Lipopolysaccharide-induced TNF-α Production Research Articles

Features of baseline and lipopolysaccharide-induced cytokine secretion in mononuclear leukocyte cultures from patients with the erythema migrans form of acute lyme borreliosis based on clinical parameters

Introduction. Features of cytokine production in mononuclear cell cultures from Lyme borreliosis patients based on clinical data remained poorly studied. The study aim was to estimate the patterns of baseline and lipopolysaccharide-induced cytokine-secretory activity of peripheral blood mononuclear leukocytes from patients with erythema migrans form of acute Lyme borreliosis based on clinical parameters. Materials and methods. Groups of 22 and 12 patients with the diagnoses of mild or moderate severity of monoinfection and co-infection with tick-borne encephalitis of Lyme borreliosis with erythema migrans were examined twice: on week 1 after disease onset and day 14. The control group included 17 healthy donors. Basal and lipopolysaccharide-induced IL-6, IL-10, and TNFα secretion levels were assessed in mononuclear leukocyte culture supernatants applying enzyme immunoassay. Statistical analysis was performed by using the Mann–Whitney U-test, Wilcoxon test, and Spearman’s rank correlation. Results. The group of moderate severity patients was clinically distinguished by severer fever and intoxication manifestations. At the disease onset, the basal TNFα, IL-6 and IL-10 secretion levels in the moderate severity patient cultures were significantly higher than in those of the other groups. After antibiotics treatment, the baseline TNFα and IL-10 levels tended to decrease. At the onset, lipopolysaccharide-induced cultures from the moderate severity patients showed significantly suppressed TNFα production and increased IL-10 secretion as compared to the other groups. Lipopolysaccharide-induced IL-6 secretion in the moderate vs. mild severity group supernatants was significantly lower. In dynamics, the induced TNFα levels in the moderate severity patients were increased to the magnitude exceeding that in the controls. Positive correlations between the IL-6 and TNFα basal levels and maximum body temperature or the C-reactive protein serum concentrations were revealed in the patients. Induced TNFα levels showed negative correlations with fever levels or with IL-10 secretion. Conclusions. It was demonstrated that basal TNFα, IL-6 and IL-10 secretion levels in the mononuclear cell cultures of acute Lyme borreliosis patients increased with the increasing disease severity. Suppression of lipopolysaccharide-induced TNFα production in the moderate severity patient cultures was presumably associated with the regulatory cytokine IL-10 effects.

In vitro fermentation of seaweed polysaccharides and tea polyphenol blends by human intestinal flora and their effects on intestinal inflammation.

A combination of polysaccharides and tea polyphenols can enhance immune activity synergistically, depending on the type and structure of polysaccharides, but the mechanism remains unknown. This study is aimed to investigate the regulating effects of different seaweed polysaccharide (ι-carrageenan, agarose) and tea polyphenol blends on intestinal flora and intestinal inflammation using an in vitro ascending-transverse-descending colon fermentation system and RAW264.7 cell model. The results showed that seaweed polysaccharides in the presence of tea polyphenol were almost completely degraded at transverse colon fermentation for 36 h. Agarose significantly enhanced the butyric acid production content by increasing the abundance of Lachnospiraceae, whereas agarose and tea polyphenol blends did not have a synergistic effect. On the contrary, ι-carrageenan and tea polyphenol blends synergistically increased the abundance of beneficial bacteria (e.g., Bacteroidetes and Bifidobacterium) and promoted the production of short-chain fatty acids (SCFAs), such as isobutyric acid. Such changes tended to alter the impacts of different seaweed polysaccharides and tea polyphenol blends on intestinal inflammation. Among them, ι-carrageenan and tea polyphenol blends were the most effective in inhibiting lipopolysaccharide-induced NO, ROS, IL-6, and TNF-α production in RAW264.7 cells, indicating the alleviated intestinal inflammation. The results suggest that the seaweed polysaccharide and tea polyphenol blends have prebiotic potential and can benefit intestinal health.

Limosilactobacillus fermentum Strain 3872: Antibacterial and Immunoregulatory Properties and Synergy with Prebiotics against Socially Significant Antibiotic-Resistant Infections of Animals and Humans.

