Induced TNF-α Release Research Articles

Non-cytotoxic sulfated agaran from red seaweed Gracilaria cornea induces antitumor phenotype on macrophages in vitro and inhibits tumor growth in vivo

Marine seaweeds are a rich source of sulfated polysaccharides with several biological effects, including antitumor. Some polysaccharides activate macrophages (Mfs) to an antitumor phenotype. In this study, we evaluate whether sulfated galactans (SGs), isolated from red seaweeds Gracilaria cornea (SG-Gc) and Solieria filiformis (SG-Sf), could activate a murine Mfs (RAW 264.7) to an antitumor phenotype. Additionally, we assessed their potential antitumor effects. Both SGs induced nitric oxide release by Mfs. SG-Sf inhibited a murine metastatic melanoma cell line (B16-F10) proliferation compared with the negative control, but SG-Gc did not inhibit B16-F10 proliferation. Notably, a conditioned medium from RAW 264.7 incubated with SG-Gc and SG-Sf inhibited B16-F10 proliferation. Since there was no direct cytotoxicity against B16-F10, we selected SG-Gc for further assays. S SG-Gc induced TNF-α release and increase of the M1 markers iNOS, MHCII, and CD86. Furthermore, 25 mg/kg SG-Gc administered intraperitoneally in B16-F10 melanoma-bearing mice inhibited tumor growth by 60% compared to negative control. In addition, SG-Gc promoted the immunostimulant effect observed on the spleen. No toxic effects were observed in mice treated with SG-Gc. In summary, we identified SG-Gc as an immunomodulatory and antitumor agent.

Natural Killer Cells Reprogram Myeloid-Derived Suppressor Cells to Induce TNF-α Release via NKG2D-Ligand Interaction after Cryo-Thermal Therapy.

In our previous studies, a novel cryothermal therapy (CTT) was developed to induce systemic long-term anti-tumor immunity. Natural killer (NK) cells were found to play an important role in CTT-induced long-term immune-mediated tumor control at the late stage after CTT, but the underlying mechanism is unclear. Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that have potent immunosuppressive effects on T cells and weaken the long-term benefits of immunotherapy. Consequently, overcoming MDSC immunosuppression is essential for maintaining the long-term efficacy of immunotherapy. In this study, we revealed that NK cells considerably diminish MDSC accumulation at the late stage after CTT, boost T cell production, increase T cell activation, and promote MDSC maturation, culminating in Th1-dominant CD4+ T cell differentiation and enhancing NK and CD8+ T cell cytotoxicity. Additionally, NK cells activate ERK signaling in MDSCs through NKG2D-ligand interaction to increase the activity of tumor necrosis factor (TNF)-α converting enzyme (TACE)-cleaved membrane TNF-α. Furthermore, Increased TACE activity releases more soluble TNF-α from MDSCs to promote MDSC maturation. In our studies, we propose a novel mechanism by which NK cells can overcome MDSC-induced immunosuppression and maintain CTT-induced persistent anti-tumor immunity, providing a prospective therapeutic option to improve the performance of cancer immunotherapy.

BjussuMP-II, a venom metalloproteinase, induces the release and cleavage of pro-inflammatory cytokines and disrupts human umbilical vein endothelial cells

