Ganoderma lucidum polysaccharides enhance CD14 endocytosis of LPS and promote TLR4 signal transduction of cytokine expression

We have previously reported that a well-characterized glycoprotein fraction containing fucose residues in an extract of Ganoderma lucidum polysaccharides (EORP) exerts certain immuno-modulation activity by stimulating the expression of inflammatory cytokines via TLR4. Continuing our studies, we have demonstrated that EORP increases the surface expression of CD14 and TLR4 within murine macrophages J774A.1 cells in vitro, and further promotes LPS binding and uptake by J774A.1 cells in a CD14-dependent fashion. Moreover, we observed the co-localization of internalized LPS with lysosome- and Golgi-apparatus markers within 5 min after J774A.1 cells stimulated with LPS. In addition, EORP pretreatment of J774A.1 cells and human blood-derived primary macrophages, followed by LPS stimulation, results in the super-induction of interleukin-1beta (IL-1) expression. Endocytosis inhibitors: such as cytochalasin D and colchicine effectively block EORP-enhanced LPS internalization by J774A.1 cells; yet they fail to decrease the LPS-induced phosphorylation of certain mitogen-activated protein kinases, and IL-1 mRNA and proIL-1 protein expression, indicating that LPS internalization by J774A.1 cells is not associated with LPS-dependent activation. Our current results could provide a potential EORP-associated protection mechanism for bacteria infection by enhancing IL-1 expression and the clearance of contaminated LPS by macrophages.     

Flavonoid Apigenin Inhibits Lipopolysaccharide-Induced Inflammatory Response through Multiple Mechanisms in Macrophages

Background

Apigenin is a non-toxic natural flavonoid that is abundantly present in common fruits and vegetables. It has been reported that apigenin has various beneficial health effects such as anti-inflammation and chemoprevention. Multiple studies have shown that inflammation is an important risk factor for atherosclerosis, diabetes, sepsis, various liver diseases, and other metabolic diseases. Although it has been long realized that apigenin has anti-inflammatory activities, the underlying functional mechanisms are still not fully understood.

Methodology and Principal Findings

In the present study, we examined the effect of apigenin on LPS-induced inflammatory response and further elucidated the potential underlying mechanisms in human THP-1-induced macrophages and mouse J774A.1 macrophages. By using the PrimePCR array, we were able to identify the major target genes regulated by apigenin in LPS-mediated immune response. The results indicated that apigenin significantly inhibited LPS-induced production of pro-inflammatory cytokines, such as IL-6, IL-1β, and TNF-α through modulating multiple intracellular signaling pathways in macrophages. Apigenin inhibited LPS-induced IL-1β production by inhibiting caspase-1 activation through the disruption of the NLRP3 inflammasome assembly. Apigenin also prevented LPS-induced IL-6 and IL-1β production by reducing the mRNA stability via inhibiting ERK1/2 activation. In addition, apigenin significantly inhibited TNF-α and IL-1β-induced activation of NF-κB.

Conclusion and Significance

Apigenin Inhibits LPS-induced Inflammatory Response through multiple mechanisms in macrophages. These results provided important scientific evidences for the potential application of apigenin as a therapeutic agent for inflammatory diseases.     

Microtubule-associated serine/threonine kinase-205 kDa and Fc gamma receptor control IL-12 p40 synthesis and NF-kappa B activation

Stimulation of murine macrophages with LPS results in the coordinated activation of a set of proinflammatory cytokines and costimulatory molecules, including TNF-alpha, IL-6, IL-1, IL-8, IL-12, and CD80. Macrophage LPS-induced synthesis of IL-12 is inhibited following FcgammaR ligation; TNF-alpha secretion is unchanged. We report that microtubule-associated serine/threonine kinase-205 kDa (MAST205) is required for LPS-induced IL-12 synthesis. RNA interference-mediated suppression of MAST205 results in the inhibition of LPS-stimulated IL-12 promoter activity and IL-12 secretion, from both J774 cells and bone marrow-derived macrophages. Similarly, dominant-negative MAST205 mutants inhibit LPS-stimulated IL-12 synthesis and NF-kappaB activation, but do not affect IL-1 or TNF-alpha signaling. Finally, macrophage FcgammaR ligation regulates MAST205 by inducing the rapid ubiquitination and proteasomal degradation of the protein.     