Limosilactobacillus fermentum strain 3872 (LF3872) was originally isolated from the breast milk of a healthy woman during lactation and the breastfeeding of a child. The high-quality genome sequencing of LF3872 was performed, and a gene encoding a unique bacteriocin was discovered. It was established that the bacteriocin produced by LF3872 (BLF3872) belongs to the family of cell-wall-degrading proteins that cause cell lysis. The antibacterial properties of LF3872 were studied using test cultures of antibiotic-resistant Gram-positive and Gram-negative pathogens. Gram-positive pathogens (Staphylococcus aureus strain 8325-4 and S. aureus strain IIE CI-SA 1246) were highly sensitive to the bacteriolytic action of LF3872. Gram-negative pathogens (Escherichia coli, Salmonella strains, and Campylobacter jejuni strains) were more resistant to the bacteriolytic action of LF3872 compared to Gram-positive pathogens. LF3872 is a strong co-aggregator of Gram-negative pathogens. The cell-free culture supernatant of LF3872 (CSLF3872) induced cell damage in the Gram-positive and Gram-negative test cultures and ATP leakage. In the in vitro experiments, it was found that LF3872 and Actigen prebiotic (Alltech Inc., Nicholasville, KY, USA) exhibited synergistic anti-adhesive activity against Gram-negative pathogens. LF3872 has immunoregulatory properties: it inhibited the lipopolysaccharide-induced production of proinflammatory cytokines IL-8, IL-1β, and TNF-α in a monolayer of Caco-2 cells; inhibited the production of IL-12 and stimulated the production of IL-10 in immature human dendritic cells; and stimulated the production of TGF-β, IFN-γ, and IgA in the immunocompetent cells of intestinal Peyer’s patches (PPs) in mice. These results indicate the possibility of creating a synbiotic based on LF3872 and a prebiotic derived from Saccharomyces cerevisiae cell wall components. Such innovative drugs and biologically active additives are necessary for the implementation of a strategy to reduce the spread of antibiotic-resistant strains of socially significant animal and human infections.

Preparation of chitosan-rosmarinic acid derivatives with enhanced antioxidant and anti-inflammatory activities

In present study, several chitosan derivatives grafted with rosmarinic acid，including chitosan-rosmarinic acid conjugate (CS-RA), chitosan rosmarinic acid salt (CSRA), N,N,N-trimethylated chitosan rosmarinic acid salt (TMCRA), and N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan rosmarinic acid salt (HACRA), were prepared and the effects of different grafting methods on bioactivity of these derivatives were investigated. The structural characterization was identified by FTIR and 1H NMR spectroscopy. The chitosan derivatives were also evaluated biologically for antioxidant, cytotoxic, and anti-inflammatory activities. Derivatives CSRA, TMCRA, and HACRA prepared through ion exchange method exhibited significant antioxidant ability. All derivatives remarkably inhibited lipopolysaccharide-induced nitric oxide and TNF-α production in RAW 264.7 cells without cytotoxicity, especially derivative TMCRA, whose inhibition rate was >50 % at only 250 μg/mL. The results suggested that chitosan quaternary ammonium derivatives grafted with rosmarinic acid possessed better antioxidant and anti-inflammatory properties than chitosan, which could be used as drug materials against oxidative and inflammation-related pathological processes.

Immune System Dysfunction Criteria in Critically Ill Children: The PODIUM Consensus Conference.

Immune system dysfunction is poorly represented in pediatric organ dysfunction definitions. To evaluate evidence for criteria that define immune system dysfunction in critically ill children and associations with adverse outcomes and develop consensus criteria for the diagnosis of immune system dysfunction in critically ill children. We conducted electronic searches of PubMed and Embase from January 1992 to January 2020, using medical subject heading terms and text words to define immune system dysfunction and outcomes of interest. Studies of critically ill children with an abnormality in leukocyte numbers or function that is currently measurable in the clinical laboratory in which researchers assessed patient-centered outcomes were included. Studies of adults or premature infants, animal studies, reviews and commentaries, case series (≤10 subjects), and studies not published in English with inability to determine eligibility criteria were excluded. Data were abstracted from eligible studies into a standard data extraction form along with risk of bias assessment by a task force member. We identified the following criteria for immune system dysfunction: (1) peripheral absolute neutrophil count <500 cells/μL, (2) peripheral absolute lymphocyte count <1000 cells/μL, (3) reduction in CD4+ lymphocyte count or percentage of total lymphocytes below age-specific thresholds, (4) monocyte HLA-DR expression <30%, or (5) reduction in ex vivo whole blood lipopolysaccharide-induced TNFα production capacity below manufacturer-provided thresholds. Many measures of immune system function are currently limited to the research environment. We present consensus criteria for the diagnosis of immune system dysfunction in critically ill children.

