Gene Expression In Murine Macrophages Research Articles

The early macrophage response to pathogens requires dynamic regulation of the nuclear paraspeckle

To ensure a robust immune response to pathogens without risking immunopathology, the kinetics and amplitude of inflammatory gene expression in macrophages need to be exquisitely well controlled. There is a growing appreciation for stress-responsive membraneless organelles (MLOs) regulating various steps of eukaryotic gene expression in response to extrinsic cues. Here, we implicate the nuclear paraspeckle, a highly ordered biomolecular condensate that nucleates on the Neat1 lncRNA, in tuning innate immune gene expression in murine macrophages. In response to a variety of innate agonists, macrophage paraspeckles rapidly aggregate (0.5 h poststimulation) and disaggregate (2 h poststimulation). Paraspeckle maintenance and aggregation require active transcription and MAPK signaling, whereas paraspeckle disaggregation requires degradation of Neat1 via the nuclear RNA exosome. In response to lipopolysaccharide treatment, Neat1 KO macrophages fail to properly express a large cohort of proinflammatory cytokines, chemokines, and antimicrobial mediators. Consequently, Neat1 KO macrophages cannot control replication of Salmonella enterica serovar Typhimurium or vesicular stomatitis virus. These findings highlight a prominent role for MLOs in orchestrating the macrophage response to pathogens and support a model whereby dynamic assembly and disassembly of paraspeckles reorganizes the nuclear landscape to enable inflammatory gene expression following innate stimuli.

Exposure of Tritrichomonas foetus to sublethal doses of metronidazole induces a specific proinflammatory response in murine macrophages.

Tritrichomonas foetus is a flagellated parasite that primarily infects the reproductive tissues of livestock, causing bovine trichomoniasis. The cytoplasmic membrane of T. foetus contains various compounds that contribute to adherence, colonization, and pathogenicity. Metronidazole (MTZ) is the main treatment for trichomoniasis, but the emergence of drug-resistant strains is a concern due to improper use and dosing. T. foetus infection induces inflammation, and macrophages are key players in the immune response. However, our understanding of the host's immune response to T. foetus is limited, and the specific mechanisms underlying these responses are not well understood. This study aimed to investigate the impact of T. foetus surface proteins from trophozoites cultured under different sublethal MTZ conditions (MTZ-treated T. foetus MPs) on macrophage activation. By analyzing cytokine levels and gene expression in murine macrophages, we demonstrated that MTZ-treated T. foetus MPs induce a specific proinflammatory response. MTZ-treated T. foetus MPs-exposed macrophages exhibited a higher NO and H2 O2 production and overexpression of iNOS and NOX-2 genes in comparison to untreated T. foetus. Additionally, MTZ-treated T. foetus MPs triggered a significant induction of the proinflammatory cytokines IL-1β, IL-6, TNF-α, and IFN-γ, as well as the overexpression of the TLR4, MyD88, and NF-κB genes on murine macrophages. The study aimed to unravel the immunological response and potential proinflammatory pathways involved in T. foetus infection and MTZ stress. Understanding the immune responses and mechanisms through which T. foetus surface proteins activate macrophages can contribute to the development of new therapeutic strategies for controlling bovine trichomoniasis.

Modified sites and functional consequences of 4-oxo-2-nonenal adducts in HDL that are elevated in familial hypercholesterolemia

