Induction Of TNF-a Research Articles

Stat3 mediates Fyn kinase-driven dopaminergic neurodegeneration and microglia activation.

The Alzheimer's disease and Parkinson's disease risk locus FYN kinase is implicated in neurodegeneration and inflammatory signaling. To investigate in vivo mechanisms of Fyn-driven neurodegeneration, we built a zebrafish neural-specific Gal4:UAS model of constitutively active FynY531F signaling. Using in vivo live imaging, we demonstrated that neural FynY531F expression leads to dopaminergic neuron loss and mitochondrial aggregation in 5 day larval brain. Dopaminergic loss coincided with microglia activation and induction of tnfa, il1b and il12a inflammatory cytokine expression. Transcriptome analysis revealed Stat3 signaling as a potential Fyn target. Chemical inhibition experiments confirmed Fyn-driven dopaminergic neuron loss, and the inflammatory response was dependent upon activation of Stat3 and NF-κB pathways. Dual chemical inhibition demonstrated that Stat3 acts synergistically with NF-κB in dopaminergic neuron degeneration. These results identify Stat3 as a novel downstream effector of Fyn signaling in neurodegeneration and inflammation.

123 Impact of local iron overload on crosstalk and phenotypes of immune and tissue cells in skin

This project focuses on elucidating the pathogenic effects of iron overload due to erythrocyte extravasation in the skin on the immune-tissue cell crosstalk leading to lipodermatosclerosis and leg ulcer in patients with chronic venous insufficiency. We generated a new mice model with local iron-overload in the skin, via ID-injection of iron-dextran (Fedx). Prussian blue staining proved that iron accumulates in the dermis and in the dWAT of these mice, like in patients’ skin. Skin of Fedx mice shows signs of lipodermatosclerosis; an increase of total cell count (p=0.001) in the dermal layer, particularly of F4/80+ macrophages (p=0.019) and PDGFRa+ fibroblasts (p<0.001), accompanied with less collagen in the deeper dermis and a reduction in the size of the dWAT (p<0.001) and of the adipocytes (p<0.001). Microscopic and gene expression analysis highlight lipolysis, the consequent reduction of lipids droplets (p=0.04), and loss of plin-1 (p=0.004) on adipocytes membrane, associated with downregulation of pro-adipogenic genes in the dWAT of Fedx mice. FACS analysis reveals a shift in F4/80+ Macrophage subtypes with the induction of TNFa (p=0.0163) and reduction of CD301b and Relm-a (both p<0.001). Upregulation of CCL2 (p=0.02), IL1ß, HMOX-1 (p=0.0001 dermis, p=0.01 dWAT) and CCR2 (p=0.01) genes, indicate for a pro-inflammatory activation in the dermal compartment. Mechanistic analysis with human cells in vitro shows that erythrocyte-uptake induces ROS and a pro-inflammatory phenotype in Macrophages. Fibroblasts in response to erythrocyte-fed macrophages resemble those phenotypes found in Fedx mice. The impact of erythrocytes on the differentiation and activation of adipocytes and their crosstalk to Macrophages and Fibroblasts is ongoing. Our data suggest that local iron-overload causes a complex skin phenotype with a maintained inflammatory status in the dermis, proliferative and less fibrogenic fibroblasts, impaired adipogenesis, and increased lipolysis.

Airway Prevotella promote TLR2-dependent neutrophil activation and rapid clearance of Streptococcus pneumoniae from the lung

Abstract This study investigates how the lung microbiome influences the early immune response to infection. Prevotella are a major component of the endogenous airway flora, and Prevotella melaninogenica is one of the most commonly isolated species in the human lung. Increased abundance of P. melaninogenica correlates with reduced infection with the bacterial pathogen Streptococcus pneumoniae, indicating a potentially beneficial role. Using a mouse co-infection model, we found that P. melaninogenica enhanced protection against S. pneumoniae, resulting in rapid clearance from the lung and improved survival. This response required recognition of P. melaninogenica lipoproteins by toll-like receptor (TLR)2, the induction of TNFa, and neutrophils, as the loss of any of these factors abrogated Prevotella-induced protection. Improved clearance of S. pneumoniae was associated with increased serine protease-mediated killing by lung neutrophils. IL-10 was also important for restraining P. melaninogenica-induced inflammation in co-infected mice. Together, these findings highlight innate immune priming by the airway microbiome as an important protective feature in the respiratory tract. Supported by the National Institute of Allergy and Infectious Diseases grant K22AI143922, an American Thoracic Society Research Program Grant, an American Lung Association Innovation Award, and a Boettcher Foundation Webb-Waring Biomedical Research Award.

