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During inflammation, the inflammasomes representing a group of multi-protein complexes trigger the biological maturation of pro-inflammatory cytokines such as interleukin-1β and interleukin-18 by proteolytic activation of caspase-1 from its inactive proforms. The individual genes encoding components of the inflammasome machinery are regulated at transcriptional and post-transcriptional levels. Once activated, they drive a wide variety of cellular responses that are necessary to mediate host defense against microbial pathogens and to guarantee tissue homeostasis. In the present work, we have studied the expression of the different inflammasomes in various primary hepatic cell subpopulations, in models of acute inflammation and during experimental liver fibrogenesis. We demonstrate that NLRP-1, NLRP-3 and AIM2 are prominently expressed in Kupffer cells and liver sinusoidal endothelial cells, moderately expressed in periportal myofibroblasts and hepatic stellate cells, and virtually absent in primary cultured hepatocytes. We found that the challenge with the lipopolysaccharides results in a time- and concentration-dependent expression of the NOD-like receptor family members NLRP-1, NLRP-3 and NLRC4/NALP4 in cultured hepatic stellate cells and a strong transcriptional activation of NLRP-3 in hepatocytes. Moreover, we detect a diverse regulatory network of the different inflammasomes in the chosen experimental models of acute and chronic liver insult suggesting that the various inflammasomes might contribute simultaneously to the outcome of inflammatory and fibrotic liver insult, irrespectively of the underlying inflammatory stimulus.
Keywords:Hepatic inflammation; Inflammasome; Animal models; Hepatocytes; Hepatic stellate cells; Kupffer cells
The inflammasomes are cytoplasmic multiprotein complexes that have recently been identified in immune cells as an important sensor of signals released by cellular injury and death. During inflammation they trigger the maturation of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and engage innate immune defenses and function as a guardian of organ homeostasis [1-5]. Likewise, cellular stress, viral or bacterial infection, free cytoplasmic DNA, or any other kind of injury leads to activation of specialized receptors resulting in the formation of these high-molecular-mass inflammasome platforms [6-9]. The assembly of these molecular platforms is unique, triggered by a variety of endogenous and exogenous signals and follows a defined chronological sequence: after recognition of pathogen-derived molecules, the inflammasomes induce the autocatalytic generation of intracellular active caspase-1 (CASP-1) that in turn induces the proteolytic cleavage and biological activation of the IL-1β- and IL-18-precursors [6,7,10]. In principal, there are four individual inflammasomes (i.e. NLRP-1, NLRP-3, NLRC4/NALP4, and AIM2) that are each composed of a sensor molecule that is a member of the family of nucleotide-binding oligomerization domain (NOD)-like receptors (NLR) [11]. In its activated form, this sensor molecule has the ability to physically interact with CASP-1 or to recruit this protease via an intermediary adaptor molecule termed apoptosis-associated speck-like protein or caspase recruitment domain (CARD) of the adaptor protein ASC [4]. Once activated, a complex network of cellular reactions is triggered leading to local and systemic (e.g. acute-phase response) inflammatory reactions, recruitment of neutrophils and platelets as well as activation of the innate immune system [7]. Furthermore, the activation of the inflammasomes is linked to host defense against microbial pathogens, in many other multifaceted diseases such as metabolic syndrome and inflammatory bowel disease. In addition, inflammasomes are relevant in the regulation of diverse important aspects of inflammation and tissue repair such as pyroptosis representing a specialized form of cell death [5]. Based on these eminent functions, it is not surprising that mutations within this family of genes are associated with severe immune diseases and supposed to be involved in tumorigenesis [12]. Most studies highlighting the regulation and function of the different inflammasome branches are presently available from lung but it is now well documented that inflammasome activation is a general phenomenon found in all organs that is also proposed to be involved in insulin signaling, β-cell function and formation of atherosclerosis [5,13].
In regard to liver it has been recently demonstrated that the NLR family members NLRP6/NALP6 and NLRP-3 in conjunction with IL-18 negatively regulate progression of non-alcoholic fatty liver disease [14] and that the application of endotoxins including lipopolysaccharide (LPS) or fatty acids results in increased IL-1β production and strong activation of the NLRP-3 inflammasome [15,16]. Moreover, it was proposed that the induction and proteolytical activation of CASP-1 during activation of inflammasomes has hepatoprotective effect, in part through regulation of cell death pathways after major trauma [17]. In line, the silencing of NLRP-3 during liver ischemia-reperfusion by small hairpin RNAs confirmed that NLRP-3 signaling is involved in progression of liver injury and that its lack can protect the liver by reducing the concentration of IL-1β, IL-18, TNF-α, and IL-6 through downregulation of CASP-1 activation and NF-κB activity in mice [18]. At the cellular level, it was proposed that the activation of inflammasome components regulate a variety of endogenous functions in hepatic stellate cells (HSC) and are required for the development of liver fibrosis [19]. However, precise activities and involved signaling pathways of individual inflammasomes in liver cells are still enigmatic and the exact determination how the inflammasomes are activated in different diseases and experimental settings remains a demanding challenge.
Here we studied the inflammasome expression in various primary hepatic cell subpopulations and in experimental models of acute and chronic inflammation and ongoing hepatic fibrogenesis. We demonstrate that NLRP-1, NLRP-3 and AIM2 are prominently expressed in Kupffer cells (KC) and liver sinusoidal endothelial cells (LSEC), moderately expressed in periportal myofibroblasts (pMF) and HSC, and virtually absent in primary cultured hepatocytes. We further demonstrate that in vitro stimulation with LPS results in a time- and concentration-dependent activation of NLRP-1, NLRP-3 and NLRP4 in cultured HSC and a strong activation of NLRP-3 in hepatocytes. In summary, we found a dynamic transcriptional regulation of the diverse inflammasomes in experimental models of acute and chronic liver insult suggesting that the various inflammasomes might contribute alone or in conjunction with each other to the outcome of liver insult.
Cell culture
Primary HSC, KC and LSEC were isolated from male Sprague–Dawley rats by a standard Nycodenz density gradient centrifugation technique and cultured as described previously [20,21]. Fully transdifferentiated myofibroblasts (MFB) were obtained by subcultivation of HSC seven days after initial plating. Primary hepatocytes were isolated after the collagenase method established by Seglen [22] and plated and cultured for one day on collagen-coated dishes in HepatoZYME-SFM medium (Gibco, St Louis, MO, USA). pMF were prepared following established protocols [23], characterized by their microscopic appearance, their positivity for fibulin-2 and cultured essentially as described previously [24]. The rat cirrhotic fat storing cell line CFSC-2G [25,26] was cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS) (Gibco), 4 mM L-Glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 1 x non essential amino acids (all from Cambrex, Verviers, Belgium).
LPS stimulation
For endotoxin stimulation, CFSC-2G were starved for 16 hr in DMEM containing 0.5% FCS (Gibco) and stimulation was done with indicated LPS concentrations in medium containing 0.2% FCS. When primary hepatocytes were stimulated with LPS, the cells were initially plated in HepatoZYME, then cultured in DMEM (10% FCS) overnight, followed by starvation for 8 h in DMEM (0.5% FCS), and stimulated with LPS as described above.
RNA isolation and qRT-PCR
RNA from primary liver cells, rat cirrhotic fat storing cell line CFSC-2G or total liver tissue was isolated by the guanidine thiocyanate/CsCl method, followed by DNAse digestion using the Purelink RNA Mini kit system (Invitrogen, Life Technologies, Darmstadt, Germany). Total RNA was quantified and 2 μg samples reverse transcribed using Superscript II reverse transcriptase and random hexamer primers (both from Invitrogen). For the individual TaqMan PCR assays, the cDNA derived from 25 ng RNA was amplified in 25-μl volume using qPCR Core Kits (Eurogentec, Cologne, Germany) and primer combinations given in Table 1. The amplification of all respective target gene sequences were done as follows: melting at 95°C for 10 min and then 40 cycles at 95°C for 15 sec and 60°C for 1 min, respectively. Normalization was done either to theexpression of GAPDH or rS6 mRNAs.
Table 1. Primers used for quantitative TaqMan analysis
SDS-PAGE and immunoblotting
Whole-cell and liver protein extracts were prepared essentially as previously described [27]. Equal amounts of proteins (20 μg/lane) were resolved in NuPAGETM Bis-Tris gels (Invitrogen) and electroblotted on a Protran membrane (Schleicher & Schuell, Dassel, Germany). The sources and concentrations of antibodies used in Western blot analysis are given in Table 2.
