Expression Of TNF-α mRNAs Research Articles

Regulation of TNF-α and NF-κB activation through the JAK/STAT signaling pathway downstream of histamine 4 receptor in a rat model of LPS-induced joint inflammation

Histamine 4 receptor (H4R) is a novel target for the pharmacological modulation of histamine-mediated immune signals during inflammatory diseases. The purpose of this study was to assess the effects of the H4R agonist 4-methylhistamine dihydrochloride (4-MeH) and antagonist JNJ7777120 (JNJ) in the inflamed rat knee. Animals were fasted for 18h before a single dose of 4-MeH or JNJ (30mg/kg) was administered intraperitoneally (i.p.), both followed by intra-articular (i.a.) injection of LPS 2h later. Blood and synovial fluid were collected after a short incubation period and TNF-α, NF-κB, and IkB-α levels were measured via flow cytometry. Additionally, we assessed the effects of H4R engagement on the expression of IL-1β, TNF-α, and NF-κB mRNAs and the protein levels of TNF-α, NF-κB, JAK-1, and STAT-3 in the inflamed knee tissue. These results revealed increased TNF-α and NF-κB expression and decreased IkB-α levels in both the LPS alone and 4-MeH treated groups in whole blood and synovial fluid. Further, IL-1β, TNF-α, and NF-κB mRNA levels were significantly increased and western blot analysis confirmed increased expression of TNF-α, NF-κB, JAK-1, and STAT-3 in both LPS and 4-MeH treatment groups. Furthermore, these increases were completely inhibited in the inflamed knee tissue of the JNJ-treated group. Thus, the inhibition of inflammatory mediators and signaling pathways by the H4R antagonist JNJ suggests the anti-arthritic importance of this molecule.

Expression of TNF-α, VEGF, and MMP-3 mRNAs in synovial tissues and their roles in fibroblast-mediated osteogenesis in ankylosing spondylitis

The aim of this study was to explore the mRNA levels of tumor necrosis factor-α (TNF-α), vessel endothelial growth factor (VEGF), and matrix metalloproteinase-3 (MMP-3) in synovial tissues in ankylosing spondylitis (AS), and to analyze the functions of these proteins in the differentiation of AS synovial tissue fibroblasts into osteoblasts (OB) and osteoclasts. Synovial tissue samples from 22 AS patients and 22 normal individuals were collected. In situ hybridization was utilized to detect TNF-α, VEGF, and MMP-3 transcripts. After counting numbers of positive cells, Spearman analysis was used to determine the correlation between transcriptional levels of the three mRNAs and the AS disease activity index (BASDAI) and the C-response protein (CRP) levels. With the addition of TNF-α, VEGF, or both factors into cultured normal synovial fibroblasts, osteocalcin (bone gla protein, BGP) secretion levels were compared. We found that expression of TNF-α, VEGF, and MMP-3 was identified exclusively in the disease group. mRNA levels were significantly positively correlated with BASDAI (r = 0.42, 0.38, and 0.47, respectively; P < 0.05) and CRP (r = 0.44, 0.34, and 0.47 respectively; P < 0.05) scores. The secretion level of BGP in normal synovial fibroblasts increased progressively with increasing concentrations of VEGF or TNF-α (P < 0.01 compared to levels before treatment). Furthermore, co-incubation using both VEGF and TNF-α significantly elevated BGP levels compared to the single addition of VEGF or TNF-α (P < 0.01). These results suggest TNF-α, VEGF, and MMP-3 might directly participate in the differentiation of fibroblasts into OBs.

Age-dependent changes in pattern recognition receptor and cytokine mRNA expression in children with otitis media with effusion

ObjectiveTo investigate age-dependent changes in expression of pattern recognition receptors (PRRs) and cytokines in pediatric OME. Materials and methodsNinety five pediatric patients with OME were divided into 4 age groups: 0–2, 2–4, 4–7, and over 7 years. The presence of bacteria, and the levels of expression of mRNAs encoding Toll-like receptor (TLRs), NOD like receptors (NLRs) and cytokines in middle ear fluid were assessed, as were their correlations with age, gender, presence of bacteria and accompanying disease. ResultsBacteria were detected in 32.6% of patients. The levels of expression of PRR and cytokine mRNAs tended to be lower in children aged 2–4 and 4–7 years. The levels of expression of TLR-2, TLR-9, NOD-1, NOD-2, IL-1, IL-6, and TNF-α mRNAs in effusion fluid were significantly lower in these two groups than in children aged 0–2 and over 7 years (p<0.05 each). The levels of expression of TLR-4, TLR-5, TLR-9, and NOD-1 mRNAs were significantly lower in culture positive than in culture negative patients (p<0.05 each). However, the expression levels of PRR and cytokine mRNAs were unrelated to gender and accompanying disease (p>0.05 each). ConclusionsThe levels of expression of PRR and cytokine mRNAs differed by age in children with OME.

