LPS-stimulated Cytokine Production Research Articles

Anti-inflammatory Activity of 8-Hydroxydaidzein in LPS-Stimulated BV2 Microglial Cells via Activation of Nrf2-Antioxidant and Attenuation of Akt/NF-κB-Inflammatory Signaling Pathways, as Well As Inhibition of COX-2 Activity.

It was demonstrated that isoflavones can cross the blood-brain barrier, making them desirable candidate agents for the prevention of neurological symptoms. 8-Hydroxydaidzein (8-OHD, 4',7,8-trihydoxyisoflavone) is an isoflavone found only in fermented soy food. Current results showed that 8-OHD inhibited LPS-stimulated production of nitric oxide (NO) and proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6, by inhibiting gene expression in BV2 microglial cells. Moreover, 8-OHD markedly quenched reactive oxygen species (ROS) and activated NF-E2-related factor 2 (Nrf2) so as to upregulate expression of Phase II enzymes, including heme oxygenase (HO)-1, NAD(P)H quinone dehydrogenase 1 (NQO1), and the modifier subunit of glutamate cysteine ligase (GCLM). 8-OHD also suppressed LPS-stimulated phosphorylation of Akt and NF-κB-p65. The anti-inflammatory activity of 8-OHD was attenuated by the HO-1 inhibitor zinc protoporphyrin (Znpp) but augmented by the PI3K/Akt inhibitor LY294002. 8-OHD also diminished LPS-induced prostaglandin E2 (PGE2) production without affecting cyclooxygenase (COX)-2 expression. In vitro assay shows that 8-OHD displayed mixed-type inhibition of COX-2 with an IC50 of 8.9 ± 1.2 μM. These data suggest that the anti-inflammatory activity of 8-OHD may be associated with the activation of Nrf2/HO-1 and attenuation of Akt/NF-κB signaling pathways as well as inhibition of COX-2 enzyme activity. In conclusion, 8-OHD, a potent Nrf2 activator, Akt/NF-κB activation suppressor, and COX-2 enzyme inhibitor, may have health-promoting effects for mitigating microglia activation and preventing neuroinflammation.

Chemical constituents from Sanguisorba officinalis L. and their inhibitory effects on LPS-stimulated pro-inflammatory cytokine production in bone marrow-derived dendritic cells.

Sanguisorba officinalis L. (Rosaceae) is a perennial herbaceous plant and its roots have been used as an important astringent medicine in Eastern Asian countries over many thousand years. In this phytochemical research, 23 compounds (1-23) were isolated from the roots of S. officinalis. Their chemical structures were identified by extensive spectroscopic methods, including 1D and 2D NMR experiments. The anti-inflammatory effects of extracts and isolated compounds were investigated by measuring the production of pro-inflammatory cytokine IL-12 p40, IL-6 and TNF-α in LPS-stimulated bone marrow-derived dendritic cell. Compounds 1, 3, 7-8, 11-14 revealed promising anti-inflammatory effects. These results suggested that some phenolic compounds and monoterpenoids from S. officinalis could be potential candidates for anti-inflammatory treatments.

Gut Microbiota-Derived Tryptophan Metabolites Modulate Inflammatory Response in Hepatocytes and Macrophages

The gut microbiota plays a significant role in the progression of fatty liver disease; however, the mediators and their mechanisms remain to be elucidated. Comparing metabolite profile differences between germ-free and conventionally raised mice against differences between mice fed a low- and high-fat diet (HFD), we identified tryptamine and indole-3-acetate (I3A) as metabolites that depend on the microbiota and are depleted under a HFD. Both metabolites reduced fatty-acid- and LPS-stimulated production of pro-inflammatory cytokines in macrophages and inhibited the migration of cells toward a chemokine, with I3A exhibiting greater potency. Inhepatocytes, I3A attenuated inflammatory responses under lipid loading and reduced the expression of fatty acid synthase and sterol regulatory element-binding protein-1c. These effects were abrogated in the presence of an aryl-hydrocarbon receptor (AhR) antagonist, indicating that the effects are AhR dependent. Our results suggest that gut microbiota could influence inflammatory responses in the liver through metabolites engaging host receptors.