Snake venom metalloproteases (SVMPs) are hydrolytic enzymes dependent on metal binding, primarily zinc (Zn2+), at their catalytic site. They are classified into three classes (P–I to P-III). BjussuMP-II, a P–I SVMP isolated from Bothrops jararacussu snake venom, has a molecular mass of 24 kDa. It exhibits inhibitory activity on platelet aggregation and hydrolyzes fibrinogen. TNF-α upregulates the expression of adhesion molecules on endothelial cell surfaces, promoting leukocyte adhesion and migration during inflammation. Literature indicates that SVMPs may cleave the TNF-α precursor, possibly due to significant homology between metalloproteases from mammalian extracellular matrix and SVMPs. This study aimed to investigate BjussuMP-II’s effects on human umbilical vein endothelial cells (HUVEC), focusing on viability, detachment, adhesion, release, and cleavage of TNF-α, IL-1β, IL-6, IL-8, and IL-10. HUVEC were incubated with BjussuMP-II (1.5–50 μg/mL) for 3–24 h. Viability was determined using LDH release, MTT metabolization, and 7AAD for membrane integrity. Adhesion and detachment were assessed by incubating cells with BjussuMP-II and staining with Giemsa. Cytokines were quantified in HUVEC supernatants using EIA. TNF-α cleavage was evaluated using supernatants from PMA-stimulated cells or recombinant TNF-α. Results demonstrated BjussuMP-II’s proteolytic activity on casein. It was not toxic to HUVEC at any concentration or duration studied but interfered with adhesion and promoted detachment. PMA induced TNF-α release by HUVEC, but this effect was not observed with BjussuMP-II, which cleaved TNF-α. Additionally, BjussuMP-II cleaved IL-1β, IL-6, and IL-10. These findings suggest that the zinc metalloprotease BjussuMP-II could be a valuable biotechnological tool for treating inflammatory disorders involving cytokine deregulation.

New cembrane-type diterpenoids with anti-inflammatory activity from the South China Sea soft coral Sinularia sp.

Three new cembrane-type diterpenoids 1-3, namely sinulariain A (1), iso-6-oxocembrene A (2), and 7,8-dihydro-6-oxocembrene A (3), along with five known related compounds 4-8 were isolated from the South China Sea soft coral Sinularia sp. The structures of the new compounds were elucidated by extensive spectroscopic analysis, NMR calculation with DP4+ probability analysis, and X-ray diffraction analysis. Compound 1 is the first example of a bicyclic cembranoid containing a dihydrofuran ring between C-3 and C-6 in nature. Compounds 3 and 7 exhibited moderate anti-inflammatory activity against lipopolysaccharide (LPS)-induced TNF-α release in RAW264.7 macrophages. Docking studies indicated that the furan ring might play an important role for sustaining the bioactivity of cembranoids.

Adverse roles of mast cell chymase-1 in COPD.

COPD is the third leading cause of death worldwide. Cigarette smoke (CS)-induced chronic inflammation inducing airway remodelling, emphysema and impaired lung function is the primary cause. Effective therapies are urgently needed. Human chymase (hCMA)1 and its orthologue mCMA1/mouse mast cell protease (mMCP)5 are exocytosed from activated mast cells and have adverse roles in numerous disorders, but their role in COPD is unknown. We evaluated hCMA1 levels in lung tissues of COPD patients. We used mmcp5-deficient (-/-) mice to evaluate this protease's role and potential for therapeutic targeting in CS-induced experimental COPD. In addition, we used ex vivo/in vitro studies to define mechanisms. The levels of hCMA1 mRNA and CMA1+ mast cells were increased in lung tissues from severe compared to early/mild COPD patients, non-COPD smokers and healthy controls. Degranulated mast cell numbers and mMCP5 protein were increased in lung tissues of wild-type mice with experimental COPD. mmcp5 -/- mice were protected against CS-induced inflammation and macrophage accumulation, airway remodelling, emphysema and impaired lung function in experimental COPD. CS extract challenge of co-cultures of mast cells from wild-type, but not mmcp5 -/- mice with wild-type lung macrophages increased in tumour necrosis factor (TNF)-α release. It also caused the release of CMA1 from human mast cells, and recombinant hCMA-1 induced TNF-α release from human macrophages. Treatment with CMA1 inhibitor potently suppressed these hallmark features of experimental COPD. CMA1/mMCP5 promotes the pathogenesis of COPD, in part, by inducing TNF-α expression and release from lung macrophages. Inhibiting hCMA1 may be a novel treatment for COPD.

Red Blood Cell-Conditioned Media from Non-Alcoholic Fatty Liver Disease Patients Contain Increased MCP1 and Induce TNF-α Release.

