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ORIGINAL RESEARCH article
Sec. Mucosal Immunity
Volume 12 - 2021 | https://doi.org/10.3389/fimmu.2021.618365
Peroxisome Proliferator-Activated Receptor Alpha Mediates the Beneficial Effects of Atorvastatin in Experimental Colitis
- 1Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil
- 2Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil
- 3Departmento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil
- 4Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro, Uberaba, Brazil
The current therapeutic options for Inflammatory Bowel Diseases (IBD) are limited. Even using common anti-inflammatory, immunosuppressive or biological therapies, many patients become unresponsive to the treatments, immunosuppressed or unable to restrain secondary infections. Statins are cholesterol-lowering drugs with non-canonical anti-inflammatory properties, whose underlying mechanisms of action still remain poorly understood. Here, we described that in vitro atorvastatin (ATO) treatment was not toxic to splenocytes, constrained cell proliferation and modulated IL-6 and IL-10 production in a dose-dependent manner. Mice exposed to dextran sulfate sodium (DSS) for colitis induction and treated with ATO shifted their immune response from Th17 towards Th2, improved the clinical and histological aspects of intestinal inflammation and reduced the number of circulating leukocytes. Both experimental and in silico analyses revealed that PPAR-α expression is reduced in experimental colitis, which was reversed by ATO treatment. While IBD patients also downregulate PPAR-α expression, the responsiveness to biological therapy relied on the restoration of PPAR-α levels. Indeed, the in vitro and in vivo effects induced by ATO treatment were abrogated in Ppara-/- mice or leukocytes. In conclusion, the beneficial effects of ATO in colitis are dependent on PPAR-α, which could also be a potential predictive biomarker of therapy responsiveness in IBD.
Statins are the most effective drugs used in lowering serum cholesterol levels, preventing or treating atherosclerosis and coronary heart disease (1, 2). They act by selectively inhibiting the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key regulatory and rate-controlling enzyme of cholesterol biosynthesis (3).
The cholesterol-lower drugs have also been shown to exert cholesterol-independent or “pleiotropic” effects, which comprise anti-inflammatory, immunomodulatory, vasodilatory, antioxidative, antithrombotic and osteomodulatory activities (4–6). Several mechanisms have been suggested to explain the wide range of statins functions and most of them arise from the inhibition of isoprenoid intermediates produced during the mevalonate pathway (6). These lipid mediators are essential to structure and function of various cell-signaling proteins, including the super family of small GTPases (e.g., Ras, Rho, Rac) associated to cell traffic, cycle, and differentiation (7, 8). Thus, statins may be used to treat other conditions not related to hypercholesterolemia, including autoimmune and inflammatory diseases (9–11). Accordingly, a few experimental and clinical studies have used different types of statins to treat Inflammatory Bowel Diseases (IBD), a group of chronic and recurrent inflammatory disorders of the digestive tract, which includes Crohn’s Disease (CD) and Ulcerative Colitis (UC) (12).
Both CD and UC share clinical similarities, but they comprise unlike characteristics to be considered as autonomous entities, such as the distribution and types of lesions in the gastrointestinal tract, the histopathology, the dynamic of immune responses and the treatment responsiveness (13–15). HMG-CoA inhibitors in experimental models of colitis have been shown to attenuate the clinical signs of disease and improve the survival rates in mice (16–18). Studies in humans are scarce, but Grip and colleagues described that CD patients treated with atorvastatin (ATO) had a reduction in both disease activity and inflammatory markers, including plasma C-reactive protein (CRP) and soluble TNF receptor II (sTNFRII) levels (19). Although some underlying mechanisms have been identified as responsible for the benefits of statins in IBD, it is still unknown whether this drug can modulate inflammation in a cholesterol-independent manner.
It has been evidenced that statins can activate peroxisome proliferator-activated receptors (PPARs), a group of three transcription factors (PPAR-α, PPAR-β/δ and PPAR-γ) involved in inflammation and metabolism (20). PPARs translocate from cytoplasm into nucleus after binding to cognate ligands, heterodimerize with retinoid-X receptor (RXR) and then bind to PPAR-responsive elements (PPREs) to activate the transcription of several target genes (20). The use of statins was showed to improve the outcome of inflammatory conditions such as experimental autoimmune encephalomyelitis (EAE) and LPS-induced acute inflammation through PPAR-α activation (21, 22). However, it is unknown whether PPAR-α plays any role in ameliorating IBD outcome after statin therapy. Therefore, here we hypothesized that statins could improve the outcome of experimental colitis by modulating PPAR-α expression or activation, which could in turn impact the resolution of the ongoing pathogenic intestinal inflammation.
Material and Methods
Male C57BL/6J wild type (WT) and transgenic Ppar-/- mice (6-8 weeks old) were bred and maintained in the animal facility at School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo. Mice were housed in controlled (25°C ± 2°C and 12h/12h light/dark cycles) and in specific pathogen free conditions along with water and food ad libitum. All experimental procedures were approved according to the guidelines of the Ethics Committee of the University of São Paulo - campus Ribeirão Preto (protocol number 10.1.499.53.8) and performed according to the criteria outlined by the Brazilian Society for Laboratory Animal Science (SBCAL).
Acute Colitis Induction and Experimental Protocol
Mice were randomly assigned to one of the three groups: untreated healthy animals, mice exposed to 3% (w/v) DSS (molecular weight: 36,000–50,000, MP Biomedicals, Illkirch, France) in drinking water and treated with vehicle (normal saline) or mice exposed to 3% DSS and treated with ATO (10mg/kg/day, Lipitor®, Pfizer, SP, Brazil). Healthy mice treated with ATO were not depicted since they did not show any difference compared to the untreated healthy group. DSS was provided ad libitum for 6 consecutive days in drinking water. ATO was suspended in normal saline and given orally to mice for 3 consecutive days from day 3 after the onset of colitis induction, once a day, until the day before euthanasia. Mice were euthanized on the 6th day after colitis induction and had the blood, intestine (colon), spleen and mesenteric lymph nodes (MLN) collected for further analysis. Colon samples were divided into smaller fragments of approximately 1 cm. Then, one of these fragments was immersed in PBS/10% formaldehyde for paraffin embedding and histopathological analysis (distal colon). The other fragments were immediately frozen in liquid nitrogen for total RNA extraction (middle colon) or for measurements of cytokines levels (distal colon) or eosinophil peroxidase (EPO; middle colon), myeloperoxidase (MPO; proximal colon) and N-acetylglucosaminidase (NAG; proximal colon) activities.
Clinical, Overall, Postmortem and Accumulated Scores
Clinical signs and overall, post-mortem or accumulated scores were performed as previously described (23). Briefly, clinical parameters such as daily weight loss (≥5% and <10%), wet anus, diarrhea, bleeding stools, hypoactivity and piloerection were evaluated and received 1 point each. Weight reduction of ≥10% compared to previous day received 2 points. The overall score was resulted of the sum of all daily clinical scores per mouse. Following the same scoring criteria used to clinical signs analysis, the post-mortem score was performed by evaluating local or diffuse diarrhea/bleeding in the colon. Local alterations received 1 point, while diffuse changes and stenosis received 2 points. The accumulated score was defined as sum of clinical score at the day of euthanasia and post-mortem score, as previously described (23).
Total Leukocytes and Differential Cell Counting
Total circulating leukocytes were counted using a Neubauer chamber and blood smears processed for Romanowsky staining (Laborclin Products for Laboratory Ltda, Pinhais, PR, Brazil) for differential cell counting.
Histology and Histopathological Analyzes
The colon fragments previously immersed in PBS/10% formalin were processed for paraffin embedding followed by microtomy of 5 μm sections, which were stained with hematoxylin and eosin. The histopathological analysis was based on a previous study with some modification (24). Briefly, mucosa thickness, lamina propria (LP) cellularity, inflammatory infiltrate and epithelial morphology were evaluated. The images were captured using both 20x and 40x objectives through a digital video camera (Evolution MP 5.0-color, Media Cybernetic, Silver Spring, MD, USA) coupled to a light microscope (Nikon, Eclipse 50i, Melville, NY, USA). An area of 1,57 mm2 from each colon sample was evaluated and morphometry was performed using the software “Image-Pro Insight” (Media Cybernetics, Rochville, MD, USA). The cells in the LP were counted and the results were expressed as number of cells per mm2.
Cytokines Quantification by ELISA
The cytokines were quantified by ELISA on cell culture supernatants and/or colon tissue lysate (IL-4, IL-6 and IL-10 - BD Biosciences, San Jose, CA, USA; IL-17A – Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions.
Myeloperoxidase (MPO), Eosinophil Peroxidase (EPO) and N-Acetylglucosaminidase (NAG) Activities Measurement
MPO, NAG and EPO activities were measured as described previously (25). For MPO assay, small colonic fragments were homogenized in buffer containing 0.1 M NaCl, 0.02 M NaPO4, and 1.015 M NaEDTA, pH 4.7 and resuspended in a hypotonic solution to erythrocytes rupture followed by three freeze-thaw cycles to lyse the remaining cells. After centrifugation, the supernatant was incubated with 3,3’,5,5’-tetramethylbenzidine (TMB Substrate Reagent Set – BD OptEIATM, San Diego, CA) at 37°C for 15 minutes, the reaction stopped using 2 N H2SO4 and the optical density (OD) measured at 450 nm. For NAG activity, the same supernatant from MPO assay was used and then incubated with 2.24 mM p-nitrophenyl-2-acetamide-β-D-glucopyranoside and 50 mM citrate buffer (pH 4.5) for 60 minutes at 37°C. The reaction was stopped using 0,2 M glycine buffer (pH 10.4) and absorbance was determined at 405 nm. For EPO measurement, colonic fragments were homogenized in HBSS buffer and then subjected to both hypotonic lysis and freeze–thaw cycles. The supernatant obtained by centrifugation was incubated with o-phenylenediamine dihydrochloride (SIGMAFAST OPD tablet set, Sigma-Aldrich, Saint-Louis, MO, USA) for 30 min at 37°C. The reaction was stopped using 2 N H2SO4 and absorbance was determined at 492 nm. All the results were expressed as optical density per gram of tissue.
RNA Extraction, Reverse Transcription, and Quantitative Real-Time PCR (qRT-PCR)
The colonic tissues were homogenized in TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) for total extraction and the mRNA was isolated using SV Total RNA Isolation System Kit (Promega, Madison, WI, EUA), according to manufacturer’s instructions. RNA purity and quantity were assessed by using a NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE, EUA). The purified RNA was used to synthetize the complementary DNA with GoScript™ Reverse Transcription System (Promega, Madison, WI, USA) according to the manufacturer´s instructions. Finally, all cDNA samples were diluted in nuclease-free water. Quantitative real-time PCR (qRT-PCR) studies were performed using GoTaq® qPCR Master Mix (Promega, Madison, WI, EUA) in a StepOnePlus™ Real-Time PCR System (Applied Biosystem, Foster City, CA, EUA). cDNA sample were mixed with solution containing forward and reverse target primers, GoTaq® qPCR Master Mix and nuclease-free water as provided by the manufacturer in 96-well MicroAmp Fast Optical reaction plates (Applied Biosystem, Foster City, CA, EUA). Gene-specific primers for mouse IL-4, IL-17A, PPAR-α and β-actin were used and their respective sequences were as follows: IL-4 - (sense) 5’-AAG AGC ATC ATG CAA ATG GA-3’, (antisense) 5’-TTA AAG CAT GGT GGC TCA GTA C-3’; IL-17A - (sense) 5’-TGC CCT CCA CAA TGA AAA GA-3’, (antisense) 5’-AAC ACG AAG CAG TTT GGG AC-3’; PPAR-α - (sense) 5’- TCA ATG CCT TAG AAC TGG ATG A-3’, (antisense) 5’- CCGATCTCCACAGCAAATTATA-3’;
β-actin - (sense) 5’-AAC GAG CGG TTC CGA TG-3’; (antisense) 5’-GGA TTC CAT ACC CAA GAA GGA-3’. All primers were obtained from Sigma-Aldrich (Saint-Louis, MO, USA). The PCR conditions were 50°C for 2 min, 95°C for 2 minutes, 40 cycles for 15 s at 95°C, 30 s at 58°C and 30 s at 72°C, followed by melting curve determination. All the qPCR assays included a negative control (without cDNA template), and the samples were run in duplicate. β-actin was used to normalize the expression levels of target genes and the results were evaluated based on the Ct value. The differential expression of mRNA was defined by the formula 2-ΔΔCt, according to previous studies (26).