Anti-inflammatory activity on mice of extract of Ganoderma lucidum grown on rice via modulation of MAPK and NF-κB pathways

Ganoderma lucidum is a popular medicinal mushroom with anti-inflammatory potential. In the present study, the aim was to determine the anti-inflammatory effect and mode of action of G. lucidum grown on germinated brown rice (GLBR) in a mouse model of colitis. It was shown that GLBR suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-stimulated macrophages and decreased the expression of COX-2, TNF-α, iNOS, IL-1β, IL-6, and IL-10 mRNAs. GLBR also inhibited activation of p38, ERK, JNK, MAPKs, and nuclear factor kappa-B (NF-κB). In a mouse model of colitis, colonic mucosal injury was evaluated using macroscopic, biochemical, and histopathological testing. Disease activity index (DAI), macroscopic score, and histological score significantly decreased upon GLBR treatment. Moreover, immunofluorescence studies indicated that DSS activates nuclear translocation of NF-κB in colon tissue, which is attenuated by GLBR extract. These findings suggest that GLBR is protective against colitis via inhibition of MAPK phosphorylation and NF-κB activation.

Stimulation of the histamine 4 receptor with 4-methylhistamine modulates the effects of chronic stress on the Th1/Th2 cytokine balance

Alterations to the immune system caused by stress have been considered to markedly increase the risk for immune-related diseases such as cancer and autoimmune disorders. We investigated the potential anti-stress effects of the histamine 4 receptor (H4R) agonist, 4-methylhistamine (4-MeH), in a murine stress model. Mice were placed in 50ml conical centrifuge tubes for 12h followed by a 12h rest. The effects of treatment with 4-MeH (30mg/kg, i.p., twice daily) for 2 days were assessed. At 2 days after physical restraint, mice were sacrificed and tissues harvested. We evaluated the effects of 4-MeH treatment on CD4+ T cell production, and intracellular IFN-γ and IL-4 expression in these cells. We also assessed IL-1β, IFN-γ, TNF-α, and IL-4 mRNA expression as well as IFN-γ, TNF-α, GITR, Ox40 and IL-4 protein expression in the spleen. The results showed that 4-MeH treatment of stressed mice results in a substantial increase in the CD4+ T cells as well as in IFN-γ production by these cells. Compared to both untreated and stressed controls. In contrast, IL-4 expression decreased significantly following 4-MeH treatment of mice. Moreover, stimulation of the H4R resulted in up-regulated expression of IL-1β, IFN-γ and TNF-α mRNAs and decreased the expression of IL-4. Western blot analysis confirmed decreased protein expression of IFN-γ, TNF-α, GITR, Ox40 and increased IL-4 in the SC group and treatment of mice with 4-MeH reversed these effects. Our results confirm the significant impact of chronic stress on T cell function and production of Th1/Th2 mediators H4R.

Toll-like receptor 2 mediates ischemia-reperfusion injury of the small intestine in adult mice.