Caffeic Acid Inhibits RANKL and TNFa-induced Osteoclastogenesis by Targeting TAK1-p44/42 MAPK

BACKGROUND: The potential of the caffeic acid in other important Receptor Activator Nuclear Factor kB Ligand (RANKL)-Tumor Necrosis Factor (TNF)a-induced osteoclastogenic signaling pathways has not been known. Therefore, the current study was conducted to explore as well as to understand the inhibition potential of caffeic acid.METHODS: RAW264.7 cells were cultured, treated with caffeic acid, RANKL and TNFa. Tartrate Resistant Acid Phosphatase (TRAP) staining was performed to detect TRAP+ osteoclast-like polynuclear cells. To detect the activity of p44/42 Mitogen Activated Protein Kinase (MAPK), Akt, and Transforming Growth Factor-β-activated Kinase (TAK)1, the phosphorylated forms of the proteins were investigated with the immunoblotting assay.RESULTS: Pre-treatment of caffeic acid inhibited the RANKL and TNFa-induced differentiation of RAW264.7 cells into TRAP+ osteoclast-like polynuclear cells. RANKL and TNFa induced phosphorylation of p44/42 MAPK at Thr202/Tyr204, phosphorylation of Akt at both Ser473 and Thr308 and phosphorylation of TAK1 at Ser412. Pre-treatment with caffeic acid prior to the RANKL and TNFa induction, inhibited the phosphorylation of MAPK, and TAK1, but not Akt.CONCLUSION: Caffeic acid might regulate the RANKL-TNFa-induced osteoclastogenic pathway in RAW264.7 by targeting TAK1, which later activation of p44/42 MAPK was abolished.KEYWORDS: caffeic acid, osteoclastogenesis, p44/42, Erk1/2, Akt, TAK1, RAW264.7

Metabolic Reprogramming of Dendritic Cells Is Controlled By the Mir-142

AbstractDendritic cells (DCs) are key regulators of both immunity and tolerance. Recent studies showed that DC activation is coupled to profound changes in DC metabolism that is characterized by increase in glycolysis. The molecular regulation of these metabolic changes remains poorly understood. MicroRNAs (miRs) have been linked to DC activation, but their role, if any, in critical regulation of DC activation by regulating metabolic reprogramming is unknown. Amongst miRs, one that can critically alters DC's response to stimulation is miR-142 (Sun et al., 2011 and 2013). Utilizing complementary loss and gain of function approaches, and through comprehensive gene arrays, metabolic and functional analyses, we found that miR-142 controls DC maturation and regulates their function. Upon stimulation, the miR-142 KO DCs showed reduced expression of surface molecules such as MHC class II, CD80, CD86 and CD40, and reduced expression of TNFa, but enhanced expression of IL-10 and IL-6 when compared to WT DCs. Priming capability for allogeneic reaction was significantly reduced in miR-142 KO DCs compared to WT DCs as demonstrated by significantly reduced T cell proliferation, expression of IL-2, IFNg and IL-17A. Both WT DCs and miR-142 KO DCs maintained glycolysis switch upon TLR stimulation as showed by similar contents of lactate production, Glut1 and HK2 induction and glucose uptake. These data are consistent with gene expression microarrays analyzed by IPA (Ingenuity Pathway Analysis) and GSEA(Gene Set Enrichment Analysis). Utilizing Seahorse technology, we observed that OCR/ECAR ratios and ATP-linked respiration in miR-142 KO DCs were significantly higher than those in WT DCs, along with an increased mitochondrial membrane potential (ΔΨM). These observations were consistent with our gene expression microarray which showed augmented OXPHOS in miR-142 KO DCs. Gene expression microarray also showed significant increase in fatty acid oxidation (FAO) pathway and enhanced expression of CPT1a, a rate-limiting enzyme of mitochondrial FAO by controlling FA mitochondrial transfer, in miR-142 KO DCs. Consistently, FAO assay showed significantly higher FAO rate in miR-142 KO DCs than that in WT DCs, which was partially, but significantly, reduced after specific knockdown of CPT1a in miR-142 KO DCs. Further investigation revealed that enhanced FAO in miR-142 KO DCs was associated with increase in FA uptake and electron transfer chain activity in miR-142 KO DCs, because of direct targeting CPT1a, FABP4, FABP5, Ndufs7 and Uqcrb. Importantly, restoration of miR-142 in miR-142 deficient DCs produced notable inhibition of protein expressions of its target genes including CPT1a, FABP4, Ndufs7 and UqcrB and induced further significant deduction in FAO, demonstrating that the miR-142 augments FAO in DCs by targeting multiple molecules to promote FA uptake (FABP4 and FABP5), FA mitochondrial transfer (CPT1a) and electron transfer chain activity (Ndufs7 and Uqcrb). Further functional analyses demonstrated that the enhanced OXPHOS and FAO in metabolic reprogramming of DCs that was controlled by miR-142 did not impair trained immunity. Finally, we evaluated the biological relevance of miR-142, and demonstrated that IL-10 expression was dual dependent on glycolysis and FAO in miR-142 KO DCs and that although expressions of IL-12 and TNFa were glycolysis-dependent in both WT and miR-142 KO DCs, FAO is negatively related to TNFa induction in miR-142 KO DCs. Our study demonstrated that targeting miR-142 in DCs may have therapeutic potential for clinic application to prevent autoimmune disease including GVHD and cancer therapy by mediating DC's activation through targeting metabolic reprogramming. DisclosuresNo relevant conflicts of interest to declare.