The lipid aldehyde 4-oxo-2-nonenal (ONE) is a highly reactive protein crosslinker derived from peroxidation of n-6 polyunsaturated fatty acids and generated together with 4-hydroxynonenal (HNE). Lipid peroxidation product-mediated crosslinking of proteins in high-density lipoprotein (HDL) causes HDL dysfunction and contributes to atherogenesis. Although HNE is relatively well-studied, the role of ONE in atherosclerosis and in modifying HDL is unknown. Here, we found that individuals with familial hypercholesterolemia (FH) had significantly higher ONE-ketoamide (lysine) adducts in HDL (54.6 ± 33.8 pmol/mg) than healthy controls (15.3 ± 5.6 pmol/mg). ONE crosslinked apolipoprotein A-I (apoA-I) on HDL at a concentration of > 3 mol ONE per 10 mol apoA-I (0.3 eq), which was 100-fold lower than HNE, but comparable to the potent protein crosslinker isolevuglandin. ONE-modified HDL partially inhibited HDL's ability to protect against lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) gene expression in murine macrophages. At 3 eq, ONE dramatically decreased apoA-I exchange from HDL, from ∼46.5 to ∼18.4% (p < 0.001). Surprisingly, ONE modification of HDL or apoA-I did not alter macrophage cholesterol efflux capacity. LC-MS/MS analysis revealed that Lys-12, Lys-23, Lys-96, and Lys-226 in apoA-I are modified by ONE ketoamide adducts. Compared with other dicarbonyl scavengers, pentylpyridoxamine (PPM) most efficaciously blocked ONE-induced protein crosslinking in HDL and also prevented HDL dysfunction in an in vitro model of inflammation. Our findings show that ONE-HDL adducts cause HDL dysfunction and are elevated in individuals with FH who have severe hypercholesterolemia.

Modeling the LPS-induced effects on transcription factor activation and gene expression in murine macrophages.

Macrophages within the liver are of particular importance for a functional defense against bacterial infection. They exhibit a complex response to lipopolysaccharide and secrete a variety of pro-inflammatory cytokines and chemokines that both coordinate the immune response and regulate activity of the macrophages, themselves. In this context, the dynamic of pathway activation and gene expression is important for a better understanding of the role of activated macrophages in healthy and diseased states. Therefore, we present a representative model of LPS-induced macrophage activation that covers the principle regulatory motifs. Based on that, we propose a simplified model with a reduced number of states and parameters that allows estimation of transcription factor activity from gene expression data and can be easily extended to describe the full spectrum of gene regulation in LPS-activated macrophages.

Analytical workflow profiling gene expression in murine macrophages.

Comprehensive and simultaneous analysis of all genes in a biological sample is a capability of RNA-Seq technology. Analysis of the entire transcriptome benefits from summarization of genes at the functional level. As a cellular response of interest not previously explored with RNA-Seq, peritoneal macrophages from mice under two conditions (control and immunologically challenged) were analyzed for gene expression differences. Quantification of individual transcripts modeled RNA-Seq read distribution and uncertainty (using a Beta Negative Binomial distribution), then tested for differential transcript expression (False Discovery Rate-adjusted p-value < 0.05). Enrichment of functional categories utilized the list of differentially expressed genes. A total of 2079 differentially expressed transcripts representing 1884 genes were detected. Enrichment of 92 categories from Gene Ontology Biological Processes and Molecular Functions, and KEGG pathways were grouped into 6 clusters. Clusters included defense and inflammatory response (Enrichment Score = 11.24) and ribosomal activity (Enrichment Score = 17.89). Our work provides a context to the fine detail of individual gene expression differences in murine peritoneal macrophages during immunological challenge with high throughput RNA-Seq.

Direct and indirect suppression of interleukin-6 gene expression in murine macrophages by nuclear orphan receptor REV-ERBα.

It is now evident that many nuclear hormone receptors can modulate target gene expression. REV-ERBα, one of the nuclear hormone receptors with the capacity to alter clock function, is critically involved in lipid metabolism, adipogenesis, and the inflammatory response. Recent studies suggest that REV-ERBα plays a key role in the mediation between clockwork and inflammation. The purpose of the current study was to investigate the role of REV-ERBα in the regulation of interleukin-6 (il6) gene expression in murine macrophages. REV-ERBα agonists, or overexpression of rev-erb α in the murine macrophage cell line RAW264 cells, suppressed the induction of il6 mRNA following a lipopolysaccharide (LPS) endotoxin challenge. Also, rev-erb α overexpression decreased LPS-stimulated nuclear factor κB (NFκB) activation in RAW264 cells. We showed that REV-ERBα represses il6 expression not only indirectly through an NFκB binding motif but also directly through a REV-ERBα binding motif in the murine il6 promoter region. Furthermore, peritoneal macrophages from mice lacking rev-erb α increased il6 mRNA expression. These data suggest that REV-ERBα regulates the inflammatory response of macrophages through the suppression of il6 expression. REV-ERBα may therefore be identified as a potent anti-inflammatory receptor and be a therapeutic target receptor of inflammatory diseases.