Toll-like receptor 2 (TLR2) recognizes conserved molecular patterns associated with both gram-negative and gram-positive bacteria, and detects some endogenous ligands. Previous studies demonstrated that in ischemia-reperfusion (I/R) injury of the small intestine, the TLR2-dependent signaling exerted preventive effects on the damage in young mice, but did not have a significant effect in neonatal mice. We investigated the role of TLR2 in adult ischemia-reperfusion injury in the small intestine. Wild-type and TLR2 knockout mice at 16 weeks of age were subjected to intestinal I/R injury. Some wild-type mice received anti-Ly-6G antibodies to deplete circulating neutrophils. In wild-type mice, I/R induced severe small intestinal injury characterized by infiltration by inflammatory cells, disruption of the mucosal epithelium, and mucosal bleeding. Compared to wild-type mice, TLR2 knockout mice exhibited less severe mucosal injury induced by I/R, with a 35%, 33%, and 43% reduction in histological grading score and luminal concentration of hemoglobin, and the numbers of apoptotic epithelial cells, respectively. The I/R increased the activity of myeloperoxidase (MPO), a marker of neutrophil infiltration, and the levels of mRNA expression of tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1), and cyclooxygenase-2 (COX-2) in the small intestine of the wild-type mice by 3.3-, 3.2-, and 13.0-fold, respectively. TLR2 deficiency significantly inhibited the I/R-induced increase in MPO activity and the expression of mRNAs for TNF-α and ICAM-1, but did not affect the expression of COX-2 mRNA. I/R also enhanced TLR2 mRNA expression by 2.9-fold. TLR2 proteins were found to be expressed in the epithelial cells, inflammatory cells, and endothelial cells. Neutrophil depletion prevented intestinal I/R injury in wild-type mice. These findings suggest that TLR2 may mediate I/R injury of the small intestine in adult mice via induction of inflammatory mediators such as TNF-α and ICAM-1.

Study on the immune response to recombinant Hsp70 protein from Megalobrama amblycephala

The expression of heat shock protein 70 (Hsp70) is induced in response to many factors including high temperature, infection, metal pollutants and toxic chemicals. In this study, Megalobrama amblycephala HSP70 promoter was cloned, and characteristic heat shock elements (HSEs) were identified in the promoter region. The recombinant M. amblycephala Hsp70 protein (rMaHsp70) was expressed and purified from Escherichia coli BL21 (DE3). To evaluate in vivo immune response of rMaHsp70, we administered intraperitoneal (IP) injection, and demonstrated that rMaHsp70 stimulated M. amblycephala immune activity by inducing the expression of HSP70, HIF-1α, HSC70, CXCR4b, TNF-α and IL-1β mRNAs in liver, headkidney, spleen and gill, as well as SOD, glutathione, lysozyme and interferon alpha proteins in serum and liver. The effect of rMaHsp70 as adjuvant against Aeromonas hydrophila was assessed by injecting a mixed vaccine of rMaHsp70 and A. hydrophila (A. hydrophila/Hsp70) into M. amblycephala, and the relative percent survival (RPS) in the A. hydrophila/Hsp70 group was 75% compared to 50% in the A. hydrophila/PBS group. Furthermore, rMaHsp70 also promoted the proliferation and suppressed apoptosis in M. amblycephala fin cells (MAF) in a dose-dependent manner. Taken together, these results suggest that rMaHsp70 can induce organic immune response and improve environmental tolerance.

Topical Administration of Dual siRNAs Using Fusogenic Lipid Nanoparticles for Treating Psoriatic-Like Plaques

Psoriasis is a chronic autoimmune skin disorder with substantial negative impact on the patient's quality of life. The present study was carried out to demonstrate the efficiency of a novel topical delivery system in the transport of two siRNAs for the treatment of psoriatic-like plaques. We designed and developed a novel fusogenic nucleic acid lipid particle (F-NALP) system containing two therapeutic nucleic acids, anti-STAT3 siRNA (siSTAT3) and anti-TNF-α siRNA (siTNF-α). Novel cationic amphiphilic lipid with oleyl chains was synthesized and used in the nanocarrier system. Therapeutic efficacies of F-NALPs were assessed using an imiquimod-induced psoriatic-like plaque model. Hydrodynamic size and surface potential of F-NALPs were 102 ± 6 nm and 32.14 ± 6.21 mV, respectively. F-NALPs delivered fluorescein isothiocyanate-siRNA to a skin depth of 360 µm. F-NALPs carrying siSTAT3 and siTNF-α significantly (p < 0.05) reduced expression of STAT3 and TNF-α mRNAs and IL-23 and Ki-67 proteins compared with solution, and was superior in comparison with Topgraf(®) (GlaxoSmithKline Pharmaceuticals Limited, Maharashtra, India). Our observations demonstrate that F-NALPs can efficiently carry siSTAT3 and siTNF-α into the dermis and combination of the two nucleic acids can synergistically treat psoriatic-like plaques.