Carbon nanotubes provoke inflammation by inducing the pro-inflammatory genes IL-1β and IL-6

Carbon nanotubes (CNTs) are largely produced and widely used because of their novel features, and the annual yield is expected to increase dramatically in the near future. Meanwhile, adverse health influences from exposure to CNTs are widely concerned, partially due to their asbestoid characteristics. In the current study, to assess the inflammatory responses and related mechanisms, we established a mouse model of chronic exposure to CNTs using intraperitoneal injection of single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs). Our results demonstrated the fibre-like pathogenic behaviors of CNTs, reflected by increased total protein content in the lavageate from peritoneal cavities and increased serum levels of inflammatory cytokines, IL-1β and IL-6. The pro-inflammatory effects of CNTs were further validated with exposure to in vitro cultured monocyte-macrophage cells, J774A.1, as SWNTs and MWNTs significantly increased the expression levels of pro-inflammatory genes IL-1β and IL-6. Collectively, our data demonstrate that SWNTs and MWNTs provoke considerable inflammation presumably due to their fibre-like shape, and further confirm the length- and size-related structure-activity relationship for CNTs in stimulating inflammatory responses.     

Effect of In Vitro and In Vivo Anakinra on Cytokines Production in Schnitzler Syndrome

IL-1 receptor antagonist anakinra is usually highly efficient in Schnitzler syndrome (SS), a rare inflammatory condition associating urticaria, fever, and IgM monoclonal gammopathy. In this study, we aimed to assess lipopolysaccharide (LPS)-induced production of inflammatory cytokines by peripheral blood mononuclear cells (PBMCs) before and after 1 month of anakinra in patients with SS. LPS-induced production of IL-1β, IL-6 and TNFα was assessed by enzyme-linked immunosorbent assay with and without anakinra in vitro, and before and after 1 month (in vivo condition) of treatment in 2 patients with SS. Spontaneous production of IL-1β, IL-6 and TNF-α by PBMCs was similar in the patients and the healthy controls and was almost undetectable. Stimulation with LPS caused a higher release of cytokines from the patients than from the healthy controls. Before in vivo anakinra start, in vitro adjunction of anakinra reduced the high LPS-induced production of IL-1β and TNFα in both patients and of IL-6 in one patient. After 1 month of treatment with anakinra, while the patients had dramatically improved, there was also a marked reduction in LPS-induced cytokines production, which was almost normalized in one patient. This study shows an abnormal LPS-induced inflammatory cytokines production in SS, which can be decreased or even normalized by in vitro and in vivo anakinra.     

Budlein A from Viguiera robusta inhibits leukocyte-endothelial cell interactions,adhesion molecule expression and inflammatory mediators release

Budlein A has been reported to exert some analgesic and anti-inflammatory properties. In this study, we have evaluated its effect on LPS-induced leukocyte recruitment in vivo and the mechanisms involved in its anti-inflammatory activity. In vivo, intravital videomicroscopy was used to determine the effects of budlein A on LPS-induced leukocyte-endothelial cell interactions in the murine cremasteric microcirculation. In vitro, the effects of budlein A on LPS-induced cytokine, chemokine and nitrites release, T-cell proliferative response as well as cell adhesion molecule expression (CAM) were evaluated. In vivo, intraperitoneal administration of budlein A (2.6 mM/kg) caused a significant reduction of LPS-induced leukocyte rolling flux, adhesion and emigration by 84, 92 and 96% respectively. In vitro, T-cell proliferative response was also affected by budlein A. When murine J774 macrophages were incubated with the sesquiterpene lactone, LPS-induced IL-1β, tumor necrosis factor-α (TNF-α) and keratinocyte-derived chemokine (KC) release were concentration-dependently inhibited. In human umbilical vein endothelial cells (HUVECs), budlein A also reduced the production of TNF-α, monocyte chemoattractant protein-1 (MCP-1), IL-8, nitrites and CAM expression elicited by LPS. Budlein A is a potent inhibitor of LPS-induced leukocyte accumulation in vivo. This effect appears to be mediated through inhibition of cytokine and chemokine release and down-regulation of CAM expression. Thus, it has potential therapeutic interest for the control of leukocyte recruitment that occurs in different inflammatory disorders.     