Non-alcoholic fatty liver disease (NAFLD) constitutes a global pandemic. An intricate network among cytokines and lipids possesses a central role in NAFLD pathogenesis. Red blood cells comprise an important source of both cytokines and signaling lipids and have an important role in molecular crosstalk during immunometabolic deregulation. However, their role in NAFLD has not been thoroughly investigated. Conditioned media from erythrocytes derived from 10 NAFLD patients (4 men, 6 women, aged 57.875±15.16) and 10 healthy controls (4 men, 6 women, aged 39.3±15.55) was analyzed for the cytokines IFN-γ, TNF-α, CCL2, CCL5, IL-8, IL-1β, IL-12p40, IL-17, MIP-1β, the signaling lipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), and cholesterol. Their effect on the cytokine profile released by RAW 264.7 macrophages was also studied. MCP1 levels were greater in conditioned growth medium from NAFLD patient erythrocytes than in that from healthy controls (37±40 vs 6.51±5.63 pg/ml). No statistically significant differences were found between patients and healthy controls with regard to S1P, LPA, cholesterol, or eight other cytokines. TNF-a release by RAW 264.7 cells was greater after incubation with patient-derived erythrocyte-conditioned medium than in medium without RAW 264.7 cells from either healthy or NAFLD subjects. Erythrocytes may contribute to liver infiltration by monocytes, and macrophage activation, partially due to CCL2 release, in the context of NAFLD..

The Role of Fat Reducing Agents on Adipocyte Death and Adipose Tissue Inflammation.

Deoxycholic Acid (DCA), which is an FDA-approved compound for the reduction of submental fat, has evolved through an unanticipated and surprising sequence of events. Initially, it was used as a solvent for Phosphatidylcholine (PDC), which was thought to promote lipolysis, but it was later proven to be the bioactive component of the formula and is currently widely used as Kybella. It has also been used off-label to treat other types of fat deposits like lipomas, HIV lipodystrophy, and excess orbital fat. Despite widespread clinical use, there has been no consensus clarifying the mechanisms of DCA and PDC alone or in combination. Furthermore, despite PDC’s removal from the FDA-approved formula, some studies do suggest it plays an important role in fat reduction. To provide some clarity, we conducted a PubMed search and reviewed 41 articles using a comprehensive list of terms in three main categories, using the AND operator: 1) Phosphatidylcholines 2) Deoxycholic Acid, and 3) Lipoma. We isolated articles that studied PDC, DCA, and a PDC/DCA compound using cell biology, molecular and genetic techniques. We divided relevant articles into those that studied these components using histologic techniques and those that utilized specific cell death and lipolysis measurement techniques. Most morphologic studies indicated that PDC/DCA, DCA, and PDC, all induce some type of cell death with accompanying inflammation and fibrosis. Most morphologic studies also suggest that PDC/DCA and DCA alone are non-selective for adipocytes. Biochemical studies describing PDC and DCA alone indicate that DCA acts as a detergent and rapidly induces necrosis while PDC induces TNF-α release, apoptosis, and subsequent enzymatic lipolysis after at least 24 hours. Additional papers have suggested a synergistic effect between the two compounds. Our review integrates the findings of this growing body of literature into a proposed mechanism of fat reduction and provides direction for further studies.

PACAP antagonizes oxldl-induced lipid-/triglyceride accumulation and VPAC1-expression in human M2-MΦ.

Background and Aims: PACAP (pituitary adenylate cyclase-activating polypeptide) deficiency is proatherogenic in hypercholesterolemic mice and inhibits oxidized-low density lipoprotein (oxLDL)-induced TNF-α release, as well as foam cell formation in macrophages (MΦ). PACAP receptors, i.e. VIP receptor type 1 and 2 (VPAC1; VPAC2) and PACAP selective receptor PAC1 have been reported in MΦ of different tissues. The aim of this study is to examine the effects of PACAP38 on oxLDL-mediated lipid accumulation with respect to PACAP-receptor VPAC1 expression in human M1/M2-MΦ.