Table 2. Antibodies used in this study
Experimental in vivo liver injury models
(i) Bile duct ligation (BDL) in rats: Male Sprague Dawley rats were subjected to bile duct ligation for 2, 7 or 14 days following a protocol previously described [28,29]. Sham operated rats that were sacrificed at the same time points served as controls. (ii) CCl4 application in rats: A total of 30 male Sprague Dawley rats at age six to eight weeks and weighing about 180–200 g were utilized for this study. The rats received intraperitoneal injections twice weekly of 1 ml/kg of CCl4 in an equal volume of mineral oil for up to 12 weeks, whereas mineral oil alone was used for 12 control animals following established protocols. Liver specimens were harvested and snap frozen and stored at −80°C for protein and RNA isolation. (iii) LPS and Concanavalin A (Con A) models in mice: Eight weeks old C57BL/6 wild type mice were subjected to a single intravenous injection of 20 mg/kg body weight Con A (Sigma) as previously described [30] or LPS (2.5 μg/g body weight). After 8 hrs (Con A) or after 2 or 6 hrs (LPS), mice were sacrificed and liver extracts prepared for qRT-PCR and Western blot analysis. All animal experiments performed were approved by the local review board according to prevailing guidelines for scientific animal experimentation.
Immunohistochemistry of liver sections
Liver tissue sections were deparaffinized and rehydrated with xylene and decreasing graded ethanol, and antigen retrieval was engendered by heating the sections in 0.01 M sodium citrate buffer (pH 6) in a microwave for 20 min. Blocking of nonspecific binding sides and antigen detection was essentially done as described elsewhere [31]. The source of the NLRP-3, AIM2, and the NALP4/NLRP4 antibodies used for this analysis are given in Table 2. Stains with normal rabbit control serum served as negative controls. The specimens were briefly counterstained with hematoxylin and representative images made at a magnification of x200 using a Nikon Eclipse 80i microscope (Nikon, Düsseldorf, Gemany) equipped with the NIS Elements Vis software (version 3.22.01).
Terminal transferase dUTP nick end-labelling assay (TUNEL)
For DNA fragmentation detection resulting from apoptotic signalling cascades, we used In Situ Cell Death Detection Kit Fluorescein (# 1684795, Roche Diagnostics, Mannheim, Germany) according to manufacturer's instructions. The presence of nicks in the DNA of cultured cells was identified by terminal deoxynucleotidyl transferase (TdT), an enzyme that catalyzes the addition of labelling dUTPs. In brief, cells were seeded on glass slides and incubated with 200 ng/ml LPS for 30 min or 16 hrs. Thereafter, slides were rinsed with PBS, cells permeabilized in 0.1% Trition, 0.1% sodium citrate for 3 min on ice, and TUNEL reaction mixture added. As a negative control, the cells were only incubated with label solution and as a positive control, the cells were incubated after permeabilization with DNAse I Mix for 10 min. Cells were analyzed by fluorescence microscopy for direct fluorescein in a Leica DMLB microscope (Leica, Wetzlar, Germany) using the DISKUS software (version 4.50.1638) obtained from Carl H. Hilgers (Königswinter, Germany).
LDH assay
The measurement of cell death was done in 96 well plate formats using the Cytotoxicity detection kit+ (LDH), version 5 (Roche) that allows detection of cell-mediated cytotoxicity and quantification of the cytotoxic potential of compounds. The assay was done essentially as outlined in the manufacturer's instruction. Briefly, 100 μl of cell-free culture supernatants obtained from equal cell numbers (seeded at a density of 2.5 x 105 cells/well in 6 well-plates) that were incubated with different concentration of LPS were incubated with 100 μl of the reaction mixture from the test system kit. After incubation for 25 min at room temperature, 50 μl stop solution was added, the plates were shacked for 10 min and the formation of formazan was measured in a Wallac 1420 Victor Multilabel Counter (Wallac Oy, Turku, Finland) equipped with a 490 nm filter and the WIACALC (version 1) software. For standardization of LDH activity, purified LDH from hog muscle (Roche, #101107085001, Lot no. 12169925) with a specific activity of 597 U/mg was included in this analysis.
Measurement of influx of neutrophils, monocytes and other immune cells in livers of mice subjected to BDL or CCl4 treatment via flow cytometry
To determine the influx of leukocytes into the livers of animals that received BDL surgery or were injected with CCl4, liver leukocyte isolates were prepared and analyzed by Fluorescence activated cell sorting (FACS). Individual leukocyte subsets were identified by their positivity for CD45 (all leukocytes), CD11b (monocytes and granulocytes), CD11b F4/80 (macrophages), and Ly-6G (neutrophils). A FACS Canto-II (BD Biosciences, Heidelberg, Germany) was used for flow cytometric analysis. The acquired data sets were analyzed by FlowJo software (TreeStar, Ashland, OR) and individual cell subsets depicted in percentage of all cells measured. The analysis shown was done from livers of animals taken 5 days after BDL surgery and 48 h after single CCl4 injection.
Statistical analysis
Statistical analyses was performed using the t-test for comparison of groups and the Kruskal-Wallis test for nonparametric multiple comparison with a statistical software program (STATGRAPHICS Plus, version 5.1). When appreciable, results are depicted with their median values. Probability values of less than 0.05 or 0.01 were considered as statistically significant. To establish the differences within individual groups, we used the Mann–Whitney test. Individual p-values for each experiment are given in Additional file 1.
Additional file 1. Statistics to Figure 2. Expression of inflammasome components in rat cirrhotic fat storing cell line CFSC-2G subjected to LPS stimulation. Statistics to Figure 3: Expression of inflammasome components in rat livers after bile duct ligation (BDL). Statistics to Figure 4: Expression of inflammasome components in rat livers after application of CCl4 . Statistics to Figure 5: Expression of inflammasomes in mice after Con A injection. Statistics to Figure 6: Expression of inflammasomes in primary murine hepatocytes after stimulation with LPS. Statistics to Suppl. Figure 3: Induction of acute phase response in mice after LPS injection.
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Establishment of quantitative real time PCR (qRT-PCR) assays for analysis of inflammasomme expression in mouse and rat
There are several key regulatory genes involved in the initiation and regulation of inflammatory pathways. To allow relative mRNA quantification of the different components in mice and rats, we have established methodologies for measurement of NLRP-3, NRLC4, AIM2, IL-1β, IL-18, ASC, TNF-α, and rS6 in rat (Additional file 2: Figure S1) and NLRP-1b, NLRP-1c, NLRP-3, NLRC4/NALP4, AIM2, IL-1β, IL-18, ASC, TNF-α, IL-6, IL-10, IFN-γ, and GAPDH in mice (Additional file 3: Figure S2). In addition, we have established a protocol for measuring CCL2/MCP-1 mRNA expression in both species (not shown). These assays are designed for quantitative analysis or mRNA expression using the TaqMan platform. The different primer combinations (Table 1) that are used for amplification are designed in a fashion that allows amplification of respective target gene sequences under same cycling conditions (melting temperature: 95°C and amplification/extension temperature 60°C) permitting a general, easy applicable testing system of respective components.
Additional file 2: Figure S1. Establishment of TaqMan tests for expression analysis of individual inflammasome genes in rats. TaqMan assays for rat NLRP-3 (A), NLRC-4 (B), AIM2 (C), IL-1β (D), IL-18 (E), ASC (F), TNF-α (G), and rS6 (H) were established. Representative melting curves for each gene are depicted. Amplification of respective target gene sequences were performed under the same cycling conditions using a melting temperature of 95°C and amplification/extension temperatures of 60°C, respectively. The individual primer combinations used in each test are depicted in Table 1.
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Additional file 3: Figure S2. Establishment of TaqMan tests for expression analysis of individual inflammasome genes in mouse. TaqMan assays for murine NLRP-1b (A), NLRP-1c (B), NLRP-3 (C), NLRC-4 (D), AIM2 (E), IL-1β (F), IL-18 (G), ASC (H), TNF-α (I), IL-6 (J), IL-10 (K), IFN-γ (L) and GAPDH (M) were established. Amplification of the different gene sequences were essentially performed under the same cycling conditions each using a melting temperature of 95°C and amplification/extension temperatures of 60°C, respectively. Representative melting curves for each gene are depicted. The individual primer combinations used in each test are depicted in Table 1.