Eupatilin inhibits lipopolysaccharide-induced expression of inflammatory mediators in macrophages

AimsEupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) is a pharmacologically active ingredients in Stillen^TM, a drug for the gastric mucosal ulcers. Eupatilin has been known to possess anti-peptic, anti-cancer, and anti-allergy activity. In this report, we defined the effect of eupatilin on the endotoxin-induced inflammation in lipopolysaccharide (LPS)-stimulated macrophages.Main methodsMouse J774A.1 cell line and mouse peritoneal macrophages were used. Gene expression and production of inflammatory mediators were determined by real-time PCR and Western blot.Key findingsEupatilin dose-dependently suppressed LPS-induced expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO). Eupatilin decreased LPS-induced expression of inflammatory mediators and pro-inflammatory cytokines such as cyclooxygenase-2, monocyte chemoattractant protein-1, tumor necrosis factor-α, interleukin (IL)-1β and IL-6. In addition, this suppression of inflammatory mediators was nuclear factor (NF)-κB dependent.SignificanceOur findings imply that eupatilin suppresses inflammatory responses by the inhibition of NF-κB signaling pathway, and downstream inflammatory mediators in endotoxin-stimulated macrophages.     

β-Glucan from Saccharomyces cerevisiae reduces lipopolysaccharide-induced inflammatory responses in RAW264.7 macrophages

Background

β-Glucans obtained from fungi, such as baker's yeast (Saccharomyces cerevisiae)-derived β-glucan (BBG), potently activate macrophages through nuclear factor κB (NFκB) translocation and activation of its signaling pathways. The mechanisms by which β-glucans activate these signaling pathways differ from that of lipopolysaccharide (LPS). However, the effects of β-glucans on LPS-induced inflammatory responses are poorly understood. Here, we examined the effects of BBG on LPS-induced inflammatory responses in RAW264.7 mouse macrophages.

Methods

We explored the actions of BBG in RAW264.7 macrophages.

Results

BBG inhibited LPS-stimulated nitric oxide (NO) production in RAW264.7 macrophages by 35–70% at concentrations of 120–200 μg/ml. BBG also suppressed mRNA and protein expression of LPS-induced inducible NO synthase (iNOS) and mitogen-activated protein kinase phosphorylation, but not NFκB activation. By contrast, a neutralizing antibody against dectin-1, a β-glucan receptor, did not affect BBG-mediated inhibition of NO production. Meanwhile, BBG suppressed Pam3CSK-induced NO production. Moreover, BBG suppressed LPS-induced production of pro-and anti-inflammatory cytokines, including interleukin (IL)-1α, IL-1ra, and IL-27.

Conclusions

Our results indicate that BBG is a powerful inhibitor of LPS-induced NO production by downregulating iNOS expression. The mechanism involves inactivation of mitogen-activated protein kinase and TLR2 pathway, but is independent of dectin-1.

General significance

BBG might be useful as a novel agent for the chemoprevention of inflammatory diseases.     

IL-10 induces gene expression in macrophages: partial overlap with IL-5 but not with IL-4 induced genes

The hypothesis that IL-10, in addition to down-regulating pro-inflammatory activities of macrophages, induces an alternative state of macrophage reactivity was tested. We therefore conducted a systematic search for genes induced by IL-10 using the method of suppression subtractive hybridisation. Of an initial 1,300 candidate clones obtained, several screening rounds led to the identification of 51 clones which were reproducibly at least twofold up-regulated in mouse J774 macrophages in response to treatment with IL-10. Of these, 41 genes were homologous to known genes involved in cell metabolism or immunoregulation, five contained novel sequences and another five were homologous to ESTs without known function. One major finding was that about 25% of the IL-10 genes were also found expressed in response to IFNgamma, and several of these also reappeared in IL-4 or IL-5 induced mRNA species. Hence, Th1 and Th2 type cytokines may elicit a common basal activation response in macrophages. The second major finding was that 57% of IL-10 induced genes reappeared in IL-5 induced mRNA but no more than 18% were also found in IL-4 induced mRNA of J774 cells. We conclude that the gene expression response to IL-10 in macrophages is partially different from the response to IL-5 and is substantially different from the response to IL-4, which suggests an unexpected diversity of biological phenotypes induced by different Th2 type cytokines.     