Differential regulation of lipopolysaccharide-induced IL-1β and TNF-α production in macrophages by palmitate via modulating TLR4 downstream signaling

Diabetic patients are susceptible to infectious diseases. Bacterial invasion activates immune cells such as macrophages through interaction between LPS and TLR4, and induces the expression of inflammatory mediators, including IL-1β and TNF-α, which play key roles in the elimination of infections. Unregulated overproduction or underproduction of these cytokines has been reported as a major factor in the development of septic shock, immune deficiency, and autoimmunity. Recent studies found that metabolic abnormalities of diabetes, such as hyperglycemia and dyslipidemia, played a major role in modulating the immune response. In this study, we studied the effects of palmitic acid (PA) pretreatment on LPS-induced IL-1β and TNF-α production and LPS-TLR4 signaling in macrophages. Compared with control, PA pretreatment significantly increased LPS-induced TNF-α production and secretion in macrophages. In contrast, LPS-induced IL-1β production and secretion was significantly suppressed by PA pretreatment. PA pretreatment did not affect the expression levels of TLR4 or Myd88, or the endocytosis of TLR4 in macrophages. However, PA pretreatment significantly suppressed the phosphorylation level and nuclear translocation of NF-κB, and the phosphorylation level of ERK1/2, whereas increased the phosphorylation levels of p38 and JNK. The activation of IKK which was upstream of NF-κB and ERK1/2 was attenuated, while the activation of TAK1 which was upstream of JNK and p38 was augmented by PA pretreatment. Inhibitors of NF-κB, MEK1/2, and p38 significantly decreased IL-1β expression, while JNK and p38 pathway inhibitors significantly inhibited TNF-α expression. The differential regulation of LPS-induced TNF-α and IL-1β production by PA was associated with cellular metabolism of PA, because inhibiting metabolism of PA with etomoxir or pretreatment with Br-PA which cannot be metabolized reversed these effects. We also showed that PA treatment increased acetylated IKK level which might contribute to the suppressed activation of IKK. The present study showed that LPS-induced production of TNF-α and IL-1β was regulated by different TLR4 downstream pathways in macrophages. PA differentially affected LPS-induced production of TNF-α and IL-1β in macrophages through differentially modulating these pathways. Further experiments will be needed to determine how these phenomena lead to the impaired immune response in patients with diabetes.

Histone Deacetylases Regulation by δ-Opioids in Human Optic Nerve Head Astrocytes.

PurposeWe determined whether δ-opioid receptor agonist (SNC-121) regulates acetylation homeostasis via controlling histone deacetylases (HDACs) activity and expression in optic nerve head (ONH) astrocytes.MethodsONH astrocytes were treated with SNC-121 (1 µM) for 24 hours. The HDAC activity was measured using HDAC-specific fluorophore-conjugated synthetic substrates, Boc-Lys(Ac)-AMC and (Boc-Lys(Tfa)-AMC). Protein and mRNA expression of each HDAC was determined by Western blotting and quantitative real-time PCR. IOP in rats was elevated by injecting 2.0 M hypertonic saline into the limbal veins.ResultsDelta opioid receptor agonist, SNC-121 (1 µM), treatment increased acetylation of histone H3, H2B, and H4 by 128 ± 3%, 45 ± 1%, and 68 ± 2%, respectively. The addition of Garcinol, a histone-acetyltransferase inhibitor, fully blocked SNC-121–induced histone H3 acetylation. SNC-121 reduced the activities of class I and IIb HDACs activities significantly (17 ± 3%) and this decrease in HDACs activities was fully blocked by a selective δ-opioid receptors antagonist, naltrindole. SNC-121 also decrease the mRNA expression of HDAC-3 and HDAC-6 by 19% and 18%, respectively. Furthermore, protein expression of HDAC 1, 2, 3, and 6 was significantly (P < 0.05) decreased by SNC-121 treatment. SNC-121 treatment also reduced lipopolysaccharide-induced TNF-α production from ONH astrocytes and glial fibrillary acidic protein immunostaining in the optic nerve of ocular hypertensive animals.ConclusionsWe provided evidence that δ-opioid receptor agonist activation increased histone acetylation, decrease HDACs class I and class IIb activities, mRNA, and protein expression, lipopolysaccharide-induced TNF-α production in ONH astrocytes. Our data also demonstrate that SNC-121 treatment decrease glial fibrillary acidic protein immunostaining in the optic nerves of animals with ocular hypertension.