Inhibitory effects of methamphetamine on mast cell activation and cytokine/chemokine production stimulated by lipopolysaccharide in C57BL/6J mice.

Previous studies have demonstrated that methamphetamine (MA) influences host immunity; however, the effect of MA on lipopolysaccharide (LPS)-induced immune responses remains unknown. Mast cells (MCs) are considered to serve an important role in the innate and acquired immune response, but it remains unknown whether MA modulates MC activation and LPS-stimulated cytokine production. The present study aimed to investigate the effect of MA on LPS-induced MC activation and the production of MC-derived cytokines in mice. Markers for MC activation, including cluster of differentiation 117 and the type I high affinity immunoglobulin E receptor, were assessed in mouse intestines. Levels of MC-derived cytokines in the lungs and thymus were also examined. The results demonstrated that cytokines were produced in the bone marrow-derived mast cells (BMMCs) of mice. The present study demonstrated that MA suppressed the LPS-mediated MC activation in mouse intestines. MA also altered the release of MC cytokines in the lung and thymus following LPS stimulation. In addition, LPS-stimulated cytokines were decreased in the BMMCs of mice following treatment with MA. The present study demonstrated that MA may regulate LPS-stimulated MC activation and cytokine production.

Associations of postpartum sleep, stress, and depressive symptoms with LPS-stimulated cytokine production among African American and White women

BackgroundPostpartum is a period of unique psychosocial stress characterized by sleep disturbance, risk for depressed mood, and heightened parenting stress. However, data on effects of these exposures on inflammatory immune function are limited. MethodsThis study examined associations among sleep, psychosocial stress (i.e., parenting stress, general perceived stress), mood (i.e., depressive symptoms), serum cytokine levels, and LPS-stimulated proinflammatory cytokine production among 69 women (32 African American, 37 White) assessed at 7–10weeks postpartum. ResultsNo associations between behavioral measures and serum cytokine levels were observed among women of either race. In African American women, but not Whites, poorer sleep quality, greater parenting stress, and greater depressive symptoms were associated with greater LPS-stimulated IL-6 and IL-8 production (ps≤0.05). Also in African Americans, greater general perceived stress was associated with greater IL-8 production, and greater depressive symptoms with greater stimulated TNF-α production (ps≤0.05). Simple mediation models highlighted the bidirectional relationship between stress and sleep in relation to inflammation among African American women. ConclusionsSignificant effects of both stress/distress and poor sleep quality on proinflammatory cytokine production during postpartum were observed uniquely among African American women. These data are consistent with an allostatic load model which predicts that conditions of chronic stress impart vulnerability to dysregulated responses to novel stressor exposures. The bidirectional nature of the stress-sleep relationship has clinical relevance. Studies examining whether interventions focused on one or both of these psychological factors during postpartum is beneficial for inflammatory profiles would be informative. In addition, examination of these models in relation to maternal health at postpartum, including delivery related wounds and other infections, is warranted.

Identification of GXXXXG motif in Chrysophsin-1 and its implication in the design of analogs with cell-selective antimicrobial and anti-endotoxin activities

Marine fish antimicrobial peptide, chrysophsin-1 possesses versatile biological activities but its non-selective nature restricts its therapeutic possibilities. Often small alterations in structural motifs result in significant changes in the properties of concerned proteins/peptides. We have identified GXXXXG motif in chrysophsin-1. Glycine residue(s) of this motif in Chrysophsin-1 was/were replaced with alanine, valine and proline residue(s). Of these, proline-substituted Chrysophsin-1 analogs exhibited significantly reduced cytotoxicity towards mammalian cells. Further, these analogs showed broad-spectrum activity against Gram-positive, Gram-negative bacteria, Methicillin-resistant Staphylococcus aureus strains and fungi and also retained antibacterial activity in presence of physiological salts, serum and at elevated temperatures indicative of their therapeutic potential. These Chrysophsin-1 analogs also inhibited lipopolysaccharide (LPS) induced pro-inflammatory responses in THP-1 cells and in murine primary macrophages. One of these single proline-substituted Chrysophsin-1 analogs inhibited LPS-stimulated pro-inflammatory cytokine production in BALB/c mice and elicited appreciable survival of mice administered with a lethal dose of LPS in a model of severe sepsis. The data for the first time showed the implication of GXXXXG motifs in functional and biological properties of an antimicrobial peptide and could be useful to design novel anti-microbial and anti-endotoxin peptides by employing this motif.

