Parthenolide inhibits TRIF-dependent signaling pathway of toll-like receptors in RAW264.7 macrophages

Toll-like receptors (TLRs) play an important role in induction of innate immune responses for host defense against invading microbial pathogens. Microbial component engagement of TLRs can trigger the activation of myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent downstream signaling pathways. Parthenolide, an active ingredient of feverfew (Tanacetum parthenium), has been used for centuries to treat many chronic diseases. Parthenolide inhibits the MyD88-dependent pathway by inhibiting the activity of inhibitor-κB kinase. However, it is not known whether parthenolide inhibits the TRIF-dependent pathway. To evaluate the therapeutic potential of parthenolide, its effect on signal transduction via the TRIF-dependent pathway of TLRs induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (poly [I:C]) was examined. Parthenolide inhibited nuclear factor-κB and interferon regulatory factor 3 activation induced by LPS or poly[I:C], and the LPS-induced phosphorylation of interferon regulatory factor 3 as well as interferon-inducible genes such as interferon inducible protein-10. These results suggest that parthenolide can modulate TRIF-dependent signaling pathways of TLRs, and may be the basis of effective therapeutics for chronic inflammatory diseases.     

Suppression of homodimerization of toll-like receptor 4 by isoliquiritigenin

Toll-like receptors (TLRs) play important inductive roles in innate immune responses for host defense against invading microbial pathogens. Activation of TLR4 by lipopolysaccharide (LPS) induces dimerization of TLR4 and, subsequently, activation of downstream signaling pathways including nuclear factor-kappa B and interferon regulatory factor 3. TLR4 dimerization may be an early regulatory event in activating signaling pathways induced by LPS. Here, biochemical evidence is reported that isoliquiritigenin, one of the major ingredients derived from licorice root, inhibits LPS-induced TLR4 dimerization resulting in inhibition of nuclear factor-kappa B and interferon regulatory factor 3 activation, and cyclooxygenase-2 and inducible nitric oxide synthase expression. These results suggest that isoliquiritigenin modulates TLR-mediated signaling pathways at the receptor level. Furthermore, these results suggest that TLRs themselves may be important targets for the prevention of chronic inflammatory diseases.     

Suppression of the TRIF-dependent signaling pathway of toll-like receptors by (E)-isopropyl 4-oxo-4-(2-oxopyrrolidin-1-yl)-2-butenoate

Toll-like receptors (TLRs) are pattern recognition receptors that recognize molecular structures derived from microbes and initiate innate immunity. TLRs have two downstream signaling pathways, the MyD88- and TRIF-dependent pathways. Dysregulated activation of TLRs is closely linked to increased risk of many chronic diseases. Previously, we synthesized fumaryl pyrrolidinone, (E)-isopropyl 4-oxo-4-(2-oxopyrrolidin-1- yl)-2-butenoate (IPOP), which contains a fumaric acid isopropyl ester and pyrrolidinone, and demonstrated that it inhibits the activation of nuclear factor kappa B by inhibiting the MyD88-dependent pathway of TLRs. However, the effect of IPOP on the TRIF-dependent pathway remains unknown. Here, we report the effect of IPOP on signal transduction via the TRIF-dependent pathway of TLRs. IPOP inhibited lipopolysaccharide- or polyinosinic-polycytidylic acid-induced interferon regulatory factor 3 activation, as well as interferon- inducible genes such as interferon inducible protein-10. These results suggest that IPOP can modulate the TRIF-dependent signaling pathway of TLRs, leading to decreased inflammatory gene expression.     

Mitogen-activated protein kinase pathway mediates DBP-maf-induced apoptosis in RAW 264.7 macrophages

Vitamin D-binding protein-macrophage-activating factor (DBP-maf) is derived from serum vitamin D binding protein (DBP) by selective deglycosylation during inflammation. In the present study, we investigated the effect of DBP-maf on RAW 264.7 macrophages and the underlying intracellular signal transduction pathways. DBP-maf increased proapoptotic caspase-3, -8, and -9 activities and induced apoptosis in RAW 264.7 cells. However, DBP, the precursor to DBP-maf did not induce apoptosis in these cells. Cell cycle analysis of DBP-maf-treated RAW 264.7 cells revealed growth arrest with accumulation of cells in sub-G(0)/G(1) phase. We also investigated the role of mitogen-activated protein kinase (MAPK) pathways in the DBP-maf-induced apoptosis of RAW264.7 cells. DBP-maf increased the phosphorylation of p38 and JNK1/2, while it decreased the ERK1/2 phosphorylation. Treatment with the p38 MAPK inhibitor, SB202190, attenuated DBP-maf-induced apoptosis. PD98059, a MEK specific inhibitor, did not show a significant inhibition of apoptosis induced by DBP-maf. Taken together, these results suggest that the p38 MAPK pathway plays a crucial role in DBP-maf-mediated apoptosis of macrophages. Our studies indicate that, during inflammation DBP-maf may function positively by causing death of the macrophages when activated macrophages are no longer needed at the site of inflammation. In summary, we report for the first time that DBP-maf induces apoptosis in macrophages via p38 and JNK1/2 pathway.     

