Expression Of Pro-inflammatory Proteins Research Articles

Early Enteral Stressors in Newborns Increase Inflammatory Cytokine Expression in a Neonatal Necrotizing Enterocolitis Rat Model

Inflammation in the premature intestine is a key factor that leads to the development of necrotizing enterocolitis (NEC). Activation of nuclear factor kappa B (NF-κB) and subsequent inflammation increases the severity of NEC. The aim of this study was to investigate the early temporal expression of inflammatory markers and activation of NF-κB in a neonatal rat model of NEC. Pre- and full-term newborn Sprague-Dawley rat pups were sacrificed at birth, 1.5, 4, 8, and 24 hours after receiving their first feed. Control pups were vaginally delivered and mother fed; NEC was induced by a combination of gavage feeding formula, hypoxia, and enteral lipopolysaccharide (LPS); and formula fed pups were fed every 4 hours with infant formula. Ileal tissue was collected for immunohistochemistry, real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. Serum was collected for cytokine content. Fold change of expression of inducible nitric oxide synthase (iNOS), interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), IL-10, NF-κB p65, and IκBα used RT-PCR. Data were analyzed by paired two-tailed t test, expressed as mean ± standard error of the mean, and p ≤ 0.05 considered significant. No histologic injury was evident in ileal sections. At 1.5 h, iNOS expression increased twofold over control in NEC pups (2.1 vs. 1.0, p ≤ 0.05) and remained elevated at 24 h (0.7 vs. 9.4, p ≤ 0.05). IL-1β and IL-6 reached a peak at 24 h in NEC tissue compared with control. IL-10 expression rose in NEC pups after 4 h of insult and remained elevated in formula and NEC stressed pups. Coincident with an increase in p65 translocation into the nucleus and a reduction of IκBα detected in the cytoplasm, increased transcription of IκBα occurs. These findings suggest that NF-κB activation initiates inflammation early in the course of NEC resulting in increased proinflammatory protein expression, underscoring the importance of the inflammatory response in this NEC model, which precedes evidence of histological injury.

Inflammatory prostaglandin E2 signaling in a mouse model of Alzheimer disease

There is significant evidence for a central role of inflammation in the development of Alzheimer disease (AD). Epidemiological studies indicate that chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD in healthy aging populations. As NSAIDs inhibit the enzymatic activity of the inflammatory cyclooxygenases COX-1 and COX-2, these findings suggest that downstream prostaglandin signaling pathways function in the preclinical development of AD. Here, we investigate the function of prostaglandin E(2) (PGE(2) ) signaling through its EP3 receptor in the neuroinflammatory response to Aβ peptide. The function of PGE(2) signaling through its EP3 receptor was examined in vivo in a model of subacute neuroinflammation induced by administration of Aβ(42) peptides. Our findings were then confirmed in young adult APPSwe-PS1ΔE9 transgenic mice. Deletion of the PGE(2) EP3 receptor in a model of Aβ(42) peptide-induced neuroinflammation reduced proinflammatory gene expression, cytokine production, and oxidative stress. In the APPSwe-PS1ΔE9 model of familial AD, deletion of the EP3 receptor blocked induction of proinflammatory gene and protein expression and lipid peroxidation. In addition, levels of Aβ peptides were significantly decreased, as were β-secretase and β C-terminal fragment levels, suggesting that generation of Aβ peptides may be increased as a result of proinflammatory EP3 signaling. Finally, deletion of EP3 receptor significantly reversed the decline in presynaptic proteins seen in APPSwe-PS1ΔE9 mice. Our findings identify the PGE(2) EP3 receptor as a novel proinflammatory, proamyloidogenic, and synaptotoxic signaling pathway, and suggest a role for COX-PGE(2) -EP3 signaling in the development of AD.