FcγRI is required for TGFβ2-treated macrophage-induced tolerance

Macrophages treated with TGFβ2 (TGFβ2-Mϕ) and antigen are highly tolerogenic in vivo, and induce antigen-specific and long-lasting tolerance in both naïve and primed mice via induction of suppressor/regulatory T cells. In this study, we examined the molecular pathways, including the requirements for Smad-dependent signaling, that are involved in the induction and function of tolerogenic TGFβ2-Mϕ. Treatment of murine macrophages with TGFβ2 induced translocation of Smad2/3 to the nucleus, and impairment of Smad3-, but not Smad2-, dependent signaling inhibited the tolerogenic function of a TGFβ2-treated murine macrophage cell line. Gene expression in murine macrophages treated with TGFβ2 was evaluated by microarray analysis. The FcγRI gene was one of a number of immune-related genes differentially expressed in TGFβ2-Mϕ, and appeared to be critical for tolerance in this system, since TGFβ2-Mϕ from FcγRI deficient mice were unable to induce tolerance. The role that FcγRI plays in TGFβ2-Mϕ-mediated tolerance is currently unclear. The results of this study provide important information about the factors that are critical for the induction of TGFβ2-Mϕ-mediated tolerance, and a better understanding of these mechanisms could lead to the development of more effective tolerance-inducing strategies for the treatment of autoimmune/inflammatory diseases.

367 Molecular Regulation of Il23a Gene Expression in Murine Macrophages and Experimental Colitis

Background: Interleukin (IL)-12 and 23 are heterodimeric cytokines composed of a common p40 subunit and p35 and p19 subunits, respectively. IL-23 is strongly implicated in the pathogenesis of inflammatory bowel disease. Aim: The aim of this study is to characterize the molecular regulation of Il23a gene expression and promoter activation in murine macrophages and experimental colitis. Results: Interferon-γ (IFN-γ) inhibits LPS-induced Il23a mRNA and IL-23 protein expression in murine bone marrow-derived macrophages (BMMs). A conserved nucleotide sequence across multiple species was identified in the promoter of the Il23a gene that contains an interferon stimulated response element (ISRE). In LPS and IFN-γ activated BMMs, chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrate that interferon regulatory factors (IRFs) interact with this ISRE. Using a 1.8 kb murine Il23a luciferase reporter plasmid, LPS induces and IFN-γ inhibits LPSinduced p19 promoter activity in BMMs. Mutations in the ISRE abrogate IFN-γ inhibition of LPS-induced promoter activity. To understand the contribution of specific IRFs, IRF-1 was inhibited in BMMs by siRNA and experiments were performed in IRF-1 deficient (-/-) macrophages. IRF-1 knockdown and IRF-1-/BMMs demonstrate increased LPS-induced IL-23 expression. Primary response genes have promoters that either exist in an open chromatin structure or undergo rapid nucleosomal remodeling. In contrast, secondary response genes with delayed induction kinetics require new protein synthesis prior to transcription initiation. Il23a expression in LPS-activated BMMs demonstrates rapid induction kinetics and unlike Il12b (established secondary response gene) is not dependent on new protein synthesis. Interestingly, LPS plus IFN-γ activated BMMs demonstrate loss of the IFN-γ inhibitory effect, suggesting IFN-γ mediated regulation of Il23a requires new protein synthesis. Il23a regulation was then studied in murine colitis models. Following intrarectal administration of TNBS, IRF-1-/mice exhibit increased inflammation and increased colonic IL-23 expression compared to wild type (WT) mice. Additionally, we demonstrate mucosal regulation of IL-23 by the enteric microbiota using germ-free and conventionalized WT and colitis-prone IL-10-/mice. Enteric bacteria induce significantly higher intestinal IL-23 expression in IL-10-/-mice compared withWTmice. Conclusions: IFN-γ and IRF-1 negatively regulate Il23a in murine macrophages and experimental colitis. Regulation of intestinal Il23a by the enteric microflora is a significant event in the initiation of chronic intestinal inflammation.