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Expression of NLR family members in primary hepatic cell subpopulations
To get a first hint of inflammasome expression in the different hepatic cell subpopulations, we isolated KC, LSEC, pMF, HSC/MFB, and hepatocytes and quantified the relative expression of NLRP-1, NLRP-3, and the interferon-inducible HIN200 family member AIM2 in these cell entities. This analysis revealed that all three genes are highest expressed in KC and LSEC, while the expression was only weak in cultured pMF and HSC/MFB (Figure 1).
Figure 1. Expression of NLRP-1, NLRP-3 and AIM2 in primary hepatic cells. Total RNA from Kupffer cells (KC) cultured for 2 days, freshly isolated sinusoidal endothelial cells (LSEC), portal fibroblasts (pF) in primary culture, hepatic stellate cells (HSC/MFB) cultured for 4 days and hepatocytes (PC) cultured for 2 days were isolated and subjected to quantitative real time PCR for NLRP-1, NLRP-3, and AIM2. In this analysis, the expression of the different genes was set to 100% in KC. The individual primers used for quantitative analysis are depicted in Table 1. For detailed statistical analysis of this set of experiments see Additional file 1.
Transcriptional regulation of inflammasome related genes in CFSC-2G after LPS challenge
It is well established that the endotoxin LPS plays an important modulator role in acute liver injury as well as chronic liver diseases. In addition, it was recently demonstrated that components of the inflammasome are expressed in immortalized and primary HSC and that the lack of ASC and NLRP-3 interfere with the process of ongoing fibrogenesis after CCl4 or thioacetamide (TAA) challenge that become apparent in reduced collagen deposition and HSC activation in mice lacking ASC or NLRP-3 [19]. To analyze the expression of relevant inflammasomes after inflammatory stimuli in more detail, we challenged cultured CFSC-2G cells with different concentrations of LPS (50, 100, and 200 ng/ml) and measured the mRNA expression of NLRP-1, NLRP-3, NLRC-4, AIM2, IL-1β, IL-18, ASC, and TNF-α after 30 min, 1 h, 2 hrs, 4 hrs, 8 hrs, and 16 hrs (Figure 2). As expected, the analysis shows a rapid increase in TNF-α expression that was highest after 1 h and strongly dependent on the concentration of LPS. In addition, we noticed a simultaneous upregulation of IL-1β expression. The expression of all four inflammasomes was not altered after 30 min of LPS challenge (Figure 2A).
Figure 2. Expression of inflammasome components in rat cirrhotic fat storing cell line CFSC-2G subjected to LPS stimulation. CFSC-2G cells were treated one day after plating with 0, 50, 100 or 200 ng/ml LPS. After 30 min (A), 1 h (B), 2 hrs (C), 4 hrs (D), 8 hrs (E), 16 hrs (F), RNA from respective cells was extracted and, the expression of NLRP-1, NLRP-3. NLRC-4, AIM2, IL-1β, IL-18, ASC, and TNF-α analysed by qRT-PCR. In this analysis, the expression of each gene without LPS stimulation was set to 1. Please note, the high expression of TNF-α at early (inlets in B and C) and IL-1β (inlet in D) and medial time points after LPS treatment. For detailed statistical analysis of this set of experiments see Additional file 1.
However, one hour after LPS stimulation, the expression of NLRP-3 was dose-dependently upregulated, while all other genes were not affected at that time point. Interestingly, two hours after LPS stimulation the expression of NLRP-1, NLRP-3, NLRC4/NALP4 were significant upregulated to a similar degree, while the expression of AIM2 was lowered in cells that received LPS (Figure 2C). The highest expression of these components at that time point was observed in cells that were triggered with 100 ng LPS/ml culture medium, while even higher concentrations did not significantly affect the expression of these genes. At later time points, we only noticed a sustained higher expression of NLRC4/NALP4 and IL-1β (Figure 2F) suggesting that the different inflammasomes and genes that trigger inflammasome activity have a well defined individual expression kinetic after stimulation with LPS.
To rule out that LPS induced cytotoxic effects in CFSC-2G cells under the chosen experimental conditions that might interfere with the expression of the individual inflammasome genes, we further measured cell death and cell lysis by use of TUNEL assay (Additional file 4: Figure S3) and a cytotoxicity detection assay that is based on measurement of lactate-dehydrogenase (LDH) activity released from the cytosol of damaged cells (Additional file 5: Figure S4). Both assays revealed that the incubation of the cells with LPS does not result in plasma-membrane damage or activation of endonucleases that would be indicative for cells that undergo apoptosis.
Additional file 4: Figure S3. Analysis of DNA fragmentation after treatment with LPS. (A) TUNEL assay in CFSC-2G cells that were stimulated in short (30 min) and long term (16 h) with 200 ng/ml LPS kept their cellular integrity. The nuclei are counterstained with DAPI.
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Additional file 5: Figure S4. Analysis of cell cytotoxicity after treatment with LPS. (A) The standard curve for measurement of LDH activity was established with a preparation of hog LDH. (B) The chosen kit system allows to measure LDH activity in a wide range from 0.001 (Background), to 5,083 (high control). (C) Measurement of LDH in cellular supernatants of CFSC-2G cells that were incubated with indicated concentrations of LPS for indicated time intervals reveal only low cellular toxicity of LPS.
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mRNA expression of inflammasomes during experimental liver insult induced by ligation of the common bile duct
To analyze the expression of inflammasome genes during the process of acute and chronic liver insult, we first monitored expression of relevant genes in rats that underwent bile duct ligation (BDL). This model represents a well established experimental model of cholestatic liver disease. We performed qRT-PCR using RNA that was isolated from rats after 2, 7 or 14 after BDL surgery and compared the expression of the different genes to those observed in livers of sham-operated control rats. This analysis revealed that the expression of all tested genes (NLRP-1, NLRP-3, NLRC4/NALP4, AIM2, IL-1β, IL-18, ASC, and TNF-α) was increased in inflamed liver at all time points during ongoing insult (i.e. fibrogenesis) suggesting that the expression of these genes is not only necessary for disease initiation but also for progression (Figure 3A). A detailed statistical analysis using the Kruskal-Wallis test for nonparametric multiple comparison revealed that the observed increased expression of NLRP-3, NLRP-1 and AIM2 reached statistical significant values (Figures 3B, 3C, 3D).
Figure 3. Expression of inflammasome components in rat livers after bile duct ligation (BDL). (A) Sprague Dawley rats were subjected to BDL and sacrificed 2 (3 animals), 7 (5 animals) or 14 (5 animals) days after surgery. The livers of 5 untreated rats were taken as controls. RNA was isolated and the expression of NLRP-1, NLRP-3. NLRC4/NALP4, AIM2, IL-1β, IL-18, ASC, and TNF-α analyzed by qRT-PCR. In this analysis, the expression of the tested genes in livers of sham operated rats (control) was set to 1. (B, C, D) Kruskal-Wallis testing of NLRP-3 (B), NLRP-1 (C), and AIM2 (D) expression during ongoing fibrogenesis induced by the BDL surgery. For detailed statistical analysis of this set of experiments see Additional file 1.
mRNA expression of inflammasomes during experimental liver insult induced by single or repeated application of CCl4
We next tested the expression of various genes of the inflammasome machinery in mice 48 hrs after CCl4 injection. Compared to mice that received oil, the single administration of CCl4 resulted in significant increased expression of ASC, TNF-α, NLRP-1, NLRP-3, NLRC4/NALP4 and AIM2 (Figure 4A, see Additional file 1 for statistics) demonstrating that the single application of CCl4 is already suitable to induce a strong inflammatory reaction in liver. Under these conditions, we also observed an elevated expression of CCL2/MCP-1 (Figure 4B) that is known to be necessary for recruitment of monocytes, memory T cells, and dendritic cells to sites of tissue injury, infection, and inflammation [32,33]. When we analyzed expression of NLRP-3 and NLRP-1 and AIM2 in livers of animals that were subjected to prolonged treatment with CCl4 for 1, 2, 4, 8 and 12 weeks, we noticed that the initial high expression of all three genes were somewhat blunted at later time points and showed a somewhat undulated expression kinetics (Figure 4C).
