and tumor necrosis factor-a, and promoted the expression of transforming growth factor-ß and interleukin-10. These results showed that Fh- Cystatin played an important role in regulating the activity of mouse macrophages. Our findings provide new insights into mechanisms underlying the immune evasion and contribute to the exploration of potential targets for the development of new drug to control F. hepatica infection. [ABSTRACT FROM AUTHOR]

Here, we determined the immunostimulatory effects of black radish (Raphanus sativus ver niger) hot water extract (BRHE) on a mouse macrophage cell line (RAW 264.7) and mouse peritoneal macrophages. We found that BRHE treatment increased cell proliferation, phagocytic activity, nitric oxide (NO) levels, cytokine production, and reactive oxygen species synthesis. Moreover, BRHE increased the expression of the following immunomodulators in RAW 264.7 cells and peritoneal macrophages: pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), iNOS, and COX-2. BRHE treatment significantly up-regulated the phosphorylation of components of the mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), Akt, and STAT3 signaling pathways. Further, the effects of BRHE on macrophages were significantly diminished after the cells were treated with the TLR2 antagonist C29 or the TLR4 antagonist TAK-242. Therefore, BRHE-induced immunostimulatory phenotypes in mouse macrophages were reversed by multiple inhibitors, such as TLR antagonist, MAPK inhibitor, and Akt inhibitor indicating that BRHE induced macrophage activation through the TLR2/4–MAPK–NFκB–Akt–STAT3 signaling pathway. These results indicate that BRHE may serve as a potential immunomodulatory factor or functional food and provide the scientific basis for the comprehensive utilization and evaluation of black radish in future applications.

Hydrophilic acylated surface proteins (HASPs) are acidic surface proteins which get localized on the surface of Leishmania parasite during infective stages through a 'non-classical' pathway. In this study, we report the heterologous expression and purification of Leishmania donovani HASPA (r-LdHASPA) in E. coli system and its partial characterization. The structural aspects of the purified protein were analyzed using CD spectroscopy and modeling studies which indicate that r-LdHASPA consists of random coils. Studies in mouse macrophage RAW264.7 cell lines indicate that r-LdHASPA enhances reactive oxygen species (ROS) production. Co-immunoprecipitation (IP) studies indicate that r-LdHASPA interacts with certain macrophage proteins which however could not be identified unambiguously. The present study provides key insights into the structural and functional aspects of an important Leishmania protein, HASPA, which we believe could be useful for further research on vaccine/drug development. [ABSTRACT FROM AUTHOR]

The relationship between bacteria and host phagocytic cells is key to the induction of immunity. To visualize and monitor bacterial infection, we developed a novel bacterial membrane permeable pH sensor for the noninvasive monitoring of bacterial entry into murine macrophages. The pH sensor was constructed using 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) as an electron-withdrawing group and aniline as an electron-donating group. A piperazine moiety was used as the pH-sensitive group. Because of the strong electron-donating and -withdrawing units conjugated in the sensing moiety M, the fluorophore emitted in the red spectral window, away from the autofluorescence regions of the bacteria. Following the engulfment of sensor-labeled bacteria by macrophages and their subsequent merger with host lysosomes, the resulting low-pH environment enhances the fluorescence intensity of the pH sensors inside the bacteria. Time-lapse analysis of the fluorescent intensity suggested significant heterogeneity of bacterial uptake among macrophages. In addition, qRT-PCR analysis of the bacterial 16 S rRNA gene expression within single macrophage cells suggested that the 16 S rRNA of the bacteria was still intact 120 min after they had been engulfed by macrophages. A toxicity assay showed that the pH sensor has no cytotoxicity towards either E. coli or murine macrophages. The sensor shows good repeatability, a long lifetime, and a fast response to pH changes, and can be used for a variety of bacteria. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Enterococci are commensal organisms in the alimentary tract. However, they can cause a variety of life-threatening infections, especially in nosocomial settings. We hypothesized that induction of cell death might enable these facultative pathogenic bacteria to evade the innate immune response and to cause infections of their host. We demonstrate that E. faecium when exposed to lysozyme induces cell death in macrophages in vitro and in vivo. Flow cytometric analyses of J774A.1 macrophages infected with E. faecium revealed loss of cell membrane integrity indicated by uptake of propidium iodide and decrease of the inner mitochondrial transmembrane potential ΔΨ. Inhibition of caspases, treatment of macrophages with cytochalasin D, or rifampicin did not prevent cells from dying, suggesting cell death mechanisms that are independent of caspase activation, bacterial uptake, and intracellular bacterial replication. Characteristics of necrotic cell death were demonstrated by both lack of procaspase 3 activation and cell shrinkage, electron microscopy, and release of lactate dehydrogenase. Pretreatment of E. faecium with lysozyme and subsequently with broad spectrum protease considerably reduced cell death, suggesting that a bacterial surface protein is causative for cell death induction. Moreover, in a mouse peritonitis model we demonstrated that E. faecium induces cell death of peritoneal macrophages in vivo. Altogether, our results show that enterococci, under specific conditions such as exposure to lysozyme, induce necrotic cell death in macrophages, which might contribute to disseminated infections by these facultative pathogenic bacteria. [ABSTRACT FROM AUTHOR]