Isolation of Leukocytes From Spleen and Mesenteric Lymph Nodes (MLN)
Spleen and MLNs were removed, crushed through a 70 μm cell strainer (BD Biosciences, Heidelberg, Germany) using a syringe plunger and then suspended in complete RPMI 1640 medium - supplemented with 3.7 g/L sodium bicarbonate, 5% heat inactivated fetal bovine serum (FBS; Gibco – Invitrogen, Ontario, Canada), 2 mM L-glutamine (Sigma-Aldrich, Steinheim, Germany), 1 mM sodium pyruvate (Sigma-Aldrich, Saint Louis, MO, USA), 0.5 mM 2-mercaptothanol (Sigma-Aldrich, Saint Louis, MO, USA), 10 mM HEPES (Sigma-Aldrich, Saint Louis, MO, USA), 100 IU/mL penicillin (Gibco-Invitrogen, Ontario, Canada) and 100 μg/mL streptomycin (Gibco – Invitrogen, ONT, CA) - for subsequent cell culture and flow cytometry assays. The spleen cells were subjected to additional step to lyse red blood cells with appropriate lysis buffer. Trypan blue dye (0.2%) exclusion tests were used to determine the number of live cells before cell culture and immunophenotyping experiments.
For phenotypic characterization of the leukocyte’s populations from spleen and MLN, monoclonal antibodies conjugated to fluorochromes were used. In brief, cells were incubated with antibodies (1x106 cells/mL) for 20 minutes at 4°C in the dark. The cells were then fixed with PBS containing 1% formaldehyde or followed by intracellular staining. All antibodies (CD3, CD4, CD8, CD11b, CD11c, CD49b, IL-17, IFN-γ) were obtained from BD Pharmigen™ (San Diego, CA, USA). Leukocytes were acquired on Canto II Flow Cytometer (BD Biosciences, San Jose, CA) and the data were analyzed using FlowJo version 6.6.3 software (TreeStar, San Carlos, CA).
Lymphocyte Proliferation and Cell Death Assays
For lymphocyte proliferation assay, the spleen cells were stained with 5 µM carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Invitrogen, Carlsbad, USA) for 10 minutes at 37°C. Subsequently, cells (2.5x106/mL) were stimulated with concanavalin A (ConA, 3 μg/mL) and incubated at 37°C/5%CO2 for 72 hours. Then, the supernatant was collected, and the cells analyzed by flow cytometry as described earlier. The results were expressed as the total number of divisions divided by the number of cells that went into division and termed as proliferation index.
Cell death (apoptosis and necrosis) was assessed using propidium iodide and annexin-fluorescein, according to manufacturer’s instructions (Annexin-V-FLUOS Staining kit - Roche, Mannhelm, Germany). Cells were acquired in flow cytometer and results interpreted as follows: double-positive cells (annexin+PI+) were considered as late apoptotic cells or necrotic cells; annexin+PI- cells were considered as early apoptotic cells; annexin-PI+ cells were considered as necrotic cells; double-negative cells (annexin-PI-) were considered as live cells. Saponin (100mM) was used as positive control of cell death.
We manually curated the Gene Expression Omnibus (GEO) repository (https://www.ncbi.nlm.nih.gov/geo/) to find transcriptome datasets of colonic regions related to “IBD (CD and UC)” in humans and “DSS-induced colitis” in mice. Expression analysis was performed based on author normalized expression values using the GEO2R software (https://www.ncbi.nlm.nih.gov/geo/geo2r/). When necessary, the normalized expression values were converted to a log2 scale for analysis. Probes that matched the same gene symbol were collapsed by taking the one with the lowest p-value. The heatmap was generated using the Morpheus software (https://software.broadinstitute.org/morpheus/) based on the log2-fold change values for each gene throughout all studies analyzed. Volcano plot was generated using limma (27). The genes depicted were significantly differentially expressed at an adjusted p-value cutoff of 0.05 (red = upregulated genes; blue = downregulated genes).
Five datasets were selected for analysis, being 4 from humans and 1 from mice. Human datasets comprised two representative expression data from UC patients and controls (GSE75214 and GSE87466) and other two from UC patients who received infliximab therapy, including responders and non-responders to drug therapy (GSE16879 and GSE12251). GSE75214 contains transcriptomic data of colonic biopsies from healthy individuals (n=11) and patients with CD (n=8) or UC (n=74). Only subjects with active disease were considered for analysis, which was confirmed by endoscopic evaluation. GSE87466 contained gene expression profiling of mucosal biopsies from controls (n=21) and patients with moderately to severely active UC (n=87). Disease activity was also endoscopically assessed. The differentially expressed genes (DEGs) in GSE75214 and GSE87466 datasets were filtered by either logFC < -1 (downregulated genes) or logFC > 1 (upregulated genes) and by adjusted p-value <0.05. Then, KEGG Pathway enrichment analysis was performed on the up and downregulated genes using the Metascape software (28). Correlation analyzes were performed between PPARA and PPARGC1A genes using data from GSE75214 and GSE87466 datasets. Based on Kolmogorov-Smirnov normality test, we selected either Pearson or Spearman analysis for the correlation analyzes.
GSE16879 contained transcriptomic data of mucosal biopsies from patients with UC before and 4-6 weeks after their first infliximab infusion. Both responders and non-responders to therapy were included in the study. Responders (n=8) and non-responders (n=16) were classified based on endoscopic and histologic findings at 4-6 weeks after first dose of infliximab treatment. GSE12251 contained transcriptomic data of UC patients 8 weeks after receiving infliximab therapy, including responders and non-responders. Responders (n=12) and non-responders (n=10) to infliximab were defined according to endoscopic and histologic mucosal healing at week 8. Mice dataset comprised one representative expression data from C57BL/6 mice subjected to 3% DSS colitis for 6 days (GSE22307). We assessed the data from normal (n=5) and sick (n=6) colonic samples.
Data Analysis and Statistics
Statistical analysis was performed using the Graphpad Prism software. In all variables normal distribution and homogeneous variance were tested. When the distribution was considered normal, and variance was homogeneous, parametric tests were used (unpaired Student’s t test or one-way ANOVA with Tukey’s post-test). In cases of non-Gaussian distribution of data, the nonparametric tests Mann–Whitney or Kruskal–Wallis (Dunn’s) were used. Results were expressed as mean ± SEM. The observed differences were considered significant when p < 0.05 (5%).
ATO Modulates Immune Responses In Vitro
To evaluate the immunomodulatory properties of statins, CFSE-stained splenocytes were stimulated with ConA in the presence or absence of ATO for 72 h. Treatment with 10 μM ATO reduced the proliferation rate, whereas no difference was observed after the use of 5 μM ATO (Figures 1A, B, respectively). Furthermore, whereas only 10 μM ATO reduced IL-6 concentrations in the cell culture supernatant (Figure 1C), the 5 μM ATO dose strikingly increased the synthesis of IL-10 in ConA-stimulated cells (Figure 1D). Then, we investigated whether the changes in both proliferation and cytokine production associated with the highest concentration of ATO were a consequence of immunomodulatory effects of the therapy or a putative induction of cell death. The frequency of dead cells (annexin+PI+, annexin-PI+ or annexin+PI-) treated with both 5 μM and 10 μM ATO was similar to that found in vehicle-treated leukocytes (Figure 1E). In addition, the use of 5 μM ATO slightly decreased the cell death of ConA-stimulated cells. Altogether the results showed that ATO modulates the proliferation and synthesis of IL-6 and IL-10 cytokines in stimulated cells without inducing cell death.
Figure 1 Immunomodulatory effects of ATO in vitro. Splenocytes from C57BL/6 WT mice were stimulated with 3 µg/mL of ConA and concomitantly treated with 5 or 10 µM ATO for 72 h. (A) Representative histograms of cell proliferation in CFSE-stained cells assessed by flow cytometry. (B) Graphic representation of proliferation index (the total number of divisions divided by the number of cells that went into division). (C, D). Quantification of IL-6 (C) and IL-10 (D) levels on the cell culture supernatants of ConA-stimulated cells that received or not ATO treatment. Dotted lines indicate cytokine production by unstimulated cells. (E) Cell death assessed by Annexin/PI staining followed by flow cytometry analysis. Double positive-staining (annexin+PI+) was considered late apoptotic or necrotic cells; annexin+PI- was considered early apoptotic cells; annexin-PI+ were the necrotic cells; double-negative cells (annexin-PI-) were considered as live splenocytes. Saponin was used as positive control of cell death. The dashed line indicates basal values from unstimulated cells. All the results are representative of 3 independent experiments that were performed, each one, with splenocytes pooled from 3 mice. ANOVA test was used followed by Tukey comparison or Kruskal–Wallis (Dunns) test. *p < 0.05. ATO, Atorvastatin; ConA, Concanavalin A; CFSE, 5(6)-carboxyfluorescein N-hydroxysuccinimidyl ester; PI, Propidium iodide. WT, Wild type.
ATO Treatment Attenuates the Clinical Signs of DSS-Induced Colitis
Since ATO showed anti-inflammatory effects in vitro, we next evaluated whether the drug would also have the same effects in vivo. For this purpose, WT mice were exposed to 3% DSS in drinking water for 6 consecutive days. After 3 days, when the clinical signs arise, mice were orally administered with vehicle or 10 mg/kg/day of ATO for 3 days, once a day. A dose-response study was conducted previously to select the best dose and route of administration of ATO (data not shown). Treatment with ATO improved clinical signs of colitis when compared to vehicle-treated mice (Figure 2A). The overall, post-mortem and accumulated scores confirmed that ATO therapy attenuated the course of colitis (Figures 2B–D, respectively).
Figure 2 ATO treatment improves the clinical outcome of experimental colitis. C57BL/6 WT mice were exposed to 3% (w/v) DSS in drinking water for 6 days. Mice received oral treatment once daily for three days (days 3-5) with saline (vehicle) or 10 mg/Kg/day ATO. (A) Clinical score during the disease course. The hatched area indicates the period of ATO treatment. (B) The sum of daily scores was termed as overall clinical score. (C) The macroscopic evaluation of colon after euthanasia was termed as post-mortem score. (D) The sum of clinical score on day six after DSS administration and post-mortem score was termed as cumulative score. (E, F) Total number of circulating white blood cells (leukocytes) (E) and differential leukocyte counts (F). The dashed line indicates values from healthy control mice without colitis induction. These results are representative of 3-5 independent experiments with 5 mice per group in each experiment. Unpaired t tests or Mann-Whitney non-parametric t test were used to determine if differences attained significant response. *p < 0.05. ATO, Atorvastatin; DSS, Dextran sodium sulfate.
The number of total circulating white blood cells was reduced in mice that received ATO (Figure 2E). A decrease in lymphocytes and monocytes was more significative, followed by a slight decrease of eosinophils (Figure 2F).
These findings pointed out that ATO improves the colitis outcome and decreases the number of circulating leukocytes, suggesting a systemic anti-inflammatory role of drug.
ATO Ameliorates Histopathological Aspects of Colon and Shifts Th17-Polarized Immune Response to Th2 Profile in the Lamina Propria (LP)
To verify if the clinical improvements induced by ATO also extended to microscopic anatomy of the colon, we proceed with the histopathological analyzes of the large intestine sections. Healthy control mice had a normal epithelial lining along with parallel and well-structured crypts (Figure 3A – left picture). The mean number of LP cells was 2916 ± 123,3 cells/mm2 (Figure 3B, dashed line). The colon of vehicle-treated DSS-exposed mice showed areas with absence of crypts interspersed with areas of preserved mucosa together with mucin-depleted, irregular and flattened superficial epithelium (Figure 3A – central picture). There was also a marked inflammatory infiltrate in the LP of DSS group treated with vehicle with a mean number of 8908 ± 191,8 cells/mm2 (Figure 3B). However, the treatment with ATO improved mucosal and crypt architecture preservation (Figure 3A – right picture), besides decreasing the inflammatory infiltrate in LP with a mean number of 4466 ± 150,6 cells/mm2 (Figure 3B).
Figure 3 ATO treatment improves the histopathological aspects of the colon, increases the eosinophil activity and IL-4 levels in the gut. C57BL/6 WT mice were exposed to 3% (w/v) DSS in drinking water for 6 days. Mice received oral treatment once daily for three days (days 3-5) with saline (vehicle) or 10 mg/Kg/day ATO. (A) Photomicrographs showing the histological analysis of colon. Asterisk indicates areas with absence of crypts. Arrows indicate cell infiltration in the LP. Original magnification: 200x and 400x. (B) Number of mononuclear cells in LP per mm2 of tissue. (C) Activity of EPO, NAG and MPO in colon. The results were expressed by OD (in nm) per gram of tissue. (D) Relative mRNA expression of IL-17A and IL-4 in colon. The dashed line indicates the values from healthy control mice without colitis induction. These results are representative of 2 or 3 independent experiments with 5 mice per group in each experiment. Unpaired t test or Mann-Whitney non-parametric t tests were used to determine if differences attained significant response. *p < 0.05. ATO, Atorvastatin; DSS, Dextran sodium sulfate; EPO, Eosinophil peroxidase; LP, Lamina propria; MLN, Mesenteric lymph nodes; MPO, Myeloperoxidase; NAG, N-Acetylglucosaminidase. OD, Optical density.