Involvement of adrenomedullin in spinal glial activation following chronic administration of morphine in rats

Adrenomedullin (AM) belongs to the calcitonin gene-related peptide (CGRP) family. Our previous studies show that chronic exposure to morphine increases spinal AM bioactivity, contributing to the development and maintenance of morphine tolerance. This study investigated the possible involvement of AM in morphine-evoked gliosis. Real-time polymerase chain reaction was performed to determine interleukin-1β (IL-1β), IL-6 and tumour necrosis factor-α (TNF-α) mRNAs in the spinal dorsal horn and cultured sensory ganglion explants. Immunohistochemistry was performed to identify spinal microglia and astrocytes. Repetitive intrathecal (i.t.) injection of morphine (20 μg) increased the expression of IL-1β, IL-6 and TNF-α mRNAs in the spinal dorsal horn. The co-administration of the selective AM receptor antagonist AM22-52 (36 μg) markedly attenuated chronic morphine-evoked increase in IL-1β and IL-6, but not TNF-α, mRNA levels. Exposure of cultured dorsal root ganglion (DRG) explants to morphine (3.3 μmol/L) for 6 days up-regulated IL-1β and IL-6 mRNA expressions. The depletion of AM gene using small interfering RNA (siRNA) approach abolished morphine-evoked increase in IL-1β and IL-6 syntheses in the cultured DRG. The blockade of AM receptors by i.t. AM22-52 also inhibited chronic morphine-evoked cell hypertrophy of microglia and astrocytes as well as an increase in OX-42 and GFAP (glial fibrillary acidic protein) immunoreactivities. Furthermore, the 6-day treatment with AM (10 μg, i.t.) induced morphological changes of microglia and astrocytes as well as an increase in IL-1β, IL-6 and TNF-α mRNA levels in the spinal dorsal horn. The present study supports the idea that up-regulation of the pronociceptive mediator AM can recruit spinal glial cells, resulting in an increase in cytokines during chronic use of morphine.

Imipramine and fluoxetine inhibit LPS-induced activation and affect morphology of microglial cells in the rat glial culture

BackgroundRecent evidence has suggested that antidepressants evoke neuroprotective and immunomodulatory effects in the brain, partly at least, by inhibiting glia activation. This study has been conducted on the lipopolysaccharide (LPS)-stimulated primary rat mixed glial cell culture in order to better recognize the influence of imipramine (a tricyclic antidepressant) and fluoxetine (a selective serotonin reuptake inhibitor) on the important balance between pro- and anti-inflammatory cytokines produced by the glial cells. Moreover, microscopic observations were made to describe the morphological alterations in the studied cell cultures exposed to the drugs. MethodsThe effect of both antidepressants on TNF-α, IL-1β and IL-10 levels was determined by ELISA. The mRNA levels of mentioned cytokines were evaluated by qRT-PCR assay. Moreover, drug influence on the LPS-stimulated level of NF-κB p65 subunit in nuclear fraction was determined by the colorimetric transcription factor assay. ResultsAfter LPS-stimulation both drugs decreased concentration of TNF-α and IL-1β in culture medium and expression of TNF-α and IL-1β mRNAs in cellular extracts. They also diminished the LPS-induced nuclear translocation of NF-κB p65 subunit. In contrast, imipramine and fluoxetine induced a few-fold weaker suppressing effect on the levels of IL-10. Parallelly to the inhibition of the LPS-induced inflammatory response, the antidepressants prevented the morphological alterations of cells elicited by LPS. Moreover, in unstimulated cultures imipramine but not fluoxetine caused transformation of microglia cells into cells with neuron-like morphology. ConclusionsImipramine and fluoxetine, by modulating glia activation, may exert anti-inflammatory effects in the CNS. It also seems that microglia cells are important target particularly for imipramine.

Inhibitory effects of Pleurotus tuber-regium mycelia and bioactive constituents on LPS-treated RAW 264.7 cells

Pleurotus tuber-regium (PTR) is an edible and medicinal mushroom found mainly in tropical and subtropical countries. In this study, we investigated the anti-inflammatory effects of PTR mycelia on RAW 264.7 macrophages. Samples of PTR mycelia were prepared by submerged fermentation. An ethanolic extract of PTR mycelia (PTRA) inhibited the production of nitric oxide (NO), TNF-α, and IL-6 in LPS-treated RAW 264.7 cells. Two compounds were purified and identified as cerevisterol (CE) and ergosta-4,6,8(14),22-tetraen-3-one (EG). Both CE and EG inhibited the production of NO, TNF-α, and PGE2 in LPS-treated RAW 264.7 cells. The expression of the iNOS and COX2 proteins was inhibited by CE and EG in a dose-dependent manner. In addition, CE and EG repressed the expression of iNOS, COX2, TNF-α, and SOCS3 mRNAs. This study revealed that an ethanolic extract of PTR mycelia exhibited anti-inflammatory activity and that CE and EG might contribute to this activity.