Amphiphilic Guanidinocalixarenes Inhibit Lipopolysaccharide (LPS)- and Lectin-Stimulated Toll-like Receptor 4 (TLR4) Signaling.

We recently reported on the activity of cationic amphiphiles in inhibiting TLR4 activation and subsequent production of inflammatory cytokines in cells and in animal models. Starting from the assumption that opportunely designed cationic amphiphiles can behave as CD14/MD-2 ligands and therefore modulate the TLR4 signaling, we present here a panel of amphiphilic guanidinocalixarenes whose structure was computationally optimized to dock into MD-2 and CD14 binding sites. Some of these calixarenes were active in inhibiting, in a dose-dependent way, the LPS-stimulated TLR4 activation and TLR4-dependent cytokine production in human and mouse cells. Moreover, guanidinocalixarenes also inhibited TLR4 signaling when TLR4 was activated by a non-LPS stimulus, the plant lectin PHA. While the activity of guanidinocalixarenes in inhibiting LPS toxic action has previously been related to their capacity to bind LPS, we suggest a direct antagonist effect of calixarenes on TLR4/MD-2 dimerization, pointing at the calixarene moiety as a potential scaffold for the development of new TLR4-directed therapeutics.

Effect of alcohol after muscle-damaging resistance exercise on muscular performance recovery and inflammatory capacity in women.

To investigate the effect of acute alcohol consumption on muscular performance recovery, assessed by maximal torque production, and on inflammatory capacity, assessed by lipopolysaccharide (LPS)-stimulated cytokine production, following muscle-damaging resistance exercise in women. Thirteen recreationally resistance-trained women completed two identical exercise bouts (300 maximal single-leg eccentric leg extensions) followed by alcohol (1.09gethanolkg-1 fat-free body mass) or placebo ingestion. Blood was collected before (PRE), and 5 (5h-POST), 24 (24h-POST), and 48 (48h-POST) hours after exercise and analyzed for LPS-stimulated cytokine production (TNF-α, IL-1β, IL-6, and IL-8 and IL-10). Maximal torque production (concentric, eccentric, isometric) was measured for each leg at PRE, 24h-POST, and 48h-POST. Although the exercise bout increased LPS-stimulated production of TNF-α (%change from PRE: 5h-POST 109%; 24h-POST 49%; 48h-POST 40%) and decreased LPS-stimulated production of IL-8 (5h-POST -40%; 24h-POST -50%; 48h-POST: -43%) and IL-10 (5h-POST: -37%; 24h-POST -32%; 48h-POST -31%), consuming alcohol after exercise did not affect this response. Regardless of drink condition, concentric, eccentric, and isometric torque produced by the exercised leg were lower at 24h-POST (concentric 106±6Nm, eccentric 144±9Nm, isometric 128±8Nm; M±SE) compared to PRE (concentric 127±7Nm, eccentric 175±11Nm, isometric 148±8Nm). Eccentric torque production was partially recovered and isometric torque production was fully recovered by 48h-POST. Alcohol consumed after muscle-damaging resistance exercise does not appear to affect inflammatory capacity or muscular performance recovery in resistance-trained women. Combined with previous findings in men, these results suggest a gender difference regarding effects of alcohol on exercise recovery.

