Tumor Necrosis Factor Synthesis Research Articles

The level of matrix metalloproteinases in the blood in a model of adjuvant-induced arthritis and its correction with antisense oligonucleotides

Rheumatoid arthritis is characterized by significant rearrangements in bone and cartilage tissues mediated by matrix metalloproteinases (MMPs). One of the directions in the development of new drugs for rheumatoid arthritis is the search for new targets, including MMPs or their inhibitors. The work aimed to determine the levels of MMP-1, 3, and 9 in the peripheral blood of laboratory rats with a model of adjuvant-induced arthritis during the correction of this condition with antisense oligonucleotides that block the synthesis of tumor necrosis factor and interleukin 6. In laboratory Wistar rats, arthritis was modeled by administering Freund's adjuvant and then correcting the disease by introducing antisense oligonucleotides subcutaneously and using electrophoresis. The level of MMP in peripheral blood was determined at the beginning of treatment, after 1, 2, and 3 months. Chronic inflammation in the experiment was accompanied by an increase in the level of MMP-1, 3, and 9 in the peripheral blood by 2, 4.5, and 0.5 times, respectively. Blocking the proinflammatory cytokines TNF-α and IL-6 with antisense oligonucleotides was effective in reducing MMP levels. In most cases, it decreased significantly faster than in the untreated arthritis model. These changes were most pronounced in groups using tocilizumab, as well as in group with an electrophoretic form of administration of a combination of two oligonucleotides.

Diminazene aceturate attenuates systemic inflammation via microbiota gut-5-HT brain-spleen sympathetic axis in male mice

The sympathetic arm of the inflammatory reflex is the efferent pathway through which the central nervous system (CNS) can control peripheral immune responses. Diminazene aceturate (DIZE) is an antiparasitic drug that has been reported to exert protective effects on various experimental models of inflammation. However, the pathways by which DIZE promotes a protective immunomodulatory effects still need to be well established, and no studies demonstrate the capacity of DIZE to modulate a neural reflex to control inflammation. C57BL/6 male mice received intraperitoneal administration of DIZE (2 mg/Kg) followed by lipopolysaccharide (LPS, 5 mg/Kg, i.p.). Endotoxemic animals showed hyperresponsiveness to inflammatory signals, while those treated with DIZE promoted the activation of the inflammatory reflex to attenuate the inflammatory response during endotoxemia. The unilateral cervical vagotomy did not affect the anti-inflammatory effect of DIZE in the spleen and serum. At the same time, splenic denervation attenuated tumor necrosis factor (TNF) synthesis in the spleen and serum. Using broad-spectrum antibiotics for two weeks showed that LPS modulated the microbiota to induce a pro-inflammatory profile in the intestine and reduced the serum concentration of tryptophan and serotonin (5-HT), while DIZE restored serum tryptophan and increased the hypothalamic 5-HT levels. Furthermore, the treatment with 4-Chloro-DL-phenylalanine (pcpa, an inhibitor of 5-HT synthesis) abolished the anti-inflammatory effects of the DIZE in the spleen. Our results indicate that DIZE promotes microbiota modulation to increase central 5-HT levels and activates the efferent sympathetic arm of the inflammatory reflex to control splenic TNF production in endotoxemic mice.

Effectiveness of Histopathological Changes of Induced Thin Layer Endometrium by Pentoxifylline and Pentoxifylline-Loaded Poly Lactic-co-Glycolic Acid on Female Rats.

Pentoxifylline (PTXF) is a vasoactive agent that plays a significant role in the treatment of thin-layer endometrium cases. The PTXF, also identified as oxpentifylline, is a member of xanthine derivatives and a competitive nonselective phosphodiesterase inhibitor leading to the elevation of intracellular cAMP, inhibition of tumor necrosis factor and leukotriene synthesis, activation of protein kinase A, and reduction of inflammation and innate immunity. Moreover, it is used as an agent to relieve muscle pain in people with peripheral artery disease (vascular irregularities). It is also an acceptable choice for the treatment of radiation-induced fibrosis. Therefore, the present study aimed to determine the advantageous impact of PTXF and PTXF-loaded poly lactic-co-glycolic acid (PLGA) on female rats after being exposed to ethanol to create a thin layer of the endometrium. For this purpose, 50 female rats were selected and divided into five groups (G1: negative normal control, G2: positive control, G3: PLGA only, G4: preference PTXF, and G5: PLGA-PTXF groups) for a 20-day treatment period. In this study, the histopathological section revealed a perfect improvement in the tissues of the uterine horn of female rats that induced endometria and were treated with PLGA-PTXF. In this group of rats, clear healing was achieved and there was an increase in the thickness of endometrium and myometrium, compared to the ordinary PTXF-treated group which had the lowest recovery characteristics. However, the positive control group underwent a significant decrease in terms of endometrium and myometrium thickness as well as vascular and glandular density. This study showed that the PTXF-loaded PLGA had the capacity to heal the thin layer of the endometrium by improving the levels of histopathological changes, especially regarding the thickness of the endometrium and myometrium more than the ordinary PTXF.