Potential of miR-25-3p in protection of chondrocytes: emphasis on osteoarthritis.

Osteoarthritis (OA) is the most prevailing musculoskeletal dysfunction triggered by lesions in synovial membranes and articular cartilage. MicroRNAs (miRNAs) have emerged as crucial regulators participated in many biological processes, such as osteoarthritis. This study was undertaken to address the role of miR-25-3p in the apoptosis of rat chondrocytes under an OA-like condition and its underlying mechanism. OA cellular model was established in rat chondrocytes by TNF-a induction. Then, qRTPCR and Western blotting were utilized for evaluation of the expressions of miR-25-3p and insulin-like growth factor-binding protein 7 (IGFBP7), CCK-8 assay for inspection of chondrocyte viability, flow cytometry for assessment of cell apoptosis rate, Western blotting for the detection of cleaved caspase-3 level and dual-luciferase reporter gene assay for verification of the targeting relationship between miR-25-3p and IGFBP7. The miR-25-3p expression was decreased and IGFBP7 was elevated in TNF-a-induced rat chondrocytes. The miR-25-3p inhibited chondrocyte apoptosis and IGFBP7 promoted apoptosis as evidenced by enhanced cell viability and suppressed cell apoptosis in OA chondrocytes after miR-25-3p overexpression or IGFBP7 knockdown. The miR-25-3p facilitated chondrocyte viability and repressed cell apoptosis in OA by negatively regulating IGFBP7. MiR-25-3p negatively regulates IGFBP7 to promote chondrocyte proliferation and restrain chondrocyte apoptosis. Our findings suggest that the regulation of IGFBP7 by miR-25-3p may emerge as a novel therapeutic regimen for OA.

Age-associated decline of monocyte insulin sensitivity in diabetic and healthy individuals.

Objective:It is unclear whether monocyte/macrophage insulin signaling in humans is affected by type 2 diabetes (T2DM), systemic insulin sensitivity, and other unknown factors.Research design and methods:Fifty-three adult volunteers (control group) not taking any medication and without cardiovascular risk factors, and 59 patients with T2DM (T2DM group) were included. Monocytes were isolated and cultured from all participants.Results:In cultured monocytes, insulin-stimulated AKT and FOXO3 phosphorylation was significantly suppressed in T2DM compared with that in the control group. Insulin-stimulated phosphorylation of AKT was significantly correlated with body mass index and serum insulin level only in the control group. In both groups, significant negative correlation between age and insulin-stimulated phosphorylation of AKT and FOXO3 was commonly observed. In the control group, lipopolysaccharide (LPS)-stimulated induction of TNFA, and NOS2 was significantly and negatively correlated with insulin-stimulated AKT phosphorylation. Age was also significantly correlated with LPS-stimulated induction of TNFA.Discussion:Aging plays an important role in the development of monocyte insulin resistance, not only in patients with T2DM but also in healthy participants. Monocyte insulin sensitivity is negatively correlated with inflammatory responses and may be helpful for subclinical risk assessment of CVDs and/or insulin resistance in participants without risk factors.

Sustained hyperammonemia induces TNF-a IN Purkinje neurons by activating the TNFR1-NF-\u03baB pathway

BackgroundPatients with liver cirrhosis may develop hepatic encephalopathy. Rats with chronic hyperammonemia exhibit neurological alterations mediated by peripheral inflammation and neuroinflammation. Motor incoordination is due to increased TNF-a levels and activation of its receptor TNFR1 in the cerebellum.The aims were to assess (a) whether peripheral inflammation is responsible for TNF-a induction in hyperammonemic rats, (b) the cell type(s) in which TNF-a is increased, (c) whether this increase is associated with increased nuclear NF-κB and TNFR1 activation, (d) the time course of TNF-a induction, and (e) if TNF-a is induced in the Purkinje neurons of patients who die with liver cirrhosis.MethodsWe analyzed the level of TNF-a mRNA and NF-κB in microglia, astrocytes, and Purkinje neurons in the cerebellum after 1, 2, and 4 weeks of hyperammonemia. We assessed whether preventing peripheral inflammation by administering an anti-TNF-a antibody prevents TNF-a induction. We tested whether TNF-a induction is reversed by R7050, which inhibits the TNFR1-NF-κB pathway, in ex vivo cerebellar slices.ResultsHyperammonemia induced microglial and astrocyte activation at 1 week. This was followed by TNF-a induction in both glial cell types at 2 weeks and in Purkinje neurons at 4 weeks. The level of TNF-a mRNA increased in parallel with the TNF-a protein level, indicating that TNF-a was synthesized in Purkinje cells. This increase was associated with increased NF-κB nuclear translocation. The nuclear translocation of NF-κB and the increase in TNF-a were reversed by R7050, indicating that they were mediated by the activation of TNFR1. Preventing peripheral inflammation with an anti-TNF-a antibody prevents TNF-a induction.ConclusionSustained (4 weeks) but not short-term hyperammonemia induces TNF-a in Purkinje neurons in rats. This is mediated by peripheral inflammation. TNF-a is also increased in the Purkinje neurons of patients who die with liver cirrhosis. The results suggest that hyperammonemia induces TNF-a in glial cells and that TNF-a released by glial cells activates TNFR1 in Purkinje neurons, leading to NF-κB nuclear translocation and the induction of TNF-a expression, which may contribute to the neurological alterations observed in hyperammonemia and hepatic encephalopathy.