O,p′-DDT induces cyclooxygenase-2 gene expression in murine macrophages: Role of AP-1 and CRE promoter elements and PI3-kinase/Akt/MAPK signaling pathways

Dichlorodiphenyltrichloroethane (DDT) has been used as an insecticide to prevent the devastation of malaria in tropical zones. However, many reports suggest that DDT may act as an endocrine disruptor and may have possible carcinogenic effects. Cyclooxygenase-2 (COX-2) acts as a link between inflammation and carcinogenesis through its involvement in tumor promotion. In the present study, we examined the effect of o,p′-DDT on COX-2 gene expression and analyzed the molecular mechanism of its activity in murine RAW 264.7 macrophages. Exposure to o,p′-DDT markedly enhanced the production of prostaglandin E 2 (PGE 2), a major COX-2 metabolite, in murine macrophages. Furthermore, o,p′-DDT dose-dependently increased the levels of COX-2 protein and mRNA. Transfection with human COX-2 promoter construct, electrophoretic mobility shift assays and DNA-affinity protein-binding assay experiments revealed that o,p′-DDT activated the activator protein 1 (AP-1) and cyclic AMP response element (CRE) sites, but not the NF-κB site. Phosphatidylinositol 3 (PI3)-kinase, its downstream signaling molecule, Akt, and mitogen-activated protein kinases (MAPK) were also significantly activated by the o,p′-DDT-induced AP-1 and CRE activation. These results demonstrate that o,p′-DDT induced COX-2 expression via AP-1 and CRE activation through the PI3-K/Akt/ERK, JNK, and p38 MAP kinase pathways. These findings provide further insight into the signal transduction pathways involved in the carcinogenic effects of o,p′-DDT.

Host Inactivation of Bacterial Lipopolysaccharide Prevents Prolonged Tolerance Following Gram-Negative Bacterial Infection

A transient state of tolerance to microbial molecules accompanies many infectious diseases. Such tolerance is thought to minimize inflammation-induced injury, but it may also alter host defenses. Here we report that recovery from the tolerant state induced by Gram-negative bacteria is greatly delayed in mice that lack acyloxyacyl hydrolase (AOAH), a lipase that partially deacylates the bacterial cell-wall lipopolysaccharide (LPS). Whereas wild-type mice regained normal responsiveness within 14 days after they received an intraperitoneal injection of LPS or Gram-negative bacteria, AOAH-deficient mice had greatly reduced proinflammatory responses to a second LPS injection for at least 3 weeks. In contrast, LPS-primed Aoah- knockout mice maintained an anti-inflammatory response, evident from their plasma levels of interleukin-10 (IL-10). LPS-primed Aoah-knockout mice experiencing prolonged tolerance were highly susceptible to virulent E. coli challenge. Inactivating LPS, an immunostimulatory microbial molecule, is thus important for restoring effective host defenses following Gram-negative bacterial infection in animals.

Apoptosis induction and attenuation of inflammatory gene expression in murinemacrophages via multitherapeutic nanomembranes

