Apurinic/Apyrimidinic Endonuclease 1 Regulates Inflammatory Response in Macrophages

The multi-functional apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) DNA repair and redox signaling protein has been shown to have a role in cancer growth and survival, however, little has been investigated concerning its role in inflammation. In this study, an APE1 redox-specific inhibitor (E3330) was used in lypopolysaccharide (LPS)-stimulated macrophages (RAW264.7). E3330 clearly suppressed secretion of inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL-6) and IL-12 and inflammatory mediators nitric oxide (NO) as well as prostaglandin E2 (PGE2) from the LPS-stimulated RAW264.7 cells. These data were supported by the down-regulation of the LPS-dependent expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) genes in the RAW264.7 cells. The effects of E3330 were mediated by the inhibition of transcription factors nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) in the LPS-stimulated macrophages, both known targets of APE1. In conclusion, pharmacological inhibition of APE1 by E3330 suppresses inflammatory response in activated macrophages and can be considered as a novel therapeutic strategy for the inhibition of tumor-associated macrophages

Mushroom \u3ci\u3eGanoderma lucidum\u3c/i\u3e Prevents Colitis- Associated Carcinogenesis in Mice

Background: Epidemiological studies suggest that mushroom intake is inversely correlated with gastric, gastrointestinal and breast cancers. We have recently demonstrated anticancer and anti-inflammatory activity of triterpene extract isolated from mushroom Ganoderma lucidum (GLT). The aim of the present study was to evaluate whether GLT prevents colitis-associated carcinogenesis in mice. Methods/Principal Findings: Colon carcinogenesis was induced by the food-borne carcinogen (2-Amino-1-methyl-6- phenylimidazol[4,5-b]pyridine [PhIP]) and inflammation (dextran sodium sulfate [DSS]) in mice. Mice were treated with 0, 100, 300 and 500 mg GLT/kg of body weight 3 times per week for 4 months. Cell proliferation, expression of cyclin D1 and COX-2 and macrophage infiltration was assessed by immunohistochemistry. The effect of GLT on XRE/AhR, PXR and rPXR was evaluated by the reporter gene assays. Expression of metabolizing enzymes CYP1A2, CYP3A1 and CYP3A4 in colon tissue was determined by immunohistochemistry. GLT treatment significantly suppressed focal hyperplasia, aberrant crypt foci (ACF) formation and tumor formation in mice exposed to PhIP/DSS. The anti-proliferative effects of GLT were further confirmed by the decreased staining with Ki-67 in colon tissues. PhIP/DSS-induced colon inflammation was demonstrated by the significant shortening of the large intestine and macrophage infiltrations, whereas GLT treatment prevented the shortening of colon lengths, and reduced infiltration of macrophages in colon tissue. GLT treatment also significantly downregulated PhIP/DSS-dependent expression of cyclin D1, COX-2, CYP1A2 and CYP3A4 in colon tissue. Conclusions: Our data suggest that GLT could be considered as an alternative dietary approach for the prevention of colitis-associated cancer

Anti-inflammatory activity of edible oyster mushroom is mediated through the inhibition of NF-κB and AP-1 signaling

<p>Abstract</p> <p>Background</p> <p>Mushrooms are well recognized for their culinary properties as well as for their potency to enhance immune response. In the present study, we evaluated anti-inflammatory properties of an edible oyster mushroom (<it>Pleurotus ostreatus</it>) <it>in vitro </it>and <it>in vivo</it>.</p> <p>Methods</p> <p>RAW264.7 murine macrophage cell line and murine splenocytes were incubated with the oyster mushroom concentrate (OMC, 0-100 μg/ml) in the absence or presence of lipopolysacharide (LPS) or concanavalin A (ConA), respectively. Cell proliferation was determined by MTT assay. Expression of cytokines and proteins was measured by ELISA assay and Western blot analysis, respectively. DNA-binding activity was assayed by the gel-shift analysis. Inflammation in mice was induced by intraperitoneal injection of LPS.</p> <p>Results</p> <p>OMC suppressed LPS-induced secretion of tumor necrosis factor-α (TNF-α, interleukin-6 (IL-6), and IL-12p40 from RAW264.7 macrophages. OMC inhibited LPS-induced production of prostaglandin E2 (PGE₂) and nitric oxide (NO) through the down-regulation of expression of COX-2 and iNOS, respectively. OMC also inhibited LPS-dependent DNA-binding activity of AP-1 and NF-κB in RAW264.7 cells. Oral administration of OMC markedly suppressed secretion of TNF-α and IL-6 in mice challenged with LPS <it>in vivo</it>. Anti-inflammatory activity of OMC was confirmed by the inhibition of proliferation and secretion of interferon-γ (IFN-γ), IL-2, and IL-6 from concanavalin A (ConA)-stimulated mouse splenocytes.</p> <p>Conclusions</p> <p>Our study suggests that oyster mushroom possesses anti-inflammatory activities and could be considered a dietary agent against inflammation. The health benefits of the oyster mushroom warrant further clinical studies.</p