Sparassis crispa Intake Improves the Reduced Lipopolysaccharide-Induced TNF-α Production That Occurs upon Exhaustive Exercise in Mice.

Our previous study showed that lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production is inhibited by acute exhaustive exercise in mice, leading to transient immunodepression after exercise. Sparassis crispa (SC), an edible mushroom, has immunopotentiative properties. This study aimed to clarify the effects of SC intake on reduced LPS-induced TNF-α production upon exhaustive exercise in mice. Male C3H/HeN mice were randomly divided into three groups: normal chow intake + resting sedentary, normal chow intake + acute exhaustive treadmill running exercise, and SC intake (chow containing 5% SC powder for 8 weeks) + the exhaustive exercise groups. Each group was injected with LPS immediately after the exhaustive exercise or rest. Plasma and tissue TNF-α levels were significantly decreased by exhaustive exercise. However, this reduction of the TNF-α level was partially attenuated in the plasma and small intestine by SC intake. Although levels of TLR4 and MyD88 protein expression were significantly decreased in tissues by exhaustive exercise, the reduction of TLR4 and MyD88 levels in the small intestine was partially attenuated by SC intake. These results suggest that SC intake attenuates exhaustive exercise-induced reduction of TNF-α production via the retention of TLR4 and MyD88 expression in the small intestine.

Interleukin-35: A Serological Biomarker for Patients with Polymyositis/Dermatomyositis.

Interleukin (IL)-35 is confirmed as an important modulator in immune response. The aim of the study was to explore the role of IL-35 in the development of polymyositis/dermatomyositis (PM/DM). Ninety-five patients with PM/DM and 30 healthy controls (HCs) were recruited. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus density gradient centrifugation. Protein IL-35, IL-17, and tumor necrosis factor (TNF)-α levels were measured using enzyme-linked immunosorbent assay method. The IL-35 serum levels in PM/DM patients were significantly higher than those in HCs and were correlated with PM/DM-related features: disease activity, muscle damage, and interstitial lung disease (ILD). Exogenous IL-35 notably suppressed lipopolysaccharide-induced IL-17 and TNF-α production in PBMCs of patients with PM/DM. Elevated serum IL-35 levels could act as a disease activity biomarker and an indicator of ILD in PM/DM. IL-35 may play an anti-inflammatory role in PM/DM.

Immunomodulating Effects Exerted by Glucans Extracted from the King Oyster Culinary-Medicinal Mushroom Pleurotus eryngii (Agaricomycetes) Grown in Substrates Containing Various Concentrations of Olive Mill Waste

We have recently demonstrated that we could enhance glucan content in Pleurotus eryngii following cultivation of the mushrooms on a substrate containing different concentrations of olive mill solid waste (OMSW). These changes are directly related to the content of OMSW in the growing substrate. Using dextran sulfate sodium (DSS)-in-flammatory bowel disease (IBD) mice model, we measured the colonic inflammatory response to the different glucan preparations. We found that the histology damaging score (HDS) resulting from DSS treatment reach a value of 11.8 ± 2.3 were efficiently downregulated by treatment with the fungal extracted glucans. Glucans extracted from stalks cultivated at 20% OMSW downregulated to a HDS value of 6.4 ± 0.5 whereas those cultivated at 80% OMSW showed the strongest effects (5.5 ± 0.6). Similar downregulatory effects were obtained for expression of various intestinal cytokines. All tested glucans were equally effective in regulating the number of CD14/CD16 monocytes from 18.2 ± 2.7% for DSS to 6.4 ± 2.0 for DSS + glucans extracted from stalks cultivated at 50% OMSW. We tested the effect of glucans on lipopolysaccharide-induced production of TNF-α, which demonstrated that stalk-derived glucans were more effective than caps-derived glucans. Isolated glucans competed with anti-Dectin-1 and anti-CR3 antibodies, indicating that they contain β-glucans recognized by these receptors. In conclusion, the most effective glucans in ameliorating IBD-associated symptoms induced by DSS treatment in mice were glucan extracts prepared from the stalk of P. eryngii grown at higher concentrations of OMSW. We conclude that these stress-induced growing conditions may be helpful in selecting more effective glucans derived from edible mushrooms.