Next, we evaluated whether the cellular composition of the infiltrate was modified following therapy with ATO. Thus, the number of eosinophils, macrophages and neutrophils in the LP was determined using indirect evaluation through EPO, NAG and MPO assays, respectively. As expected, there was an increase in the activity of colonic neutrophils and macrophages in DSS-exposed mice treated with vehicle when compared to healthy controls (Figure 3C). Interestingly, the ATO-treated group increased the activity of LP-infiltrating eosinophils, while no changes were detected in macrophages and neutrophils.
Studies regarding experimental models of colitis have described that acute DSS-induced colitis have a predominant Th1/Th17 immune response, while in the chronic phase the inflammation shifts to a Th2 profile (29). Therefore, we investigated whether the different distribution of cells in MLN and spleen was related to a putative cytokine modulation induced by ATO treatment. Interestingly, ATO increased the frequency of CD4+IL-17+ T lymphocytes in the spleen and MLN, whereas there was a reduction in CD4+IFN-γ+ T lymphocytes in MLN (Supplementary Figures 1A, B). Moreover, we observed a shift in cytokine profile towards a Th2 response after treatment with ATO, by detecting an enhancement of IL-4 at the expense of IL-17 expression in the colon (Figure 3D).
Together, the results suggest that ATO improves the morphological aspects of the colon, decreases the number of local inflammatory cell infiltrate, and switch the local responses to a Th2-prone profile, which could be related to immune regulation in the gut during the acute phase of colitis.
Modification of Immune Cell Subpopulations in Secondary Lymphoid Organs
Because the number and cell types in the colon were changed after treatment using ATO, we next investigated whether these alterations could be observed in secondary lymphoid organs. The total number of cells in spleen and mesenteric lymph nodes (MLN) was not different between the ATO or vehicle-treated groups. We identified an increased frequency of both CD11b+ and CD11b+CD11c+ monocyte-derived cells, CD8+ T lymphocytes, CD3+CD49b+ NKT cells and CD3-CD49b+ NK cells in the spleen of ATO-treated mice (Figure 4A). In addition, a marked reduction in the frequency of monocyte-derived CD11c+ cells were also observed. In turn, ATO therapy decreased the frequency of CD4+ T lymphocytes and increased both CD11c+ cells and CD8+ T lymphocytes in the MLN (Figure 4B). These results indicated that ATO has both systemic and local effects on immune cells distribution during the DSS-induced colitis.
Figure 4 ATO treatment modifies the frequency of several immune cell subpopulations in spleen and MLN. C57BL/6 WT mice were exposed to 3% (w/v) DSS in drinking water for 6 days. Mice received oral treatment once daily for three days (days 3-5) with saline (vehicle) or 10 mg/Kg/day ATO. (A, B) The frequency (%) of several immune cell subtypes were assessed by flow cytometry in both spleen (A) and MLN (B). Immune cell subsets were determined by expression of surface markers, including: CD11b, CD11c, CD3, CD4, CD8 and CD49b. The dashed line indicates the values from healthy control mice without colitis induction. These results are representative of 3 independent experiments with 5 mice per group. Unpaired t test or Mann-Whitney non-parametric t tests were used to determine if differences attained significant response. *p < 0.05. ATO, Atorvastatin; DSS, Dextran sodium sulfate; MLN, Mesenteric lymph nodes.
PPAR-α Expression Correlates to Disease Severity
Lovett-Racke et al. showed that Th2 cytokine production may be elicited by activating PPAR-α that was sufficient to improve the EAE (30). Then, we investigated if mRNA expression of Ppara was modified by ATO treatment. In fact, Ppara expression was increased in colon of mice treated with ATO compared to vehicle-treated group (Figure 5A). Moreover, our experiments and in silico analyzes using normalized GEO datasets (GSE22307) showed that Ppara expression was decreased in the colon of DSS-exposed mice (Figures 5A, B, respectively). Similar results were found in patients with UC and CD (Figures 5C, E) using GEO datasets (GSE75214/GSE87466). Furthermore, while experimental colitis upregulated inflammatory genes in the colon, including Tnfa, Il6 and Ifng (Figure 5B), UC patients also upregulated IL17A and IL10, followed by a slight increase in IL4 and mevalonate kinase (MVK) expression (Figure 5D). The pathway enrichment analysis confirmed that IL-17 signaling pathway is upregulated in UC patients (Figure 5C). Importantly, downregulation of Ppara was accompanied by a reciprocal decrease of Ppargc1a (Figure 5B), a gene that encodes the transcription factor peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α and highly associated with PPAR-α functions (Figure 5B). PPARGC1A was not only decreased in UC and CD patients but also had a strong correlation with PPARA expression (Figures 5F, J, K). To support our hypothesis, we also found that other PPARA-related genes were downregulated in both UC and CD patients, such as PPARGC1B, ACOX and HMGCS2, which codify PGC-1β, and the enzymes Acyl-CoA oxidase 1 and 3-Hydroxy-3-Methylglutaryl-CoA Synthase 2, respectively (Figures 5G-I).
Figure 5 PPAR-α expression is decreased in the inflamed intestine and restored by ATO treatment. (A) C57BL/6 WT mice were exposed to 3% (w/v) DSS in drinking water for 6 days. Mice received oral treatment once daily for three days (days 3-5) with saline (vehicle) or 10 mg/Kg/day ATO. Middle colon fragments were used for qRT-PCR analysis and the differential expression of mRNA was defined by the formula 2-ΔΔCt. The dashed line indicates the values from healthy control mice without colitis induction. These results are representative of 2 independent experiments with 3-4 mice per group, in each experiment. (B) Data were obtained from available dataset under GEO accession GSE22307. Volcano plot shows upregulated (red) and downregulated genes (blue) significantly and differentially expressed at an adjusted p-value cutoff of 0.05. (C). Differentially expressed genes were obtained from GSE75214 and GSE87466 datasets. They were filtered by either logFC<-1 (downregulated genes) or logFC>1 (upregulated genes) and by adjusted p value<0.05. Then, KEGG Pathway enrichment analysis was performed on the up and downregulated genes using the Metascape software. (D) Heatmap showing the relative colonic PPARA expression between healthy individuals and UC patients (GEO accession GSE75214 and GSE87466), and between responder or non-responder UC patients to infliximab therapy (GEO accession GSE16879 and GSE12251). The expression of inflammatory genes as well as enzymes related to both PPAR signaling and mevalonate pathways in the colon are also depicted. Expression data were preprocessed using a log2 transformation and normalization approach. (E–I). PPARα-related gene expression from colon of healthy, CD and UC patients (GEO accession GSE75214). Expression data were preprocessed using a log2 transformation and normalization approach. (J, K). Correlation analyzes between PPARA and PPARGC1A genes using data from GSE75214 and GSE87466 datasets. Based on Kolmogorov-Smirnov normality test, we selected either Pearson or Spearman analysis for the correlation analyzes. *p<0.05. Unpaired t tests or Mann-Whitney non-parametric t test were used for 2 groups, while ANOVA test was used for 3 or more groups followed by Tukey comparison or Kruskal–Wallis (Dunns) test to determine if differences attained significant response. ACOX, Acyl-CoA oxidase 1; CT, control; DSS, Dextran sodium sulfate; HMGCS2, 3-Hydroxy-3-Methylglutaryl-CoA Synthase 2; MVK, Mevalonate kinase; NR, Non-responder to Infliximab therapy; PPAR-α/Ppara/PPAR, Peroxisome proliferator-activated receptor alpha; Ppargc1a/PPARGC1A, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PPARGC1B, Peroxisome proliferator-activated receptor gamma coactivator 1-beta; R, Responder to infliximab therapy; UC, Ulcerative colitis.
Interestingly, the comparison between UC patients’ responders and non-responders to infliximab therapy, a gold standard drug to treat moderate to severe disease (31), showed that treatment responsiveness was directly associated with the upregulation of PPARA, IL4, PPARGC1A and MVK (Figure 5D). As expected, infliximab-responsive UC patients downregulated the expression of inflammatory genes in the gut compared to non-responders (Figure 5D). Altogether, these results suggest that one of the non-canonical mechanisms mediated by ATO could be the modulation of PPAR-α signaling pathway.
Genetic Deletion of PPAR-α Abrogates the Beneficial Effects of ATO In Vitro and In Vivo
To verify whether PPAR-α mediates the improvement found in ATO-treated group, Ppara-/- mice were subjected to the same protocol of colitis induction and treatment. The Ppara deletion abrogated the amelioration induced by ATO in DSS-exposed mice (Figure 6A). Moreover, ATO-treated Ppara-/- mice did not decrease the number of total circulating leukocytes nor lymphocytes, while the reduction of blood monocytes seemed to be partially dependent on PPAR-α in ATO-treated mice (Figures 6B, C). Importantly, Ppara-/- mice treated with ATO were also unable to increase the IL-4 production in the colon as compared to WT mice treated with ATO, although they slightly decreased IL-17 (Figures 6D, E, respectively). In accordance with the in vitro results on ATO-induced IL-10 production, ATO-treated WT mice also increased IL-10 levels in the gut following therapy, while Ppara-/- mice showed a modest cytokine augment (Figure 6F).
Figure 6 PPAR-α deficiency abrogates the ATO-mediated effects in both experimental colitis and ConA-stimulated splenocytes in vitro. (A) WT and Ppara-/- mice were exposed to 3% (w/v) DSS in drinking water for 6 days. Mice received oral treatment once daily for three days (days 3-5) with saline (vehicle) or 10mg/Kg/day of ATO. In (B) total number of circulating white blood cells (leukocytes) and (C) differential leukocyte counts. (D-F) Quantification of IL-4 (D), IL-17 (E) and IL-10 (F) levels in colon tissue lysates. (G–I) Splenocytes from C57BL/6 WT and Ppar-/- naïve mice were stimulated with 3 µg/mL ConA and concomitantly treated with 5 or 10 µM of ATO for 72h. IL-6 (G) and IL-10 (H) cytokines quantification in the supernatant was performed by ELISA. The proliferation of CFSE-stained cells was evaluated by flow cytometry and expressed as proliferation index (I). The dashed lines indicate values from healthy control WT or Ppara-/- mice without colitis induction or unstimulated cells. These results in A-F are representative of 3 independent experiments with 5 mice per group. The data from graphs G-I are representative of 2 independent experiments that were performed, each one, with splenocytes pooled from 3 WT or Ppara-/- mice. ANOVA test was used followed by Tukey comparison or Kruskal–Wallis (Dunns) test to determine if differences attained significant response. *p < 0.05. ATO, Atorvastatin; ConA, Concanavalin A; DSS, Dextran sodium sulfate; LP, Lamina propria; MLN, Mesenteric lymph nodes; PPAR-α/Ppara, Peroxisome proliferator-activated receptor alpha; WT, wild type.
Finally, ConA-stimulated Ppara-/- splenocytes did not respond to 5 or 10 μM ATO as seen by the maintenance of increased IL-6 levels and the reduced ability to produce IL-10 after treatment (Figures 6G, H, respectively). However, ConA-stimulated Ppara-/- splenocytes had similar proliferation index compared to WT cells when treated with 5 and 10 μM ATO (Figure 6I), indicating that cell proliferation regulated by ATO is independent on PPAR-α functions.
Altogether, our results suggest that the anti-inflammatory role of ATO in colitis seems broadly dependent on the PPAR-α effects and colonic PPAR-α expression is a potential candidate to predict the outcome of IBD treatment.
Current treatments for IBD are focused on the control of inflammatory responses. For this purpose, different strategies and classes of anti-inflammatory or immunosuppressive drugs are used to treat both CD and UC. Consequently, most of patients become either immunosuppressed or unable to restrain secondary infections, besides the occurrence of non-responsiveness to the treatments. This refractoriness to drugs contributes to persistent inflammation, increasing the severity of disease. Thus, more efficient therapies have been investigated thoroughly the last decades. Despite the positive effects of statins in the outcome of IBD are not considered as a recent discovery (16–18, 32, 33), their underlying mechanisms remain poorly understood.