Figure 4. Expression of inflammasome components in rat livers after application of CCl4. (A) Sprague Dawley rats received one single application of CCl4 or oil. 48 hrs later RNA was prepared and analyzed for expression of IL-1α, IL-1β, IL-6, IL-10, IL-18, IFN-γ, ASC, TNF-α, NLRP-1, NLRP-3, NLRC-4 and AIM2 by qRT-PCR. Details on primer combinations are given in Table 1. (B) The same set of RNAs from (A) was analyzed for expression of CCL-2. (C) The livers of Sprague Dawley rats (3 animals/group) that received one single (1x) or twice weekly intraperitoneal injections of 1 ml/kg BW of CCl4 in an equal volume of mineral oil for 1, 2, 4, 8, or 12 weeks were sampled, RNA extracted, and the expression of NLRP-3 (C), NLRP-1 (D), and AIM2 (E) analyzed by qRT-PCR. Representative melting curves for the expression studies of the three genes investigated are depicted in the lower panels. For detailed information about primer combinations and statistical analysis of this set of experiments see Table 1 and Additional file 1.
Protein expression of inflammasomes during experimental liver insult
To test if the observed mRNA expression kinetics of the different inflammasomes after administration of CCl4 or BDL surgery was also reflected at the protein level, we performed immunohistochemistry for NALP3, NALP1, AIM2, and NLRC4/NALP4 (Figures 5 and 6). In liver sections that were prepared from animals that received either BDL for 5 days, 2 weeks, and 4 weeks or subjected to CCl4 for 1 week, 2 weeks or 4 weeks, we found that hepatocytes stained weakly positive for NLRP-3 (Figure 5 A). In addition, another cell population that was most likely KC stained strongly positive for NLRP-3. In contrast, NLRP-3 was virtually absent in liver sections taken from untreated controls. Similar results were found for NALP1 (Figure 5B), AIM2 (Figure 6A), and NLRC4/NALP4 (Figure 6B) suggesting that there is (i) either an intensive influx of cells into liver with capacity to express these proteins, (ii) an increased expression of inflammasome proteins in liver residential cells (e.g. KC), or (iii) both cellular influx and enhanced expression by hepatic cell subpopulations. By FACS-based analysis of hepatic leukocyte isolates, we were able to demonstrate that in both models (i.e. BDL, CCl4) significant quantities of leukocytes infiltrated the liver (Figure 7A). While the quantities of monocytes/granulocytes (Figure 7B) were higher in the CCl4 model, livers of BDL animals showed a marked increase of macrophages (Figure 7C), while the number of neutrophils was higher in both models compared to untreated control livers (Figure 7D).
Figure 5. Hepatic expression of NLRP-3 and NLRP-1 in rats after administration of CCl4 or BDL surgery. Liver specimen from animals that received CCl4 or underwent BDL for indicated time points were stained with antibodies specific for NLRP-3 (A) or NLRP-1 (B). Section from control animals and a stain with an unspecific rabbit IgG served as controls in this analysis.
Figure 6. Hepatic expression of AIM2 and NALP4/NLRC4 in rats after administration of CCl4 or BDL surgery. Liver specimen from animals that received CCl4 or underwent BDL for indicated time points were stained with antibodies specific for AIM2 (A) or NALP4/NLRC4 (B). Section from control animals and a stain with an unspecific rabbit IgG served as controls in this analysis.
Figure 7. Influx of neutrophils, monocytes and other immune cells in livers of mice subjected to either BDL or CCl4 treatment. (A) Total number of leukocytes in liver cell extracts was identified by their positivity for CD45. (B) The numbers of monocytes and granulocytes were measured by their positivity for CD11b. (C) Macrophages were identified by their positivity for CD11b and F4/80. (D) The total number of neutrophils was measured by their positivity for Ly-6G. The analysis was done from livers of animals taken 5 days after BDL surgery and 48 h after single injection of CCl4.
Expression of inflammasomes and signaling cascades activated during acute inflammatory stimuli
All these previous findings described above demonstrate that various stimuli that induce liver damage are in principle suitable to transcriptional activate the different inflammasome branches simultaneously suggesting that the subsequent molecular pathways regulated by these pathways are highly complex especially at later time points when secondary effects come along. Therefore, we decided to exemplary analyze NLRP-3 expression during the acute phase response. This reaction occurs soon after setting of inflammatory stimuli and represents a complex series of reactions resulting in the production of inflammation-associated cytokines and diverse acute phase proteins. For induction we either used the application of LPS or Concanavalin A (Con A) for 6 hrs (LPS) or 8 hrs (Con A). When we analyzed the expression of NLRP-3 in liver protein extracts from animals that received LPS, we found that the endotoxin induced expression of NLRP-3 at the protein level (Figure 8A). The LPS injection further resulted in a significant induction of the acute phase protein Lipocalin 2 (LCN2) and correlated well with the phosphorylation of the Signal Transducers and Activators of Transcription-1 (STAT1) that is involved in mediating the acute phase response via transcriptional activation of cytokine inducible genes. In line with this activation, we found that the LPS injection resulted in a significant phosphorylation of the JNK-MAPK and NF-κB/p65 and an increased expression of caspase-3. Significant phosphorylation of STAT-3 was already observed at 2 hrs after LPS injection (not shown) confirming well established findings that both STAT-1 and STAT-3 have critical roles in the control of systemic inflammation and acute phase response [34,35]. The induction of the acute phase response was further confirmed by quantitative analysis of various cytokine mRNA expression measuring after 2 and 6 hrs after application of LPS (Additional file 6: Figure S5A) and increased expression of CCL2/MCP-1 (Additional file 6: Figure S5B).
Additional file 6: Figure S5. Induction of acute phase response in mice after LPS injection. (A) Mice were subjected to LPS stimulation and livers samples were taken after 2 and 6 hours. The expression of pro-inflammatory, anti-inflammatory cytokines as well as indicated inflammasome components was analyzed by qRT-PCR. (B) Expression analysis of the monocyte chemotactic protein (MCP-1, CCL2) in livers after LPS injection. For detailed statistical analysis of this set of experiments see Additional file 1.
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Figure 8. Expression of inflammasomes in mice after LPS and Con A injection. (A) Liver extracts were prepared from control mouse (4 animals) or animals that received intraperitoneal injection of LPS (2.5 μg/g body weight) for 2 (2 animals) or 6 hrs (5 animals). Equal protein amounts (100 μg) were then subjected to SDS-PAGE and analyzed in Western blot for expression of NLRP-3, Caspase 3, pNF-κB p65, total NF-κB p65, Lipocalin-2 (LCN2), pJNK, total JNK, pSTAT1, total STAT1, pSTAT3, total STAT3, GAPDH, and β-actin. A representative blot is shown for two controls and two animals that received LPS for 2 hrs. (B) Liver extracts were prepared from control mouse or animals that received intraperitoneal injection of Con A (20 μg/g body weight) for 8 hrs. Equal protein amounts were then subjected to SDS-PAGE and analyzed for expression of NLRP-3, Lipocalin-2 (LCN2), pJNK, total JNK, pSTAT1, total STAT1, pSTAT3, total STAT3, and GAPDH. The antibodies used in this study are given in Table 2. (C) RNA was isolated from livers of mice that were treated with Con A for 8 hrs or from 2 controls and 4 Con A-treated mice and analyzed for expression of NLRP-3, NLRC-4, AIM2, NLRP-1, IL-1β, IL-18, and ASC. (D) The same set of RNAs was tested for TNF-α expression. For detailed statistical analysis of this set of experiments see Additional file 1.
In the other acute model (i.e. Con A), we again found a strong upregulation of NLRP-3 that correlated well with increased expression of LCN2, caspase-3 and elevated phosphorylation of JNK and STAT-1 (Figure 8B). In this model, the increase of NLRP-3 expression seems to be the result of an increased transcriptional activation of the Nlrp-3 gene (Figure 8C) and correlated well with the expression of IL-1β and TNF-α that are indicative for the acute phase response that is triggered by the LPS stimulus (Figure 8D). Interestingly, the expression of NLRC4/NALP4 and AIM2 was reduced after application of Con A, while NLRP-1 gene expression was unaffected.
Induction of inflammasome expression in primary cultured hepatocytes and KC
In the initial analysis, we found that the expression of inflammasomes in primary cultured hepatocytes is rather low or even not detectable (cf. Figure 1). However, we noticed in vivo that hepatocytes stained positive for NLRP-3 when animals were subjected to BDL (cf. Figure 3E) suggesting that hepatocytes should in principal be also able to respond to inflammatory stimuli with expression of NLRP-3 and other inflammasome-associated proteins. To test this in more detail, we serum-starved primary isolated murine hepatocytes to avoid influences of serum factors and stimulated them with different concentration of LPS for various time intervals and measured the expression of respective genes (Figure 9).