Mechanism of matrix metalloproteinase axis-induced neointimal growth

Tumor necrosis factor-α, platelet-derived growth factor, matrix metalloproteinases 9 and 2 have very important roles in neointimal hyperplasia, which develops after endovascular injury. However, the relationships among the four factors in inducing neointimal hyperplasia are unclear. Here, we used a mouse model of femoral arterial transluminal wire injury, and examined neointimal hyperplasia within the 28days that followed the injury. We confirmed that the neointima kept growing during the 28days, and found that expression of TNF-α and PDGF mRNAs in femoral arteries peaked within 24h after injury. However, MMP9 mRNA expression peaked 7days, and MMP2 mRNA expression peaked 28days after injury. Then, we administered exogenous TNF-α or PDGF to the peri-femoral artery following an injury, and found that exogenous TNF-α led to significantly more neointimal hyperplasia during the first 2weeks, and PDGF led to increased neointimal hyperplasia during the second 2weeks after injury. We also used the model of femoral artery injury in MMP9- or MMP2-deficient (MMP9−/− or MMP2−/−) mice. We found that neointimal hyperplasia was reduced in MMP9−/− mice during the first 2weeks after injury, and neointimal hyperplasia was reduced in MMP2−/− mice during the second 2weeks after injury. When TNF-α or PDGF was administered to the peri-femoral artery immediately after injury, TNF-α did not promote neointimal hyperplasia in MMP9−/− mice during the first 2weeks after injury but did in MMP2−/− mice, and PDGF did not promote neointimal hyperplasia in MMP2−/− mice during the second 2weeks after injury but did in MMP9−/− mice. We used an in vitro system to treat vascular smooth muscle cells (VSMCs) with TNF-α or PDGF; TNF-α induced MMP9, but not MMP2, expression at a fast reaction speed, while PDGF induced MMP2, but not MMP9, expression at a slow reaction speed. Meanwhile, TNF-α induced VSMC migration in a MMP9-dependent manner, and PDGF induced VSMC proliferation in a MMP2-dependent manner. Taken together, our studies elucidated the axis of TNF-α-MMP9-VSMC migration and PDGF-MMP2-VSMC proliferation, both of which contributed to the mechanism of neointimal hyperplasia formation.

Alleviation of alcoholic liver injury by betaine involves an enhancement of antioxidant defense via regulation of sulfur amino acid metabolism

Previous studies suggested that the hepatoprotective activity of betaine is associated with its effects on sulfur amino acid metabolism. We examined the mechanism by which betaine prevents the progression of alcoholic liver injury and its therapeutic potential. Rats received a liquid ethanol diet for 6 wk. Ethanol consumption elevated serum triglyceride and TNFα levels, alanine aminotransferase and aspartate aminotransferase activities, and lipid accumulation in liver. The oxyradical scavenging capacity of liver was reduced, and expression of CD14, TNFα, COX-2, and iNOS mRNAs was induced markedly. These ethanol-induced changes were all inhibited effectively by betaine supplementation. Hepatic S-adenosylmethionine, cysteine, and glutathione levels, reduced in the ethanol-fed rats, were increased by betaine supplementation. Methionine adenosyltransferase and cystathionine γ-lyase were induced, but cysteine dioxygenase was down-regulated, which appeared to account for the increment in cysteine availability for glutathione synthesis in the rats supplemented with betaine. Betaine supplementation for the final 2 wk of ethanol intake resulted in a similar degree of hepatoprotection, revealing its potential therapeutic value in alcoholic liver. It is concluded that the protective effects of betaine against alcoholic liver injury may be attributed to the fortification of antioxidant defense via improvement of impaired sulfur amino acid metabolism.