Meloxicam Inhibits Apoptosis in Neurons by Deactivating Tumor Necrosis Factor Receptor Superfamily Member 25, Leading to the Decreased Cleavage of DNA Fragmentation Factor Subunit α in Alzheimer's Disease.

Neuronal apoptosis is considered to be a critical cause of Alzheimer's disease (AD). Recently, meloxicam has shown neuroprotective effects; however, the inherent mechanisms are highly overlooked. Using APP/PS1 transgenic (Tg) mice as in vivo animal models, we found that meloxicam inhibits apoptosis in neurons by deactivating tumor necrosis factor receptor superfamily member 25 (TNFRSF25), leading to the suppression of the expression of fas-associated protein with death domain (FADD) and the cleavage of DNA fragmentation factor subunit α (DFFA) and cysteine aspartic acid protease-3 (caspase 3) via β-amyloid protein (Aβ)-depressing mechanisms. Moreover, the meloxicam treatment blocked the effects of β-amyloid protein oligomers (Aβo) on stimulating the synthesis of tumor necrosis factor α (TNF-α) and TNF-like ligand 1A (TL1A) in neuroblastoma (N) 2a cells. TNF-α and TL1A induce apoptosis in neurons via TNFR- and TNFRSF25-dependent caspase 3-activating mechanisms, respectively. Knocking down the expression of TNFRSF25 blocked the effects of TL1A on inducing apoptosis in neurons by deactivating the signaling cascades of FADD, caspase 3, and DFFA. Consistently, TNFRSF25 shRNA blocked the effects of Aβo on inducing neuronal apoptosis, which was corroborated by the efficacy of meloxicam in inhibiting Aβo-induced neuronal apoptosis. By ameliorating neuronal apoptosis, meloxicam improved memory loss in APP/PS1 Tg mice.

Post Covid-19 pulmonary fibrosis: A review article

COVID-19 also known as Coronavirus disease 2019 is an infectious disease caused by the highly contagious pathogen SARS-CoV-2.Various studies have found that 70–80% of patients who recovered from COVID-19 continue to have at least one or more symptoms even after being pronounced COVID-free. Pulmonary fibrosis is a severe respiratory illness consequence. It is defined by the lungs' failure to repair the injured alveolar epithelium, the persistence of fibroblasts, and the excessive deposition of collagen and other extracellular matrix components. An initial phase of lung injury is followed by acute inflammation and an effort at healing. Oxygen toxicity and ventilator-induced lung injury are two iatrogenic variables that may contribute to the fibrosis seen in survivors of severe COVID19 pneumonia. Based on scientific evidence and demographic findings, Pirfenidone and Nintedanib, used alone or in combination with anti-inflammatory agents and this combination is effective in preventing serious complications during COVID-19 infection. Pirfenidone inhibits TGF-β stimulated collagen synthesis and the synthesis of tumor necrosis factor, interferon-gamma, interleukin- 1beta, and interleukin-6 resulting in anti-inflammatory action. Depending on the concentration, pirfenidone has been demonstrated to have antioxidant capabilities. Nintedanib saw inhibitory action on pro-fibrotic mediators like platelet-derived growth factor and fibroblast growth factor, transforming growth factor-beta, and vascular endothelial growth factor. Who were already taking antifibrotic therapy saw a reduction in the number of acute exacerbations of IPF. Thus, Nintedanib and Pirfenidone both drugs slowed down the progression of Lung Fibrosis in patients over 18yr of age. However, more studies are required for usage of antifibrotics in Non- IPF patients.

Anti-Inflammatory Effects of Mitrephora sirikitiae Leaf Extract and Isolated Lignans in RAW 264.7 Cells.