Lipid raft-dependent endocytosis negatively regulates responsiveness of J774 macrophage-like cells to LPS by down regulating the cell surface expression of LPS receptors

Acting through CD14 and TLR4/MD-2, lipopolysaccharide (LPS) triggers strong pro-inflammatory activation of macrophages, which, if not appropriately controlled, may lead to lethal septic shock. Therefore, numerous mechanisms of negative regulation of responses to LPS exist, but whether they include down-regulation of LPS receptors is not clear. We have found that in J774 cells, the clathrin-dependent endocytic pathway enables activation of TRIF-dependent TLR4 signaling within endosomes, but is not associated with the down-regulation of TLR4 or CD14 surface expression. In contrast, lipid raft-dependent endocytosis negatively regulates the basal cell surface expression of LPS receptors and, consequently, responsiveness to LPS. Together with observations that treatments, known to selectively disrupt lipid rafts, do not inhibit LPS-stimulated cytokine production, our results suggest that lipid rafts may serve as sites in which LPS receptors are sorted for endocytosis, rather than being platforms for the assembly of TLR4-centered signaling complexes, as suggested previously.

Myeloid Mixed Lineage Kinase 3 Contributes to Chronic Ethanol-Induced Inflammation and Hepatocyte Injury in Mice.

Proinflammatory activity of hepatic macrophages plays a key role during progression of alcoholic liver disease (ALD). Since mixed lineage kinase 3 (MLK3)-dependent phosphorylation of JNK is involved in the activation of macrophages, we tested the hypothesis that myeloid MLK3 contributes to chronic ethanol-induced inflammatory responses in liver, leading to hepatocyte injury and cell death. Primary cultures of Kupffer cells, as well in vivo chronic ethanol feeding, were used to interrogate the role of MLK3 in the progression of liver injury. Phosphorylation of MLK3 was increased in primary cultures of Kupffer cells isolated from ethanol-fed rats compared to cells from pair-fed rats. Kupffer cells from ethanol-fed rats were more sensitive to LPS-stimulated cytokine production; this sensitization was normalized by pharmacological inhibition of MLK3. Chronic ethanol feeding to mice increased MLK3 phosphorylation robustly in F4/80(+) Kupffer cells, as well as in isolated nonparenchymal cells. MLK3(-/-) mice were protected from chronic ethanol-induced phosphorylation of MLK3 and JNK, as well as multiple indicators of liver injury, including increased ALT/AST, inflammatory cytokines, and induction of RIP3. However, ethanol-induced steatosis and hepatocyte apoptosis were not affected by MLK3. Finally, chimeric mice lacking MLK3 only in myeloid cells were also protected from chronic ethanol-induced phosphorylation of JNK, expression of inflammatory cytokines, and increased ALT/AST. MLK3 expression in myeloid cells contributes to phosphorylation of JNK, increased cytokine production, and hepatocyte injury in response to chronic ethanol. Our data suggest that myeloid MLK3 could be targeted for developing potential therapeutic strategies to suppress liver injury in ALD patients.

Adaptation of the inflammatory immune response across pregnancy and postpartum in Black and White women

Pregnancy is a period of considerable physiological adaption in neuroendocrine, cardiovascular, as well as immune function. Understanding of typical changes in inflammatory immune responses during healthy pregnancy is incomplete. In addition, despite considerable racial difference in adverse pregnancy outcomes, data are lacking on potential racial differences in such adaptation. This repeated measures prospective cohort study included 37 Black and 39 White women who provided blood samples during early, mid-, and late pregnancy and 8–10 weeks postpartum. Peripheral blood mononuclear cells were incubated with lipopolysaccharide (LPS) for 24h and supernatants assayed by electrochemiluminescence to quantify interleukin(IL)-6, tumor necrosis factor(TNF)-α, IL-1β, and IL-8 production. While no changes were observed in IL-8 production over time, significant increases in IL-6, TNF-α, and IL-1β production were observed from early to late pregnancy, with subsequent declines approaching early pregnancy values at postpartum (ps<0.05). Overall, inflammatory response patterns were highly similar among Black versus White women. However, Black women had greater TNF-α production during mid-pregnancy (p=0.002) and marginally lower IL-1β production at postpartum (p=0.054). These data show a clear trajectory of change in the inflammatory immune response across pregnancy and postpartum. In this cohort of generally healthy women, Black and White women exhibited minimal differences in LPS-stimulated cytokine production across the perinatal period. Future prospective studies in Black and White women with healthy versus adverse outcomes (e.g., preeclampsia, preterm birth) would inform our understanding of the potential role of immune dysregulation in pregnant women and in relation to racial disparities in perinatal health.