Here, we described that ATO treatment improved several clinical aspects of experimental colitis and promoted a shift from Th17 responses towards to Th2 in the gut of mice exposed to DSS. This shift to Th2 immune response has been described as an important mechanism to ameliorate and prevent several inflammatory conditions. Nath et al. observed that lovastatin treatment induced IL-4 production and GATA3 stabilization in Th2 cells, improving the outcome of EAE (34). Indeed, our experiments and in silico analyses revealed, respectively, that both Il4 expression and IL-4 production at protein level were increased in the colon of ATO-treated mice, while responsiveness to biological therapy was associated with increased IL4 expression over IL17, IFNG and other pro-inflammatory genes. Furthermore, the increased activity of eosinophils in colonic LP corroborated the Th2-skewed response after ATO therapy since they are source of IL-4 production in the gut and closely associated to Th2 cells (35).
Lovett-Racke et al. suggested that PPAR-α activation mediates IL-4 synthesis to prevent clinical signs of EAE (30). These results urged us to investigate if PPAR-α was related to the positive effects mediated by ATO therapy. We found that both experimental mice and IBD patients in fact decreased the colonic PPAR-α expression and the full deletion of Ppara abrogated the ATO-induced clinical improvements. Importantly, ATO treatment was able to increase the colonic Ppara levels in colitis-induced mice, while infliximab-responder UC patients showed an augmented PPARA expression compared to non-responder UC patients, suggesting that PPAR-α can be a promising predictive biomarker for clinical responsiveness.
Although we suggest that both infliximab and ATO treatments are associated with PPAR-α upregulation and signaling, these medicines have significantly different pharmacological characteristics and clinical implications. Infliximab is an effective chimeric IgG1 anti-TNF-α monoclonal antibody used to treat moderate to severe IBD patients (31). However, this biological agent has several limitations, including high rates of unresponsiveness and drug discontinuation mainly because of antibodies’ production against infliximab or severe adverse effects (31). ATO, in turn, is a relatively safe drug, orally administrated, economically advantageous, accessible, and less toxic than other drugs (36), increasing the patient adherence. Indeed, our study showed that even with a damaged intestine, ATO was effective in restraining the colitis progression indicating that drug absorption and its consecutive bioavailability were probably not affected. It is also important to highlight that DSS is a chemical model of colitis, but offer several advantages over other experimental methods, including its use to test new treatment options for IBD (37).
Considering the analysis of other genes that could be associated to PPAR-α signaling, mice with experimental colitis, CD and UC patients downregulated Ppargc1a/PPARGC1A expression. The product of this gene is PGC-1α, a protein involved with lipid metabolism and mitochondrial biogenesis (38). Infliximab-responder UC patients upregulated PPARGC1A expression, suggesting that cell metabolism and, consequently, the cell function can be restored after biological treatment. We also evaluated the expression of other PPAR-α-related genes in both UC and CD patients, including PPARGC1B, ACOX (39) and HMGCS2 (40), all of which were downregulated, indicating that PPAR-α pathway is impaired in IBD, and confirmed by pathway enrichment analysis. The upregulation of MVK in infliximab-responder UC patients, an important enzyme of mevalonate pathway, also indicates that cholesterol synthesis inhibition is not essential for better outcomes of disease, corroborating our hypothesis that ATO plays cholesterol-independent anti-inflammatory roles.
In addition to the immunomodulatory capacity of statins, these drugs also present an immunosuppressive activity, which could be explained by its effects on lowering the number of cells in both gut infiltrate and circulating leukocytes, which were then abrogated in the absence of PPAR-α. In accordance, statins have also been successfully used in cardiac transplantation to reduce the occurrence of organ rejection and hemodynamic disturbances besides the effects of increasing survival rates (41, 42). Additionally, the decreased number of circulating lymphocytes and monocytes could be a consequence of a described action of statins over both LFA-1 and MCP-1, respectively, impairing their homing and recirculation (43, 44). Furthermore, although we did not observe differences in the activity of colonic macrophages (NAG) between vehicle- and ATO-treated mice in our study, we were not able to determine if the increased local levels of IL-4 induced macrophage polarization to a predominant anti-inflammatory M2 phenotype.
Importantly, while several studies have shown that PPAR-α activation is important to decrease the colitis severity in several experimental models (45–48), Qi et al. observed that Ppar-/- mice as well as those treated with fenofibrate were protected from TNBS- and DSS-induced colitis, and Salmonella Typhi-induced intestinal inflammation (49). We hypothesized that these discrepancies could be a result of different background strains that can cause distinct metabolic response and microbiome composition as previously described (50).
ATO treatment significantly increased the frequency of CD11b-CD11c+ DCs in the MLN, while increased CD11b+CD11c+ DCs in the spleen. Studies show that CD11b-CD11c+ DCs promotes CD8+ T cell responses (51), corroborating the increased frequency of CD8+ T cells observed in both spleen and MLN of ATO-treated mice. Cytotoxicity induced by CD8+ T cells is important to restrain intestinal inflammation during colitis and requires direct interaction with B cells (52). The absence of CD8+ T lymphocytes increases intestinal inflammation. Current metabolic approaches also show that PPAR-α is important to preserve CD8+ T lymphocytes function (53).
The in vitro evaluation showed that ATO plays anti-inflammatory role by two different mechanisms independent on cell death, but dependent on drug concentration, i.e., whereas low dose increases regulatory cytokines such as IL-10, high dose decreases both proliferation and proinflammatory activity in cells. Both IL-6 and IL-10 play an important role in IBD. While the former is increased in IBD and closely related to the differentiation of Th17 cells (54), the lack of IL-10 predisposes to IBD development (55, 56). Although statins have been associated with a decrease in pro-inflammatory cytokines and IL-10 increase (57, 58), no study so far showed their dependency on statin concentration. Moreover, PPAR-α agonists have been described to decrease IL-6 levels in the gut of DSS-exposed mice (45). However, the association between PPAR-α and IL-10 have not been deeply investigated yet, and our study is original by showing this connection not only in vitro, but also in vivo, thus requiring further investigation. Thus, our experimental results lay at conjunction to in silico analysis, indicating that PPAR-α signaling mediates ATO mechanisms that could be explored through clinical managements.
Finally, our study had some limitations regarding the impossibility to perform experiments to demonstrate the direct relationship between ATO and PPAR-α. Even though, previous studies indicate that ATO promotes both upregulation and activation of PPAR-a and that its metabolic and anti-inflammatory effects are mediated by COX-2 enzyme and protein kinase C (PKC) signaling pathway (21, 59, 60). Therefore, the determination of the underlying mechanism of statin-induced modulation of PPAR-a expression and activity, besides the experimental confirmation of the bioinformatic analyses should be performed in future studies of the group.
In conclusion, our results suggest that PPAR-α is essential to the clinical benefits of ATO treatment and it has potential as a predictive biomarker of therapy responsiveness in IBD.
Data Availability Statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/geo/, GSE75214; https://www.ncbi.nlm.nih.gov/geo/, GSE87466; https://www.ncbi.nlm.nih.gov/geo/, GSE16879; https://www.ncbi.nlm.nih.gov/geo/, GSE12251; and https://www.ncbi.nlm.nih.gov/geo/, GSE22307.
The animal study was reviewed and approved by Ethics Committee of the University of São Paulo - campus Ribeirão Preto (protocol number 10.1.499.53.8).
PB designed the project, acquired the data, interpreted and assessed the data, and wrote the manuscript. HS-C provided technical support and performed a critical revision of the manuscript. VN provided significant technical support. MD-S, VA, GB, and CR provided technical support. BG assisted with bioinformatics analysis. JC performed the histological analysis. AN contributed to the project design. CC contributed to the project design, checked the statistical analysis, and edited the manuscript. All authors contributed to the article and approved the submitted version.
The research leading to these results has received support from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant numbers 310174/2016-3, 309583/2019-5, 141050/2013-6, 482390/2013-1, 311882/2013-7) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; grant numbers 2010/20162-7, 2013/11042-6, 2017/08651-1 and 2021/08404-0).
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2021.618365/full#supplementary-material
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49. Qi Y, Jiang C, Tanaka N, Krausz KW, Brocker CN, Fang ZZ, et al. PPARalpha-Dependent Exacerbation of Experimental Colitis by the Hypolipidemic Drug Fenofibrate. Am J Physiol Gastrointestinal Liver Physiol (2014) 307(5):G564–73. doi: 10.1152/ajpgi.00153.2014
50. Fontaine DA, Davis DB. Attention to Background Strain Is Essential for Metabolic Research: C57BL/6 and the International Knockout Mouse Consortium. Diabetes (2016) 65(1):25–33. doi: 10.2337/db15-0982
51. Turley SJ, Fletcher AL, Elpek KG. The Stromal and Haematopoietic Antigen-Presenting Cells That Reside in Secondary Lymphoid Organs. Nat Rev Immunol (2010) 10(12):813–25. doi: 10.1038/nri2886
52. McPherson M, Wei B, Turovskaya O, Fujiwara D, Brewer S, Braun J. Colitis Immunoregulation by CD8+ T Cell Requires T Cell Cytotoxicity and B Cell Peptide Antigen Presentation. Am J Physiol Gastrointestinal Liver Physiol (2008) 295(3):G485–92. doi: 10.1152/ajpgi.90221.2008
53. Zhang Y, Kurupati R, Liu L, Zhou XY, Zhang G, Hudaihed A, et al. Enhancing CD8(+) T Cell Fatty Acid Catabolism Within a Metabolically Challenging Tumor Microenvironment Increases the Efficacy of Melanoma Immunotherapy. Cancer Cell (2017) 32(3):377–91.e9. doi: 10.1016/j.ccell.2017.08.004
54. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B. TGFbeta in the Context of an Inflammatory Cytokine Milieu Supports De Novo Differentiation of IL-17-Producing T Cells. Immunity (2006) 24(2):179–89. doi: 10.1016/j.immuni.2006.01.001
55. Rubtsov YP, Rasmussen JP, Chi EY, Fontenot J, Castelli L, Ye X, et al. Regulatory T Cell-Derived Interleukin-10 Limits Inflammation at Environmental Interfaces. Immunity (2008) 28(4):546–58. doi: 10.1016/j.immuni.2008.02.017
56. Tagore A, Gonsalkorale WM, Pravica V, Hajeer AH, McMahon R, Whorwell PJ, et al. Interleukin-10 (IL-10) Genotypes in Inflammatory Bowel Disease. Tissue Antigens (1999) 54(4):386–90. doi: 10.1034/j.1399-0039.1999.540408.x
57. Stumpf C, Petzi S, Seybold K, Wasmeier G, Arnold M, Raaz D, et al. Atorvastatin Enhances Interleukin-10 Levels and Improves Cardiac Function in Rats After Acute Myocardial Infarction. Clin Sci (Lond) (2009) 116(1):45–52. doi: 10.1042/CS20080042
58. Rezaie-Majd A, Maca T, Bucek RA, Valent P, Muller MR, Husslein P, et al. Simvastatin Reduces Expression of Cytokines Interleukin-6, Interleukin-8, and Monocyte Chemoattractant Protein-1 in Circulating Monocytes From Hypercholesterolemic Patients. Arterioscler Thromb Vasc Biol (2002) 22(7):1194–9. doi: 10.1161/01.ATV.0000022694.16328.CC
59. Roglans N, Sanguino E, Peris C, Alegret M, Vazquez M, Adzet T, et al. Atorvastatin Treatment Induced Peroxisome Proliferator-Activated Receptor Alpha Expression and Decreased Plasma Nonesterified Fatty Acids and Liver Triglyceride in Fructose-Fed Rats. J Pharmacol Exp Ther (2002) 302(1):232–9. doi: 10.1124/jpet.302.1.232
60. Yano M, Matsumura T, Senokuchi T, Ishii N, Murata Y, Taketa K, et al. Statins Activate Peroxisome Proliferator-Activated Receptor Gamma Through Extracellular Signal-Regulated Kinase 1/2 and P38 Mitogen-Activated Protein Kinase-Dependent Cyclooxygenase-2 Expression in Macrophages. Circ Res (2007) 100(10):1442–51. doi: 10.1161/01.RES.0000268411.49545.9c
Keywords: Inflammatory bowel diseases, ulcerative colitis, crohn’s disease, therapy, statins, dextran sodium sulfate
Citation: Basso PJ, Sales-Campos H, Nardini V, Duarte-Silva M, Alves VBF, Bonfá G, Rodrigues CC, Ghirotto B, Chica JEL, Nomizo A and Cardoso CRB (2021) Peroxisome Proliferator-Activated Receptor Alpha Mediates the Beneficial Effects of Atorvastatin in Experimental Colitis. Front. Immunol. 12:618365. doi: 10.3389/fimmu.2021.618365
Received: 16 October 2020; Accepted: 20 July 2021;
Published: 09 August 2021.
Edited by:Mats Bemark, University of Gothenburg, Sweden
Reviewed by:Li Yang, East Carolina University, United States
Makoto Makishima, Nihon University, Japan
Copyright © 2021 Basso, Sales-Campos, Nardini, Duarte-Silva, Alves, Bonfá, Rodrigues, Ghirotto, Chica, Nomizo and Cardoso. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Cristina Ribeiro de Barros Cardoso, email@example.com