Lipopolysaccharide Adsorbed to the Bio-Corona of TiO2 Nanoparticles Powerfully Activates Selected Pro-inflammatory Transduction Pathways.

It is known that the adsorption of bioactive molecules provides engineered nanoparticles (NPs) with novel biological activities. However, the biological effects of the adsorbed molecules may also be modified by the interaction with NP. Bacterial lipopolysaccharide (LPS), a powerful pro-inflammatory compound, is a common environmental contaminant and is present in several body compartments such as the gut. We recently observed that the co-incubation of LPS with TiO2 NPs markedly potentiates its pro-inflammatory effects on murine macrophages, suggesting that, when included in a NP bio-corona, LPS activity is enhanced. To distinguish the effects of adsorbed LPS from those of the free endotoxin, a pellet fraction, denominated P25/LPS, was isolated by centrifugation from a mixture of P25 TiO2 NP (128 µg/ml) and LPS (10 ng/ml) in the presence of fetal bovine serum. Western blot analysis of the pellet eluate indicated that the P25/LPS fraction contained, besides proteins, also LPS, pointing to the presence of LPS-doped NP. The effects of adsorbed or free LPS were then compared in Raw264.7 murine macrophages. RT-PCR was used to evaluate the induction of cytokine genes, whereas active, phosphorylated isoforms of proteins involved in signaling pathways were assessed with western blot. At a nominal LPS concentration of 40 pg/ml, P25/LPS induced the expression of both NF-κB and IRF3-dependent cytokines at levels comparable with those observed with free LPS (10 ng/ml), although with different time courses. Moreover, compared to free LPS, P25/LPS caused a more sustained phosphorylation of p38 MAPK and a more prolonged induction of STAT1-dependent genes. Cytochalasin B partially inhibited the induction of Tnfa by P25/LPS, but not by free LPS, and suppressed the induction of IRF3-dependent genes by either P25/LPS or free LPS. These data suggest that, when included in the bio-corona of TiO2 NP, LPS exhibits enhanced and time-shifted pro-inflammatory effects. Thus, in assessing the hazard of NP in real life, the enhanced effects of adsorbed bioactive molecules should be taken into account.

The aliens inside human DNA: HERV-W/MSRV/syncytin-1 endogenous retroviruses and neurodegeneration.

The human genome contains remnants of ancestral retroviruses now endogenously transmitted, called human endogenous retroviruses (HERVs). HERVs can be variably expressed, and both beneficial and detrimental effects have described. This review focuses on the MSRV and syncytin-1 HERV-W elements in relationship to neurodegeneration in view of their neuro-pathogenic and immune-pathogenic properties. Multiple sclerosis (MS) and a neurodegenerative disease (neuroAIDS) are reported in this review. In vivo studies in patients and controls for molecular epidemiology and follow-up studies are reviewed, along with in vitro cellular studies of the effects of treatments and of molecular mechanisms. HERV-W/MSRV has been repeatedly found in MS patients (in blood, spinal fluid, and brain samples), and MRSV presence/load strikingly parallels MS stages and active/remission phases, as well as therapy outcome. The DNA of MS patients has increased MSRVenv copies, while syncytin-1 copies are unchanged in controls. Presence of MSRV in the spinal fluid predicted the worst MS progression, ten years in advance. The Epstein-Barr virus (EBV) activates HERV-W/MSRV both in vitro and in vivo. With respect to neuroAIDS, the HIV transactivator of transcription (Tat) protein activates HERV-W/MSRV in monocytes/macrophages and astrocytes indirectly by interaction with TLR4 and induction of TNFa. HERV-W/MSRV can be considered a biomarker for MS behavior and therapy outcome. Regarding MS pathogenesis, we postulate the possibility for EBV of an initial trigger of future MS, years later, and for MSRV of a direct role of effector of neuropathogenesis during MS. Additionally, HERV-W/MSR/syncytin-1 activation by HIV Tat could contribute to the HIV-related neurodegeneration.