BF02, a recombinant TNFR2 fusion protein, alleviates adjuvant arthritis by regulating T lymphocytes in rats

To investigate the therapeutic effects of BF02 on adjuvant arthritis (AA) in rats and the regulatory effects of BF02 on T lymphocyte function. SD rats received a single intradermal injection of Freund's complete adjuvant emulsion into the right hind metatarsal footpad. After the onset of AA, the rats were injected BF02 (1, 3, or 9 mg/kg, sc) every 3 d for a total of 15 d. Intragastric administration of methotrexate (MTX, 0.5 mg/kg, every 3 d for a total of 15 d) was taken as the positive control drug. Arthritis index, swollen joint count, ankle joint histopathology, spleen histopathology and the paw radiography were used for evaluating the drug effects on AA rats. T lymphocyte function was assessed by measuring T lymphocyte cytokine levels, IL17 and TNF-α mRNA expression levels, and percentage of T lymphocyte subsets. In the AA rats, remarkable secondary inflammatory responses exhibited, accompanied by significantly higher levels of IL-1, IL-6, TNF-α, IL-17, LTα, RANKL, and MMP-13. The expression of IL17 and TNF-α mRNAs was also substantially higher than in normal rats. The percentages of CD3(+)CD4(+) and CD4(+)CD25(+) T lymphocytes were increased, whereas the percentages of CD4(+)CD62L(+) and CD4(+)CD25(+)FoxP3(+) T lymphocytes were decreased. Treatment of the AA rats with BF02 (9 mg/kg) or MTX significantly decreased the arthritis index, swollen joint count and arthritis global assessment. Moreover, both BF02 (9 mg/kg) and MTX significantly inhibited T lymphocyte proliferation, and blocked the above mentioned aberrance in T lymphocyte cytokine levels, IL17 and TNF-α mRNA expression, and percentages of T lymphocyte subsets. BF02 exerts therapeutic effects on AA rats via the regulation of T lymphocytes.

The influence of oral bacteria on tissue levels of Toll-like receptor and cytokine mRNAs in feline chronic gingivostomatitis and oral health

Feline chronic gingivostomatitis (FCGS) is an inflammatory disease of the oral cavity that causes severe pain and distress in affected cats. Treatment methods are currently very limited. The aims of this study were to assess the feline innate immune response by investigating the levels of cytokine and Toll-like receptor (TLR) mRNAs in tissue biopsies of cats with and without FCGS, and to relate this to the presence or absence of putative oral pathogens identified previously within these cats. Mucosal biopsies were collected from 28 cats with FCGS and eight healthy cats. The levels of TLR (TLR2, TLR3, TLR4, TLR7, TLR9) and cytokine (IL-1β, IL-4, IL-6, IL-10, IL-12, TNF-α, IFN-γ) mRNA was determined using quantitative PCR. In the FCGS group a statistically significant increase was seen in TLR2, TLR7, TNF-α, IFN-γ, IL-1β and IL-6 mRNA levels compared to the healthy group. In cats where Tannerella forsythia was present, statistically significant increases were seen in TLR2, TLR4, TLR7, TLR9, TNF-α and IL-1β mRNA levels compared to cats where this putative pathogen was absent. Statistically significant increases in mRNA expression were also seen in cats harbouring feline calicivirus (FCV) (TLR2, IL-1β, IL-6, IFN-γ) and Porphyromonas circumdentaria (TLR2, TLR3) compared to cats where these putative pathogens were absent. Pasteurella multocida subsp. multocida and Pseudomonas sp. did not significantly alter the expression of any TLR or cytokine mRNAs when compared to animals who tested negative for these species, while cats colonised with P. multocida subsp. septica demonstrated a statistically significant reduction in the expression of TLR7, TNF-α and IFN-γ mRNAs compared to cats free of this species. The expression of mRNA for several TLRs and cytokines is elevated in FCGS. A positive correlation was observed between clinical disease severity and the presence of FCV (p=0.001; Rho=0.58). Although the number of cats harbouring T. forsythia was low by comparison, 80% of samples in which it was present were from cases with the highest clinical disease severity. Positive correlations with clinical disease severity were seen for TLR2 (p=0.00086), TLR7 (p=0.049), TNF-α (p=0.027), IFN-γ (p=0.0015), IL-1β (p=0.004) and IL-6 (p=0.00001) mRNAs. The putative pathogens FCV and T. forsythia may be important in stimulating a host immune response to FCGS and may play a role in the pathogenesis of this disease.