Selective Class I Histone Deacetylase Inhibition Suppresses Hypoxia-Induced Cardiopulmonary Remodeling Through an Antiproliferative Mechanism

Histone deacetylase (HDAC) inhibitors are efficacious in models of hypertension-induced left ventricular heart failure. The consequences of HDAC inhibition in the context of pulmonary hypertension with associated right ventricular cardiac remodeling are poorly understood. This study was performed to assess the utility of selective small-molecule inhibitors of class I HDACs in a preclinical model of pulmonary hypertension. Rats were exposed to hypobaric hypoxia for 3 weeks in the absence or presence of a benzamide HDAC inhibitor, MGCD0103, which selectively inhibits class I HDACs 1, 2, and 3. The compound reduced pulmonary arterial pressure more dramatically than tadalafil, a standard-of-care therapy for human pulmonary hypertension that functions as a vasodilator. MGCD0103 improved pulmonary artery acceleration time and reduced systolic notching of the pulmonary artery flow envelope, which suggests a positive impact of the HDAC inhibitor on pulmonary vascular remodeling and stiffening. Similar results were obtained with an independent class I HDAC-selective inhibitor, MS-275. Reduced pulmonary arterial pressure in MGCD0103-treated animals was associated with blunted pulmonary arterial wall thickening because of suppression of smooth muscle cell proliferation. Right ventricular function was maintained in MGCD0103-treated animals. Although the class I HDAC inhibitor only modestly reduced right ventricular hypertrophy, it had multiple beneficial effects on the right ventricle, which included suppression of pathological gene expression, inhibition of proapoptotic caspase activity, and repression of proinflammatory protein expression. By targeting distinct pathogenic mechanisms, isoform-selective HDAC inhibitors have potential as novel therapeutics for pulmonary hypertension that will complement vasodilator standards of care.

Inhibitory Effect of Lignans from Myristica fragrans on LPS-induced NO Production in RAW264.7 Cells

Inflammation is a beneficial host response to foreign challenge or tissue injury that leads ultimately to the restoration of tissue structure and function. Inflammation involves a complex web of intercellular cytokine signals. Activated monocytes and/or macrophages release a variety of inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), reactive oxygen species, prostaglandin E2 (PGE2) and nitric oxide (NO). A normal inflammatory response is self-limiting and involves the down-regulation of pro-inflammatory protein expression, increased expression of anti-inflammatory proteins and a reversal in the vascular changes that facilitated the initial immune cell recruitment process. Nitric oxide (NO) is a short-living free radical that is produced from L-arginine by catalytic reaction of NO synthases (NOSs) within mammalian immune, cardiovascular and neural systems, where it functions as a signaling or cytotoxic molecule. Cyclooxygenase (COX), an enzyme also known as prostaglandin (PG) H synthase (EC 1.14.99.1), converts arachidonic acid to prostaglandins, which play a crucial role as mediators of inflammatory responses. Myristica fragrans Houtt (Myristicaceae) is an aromatic evergreen tree cultivated in South Africa India, and other tropical countries. The seed and aril of the fruit of M. fragrans are commonly known as nutmeg and mace, respectively. Since the Middle Ages, nutmeg has been used as a carminative, stimulant, narcotic, emmenagogue and abortifacient. Nutmeg is also prescribed for medicinal purposes in Asia to treat many diseases, such as rheumatism, muscle spasm, decreased appetite and diarrhea. It has shown antioxidant, anti-inflammatory, protein tyrosine phosphatase 1B inhibitory, hepatoprotective and acetylcholine esterase inhibitory activities. In continuing investigation for discovering inhibitors on LPS-induced NO production in macrophage RAW264.7 cells, studies of the EtOAc-soluble fraction from the seeds of M. fragrans resulted in the isolation of a new lignan (1), along with five known ones (2-6). This paper describes the isolation and structural elucidation of the isolates, as well as the evaluation of their inhibitory activity on LPS-induced NO production in macrophage RAW264.7 cells. The MeOH extract of the seeds of M. fragrans was partitioned into hexane-, EtOAcand n-BuOH-soluble fractions. Chromatographic purification of the ethyl acetate fraction led to the isolation of six compounds (1-6) (Fig. 1). The structures of known compounds were identified as (8R,8'S)-

Bioactive Cembranoids from the Soft Coral Sinularia crassa

Eight new cembranoids, crassarines A–H (1–8) were isolated from the Formosan soft coral Sinularia crassa. Compounds 1–3 represent the rare cembranoids with a 1,12-oxa-bridged tetrahydrofuran ring, while 4 and 5 are the firstly discovered 1,11-oxa-bridged tetrahydropyranocembranoids. The absolute configuration of 6 was determined using the Mosher’s method. Compounds 6 and 8 were found to significantly inhibit the expression of both pro-inflammatory iNOS and COX-2 proteins at 10 μM, respectively, while compounds 4–8 were found to be non-cytotoxic toward the selected human liver cancer cells.