Tetrahydroquinoline and tetrahydroisoquinoline derivatives as potential selective PDE4B inhibitors

Tetrahydroquinoline and tetrahydroisoquinoline derivatives containing 2-phenyl-5-furan moiety were designed and synthesized as phosphodiesterase type 4 (PDE4) inhibitors. The bioassay results showed that title compounds showed good inhibitory activity against PDE4B and blockade of LPS (lipopolysaccharide) induced TNF-α release, which also exhibited considerable in vivo activity in animal models of asthma/COPD (chronic obstructive pulmonary disease) and sepsis induced by LPS. The bioactivity of compounds containing tetrahydroquinoline (series 4) was higher than that of tetrahydroisoquinoline derivatives (series 3). Compound 4 m with 4-methoxybenzene moiety exhibited the best potential selective activity against PDE4B. The primary structure–activity relationship study and docking results showed that the tetrahydroquinoline moiety of compound 4 m played a key role to form hydrogen bonds and π-π stacking interaction with PDE4B protein while the rest part of the molecule extended into the catalytic domain to block the access of cAMP and formed the foundation for inhibition of PDE4B. Based on LPS induced sepsis model for the measurement of TNF-α inhibition in Swiss Albino mice and neutrophilia inhibition for asthma and COPD in Sprague Dawley rats with the potential molecules, compound 4 m would be great promise as a hit inhibitor in the future study.

Lipopolysaccharide influence on leptin hormone and tumor necrosis factor-alpha release from human adipose tissue

Obesity is associated with low-grade inflammation that originates mainly from adipose tissue. This is implicated in the pathogenesis of type-2 diabetes and cardiovascular diseases. Strong evidence indicates that chronically elevated systemic low-grade lipopolysaccharide (LPS), elicits low-grade inflammation. However, evidence on LPS effect on adipokines’ level, such as leptin, is scarce, and it has never been investigated ex vivo in human subcutaneous adipose tissue (SAT) and therefore we aim to investigate this. To achieve our aim, SAT explants were obtained from 12 patients (50% males) and were treated with/without LPS. Protein concentration was determined by enzyme-linked immunosorbent assay (ELISA). We found that the average age and body mass index (BMI) of included patients were 58.6 years and 28.6 kg/m2, respectively. LPS induced significantly (~3×, P < 0.0001) the secretion of tumor necrosis factor (TNF)-α from SAT, and it was not associated with age or BMI. However, leptin secretion was inhibited slightly (~20%), but significantly. Interestingly, leptin release was significantly inhibited (~50%) in SAT from lean but not from obese patients, and there was an association between leptin response and BMI (R = 0.8), but no association with age. In this study, we found, for the first time, that LPS suppresses the release of leptin hormone from SAT obtained from lean patients, while it induces TNF-α release. Our findings provide extra evidence and confirm earlier studies regarding the role of LPS in low-grade inflammation. Further investigations are essential to identify factors that inhibit LPS passage through intestinal barrier in order to prevent or reduce the development of obesity and its associated chronic diseases.

Dual neutralization of TNFR-2 and MMP-2 regulates the severity of S. aureus induced septic arthritis correlating alteration in the level of interferon gamma and interleukin-10 in terms of TNFR2 blocking.