The realization of optimized therapeutic delivery is impaired by the challenge oflocalized drug activity and by the dangers of systemic cytotoxicity which oftencontribute to patient treatment complications. Here we demonstrate the blockcopolymer-mediated deposition and release of multiple therapeutics which include anLXRα/β agonist 3-((4-methoxyphenyl)amino)-4-phenyl-1-(phenylmethyl)-1H-pyrrole-2,5-dione (LXRa)and doxorubicin hydrochloride (Dox) at the air–water interface via Langmuir–Blodgettdeposition, as well as copolymer-mediated potent drug elution toward the Raw264.7 murine macrophage cell line. The resultant copolymer–therapeutic hybridserves as a localized platform that can be functionalized with virtually any drugdue to the integrated hydrophilic and hydrophobic components of the polymerstructure. In addition, the sequestering function of the copolymer to anchor thedrugs to implant surfaces can enhance delivery specificity when compared tosystemic drug administration. Confirmation of drug functionality was confirmedvia suppression of the interleukin 6 (Il-6) and tumor necrosis factor alpha(TNFα) inflammatory cytokines (LXRa), as well as DNA fragmentation analysis (Dox).Furthermore, the fragmentation assays and gene expression analysis demonstrated theinnate biocompatibility of the copolymeric material at the gene expression level via theconfirmed absence of material-induced apoptosis and a lack of inflammatory geneexpression. This modality enables layer-by-layer control of agonist and chemotherapeuticfunctionalization at the nanoscale for the localization of drug dosage, while simultaneouslyutilizing the copolymer platform as an anchoring mechanism for drug sequestering, all withan innate material thickness of 4 nm per layer, which is orders of magnitude thinner thanexisting commercial technologies. Furthermore, these studies comprehensively confirmedthe potential translational applicability of copolymeric nanomaterials as localizedmultitherapeutic thin film platforms.

TNF-induced gene expression oscillates in time

With only few exceptions that include Hes-1 p53, and IκB, the expression of genes has never been shown to be oscillatory. Here, we show that the inflammatory cytokine TNF triggers oscillations in >5000 genes. We utilize microarrays at 30-min intervals to analyze the pattern of global gene expression in murine macrophages. We find that 15% of genes in the genome underwent a significant >3-fold increase in expression, with 89% of these displaying oscillations at frequencies as low as every 50 min. We analyze further two sub-clusters of genes that either began oscillating early or after a lag phase. Through the use of quantitative PCR, we confirm the oscillations and show that the oscillations are continuous. Moreover, we show that these continuous oscillations are not unique to TNF, but that related cytokines such as RANK-L produces oscillations with a unique induction profile. In the two papers accompanying this one, we analyze the mechanism of these oscillations and find that TNF also triggers oscillations in the phosphorylation of MAP kinases, and that these oscillations combine to recruit transcription factors to promoters in a cyclical fashion. The results presented here suggest that gene transcription is a highly dynamic processes, with thousands of genes displaying rapid (<60 min) oscillations over time. Considering this dynamism, time-resolved measurements of gene transcription should become the experimental norm.

Equol inhibits nitric oxide production and inducible nitric oxide synthase gene expression through down-regulating the activation of Akt

In the present study, we report the inhibitory effect of equol on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression in murine macrophages. In vivo administration of equol (i.p.) attenuated NO production by peritoneal adherent cells isolated from lipopolysaccharide (LPS)-treated mice. Equol dose-dependently inhibited the LPS-induced production of NO in isolated peritoneal adherent cells and RAW 264.7 cells. The mRNA expression of iNOS was also blocked by equol in LPS-stimulated RAW 264.7 cells. Further study demonstrated that the LPS-induced activation of Akt was suppressed by equol in RAW 264.7 cells while the activation of ERK, SAPK/JNK and p38 MAP kinase was not affected. Equol also blocked LPS-induced NF-κB activation. Moreover, the LPS-induced NO production and NF-κB activation was inhibited by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase/Akt pathway, in RAW 264.7 cells. These results suggest that equol might inhibit NO production and iNOS gene expression, at least in part, by blocking Akt activation and subsequent down-regulation of NF-κB activity.

Lipopolysaccharide-induced gene expression in murine macrophages is enhanced by prior exposure to oxLDL