Abstract: Oxidative modification of low density lipoproteins is thought to play a pivotal role in the development and exacerbation of atherosclerosis and atherogenesis, and is believed to be closely associated with alterations in the vascular production of nitric oxide (NO). Previous work has shown that several products emerging from lipid peroxidation (e.g. lipid hydroperoxides, lysophospholipids, oxidized cholesterol) are able to reduce NO production in macrophages. The naturally occurring oxidant hypochlorite has been shown to be responsible for the in vivo formation of hypochlorite-oxidized LDL and such OxLDL are known to lack lipid peroxidation products. In this work we demonstrate that hypochlorite-oxidized LDL mediate profound effects on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages. By means of the membrane-permeable NO indicator 4,5-diaminofluorescein diacetate, we are able to show decreased levels of intracellular authentic nitric oxide following incubation with hypochlorite-oxidized LDL. The observed effects are dose-dependent and comparable to results obtained in the presence of the NOS inhibitor N G-monomethyl-l-arginine. This marked reduction of intracellular NO is accompanied by a dose-dependent inhibition of inducible nitric oxide synthase (iNOS) protein and mRNA expression. Furthermore, hyp-OxLDL lead to the generation of peroxynitrite, thereby also reducing bioavailability of NO. By mediating these effects on production and bioavailabiltiy of NO, hyp-OxLDL might also contribute to atherogenesis by reducing the antiatherogenic effects of nitric oxide. [Copyright &y& Elsevier]

Abstract: Trace amines such as tyramine, octopamine and β-phenylethylamine bind with high affinity to the mammalian trace amine-associated receptor 1 (Taar1), potentially activating G-proteins in the synaptic membranes of target neurons. Recently there has been significant interest in Taar1, since this receptor can bind certain psychoactive drugs of abuse such as Ecstasy (3,4-methylenedioxymethamphetamine). Surprisingly, Ecstasy has been shown to alter responses of immune cells, and we questioned whether Taar receptors might be responsible for this effect. Using sensitive and quantitative RT-PCR assays, we found no detectable expression of Taar mRNA in bone marrow, or in primary cultures of mouse macrophages and dendritic cells whether quiescent or activated by exposure to lipopolysaccharide or the mouse gamma herpesvirus-68 (γHV-68). Mouse B cells and NK cells isolated from spleen, however, showed expression of several Taar mRNA species. Taar mRNA expression was also upregulated in human peripheral blood lymphocytes following in vitro stimulation with PHA. These studies represent the first to define expression of the mRNAs encoding these trace amine receptors in leukocytes. [Copyright &y& Elsevier]