Immunohistochemical expression of proinflammatory cytokines IL-1β, IL-6, TNF-α and involvement of COX-2, quantitatively confirmed by Western blot analysis, in Wernicke's encephalopathy

Selective cerebral vulnerability is a major consequence of Wernicke's encephalopathy (WE), in which focal areas of the brain exhibit symmetrical profound neuronal loss and accompanying gliosis, occurring most frequently in diencephalic regions such as the thalamus and the mammillary bodies. Many processes have been proposed to explain the selective cerebral vulnerability and the focal neuronal cell death in Wernicke's encephalopathy. There are several mechanisms which are common to the pathophysiology of encephalopathies caused by thiamine deficiency (TD). Recently, emphasis is being placed on deficit in mitochondrial oxidative metabolism, oxidative/nitrosative stress, and the release of proinflammatory cytokines such as IL-1β, IL-6, and tumor necrosis factor-α (TNF-α). Cyclooxygenase-2 (COX-2) plays major roles in regulating brain damage and inflammation. Here we present two fatal cases of non-alcohol associated WE. The immunohistochemical study revealed increased proinflammatory cytokine immunoreactivity in the neurons of the mammillary bodies and medial thalamus, and in the periaqueductal regions, compared with basal constitutive levels of expression in the frontal cortex. Positive (WE cases) and negative (immediate trauma deaths) case-controls were used to confirm the results. TD induced IL-1β proteins weakly, while moderate increase was observed for TNF-α and IL-6. Immunofluorescence analysis by confocal microscopy confirmed the staining results for immunoreactivity in WE brains. Further, the induction of proinflammatory cytokine protein expression levels was quantified by Western blot analysis.

Characterisation of amyloid-induced inflammatory responses in the rat retina

Amyloid-induced inflammation is thought to play a critical and early role in the pathophysiology of Alzheimer's disease. As such, robust models with relevant and accessible compartments that provide a means of assessing anti-inflammatory agents are essential for the development of therapeutic agents. In the present work, we have characterised the induction of inflammation in the rat retina following intravitreal administration of amyloid-beta protein (Aβ). Histology and mRNA endpoints in the retina demonstrate Aβ1-42-, but not Aβ42-1-, induced inflammatory responses characterised by increases in markers for microglia and astrocytes (ionised calcium-binding adaptor molecule 1 (iba-1), GFAP and nestin) and increases in mRNA for inflammatory cytokines and chemokines such as IL1-β, MIP1α and TNFα. Likewise, analysis of vitreal cytokines also revealed increases in inflammatory cytokines and chemokines, including IL1-β, MIP1α and MCP1, induced by Aβ1-42 but not Aβ42-1. This profile of pro-inflammatory gene and protein expression is consistent with that observed in the Alzheimer's disease brain and suggest that this preclinical model may provide a useful relevant tool in the development of anti-inflammatory approaches directed towards Alzheimer's disease therapy.