Abstract A112: Observed separation of survival curves by WBC in CBP501-treated patients leads to experimental identification of macrophage functions suppressed by CBP501

Abstract The CBP501 calmodulin-binding peptide is an anti-cancer drug candidate that has completed a Phase II clinical trial for patients with non-small cell lung carcinoma (NSCLC). CBP501 was also previously identified as a unique G2 checkpoint-directed agent and as an enhancer of cisplatin (CDDP) uptake. In a post-trial analysis of the NSCLC patients, it was found that the survival was statistically significantly different between subpopulations with normal white blood cell counts (WBC) (WBC &amp;lt; 8000) and high WBC (WBC &amp;gt; 8000). Notably, treatment by CDDP with or without CBP501 had no effect on the neutrophil oxidative burst or phagocytosis Here, we show that CBP501 inhibits several functions of macrophages. The LPS-stimulated production of several cytokines (IL-6. TNF-alpha and IL-10) by macrophages were suppressed by CBP501 treatment in vitro. The population of potential cancer stem-like cells was reduced by CBP501 in co-cultures of macrophages and tumor cell lines. In human macrophages isolated from peripheral blood-derived monocytes or THP-1 monocytic cells, phagocytic activity induced by IgG-conjugated beads was suppressed by in vitro CBP501 treatment. The neutrophil extracellular traps (NETs), which are supposed to be cleared by macrophages, were increased by CBP501 in response to leukocyte stimulation. Thrombotic tendency was increased in LPS- and CBP501-treated mice. These results suggest that the inhibition of macrophages by CBP501 could explain the observed dramatic separation of survival curves by WBC in patients with NSCLC. Many reports (Roy Noy and Jeffrey W. Pollard Immunity. 2014) indicate that tumor associated macrophages (TAMs) promote metastasis. To see if CBP501 might affect this relationship between macrophage activity and metastasis, we performed in vivo mouse study of mice bearing subcutaneous Lewis lung carcinomas. Results showed reduced metastasis to the lung upon CBP501 administration. Further investigation is underway to clarify the role of CBP501 in modulating the earlier characterized TAM-promoted metastasis. Citation Format: NAOKI MINE, Sayaka Yamamoto, Naoya Saito, Takuji Sato, Keiichi Sakakibara, Donald W. Kufe, Daniel D. Von Hoff, Takumi Kawabe. Observed separation of survival curves by WBC in CBP501-treated patients leads to experimental identification of macrophage functions suppressed by CBP501. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A112.

The effect of post‑resistance exercise alcohol ingestion on lipopolysaccharide‑stimulated cytokines.

To examine the effect of post-resistance exercise alcohol ingestion on lipopolysaccharide (LPS)-stimulated production of IFNγ, TNF-α, IL-1β, IL-6, IL-8, and IL-10. Recreationally resistance-trained men (n = 10, 25 ± 3 year, 177 ± 7 cm, 83.8 ± 15.7 kg, 14.8 ± 8.5% body fat) and women (n = 8, 23 ± 2 year, 161 ± 3 cm, 59.5 ± 6.0 kg, 26.5 ± 3.0% body fat) completed two identical heavy back squat sessions (6 × 10 at 80% 1 repetition maximum) followed by ingestion of either an alcohol (ALC; 1.09 g ethanol · kg fat-free mass(-1)) or water (PLA) drink. Blood samples were collected before exercise (PRE), and at 3 h (3 h), and 5 h (5 h) after exercise, stimulated with LPS, and analyzed for IFNγ, TNF-α, IL-1β, IL-6, IL-8, and IL-10 concentrations. There were no drink conditions by time effects for IFNγ, TNF-α, IL-1β, or IL-10. Regardless of condition, resistance exercise induce an increase in IFNγ, TNF-α, and IL-1β at 5 h compared to PRE but a decrease in IL-10 at 3 and 5 h compared to PRE. For ALC, IL-8 was reduced at 5 h compared to PLA. From PRE to 3 h, IL-6 was reduced for ALC but increased for PLA; resistance exercise induced an increase in IL-6 for both conditions at 5 h. Heavy resistance exercise increased production of IFNγ, TNF-α, IL-1β, and Il-6 and decreased production of IL-10. Alcohol ingestion after resistance exercise affected aspects of inflammatory capacity (IL-6 and IL-8 production). It appears that some of the effects previously observed for alcohol ingestion alone on the LPS-stimulated cytokine production were overwhelmed by the response to resistance exercise.