Mitrephora sirikitiae Weeras., Chalermglin & R.M.K. Saunders has been reported as a rich source of lignans that contribute to biological activities and health benefits. However, cellular anti-inflammatory effects of M. sirikitiae leaves and their lignan compounds have not been fully elucidated. Therefore, this study aimed to investigate the anti-inflammatory activities of methanol extract of M. sirikitiae leaves and their lignan constituents on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 mouse macrophage cells. Treatment of RAW 264.7 cells with the methanol extract of M. sirikitiae leaves and its isolated lignans, including (−)-phylligenin (2) and 3′,4-O-dimethylcedrusin (6) significantly decreased LPS-induced prostaglandin E2 (PGE2) and nitric oxide (NO) productions. These inhibitory effects of the extract and isolated lignans on LPS-induced upregulation of PGE2 and NO productions were derived from the suppression of cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) production, respectively. In addition, treatment with 2-(3,4-dimethoxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (3) and mitrephoran (5) was able to suppress LPS-induced tumor necrosis factor alpha (TNF-α) secretion and synthesis in RAW 264.7 cells. These results demonstrated that M. sirikitiae leaves and some isolated lignans exhibited potent anti-inflammatory activity through the inhibition of secretion and synthesis of PGE2, NO, and TNF-α.

Sulfasalazine: a risk factor for severe COVID-19?

Sulfasalazine, a disease-modifying antirheumatic drug (DMARD), has a well established role in the treatment of patients with rheumatoid arthritis, spondyloarthritis, and inflammatory bowel disease—diseases in which therapeutic benefit may primarily derive from the drug's inhibitory effect on tumour necrosis factor (TNF) and prostaglandin synthesis. In view of this mechanism, it is of note that several independent studies identified baseline treatment with sulfasalazine or mesalamine as a major risk factor (second only to rituximab and other B cell-depleting therapies) for severe COVID-19 in patients with autoimmune and rheumatic diseases.

Acute Effect of Caffeine on the Synthesis of Pro-Inflammatory Cytokines in the Hypothalamus and Choroid Plexus during Endotoxin-Induced Inflammation in a Female Sheep Model.

This study was designed to determine the effect of acute caffeine (CAF) administration, which exerts a broad spectrum of anti-inflammatory activity, on the synthesis of pro-inflammatory cytokines and their receptors in the hypothalamus and choroid plexus (ChP) during acute inflammation caused by the injection of bacterial endotoxin—lipopolysaccharide (LPS). The experiment was performed on 24 female sheep randomly divided into four groups: control; LPS treated (iv.; 400 ng/kg of body mass (bm.)); CAF treated (iv.; 30 mg/kg of bm.); and LPS and CAF treated. The animals were euthanized 3 h after the treatment. It was found that acute administration of CAF suppressed the synthesis of interleukin (IL-1β) and tumor necrosis factor (TNF)α, but did not influence IL-6, in the hypothalamus during LPS-induced inflammation. The injection of CAF reduced the LPS-induced expression of TNF mRNA in the ChP. CAF lowered the gene expression of IL-6 cytokine family signal transducer (IL6ST) and TNF receptor superfamily member 1A (TNFRSF1) in the hypothalamus and IL-1 type II receptor (IL1R2) in the ChP. Our study on the sheep model suggests that CAF may attenuate the inflammatory response at the hypothalamic level and partly influence the inflammatory signal generated by the ChP cells. This suggests the potential of CAF to suppress neuroinflammatory processes induced by peripheral immune/inflammatory challenges.

Expression of mRNA TNFα and level of protein TNFα after exposure sCD40L in bone marrow mononuclear cells of myelodysplastic syndromes.

Cytopenia is the primary phenomenon in myelodysplastic syndrome (MDS) amidst hypercellular bone marrow. The soluble CD40 ligand (sCD40L) is considered as a cytokine that can trigger synthesis of tumor necrosis factor α (TNFα) that promotes apoptosis. The objective of this study is to prove that recombinant human sCD40L (rh-sCD40L) exposure on bone marrow mononuclear cells (BMMC) MDS increases TNFα expression at mRNA level and at protein level. BMMC from MDS patients whom diagnosed and classified using the WHO 2008 criteria, were exposed to rh-sCD40L and antiCD40L. The expressions of TNFα mRNAs were quantified by qRT-PCR, level of TNFα were measured using the ELISA method. Exposure of rh-sCD40L significantly increased the expression of TNFα mRNA. The similar exposure also significantly increased the level of TNFα compared to controls. TNFα mRNA expression on BMMC in MDS samples exposed to rh-sCD40L is 3.32 times compared to TNFα mRNA expression without exposure. level of TNFα in supernatant media exposed to rh-sCD40L in MDS samples was higher than that of control samples which were 44.44 and 4.85 pg/mL, P=0.018. The sCD40L plays a role in increasing the synthesis of TNFα in mRNA level and protein level in BMMC MDS.