Figure 9. Expression of inflammasomes in primary murine hepatocytes after stimulation with LPS. Primary hepatocytes in culture were stimulated one day after isolation with 50, 100, 200 and 400 ng/ml LPS or left untreated. After indicated times (30 min, 1 h, 2 hrs, 4 hrs) RNA was isolated from respective cells and tested for expression of NLRP-1 (A), NLRP-3 (B), NLRC-4 (C), AIM2 (D), IL-1β (E), IL-18 (F), ASC (G), and TNF-α (H) by qRT-PCR. For detailed statistical analysis of this set of experiments see Additional file 1.
While the expression of NLRP-1 (Figure 9A), NLRC4/NALP4 (Figure 9C), AIM2 (Figure 9D), and ASC (Figure 9G) was nearly unaffected 30 min after stimulation with 50-400 ng LPS/ml medium, the expression of all four genes were more or less suppressed at later time points. In contrast, the LPS challenge resulted in a drastic upregulation of NLRP-3 expression (up to 100 fold two hours after stimulation) at all concentrations tested (Figure 9B). This expression pattern correlated well with the expression of IL-1β (Figure 9E) and TNF-α (Figure 9H). The analysis further revealed that the mRNA quantity of caspase-1 that represents the major protease necessary to activate the inflammasome machinery is also highest at 2 hrs, while the expression of IL-18 in hepatocytes is unaffected after LPS challenge.
In this scenario it is most likely that liver residential KC are the major source of IL-1β and TNF-α because it is generally assumed and confirmed by our analysis (Additional file 7: Figure S6) that primary isolated KC have a high capacity to express these pro-inflammatory cytokines after LPS stimulation.
Additional file 7: Figure S6. Expression analysis of Kupffer cells after stimulation with LPS. Primary KC were stimulated with indicated concentration of LPS for 2 hrs and the expression of (A) NLRP-1, NLRC4/NALP4, AIM2, IL-18, ASC and (B) NLRP-3, IL-1β and TNF-α determined by quantitative RT-PCR. In this analysis, the target gene expression without stimulation with LPS was set to 1.
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There is no doubt that the understanding of inflammasome regulation and function will potentially offer great opportunities to interfere with the process of inflammation, fibrogenesis and tumorigenesis. Irrespectively of the inflammatory stimuli analyzed, the activation of the inflammasome machinery is a well orchestrated process in which pattern recognitions receptors recognize distinct danger signals and in turn activate signaling pathways that subsequently initiate the inflammatory response resulting in activation of different pathways such as NF-κB and MAPK and culminating in transcriptional activation of a large number of different inflammation-associated genes. It is superfluous to mention and confirmed in our study that this regulatory network is highly complex and that the individual inflammasome protein complexes might be simultaneously expressed and activated in the same cell type at the same time in the inflamed tissue. Recent studies have characterized and classified distinct molecular agents and pathways for several sensor proteins and have identified a multitude of inflammatory ligands of both endogenous and exogenous origin that drive inflammasome activities in healthy and diseased organs [5]. However, there is only limited knowledge of inflammasome regulation and function in healthy liver and various liver diseases.
To allow quantification of mRNAs of genes that are directly linked to the activity of the inflammasomes, we have established qRT-PCR assays for mRNA quantification of NLRP-3, NLRP-1, NLRC4/NALP4, AIM2, IL-1β, IL-18, ASC, TNF-α, IL-6, IL-10 in mice and rat (Additional file 2: Figure S1, Additional file 3: Figure S2).
We have shown that the expression of NLRP-1, NLRP-3 and AIM2 in cultured primary cell population is mainly restricted to KC, LSEC and pMF, while the expression in HSC is only low and virtually absent in primary cultured hepatocytes (Figure 1). However, the challenge with LPS demonstrates that the expression of respective genes involved in formation of inflammasomes can be induced to high levels in HSC/MFB (Figure 2) and hepatocytes (Figure 6) suggesting that all liver cell types tested are in principal able to mediate inflammasome activities. Interestingly, NLRP-3, NLRP-1 and NLRC4/NALP4 are inducible in CFSC-2G in response to LPS stimulation (Figure 2) demonstrating that the different inflammasome branches can be simultaneously activated at the same time in this cell entity. However, during activation there seems to be a clear sequential order because NLRP-3 expression was found to be highest already one hour after LPS challenge, while the elevated expression of NLRP-1 and NLRC4/NALP4 followed one hour later.
During experimental liver insult induced by ligature of the common bile duct (BDL), the expression of all four core inflammasomes (i.e. NLRP-1, NLRP-3, NLRC4/NALP4, and AIM2) were simultaneously activated at the mRNA (Figures 3) and protein level (Figures 5 and 6). Also the single or repeatedly application of CCl4 resulted in a simultaneous increase of all four inflammasomes in liver (Figure 4, Figures 5 and 6) suggesting that inflammatory stimuli induce a highly complex network of biological responses in residential and infiltrating cells in which all inflammatory branches are integrated.
When mice were injected with LPS, the expression of NLRP-3 was induced at both mRNA and protein levels (Figure 5A) confirming previous reports [16]. LPS is a prototypical ligand for the Toll like receptor 4 (TLR4) that upon activation induces the production of pro-inflammatory cytokines through activation of the NF-κB pathway [36,37]. In addition, we observed a strong induction/activation (i. e. phosphorylation) of other genes in that disease model which are involved in inflammatory reactions and recovery from endotoxic shock including LCN2, caspase-3, NF-κB, JNK, pSTAT1, and STAT3 [35,38]. A comparable activation pattern of all these genes was observed when normal liver was challenged with Con A (Figure 5B, 5C). Since Con A injection leads to immune-mediated liver injury and release of several cytokines (e.g. TNF-α, IFN-γ) triggering liver damage [39], these findings suggest that irrespectively of the stimuli triggering the inflammatory response, the subsequent changes within the liver end up in similar molecular alterations and correlate with the activation of the NLRP-3 inflammasome.
Presently, we do not know if the induction/activation of the diverse target genes and pathways occur in an orchestrated way with NLRP-3 or if the activation of these genes is mandatory to stimulate NLRP-3 expression. However, based on our experimentation, it is reasonable to speculate that the expression of inflammasome components is directly linked to the activation of NF-κB. It is known that under the condition of BDL, rats have an overall constitutive activation of NF-κB in the liver [40]. Also carbon tetrachloride exposure in mice leads to activation of NF-κB [41]. Likewise, the application of the lectin Con A and endotoxin LPS induces activation and nuclear translocation of NF-κB [36,42]. Therefore, it will be interesting to test if the administration of inhibitors of canonical or non-canonical NF-κB signaling such as the thiol-reactive quinol and putative thioredoxin inhibitor PMX464 or the lack of factors necessary to activate/phosphorylate NF-κB is suitable to interfere or blunt expression of inflammasome genes.
The finding that purified hepatocytes alone do not express inflammasome components (Figure 1) but induce their transcription after appropriate challenge with inflammatory stimuli such as LPS (Figure 6) demonstrate that the presence of immune cells per se is not necessary to mediate respective responses in vitro. However, it will be essential to analyze in vivo in more detail if the initiation of inflammasome activity in inflamed liver tissue is mainly triggered by influx of neutrophils, monocytes and other immune cells, is a capacity of liver residential cells or is the outcome of both processes. Based on our findings, we suggest that infiltrating cells as well as liver residential cells have capacity to induce inflammasome expression after appropriate trigger within the liver. Most likely, all primary hepatic cell entities are capable to induce inflammasome expression and act in conjunction with infiltrating cells that may vary in the different experimental settings.
The fact that the immortalized cirrhotic fat storing cell line CFSC-2G induces the inflammasome machinery after challenge with LPS (Figure 2) further demonstrates that immortalization is not sufficient to blunt inflammasome activity.
Definitely, we are still at the beginning in understanding the regulation of inflammasomes in different disease models and far away to understand the functions of the individual components. The fact that the expression of the different inflammasome branches in liver become simultaneously activated during hepatic inflammation and their linkage to the activation of general molecular transcriptions factors (e.g. NF-κB) further strengthens the notion that there are several master key switches of inflammasome activity. It will be interesting and challenging to unravel these interactions and to identify specific regulatory control points that might be suitable for pharmacological intervention.