The role of C1q in recognition of apoptotic epithelial cells and inflammatory cytokine production by phagocytes during Helicobacter pylori infection.

BackgroundGastric epithelial cells (GECs) undergo apoptosis during H. pylori infection and phagocytes within the mucosa engulf these cells. The recognition and clearance of apoptotic cells is a multifactorial process, enhanced by the presence of various bridging molecules and opsonins which are abundant in serum. However, it is not clear how recognition or clearance may differ in the context of H. pylori infection induced apoptosis. In addition, efferocytosis of sterile apoptotic cells is known to confer anti-inflammatory properties in the engulfing phagocyte, however it is unknown if this is maintained when phagocytes encounter H. pylori-infected cells. Thus, the ability of macrophages to bind and engulf gastric epithelial cells rendered apoptotic by H. pylori infection and the association of these interactions to the modulation of phagocyte inflammatory responses was investigated in the absence and presence of serum with a particular focus on the role of serum protein C1q.MethodsControl (uninfected) or H. pylori-infected AGS cells were co-cultured with THP-1 macrophages in the presence or absence of serum or serum free conditions + C1q protein (40–80 μg/mL). Binding of AGS cells to THP-1 macrophages was assessed by microscopy and cytokine (IL-6 and TNF-α) release from LPS stimulated THP-1 macrophages was quantified by ELISA.ResultsWe show that macrophages bound preferentially to cells undergoing apoptosis subsequent to infection with H. pylori. Binding of apoptotic AGS to THP-1 macrophages was significantly inhibited when studied in the absence of serum and reconstitution of serum-free medium with purified human C1q restored binding of macrophages to apoptotic cells. Co-culture of sterile apoptotic and H. pylori-infected AGS cells both attenuated LPS-stimulated cytokine production by THP-1 macrophages. Further, direct treatment of THP-1 macrophages with C1q attenuated LPS stimulated TNF-α production.ConclusionsThese studies suggest that C1q opsonizes GECs rendered apoptotic by H. pylori. No differences existed in the ability of infected or sterile apoptotic cells to attenuate macrophage cytokine production, however, there may be a direct role for C1q in modulating macrophage inflammatory cytokine production to infectious stimuli.Electronic supplementary materialThe online version of this article (doi:10.1186/s12950-015-0098-8) contains supplementary material, which is available to authorized users.

LPS-Stimulated Whole Blood Cytokine Production Is Not Related to Disease Behavior in Patients with Quiescent Crohn's Disease.