Effects of soy protein on alcoholic liver disease in rats undergoing ethanol withdrawal

ObjectiveThis investigation attempted to clarify the effects of soy protein on alcoholic liver disease (ALD) in rats undergoing ethanol withdrawal. MethodsAlcoholic liver disease was induced in rats by administration of a low-carbohydrate ethanol liquid diet for 12 weeks, after which the ethanol was withdrawn and the rats were divided into two experimental groups: a control group (EC group) and a soy protein group (EP group) for 4 weeks. ResultsAfter the 12-week ALD-inducing period, the ethanol group had significantly higher hepatic lipid accumulation, oxidative stress and inflammation. We found that the EP group had significantly lower hepatic lipids, malondialdehyde, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, hydroxyproline levels and myeloperoxidase activity compared to the EC group. Moreover, the fecal total cholesterol and total lipids were higher in the EP group. Expression of the hepatic cytochrome P450 2E1 (CYP2E1) protein in the EP group was significantly lower than that in the EC group, and the hepatic peroxisome proliferator-activated receptor (PPAR) α and cytochrome P450 4A (CYP4A) protein expressions in the EP group were significantly higher than those in the EC group. In the histopathological analysis, we also found that soy protein ameliorated fat accumulation in the liver. ConclusionThese results suggest that soy protein may improve alcohol-induced lipid accumulation, oxidative stress and inflammation by decreasing proinflammatory cytokines and CYP2E1 protein expression and by increasing PPARα and CYP4A protein expressions and fecal lipid excretion, thereby producing beneficial effects on ALD during ethanol withdrawal.

Loss of poly(ADP-ribose) polymerase 1 attenuates renal fibrosis and inflammation during unilateral ureteral obstruction

Poly(ADP-ribose) polymerase 1 (PARP1) contributes to necrotic cell death and inflammation in several disease models; however, the role of PARP1 in fibrogenesis remains to be defined. Here, we tested whether PARP1 was involved in the pathogenesis of renal fibrosis using the unilateral ureteral obstruction (UUO) mouse model. UUO was performed by ligation of the left ureter near the renal pelvis in Parp1-knockout (KO) and wild-type (WT) male mice. After 10 days of UUO, renal PARP1 expression and activation were strongly increased by 6- and 13-fold, respectively. Interstitial fibrosis induced by UUO was significantly attenuated in Parp1-KO kidneys compared with that in WT kidneys at 10 days, but not at 3 days, based on collagen deposition, α-smooth muscle actin (α-SMA), and fibronectin expression. Intriguingly, the UUO kidneys in Parp1-KO mice showed a dramatic decrease in infiltration of neutrophil and reduction in expression of proinflammatory proteins including intercellular adhesion molecule-1, tumor necrosis factor-α, inducible nitric oxide synthase, and toll-like receptor 4 as well as phosphorylation of nuclear factor-κB p65, but not transforming growth factor-β1 (TGF-β1) at both 3 and 10 days. Pharmacological inhibition of PARP1 in rat renal interstitial fibroblast (NRK-49F) cell line or genetic ablation in primary mouse embryonic fibroblast cells did not affect TGF-β1-induced de novo α-SMA expression. Parp1 deficiency significantly attenuated UUO-induced histological damage in the kidney tubular cells, but not apoptosis. These data suggest that PARP1 induces necrotic cell death and contributes to inflammatory signaling pathways that trigger fibrogenesis in obstructive nephropathy.

The Effect of A2A Adenosine Receptor Activation on C-C Chemokine Receptor 7 Expression in Human THP1 Macrophages During Inflammation

C-C chemokine receptor 7 (CCR7) and its chemoattractant agonist CCL21 promote cell migration and expression of pro-inflammatory proteins in an atherogenic environment. Since A(2A) adenosine receptor activation reduces migration and inflammatory effects, we examined its effect on CCR7 expression and migration. CCR7 protein expression decreased by about a third in macrophages treated with A(2A) receptor agonist CGS 21680 (p = 0.028, n = 7) and was reversed with antagonist, although mRNA levels increased twofold (p = 0.001, n = 3). Furthermore, macrophages treated with CGS 21680 showed a significant decrease in migration (p = 0.0311, n = 7). These results suggest that A(2A) adenosine receptor activation not only modulates CCR7 expression in both normal and inflammatory environments but also regulates macrophage migration to CCR7-specific chemoattractants.