Abstract: Exposure to complex mixtures of bacteria and fungi in moisture-damaged buildings is a potential cause of inflammatory related symptoms among occupants. The present study assessed interactions between two characteristic moldy house microbes Streptomyces californicus and Stachybotrys chartarum. Differences in cytotoxic and inflammatory responses in mouse (RAW264.7) macrophages were studied after exposure to the spores of co-cultivated microbes, the mixture of separately cultivated spores, and the spores of either of these microbes cultivated alone. The RAW264.7 cells were exposed to six doses (1 × 104 to 3 × 106 spores/ml) for 24 h, and the time course of the induced responses was evaluated after 4, 8, 16, and 24 h of exposure (1 × 106 spores/ml). The cytotoxic potential of the spores was characterized by the MTT test, DNA content analysis, and enzyme assay for caspase-3 activity. The production of cytokines (IL-1β, IL-6, IL-10, TNFα, and MIP2) was measured immunochemically and nitric oxide by the Griess method. Co-cultivation increased the ability of the spores to cause apoptosis by more than 4-fold and the proportion of RAW264.7 cells at the G2/M stage increased nearly 2-fold when compared to the response induced by the mixture of spores. In contrast, co-cultivation decreased significantly the ability of the spores to trigger the production of NO and IL-6 in RAW264.7 cells. In conclusion, these data suggest that co-culture of S. californicus and S. chartarum can result in microbial interactions that significantly potentiate the ability of the spores to cause apoptosis and cell cycle arrest in mammalian cells. [Copyright &y& Elsevier]

Inhalation of fungal spores may cause inflammation and respiratory diseases, such as bronchitis, allergic alveolitis, and asthma. Alveolar macrophages provide the first line of defense in the respiratory tract. To examine the cellular mechanisms involved in Aspergillus fumigatus-induced airway inflammation, mouse macrophage cell line (RAW 264.7) cells were exposed for 2 h or 6 h to graded doses of A. fumigatus spores that were either alive or heat-killed. Furthermore, the ability of the cells to phagocytize the spores was visualized by electron microscopy. Expression of selected cytokines and chemokines was assessed by a real time quantitative PCR method and by enzyme-linked immunoabsorbent assay (ELISA) after exposure. A significant increase in mRNA expression of TNF-α, MIP-1α, MIP-1β, and MCP-1 was observed with a maximal induction at 6 h after exposure to the highest (1×107) concentration of live spores. Similar response was not detected with heat-killed spores in the expression of chemokines and cytokines, even though there were no differences between the phagocytosis of live and heat-killed spores. These results suggest that exposure to live spores of A. fumigatus can modulate the expression of proinflammatory cytokines and chemokines in mouse macrophages and thus influence the development of inflammatory processes in the airways. [Copyright &y& Elsevier]

Previous data have shown that a citrus flavanone, hesperidin possesses chemopreventive efficacies. We designed to investigate the inhibitory effect of hesperidin on lipopolysaccharide (LPS)-induced over-expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) proteins, over-production of prostaglandin E2 (PGE2) and nitric oxide (NO) using mouse macrophage cells. Treatment with hesperidin suppressed production of PGE2, nitrogen dioxide (NO2), and expression of iNOS protein. In the case of COX-2, hesperidin did not affect the protein level. Our data indicate hesperidin as a COX-2 and iNOS inhibitor, which might be related to the anti-inflammatory and anti-tumorigenic efficacies. [Copyright &y& Elsevier]

SUMMARY Unmethylated CpG motif in synthetic oligodeoxynucleotide (CpG ODN) or bacterial DNA is well recognized for its role in innate immunity, including enhancing production of NO and cytokines by macrophages. In the present study, we demonstrated the effect of CpG ODN on the phagocytic uptake of bacteria by macrophages. Flow cytometric analysis of mouse macrophages (RAW 264·7) incubated with fluorescein isothiocyanate (FITC)-labelled Burkholderia pseudomallei , Salmonella enterica serovar Typhi or Escherichia coli showed that CpG ODN increased the uptake of these bacteria by mouse macrophages. The enhancement of bacterial uptake by CpG ODN was concentration-dependent. The increase of bacterial uptake by CpG ODN-activated macrophages shown above is consistent with the result of bacteria internalization study using a standard antibiotic protection assay. There was also an increase in the rate and degree of multi-nucleated giant cell formation, phenomena which have been shown previously to be unique when the cells were infected with B. pseudomallei . These observations may provide significant insights for future investigation into host cell–pathogen interaction. [ABSTRACT FROM AUTHOR]