IntroductionCrohn’s disease (CD) is a chronic inflammatory disease in which cytokines play a pivotal role in the induction and maintenance of inflammation. Innate cytokine production is genetically determined and varies largely between individuals; this might impact the severity of inflammation. We aimed to assess whether ex-vivo endotoxin-stimulated levels of cytokines could be associated with disease phenotype.MethodsPatients with quiescent CD (Harvey-Bradshaw Index ≤ 4 and negative inflammation markers) who were not using immunomodulating drugs or biologicals were eligible. Historical disease characteristics (localization, behavior, number of bowel resections, drug history, extra-intestinal symptoms) were extracted from medical records. We measured cytokine levels (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-10) in supernatants of lipopolysaccharide (LPS) -stimulated whole blood cultures and correlated these with disease characteristics and age- and sex-matched healthy controls. In addition, we analyzed whether single nucleotide polymorphisms (SNPs) in the promoter region of the TNF-α gene were related to TNF-α levels.ResultsWe included 75 patients with CD and 24 healthy controls. Six patients were excluded because of increased inflammation markers resulting in a total of 69 patients. The mean age (SD) of patients with CD was 51.2 (12.3) years with a mean (SD) disease duration of 24.1 (11.5) years. Disease localization, peri-anal involvement and behavior were not related to LPS-stimulated TNF-α, IL-1β, IL-6 or IL-10 levels. In addition, combination of localization with behavior to differentiate mild from severe disease type showed no significant differences. TNF-α levels were higher in patients with CD (428 pg/ml IQR [267-468]) compared to healthy controls (459 pg/ml IQR [364-570], p=0.02). We found no associations between SNPs in the promoter region and TNF-α levels.ConclusionIn this study, innate cytokine production of TNF-α, IL-1β, IL-6 and IL-10 was not related to historical disease characteristics or disease severity in patients with quiescent CD. These findings suggest that genetically determined levels of these cytokines obtained from LPS-stimulated whole blood cultures are not linked with disease behavior or severity.

Effects of Standardized Wrightia tinctoria and Stereospermum suaveolens Extracts on Various Immuno-Inflammatory Parameters

Effects of standardized Wrightia tinctoria seeds extract (WTSE) and Stereospermum suaveolens root extract (SSRE) on erythrocyte membrane stabilization, mast cell protection, nitric oxide, and pro-inflammatory cytokines production were evaluated by in vitro methods. WTSE and SSRE were standardized against chlorogenic acid and lapachol, respectively, using reverse-phase high-performance liquid chromatography. Standardized WTSE and SSRE inhibited erythrocyte membrane lysis, mast cell degranulation, and LPS-stimulated nitric oxide and pro-inflammatory cytokine (IL-β, IL-6 and TNF-α) production by RAW 264.7 macrophage cells in a dose-dependent manner.

Hepatitis B e antigen perturbs the LPS-stimulated production of inflammatory cytokines by mononuclear-derived dendritic cells

To investigate whether hepatitis B e antigen (HBeAg) can modulate the ability of dendritic cells (DCs) to produce inflammatory cytokines (IL-12/IL-6) upon stimulation in vitro. Purified adherent mononuclear cells isolated by Ficoll-hypaque density gradient centrifugation were cultured in complete medium containing granulocyte macrophage colony-stimulating factor plus interleukin (IL)-4 to generate immature (i)DCs. Microscopic analysis and flow cytometry were performed to define the phenotypic characteristics of the iDCs. Then, different concentrations (1, 2 and 5 mug/ml) of HBeAg were added to the culture medium and for 24 hrs of incubation. To induce iDCs' maturation, the various groups of cells were incubated for 24 hrs in differentiation culture with lipopolysaccharide (LPS). Effects on secreted inflammatory cytokines were determined by enzyme-linked immunosorbent assay of the cells' supernatants. All concentrations of HBeAg led to significant reductions in IL-6 (all P less than 0.05). Similar significant reduction trends were seen for IL-12 at the HBeAg concentrations of 2 and 5 mug/ml (both P less than 0.05), but not at the 1 mug/ml concentration. HBeAg may suppress the production of cytokines from DCs; this mechanism may contribute to the immune escape of HBV that supports persistent infection.