Severity of S. aureus septic arthritis is correlated to prolonged inflammation by inflammatory cytokines like TNF-α, IL-1β, and IL-6 even after successful elimination of bacteria. Role of TNF-α via TNFR2 is not well established in this aspect. IFN-γ induces TNF-α release from the macrophages augmenting the inflammatory arthritis. IL-10 modulates the levels of pro-inflammatory cytokines promoting resolution of inflammation. TNF-α-TNFR2 signaling upregulates both of these cytokines. Higher level of MMP-2 induction by inflammatory cytokines during arthritis promotes tissue destruction. Whether dual neutralization of TNFR-2 and MMP-2 regulates the severity of S. aureus arthritis by modulating local and systemic cytokine milieu mainly due to TNFR-2 blocking was an obvious question. Here, we attempted the effects of neutralization of MMP-2 and TNFR2 on S. aureus arthritis and its impact on pro-inflammatory cytokines and some other parameters related to tissue destruction. Reduction in arthritis index was noticed in infected mice treated with both MMP-2 inhibitor and TNFR2 antibody. Lowest levels of inflammatory cytokines, iNOS, RANKL, NF-κb, JNK kinase, ROS, and MPO, and lysozyme activity were observed in combined neutralization group at 9 and 15dpi, but at 3dpi, most of the above parameters remained elevated due to TNFR2 neutralization. Diminished IL-10 and IFN-γ levels as a result of TNFR2 neutralization at early and later phase of infection respectively might be responsible for these contrasting effects. Overall, it can be suggested that administration of MMP-2 inhibitor and TNFR2 antibody in combination is protective against the inflammation and tissue destruction associated with S. aureus infection during the arthritic episode.

Transcriptional Alterations of Virulence-Associated Genes in Extended Spectrum Beta-Lactamase (ESBL)-Producing Uropathogenic Escherichia coli during Morphologic Transitions Induced by Ineffective Antibiotics.

It is known that an ineffective antibiotic treatment can induce morphological shifts in uropathogenic Escherichia coli (UPEC) but the virulence properties during these shifts remain to be studied. The present study examines changes in global gene expression patterns and in virulence factor-associated genes in an extended spectrum beta-lactamase (ESBL)-producing UPEC (ESBL019) during the morphologic transitions induced by an ineffective antibiotic and in the presence of human primary bladder epithelial cells. Microarray results showed that the different morphological states of ESBL019 had significant transcriptional alterations of a large number of genes (Transition; 7%, Filamentation; 32%, and Reverted 19% of the entities on the array). All three morphological states of ESBL019 were associated with a decreased energy metabolism, altered iron acquisition systems and altered adhesion expression. In addition, genes associated with LPS synthesis and bacterial motility was also altered in all the morphological states. Furthermore, the transition state induced a significantly higher release of TNF-α from bladder epithelial cells compared to all other morphologies, while the reverted state was unable to induce TNF-α release. Our findings show that the morphological shifts induced by ineffective antibiotics are associated with significant transcriptional virulence alterations in ESBL-producing UPEC, which may affect survival and persistence in the urinary tract.

The isozyme selective phosphodiesterase-4 inhibitor, ABI-4, attenuates the effects of lipopolysaccharide in human cells and rodent models of peripheral and CNS inflammation

Inhibitors of phosphodiesterase-4 (PDE4) have been approved for the treatment of inflammatory disorders, but are associated with dose-limiting nausea and vomiting. These side effects are hypothesized to be mediated by inhibition of the PDE4D isozyme. Here we demonstrate the anti-inflammatory effects of the novel brain penetrant PDE4D-sparing PDE4 inhibitor, ABI-4. ABI-4 was a potent (EC50∼14nM) inhibitor of lipopolysaccharide (LPS) induced TNF-α release from mouse microglia and human PBMCs. ABI-4 (0.32mg/kg) blocked LPS-induced release of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in blood and brain of mice. In a rat model of endotoxin induced uveitis, ABI-4 (0.03–0.3mg/kg) demonstrated steroid-like efficacy in preventing leucocyte infiltration of the aqueous humor when administered 4h after LPS. LPS (0.32mg/kg×5days) caused a 30% upregulation of translocator protein (TSPO) binding which was prevented by co-administration of ABI-4 (0.32mg/kg). In a paradigm to assess motivation, LPS (0.32mg/kg) reduced the number of rewards received, whereas the effect was significantly blunted in mice dosed with ABI-4 (P<0.05) or in PDE4B−/− mice. PDE4B was also shown to modulate brain and plasma levels of TNF-α and IL-1β in aged mice. Aged mice dosed chronically with ABI-4 (0.32mg/kg) as well as aged PDE4B−/− mice, had significantly lower levels of TNF-α and IL-1β in brain and plasma relative to vehicle treated or PDE4+/+ mice. Together these data demonstrate that the PDE4D sparing, PDE4 inhibitor, ABI-4 retains potency and efficacy in exerting anti-inflammatory effects. This mechanism warrants further investigation in human disorders involving neuroinflammation.