Uptake of modified lipoproteins by macrophages results in the formation of foam cells. We investigated how foam cell formation affects the inflammatory response of macrophages. Murine bone marrow-derived macrophages were treated with oxidized LDL (oxLDL) to induce foam cell formation. Subsequently, the foam cells were activated with lipopolysaccharide (LPS), and the expression of lipid metabolism and inflammatory genes was analyzed. Furthermore, gene expression profiles of foam cells were analyzed using a microarray. We found that prior exposure to oxLDL resulted in enhanced LPS-induced tumor necrosis factor (TNF) and interleukin-6 (IL-6) gene expression, whereas the expression of the anti-inflammatory cytokine IL-10 and interferon-beta was decreased in foam cells. Also, LPS-induced cytokine secretion of TNF, IL-6, and IL-12 was enhanced, whereas secretion of IL-10 was strongly reduced after oxLDL preincubation. Microarray experiments showed that the overall inflammatory response induced by LPS was enhanced by oxLDL loading of the macrophages. Moreover, oxLDL loading was shown to result in increased nuclear factor-kappaB activation. In conclusion, our experiments show that the inflammatory response to LPS is enhanced by loading of macrophages with oxLDL. These data demonstrate that foam cell formation may augment the inflammatory response of macrophages during atherogenesis, possibly in an IL-10-dependent manner.

Contribution of Interferon-β to the Murine Macrophage Response to the Toll-like Receptor 4 Agonist, Lipopolysaccharide

Interferon-beta (IFN-beta) has been identified as the signature cytokine induced via the Toll-like receptor (TLR) 4, "MyD88-independent" signaling pathway in macrophages stimulated by Gram-negative bacterial lipopolysaccharide (LPS). In this study, we analyzed the responses of macrophages derived from wild-type (IFN-beta(+/+)) mice or mice with a targeted mutation in IFN-beta (IFN-beta(-/-)) to the prototype TLR4 agonist, Escherichia coli LPS. A comparison of basal and LPS-induced gene expression (by reverse transcription-PCR, real-time PCR, and Affymetrix microarray analyses) resulted in the identification of four distinct patterns of gene expression affected by IFN-beta deficiency. Analysis of a subset of each group of differentially regulated genes by computer-assisted promoter analysis revealed putative IFN-responsive elements in all genes examined. LPS-induced activation of intracellular signaling molecules, STAT1 Tyr-701, STAT1 Ser-727, and Akt, but not p38, JNK, and ERK MAPK proteins, was significantly diminished in IFN-beta(-/-) versus IFN-beta(+/+) macrophages. "Priming" of IFN-beta(-/-) macrophages with exogenous recombinant IFN-beta significantly increased levels of LPS-induced gene expression for induction of monocyte chemotactic protein 5, inducible nitric-oxide synthase, IP-10, and IL-12 p40 mRNA, whereas no increase or relatively small increases were observed for IL-1beta, IL-6, monocyte chemotactic protein 1, and MyD88 mRNA. Finally, IFN-beta(-/-) mice challenged in vivo with LPS exhibited increased survival when compared with wild-type IFN-beta(+/+) controls, indicating that IFN-beta contributes to LPS-induced lethality; however, not to the extent that one observes in mice with more complete pathway deficiencies (e.g. TLR4(-/-) or TRIF(-/-) mice). Collectively, these findings reveal unanticipated regulatory roles for IFN-beta in response to LPS in vitro and in vivo.

Mycobacterium paratuberculosis, Mycobacterium smegmatis, and lipopolysaccharide induce different transcriptional and post-transcriptional regulation of the IRG1 gene in murine macrophages

Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic enteritis in ruminants. In addition, MAP is presently the most favored pathogen linked to Crohn's disease. In this study, we were interested in dissecting the molecular mechanisms of macrophage activation or deactivation after infection with MAP. By subtractive hybridization of cDNAs, we identified the immune-responsive gene 1 (IRG1), which was expressed substantially higher in lipopolysaccharide (LPS)-stimulated than in MAP-infected murine macrophage cell lines. A nuclear run-on transcription assay revealed that the IRG1 gene was activated transcriptionally in LPS-stimulated and MAP-infected macrophages with higher expression in LPS-stimulated cells. Analysis of post-transcriptional regulation demonstrated that IRG1 mRNA stability was increased in LPS-stimulated but not in MAP-infected macrophages. Furthermore, IRG1 gene expression of macrophages infected with the nonpathogenic Mycobacterium smegmatis differed from those of LPS-stimulated and MAP-infected macrophages. At 2 h postinfection, M. smegmatis-induced IRG1 gene expression was as low as in MAP-infected, and 8 h postinfection, it increased nearly to the level in LPS-stimulated macrophages. Transient transfection experiments revealed similar IRG1 promoter activities in MAP- and M. smegmatis-infected cells. Northern analysis demonstrated increased IRG1 mRNA stability in M. smegmatis-infected macrophages. IRG1 mRNA stabilization was p38 mitogen-activated protein kinase-independent. Inhibition of protein synthesis revealed that constitutively expressed factors seemed to be responsible for IRG1 mRNA destabilization. Thus, our data demonstrate that transcriptional and post-transcriptional mechanisms are responsible for a differential IRG1 gene expression in murine macrophages treated with LPS, MAP, and M. smegmatis.