Protective effect of ellagic acid, a natural polyphenolic compound, in a murine model of Crohn's disease

Current epidemiological and experimental studies support a beneficial role of dietary polyphenols in several gastrointestinal diseases, including inflammatory bowel disease. The aim of this study was to gain a better understanding of the effects of a naturally occurring polyphenol, ellagic acid, present in some fruits such as pomegranate, raspberries and nuts among others, in an experimental murine model of Crohn's disease by intra-colonic administration of TNBS in rats. Analysis of the lesions were carried out by macroscopic and histological technics. Inflammation response was assessed by histology and myeloperoxidase activity. iNOS and COX-2 are upregulated by MAPKs and NF-κB nuclear transcription factor in intestinal epithelial cells thus, we determined the expression of iNOS, COX-2 and the involvement of the p38, JNK, ERK1/2 MAPKs and NF-κB signalling in the protective effect of EA by western blotting. Oral administration of EA (10–20 mg/kg) diminished the severity and extension of the intestinal injuries induced by TNBS although there was no observed a significant dose–response. In addition, EA increased mucus production in goblet cells in colon mucosa, decreased neutrophil infiltration and pro-inflammatory proteins COX-2 and iNOS overexpression. Also EA was capable of reducing the activation of p38, JNK and ERK1/2 MAPKs, preventing the inhibitory protein IκB-degradation and inducing an inhibition of the nuclear translocation level of p65 in colonic mucosa. In conclusion, EA reduces the damage in a rat model of Crohn's disease, alleviates the oxidative events and returns pro-inflammatory proteins expression to basal levels probably through MAPKs and NF-κB signalling pathways.

Circulating microparticles from septic shock patients exert differential tissue expression of enzymes related to inflammation and oxidative stress*

Septic shock is characterized by hypotension and multiple organ failure after infection of microorganisms. Septic shock patients display high levels of circulating microparticles. These are small vesicles released from the plasma membrane of activated or apoptotic cells. Here, we have investigated the effects of in vivo injection of microparticles from nonseptic or septic subjects on protein expression in mouse tissues. Prospective, controlled experiments. Animal basic science laboratory. Male Swiss mice were randomly assigned to one of two groups: 11 animals injected with microparticles isolated from healthy subjects and 15 animals injected with microparticles isolated from septic patients. Microparticles were extracted from whole blood of septic and nonseptic subjects and were intravenously injected in mice. After 24 hrs, mice were killed and heart, lungs, liver, and kidneys were isolated for Western blot assays. Organs were also used for direct measurements of nitric oxide and superoxide anion production by electron paramagnetic resonance. In heart and lungs, microparticles from septic shock patients increased the expression of endothelial and inducible nitric oxide synthases, cyclooxygenase-2, and nuclear factor-κB. However, extracellular superoxide dismutase was only increased in the heart. These effects were associated either with a greater oxidative or nitrative stress in heart and lungs, without affecting nitric oxide production. The liver exhibited an increase in oxidative stress linked to decreased endothelial nitric oxide synthase and manganese superoxide dismutase expression. However, cyclooxygenase-2 expression and IκBα phosphorylation were decreased. Septic microparticles did not change superoxide anion and nitric oxide productions in kidneys. Results suggest that microparticles from septic shock patients exert pleiotropic and differential effects depending on target tissues with regard to the expression of proinflammatory proteins related with nitrative and oxidative stresses. Thus, microparticles might participate in organ dysfunction observed in septic shock patients.

Niaspan reduces high-mobility group box 1/receptor for advanced glycation endproducts after stroke in type-1 diabetic rats