TNF Production and Release from Microglia via Extracellular Vesicles: Impact on Brain Functions.

Tumor necrosis factor (TNF) is a pleiotropic cytokine powerfully influencing diverse processes of the central nervous system (CNS) under both physiological and pathological conditions. Here, we analyze current literature describing the molecular processes involved in TNF synthesis and release from microglia, the resident immune cells of the CNS and the main source of this cytokine both in brain development and neurodegenerative diseases. A special attention has been given to the unconventional vesicular pathway of TNF, based on the emerging role of microglia-derived extracellular vesicles (EVs) in the propagation of inflammatory signals and in mediating cell-to-cell communication. Moreover, we describe the contribution of microglial TNF in regulating important CNS functions, including the neuroinflammatory response following brain injury, the neuronal circuit formation and synaptic plasticity, and the processes of myelin damage and repair. Specifically, the available data on the functions mediated by microglial EVs carrying TNF have been scrutinized to gain insights on possible novel therapeutic strategies targeting TNF to foster CNS repair.

Inhibitors of Tumor Necrosis Factor Synthesis as a New Approach for theTreatment of Rheumatoid Arthritis

Objective: The treatment of rheumatoid arthritis (RA) is based on the inhibition of TNF. Here we evaluated whether drugs that might inhibit TNF, such as pentoxifylline (PTX), rupatadine (RUP), rolipram (ROL) and thalidomide (THA), could be an alternative for RA treatment. Methods: In wistar male rats the changes in paw thickness, plasma TNF and by the activity of basic aminopeptidase (APB) in soluble fraction of synovial tissue and peripheral blood mononuclear cells (PBMC) evaluated after daily injection for 30 days were taken as anti-inflammatory outputs, while hepatotoxicity was assessed by measuring plasma alanine transaminase (ALT) and aspartate transaminase (AST) activity. The content of IL1-?, IL-6 in serum and synovial fluid and the histology of the injured tissue were determined only for ROL, THA and ROL+THA. Prednisolone was used as a standard drug. Results: Collagen treatment induced paw thickness, histological changes in the tibiotarsal joint, increase in synovial fluid of both cytokines and synovial tissue of APB activity. Furthermore, the APB activity in PBMC was reduced and ALT and AST activity were enhanced. The most effective drug schedule in reducing arthritis induced changes described above, as well as recovering from control levels TNF, IL1-β, APB in synovial tissue and AST activities were THA and the association of ROL and THA. However, only THA alone reduced the levels of ALT. Conclusion: The synthesis of TNF in RA models can be blocked by drugs acting at different targets. We show that THA and THA+ROL emerges as simple and effective therapeutic alternatives for RA.

Transcutaneous Stimulation of Auricular Branch of the Vagus Nerve Attenuates the Acute Inflammatory Response After Lung Lobectomy.

Systemic inflammation is a potentially debilitating complication of thoracic surgeries with significant physical and economic morbidity. There is compelling evidence for the role of thecentral nervous system inregulating inflammatory processes through humoral mechanisms. Activation of theafferent vagus nerve by cytokines triggers anti-inflammatory responses. Peripheral electrical stimulation of the vagus nerve in vivo during lethal endotoxemia in rats inhibited tumor necrosis factor synthesis and prevented shock development. However, the vagal regulatory role of systemic inflammation after lung lobectomy is unknown. One hundred patients who underwent lobectomy via thoracotomy were recruited and equally randomized to treated group or controls. Intermittent stimulation of the auricular branch of vagus nerve in the triangular fossa was applied in the treated group using neurostimulator V (Ducest®, Germany), starting 24h preoperatively and continued till the 4th postoperative day (POD). Inflammatory interleukins (IL) were analyzed using ELISA preoperatively, on the 1st and 4th POD. On the 1st POD, patients who underwent neurostimulation had reduced serum concentrations of CRP (p = 0.01), IL6 (p = 0.02) but elevated IL10 (p = 0.03) versus controls. On the 4th POD, serum concentrations of CRP, IL6 and IL10 were similar in both groups. Moreover, the treated group was associated with lower incidence of pneumonia (p = 0.04) and shorter hospitalization time (p = 0.04) versus controls. Modulations in the brain stem caused by noninvasive transcutaneous stimulation of the vagus nerve after lung lobectomy attenuate the acute postsurgical inflammatory response by the regulation of IL6 and IL10, resultingin reduced incidence of postoperative pneumonia and short hospitalization time. NCT03204968.