Abstract 19235: Infiltrating Macrophage-Derived Angiopoietin-Like Protein2 (Angptl2) Accelerates Abdominal Aortic Aneurysm by Inducing Chronic Inflammation and ECM Degradation

Abdominal Aortic Aneurysm (AAA) is characterized by aneurysmal morphological changes with low-grade chronic inflammation of atherosclerotic aortic walls. However, molecular mechanism has not been elucidated well. We recently reported that Angptl2 mediates various chronic inflammatory conditions, such as rheumatoid arthritis, dermatomyositis, cancer, and obesity-related metabolic disorder. In this study, we examined whether Angptl2 contributes to the pathogenesis of AAA formation. Firstly, we found expression of Angptl2 and pro-inflammatory cytokines mRNA in aneurysmal lesion of AAA patients were significantly increased compared to the non-dilating lesion. Angptl2 protein was abundantly expressed in infiltrating macrophages but not expressed in other inflammatory cells. We next examined whether Angptl2 deficiency affects the AAA formation in Angptl2-knockout (KO) mice. Interestingly, on day 28 after surgery, the sizes of AAA in Angptl2 KO mice were significantly decreased compared to wild-type mice (p&lt;0.01). And expression of TNF-α, IL-1β, IL-6 and MMP-9 mRNAs in tissues from the aortic wall of CaCl 2 -treated Angptl2 KO mice was markedly decreased compared with that seen in wild-type mice. To identify the culprit cells secreting Angptl2 in AAA lesion, we transplanted bone marrow (BM) cells from Angptl2 KO mice or littermate wild-type controls into lethally irradiated wild-type recipients. The sizes of AAA in the recipients of Angptl2-deficient BM cells were significantly attenuated compared to those in the recipients of wild-type control BM cells (p&lt;0.01). Finally, to investigate whether macrophage-derived Angptl2 could alter expression levels of proinflammatory cytokines and MMP-9 in macrophages, we analyzed expression of pro-inflammatory cytokines in thioglycollate(TGC)-induced peritoneal inflammatory macrophages. Significant decreases in TNF-α, IL-1β, IL-6 and MMP-9 expression were observed in macrophages from Angptl2 KO mice compared with similarly treated wild-type mice. These results suggest that infiltrating macrophage-derived Angptl2 promotes expression of proinflammatory cytokines and MMP-9 in these cells, resulting in progression of AAA pathogenesis.

Anti-inflammatory/anti-amyloidogenic effects of plasmalogens in lipopolysaccharide-induced neuroinflammation in adult mice

BackgroundNeuroinflammation involves the activation of glial cells in neurodegenerative diseases such as Alzheimer’s disease (AD). Plasmalogens (Pls) are glycerophospholipids constituting cellular membranes and play significant roles in membrane fluidity and cellular processes such as vesicular fusion and signal transduction.MethodsIn this study the preventive effects of Pls on systemic lipopolysaccharide (LPS)-induced neuroinflammation were investigated using immunohistochemistry, real-time PCR methods and analysis of brain glycerophospholipid levels in adult mice.ResultsIntraperitoneal (i.p.) injections of LPS (250 μg/kg) for seven days resulted in increases in the number of Iba-1-positive microglia and glial fibrillary acidic protein (GFAP)-positive astrocytes in the prefrontal cortex (PFC) and hippocampus accompanied by the enhanced expression of IL-1β and TNF-α mRNAs. In addition, β-amyloid (Aβ3–16)-positive neurons appeared in the PFC and hippocampus of LPS-injected animals. The co-administration of Pls (i.p., 20 mg/kg) after daily LPS injections significantly attenuated both the activation of glial cells and the accumulation of Aβ proteins. Finally, the amount of Pls in the PFC and hippocampus decreased following the LPS injections and this reduction was suppressed by co-treatment with Pls.ConclusionsThese findings suggest that Pls have anti-neuroinflammatory and anti-amyloidogenic effects, thereby indicating the preventive or therapeutic application of Pls against AD.

Smooth Muscle-specific Expression of Calcium-independent Phospholipase A2β (iPLA2β) Participates in the Initiation and Early Progression of Vascular Inflammation and Neointima Formation