In regard to liver NLRP-1, NLRP-3 and AIM2 are most prominently expressed in Kupffer cells and liver sinusoidal endothelial cells, moderately expressed in periportal myofibroblasts and hepatic stellate cells, and virtually absent in primary cultured hepatocytes. In vitro, the challenge of cultured hepatic stellate cells with lipopolysaccharides results in a time- and concentration-dependent upregulation of NLRP-1, NLRP-3 and NLRC4/NALP4, while hepatocytes respond mainly with a strong transcriptional activation of NLRP-3. Our data using different experimental animals of inflammatory liver insult further indicates that the various inflammasome components contribute simultaneously to the outcome of inflammatory liver disease, irrespectively of the underlying inflammatory stimuli. The fact that the stimulation of all inflammasome branches in the different disease models leads to simultaneous activation of general molecular transcriptions factors (e.g. NF-κB) further strengthens the notion that there are several master key switches regulating inflammasome activity.
BDL: Bile duct ligation; CASP-1: Caspase-1; Con A: Concanavalin A; FACS: Flurorescence-activated cell sorting; HSC: Hepatic stellate cell(s); KC: Kupffer cell(s); LPS: Lipopolysaccharides; LSEC: Liver sinusoidal endothelial cell(s); MFB: Myofibroblasts; NLR: Nucleotide-binding oligomerization domain (NOD)-like receptors; PC: Parenchymal cell(s), i. e. hepatocyte(s); pMF: periportal myofibroblast(s).
Competing interests
The authors declare that they have no competing interest.
Authors' contributions
SGB, EBK, LT, and UH performed the experiments and designed figures; RW designed the study and drafted the manuscript. All authors read and approved the final manuscript.
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB/TRR57, P13) and a grant from the IZKF Aachen. The authors greatly acknowledge the help of Carmen Tag and Sibille Sauer-Lehnen (SFB(TRR57 Q3) for technical assistance in animal experimentation and isolation of primary liver cells. The authors thank Frank Tacke (Department of Internal Medicine III, UKA) for help in performance the LPS and Con A experimentation and are indebted to all members of lab and for helpful discussions.
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Opposition to new law is growing, says expertComment on this story
The new mineral resources law, which awaits the president's signature, faces growing opposition as the mines minister seeks a review, according to law firm Webber Wentzel.
Proposed changes to the 2002 Mineral and Petroleum Resources Development Act include giving the state the right to a free 20 percent stake in all new energy ventures and to change how projects are awarded. Firms such as Exxon Mobil, Anadarko Petroleum and Total say the law, passed by Parliament and the higher National Council of Provinces this year, will undermine their businesses and deter investment.
Mineral Resources Minister Ngoako Ramatlhodi, sworn in last month, said President Jacob Zuma must hold the bill so legislators address some clauses, the Financial Times reported on Wednesday. Another option could be to draft a bill for oil and gas, separate from mining, Ramatlhodi said.
"Certainly it created a real damper on exploration activity, the bill, and we heard from a lot of clients that everything was being put on hold," Peter Leon, the head of Africa mining and energy projects at Webber Wentzel, said yesterday.
"I think the government has realised that, hence the about-turn by the new minister."
Ramatlhodi was planning a meeting of the ANC's economic committee to go through the bill on Monday.
Exxon, Anadarko and Royal Dutch Shell have begun prospecting in South Africa's waters over recent years as new technology boosts their ability to find and pump oil from deep beneath the seabed. Anadarko said in April it halted spending on exploration in the continent's second-biggest economy because of the legislation.
Zuma was obliged to sign a bill unless there was a question about its constitutionality, which there was, Leon said.
"The bill was not correctly passed by the National Council of Provinces because they never held public hearings" in each of South Africa's nine regions, he said.
The DA "and the Legal Resources Centre wrote to the president and asked him not to sign the bill into law and I understand that he has taken legal advice on that.
"Now that the mines minister has weighed in as well, that will have another impact," Leon said." – Bloomberg
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Tim Kasher performs "Opening Night" at Ink Tank Merch in Omaha, Neb.
Workers screen print Tim Kasher posters during the performance.
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Hans Zimmer will score "Superman"
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December 7 2010, 01:38 AM
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Re: Hans Zimmer will score "Superman"
Yeah, I'm not really that optimistic about this project either to be honest. The fact that Chris Nolan has basically handed it off to Snyder and it's now his film now also disappoints me, since I thought Nolan would have been just a wee bit more involved, but I guess not as he's focusing on
The Dark Knight Rises
(and admittedly, as he should).
I'm just not a really big fan of Zack Snyder and his directorial sensibilities, so even though I'm somewhat hopeful because of Goyer's script and Nolan's initial involvement as a producer, my optimism is waning fast.
"Please... We need you to hope again... " - Professor Charles Xavier,
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With Sheriff Lee Baca's unexpected retirement, the race for Sheriff has turned in to a free for all. For the first time in the last 50 years, there will be no incumbent in the race which opens up the playing field for several entrants. Retired Sheriff Commander and Baca critic Bob Olmstead has announced his candidacy to replace the County's top cop. He joined the Morning Answer to discuss Baca's retirement and the upcoming race.
Brian Whitman wasted no time getting to the point. "Now please let's not – don't make this the moment where you say the obligatory things you don't believe, like Lee Baca's a good guy and we want to thank him for his public service," requested Brian. "You're clicking your heels over there Bob, c'mon!"
"You know, this is time," declared Olmstead. "This is time for him to step down, this is time the citizens and men and women of the LA County Sheriff's Department – need new leadership and I'm anxious to step in to this race and make the necessary changes."
Co-host Elisha Krauss asked Olmstead if he thought things would improve now that Lee Baca is out. Olmstead replied that there are significant issues that remain. "Let me put it to you this way," Olmstead explained, "the jail abuse, the questionable hiring practices, the obstruction of justice – this stuff didn't occur in a void. This stuff occurred because the second level, the third level of command leadership is vacant. They have supervisory cowardism, the fail to step up and take this stuff on."
Co-host Ben Shapiro brought up a recent LA Times editorial that called for widespread changes in the department beyond Sheriff Baca. He expressed concern because the crime fighting record is actually quite good with crime way down.
"Yeah but unfortunately the crime rates are up in the LA County Sheriff's Department!" exclaimed Olmstead. "We just had 18 people arrested – we're probably going to have 20 to 40 more is the speculation."
Brian Whitman then pressed Olmstead on who he thought was the front-runner of the race. "You know I'm the front runner and I take this from the standpoint on the act of goodness. Nobody that is running right now has come out of the whistleblower. Nobody that has come out now is considered the true reformer… My poll indicated that when we looked in to my numbers tripled when they said 'Bob Olmstead is the whistleblower.'"
Click on the player above to hear the complete interview between Sheriff candidate Bob Olmstead and the Morning Answer crew.
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Jill Abramson Takes ~2 Minutes to Land On Her Feet
Here is your perhaps unnecessary reminder that even high-powered successful "lean in"-type businesswomen like Jill Abramson zigzag. The ex-Managing Editor of the New York Times has accepted a position helping students hone their narrative non-fiction skills at Harvard:
She will be a visiting lecturer in the Department of English for the 2014-15 academic year, the university said in a statement, and will teach in the fall and spring semesters. In the statement, Ms. Abramson said she was "honored and excited." Narrative nonfiction, she said, "is more important than ever. Its traditions and how it is changing in the digital transition are fascinating areas of study." Ms. Abramson previously taught journalism seminars at Yale and Princeton.
At a commencement address at Wake Forest University just days after she was fired, Ms. Abramson spoke of an uncertain future. "What's next for me?" she said to the graduates. "I don't know. So I'm in exactly the same boat as many of you. Like you, I'm a little scared, but also excited." Ms. Abramson, a 1976 Harvard College graduate, has a tattoo featuring the school's H logo, as well as a tattoo of the T from The New York Times's masthead.
If we had to life-map ourselves in tattoos, my own more varied collection would include a lion, a phoenix, a couple of corporate insignias, two logos for workplaces that no longer exist, a plant to symbolize the time I worked in the greenhouse of the National Zoo, a Danish kroner for my semester abroad, a subway token to demonstrate my feverish gratitude that I live in a place that lets me not have to drive, a quill pen, and the Kurt Vonnegut quote, "We are what we pretend to be, so we must be very careful what we pretend to be."
Anyway, Jill Abramson is going to teach! Good job hiring her, Harvard. Now how long before one of her smart-ass students writes "pushy" on her evaluations, do you think?