Peroxisome proliferator-activated receptor β/δ and γ agonists differentially affect prostaglandin E2 and cytokine synthesis and nutrient transporter expression in porcine trophoblast cells during implantation

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor family of ligand-dependent transcription factors. PPARs have been shown to be important regulators of female reproductive functions, including conceptus development and placenta formation. This study examines the effect of PPARβ/δ and PPARγ agonists and antagonists on (1) the synthesis of prostaglandin (PG) E2, interleukin (IL) 6, interferon (IFN) γ, and tumor necrosis factor (TNF) α and (2) the mRNA expression of genes encoding nutrient transporters and/or binding proteins in Day 15 conceptus trophoblast cells. The study also examines whether PPAR agonist-modulated IL6, IFNγ, and TNFα secretion is mediated via mitogen-activated protein kinase (MAPK) pathways. Trophoblast cells were exposed to L-165,041 (a PPARβ/δ agonist) or rosiglitazone (a PPARγ agonist) in the presence or absence of GSK3787 (a PPARβ/δ antagonist) or GW9662 (a PPARγ antagonist) or in the presence or absence of U0126 (a MAPK inhibitor). Rosiglitazone stimulated PGE synthase and IFNG mRNA expression in trophoblast cells and enhanced PGE2 concentrations in the incubation medium. Moreover, cells treated with rosiglitazone exhibited increased abundance of the solute carrier organic anion transporter family member 2A1 (SLCO2A1, a PG transporter) and of fatty acid binding protein (FABP) 5 transcripts. All these effects were abolished by the addition of GW9662, which indicates that the action of rosiglitazone is PPARγ-dependent in the studied cells. L-165,041 inhibited TNFα synthesis and decreased the mRNA expression of FABP3 and IL6 in trophoblast cells. However, this effect was not abolished by the addition of GSK3787 into the incubation medium, suggesting that L-165,041 action is independent of PPARβ/δ. The inhibitory effect of L-165,041 on TNFα concentration and the stimulatory effect of rosiglitazone on IFNγ accumulation in the medium were not observed in the presence of the MAPK inhibitor, suggesting that the action of both agonists may be mediated by MAPKs. In conclusion, PPARβ/δ and PPARγ agonists are differentially involved in the trophoblast expression of genes related to conceptus development and implantation in pigs. Furthermore, L-165,041 and rosiglitazone may have PPAR-dependent and -independent effects in conceptus trophoblast cells.

Serum Visfatin does not seem to be a Useful Marker to Guide Glucocorticoid Substitution in Adrenal Insufficiency.

Primary adrenal insufficiency (Addison's disease, AD) requires lifelong steroid substitution. Excess exogenous glucocorticoids promote abdominal obesity, insulin-glucose imbalance, and hypertension. Reliable markers of the adequate glucocorticoid replacement are lacking. Visfatin is a pro-inflammatory adipokine, with enzymatic activity of nicotinamide phosphoribosyltransferase. It enhances leukocyte function and synthesis of tumour necrosis factor α (TNFα) and interleukin-6 (IL-6). Serum visfatin is elevated in autoimmunity, but also in obesity, insulin resistance, and metabolic syndrome. This study was aimed to investigate whether serum visfatin could guide the glucocorticoid substitution in AD. Biochemical analyses were performed in 96 patients with AD (mean age 43.3±14.9 years) and 91 controls (43.5±12.5 years). Visfatin level was significantly elevated in patients with AD compared to controls (p<0.0001). Higher circulating IL-6 was also detected among subjects with AD (p=0.006). In AD, visfatin level was positively correlated with IL-6 (p=0.014), TNFα (p=0.001), body mass (p=0.015), fasting insulin (p=0.001) and HOMA-IR (p=0.001). No relationship was noticed with daily hydrocortisone (p=0.096) and urinary free cortisol excretion (p=0.499). Only the correlations with IL-6 and fasting insulin survived multiple regression analysis (p=0.049 and p=0.005, respectively). Additionally, positive correlation between visfatin and autoantibodies to 21-hydroxylase was noted (p=0.005). In the control group serum visfatin was correlated with IL-6 (p=0.009) and TNFα (p=0.0002). The current study reveals elevated serum visfatin in autoimmune AD. Visfatin does not seem a useful marker of the glucocorticoid replacement, although it correlates with fasting insulin and pro-inflammatory molecules. Further functional analyses are warranted to elucidate the role of visfatin in autoimmunity.