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A Colorado lawmaker is calling for change to the state's civil forfeiture laws after Denver7 Investigates uncovered cases where people lost money seized from them during arrests even though they were not convicted.
"Civil forfeiture is set up to ensure that if the bad guy is using stuff for the wrong reasons, they shouldn't be able to keep that, if it's within the commission of a crime," said Republican state senator Kevin Lundberg. "[But] there are a couple of loopholes that are in there where the authorities can take the property and the person who is not convicted, but is simply accused, may lose that."
Edwin Couse is one of those who lost property during an arrest for which he was never convicted. In 2013, Couse and his wife started a membership organization to distribute medical marijuana. He told Denver7 Investigates he set it up with the guidance of his lawyer, and believed it was all legal, but it still became the target of police.
"We're sitting there, it's first thing in the morning, we're just drinking our coffee," Couse remembered. "They knock on the door, they say it's the police. I open the door and then they kick it open really, and they barge their way in. If you've never experienced an arrest of that nature where there's guns in your face and things like that, it is very scary."
The district attorney charged Couse with possessing and distributing marijuana. During the arrest, Couse said police took cell phones, cameras and about $1,800 in cash. The following year, the district attorney dismissed all of the charges "in the interest of justice." But Couse said he never saw any of the cash or items seized from him that day again.
"What did they return to you?" Denver7 Investigates asked Couse.
"Nothing, absolutely nothing," he responded.
The law allows police to take cash and property like cars during arrests if they believe the property is the product of a crime. The district attorney then begins two separate processes: prosecuting the suspect in criminal court, and seizing the money in civil court.
While one section of the law says property should not be forfeited unless and until the suspect is convicted, it does allow for exceptions.
"The civil process is totally separate from the criminal action," said Lamar Sims, chief deputy district attorney in Denver. "The law requires that the civil action be filed within a certain time after law enforcement takes custody or starts the civil process."
Sims said the law typically does not allow his office to wait for a criminal case to be resolved because it is required to file civil forfeiture lawsuits within 63 days of seizing the property. The lawsuits give suspects 21 days to respond. If they do not, the prosecutor can move for summary judgment -- closing the civil case and distributing the money, typically before the criminal case has ever gone before a jury.
Couse said he did not respond to the civil case and he believes he was in jail at the time. He said he believed it would be too costly to hire an attorney to fight the case.
"It would cost me more than the $1,800, I can guarantee you that," Couse said.
"I don't like that. I don't think Colorado law should be set up working that way," Sen. Lundberg said. "Justice from the civil government side should be punishing the wrongdoer but holding the citizen innocent until proven guilty."
When Marcus Cosby was arrested on drug charges in 2010, police seized $1,500 in cash he said belonged to his mother. Cosby said he filed a handwritten response to the city's forfeiture lawsuit, but he filed too late.
"I fought the criminal charges for about a year or so and eventually all the charges were dropped," Cosby said. "All the original charges were dropped from when the money was confiscated, so where's my money?"
Cosby's civil case was closed on summary judgment when he did not respond on time to the filing. He had a history of drug convictions and he believes authorities used his past against him to keep his cash.
"A lot of people are scared to go after the police," Cosby said. "They feel like that it's big force and they can't compete against it. ... But it's kind of like when you're in a situation where you don't have anything to risk, and you have everything to lose, and you try to get what belongs to you ... it really doesn't matter."
Sims encouraged anyone who feels they have had money or property confiscated unjustly in Denver to call the district attorney's office and ask for a review of their case.
Sims emphasized that people can and do avoid losing their property before the conclusion of their criminal case by simply filing a response to the civil action within the required 21-day period.
In Denver, much of the money confiscated from suspects goes into a fund for prosecutor and police training and equipment. Lundberg said a 2015 bill to reform civil asset forfeiture laws met with resistance from law enforcement.
"They need to come and tell us why the legislation is not going to work right, but I was less than pleased with the attitude I heard. Because they said: 'well, we give them due process.' Which simply means we stick to the procedures that are in place. But if the end result is the citizen who was not convicted of a crime loses their property, that's not right. That needs to be cured. They push back because they say 'Look at all the neat stuff we got from these takings,'" Lundberg said.
City records show the Denver Police Department has used confiscation funds for everything from personal protective equipment to gun lockers to hostage negotiation training. But Denver7 Investigates also uncovered more than $120,000 in confiscation spending by the DPD's media relations team, including buying high-end video editing equipment, advertising for the department's Facebook page, and paying close to $2,500 to submit entries to the local Emmy competition for department-produced videos.
Nick Rogers, the president of the Denver Police Protective Association, called the Emmy entries a misuse of funds which were earmarked for police training. DPD deputy chief Matt Murray justified the spending in an interview with Denver7 Investigates.
"I think the community would agree ... using the proceeds of crime to become the gold standard in communication by law enforcement is truly, I think, a great thing," Murray said.
"Just on the face of it, I don't think it makes sense," Lundberg said when asked about DPD's Emmy expenditures. "I'll let the people decide whether they think that's appropriate or not."
The day after Denver7's initial investigation aired, the department revealed it was spending another $3,230 on entries and memberships for the 2016 Emmys. This time, though, the department said it would not be using public funds and the nonprofit Denver Police Foundation would be paying the bill. Taxpayers did however pay $650 in January of 2016 for DPD to enter its videos into the Edward R. Murrow Awards, a journalism competition.