An Estrogen Receptor Dependent Mechanism of Oroxylin A in the Repression of Inflammatory Response

Oroxylin A, a natural flavonoid, is one of the main bioactive compounds that underlie the anti-inflammatory effect of the medicinal herb Scutellaria baicalensis Georgi widely used in southeastern Asia; however, the molecular mechanisms for the therapeutic benefits remain largely unclear. In this study, we found that Oroxylin A induces estrogen-responsive gene expression and promoter activity. In macrophages, Oroxylin A treatment significantly attenuates lipopolysaccharide (LPS)-induced but not basal inflammatory response, including nitric oxide (NO) production and the expression of inflammatory mediators (i.e., iNOS and COX-2) and cytokines (i.e., TNF-α, IL-1β, and IL-6), in an estrogen receptor (ER)-dependent manner. Oroxylin A treatment also dramatically decreases LPS-induced secretion of pro-inflammatory cytokines. Furthermore, the downregulation of all these inflammatory parameters by Oroxylin A was abolished when cells were pretreated with specific ER antagonist. Thus, Oroxylin A is a novel phytoestrogen and exhibits anti-inflammatory effects that are mediated by ER activity.     

Maternal LPS induces cytokines in the amniotic fluid and corticotropin releasing hormone in the fetal rat brain

Perinatal infections are a risk factor for fetal neurological pathologies, including cerebral palsy and schizophrenia. Cytokines that are produced as part of the inflammatory response are proposed to partially mediate the neurological injury. This study investigated the effects of intraperitoneal injections of lipopolysaccharide (LPS) to pregnant rats on the production of cytokines and stress markers in the fetal environment. Gestation day 18 pregnant rats were treated with LPS (100 microg/kg body wt i.p.), and maternal serum, amniotic fluid, placenta, chorioamnion, and fetal brain were harvested at 1, 6, 12, and 24 h posttreatment to assay for LPS-induced changes in cytokine protein (ELISA) and mRNA (real-time RT-PCR) levels. We observed induction of proinflammatory cytokines interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha) as well as the anti-inflammatory cytokine IL-10 in the maternal serum within 6 h of LPS exposure. Similarly, proinflammatory cytokines were induced in the amniotic fluid in response to LPS; however, no significant induction of IL-10 was observed in the amniotic fluid. LPS-induced mRNA changes included upregulation of the stress-related peptide corticotropin-releasing factor in the fetal whole brain, TNF-alpha, IL-6, and IL-10 in the chorioamnion, and TNF-alpha, IL-1 beta, and IL-6 in the placenta. These findings suggest that maternal infections may lead to an unbalanced inflammatory reaction in the fetal environment that activates the fetal stress axis.     

Ceramide and ceramide 1-phosphate are negative regulators of TNF-α production induced by lipopolysaccharide