Reagentless Modified Water and Its Biological Activity

It is established empirically that bidistilled water acquires anomalous physicochemical properties when placed in strong spatially inhomogeneous physical fields. In particular, the dependences of the redox potential (RP) on рН and the dipole moment on temperature, as well as the occurrence of metastability, cannot be described within classical concepts. A model is proposed to explain qualitatively these anomalous properties. The pH of reagentless modified water (RMW) free of alkaline elements may reach 9 to 12. The electrical conductivity of this water exceeds that of its chemical analog by a factor of 8 to 30. The acid-base properties of RMW are determined by the concentration ratio of solvated protons and hydroxyls that do not enter the composition of cluster ions. In other words, RMW free of alkaline elements exhibits alkalinity. This water is shown to be biologically active: it increases the synthesis of tumor necrosis factor by immune-competent cells (both in vivo and in vitro) by a factor of 3 to 4, leads to inhibition of reverse transcriptase of human immunodeficiency virus, and promotes spontaneous hydrolysis of proteins in solutions.

NEUROIMMUNE MECHANISMS OF COGNITIVE IMPAIRMENT AT AFFECTIVE DISORDERS

The aim of this study was the identification of the neuroimmune mechanisms of cognitive impairment on the basis of investigation of an of cognitive disorders association with functional activity of blood mononuclear cells and the synthesis of tumor necrosis factor - α (TNF-α) in patients with affective disorders in the form of depressive reactions and depression.TNF-α expression level in blood mononuclear cells of patients conducted with using the reverse transcriptase polymerase chain reaction method. The proliferative activity of blood mononuclear cells was investigated by a standard method based on the inclusion of a radioactive label. Cognitive function was assessed on the basis of perception, memory, praxis, speech, and control function. The severity of affective symptoms was measured by the Hamilton scale. It was found that patients with depressive reactions were characterized by the mild non-elemental cognitive impairment; while, in patients with depression, more severe non-elemental cognitive impairments in the form of decreased attention, memory, and daily activity were observed. In patients with affective disorders in the form of depression, an increase in the synthesis of TNF-α in blood mononuclear cells was detected, both an increase of the expression frequency of its gene and an increase in mRNA level, as well as an increase of the proliferative activity of blood mononuclear cells, which indicates the presence of immune system dysfunction in this category of patients.Thus, in patients with depression, activation of the TNF-α synthesis in blood mononuclear cells occurs: an increase in the frequency of its expression and an increase of the level of mRNA; these changes are accompanied by the increasing of immune cells functional activity and moderate non-delicate cognitive disorder in the form of cognitive impairment.

In vitro cytokine synthesis in unstimulated and mitogen-stimulated peripheral blood mononuclear cells from individuals with schizophrenia

Increasing evidence has shown that the immune system is involved in the schizophrenia development, with alterations in immune cell reactivity being one possible factor contributing to its pathogenesis. The purpose...

Human TNF-Luc reporter mouse: A new model to quantify inflammatory responses

Tumour necrosis factor (TNF) is a key cytokine during inflammatory responses and its dysregulation is detrimental in many inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. Here, we used a bacterial artificial chromosome (BAC) construct that expresses luciferase under the control of the human TNF locus to generate a novel transgenic mouse, the hTNF.LucBAC strain. In vitro stimulation of hTNF.LucBAC cells of different origin revealed a cell specific response to stimuli demonstrating the integrated construct’s ability as a proxy for inflammatory gene response. Lipopolysaccharide was the most potent luciferase inducer in macrophages, while TNF was a strong activator in intestinal organoids. Lipopolysaccharide-induced luciferase activity in macrophages was downregulated by inhibitors of NF-κB pathway, as well as by Interleukin-10, a known anti-inflammatory cytokine. Moreover, the transgene-dependent luciferase activity showed a positive correlation to the endogenous murine soluble TNF secreted to the culture medium. In conclusion, the hTNF.LucBAC strain is a valuable tool for studying and screening molecules that target TNF synthesis and will allow further functional studies of the regulatory elements of the TNF locus.