domain: politics


No. The O2 sensor is only in effect once the computer goes into closed loop mode (usually after engine is up to temperature)
A defective O2 sensor will not prevent the engine from starting. The engine will start and run albeit not as it should.
Yes it can cause a no start condition.
From a cold start, yes it can.
It's called VATS bypass and you can get them on eBay cheap.
I have a 240 and with the knock sensor unplugged it will still start so I would have to say no.
yes this will stop car from starting. a bad cam sensor will also stall engine at any speed.the sensor is easy to replace.
yes aas no pulse signal would be sent there for no start.
A throttle position sensor might keep the engine from starting. Any number of things can cause a vehicle not to start. The starter and the gas line sensors are also a possible culprit.
Yes. It can cause hard starting on some vehicles (fuel pulse out of time) and no-start due to no fuel or no spark in others.
"Yes. its a maf sensor (mass air flow sensor) and it will not let it start it will mostly just crank n crank over or start for a min with spiradic idling n shut off" Actually, there is such thing as a MAP sensor (manifold absolute pressure) but in some vehicles (especially Chrysler-Daimler) i believe, it can make the vehicle not start.
The MAP sensor - another frequent cause of stalling or non-starting. This measures the air pressure in the manifold. You can test it - if your car won't start, just unplug the electrical connection from the MAP sensor. If it starts, replace the sensor. MAP sensor details. (It may act as though the fuel pump is not working properly - starting, then quickly dying).
Replace the engine coolant temp sensor.
No it will not start need to change the cam sensor & crank sensor!
cold start sensor or weak fuel pump
Symptoms of a bad crank sensor is hard starting, no starting or rough idle. If you're able to start the engine, and it runs for a few minutes or seconds and then dies out, most likely it's a bad crank position sensor. Also, there may be no spark in some of the spark plugs.
Fuel pressure should be around 40. If the injectors are not getting power it will not start. Start with a crank sensor first.
i am looking were the cooled start sensor is on a 94 Saturn is at
A bad camshaft sensor can cause intermittent starting. The car may just shut off while driving. It also may not start or could shudder and backfire.
Yes it does have a cold start sensor. It is located on top of a throttle body.
It is possible that the 1992 Aerostar has a faulty distributor cap. It is likely that moisture is getting into the housing of the distribution and preventing it from starting.
Crank angle sensor or coolant temprature sensor are the two favourites that will cause a non start
i had something like this happen - and it was the ignition control module located in the distributor
Not starting when hot, or stalling ang bucking after driving or idleing in hot weather. pour cold water on sensor after no start condition to test it ( on back of head )
Yes. Cam sensor will determine the rotation of the camshaft, and if it does not sense it 'turning' the sensor will not allow the engine to start (it protects damaging the engine). Faulty camshaft or crankshaft sensors will stall engine and prevent restart until replaced.