Oxytocin Does Not Attenuate the ex vivo Production of Inflammatory Cytokines by Lipopolysaccharide-Activated Monocytes and Macrophages from Healthy Male and Female Donors

Background: Oxytocin (OT) is a neuropeptide shown to attenuate inflammatory responses in both humans and animals, but the specific mechanism underlying these actions has not yet been identified. Preliminary research in humans suggests that monocytes (MOs) and macrophages (MPs) could be the target of anti-inflammatory actions of OT. Here, we present a series of ex vivo experiments in human MOs and MPs, testing whether OT attenuates the cytokine responses of these cells to a common bacterial product, lipopolysaccharide (LPS). Methods: MO experiments were conducted using blood samples taken from healthy volunteers after obtaining informed consent. MPs were purchased frozen from a cell supplier. All samples were cultured under standard conditions: for 6 h at 37°C in a 5% CO₂ atmosphere. A number of variables were considered: volunteer sex, method of MO isolation, LPS concentration, OT concentration, preincubation with OT, cytokines measured, and method of cytokine measurement. Results: Regardless of the specific conditions, no attenuation of LPS-stimulated cytokine production by OT was observed in either MOs or MPs. Conclusion: OT does not attenuate MO or MP inflammatory cytokine production following LPS stimulation. The previously observed anti-inflammatory properties of OT may be attributable to effects on other classes of immune cells or actions in other lymphoid compartments. Alternatively, the effects of OT on inflammation could be secondary to other neurohormonal changes it elicits.

Genome-Wide Association Study of Genetic Variants in LPS-Stimulated IL-6, IL-8, IL-10, IL-1ra and TNF-α Cytokine Response in a Danish Cohort

BackgroundCytokine response plays a vital role in various human lipopolysaccharide (LPS) infectious and inflammatory diseases. This study aimed to find genetic variants that might affect the levels of LPS-induced interleukin (IL)-6, IL-8, IL-10, IL-1ra and tumor necrosis factor (TNF)-α cytokine production.MethodsWe performed an initial genome-wide association study using Affymetrix Human Mapping 500 K GeneChip® to screen 130 healthy individuals of Danish descent. The levels of IL-6, IL-8, IL-10, IL-1ra and TNF-α in 24-hour LPS-stimulated whole blood samples were compared within different genotypes. The 152 most significant SNPs were replicated using Illumina Golden Gate® GeneChip in an independent cohort of 186 Danish individuals. Next, 9 of the most statistical significant SNPs were replicated using PCR-based genotyping in an independent cohort of 400 Danish individuals. All results were analyzed in a combined study among the 716 Danish individuals.ResultsOnly one marker of the 500 K Gene Chip in the discovery study showed a significant association with LPS-induced IL-1ra cytokine levels after Bonferroni correction (P<10−7). However, this SNP was not associated with the IL-1ra cytokine levels in the replication dataset. No SNPs reached genome-wide significance for the five cytokine levels in the combined analysis of all three stages.ConclusionsThe associations between the genetic variants and the LPS-induced IL-6, IL-8, IL-10, IL-1ra and TNF-α cytokine levels were not significant in the meta-analysis. This present study does not support a strong genetic effect of LPS-stimulated cytokine production; however, the potential for type II errors should be considered.

Flavonoids from Astragalus membranaceus and their inhibitory effects on LPS-stimulated pro-inflammatory cytokine production in bone marrow-derived dendritic cells

Radix Astragali (Astragalus membranaceus) is an important traditional Chinese medicine that is widely used as a tonic to enhance the body's natural defense mechanisms. In this phytochemical study, 12 flavonoids, isoliquiritigenin (1), liquiritigenin (2), calycosin (3), calycosin 7-O-β-D-glucoside (4), formononetin (5), formononetin 7-O-β-D-glucoside (6), daidzein (7), daidzein 7-O-β-D-glucoside (8), methylnissolin (9), methylnissolin 3-O-β-D-glucoside (10), isomucronulatol (11), and isomucronulatol 7-O-β-D-glucoside (12), were isolated from the roots of A. membranaceus. Their structures were elucidated by comparing spectroscopic data with reported values. The effects of the isolated compounds on lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells were investigated. Compounds 1 and 2 exhibited significant inhibitory effects on LPS-induced IL-6 and IL-12 p40 production, with IC50 values ranging from 2.7 to 6.1μM. Compound 1 also showed a moderate inhibitory effect on LPS-stimulated production of TNF-α with an IC50 value of 20.1μM. Further studies of the potential anti-inflammatory effects and benefits of flavonoids from A. membranaceus are warranted.