High-mobility group box 1 (HMGB1), an active receptor for advanced glycation endproducts (RAGE), functions as a potent proinflammatory cytokine-like factor that contributes to the pathogenesis of vasculature. Diabetes mellitus (DM) is associated with accelerated development of both microvascular and macrovascular disease and increases the risk of ischemic stroke. Using a model of streptozotocin-induced type-1 diabetes (T1DM) in rats, we investigated the changes in HMGB and RAGE and tested the effects of Niaspan, a slow release form of niacin, on the expression of pro-inflammatory proteins in rats after stroke. T1DM rats were subjected to transient middle cerebral artery occlusion (MCAo) and treated without or with Niaspan (40 mg/kg) daily for 14 days after MCAo. Non-streptozotocin rats (WT) were also subjected to MCAo. Immunostaining for inflammatory mediators including HMGB1, RAGE, matrix metalloproteinase-9 (MMP-9) and toll-like receptor 4 (TLR4) immunostaining (n=8/group) and Western blotting (n=4/group) were performed. Compared to WT-MCAo rats, T1DM-MCAo rats showed an increased expression of HMGB1 (0.82±0.07 vs. 1.81±0.98, P<0.05), RAGE (1.31±0.22 vs. 3.77±0.72, P<0.05), MMP-9 (0.74±0.08 vs. 1.61±0.09, P<0.05) and TLR4 (2.85±0.22 vs. 6.72±0.44, P<0.05) after stroke. Niaspan treatment significantly attenuated the expression of HMGB1 (1.80±0.98 vs. 1.31±0.01, P<0.05), RAGE (3.77±0.71 vs. 1.78±0.45, P<0.05), MMP-9 (1.61±0.09 vs. 0.97±0.07, P<0.05) and TLR4 (6.72±0.44 vs. 2.28±0.43, P<0.05) in the ischemic brain in T1DM-MCAo rats. T1DM increases HMGB1/RAGE, TLR4 and MMP-9 expression after stroke. Niaspan treatment of stroke in T1DM rats inhibits HMGB1/RAGE, TLR4 and MMP-9 expression which may contribute to the reduced inflammatory response after stroke in T1DM rats.

Phellinus baumii ethyl acetate extract inhibits lipopolysaccharide-induced iNOS, COX-2, and proinflammatory cytokine expression in RAW264.7 cells

Mushrooms are valuable sources of biologically active compounds possessing anticancer, antiplatelet, and anti-inflammatory properties. Phellinus baumii is a mushroom used in folk medicine for a variety of human diseases. However, its potential anti-inflammatory effect has remained unclear. Therefore, we studied the effect of P. baumii ethyl acetate extract (PBEAE) on inflammatory mediator and proinflammatory cytokine protein and/or mRNA expression levels using the nitric oxide (NO) assay, enzyme immunoassay (EIA), western blot, and reverse transcription polymerase chain reaction (RT-PCR) in lipopolysaccharide (LPS)-stimulated macrophage like RAW264.7 cells. PBEAE markedly inhibited NO generation and prostaglandin E(2) (PGE(2)) synthesis in a concentration-dependent pattern without any cytotoxic effect at the concentration range used. PBEAE also suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression. In addition, LPS-induced iNOS and COX-2 mRNA expression levels were dose-dependently inhibited by PBEAE pretreatment. Furthermore, PBEAE attenuated the mRNA expression levels of proinflammatory cytokines, specifically interleukin (IL)-1β, IL-6, and granulocyte macrophage colony-stimulating factor (GM-CSF), in a concentration-dependent fashion. Our study suggests that P. baumii might exhibit anti-inflammatory properties by downregulating proinflammatory mediators. Thus, further study on compounds isolated from PBEAE is warranted to investigate the associated molecular mechanisms and identify the potential therapeutic targets.

Free heme is a danger signal inducing expression of proinflammatory proteins in cultured cells derived from normal rat hearts

Endogenous molecules from damaged tissue act as danger signals to trigger or amplify the immune/inflammatory response. In this study, we examined whether free heme induced pro-inflammatory proteins in cultured cells derived from normal hearts and investigated the cells targeted by heme, together with its mechanism of action in these cells. We cultured collagenase-isolated heart-derived cells from normal rats and examined whether free heme induced pro-inflammatory proteins, reactive oxygen species (ROS) production and NF-κB activation, by quantitative RT-PCR, ELISA and flow cytometry. Free heme increased mRNA of various pro-inflammatory proteins in cultured cardiac resident cells (CCRC) (at least 100-fold) and induced intracellular ROS formation. Approximately 85–90% of CCRC are fibroblast/smooth muscle cells and 10–15% are CD11bc-positive macrophages; therefore to examine individual target cells, macrophage-deleted (CD11bc-negative) CCRC, primary cultured cells (cardiac fibroblasts, arterial smooth muscle cells and cardiac microvascular endothelial cells) and macrophage cells lines (NR8383) were similarly treated. Free heme activated NF-κB and induced expression of some pro-inflammatory proteins, including IL-1 and TNF-α in NR8383. On the other hand, macrophage-deleted CCRC strongly increased expression of these proteins on treatment with IL-1 or TNF-α, but not free heme. Induction of expression of pro-inflammatory proteins by free heme was not inhibited by intracellular ROS reduction, but by protease and proteasome inhibitors capable of regulating NF-κB. These data suggest that free heme strongly induces various pro-inflammatory proteins in injured hearts through NF-κB activation in cardiac resident macrophages and through cross-talk between macrophages and fibroblast/smooth muscle cells mediated inter alia by IL-1, TNF-α.