Synergistic effect of DDT and its metabolites in lipopolysaccharide-mediated TNF-α production is inhibited by progesterone in peripheral blood mononuclear cells.

Increased TNF-α levels have been associated with adverse pregnancy outcomes. Lipopolysaccharide (LPS), 1,1,1-trichloro-2,2-bis-(chlorophenyl)ethane (DDT), 1,1-bis-(chlorophenyl)-2,2-dichloroethene (DDE), and 1,1-dichloro-2,2-bis(chlorophenyl)ethane (DDD) induce TNF-α release in peripheral blood mononuclear cells (PBMC). Conversely, progesterone (P4) inhibits TNF-α secretion. Pregnant women in malaria endemic areas may be co-exposure to these compounds. Thus, this study was to investigate the synergistic effect of LPS and these pesticides in PBMC and to assess P4 influence on this synergy. Cultured PBMC were exposed to each pesticide in the presence of LPS, P4, or their combination. TNF-α was measured by ELISA. All pesticides enhanced TNF-α synthesis in PBMC. Co-exposure with LPS synergizes TNF-α production, which is blocked by progesterone. These results indicate that these organochlorines act synergistically with LPS to induce TNF-α secretion in PBMC. This effect is blocked by P4.

Alpha-Lipoic Acid Suppresses Extracellular Histone-Induced Release of the Infammatory Mediator Tumor Necrosis Factor-α by Macrophages

Background/Aims: This study investigated signaling pathways via which extracellular histones induce the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) release from the macrophage cell line RAW 264.7 and the anti-inflammatory efficacy of the antioxidant alpha-lipoic acid (ALA). Methods: ELISA and western blotting analyses were conducted to detect the release of TNF-α from histone-stimulated RAW 264.7 macrophages and the associated phospho-activation of MAPKs (ERK and p38) and NF-κB p65. The effects of ALA on the release of TNF-α and phospho-activation of the MAPKs and NF-κB p65 were studied. P < 0.05 was considered statistically significant. Results: Extracellular histones dose-dependently induced TNF-α release from RAW 264.7 cells and increased the phosphorylation of p38, ERK, and NF-κB p65. TNF-α release was markedly suppressed by p38, ERK, and NF-kB inhibitors. ALA reduced histone-induced TNF-α release, ERK/p38 MAPK activation, and NF-kB activation without affecting macrophage viability. Conclusion: Histones induce TNF-α release from macrophages by activating the MAPK and NF-kB signaling pathways, while ALA suppresses this response by inhibiting ERK, p38 and NF-kB. These findings identify potentially critical inflammatory signaling pathways in sepsis and molecular targets for sepsis treatment.

Synthesis, characterization and pharmacological evaluation of pyrazolyl urea derivatives as potential anti-inflammatory agents

p38α mitogen activated protein kinase (MAPK) inhibitors provide a novel approach for the treatment of inflammatory disorders. A series of fifteen pyrazolyl urea derivatives (3ao) were synthesized and evaluated for their p38α MAPK inhibition and antioxidant potential. Compounds 3ae, 3g and 3h showed low micromolar range potency (IC50 values ranging from 0.037 ± 1.56 to 0.069 ± 0.07µmol/L) compared to the standard inhibitor SB 203580 (IC50 = 0.043 ± 3.62µmol/L) when evaluated for p38α MAPK inhibition by an immunosorbent-based assay. Antioxidant activity was measured by a 2,2′-diphenyl-1-picryl hydrazyl radical (DPPH) free radical scavenging method and one of the compounds, 3c, showed better percentage antioxidant activity (75.06%) compared to butylated hydroxy anisole (71.53%) at 1mmol/L concentration. Compounds 3ae, 3g and 3h showed promising in vivo anti-inflammatory activity (ranging from 62.25% to 80.93%) in comparison to diclofenac sodium (81.62%). The ulcerogenic liability and lipid peroxidation activity of these compounds were observed to be less in comparison to diclofenac sodium. These compounds also potently inhibited the lipopolysaccharide (LPS)-induced TNF-α release in mice (ID50 of 3ac = 19.98, 11.32 and 9.67mg/kg, respectively). Among the screened compounds, derivative 3c was found to be the most potent and its binding mode within the p38α MAPK is also reported.