Regulation of Hypoxia-Induced Inflammatory Responses and M1-M2 Phenotype Switch of Primary Rat Microglia by Sex Steroids

Microglia cells are the primary mediators of the CNS immune defense system and crucial for the outcome of shaping inflammatory responses. They are highly dynamic, moving constantly, and become activated by neuronal signaling under pathological conditions. They fulfill a dual role by not only regulating local neuroinflammation but also conferring neuronal protection. Gonadal steroids are known to exert anti-inflammatory effects in the CNS. Recently, we have shown that the microglial-like cell line BV-2 is hypoxia-sensitive and regulated by gonadal steroids. The present study used primary rat cerebral cortex-derived microglia to analyze whether this cell type directly perceive and respond to acute hypoxia. Second, we investigated whether 17β-estradiol (E2) and progesterone (P) interfere with hypoxia-induced changes. Short-term hypoxia increased the expression of a subset of pro-inflammatory (TNFa, IL1b) and oxidative stress-related (Hif1a) genes. The induction of TNFa and IL1b was counteracted by P. Hypoxia shifted the primary microglia to the pro-inflammatory M1 phenotype. The administration of E2 and P favored the neuroprotective M2 phenotype. Our findings extend previous data obtained with BV-2 cells and show that the primary microglia directly perceive hypoxia which increase their inflammatory activity. Both steroid hormones directly and indirectly interact with the microglia cells by reducing the inflammatory scenario and stimulating neuroprotection.

Different Responses of RT‐PCR Amplifications after Tumor Necrosis alpha (TNFa) Induction at Nuclear Factor kappa B (NFkB) Gene Silenced LNCaP Cells

Prostate cancer is the second leading reason of morbidity and mortality in men in the Western World. Androgen receptor positive and androgen responsive lymph‐node metastasis of prostate cancer cell line LNCaP was used in all further experiments. Western with NFkB specific antibody was done after TNFa induction at LNCaP cells and a significant increase was taken.LNCaP cells transfected with siNFkB construct for 1,2 and 4 days and induced with TNFa for 24 hours. RNAisolation, cDNA conversion and RT‐PCR amplifications were done with apoptosis related gene primers such as p53, Bcl‐2, STAMP family primers. Chromatine immunoprecipitation (ChIP) assay was done with NFkB specific antibody before and after TNF induction at LNCaP and normal prostate cell line WPMY1. Since both cell lines express STAMP1 and 2, promoter region located NFkB response‐elements were identified and shown with specific primers. However, no change was shown at STAMP2 RT‐PCR amplification at −/+ TNFa native/siNFkB; STAMP1 showed a significant decrease with TNFa induction, while a significant increase with siNFkB.Survival gene NFkB silencing caused anti‐apoptotic STAMP1 increase that repress p53, together with MDM2.Gene silencing differed a panel of cancer regulatory genes.

Abstract 3732: New aspirin analogs for targeted therapy of ER+ breast cancers with activated NFkB inflammatory pathway

Abstract Approximately 75% of breast cancers express estrogen receptor (ER), classified as either ER+ luminal A or luminal B. Five year survival rate of luminal B breast cancers is ∼50% compared to ∼95% for luminal A, making luminal B subtype a target for better understanding the mechanisms of carcinogenicity and improved therapy. Inflammation and activation of its nuclear factor NFkB, is linked clinically and epidemiologically to cancer. Activation of NFkB in a subset of ER+ cancers is associated with poor prognosis and resistance to therapy. We were the first to show synergy between ER and NFkB in a specific gene signature that highly correlates with luminal B ER+ breast cancers, suggesting a potential functional role for NFkB inhibition in this subset. Based on these findings, we hypothesized that co-targeting ER and NFkB may achieve clinical efficacy in luminal B breast cancer. To target the ER, a selective estrogen receptor modulator (SERM) was employed. To target NFkB pathway, an NSAID-based therapy would be safe and, based on epidemiological data, is suggested to be chemopreventive in breast cancer. For NFkB targeting, we characterized a family of aspirin (ASA)-based analogs (x-ASA) that also serve as pro-drugs for quinone methide formation. We found that x-ASA analogs inhibited TNFa induction of NFkB via a reporter assay in a dose-dependent fashion in MCF-7 cells, and also shut down several genes typically up-regulated by the ER-NFkB crosstalk. Next we tested the co-targeting approach in mammosphere formation assays: the combination significantly reduced overall mammosphere number and size. While the mechanisms of action remains to be elucidated, the aspirin analogs in combination with a SERM show promise in treating ER+ breast cancers with activated NFkB where standard endocrine therapy alone fails. 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 3732. doi:1538-7445.AM2012-3732