Saccharomyces cerevisiae Modulates Immune Gene Expressions and Inhibits ETEC-Mediated ERK1/2 and p38 Signaling Pathways in Intestinal Epithelial Cells

BackgroundEnterotoxigenic Escherichia coli (ETEC) infections result in large economic losses in the swine industry worldwide. ETEC infections cause pro-inflammatory responses in intestinal epithelial cells and subsequent diarrhea in pigs, leading to reduced growth rate and mortality. Administration of probiotics as feed additives displayed health benefits against intestinal infections. Saccharomyces cerevisiae (Sc) is non-commensal and non-pathogenic yeast used as probiotic in gastrointestinal diseases. However, the immuno-modulatory effects of Sc in differentiated porcine intestinal epithelial cells exposed to ETEC were not investigated.Methodology/Principal FindingsWe reported that the yeast Sc (strain CNCM I-3856) modulates transcript and protein expressions involved in inflammation, recruitment and activation of immune cells in differentiated porcine intestinal epithelial IPEC-1 cells. We demonstrated that viable Sc inhibits the ETEC-induced expression of pro-inflammatory transcripts (IL-6, IL-8, CCL20, CXCL2, CXCL10) and proteins (IL-6, IL-8). This inhibition was associated to a decrease of ERK1/2 and p38 MAPK phosphorylation, an agglutination of ETEC by Sc and an increase of the anti-inflammatory PPAR-γ nuclear receptor mRNA level. In addition, Sc up-regulates the mRNA levels of both IL-12p35 and CCL25. However, measurement of transepithelial electrical resistance displayed that Sc failed to maintain the barrier integrity in monolayer exposed to ETEC suggesting that Sc does not inhibit ETEC enterotoxin activity.Conclusions Sc (strain CNCM I-3856) displays multiple immuno-modulatory effects at the molecular level in IPEC-1 cells suggesting that Sc may influence intestinal inflammatory reaction.

Gene Therapy for Allergic Airway Diseases

Airway diseases such as allergic asthma and rhinitis are characterized by a T-helper type 2 (Th2) response. Treatment of allergic airway diseases is currently limited to drugs that relieve disease symptoms and inflammation. In the search for new therapeutics, efforts have been made to treat allergic airway disease with gene therapy, and many preclinical studies have demonstrated its impressive potential. Most strategies focus on blocking the expression of proinflammatory proteins or transcription factors involved in the disease pathogenesis using antisense oligonucleotides, DNAzymes, small interfering RNA, or blocking of microRNAs using antagomirs. Changing the Th1/Th2 balance by overexpressing Th1-stimulating factors is another treatment option. Although the proof of concept is convincing in animal models, progress in humans remains limited. In this review, we focus on preclinical models to describe the recent developments and major breakthroughs for treating allergic airway diseases with gene therapy.