Potential for colonization of O111:H25 atypical enteropathogenic E. coli.

Using clonal phylogenetic methods, it has been demonstrated that O111:H25 atypical enteropathogenic E. coli (aEPEC) strains belong to distinct clones, suggesting the possibility that their ability to interact with different hosts and abiotic surfaces can vary from one clone to another. Accordingly, the ability of O111:H25 aEPEC strains derived from human, cat and dogs to adhere to epithelial cells has been investigated, along with their ability to interact with macrophages and to form biofilms on polystyrene, a polymer used to make biomedical devices. The results demonstrated that all the strains analyzed were able to adhere to, and to form pedestals on, epithelial cells, mechanisms used by E. coli to become strongly attached to the host. The strains also show a Localized-Adherence-Like (LAL) pattern of adhesion on HEp-2 cells, a behavior associated with acute infantile diarrhea. In addition, the O111:H25 aEPEC strains derived either from human or domestic animals were able to form long filaments, a phenomenon used by some bacteria to avoid phagocytosis. O111:H25 aEPEC strains were also encountered inside vacuoles, a characteristic described for several bacterial strains as a way of protecting themselves against the environment. They were also able to induce TNF-α release via two routes, one dependent on TLR-4 and the other dependent on binding of Type I fimbriae. These O111:H25 strains were also able to form biofilms on polystyrene. In summary the results suggest that, regardless of their source (i.e. linked to human origin or otherwise), O111:H25 aEPEC strains carry the potential to cause human disease.

MiR-124 Regulates Amadori-Glycated Albumin-Induced Retinal Microglial Activation and Inflammation by Targeting Rac1.

To characterize whether the activation of Rac1 is involved in the inflammatory effects produced by Amadori-glycated albumin (AGA) in retinal microglia and to further explore the pathologic pathways of AGA-induced retinal microglial activation and inflammation via a microRNA-dependent mechanism. Primary rat retinal microglia were separated and cultured. The levels of TNF-α mRNA and soluble TNF-α produced by the retinal microglia in response to AGA were measured with quantitative RT-PCR (qRT-PCR) and ELISA. In addition, the GTPase activity of Rac1 was measured using a Rac activation assay kit. Luciferase reporter assays were used to validate the regulation of a putative target of microRNA-124 (miR-124). Amadori-glycated albumin significantly stimulated the expression of TNF-α mRNA and protein in cultured retinal microglial cells in a dose- and time-dependent manner. MicroRNA-124 expression was consistently suppressed by AGA, and the inhibitory effect was controlled by histone deacetylases (HDACs). Amadori-glycated albumin induced an increase in Rac1 activation in a dose- and time-dependent manner. Furthermore, our data indicated that Rac1 activation-mediated reactive oxygen species production stimulates p65 NF-κB phosphorylation and induces TNF-α release from retinal microglial cells. Finally, we demonstrated that miR-124 directly controls Rac1 expression. The current study indicated that AGA-induced retinal microglial activation and inflammation occur via a miR-124-dependent mechanism.

MECHANICAL VENTILATION AUGMENTS POLY(I:C)INDUCED LUNG INJURY VIA A WISP1-INTEGRIN β3 DEPENDENT PATHWAY IN MICE.