text: Increased βcatenin activity correlates with leukemia stem cell expansion and disease progression in persistent myeloid leukemia (CML). of Gsk3β and consequent stabilization of βcatenin in these cells. In keeping with this Bcr-abl+ cells exhibited a Fap1-reliant upsurge in βcatenin activity. Our research identified Pazopanib HCl Fap1-reliant Gsk3β inactivation being a molecular system for elevated βcatenin activity in CML. treatment of Fas-resistant cells with this peptide boosts awareness to Fas-induced apoptosis (2-4). Fap1 also interacts with various other protein including RapGEF6 and adenomatous polyposis coli (Apc) (5 6 Fap1 interacts using the C termini of the companions through the same domains that mediates Fas connections. The functional need for these Fap1 connections is unidentified and Fas may be the just known substrate for Fap1-PTP activity. Chronic myeloid leukemia (CML) is normally seen as a translocations regarding chromosomes 9 and 22 that generate the Bcr-abl oncoprotein. Prior investigations characterized the CML leukemia stem cell (LSC) being a granulocyte-monocyte progenitor (GMP) with an increase of βcatenin activity (7 8 These researchers found a link between raising βcatenin activity in the bone tissue marrow and disease development in individual CML (7). The individual CML LSC can be characterized by reduced appearance from the interferon consensus series binding proteins (Icsbp generally known as interferon regulatory aspect 8) (9)3. In murine bone tissue marrow transduction using a Bcr-abl appearance vector reduces Icsbp appearance and (11-13). Re-expression of Icsbp in Bcr-abl+ murine bone tissue marrow reduces myeloproliferation and delays blast turmoil (12). Mice with gene disruption phenocopy some areas of CML recommending that Icsbp regulates focus on genes that are highly relevant to proliferation and success of CML LSCs (14 15 In keeping with this hypothesis we discovered that Icsbp represses transcription nor Wnt signaling are located consistently within this disease (18). Nevertheless we driven previously that Icsbp affects βcatenin activity by regulating the gene (13). Development arrest-specific (Gas2) is normally a calpain inhibitor and βcatenin is normally a calpain substrate (19). We discovered that elevated Gas2 appearance in Bcr-abl+ myeloid progenitor cells added to balance and activity of βcatenin (13). Within this research we investigate the hypothesis that Fap1 also stabilizes βcatenin in Bcr-abl+ cells by interfering using the Apc-Gsk3β-βcatenin complicated. Fap1 might exert this impact by preventing set up of the complicated or by inactivating (dephosphorylating) an element proteins. If this hypothesis is normally correct therapeutic concentrating on of Fap1 might boost Fas awareness and Pazopanib HCl lower βcatenin activity impacting disease development in CML. EXPERIMENTAL Techniques Proteins and Plasmids Appearance Vectors The Icsbp cDNA was extracted from Dr. Ben Zion-Levi (Technion Haifa Israel). The full-length cDNA was generated by PCR and subcloned in to the mammalian appearance vector pcDNA (Stratagene La Jolla CA) as defined (20). The Bcr-abl (p210) cDNA in the MiGR1 retroviral vector was extracted from Dr. Ravi Bhatia (Town of Hope Country wide INFIRMARY Duarte CA) and was subcloned in to the pcDNA appearance vector. The cDNA for Gsk3β was extracted from and subcloned in to the pcDNAamp appearance vector. A kind of Gsk3β with mutation of Tyr-216 to Asp was generated by PCR-based mutagenesis as defined (21). Plasmids and Reporter Constructs The TopFlash and FopFlash reporter constructs had been bought from Millipore (Billerica MA). TopFlash includes three copies of the consensus binding site for βcatenin/Tcf-Lef associated with a Pazopanib HCl minor promoter and a luciferase reporter. FopFlash is normally a similar Pazopanib HCl build but using a mutation that abolishes βcatenin/Tcf-Lef binding. Myeloid Cell Series Culture The individual myelomonocytic leukemia cell series U937 Pazopanib HCl (22) was extracted from Andrew Kraft (Hollings Cancers Center Rabbit Polyclonal to OR4F4. Medical School of SC Charleston SC). Cells had been maintained as defined (21). Murine Bone tissue Marrow Culture Pet research were performed regarding to a process approved by the pet Care and Make use of Committees of Northwestern School and Jesse Dark brown Veterans Affairs INFIRMARY. Bone tissue marrow mononuclear cells were extracted from the femurs of Icsbp or WT?/? C57/BL6 mice. Sca1+ cells had been separated using the Pazopanib HCl Miltenyi magnetic bead program according to.

domain: biology


Research Output per year
Correlation between properties of oxide compounds formed on contact surfaces of cutting tools and cutting tool wear resistanceNesterenko, V. P., Lasukov, A. A. & Retyunskiy, O. Y., 1 Jan 2018, Materials and Processing Technology II. Chinakhov, D. A. & Chinakhov, D. A. (eds.). Trans Tech Publications Ltd, p. 141-147 7 p. (Materials Science Forum; vol. 927 MSF).
Research output: Chapter in Book/Report/Conference proceeding › Chapter


text:
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Elizabeth A. From ring-tailed coatis to majestic giraffes, endangered tigers to giant anteaters, curious meerkats to pygmy hippos, its acre park is home to an incredible range of species, in beautiful landscaped surroundings.
I agree. Not Enabled. Listening to students and showing interest in what is being said is essential.

domain: literature


'Jumanji: Welcome to the Jungle' Has a Confusing Message for Teenagers | Hollywood Reporter
's main source of novelty is its deviation from its source material's amidship premise. While the first film — and Van Allsburg's book — followed children who play a deadly lumber game that introduces timberland animals and raw elements into the "real" world, 's version of the game, players reside the bodies of characters equal Dwayne Johnson's charismatic adventurer Smolder Bravestone and Kevin Hart's whiny zoologist Franklin "Moose" Finbar. Unfortunately, the makers of don't do thing deliberate or flat ingenious with this fun body-swap premise.
Here Are Shuri’s Best ‘Black Panther’ One-Liners
Shuri is the closest thing we feature to being in a sort out book with the Obama daughters. With her micobraids and biting one-liners, she’s the film maker princess we deserve: young, gifted, black, and already rolling her thought at your ignorance. As Wakanda’s princess, Letitia inventor is 2018’s premier true breakout.
It Follows movie meaning: Director David Robert Mitchell and star Maika Monroe on how they turned the movie's retrograde premise into the realest, scariest teen horror in years.
When you change it, people official document ask what it’s about—and on its own, the premise sounds absurdly retrograde. The movie centers on Jay (Maika Monroe, reprising final daughter duties after ), who drifts done manicured Detroit suburbs in an undetermined adolescent haze. She’s eyesight a boy, who seems sweet, if a small peculiar: At one point, on a day in a historic movie house, he dead asks to go away afterward inform out a class in a yellowed dress, refusing to say why.


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Fashionable interior design is a trend followed not solely within the U.S. but in nearly all economically buoyant nations in the world. Welcome. three. Backyard Residence Interiors oﬀers start-to-ﬁnish, full inside design & transforming. Pamela and Bill Fletcher, a husband and spouse design and building staff, have been creating beautiful spaces for over 30 years. Embellish the Within Your House Make your furnishings look Halloween-prepared. Mild up your property. Play up the pumpkins. Add just a few specimen jars to your own home. Have spooky partitions. Cowl your private home with creepy surprises. Dream Plan. Dream Plan is a free house design software program which helps you to create 3D models of your own home and in addition of your yard or backyard and is flexible in the sense you could create walls, add crops to yards, etc. Fourth, it offers an opportunity to determine persistently darkish areas or components of spaces sufficiently distant from a pure light supply so as to be thought of unlighted, or not penetrated, by a natural mild supply, e.g., an area considerably back from the daylight from a coated porch, an exceptionally deep interior house.
Nov 23, 2020. Jun 21, 2020 – Discover Natalie Shriver’s board “A Frame”, adopted by 1987 people on Pinterest. See more ideas a couple of frame house, a body cabin, structure. Framing footage of your own and your family members for decorating the walls of your workplace and home is widespread, but framing a garden and using it to brighten your wall is something very inventive and outstanding. These vertical gardens can act as a masterpiece of your front room or reception, helping you to bring a change within the look of your property. 2 days ago. House design knowledgeable and star of HGTV’s Flip or Flop and Christina on the Coast, Christina Anstead, is partnering with Spectra House Furnishings to create Christina Dwelling Designs, a simple-living way of life collection designed for today’s busy families.
The Inside Designer eight.0 is a 2D and 3D drawing software and modeler optimized for dwelling inside design with over one thousand pattern inside plans to get you inspired by. Designing your interiors gets aided with an integral library containing over 5000 frequent house gadgets like furnishings, lighting, home equipment, shelves and cupboards, windows, material textures and wall paper designs to mention just some. Residing Rooms. Our massive interior design ideas for residing rooms part is very large and is damaged down into multiple rooms which can be living areas” in nature and embrace formal dwelling rooms, family rooms, great rooms, man caves, sunrooms, dwelling libraries, house bars and game rooms. Sometimes, there may be just one portray on the wall however its size is sort of of mural proportions. As well as, the patterns used in the art work are almost at all times very highly effective and placing. Different proponents of modern inside design may choose an imposing piece of sculpture as an alternative of a painting, however in any case, these accent items are purposely meant to grab the guest’s attention.
Bathrooms are a natural setting for the Tropical Island theme. Equipment can easily be positioned in most retail and department shops. You could find everything from toothbrush holders to wall paper in quite a lot of island designs. A well-liked trend in toilet designs is the usage of glass bowl sinks. These are perfect for the Tropical Island theme and add beautiful magnificence to the room. As you add residence decorations to your house venture see and store for unique art and framing, furniture, youngsters furniture custom furniture and cabinets, rugs, lighting, Home appliances and House accessories, every little thing for your residence and Backyard. Comply with others whom share a similar model desire as yourself. Agreeable Gray. Agreeable Gray is another go-to farmhouse paint shade and is in the greige color household. A novel blend of gray and beige, Agreeable Grey works nicely in open areas, working with most decor.
Nov 22, 2020. The actress’s cool house interiors will aid you get diverse home decor ideas. From choosing the shades of the partitions to picking for wallpapers and tables and other furniture, the ornament and theme of the home are simply superb. Total makeover or redecorating of your home is needed if you’re not comfortable with your home anymore. Seek advice and opinion from family and friends about how they assume the home could be made even higher. In case you have determined to design your own Japanese garden, you’ll now need to know exactly what a Japanese garden entails. Mainly each Japanese garden ought to have stones, water and vegetation and a bridge can be essential. They are designed to be extremely pleasurable on the attention and so hold that in mind when it comes to designing the place you want all the pieces to go.