Cataract Surgery Induces Retinal Pro-inflammatory Gene Expression and Protein Secretion

To investigate the effect(s) of cataract surgery on the expression of pro-inflammatory genes and proteins in the retina using an experimental rodent model. An extracapsular lens extraction was performed in one eye of C57BL/6 mice (n = 24); the contralateral unoperated eyes (n = 24) as well as eyes from unoperated animals (n = 9) served as controls. The neurosensory retina and retinal pigment epithelium (RPE)/choroid were collected postoperatively. Expression of genes involved in the acute inflammatory/injury response, including IL-1β, fibroblast growth factor, transforming growth factor β, chemokine CCL2, SDF-1, and complements C3, C4, and factor B (CFB), were examined by real-time PCR and, selectively, by immunohistochemistry. The expression of IL-1β and CCL2 genes was markedly upregulated (>20-fold, P < 0.01) in the neurosensory retina 30 minutes postoperatively and maintained for the 2-week postoperative period of observation; IL-1β expression was also upregulated in RPE/choroid. The expression of complement C3 (>5-fold) and CFB (>30-fold) genes in the neurosensory retina was also significantly upregulated (P < 0.01 in both cases). Increased IL-1b, CCL2, and CFB as well as enhanced C5b-9 immunostaining were observed by confocal microscopy. In rodents, lens extraction elicited an acute pro-inflammatory gene and protein response in the posterior segment of the eye, indicating induction of the inflammasome as well as complement activation, as occurs in the "danger" response. A similar response in humans might explain the pathogenesis of cataract surgery-associated retinal complications such as cystoid macular edema.

Proteomic analysis of the anti‐inflammatory action of minocycline

Minocycline possesses anti-inflammatory properties independently of its antibiotic activity although the underlying molecular mechanisms are unclear. Lipopolysaccharide (LPS)-induced cytokines and pro-inflammatory protein expression are reduced by minocycline in cultured macrophages. Here, we tested a range of clinically important tetracycline compounds (oxytetracycline, doxycycline, minocycline and tigecycline) and showed that they all inhibited LPS-induced nitric oxide production. We made the novel finding that tigecycline inhibited LPS-induced nitric oxide production to a greater extent than the other tetracycline compounds tested. To identify potential targets for minocycline, we assessed alterations in the macrophage proteome induced by LPS in the presence or absence of a minocycline pre-treatment using 2-DE and nanoLC-MS. We found a number of proteins, mainly involved in cellular metabolism (ATP synthase β-subunit and aldose reductase) or stress response (heat shock proteins), which were altered in expression in response to LPS, some of which were restored, at least in part, by minocycline. This is the first study to document proteomic changes induced by minocycline. The observation that minocycline inhibits some, but not all, of the LPS-induced proteomic changes shows that minocycline specifically affects some signalling pathways and does not completely inhibit macrophage activation.

Aldosterone-induced kidney injury is mediated by NFκB activation

BackgroundAldosterone induces inflammation and fibrosis in the kidney, while nuclear factor κB (NFκB) plays key roles in inflammation mediated by various cytokines. Here, we determined the roles of NFκB activation in aldosterone-induced kidney injury.MethodsWe used unilaterally nephrectomized rats with or without continuous aldosterone infusion and 0.9% saline as drinking water for 3 weeks. IMD-1041, an IKKβ inhibitor, and spironolactone were orally administered to inhibit NFκB and mineralocorticoid receptor, respectively.ResultsThe aldosterone-infused rats exhibited severe kidney injury, hypertension, and increased expression of pro-inflammatory and fibrotic proteins, osteopontin, fibrinogen, collagen type I, and PAI-1. Western blotting confirmed NFκB activation by aldosterone by the increased amount of p65 in the nuclear fraction of the kidney, and oral IMD-1041 prevented the kidney injury and lessened the increase in pro-inflammatory and fibrotic proteins without significant changes in blood pressures. In addition, changes in angiotensin-converting enzyme 2 (ACE2), which has been found to act as a protective factor in various kidney injury models, were examined. Immunofluorescence studies revealed the presence of ACE2 in the brush-border membrane of the proximal convoluted tubules and markedly blunted ACE2 staining in aldosterone-infused rats. The decrease in amount of ACE2 protein was confirmed by Western blotting, and IMD-1041 also prevented the decrease in ACE2. The administration of spironolactone also abolished the effects of aldosterone.ConclusionOur results suggest that aldosterone induces kidney injury via activation of NFκB and mineralocorticoid receptor, and that decreased ACE2 expression may play an important role in aldosterone-induced kidney injury.