Hypochlorite-oxidized low-density lipoprotein upregulates CD36 and PPARγ mRNA expression and modulates SR-BI gene expression in murine macrophages

The uptake of oxidized low-density lipoprotein (oxLDL) by scavenger receptors of macrophages with resulting foam cell formation is considered a critical event in atherogenesis. Since hypochlorite-oxidized LDL (HOCl-LDL) has been shown to be recognized by macrophages and evidence was provided that HOCl-LDL is internalized via class B scavenger receptors CD36 and SR-BI, the regulatory relationships between CD36, SR-BI, and the nuclear transcription factor PPARgamma in murine macrophages (RAW 264.7) on exposure to HOCl-LDL were examined. Using the highly sensitive real-time RT-PCR we could demonstrate that HOCl-LDL upregulated CD36 and PPARgamma levels dose- and time dependently while modulating SR-BI message levels differently in dependence on HOCl-LDL concentration and incubation time. On exposure of macrophages to HOCl-LDL but not native LDL in varying concentrations, a significant positive correlation between CD36 and PPARgamma (rho = 0.603, p = 0.001) was observed indicating the presence of a positive feedback mechanism by which HOCl-LDL could promote its own uptake. The transcriptional expression of SR-BI in macrophages was not significantly related to PPARgamma mRNA levels after treatment with HOCl-LDL suggesting a differential regulation of the two members of the scavenger receptor class B family in response to HOCl-LDL.

Glabridin, an Isoflavan from Licorice Root, Inhibits Inducible Nitric-Oxide Synthase Expression and Improves Survival of Mice in Experimental Model of Septic Shock

(R)-4-(3,4-Dihydro-8,8-dimethyl)-2H,8H-benzo[1,2-b:3,4-b']dipyran-3yl)-1,3-benzenediol (glabridin), a flavonoid present in licorice extract, is known to have antimicrobial, anti-inflammatory, and cardiovascular protective activities. In the present study, we report the inhibitory effect of glabridin on nitric oxide (NO) production and inducible nitric oxide (iNOS) gene expression in murine macrophages. Glabridin attenuated lipopolysaccharide (LPS)-induced NO production in isolated mouse peritoneal macrophages and RAW 264.7 cells, a mouse macrophage-like cell line. Moreover, iNOS mRNA expression was also blocked by glabridin treatment in LPS-stimulated RAW 264.7 cells. Further study demonstrated that the LPS-induced nuclear factor (NF)-kappaB/Rel DNA binding activity and NF-kappaB/Rel-dependent reporter gene activity were significantly inhibited by glabridin in RAW 264.7 cells and that this effect was mediated through the inhibition of inhibitory factor-kappaB degradation and p65 nuclear translocation. Moreover, reactive oxygen species generation was also suppressed by glabridin treatment in RAW 264.7 cells. In contrast, the activity of mitogen-activated protein kinases was unaffected by glabridin treatment. In animal model, in vivo administration of glabridin increased the rate of survival of LPS-treated mice and inhibited LPS-induced increase in plasma concentrations of nitrite/nitrate and tumor necrosis factor-alpha. Collectively, these data suggest that glabridin inhibits NO production and iNOS gene expression by blocking NF-kappaB/Rel activation and that this effect was mediated, at least in part, by inhibiting reactive oxygen species generation. Furthermore, in vivo anti-inflammatory effect of glabridin suggests a possible therapeutic application of this agent in inflammatory diseases.