Chemical components and bioactivity evaluation of extracts from pear (Pyrus UssuriensisMaxim) fruit

In order to discover the bioactive metabolites of Pyrus ussuriensis Maxim, the systematic chemical and pharmacological investigations were performed. The ethyl acetate extract of the plant displayed activities against ear edema in mice, with an inhibition rate of 17.1%, at a dose of 400mg/kg in vivo. Meanwhile, the ethyl acetate extract showed the best apophlegmatic (inhibition: 49.9%) and antitussive effects (inhibition: 34%) at 800mg/kg in vivo tests, and inhibition of NO production (IC50=0.06 μg/mL). Subsequently, twelve compounds were isolated from the ethyl acetate extract and identified as friedelin (1), daucosterol (2), β-sitosterol (3), betulinic acid (4), oleanolic acid (5), ursolic acid (6), 2-O-buty-1-O’-(2-ethylhexyl) benzene-1,8-dicarboxylate (7), Narcissin (8), 3-hydroxy-3-carboxyl-glutaric acid dimethyl ester (9), 2-hydroxyl -butanediol icacid-4-methyleste (10), myristic acid (11), and (Z)-11-hexadecen-1-ol acetate (12). Among them, compounds 1, 2, 4, 5, 6, and 11 exhibited moderate indirect inhibitory effects on lipopolysaccharide-induced NO and TNF-α production. Practical applications Pyrus ussuriensis Maxim is an edible and medical fruit with many functional properties. Unfortunately, few literatures about bioactive components of this fruit have been investigated. The result indicated that P. ussuriensis have obvious anti-inflammatory, antitussive, and apophlegmatic activities, which were associated with the folk uses of this fruit. Furthermore, this work also suggests a potential use of P. ussuriensis as an important source of natural anti-inflammatory agents and they may also be considered as a functional food.

Interleukin (IL)-6 Inhibits IL-27- and IL-30-Mediated Inflammatory Responses in Human Monocytes.

Interleukin (IL)-30, the IL-27p28 subunit of the heterodimeric cytokine IL-27, acts as an antagonist of IL-27 and IL-6 signaling in murine cells via glycoprotein 130 (gp130) receptor and additional binding partners. Thus far, functions of IL-30 have not been fully elucidated in human cells. We demonstrate that like IL-27, IL-30 upregulated TLR4 expression to enhance lipopolysaccharide-induced TNF-α production in human monocytes; however, these IL-30-mediated activities did not reach the same levels of cytokine induction compared to IL-27. Interestingly, IL-30- and IL-27-mediated interferon-γ-induced protein 10 (IP-10) production required WSX-1 engagement and signal transducer and activator of transcription (STAT) 3 phosphorylation; furthermore, IL-30 induced STAT phosphorylation after 16 h, whereas IL-27 induced STAT phosphorylation within 30 min. This prompted us to examine if a secondary mediator was required for IL-30-induced pro-inflammatory functions, and hence we examined IL-6-related molecules. Combined with inhibition of soluble IL-6 receptor α (sIL-6Rα) and data showing that IL-6 inhibited IL-30/IL-27-induced IP-10 expression, we demonstrate a role for sIL-6Rα and gp130 in IL-30-mediated activity in human cells.

Exosomes isolated from sera of mice fed Lactobacillus strains affect inflammatory cytokine production in macrophages in vitro

Orally administered Lactobacillus strains, including L. plantarum No.14 and L. rhamnosus GG, reportedly reduce inflammatory cytokine production in mice. The present study tested our idea that circulating exosomes mediate the action of Lactobacillus strains. The lipopolysaccharide-induced production of TNF-α and IL-6 in vitro was attenuated in peritoneal exudate cells (PECs) isolated from C57BL/6N mice that had been fed L. plantarum No.14. When PECs were cultured for 24 h with exosomes isolated from the serum of mice fed L. plantarum No.14 or L. rhamnosus GG, accumulation of both TNF-α and of the corresponding mRNA was lowered. Growth in the presence of these exosomes also decreased the production of TNF-α and IL-6 by the murine macrophage cell line RAW264.7. In contrast, supplementation with exosome-depleted serum of mice fed L. plantarum No.14 or L. rhamnosus GG failed to affect the production of TNF-α and IL-6 by RAW264.7 cells. When PECs and RAW264.7 cells were cultured for 24 h with PKH67-labeled exosomes isolated from murine serum, fluorescent signal was observed inside the cells, suggesting that these cells incorporate serum exosomes. We propose that the anti-inflammatory activity of orally administered L. plantarum No.14 and L. rhamnosus GG is mediated, at least in part, by circulating exosomes.