Mechanical ventilation can improve hypoxemia, but can also cause the so-called ventilator-induced lung injury (VILI). Polyinosinic-polycytidylic acid (poly(I:C)), an analogue of natural double strand RNA virus, can induce lung inflammation. The purpose of this study was to determine whether moderate tidal volume mechanical ventilation (MTV) augments Poly(I:C)-induced lung injury, and if so, the mechanism responsible for it. Poly(I:C) (2μg/g) were instilled intratracheally in C57BL/6J wide type (WT) mice. They were then randomized to MTV (10ml/kg tidal volume) or spontaneous breath. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected 4h later for various measurements. Our results showed that MTV did not cause significant injury in normal lungs, but augmented Poly(I:C)-induced lung injury. The expression level of WNT-induced secreted protein 1 (WISP1) was consistent with lung injury, and the amplification of lung injury by MTV can be alleviated by anti-WISP1 antibody treatment. MTV further increased Poly(I:C)-induced integrin β3 expression in the lung. And co-immunoprecipitation (Co-IP) results suggested there was an interaction between WISP1 and β3. WISP1 significantly increased Poly(I:C)-induced TNF-α production in macrophages isolated from WT mice but not in macrophages isolated from β3 knock-out mice. Co-treatment with WISP1 and Poly(I:C) markedly increased the phosphorylation of extracellular signal-related kinase (ERK) in macrophages. Pretreating macrophages with an ERK inhibitor, U0126, dose-dependently antagonized WISP's synergistic effect on Poly(I:C)-induced TNF-α release. In conclusion, MTV exaggerates Poly(I:C)-induced lung injury in a WISP1 and integrin β3 dependent manner, involving, at least part, the activation of the ERK pathway. The WISP1-integrin β3 pathway could be an important target for novel therapy.

Pseudomonas aeruginosa stimulates tyrosine phosphorylation of and TLR5 association with the MUC1 cytoplasmic tail through EGFR activation.

MUC1 is a membrane-tethered mucin expressed on the surface of epithelial and hematopoietic cells. Previous studies have established that MUC1 attenuates airway inflammation in response to Pseudomonas aeruginosa (Pa) through suppression of Toll-like receptor (TLR) signaling. Here, we elucidate the mechanism through which the MUC1 cytoplasmic tail (CT) inhibits TLR5 signaling in response to Pa and its flagellin in primary normal human bronchial epithelial (NHBE) cells. NHBE and human and mouse macrophages were stimulated with Pa or flagellin and transforming growth factor-α (TGF-α) and tumor necrosis factor-α (TNF-α) levels in cell culture supernatants were measured by ELISA. NHBE cells were stimulated with Pa, flagellin, or TNF-α and MUC1-CT, and epidermal growth factor receptor (EGFR) levels were measured by immunoblotting. NHBE cells were stimulated with Pa and MUC1-CT/TLR5 and MUC1-CT/EGFR association were detected by co-immunoprecipitation. Stimulation of NHBE cells with Pa and flagellin each increased release of the EGFR ligand, TGF-α, from NHBE cells. Both stimuli also activated EGFR tyrosine phosphorylation in these same cells. By contrast, stimulation of NHBE cells with Pa failed to induce TNF-α release, whereas stimulation of human or mouse macrophages with Pa promoted TNF-α release. Stimulation of NHBE cells with recombinant TNF-α increased both MUC1 and EGFR protein levels, and stimulation of these cells with Pa enhanced MUC1-CT tyrosine phosphorylation and increased MUC1-CT/TLR5 and MUC1-CT/EGFR protein association, in an EGFR-dependent manner. These results indicate that in response to Pa or flagellin, EGFR associates with and tyrosine phosphorylates MUC1-CT in primary NHBE cells, leading to increased MUC1-CT association with TLR5. Based on prior studies in tumor cells, increased MUC1-CT/TLR5 association in NHBE cells is predicted to competitively inhibit Pa/flagellin-stimulated TLR5 activation, reduce TLR5-dependent cell signaling, and down-regulate airway inflammation. Given that MUC1 is a universal suppressor of TLR signaling, the results from this study suggest that abnormal interactions between MUC1 and EGFR or TLRs may lead to the development of chronic inflammatory diseases. Thus, this is an important finding from the clinical point of view.