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Cinema buffs and renaissance dweebs take note: Rob Reiner's cinematic masterpiece The Princess Bride is celebrating 25 years. Gen X-ers fondly... More >>
One pill makes you larger, and one pill makes you incredibly horny. But if jail time is in the cards for suspects Cody Horn, 29, of Yorba Linda and Troy Olson, 44, of Norco, it's the former pill ... More >>
See Update No. 2 at the end of this post on ex-Officer Joe Wolfe being arraigned today on manslaughter and excessive-force charges.See Update No. 1 on a media report that the Grand Jury will convene t... More >>
For the uninitiated, metalcore music can sound like an indecipherable cacophony of screams and morse code drumming. But once in a while, a band... More >>
Continuing their residency at La Cave, Sega Genecide brings the noise—'90s style. Featuring members of OC's the Gromble, they cover songs... More >>
Blackfeet Braves' languid guitar sounds and reverb- laden fretwork call to mind peeling waves at deserted point breaks and smoky beach parties.... More >>
[See bottom of post for update.]ORIGINAL POST, Sept. 21, 3 p.m.: After more than a year of police brutality protests that launched a successful city recall election and saw two Fullerton police office... More >>
Head on down to Fashion Island for the taste of Newport. Though the festival is primarily focused on the passion of foodies, New Wave aficionados... More >>
John Huelsman, stepfather of former Fullerton Police Officer Jay Cicinelli, went on KFI-AM 640 this afternoon to blast the city council for its decision to clear Kelly Thomas of wrongdoing on the... More >>
Dan Hughes, Fullerton's acting chief of police, delivered a statement last night to the city council, effectively clearing mentally ill homeless Kelly Thomas of wrongdoing before cops beat him t... More >>
No that's not Interpol you're hearing. That icy, disaffected voice is in fact Justin Warfield, one half of the duo She Wants Revenge. This... More >>
The Planet Pluto is an indie band with an eclectic sound. Though perhaps they didn't get the memo about the celestial body, which was cut from the... More >>
How do the Swedes manage to nail American culture so deftly? We don't have the answer, but we can tell you that watching the Hives will give you a... More >>
Ron Thomas is claiming another victory for his son Kelly who died following a merciless police beating last year at the Fullerton Transportation Center. In an email sent out to media outlets last nigh... More >>
Featuring AFI members Davey Havok and Jade Punget Blaqk Audio brings the electric dancey goodness. Though the sound is significantly more... More >>
Once again it's time for the grand jesters of punk to thrill audiences with their hi-octane shenanigans. Donning menacing clown face paint and... More >>
Fullerton police are asking for the public's help in the case of 36-year-old La Habra man Daniel Olender, who died from injuries sustained the morning of Sept. 2 in front of Hero's Bar and Grill. As t... More >>
Known to many for their cover of Don Henley's "The Boys of Summer," The Ataris are in Orange County to play a show. Though they hail from... More >>
The Fresh & Onlys have concocted, seemingly in a garage, an indie-style of music that deftly melds haunting guitar work with pop sensibilities. ... More >>
The Discovery Science Center has flipped its script and is offering an exhibit geared toward the practically-minded science enthusiast. This time,... More >>
Currently based in Dana Point, Micah Brown is a homegrown talent who makes OC proud. The recipient of past honors by the Orange County Music... More >>
Coming from Omaha by way of Ingelwood CA, Pigeon John started rapping in the '90s. This was a golden time for the genre and John, aka John Dust,... More >>
See Also:*FYF Fest 2012 Review: Day One*The Growlers Announce New Album Release* Pacific Festival: The Dunes AnnouncedImagine our surprise after seeing the Growlers' Saturday night performance at FYF,... More >>
Don Carlos is best known for his association with legendary reggae act Black Uhuru. With a sound both catchy and anthemic, his voice resonates ... More >>
Head on down to the Coach House for some reggae of the roots variety. Hailing from St. Croix in the Virgin Islands, Midnite has been strumming up... More >>


text: France Bans Fracking; New York Set to Un-Ban It Controversial process extracts natural gas from ground By Kevin Spak, Newser User Posted Jul 1, 2011 2:15 PM CDT STORY COMMENTS (18) (Newser) – France became the first country to ban fracking yesterday—even as reports surfaced that New York was about to lift its de facto moratorium on the controversial practice of extracting natural gas from the earth. The French vote split along party lines, but the opposition largely came from the Socialist Party, which didn't think the ban went far enough because it contains loopholes that allow the exploitation of oil shale deposits by other means, Scientific American reports. New York, meanwhile, has had an informal ban on the process since 2008, but in a surprise move is expected to lift it soon in most places, CNN reports. Fracking is a means of drilling for natural gas that involves shooting water and chemicals into the ground. New York's new rules will ban the practice in state parks and watershed areas, but otherwise allow it. Andrew Cuomo's office is thought to be onboard with the plan, but hasn't said so publicly. Fracking opponents protest before the Tom Corbett inauguration to become the 46th governor of Pennsylvania at the state capitol in Harrisburg, Pa., Tuesday, Jan. 18, 2011. (AP Photo/Matt Rourke) ADVERTISEMENT(Click Prev or Next to continue viewing images.) « Prev« Prev | Next »Next » Slideshow Scientific American (Source Grid ») CNN (Source Grid ») US • Money • Science • World France • New York • Andrew Cuomo • fracking • hydrofracking CLICK BELOW TO VOTE 7% 1% 15% 48% 19% 11% RETURN TO THE STORY To report an error on this story, notify our editors.


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Teleka Patrick was last seen alive Dec. 5 at Borgess Medical Center in Kalamazoo.
Marvin Sapp / Palace Sports & Entertainment
A Maryland-based religious activist group is asking gospel singer Marvin Sapp to prove his Christianity by making peace with Dr. Teleka Patricks family and holding a vigil Saturday in her memory.
The Pray at the Pump Movement is also making a broad appeal for churches in Michigan and elsewhere to pray for Patrick.
Patrick had been missing since December and her body was found April 6 in a northern Indiana lake, officials confirmed earlier this week. The Kalamazoo physician appears to have drowned. Investigators found no signs of trauma or foul play on the body, and the official cause and manner of death are pending toxicology results.
■ Related: Autopsy shows missing Kalamazoo doctor Teleka Patrick may have drowned
Before her disappearance, Sapp filed a personal protection order against her last September, saying she was stalking him. He says she contacted him hundreds of times, but he never responded.
■ Related: Report: Missing Kalamazoo doctor Teleka Patrick was stalking gospel singer
Authorities have publicly stated Sapp was not involved in her disappearance.
Maryland resident Rocky Twyman, founder of the Pray at the Pump Movement, said in a phone interview he believes it would help her family get closure if Sapp reached out to them to help them grieve and begin the healing process. The group organizes prayer vigils across the country.
This afternoon, PAPM will pray for Patricks family at the Havre de Grace Seventh-day Adventist Haitian Church in Rockville, Md.
Twyman said his organization has reached out to Sapp before regarding a possible fund-raiser event for Patrick, but received no response. Sapp could not be reached for comment Friday night.
Twyman said the group was in the process of contacting churches in Michigan as well. PAPM held a special vigil for Patrick in front of the Washington, D.C., FBI Headquarters on New Years Eve.
Were asking churches all over to just pray, all denominations, to pray for her family, he said. And pray other families who are facing these mental health issues.
Contact Katrease Stafford: email@example.com, 313-223-4759. Follow her on Twitter @KatreaseS


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The Boy Who Allegedly Went to Heaven and Returned
Regardless of what you may think of The Bible or the theory of a supreme being such as "God" or "Jesus" what are your thoughts on the afterlife?
You find that some people who don't believe in God, Jesus or The Bible still believe that we go to an afterlife when we die. I believe that it's embedded into our minds that when we die we still exist.
So would you believe a child, aged 11 (who was actually 7 at the time of the alleged event) died and witnessed heaven himself? or would you believe that his claims were fabricated by his family to sell books or the somewhat more logical explanation that he was dreaming. Little boys have big imaginations.