Infliximab Selectively Modulates the Circulating Blood Monocyte Repertoire in Crohn's Disease.

Infliximab (IFX), an anti-tumour necrosis factor alpha (TNFα) monoclonal antibody, provides clinical benefits in treating Crohn's disease (CD) but its mechanisms of action are not fully elucidated. This study investigated blood monocyte repertoires and the acute effects of IFX infusion on monocyte subset phenotype and function in IFX-treated patients with CD. Monocytes and monocyte subsets were enumerated and phenotypically characterized by multicolor flow cytometry in freshly isolated blood from healthy controls (n = 21) and patients with CD treated with (IFX, n = 24) and without (non-IFX, n = 20) IFX. For the IFX-CD group, blood was sampled immediately before (tough-IFX) and after (peak-IFX) infusion. Monocyte responses to lipopolysaccharide were analyzed by whole-blood intracellular cytokine staining. Non-IFX and IFX-CD patients had increased numbers of intermediate (CD14CD16) monocytes compared with healthy controls, whereas classical (CD14CD16) and nonclassical (CD14CD16) monocytes were numerically reduced in the IFX-CD group alone. In all groups, monocyte subsets expressed high surface levels of transmembrane (tm)TNFα. After IFX infusion, a significant reduction in monocyte numbers occurred. Post-IFX monocytopenia was proportionately greatest for classical and intermediate subsets, correlated with postinfusion IFX levels and was not associated with monocyte apoptosis. In contrast, lipopolysaccharide-induced production of TNFα and IL-12 by monocytes was significantly reduced in peak-IFX compared with trough-IFX blood samples. Actively managed CD is associated with monocyte repertoire skewing suggestive of chronic inflammatory stimulation. Infused IFX acutely targets monocytes, likely by binding to tmTNFα, resulting in a non-apoptosis-related decline in circulating monocyte numbers and blunting of the inflammatory response of monocytes remaining in the blood.

STIMs and Orai1 regulate cytokine production in spinal astrocytes.

BackgroundOur previous study demonstrated that a store-operated calcium channel (SOCC) inhibitor (YM-58483) has central analgesic effects. However, the cellular and molecular mechanisms of such effects remain to be determined. It is well-known that glial cells play important roles in central sensitization. SOC entry (SOCE) has been implicated in many cell types including cortical astrocytes. However, the role of the SOCC family in the function of astrocytes has not been determined. Here, we thoroughly investigated the expression and the functional significance of SOCCs in spinal astrocytes.MethodsPrimary cultured astrocytes were prepared from neonatal (P2–P3) CD1 mice. Expressions of mRNAs and proteins were respectively assessed by real-time PCR and Western blot analysis. SOCE was measured using a calcium imaging system. Live-cell STIM1 translocation was detected using a confocal microscope. Cytokine levels were measured by the enzyme-linked immunosorbent assay.ResultsWe found that the SOCC family is expressed in spinal astrocytes and that depletion of calcium stores from the endoplasmic reticulum by cyclopiazonic acid (CPA) resulted in a large sustained calcium entry, which was blocked by SOCC inhibitors. Using the siRNA knockdown approach, we identified STIM1 and Orai1 as primary components of SOCCs in spinal astrocytes. We also observed thapsigargin (TG)- or CPA-induced puncta formation of STIM1 and Orai1. In addition, activation of SOCCs remarkably promoted TNF-α and IL-6 production in spinal astrocytes, which were greatly attenuated by knockdown of STIM1 or Orai1. Importantly, knockdown of STIM2 and Orai1 dramatically decreased lipopolysaccharide-induced TNF-α and IL-6 production without changing cell viability.ConclusionsThis study presents the first evidence that STIM1, STIM2, and Orai1 mediate SOCE and are involved in cytokine production in spinal astrocytes. Our findings provide the basis for future assessment of SOCCs in pain and other central nervous system disorders associated with abnormal astrocyte activities.