LPS is a constituent of cell walls of Gram-negative bacteria that, acting through the CD14/TLR4 receptor complex, causes strong proinflammatory activation of macrophages. In murine peritoneal macrophages and J774 cells, LPS at 1-2 ng/ml induced maximal TNF-α and MIP-2 release, and higher LPS concentrations were less effective, which suggested a negative control of LPS action. While studying the mechanism of this negative regulation, we found that in J774 cells, LPS activated both acid sphingomyelinase and neutral sphingomyelinase and moderately elevated ceramide, ceramide 1-phosphate, and sphingosine levels. Lowering of the acid sphingomyelinase and neutral sphingomyelinase activities using inhibitors or gene silencing upregulated TNF-α and MIP-2 production in J774 cells and macrophages. Accordingly, treatment of those cells with exogenous C8-ceramide diminished TNF-α and MIP-2 production after LPS stimulation. Exposure of J774 cells to bacterial sphingomyelinase or interference with ceramide hydrolysis using inhibitors of ceramidases also lowered the LPS-induced TNF-α production. The latter result indicates that ceramide rather than sphingosine suppresses TNF-α and MIP-2 production. Of these two cytokines, only TNF-α was negatively regulated by ceramide 1-phosphate as was indicated by upregulated TNF-α production after silencing of ceramide kinase gene expression. None of the above treatments diminished NO or RANTES production induced by LPS. Together the data indicate that ceramide negatively regulates production of TNF-α and MIP-2 in response to LPS with the former being sensitive to ceramide 1-phosphate as well. We hypothesize that the ceramide-mediated anti-inflammatory pathway may play a role in preventing endotoxic shock and in limiting inflammation.     

Il-10 is a central regulator of cyclooxygenase-2 expression and prostaglandin production

IL-10 is a potent anti-inflammatory and immune regulatory cytokine. IL-10(-/-) mice produce exaggerated amounts of inflammatory cytokines when stimulated with LPS, indicating that endogenous IL-10 is a central regulator of inflammatory cytokine production in vivo. PGs are lipid mediators that are also produced in large amounts during the inflammatory response. To study the role of IL-10 in the regulation of PG production during the acute inflammatory response, we evaluated LPS-induced cyclooxygenase (COX) expression and PG production in wild-type (wt) and IL-10(-/-) mice. LPS-induced PGE(2) production from IL-10(-/-) spleen cells was 5.6-fold greater than that from wt spleen cells. LPS stimulation resulted in the induction of COX-2 mRNA and protein in both wt and IL-10(-/-) spleen cells; however, the magnitude of increase in COX-2 mRNA was 5.5-fold greater in IL-10(-/-) mice as compared with wt mice. COX-1 protein levels were not affected by LPS stimulation in either wt or IL-10(-/-) mice. Neutralization of IFN-gamma, TNF-alpha, or IL-12 markedly decreased the induction of COX-2 in IL-10(-/-) spleen cells, suggesting that increased inflammatory cytokine production mediates much of the COX-2 induction in IL-10(-/-) mice. Treatment of IL-10(-/-) mice with low doses of LPS resulted in a marked induction of COX-2 mRNA in the spleen, whereas wt mice had minimal expression of COX-2 mRNA. These findings indicate that, in addition to IL-10's central role in the regulation of inflammatory cytokines, endogenous IL-10 is an important regulator of PG production in the response to LPS.     

Clinical outcome following acute ischaemic stroke relates to both activation and autoregulatory inhibition of cytokine production

Background

As critical mediators of local and systemic inflammatory responses, cytokines are produced in the brain following ischaemic stroke. Some have been detected in the circulation of stroke patients, but their role and source is unclear. Focusing primarily on interleukin(IL)-1-related mechanisms, we serially measured plasma inflammatory markers, and the production of cytokines by whole blood, from 36 patients recruited within 12 h and followed up to 1 year after acute ischaemic stroke (AIS).

Results

Admission plasma IL-1 receptor antagonist (IL-1ra) concentration was elevated, relative to age-, sex-, and atherosclerosis-matched controls. IL-1β, soluble IL-1 receptor type II, tumour necrosis factor (TNF)-α, TNF-RII, IL-10 and leptin concentrations did not significantly differ from controls, but peak soluble TNF receptor type I (sTNF-RI) in the first week correlated strongly with computed tomography infarct volume at 5–7 days, mRS and BI at 3 and 12 months. Neopterin was raised in patients at 5–7 d, relative to controls, and in subjects with significant atherosclerosis. Spontaneous IL-1β, TNF-α and IL-6 gene and protein expression by blood cells was minimal, and induction of these cytokines by lipopolysaccharide (LPS) was significantly lower in patients than in controls during the first week. Minimum LPS-induced cytokine production correlated strongly with mRS and BI, and also with plasma cortisol.