domain: artistic


text:
The Miseducation of Indigenous People
The Miseducation of Indigenous People offers readings, lesson plans, and student activities on the US government’s policies designed to “educate,” “Christianize,” and “civilize” Indigenous children. Students will interpret historical facts, photographs, reports, quotes, video clips, and more to analyze forced assimilation or “miseducation” of Indigenous children, and how they and their cultures have survived despite such adversity. Designed for high school students, the materials can be easily adapted for a college survey course.
Tags: Canada United States Lecture, Podcast, & Documentary Primary Sources Readings Teaching Guide Cultural Human Rights & Violations Native American & Indigenous Visual Culture 1800-1900 1900-1945 1945-Present
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domain: library


AUSTIN, Texas (Aug. 4, 2013) -- Daniel Northington fired rounds of 66-68-70-70--274 to win by three shots at the 2013 Austin City Men's Championship.
Northington trailed by two shots after Round 1 but made it up with solid rounds the final three days.
Brian Noonan was runner-up, shooting a final-round 67 to sneak into second place.
Justin Williams was third, five back of Northington and one ahead up Jonathan Alden and Grady Bruce. Alden led after Round 1 with a 64.


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A brand new Eastenders set has opened at Elstree Studios, it's been confirmed.
BBC Eastenders shared the exciting news with its 722,000 Instagram followers.
The account confirmed that the new Walford will allow the production teams to explore new areas on screen.
It will also mean writers, producers and directors will have greater freedom to "drive further creativity".
Soap lovers will hopefully be able to see the new set on their screens within the next couple of months.
Filming began yesterday (Tuesday, January 11) on the new and improved set, including in the famous Queen Vic.
The BBC soap opera has been based at the BBC Elstree Centre since day one in 1985.
The soap is based in the fictional London borough of Walford in the east end, with Albert Square host to countless drama over the decades.
Back in January 2014, it was announced that work was underway to prepare a new site slightly east of the then-existing location for an expanded Albert Square.
It was set to include higher quality sets with a build cost estimated to be £15m.
At the time, it was confirmed the new set would replicate Walford as it currently stands but would be 20 per cent bigger.
In 2016, the works were delayed by two years, with the earliest completion date being given as October 2020.
It's now all good to go though and the new set should be due to air in spring 2022.
The former Albert Square set was built in 1984 with an intention to be used for two years and was no longer “fit for purpose,” the BBC has said.
The soap returned to full-length episodes at the beginning of last year after a production break during the Covid-19 pandemic.
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domain: news


text:
Hai-Na-Nu "Nooligan" Saulque
is an artist, comic creator and graphic designer based in Sacramento, Ca. An old soul in a young body, HaiNaNu finds inspiration in times past. From the cartoons of his youth to films he missed by decades, from golden age comics to classic newspaper strips, you will find a creator who has immersed himself in the what he loves. His style is contemporary with a flare of nostalgia.

domain: entertainment


text:
This beautiful Aztec-inspired cuff is hand-fabricated and has a sleek, high polish look that's great for everyday wear or a night out. [Turquoise colors may vary slightly, as each stone is one-of-a-kind.]
.925 Sterling Silver
5.3mm Band Width
13.3 x 9.5mm Pear-Shaped Turquoise (x2)
Turquoise mined in Arizona
BIRTHSTONES & ANNIVERSARIES
Turquoise is the traditional birthstone for the month of December and the gem of the 11th wedding anniversary.

domain: design


text:
This was the day that I dropped of this special portrait of my friend Melissa's dog, Browning. Hated to say goodbye to this sweet dog. Melissa selected a perfect frame with the help of Victoria at Thompson's Frame Factory on Hwy 280 in Birmingham, AL. I've never been disappointed with the results there. If you go, ask for Victoria and tell her Becky sent you. She'll give you my discount!
Based in Birmingham, AL, I love creating portraits in charcoal and pastels. Join me on my creative journey, inspired by the people, places, and pets I've met along the way!


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RICHMOND, Va. — Lawmakers last month killed legislation that would give localities the ability to ban the use of coal tar sealer, a product that environmental and health agencies say can negatively impact the aquatic and human life.
A House of Delegates subcommittee voted 5-4 to table House Bill 949sponsored by Del. Kathy KL Tran, D-Fairfax.
The original bill sought to ban the sealant statewide, but an amendment gave localities the option to ban it instead. Violators of the law would have paid a fine of $250.
Coal tar sealer is a viscous black liquid sprayed or painted onto the asphalt pavement. It usually contains 20-35% coal tar or coal tar pitch, which is carcinogenic according to the US Department of Health and Human Services.
Critics of the bill say the ban is unnecessary, while supporters argue it could protect human and aquatic life from dangerous chemicals.
Chesapeake Bay Foundation senior scientist Joe Wood supported the bill. He said giving localities the option to ban coal tar sealants is a win-win situation and doesn’t negatively impact anyone.
“You get cancer reduction for kids and people, cleaner waterways, fewer fish with cancer, and better oysters,” Wood said. “There are other products that are basically the same price and work just as well.”
Of the. Tony Wilt, R-Rockingham, voted to table the bill despite the introduction of the same legislation in 2018. He was hesitant to move forward with Tran’s bill after talking with the Department of Environmental Quality about the sealant’s environmental impact, Wilt said in an email.
“Their contribution to pollution levels in our waterways was not as significant as most other problems,” Wilt said.
Wilt heard various concerns in 2018 that a ban on coal tar sealants might not be “appropriate or necessary policy”, and said he “voluntarily withdrew the bill and decided not to go ahead”.
Someone who lives next to a coal tar pavement is 38 times more likely to get cancerand much of this risk occurs during infancy, according to a report by the US Geological Survey.
Coal tar pitch is a source of a chemical called polycyclic aromatic hydrocarbon, or PAH, which has been used to seal the asphalt surfacing of parking lots, driveways and playgrounds, according to the USGS and the department. US Health and Human Services Department.
Coal tar sealer wears over time due to friction with vehicle tires. This produces fine dust that causes the particles to be washed off by rain into bodies of water, blown into the air or tracked into homes, depending on the USGS.
Exposure to PAHs, along with other contributing circumstances, can also cause DNA damage and lower IQ in children, according to studies from peer-reviewed scientific journals Neurotoxicology and teratology and Oncotarget.
Robb Archie, a third-generation sealant industry entrepreneur from Nevada who has worked with coal tar for decades, supports the bill. Archie has developed a PAH-free asphalt emulsion sealant formula. He said he has addressed similar pavement sealant bills in other state legislatures.
“It’s not a Republican or Democrat issue,” Archie said. “It’s a question of humanity.”
The chemicals have been found in Lafayette River oysters for years, although cleanup efforts have reduced levels of chemical contaminants. According to Virginia Department of Health. High levels of cancer in the Killifish have also been linked to PAH contamination in the river, according to 2020 State of the Elizabeth River Dashboard. The chemicals are serious contaminants to the Chesapeake Bay watershed, according to the Environmental Protection Agency.
Of the. Eileen Filler-Corn, D-Fairfax, praised Tran for bringing the bill to the committee’s attention.
“Just reading more about it and hearing more about it is shocking and disturbing at best,” Filler-Corn said during the committee meeting.
Brett Vassey, CEO of the Virginia Manufacturers Association, opposed the bill. The US Department of Health and Human Services did not include coal tar pavement products in its 14th report on carcinogens, Vassey said at the meeting. the HHS currently lists coal tar and coal tar pitches as carcinogens.
Vassey added that this would be the first time Virginia has delegated a science-based decision on the sale of a commercially and legally authorized product to a local government.
Washington, Minnesota and Washington, DC have banned the sale of sealants containing coal tar, along with cities and counties in Illinois, Texas, New York and Maryland, according to United States today. Maine and New York have statewide bans coming into effect soon.
According to Minnesota Pollution Control Agency.
“We take action when it matters, in recognition of protecting the health of Virginians and our wildlife,” Tran said. “I hope we can continue this conversation in the future and we have this recognition to give this tool to localities.”
By Meghan McIntyre
Capital News Service
Capital News Service is a program of the Robertson School of Media and Culture at Virginia Commonwealth University. Students in the program provide state government coverage for a variety of media outlets in Virginia.


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