Human amnion mesenchymal cells inhibit lipopolysaccharide-induced TNF-α and IL-1β production in THP-1 cells.

BackgroundHuman amnion mesenchymal cells (hAMCs), isolated from the amniotic membrane of human placenta, are a unique population of mesenchymal stem cells. Recent studies demonstrated that hAMCs could inhibit the activities and functions of several immune cells. However, their effect on inflammatory macrophages is largely unknown. This study investigated the effect of hAMCs on expression of inflammatory cytokines and mitogen-activated protein kinases (MAPKs)/NF-κB pathway in human THP-1 macrophages induced by lipopolysaccharide (LPS).ResultsThe levels of TNF-α and IL-1β secreted by LPS- stimulated THP-1 cells were increased significantly compared with those in the control group. After co-culture with different numbers of hAMCs, the levels of TNF-α and IL-1β in LPS-stimulated THP-1 cells were significantly reduced compared with the LPS group. The mRNA expression of TNF-α and IL-1β were also markedly inhibited. Moreover, treating LPS-stimulated THP-1 cells with hAMCs supernatants could also suppress TNF-α and IL-1β production in THP-1 cells. Important signaling pathways involved in the production of TNF-α and IL-1β were affected by hAMCs co-culture: hAMCs remarkably suppressed NF-κB activation and down-regulated the phosphorylation of ERK and JNK in LPS- stimulated THP-1 cells.ConclusionsHuman amnion mesenchymal cells inhibited the production of TNF-α and IL-1β secreted by LPS-stimulated THP-1 cells, partly through the suppression of NF-κB activation and ERK and JNK phosphorylation.

Botulinum neurotoxin type A induces TLR2-mediated inflammatory responses in macrophages.

Botulinum neurotoxin type A (BoNT/A) is the most potent protein toxin and causes fatal flaccid muscle paralysis by blocking neurotransmission. Application of BoNT/A has been extended to the fields of therapeutics and biodefense. Nevertheless, the global response of host immune cells to authentic BoNT/A has not been reported. Employing microarray analysis, we performed global transcriptional profiling of RAW264.7 cells, a murine alveolar macrophage cell line. We identified 70 genes that were modulated following 1 nM BoNT/A treatment. The altered genes were mainly involved in signal transduction, immunity and defense, protein metabolism and modification, neuronal activities, intracellular protein trafficking, and muscle contraction. Microarray data were validated with real-time RT-PCR for seven selected genes including tlr2, tnf, inos, ccl4, slpi, stx11, and irg1. Proinflammatory mediators such as nitric oxide (NO) and tumor necrosis factor alpha (TNFα) were induced in a dose-dependent manner in BoNT/A-stimulated RAW264.7 cells. Increased expression of these factors was inhibited by monoclonal anti-Toll-like receptor 2 (TLR2) and inhibitors specific to intracellular proteins such as c-Jun N-terminal kinase (JNK), extracellular signal–regulated kinase (ERK), and p38 mitogen–activated protein kinase (MAPK). BoNT/A also suppressed lipopolysaccharide-induced NO and TNFα production from RAW264.7 macrophages at the transcription level by blocking activation of JNK, ERK, and p38 MAPK. As confirmed by TLR2-/- knock out experiments, these results suggest that BoNT/A induces global gene expression changes in host immune cells and that host responses to BoNT/A proceed through a TLR2-dependent pathway, which is modulated by JNK, ERK, and p38 MAPK.

Anti‐inflammatory features of the amino acid‐based formula Neocate

Amino acid-based formulas (AAF) are used for the dietary management of cow’s milk allergy and multiple food protein intolerances. A recent study showed that intake of AAF, in addition to a milk-, egg-, wheat- and soy-free diet, results in a reduced colonic inflammatory status in pediatric patients (≤ 5 years, both control and formula group were negative for eosinophilia, [1] ). The present study aimed to evaluate the immune modulating effect of an amino acid-based formula (Neocate) and its pure total amino acid fraction. Human peripheral blood mononuclear cells (PBMCs) were stimulated for 20 hours with lipopolysaccharide (LPS) in the presence and absence of the complete formula, its amino acid fraction, or individual amino acids. Cytokine production was analyzed in the culture supernatant, by ELISA. The influence on CXCL8-induced neutrophil chemotaxis was measured using Boyden chambers. AAF and its amino acid fraction significantly inhibit the LPS-induced TNFα production in a concentration dependent manner. However, the LPS-induced IL-8 production was not affected. In addition to the tested complete formula and amino acid fraction, the single amino acid glycine is able to inhibit the LPS-induced TNFα production. Other amino acids tested, including taurine, lysine and glutamine did not affect the LPS induced TNFα production. CXCL8-induced neutrophil chemotaxis was inhibited by the amino acids fraction and glycine but not by the complete amino acid-based formula. The present study demonstrates that the amino acids in Neocate are able to dampen specific inflammatory responses. This effect might be mediated through specific amino acids or combinations thereof, including glycine. These in vitro studies show that the anti-inflammatory capacity of amino acid-based formulas (as indicated in [1] ) could, at least partially, be induced by the amino acid composition of the formula used.