Conclusion

Absence of spontaneous whole blood gene activation or cytokine production suggests that peripheral blood cells are not the source of cytokines measured in plasma after AIS. Increased plasma IL-1ra within 12 h of AIS onset, the relationship between sTNF-RI and stroke severity, and suppressed cytokine induction suggests early activation of endogenous immunosuppressive mechanisms after AIS.     

Red blood cell lysate modulates the expression of extracellular matrix proteins in dermal fibroblasts

Trichinella spiralis is a zoonotic nematode and food borne parasite and infection with T. spiralis leads to suppression of the host immune response and other immunopathologies. The excretory/secretory (ES) products of T. spiralis play important roles in the process of immunomodulation. However, the mechanisms and related molecules are unknown. Macrophages, a target for immunomodulation by the helminth parasite, play a critical role in initiating and modulating the host immune response to parasite infection. In this study, we examined the effect of ES products from different stages of T. spiralis on modulating J774A.1 macrophage activities. ES products from different stages of T. spiralis reduced the capacity of macrophages to express pro-inflammatory cytokines (tumor necrosis factor α, interleukin-1β , interleukin-6 , and interleukin-12) in response to lipopolysaccharide (LPS) challenge. However, only ES products from 3-day-old adult worms and 5-day-old adult worms/new-born larvae significantly inhibited inducible nitric oxide synthase gene expression in LPS-induced macrophages. In addition, ES products alone boosted the expression of anti-inflammatory cytokines interleukin-10 and transforming growth factor-β and effector molecule arginase 1 in J774A.1 macrophages. Signal transduction studies showed that ES products significantly inhibited nuclear factor-κB translocation into the nucleus and the phosphorylation of both extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in LPS-stimulated J774A.1 macrophages. These results suggest that ES products regulate host immune response at the macrophage level through inhibition of pro-inflammatory cytokines production and induction of macrophage toward the alternative phenotype, which maybe important for worm survival and host health.     

Acrolein inhalation suppresses lipopolysaccharide-induced inflammatory cytokine production but does not affect acute airways neutrophilia

Acrolein is a reactive unsaturated aldehyde that is produced during endogenous oxidative processes and is a major bioactive component of environmental pollutants such as cigarette smoke. Because in vitro studies demonstrate that acrolein can inhibit neutrophil apoptosis, we evaluated the effects of in vivo acrolein exposure on acute lung inflammation induced by LPS. Male C57BL/6J mice received 300 microg/kg intratracheal LPS and were exposed to acrolein (5 parts per million, 6 h/day), either before or after LPS challenge. Exposure to acrolein either before or after LPS challenge did not significantly affect the overall extent of LPS-induced lung inflammation, or the duration of the inflammatory response, as observed from recovered lung lavage leukocytes and histology. However, exposure to acrolein after LPS instillation markedly diminished the LPS-induced production of several inflammatory cytokines, specifically TNF-alpha, IL-12, and the Th1 cytokine IFN-gamma, which was associated with reduction in NF-kappaB activation. Our data demonstrate that acrolein exposure suppresses LPS-induced Th1 cytokine responses without affecting acute neutrophilia. Disruption of cytokine signaling by acrolein may represent a mechanism by which smoking contributes to chronic disease in chronic obstructive pulmonary disease and asthma.     

N-acetyl tryptophan glucopyranoside (NATG) as a countermeasure against gamma radiation-induced immunosuppression in murine macrophage J774A.1 cells

Radiation exposure to immune system induces imbalance in cytokines expression involved in Th1/Th2 homeostasis perturbations. In the present study, N-acetyl tryptophan glucoside (NATG), a bacterial secondary metabolite, was evaluated for its possible radioprotective potential to immune system using J774A.1 murine macrophages. In this study, expression of IFN-γ, TNF-α, IL-10, IL-2, IL-12, IL-13 and IL-17A cytokines was analyzed in irradiated and NATG pretreated cells using ELISA assay. Results of the study indicated that irradiated macrophages (NK-1R^+?cells) pretreated with NATG showed higher (p?相似文献