Long non‑coding RNA DILC is involved in sepsis by modulating the signaling pathway of the interleukin‑6/signal transducer and activator of transcription 3/Toll‑like receptor 4 axis.

Sepsis is characterized by systemic inflammatory responses. In the present study, the role of deleted in liver cancer 1 (DILC), interleukin (IL)‑6, signal transducer and activator of transcription 3 (STAT3), and Toll‑like receptor 4 (TLR4) in the pathogenesis of sepsis was investigated. Reverse transcription‑quantitative polymerase chain reaction analysis and western blotting were performed to evaluate the effects of lipopolysaccharide (LPS) on the expression of DILC, IL‑6, STAT3, and TLR4, in addition to the effects of DILC and IL‑6 on the synthesis of tumor necrosis factor (TNF‑α), chemokine ligand 5 (CCL5), E‑selectin and C‑X‑C motif chemokine receptor 1 (CXCR1). In addition, the regulatory association between DILC, IL‑6, STAT3 and TLR4 was investigated. LPS reduced the expression level of DILC, and enhanced the expression of IL‑6, STAT3 and TLR4. DILC directly and negatively regulated the synthesis of IL‑6, as demonstrated by the markedly decreased luciferase activity in cells transfected with a wild‑type DILC plasmid. On the other hand, compared with the scramble control, DILC and IL‑6 small interfering (si)RNAs significantly suppressed the expression of IL‑6, STAT3 and TLR4. In addition, DILC siRNA enhanced the expression of IL‑6, STAT3 and TLR4, whereas the expression levels of TNF‑α, CCL5, E‑selectin and CXCR1 in LPS‑treated THP‑1 cells were downregulated following transfection with DILC and IL‑6 siRNAs. In patients with sepsis, DILC expression was inhibited, although the expression levels of IL‑6, STAT3 and TLR4 were upregulated. In addition, the expression levels of TNF‑α, CCL5, E‑selectin and CXCR1 in patients with sepsis were higher compared with normal subjects. Therefore, DILC may mediate the crosstalk between the cascades of IL‑6/STAT3 and TNF‑α signaling, indicating that DILC may act as a prognostic biomarker of sepsis, and may serve as a potential therapeutic target for the treatment of sepsis.

SP and IL-33 together markedly enhance TNF synthesis and secretion from human mast cells mediated by the interaction of their receptors

The peptide substance P (SP) and the cytokine tumor necrosis factor (TNF) have been implicated in inflammatory processes. Mast cells are recognized as important in inflammatory responses. Here, we report that IL-33 (30 ng/mL), a member of the IL-1 family of cytokines, administered in combination with SP (1 µM), markedly increase (by 1,000-fold) TNF gene expression in cultured human LAD2 and primary mast cells derived from umbilical cord blood. SP (0.01-1 μM) and IL-33 (1-100 ng/mL) in combination also greatly stimulate TNF secretion (by 4,500-fold). Pretreatment of LAD2 cells with two different neurokinin-1 (NK-1) receptor antagonists and siRNA inhibits TNF secretion by 50% (P < 0.001) when stimulated by SP and IL-33. Pretreatment of LAD2 cells with a neutralizing antibody for IL-33 receptor, ST2, inhibits TNF secretion by 50% (P < 0.001), and ST2 siRNA decreases TNF secretion by 30% (P < 0.05), when stimulated by SP and IL-33. Surprisingly, NK-1 antagonists also inhibit 50% of TNF secretion (P < 0.001) when stimulated only by IL-33, and ST2 receptor reduction also decreases SP-stimulated TNF secretion by 30% (P < 0.05), suggesting an interaction between NK-1 and ST2 receptors. Moreover, IL-33 increases NK-1 gene and surface protein expression, as well as IKβ-α phosphorylation. Pretreatment of LAD2 cells with 5,7,3',4'-tetramethoxyflavone (methoxyluteolin) (1-100 μM) inhibits (P < 0.001) TNF gene expression (98%) and secretion (64%) at 50 µM and phosphorylation of p-IKB-α at 1 μM when stimulated by SP and IL-33. These findings identify a unique amplification process of TNF synthesis and secretion via the interaction of NK-1 and ST2 receptors inhibitable by methoxyluteolin.