Identification of signaling components required for the prediction of cytokine release in RAW 264.7 macrophages

Background  

Release of immuno-regulatory cytokines and chemokines during inflammatory response is mediated by a complex signaling network. Multiple stimuli produce different signals that generate different cytokine responses. Current knowledge does not provide a complete picture of these signaling pathways. However, using specific markers of signaling pathways, such as signaling proteins, it is possible to develop a 'coarse-grained network' map that can help understand common regulatory modules for various cytokine responses and help differentiate between the causes of their release.     

Involvement of the p38 MAPK signaling cascade in stress response of RAW 264.7 macrophages

The role of the p38 MAPK signaling cascade was studied in stress response of RAW 264.7 macrophages to extremely low-intensity centimeter microwaves. Irradiation stimulated production of a number of cytokines (IL-1, IL-6, TNF-α, INF-γ and IL-10), as well as induced activation of the signaling cascades NF- κB and p38 MAPK, and enhanced expression of Hsp72 heat shock protein. In the presence of the cascade p38 MAPK inhibitor (p38 MAP kinase inhibitor XI), the stimulating effects of electromagnetic waves were abrogated either completely (for NF-κB and Hsp72) or partially (for p38 MAPK and cytokines). The results obtained are indicative of a high sensitivity of the signaling cascade p38 MAPK to the effect of low-intensity physical fields.     

Magnolol stimulates lipolysis in lipid-laden RAW 264.7 macrophages

This study investigated the effect of magnolol, a compound isolated from Magnolia officinalis, on lipolysis in lipid-laden RAW 264.7 macrophages. Treatment of macrophages with magnolol led to dissolution of lipid droplets. This phenomenon was accompanied by a dose-dependent release of glycerol and cholesterol and a concomitant reduction in intracellular levels of glycerol and cholesterol. Furthermore, adipose differentiation-related protein (ADRP), a lipid droplet-associated protein, was down-regulated by magnolol in a dose- and time-dependent manner by Western blot analysis. Immunofluorescence studies also showed that ADRP became detached from the surface of lipid droplets after magnolol treatment. The lipolytic effect of magnolol was not mediated through the cAMP-protein kinase A (PKA) system, an authentic lipolytic pathway for macrophages, since magnolol did not induce an increase of intracellular cAMP levels, and pretreatment with either of PKA inhibitors, PKI and KT5720, did not abrogate the lipolytic response to magnolol. We conclude that magnolol induce-lipolysis of lipid-laden macrophages by down-regulation of ADRP expression and detachment of ADRP from the lipid droplet surface by a cAMP-independent mechanism. Lipolysis of lipid-laden macrophages may occur when the amount of ADRP on the surface of lipid droplets is not enough to stabilize the lipid droplets.     

Anti-inflammatory activity of c-phycocyanin in lipopolysaccharide-stimulated RAW 264.7 macrophages

C-phycocyanin (C-PC), found in blue green algae, is often used as a dietary nutritional supplement. C-PC has been found to have an anti-inflammatory activity and exert beneficial effect in various diseases. However, little is known about its mechanism of action. Overproduction of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) plays an important role in the pathogenesis of inflammation. The aim of this study was to determine whether C-PC inhibits production of nitrite, an index of NO, and iNOS expression in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Our results indicated that C-PC significantly inhibited the LPS-induced nitrite production and iNOS protein expression accompanied by an attenuation of tumor necrosis factor-alpha (TNF-alpha) formation but had no effect on interleukin-10 production in macrophages. Furthermore, C-PC also suppressed the activation of nuclear factor-kappaB (NF-kappaB) through preventing degradation of cytosolic IkappaB-alpha in LPS-stimulated RAW 264.7 macrophages. Thus, the inhibitory activity of C-PC on LPS-induced NO release and iNOS expression is probably associated with suppressing TNF-alpha formation and nuclear NF-kappaB activation, which may provide an additional explanation for its anti-inflammatory activity and therapeutic effect.     