Whether group VIA phospholipase A(2) (iPLA(2)β) is involved in vascular inflammation and neointima formation is largely unknown. Here, we report that iPLA(2)β expression increases in the vascular tunica media upon carotid artery ligation and that neointima formation is suppressed by genetic deletion of iPLA(2)β or by inhibiting its activity or expression via perivascular delivery of bromoenol lactone or of antisense oligonucleotides, respectively. To investigate whether smooth muscle-specific iPLA(2)β is involved in neointima formation, we generated transgenic mice in which iPLA(2)β is expressed specifically in smooth muscle cells and demonstrate that smooth muscle-specific expression of iPLA(2)β exacerbates ligation-induced neointima formation and enhanced both production of proinflammatory cytokines and vascular infiltration by macrophages. With cultured vascular smooth muscle cell, angiotensin II, arachidonic acid, and TNF-α markedly induce increased expression of IL-6 and TNF-α mRNAs, all of which were suppressed by inhibiting iPLA(2)β activity or expression with bromoenol lactone, antisense oligonucleotides, and genetic deletion, respectively. Similar suppression also results from genetic deletion of 12/15-lipoxygenase or inhibiting its activity with nordihydroguaiaretic acid or luteolin. Expression of iPLA(2)β protein in cultured vascular smooth muscle cells was found to depend on the phenotypic state and to rise upon incubation with TNF-α. Our studies thus illustrate that smooth muscle cell-specific iPLA(2)β participates in the initiation and early progression of vascular inflammation and neointima formation and suggest that iPLA(2)β may represent a novel therapeutic target for preventing cardiovascular diseases.

Role of the junctional epithelium in periodontal innate defense and homeostasis

The junctional epithelium provides the front-line defense against periodontal bacterial infection. The migration of neutrophils into the junctional epithelium might represent a protective reaction against bacterial infections. However, neutrophils penetrate into the junctional epithelium even under sterile conditions. In this study, we analyzed and compared the number of neutrophils and the cytokine expression related to neutrophil migration in the junctional epithelium in conventional and germ-free mice. Germ-free and conventional ICR mice were used at 12 wk of age. Frozen sections were used for the detection of Gr-1, macrophage inflammatory protein-2 (MIP-2/CXCL2) and proliferating cell nuclear antigen-positive cells in the two groups of mice. Laser capture microdissection and RT-PCR analysis were used to evaluate the expression of keratinocyte-derived chemokine (KC/CXCL1), MIP-2, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) mRNAs in the two groups of mice. Morphometric examination indicated an increase in the area of the junctional epithelium upon bacterial infection. Immunohistochemical studies also detected an increased number of neutrophils in the junctional epithelium upon bacterial infection. Higher up-regulation of KC and MIP-2 were detected in the junctional epithelium of conventional mice than in germ-free mice, whereas the expression of Il-1β and Tnfα mRNAs was not affected. Junctional epithelium cells constitutively expressed several types of chemokines and cytokines and the expression of chemokines was augmented by bacterial infection. Therefore, the constitutive expression of cytokines in junctional epithelium might be related to the morphological and functional homeostasis of the junctional epithelium in addition to the defense against the bacterial infection.

Abstract 1616: Preventive effects of EGCG on diethylnitrosamine-induced liver tumorigenesis in obese and diabetic C57BL/KsJ-db/db mice

Abstract Background and aims: Obesity and related metabolic abnormalities, including insulin resistance and a state of chronic inflammation, increase the risk of hepatocellular carcinoma (HCC). Abnormal activation of the IGF/IGF-1R axis is also involved in obesity-related liver tumorigenesis. In the present study, we examined the effects of (-)-epigallocatechin gallate (EGCG), a major biologically active component of green tea, on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in C57BL/KsJ-db/db (db/db) obese mice. Methods: Male db/db mice were given tap water containing 40 ppm DEN for 2 weeks and then they received drinking water containing 0.1% EGCG for 34 weeks. Results: At sacrifice, drinking water with EGCG significantly inhibited the development of liver cell adenomas in comparison to the control EGCG-untreated group. EGCG inhibited the phosphorylation of the IGF-1R, ERK, Akt, GSK-3β, Stat3, and JNK proteins in the livers of experimental mice. The serum levels of insulin, IGF-1, IGF-2, free fatty acid, and TNF-α were all decreased by drinking EGCG. EGCG also decreased the expression of TNF-α, IL-6, IL-1α, and IL-18 mRNAs in the livers. In addition, EGCG improved liver steatosis and activated the AMPK protein in the liver. Conclusion: EGCG prevents obesity-related liver tumorigenesis by inhibiting the IGF/IGF-1R axis, improving hyperinsulinemia, and attenuating chronic inflammation. EGCG, therefore, may be useful in the chemoprevention of liver tumorigenesis in obese individuals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1616. doi:1538-7445.AM2012-1616