In a Xenograft Mouse Model, Bethanechol, a Muscarinic Receptor Agonist, Attenuates the Growth of Human Colon Cancer Cells

hSRBC is a recently identified tumor suppressor located at the chromosomal region 11p15.4. hSRBC expression is frequently downregulated in many types of malignancies including colorectal cancer due to the aberrant promoter CpG sites hypermethylation. The hSRBC activation induces cell cycle arrest and apoptosis by enhancing the protein stability of p53. However, the molecular basis of hSRBC activation and its regulation has been poorly understood. To explore the implication of hSRBC abnormality in colorectal tumorigenesis and the molecular mechanism underlying its tumor suppressive function, the present study tested the possible regulation of hSRBC by TNFα.TNFα regulation of hSRBC expression was examined using semi-quantitative RT-PCR and immunoblot assays. It was found that mRNA expression of hSRBC is strongly activated by TNFa in a doseand time-associated manner in various human colonic epithelial cells, includingHCT15 andHT-29. An immunoblot assay also revealed that hSRBC protein expression is increased following TNFa treatment. TNFa induction of hSRBC was blocked by pretreatment of the cells with the NFkB inhibitor BAY11-7082 or transfection with a dominant negative mutant form of IkB, indicating that TNFα-induced hSRBC expression is mediated via the NFkB pathway. Flow cytometric analysis of sub-G1 phase using hSRBC-nonexpressing HCT15 cells revealed that ectopic expression of hSRBC greatly enhances apoptotic response to TNFα. Collectively, these data strongly suggest that hSRBC might be a novel transcription target of TNFα and play an important role in cellular response to TNFα. TNFα regulation of hSRBC expression was examined using semi-quantitative RT-PCR and immunoblot assays. It was found that mRNA expression of hSRBC is strongly activated by TNFα in a doseand time-associated manner in various human colonic epithelial cells, including HCT15 and HT-29. An immunoblot assay also revealed that hSRBC protein expression is increased following TNFa treatment. TNFa induction of hSRBC was blocked by pretreatment of the cells with the NFkB inhibitor BAY11-7082 or transfection with a dominant negative mutant form of IkB, indicating that TNFα-induced hSRBC expression is mediated via the NFkB pathway. Flow cytometric analysis of sub-G1 phase using hSRBC-nonexpressing HCT15 cells revealed that ectopic expression of hSRBC greatly enhances apoptotic response to TNFα. Collectively, these data strongly suggest that hSRBC might be a novel transcription target of TNFα and play an important role in cellular response to TNFα.

In Vitro Exploratory Studies of Haptoglobin Polymorphisms and Their Effect on Cytokine Release from Cultured Mononuclear Cells in Sickle Cell Disease.

The deleterious effects of hemolysis through its end product cell free hemoglobin(Hb) as a nitric oxide (NO) scavenger is well established and has been incriminated in the pathogenesis of some complications of SCD such as pulmonary hypertension, leg ulcers, renal dysfunction, and possibly stroke. These observations have led some investigators to hypothesize that these complications form a subphenotype of SCD related predominantly to hemolysis and endothelial dysfunction. In hemolytic states Hb released from RBC complexes with Haptoglobin (Hp) and is removed from the circulation by macrophages and monocytes through binding CD163, the Hb scavenger receptor expressed on these cells. When the binding capacity of Hp is exceeded, the concentration of free Hb rises in the plasma. Hp is a polymorphic protein encoded by a gene on chromosome 16q2.2; there are two allelic variants, Hp 1 and Hp 2. Hp 2 is believed to have resulted from an intragenic duplication event, leading to an elongated Hp a-chain. Individuals homozygous for the long a2 chain express large multimeric molecules (Hp 2-2). During the past decade, a considerable body of evidence has accumulated suggesting that Hp-2 allele is a major susceptibility gene for the development of vascular complications (coronary artery restenosis and development of cardiovascular disease) especially in diabetic patients. It has been hypothesized that the Hb-Hp2 complexes have a 10-fold greater affinity for the CD 163 receptor, and the binding of Hb-Hp2 complexes generates a more powerful inflammatory response with a more prominent cytokine release. Recently, we performed a preliminary analysis of the distribution of Hp1 and Hp2 alleles among pediatric and adult SCD patients and reported a significantly higher allele frequency for Hp2 among pediatric patients, suggesting a survival advantage for carriers of Hp1 allele (Yaun et al, Blood, 2005). We now report the results of an exploratory in vitro study of cytokine release from purified mononuclear cells obtained from a normal control and an SCD patient following exposure to Hb-Hp1-1 and Hb-Hp2-2 complexes. Mononuclear cells (106/well) isolated by Ficoll-Hypaque density gradient were incubated with Hb A-Hp1-1, Hb A-Hp2-2, Hb S Hp1-1, and Hb S-Hp2-2 complexes with a 1:1 ratio (wt/wt) at a final concentration of 1 mg/ml. After 24 hr incubation at 37°C, the supernatants collected after centrifugation were used for cytokine assays by a multiplex bead method. A blank (medium only), Hb A and Hb S without Hp were also incubated with mononuclear cells. Multiplex bead assays showed that cytokine release (GM-CSF, IL-1b, IL-6, IL-10, and TNFa) was much higher (3–12 fold) from both the control and SCD mononuclear cells upon exposure to Hb-Hp2-2 complexes, but much less or no effect by Hb-Hp1-1. The fold induction of TNFa and IL-1b was much higher in SCD cells than in control cells. There was no significant difference between Hb A and Hb S in terms of cytokine release when they were complexed with either Hp1-1 or Hp2-2, suggesting that the cytokine release was predominantly related to Hp type but not to Hb. Pure Hb A and Hb S increased cytokines over the control (blank) but to a significantly smaller extent than Hb (A or S) Hp-2-2 complexes.These preliminary results are confirmatory of a deleterious effect of the Hp2-2 genotype through a more pronounced inflammatory response and are suggestive of a potential novel mechanism whereby hemolysis could result in adverse outcomes related to Hp polymorphisms. If confirmed in larger studies and through phenotypic associations, attenuation of this response via anti-inflammatory modalities may provide a therapeutic strategy. [Display omitted]