Mouse IL-2/CD25 Fusion Protein Induces Regulatory T Cell Expansion and Immune Suppression in Preclinical Models of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is associated with an IL-2-deficient state, with regulatory T cells (Tregs) showing diminished immune regulatory capacity. A low dose of IL-2 has shown encouraging clinical benefits in SLE patients; however, its clinical utility is limited because of the requirement of daily injections and the observation of increase in proinflammatory cytokines and in non-Tregs. We recently showed that a fusion protein of mouse IL-2 and mouse IL-2Rα (CD25), joined by a noncleavable linker, was effective in treating diabetes in NOD mice by selectively inducing Treg expansion. In this report, we show that mouse IL-2 (mIL-2)/CD25 at doses up to 0.5 mg/kg twice a week induced a robust Treg expansion without showing signs of increase in the numbers of NK, CD4 Foxp3 , or CD8 T cells or significant increase in proinflammatory cytokines. In both NZB × NZW and MRL/lpr mice, mIL-2/CD25 at 0.2-0.4 mg/kg twice a week demonstrated efficacy in inducing Treg expansion, CD25 upregulation, and inhibiting lupus nephritis based on the levels of proteinuria, autoantibody titers, and kidney histology scores. mIL-2/CD25 was effective even when treatment was initiated at the time when NZB × NZW mice already showed signs of advanced disease. Furthermore, we show coadministration of prednisolone, which SLE patients commonly take, did not interfere with the ability of mIL-2/CD25 to expand Tregs. The prednisolone and mIL-2/CD25 combination treatment results in improvements in most of the efficacy readouts relative to either monotherapy alone. Taken together, our results support further evaluation of IL-2/CD25 in the clinic for treating immune-mediated diseases such as SLE

GLT suppresses PhIP/DSS induced formation of colon tumors and inhibits focal hyperplasia and ACF formation.

<p>(A) Schematic of the animal treatment. The details of the treatment are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047873#s4" target="_blank"><i>Materials and Methods</i></a>. (B) Body weight of control animals (black circle), animals treated with PhIP (white square), DSS (black square), PhIP/DSS (white circle), PhIP/DSS+GLT 100 mg/kg of body weight (black triangle), and PhIP/DSS+GLT 500 mg/kg of body weight (white traingle) during the experiment. (C) H&E staining of representative samples from animal experiments described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047873#pone-0047873-g001" target="_blank">Figure 1A</a>. (D) Focal hyperplasia was evaluated by the histological analysis after H&E staining in colon tissue samples as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047873#s4" target="_blank"><i>Materials and Methods</i></a>. Results are means ± SD (n = 6–9 mice/per group). (E) ACF formation was evaluated after methylene blue staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047873#s4" target="_blank"><i>Materials and Methods</i></a>. Results are means ± SD (n = 10 foci/3 mice/per group), *p<0.05 by ANOVA.</p

GLT prevents PhIP/DSS induced formation of colon tumors.

<p>(A) Schematic of the preventive experimental protocol. The details of the treatment are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047873#s4" target="_blank"><i>Materials and Methods</i></a>. (B) H&E staining of representative samples from animal experiments.</p

Effect of GLT on PhIP/DSS-induced expression of CYP1A2, CYP3A4 and CYP3A1 in colon tissue.

<p>Quantification of (A) CY1A2, (B) CYP3A4, and (C) CYP 3A1 positive cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047873#s4" target="_blank"><i>Materials and Methods</i></a>. Box plots represent 5^th/10^th percentiles, horizontal bars represent median values, and whiskers indicate minimum to maximum values. Significant differences (*p<0.05) were observed among (A) CYP1A2: PhIP/DSS vs. control, PhIP/DSS vs PhIP/DSS+GLT 100 (B) CYP3A4: PhIP/DSS vs. control, PhIP/DSS vs PhIP/DSS+GLT 100, and PhIP/DSS vs. PhIP/DSS+GLT 100, and (C) CYP3A1: PhIP/DSS vs. control.</p

Effect of GLT on the liver function and serum glucose.

<p>Values are Mean ± S.D. (n = 6), ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase. No significant difference from the control group.</p

Effect of GLT on the PhIP/DSS induced colon carcinogenesis and inflammation.

<p>Tumor incidence are summarized using percentage of animals with tumors and compared across groups using Fisher's exact test and the Bonferonni correction for multiple comparisons: ^a p<0.001 PhIP/DSS vs control, PhIP, DSS; ^b p<0.02 PhIP/DSS+500 GLT vs PhIP/DSS.</p><p>Tumor multiplicity are summarized using mean ± SD and compared across all group using Kruskal-Wallis one way analysis of variance on ranks and the Dunn's method for the multiple comparisons: ^a p<0.05 PhIP/DSS vs control PhIP, DSS; ^b p<0.05 PhIP/DSS+500 GLT vs PhIP/DSS.</p><p>Neoplastic index is summarized using median (min, max) and compared across groups using the Kruskal-Wallis test. Comparisons of each group to control performed using Mann-Whitney U tests with significance levels adjusted using the Bonferroni correction: ^a p<0.001 control vs PhIP; ^b p<0.001 control vs PhIP/DSS.</p><p>Data for colon length summarized using mean ± SD and compared across all group using ANOVA and Dunnett's post hoc test: ^ap<0.001 control vs DSS, control vs PhIP/DSS; ^bp<0.001 PhIP/DSS vs PhIP/DSS+100 GLT, PhIP/DSS vs PhIP/DSS+500 GLT.</p