Para-normality always intrigued me as a child and well into my adult years I would consider anything that is "mysterious but possible" to be absolutely amazing so its no wonder that when I saw this book in a local store I had to pick it up and take a look at the cover which read:
Heaven Is for Real is the true story of the four-year old son of a small town Nebraska pastor who during emergency surgery slips from consciousness and enters heaven. He survives and begins talking about being able to look down and see the doctor operating and his dad praying in the waiting room. The family didn't know what to believe but soon the evidence was clear. Colton said he met his miscarried sister, whom no one had told him about, and his great grandfather who died 30 years before Colton was born, then shared impossible-to-know details about each. He describes the horse that only Jesus could ride, about how "reaaally big" God and his chair are, and how the Holy Spirit "shoots down power" from heaven to help us.
Colton's father is somewhat conservative and traditional according to the book. I've noted the following:
– If you don't have Jesus in your heart, you go to hell.
– Everybody in heaven has wings, except Jesus. The boy's grandpa had "huge wings".
– God zaps pastors with power when they preach.
– Angels have swords to keep demons out of heaven.
– Soon there will be a big war between "bad people", demons and monsters against believers, God and Jesus. Jesus wins and throws Satan into hell. Firstly, how can you not believe in Jesus if you died and rose to heaven and then witnessed Jesus for himself? What happened to the good old "I'll believe it when I see it" theory?
Feel free to correct me but would pastors need to preach in heaven?Surely all of their work on earth is done and they have an eternity to relax?
I don't really know how much I believe the story but as much as I don't doubt the possibility, this really seemed like a bid to attract people to changing their ways and possibly going to church, however it's definitely a decent read if your into the whole "life after death" concept and it will certainly raise your eyebrows at times. In an eerily convincing way, this story has little evidence to back it up but its all down to the reader.


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The state is giving out $189,550 in used motor oil collection grants.
Of the 15 counties set to benefit from the funds, Roane County is receiving the second highest allotment at $24,600.
According to a press release from the Tennesssee Department of Environment and Conservation, Tennesseans who change their own motor oil produce more than 1 million gallons of used oil every year.
If not properly disposed, the oil can have a negative impact on the environment.
Eric Glenn Gallaher has retained high-profile Knoxville attorney Bruce Poston in his reckless homicide case.
Gallaher is accused of killing David Lee Harvey, 46, outside the Grill & Pub last July. He allegedly punched Harvey after a minor fender bender in the parking lot.
"There is probable cause to believe ... Gallaher's deliberate act of punching Harvey did cause his death," court records state.
Human remains found in Loudon County last week are not connected to a Roane County cold case.
Sheriff Jack Stockton was hopeful the remains belonged to Karen Beard, who hasn't been seen since 1991.
"It's very disappointing," he said. "I was hoping that would put an end to the case."
Stockton said authorities were able to determine the remains were not Beard's through forensics.
"The DNA didn't match, and dental records didn't match," he said.
By MIKE GIBSON
Kingston is a justifiably proud city: It is eminently livable, soundly managed and blessed with a seemingly matchless combination of natural beauty and natural resources.
But there's one area where Kingston runs a distant second to many of its small-town counterparts — that is, when it comes to its aging, space-deficient city hall.
Harriman resident and Roane County Commissioner Randy Ellis said Tuesday he toyed with a run for state representative, but it won't be happening.
Ellis was prodded to run after redistricting split Roane into two state representative districts.
"Although it was very tempting to run for the Tennessee House of Representative for the 32nd District," Ellis said, "after much prayer and consultation with my family I have decided I will not be running."
Consumer rights activist Mary Mancini, executive director of Tennessee Citizen Action, will speak Friday on the Tennessee legislative issues surrounding voter ID laws and their effect on your right to vote. Her program is open to the public and sponsored by the Roane County Democratic Party.
It will be from 6 to 7 p.m. at the Roane County Park meeting center.
Roane County News latest columnist. Josephine McKinney, is no stranger to writing.
McKinney, 90, has been writing for about 50 years. Her "Round Rockwood" society column is being moved from the Rockwood Times to every Monday's edition of the Roane County News.
The newspapers Rockwood Times and the Harriman Record, which were combined into a single weekly about a year ago, are being discontinued. Most of the content is being moved into the Roane County News.
State Sen. Ken Yager of Harriman is seeking local students to work as pages in the State Senate.
Page duties consist of anything from making copies, running errands, to distributing legislative information to members of the General Assembly.
They report to the chief clerk of the House and Senate.
"This is the best civics lesson I can imagine," said Yager. "Student pages are right there with us on the Senate floor.
They get a unique front-row view of the lawmaking process."
Copyright © 2015
The Roane County News is your source for local news, sports, events, and information in Roane County and Kingston, Tennessee, and the surrounding area.


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The Home Depot retails its wares, ranging from building supplies to appliances to tools and almost everything in between, in all 50 states and various foreign nations via its 2,248 big box stores. It also peddles the homosexual agenda while rejecting out of hand a petition signed by half a million valued shoppers who objected to that stance.Â
  Ever since its surprising corporate choice of Frank Blake as chairman and CEO in 2007, the Atlanta-based company under Blake's direction has waged an unrelenting advocacy campaign not for new customers but for the homosexual lifestyle. Blake had virtually no retail experience before his selection, which leads one to believe he has a good, influential buddy on Home Depot's Board of Directors.Â
Two years ago, Blake survived an ouster move arising from an employee-abuse scandal involving unethical conduct related to the company's stringent Code of Conduct when he essentially maintained he was oblivious of the abuses despite his "full and documented knowledge." (http://bit.ly/r0QY2T) Â
Good buddies and bottom lines go a long way at The Home Depot.Â
Apparently, ethics aren't Frank Blake's strong suit although he does seem to feel a deep sense of ethical responsibility toward the gay community. Â
According to the authoritative American Family Association (AFA), instead of opting for a course of neutrality on controversial issues, a course most publicly-traded corporations in the United States wisely and rightly elect, Frank Blake's Home Depot engages in promoting homosexuality.Â
Among its promotional, and discriminatory, activities:
• In June, 2011, Home Depot marched in the Boston Gay Pride Parade.
• Home Depot placed pro-homosexual displays in store break rooms, while rejecting requests from employees to recognize Christian Heritage month as part of its commitment to "diversity.â€
• In June 2010, Home Depot set up a "Kids Workshop†at several gay festivals and parades.
• In 2009, Home Depot allowed employees to wear "gay†buttons on their aprons, but fired an employee for wearing a "One Nation Under God†button.
• Home Depot paid to print thousands of color brochures during the 2010 Atlanta Gay Pride Festival with a schedule and map of hundreds of gay pride activities and booths. The brochure prominently featured the promotion for a gay marriage "Commitment Ceremony†at the event.
• In 2009, Home Depot gave over $5,000 to be a major sponsor of the Nashville Gay Pride Festival. It also sponsored parades in Atlanta; Kansas City; Portland, Maine; and San Diego.
• Home Depot financially supports Human Rights Campaign, the nation's leading organization pushing for homosexual marriage in America. Â
  Lest we forget the abovementioned petition, "In May, 2011, Home Depot Chairman Frank Blake summarily dismissed 500,000 petition signatures presented to him at the company's annual shareholder's meeting in Atlanta. Mr. Blake stated the company would continue supporting homosexual activist groups and activities." (http://bit.ly/kXt60S) Â
There is nothing inherently wrong in any individual or any corporation in America supporting any cause. It is grossly wrong and un-American to discriminate against employees and the general public who maintain opposing views.Â
The Home Depot's (NYSE: HD) stockholders will have to decide whether they wish to continue to hold or sell their shares in a biased company. The public can express their objections by signing the petition to boycott Home Depot at the AFA website cited above, http://bit.ly/kXt60S      Â
Do keep in mind, Lowes has better service, higher quality products, and non-judgmental corporate policies. (In the interests of full disclosure, I don't own either Home Depot or Lowes stock.)Â


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Sherman Minton Bridge shutdown threatens transportation, one of Louisville area's key industrial draws
Sep. 24, 2011
Louisville, Kentucky • Southern Indiana
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Sherman Minton Bridge shutdown threatens transportation, one of Louisville area's key industrial draws
Louisville's economy has been built largely on being able to reach 60 percent of the U.S. population in a day's drive � making and moving goods around the country, as well as around the world,
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What is APA?
Advanced Processor Architectures (APA) is a scalable distributed memory and processor system that is configurable and reconfigurable at the device, fabric and systems level statically and dynamically. APA's architecture uses patented technologies that dramatically reduce the operating and manufacturing costs, size, weight and power required for high performance processing while providing unprecedented reliability, through the use of continuous communication self testing and redundant operations.
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