Contribution of Interferon-β to the Murine Macrophage Response to the Toll-like Receptor 4 Agonist, Lipopolysaccharide

Interferon-beta (IFN-beta) has been identified as the signature cytokine induced via the Toll-like receptor (TLR) 4, "MyD88-independent" signaling pathway in macrophages stimulated by Gram-negative bacterial lipopolysaccharide (LPS). In this study, we analyzed the responses of macrophages derived from wild-type (IFN-beta(+/+)) mice or mice with a targeted mutation in IFN-beta (IFN-beta(-/-)) to the prototype TLR4 agonist, Escherichia coli LPS. A comparison of basal and LPS-induced gene expression (by reverse transcription-PCR, real-time PCR, and Affymetrix microarray analyses) resulted in the identification of four distinct patterns of gene expression affected by IFN-beta deficiency. Analysis of a subset of each group of differentially regulated genes by computer-assisted promoter analysis revealed putative IFN-responsive elements in all genes examined. LPS-induced activation of intracellular signaling molecules, STAT1 Tyr-701, STAT1 Ser-727, and Akt, but not p38, JNK, and ERK MAPK proteins, was significantly diminished in IFN-beta(-/-) versus IFN-beta(+/+) macrophages. "Priming" of IFN-beta(-/-) macrophages with exogenous recombinant IFN-beta significantly increased levels of LPS-induced gene expression for induction of monocyte chemotactic protein 5, inducible nitric-oxide synthase, IP-10, and IL-12 p40 mRNA, whereas no increase or relatively small increases were observed for IL-1beta, IL-6, monocyte chemotactic protein 1, and MyD88 mRNA. Finally, IFN-beta(-/-) mice challenged in vivo with LPS exhibited increased survival when compared with wild-type IFN-beta(+/+) controls, indicating that IFN-beta contributes to LPS-induced lethality; however, not to the extent that one observes in mice with more complete pathway deficiencies (e.g. TLR4(-/-) or TRIF(-/-) mice). Collectively, these findings reveal unanticipated regulatory roles for IFN-beta in response to LPS in vitro and in vivo.

TNF-alpha and TNF Receptor Type p75 are Induced by Recombinant Hepatitis C Virus Proteins in Peripheral Mononuclear Blood Cells

Recent studies showed an activation of the TNF system in patients with chronic Hepatitis C Virus (HCV) infection correlating with the severity of the disease. Peripheral blood mononuclear cells (PBMC), representing extrahepatic sites for HCV replication and a source of inflammatory cytokines play a central role in the regulation of immune-mediated response. The aim of this project was to imitate the very first contact with HCV of healthy individuals. To characterize the innate and primary response of PBMC to HCV components, we studied the cellular induction of TNF-alpha and TNF specific receptors TNFR–p55, -p75 after stimulation with recombinant HCV proteins in vitro. PBMC of 14 healthy individuals were incubated with recombinant HCV proteins from different core regions and non-structural (NS) proteins (NS3 and NS4) at optimal concentrations. TNF-alpha, soluble TNF receptors -p55 and -p75 were determined by enzyme-linked-immuno-assays and synthesis of specific mRNA was analyzed by RT-PCR technique. The most potent specific stimulus was the HCV core derived protein aa 1–83 with induction of TNF-a (5,8±1,9 ng/ml; p<0.001) and TNFR-p75 (3,9±0.8 ng/ml; p<0.001), respectively. The second most important inducer was NS4, followed by core and NS3. Protein synthesis was adequately paralleled by specific mRNA induction. The data show that stimulation of immunocompetent PBMC with HCV proteins leads to a specific enhancement of the TNFR-p75 protein as compared to TNF-p55. The corresponding antagonist TNF-alpha and its receptor TNFR-p75 are therefore not only involved in the pathogenesis of chronic HCV infection but already in the very early onset of the infection.