SUMMARY Synthetic CpG containing oligodeoxynucleotide (CpG ODN) is recognized for its ability to activate cells to produce several cytokines, such as IL-12 and TNF-α. In the present study we have demonstrated that CpG ODN 1826, known for its immunostimulatory activity in the mouse system could, by itself, induce nitric oxide (NO) and inducible nitric oxide synthase (iNOS) production from mouse macrophage cell line (RAW 264·7). Neutralizing antibody against TNF-α was not able to inhibit NO or iNOS production from the CpG ODN 1826-activated macrophages, suggesting that although the TNF-α was also produced by CpG ODN-activated macrophages, the production of iNOS was not mediated through TNF-α. Although both CpG ODN 1826 and lipopolysaccharide (LPS) were able to stimulate NO and iNOS production, the exposure time required for maximum production of NO and iNOS for the CpG ODN 1826-activated macrophages was significantly longer than those activated with LPS. These results were due probably to a delay of NF-κB translocation, as indicated by the delay of IκBα degradation. Moreover, the fact that chloroquine abolished NO and iNOS production from the cells treated with CpG ODN 1826 but not from those treated with LPS suggested that the induction of NO and iNOS production from the cells stimulated with CpG ODN (1826) also required endosomal maturation/acidification. [ABSTRACT FROM AUTHOR]

Dog roundworm (Toxocara canis) is the major causative agent of toxocarosis, a parasitic disease of both veterinary and medical importance. Knowledge gaps in fundamental and applied aspects hinder the control of this important zoonotic disease. To have a better understanding of Toxocara infection and host immune responses, mouse macrophages were exposed to excretory/secretory (ES) proteins released by adult worms of T. canis in vitro. The messenger RNA transcription and protein expression of nucleotide-binding oligomerization domain-containing protein 1 (NOD1), receptor interacting protein 2 (RIP2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in macrophages were analysed using quantitative real-time PCR (qRT-PCR) and Western blot. The levels of tumour necrosis factor alpha (TNF-ɑ), interleukin-1 beta (IL-1β) and IL-6 released by the stimulated macrophages were analysed using enzyme-linked immunosorbent assay. It was found that 20 μg/mL ES proteins of adult T. canis induced the expression of NOD1, RIP2 and NF-κB in mouse macrophages at both transcriptional and translational levels after 9 h of incubation in vitro. Incubation with 20 μg/mL ES proteins also modulated the production of pro-inflammatory cytokines TNF-ɑ, IL-1β and IL-6 by the macrophages. Taken together, ES proteins of adult T. canis appeared to be able to affect the macrophage NOD1-RIP2-NF-κB signalling pathway, which might play a role in regulating the production of proinflammatory cytokines. Further investigation of these aspects should lead to a better understanding of immune recognition of and modulation by Toxocara canis in host animals. This figures represent Effect of excretory/secretory adult worm proteins of Toxocara canis on the protein expression of NOD1, RIP2 and NF-κB in mouse peritoneal macrophages. (A) Western blot analysis indicates slight protein expression changes between treated (20 μg/ml ES proteins of T. canis) and untreated (blank control) macrophages after 9 h of incubation; (B) Bar chart shows the relative amount of proteins NOD1, RIP2 and NF-κB in mouse peritoneal macrophages in response to 20 μg/ml ES proteins of T. canis in vitro. Student t-test is used to analyse the difference between treatment and control. * indicates statistical significance P < 0.05; ** indicates statistical significance P < 0.01. [Display omitted] • ES proteins of Toxocara canis stimulated macrophages of mice in vitro. • T. canis ES proteins promoted NOD1, RIP2 and NF-κB macrophages of mice at both transcriptional and translational levels. • T. canis ES proteins regulated the production of cytokines TNF-α, IL-1β and IL-6. [ABSTRACT FROM AUTHOR]