NODs信号通路在RAW264.7细胞抗烟曲霉菌刺激中的作用

【目的】通过培养RAW264.7细胞,并运用siRNA沉默NOD2基因来研究NODs信号通路在体外抗烟曲霉中的作用。【方法】体外培养RAW264.7细胞,接种2×105个/孔细胞于六孔板中,分为正常对照组(N)和正常沉默组[NOD2(RNAi),正常+烟曲霉孢子刺激组(N+Af)和正常沉默+烟曲霉孢子刺激组[NOD2(RNAi)+Af],每组三复孔。通过RT-PCR法检测细胞中NOD1、NOD2、RIP2 mRNA表达;Western blot法检测细胞中分泌蛋白TNF-α表达。【结果】与N组比较,N+Af组NOD1、NOD2 mRNA和TNF-α蛋白表达显著上升。与阴性对照组(Nctrol)相比,NOD2(RNAi)组NOD2 mRNA表达明显受到抑制,沉默效果达到80%以上,说明RAW264.7细胞中NOD2基因被成功沉默。与NOD2(RNAi)组比较,NOD2(RNAi)+Af组NOD1、RIP2 mRNA和TNF-α蛋白表达小幅上升,但无显著性差异(P>0.05)。与NOD2基因沉默前比较发现:与N组比较,NOD2(RNAi)组,TNF-α蛋白表达显著性升高(P<0.05)。与N+Af组比较,NOD2(RNAi)+Af组,TNF-α蛋白显著性降低(P<0.05);NOD1、RIP2 mRNA在各组中表达均未见显著性差异。【结论】NODs信号通路在RAW264.7细胞抗烟曲霉中发挥作用,尤以NOD2的作用较突出。     

Oleic acid-induced PKC isozyme translocation in RAW 264.7 macrophages

Fatty acids are important second messengers that mediate various cellular functions, but their role in the formation of macrophage foam cells is not known. High plasma levels of oleic acid (OA) in obese patients are often associated with a high risk for atherosclerosis. In this study, we investigated the protein kinase C (PKC) isozymes involved in OA-induced lipid accumulation in RAW 264.7 macrophages. The results show that OA induces translocation of PKC alpha, beta1, and delta from the cytosol to the cell membrane 5 min after the treatment. After 16 h incubation with OA, PKC delta was found to be colocalized with adipose differentiation-related protein (ADRP) on the surface of lipid droplets, but immunoprecipitation experiments showed that PKC delta was not biochemically associated with ADRP. After 16 h incubation with OA plus phorbol 12-myristate 13-acetate (PMA), PKC delta staining on the lipid droplet surface was not seen, whereas the accumulation of lipid droplets was unaffected. Furthermore, downregulation of PKC delta was confirmed by immunoblotting. These results demonstrate possible involvement of specific PKC isozymes in the early phase of lipid accumulation, possibly during the uptake of OA.     

beta-Sitosterol modulates antioxidant enzyme response in RAW 264.7 macrophages

Uncontrolled production of reactive oxygen species contributes to the pathogenesis of diseases such as cancer and cardiovascular disorders. Olive oil exerts remarkable preventive effects on the development of these diseases, which may be due to the action of various components of the olive oil. In fact, several findings suggest that minor components, like phytosterols such as beta-sitosterol, are responsible, at least in part, for these beneficial effects. Our results show that beta-sitosterol reverts the impairment of the glutathione/oxidized glutathione ratio induced by phorbol esters in RAW 264.7 macrophage cultures. These data can be correlated with the increase in manganese superoxide dismutase and glutathione peroxidase activities and the decrease in catalase activity. We also demonstrate that the effects of beta-sitosterol on antioxidant enzymes depend on the estrogen/phosphatidylinositol 3-kinase pathway.     

Chitinous materials inhibit nitric oxide production by activated RAW 264.7 macrophages

Chitinous materials have been studied in wound healing and artificial skin substitutes for many years. Nitric oxide (NO) has been shown to contribute to cytotoxicity in cell proliferation during inflammation of wound healing. In this study, we examined the effect of chitin and its derivatives on NO production by activated RAW 264.7 macrophages. Chitin and chitosan showed a significantly inhibitory effect on NO production by the activated macrophages. Hexa-N-acetylchitohexaose and penta-N-acetylchitopentaose also inhibited NO production but with less potency. However, N-acetylchitotetraose, -triose, -biose, and monomer of chitin, N-acetylglucosamine and glucosamine had little effect on NO production by the activated cells. These results suggest that the promotive effect of chitinous material on wound healing be related, at least partly, to inhibit NO production by the activated macrophages.     