Immunomodulatory Effects of Lactic Acid Bacteria on Human Peripheral Blood Mononuclear Cells

The aim of study was to evaluate the effect of a mixture of three strains (administered in vitro ) of lactic acid bacteria ( Lactobacillus acidophilus, Lactobacillus delbrueckii ssp. bulgaricus, Bifidobacterium bifidum ) designed as Trilac, representing a part of normal human microflora on: 1) human peripheral blood mononuclear cells (PBMC) proliferation, 2) profile of cytokine synthesis and 3) expression of monocyte and lymphocyte receptors, known to be involved in non-specific and specific immune responses. Trilac induced only a moderate proliferative response (about 10-20% of PHA-induced level) in PBMC. The CD4+ T cell was the responder subset. Trilac was a rather poor inducer of IFN-g and IL-12, simultaneously inducing relatively higher expression of IL-10 and TNF-a. Trilac also induced an increase in the expression of some monocyte surface receptors (CD14, IL-2R, HLA-DR, ICAM-1, CD80), simultaneously having only slight effect on the expression of lymphocyte surface receptors, as determined by double colour flow cytometry. We conclude that stimulation of PBMC with commensal lactic acid bacteria ongoing in Trilac resulted in a characteristic pattern of immune response manifested by: a) restriction of proinflammatory activities (IL-10 prevailed over IL-12 and IFN-g), b) significant activation of monocytes but rather slight T-cell induction. Hence, Trilac-induced immune activities may indicate its potential ability for limitation of pathogen-induced pro-inflammatory responses in microflora (via IL-10 action) and simultaneous induction of antimicrobial effect (via TNF-a induction and monocyte activation). Key words : lactic acid bacteria, lymphocytes, monocytes, cytokines, co-stimulatory receptors.

Leptospira interrogans and Leptospira peptidoglycans induce the release of tumor necrosis factor a from human monocytes

Elevated plasma concentrations of the cytokine tumor necrosis factor α (TNFα ) have been observed in patients affected by leptospirosis. In this study we found that a preparation of peptidoglycan of Leptospira interrogans, serovar copenhageni, was able to induce the release of TNFa from peripheral blood mononuclear cells. TNFa induction occurred in a dose dependent manner and was not affected by the endotoxin inhibitor polymixin B. This is the first report on induction of TNFa release by a peptidoglycan of spirochetes. Our findings are consistent with existing clinical data and provide a potential mechanism for TNFa production.

The mapping of HIV-1 gpl60 epitopes required for interleukin-1 and tumor necrosis factor a production in glial cells

The pathology in central nervous system (CNS) AIDS suggests that direct infection with HIV-1 is not required for changes in glia and neurons. Induction of a variety of pathological responses in vitro in rodent brain cultures also suggests that CD4 is not the receptor for HIV-1 in the brain, given that human and rodent CD4 are not homologous. This implies that the epitopes on HIV-1 which bind glia and activate them are novel, non-CD4-binding domains. We have therefore mapped the envelope (env) regions required for production in rat glial cultures of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFα) which we hypothesize are important in CNS AIDS. Serially truncated deletion mutants from the gp120/gp41 carboxy terminus representing folded, glycosylated recombinant env proteins were expressed in HeLa cells via a vaccinia virus vector. These proteins, linear gp120/gp41 peptides, as well as polyclonal and monoclonal antibodies reactive to defined regions of gp120/gp41 were used to map the epitopes involved in production of IL-1 and TNFα. Compared to HeLa cell and wild-type vaccinia virus controls, the vaccinia recombinant env protein gp160 containing cleaved gp120 and gp41 induced both IL-1 and TNFa. If gp160 was not cleaved into gp120 and gp41, IL-1 but not TNFa induction was reduced. Peak production of TNFa by gp120/gp41 was at 4 h while IL-1 production was still significantly elevated at 44 h at the highest concentrations of env protein. Using the truncation deletions, the V3 loop of gp120 appeared to be critical for IL-1. Glycosylation and folding of V3 is probably important in IL-1 induction since a V3 peptide was not as active. While removal of glycosylated, folded V4 and C4 regions had no effect on IL-1, linear peptides in the region from the V4 loop to the C4 domain were strong inducers of IL-1. Non-glycosylated, linear V4 loop peptide induced more IL-1 than the V4 in protein generated in HeLa cells, suggesting that glycosylation and/or conformational structures sequester V4 inducer epitopes. Using the truncation deletions, the carboxy terminus region (V4-C5) of gp120 as well as gp41 were shown to be critical for TNFa production. Peptides representing linear epitopes in the V3 loop, C5 domain of gp120, and the ectodomain of gp41 were all strong inducers of TNFα; a protein representing almost the entire gp41 was the strongest inducer of TNFα. This suggests both conformational and linear determinants induce TNFα. Glycosylated, folded VI-3 and C1-3, exclusive of other gp120 domains, did not induce TNFα production. These data were further confirmed by antibody inhibition of env regions. Cytokine induction by env proteins was transcriptionally regulated, since messenger RNA levels were increased as determined by Northern blot analysis. In summary, virus infection is not required and gp160 epitopes, other than the classical CD4 binding domains, are involved in induction of IL-1 and TNFa in glial cell cultures. These data suggest that HIV-1 env proteins in their native form, as well as those that are partially denatured (linearized or non-glycosylated) could induce two proinflammatory cytokines which are possibly important in CNS AIDS.