Biologic evaluation of curcumin and structural derivatives in cancer chemoprevention model systems

Curcumin is a natural product widely used as a spice in food. It has been shown to inhibit cyclooxygenase (COX)-1 and -2 and to suppress lipopolysaccharide-induced COX-2 and iNOS gene expression. In the present study, curcumin and 22 of its derivatives were evaluated for their chemopreventive potential. Based on COX-2 inhibition, curcumin (IC 50 = 15.9 μM), 1,7- bis(3-fluoro-4-hydroxyphenyl)-1,6-heptadiene-3,5-dione ( 19) (IC 50 = 23.7 μM) and 2,6- bis(3-fluoro-4-hydroxybenzylidene)cyclohexanone ( 23) (IC 50 = 5.5 μM) were found to be most potent. Tricyclic derivatives 2,6- bis(4-hydroxy-3-methoxybenzylidene)cyclohexanone ( 10), 2,6- bis(4-hydroxy-3,5-dimethoxybenzylidene)cyclohexanone ( 13) and 2,5- bis(4-hydroxy-3,5-dimethoxybenzylidene)cyclopentanone ( 21) inhibited LPS-induced COX-2 and iNOS gene expression in murine macrophages with potency equal to curcumin. RT-PCR experiments demonstrated suppression of COX-2 and iNOS gene expression occurred at the transcriptional level. The most active compounds in the macrophage assays, 13 and 23, were also the most cytotoxic, however. Topical application of curcumin, 10, 13, 21, and 6, a methoxy derivative of curcumin, showed strong inhibition of 12- O-tetradecanoyl-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity in mouse skin. These data suggest that structural elements responsible for COX-1 and COX-2 inhibition do not correlate well with those responsible for inhibiting COX-2 and iNOS gene expression, but elements capable of inhibiting COX-2 and iNOS gene expression also contribute to inhibition of TPA-induced ODC activity. The most potent compounds in these assays, 10, 13 and 21, as well as curcumin, were further evaluated for inhibition of 7,12-dimethylbenz( a)anthracene (DMBA)-induced preneoplastic lesion formation in a mouse mammary organ culture model, and dose-dependent responses were observed. Most potent effects were at concentrations between 1 and 5 μM for 10, 13 and 21, and at 10 μM for curcumin. These data demonstrate the substitution pattern on the aromatic moiety is especially crucial for activity.

HMG-CoA reductase inhibitors induce COX-2 gene expression in murine macrophages: role of MAPK cascades and promoter elements for CREB and C/EBPβ

Except functioning as lipid-lowering agents, HMG-CoA inhibitors, statins, are good tools to clarify the signaling role of small G proteins. In this study, we found in murine RAW264.7 macrophages, statins within 1–30 μM stimulated COX-2 gene transcription and PGE 2 formation, displaying potencies as lovastatin > fluvastatin > atorvastatin >> pravastatin. Transfection experiments with COX-2 promoter construct showed the necessity of C/EBPβ and CRE promoter sites, but not NF-κB promoter site. Effects of statins on the activation of COX-2 promoter, induction of COX-2 protein, and PGE 2 production were all prevented by mevalonate and prenylated metabolites, FPP and GGPP. Consistent with the effect of statins, manumycin A, farnesyltransferase inhibitor, and geranylgeranyltransferase inhibitor increased PGE 2 production and COX-2 induction. Likewise, toxin B, an inhibitor of Rho family members, caused a prominent COX-2 induction. Results also indicated that tyrosine kinase, ERK, and p38 MAPK play essential roles in statin action. Taken together, these results not only demonstrate a unique action of statins in the upregulation of COX-2 expression in macrophages, but also suggest a negative role controlled by small G proteins in COX-2 gene regulation. Removal of this negative control by impairing G protein prenylation with statins leads to MAPKs activation and promotes COX-2 gene expression through the activation at CRE and C/EBPβ sites.