Modulation of lipopolysaccharide-induced neuronal response by activation of the enteric nervous system.

BackgroundEvidence continues to mount concerning the importance of the enteric nervous system (ENS) in controlling numerous intestinal functions in addition to motility and epithelial functions. Nevertheless, little is known concerning the direct participation of the ENS in the inflammatory response of the gut during infectious or inflammatory insults. In the present study we analyzed the ENS response to bacterial lipopolysaccharide, in particular the production of a major proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α).MethodsTNF-α expression (measured by qPCR, quantitative Polymerase Chain Reaction) and production (measured by ELISA) were measured in human longitudinal muscle-myenteric plexus (LMMP) and rat ENS primary cultures (rENSpc). They were either treated or not treated with lipopolysaccharide (LPS) in the presence or not of electrical field stimulation (EFS). Activation of extracellular signal-regulated kinase (ERK) and 5’-adenosine monophosphate-activated protein kinase (AMPK) pathways was analyzed by immunocytochemistry and Western blot analysis. Their implications were studied using specific inhibitors (U0126, mitogen-activated protein kinase kinase, MEK, inhibitor and C compound, AMPK inhibitor). We also analyzed toll-like receptor 2 (TLR2) expression and interleukin-6 (IL-6) production after LPS treatment simultaneously with EFS or TNF-α-neutralizing antibody.ResultsTreatment of human LMMP or rENSpc with LPS induced an increase in TNF-α production. Activation of the ENS by EFS significantly inhibited TNF-α production. This regulation occurred at the transcriptional level. Signaling analyses showed that LPS induced activation of ERK but not AMPK, which was constitutively activated in rENSpc neurons. Both U0126 and C compound almost completely prevented LPS-induced TNF-α production. In the presence of LPS, EFS inhibited the ERK and AMPK pathways. In addition, we demonstrated using TNF-α-neutralizing antibody that LPS-induced TNF-α production increased TLR2 expression and reduced IL-6 production.ConclusionsOur results show that LPS induced TNF-α production by enteric neurons through activation of the canonical ERK pathway and also in an AMPK-dependent manner. ENS activation through the inhibition of these pathways decreased TNF-α production, thereby modulating the inflammatory response induced by endotoxin.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0202-7) contains supplementary material, which is available to authorized users.

Novel pyrrolo[2,3-d]pyrimidines and pyrrolo[2,3-b]pyridines: design, synthesis, and in vivo TNF-α inhibitory activity

Novel pyrrolo[2,3-d]pyrimidines 5a–j, 6a–j and pyrrolo[2,3-b]pyridines 7a–h; incorporating the common vicinal diaryl motif of tumor necrosis factor-α (TNF-α) inhibitors, were synthesized starting from 2-amino-pyrrole-3-carbonitriles 1a–h. The structures of synthesized compounds were elucidated by spectral data (IR, NMR, and MS) and elemental analyses. Representative compounds were evaluated for their ability to inhibit lipopolysaccharide-induced TNF-α production in vivo in rat at 25 mg/kg p.o. Structure activity relationships are described. The pyrrolo[2,3-d]pyrimidines displayed better inhibitory activity than the pyrrolo[2,3-b]pyridines. The most potent among the biologically tested compounds was the pyrrolopyrimidine 5h (N-(4-ethoxyphenyl)-2-(4-oxo-6-phenyl-7-(pyridine-4-yl)-4H-pyrrolo[2,3-d]pyrimidin-3(7H)-yl)acetamide),showing TNF-α inhibitory activity (96 %) comparable to that of dexamethasone (91 %).