Oxidative stress reprograms lipopolysaccharide signaling via Src kinase-dependent pathway in RAW 264.7 macrophage cell line

Oxidative stress generated during ischemia/reperfusion injury has been shown to augment cellular responsiveness. Whereas oxidants are themselves known to induce several intracellular signaling cascades, their effect on signaling pathways initiated by other inflammatory stimuli remains poorly elucidated. Previous work has suggested that oxidants are able to prime alveolar macrophages for increased NF-kappa B translocation in response to treatment with lipopolysaccharide (LPS). Because oxidants are known to stimulate the Src family of tyrosine kinases, we hypothesized that the oxidants might contribute to augmented NF-kappa B translocation by LPS via the involvement of Src family kinases. To model macrophage priming in vitro, the murine macrophage cell line, RAW 264.7, was first incubated with various oxidants and then exposed to low dose LPS. These studies show that oxidant stress is able to augment macrophage responsiveness to LPS as evidenced by earlier and increased NF-kappa B translocation. Inhibition of the Src family kinases by either pharmacological inhibition using PP2 or through a molecular approach by cell transfection with Csk was found to prevent the augmented LPS-induced NF-kappa B translocation caused by oxidants. Interestingly, while Src kinase inhibition was able to prevent the LPS-induced NF-kappa B translocation in oxidant-treated macrophages, this strategy had no effect on NF-kappa B translocation caused by LPS in the absence of oxidants. These findings suggested that oxidative stress might divert LPS signaling along an alternative signaling pathway. Further studies demonstrated that the Src-dependent pathway induced by oxidant pretreatment involved the activation of phosphatidylinositol 3-kinase. Involvement of this pathway appeared to be independent of traditional LPS signaling. Together, these studies provide a novel potential mechanism whereby oxidants might prime alveolar macrophages for altered responsiveness to subsequent inflammatory stimuli and suggest different cellular targets for immunomodulation following ischemia/reperfusion.     

Cyclic AMP signaling enhances lipopolysaccharide sensitivity and interleukin‐33 production in RAW264.7 macrophages

While it has been suggested that IL‐33 plays pathogenic roles in various disorders, the factors that stimulate IL‐33 production are poorly characterized. In the present study, the effect of cyclic adenosine monophosphate (cAMP) signaling on IL‐33 production in RAW264.7 macrophages in response to various doses of LPS was examined. High‐dose LPS treatment induced IL‐33 and TNF protein production in RAW264.7 macrophages. In contrast, low‐dose LPS failed to induce IL‐33 production while significantly inducing TNF production. In the presence of the membrane‐permeable cAMP analog 8‐Br‐cAMP, low‐dose LPS induced vigorous IL‐33 production. This phenomenon was consistent with amounts of mRNA. Similarly, the cAMP‐increasing agent adrenaline also enhanced the sensitivity of RAW264.7 macrophages to LPS as demonstrated by IL‐33 production. The protein kinase A (PKA) inhibitor H89 blocked the effects of 8‐Br‐cAMP and adrenaline on IL‐33 production, suggesting that PKA is involved in IL‐33 induction. Taken together, cAMP‐mediated signaling pathway appears to enhance the sensitivity of RAW264.7 macrophages to LPS with respect to IL‐33 production. Our findings suggest that stress events and the subsequent secretion of adrenaline enhance macrophage production via IL‐33; this process may be associated with the pathogenesis of various disorders involving IL‐33.     

Anti-inflammatory effect of pyroglutamyl-leucine on lipopolysaccharide-stimulated RAW 264.7 macrophages

Aims

Food-derived peptides have been reported to yield a variety of health promoting activities. Pyroglutamyl peptides are contained in the wheat gluten hydrolysate. In the present study, we investigated the effect of pyroglutamyl dipeptides on the lipopolysaccharide (LPS)-induced inflammation in macrophages.

Main methods

RAW 264.7 macrophages were treated with LPS and various concentrations of pyroglutamyl-leucine (pyroGlu-Leu), -valine (pyroGlu-Val), -methionine (pyroGlu-Met), and -phenylalanine (pyroGlu-Phe). Cell viability/proliferation and various inflammatory parameters were measured by the established methods including ELISA and western blotting. The binding of fluorescein isothiocyanate-labeled LPS to RAW 264.7 cells was also measured fluorescently.

Key findings

All the tested dipeptides significantly inhibited the secretion of nitric oxide, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 from LPS-stimulated RAW 264.7 macrophages. Above all, pyroGlu-Leu inhibited the secretion of all these inflammatory mediators even at the lowest dose (200 μg/ml). PyroGlu-Leu dose-dependently suppressed IκBα degradation and MAPK (JNK, ERK, and p38) phosphorylation in LPS-stimulated RAW 264.7 cells. On the other hand, it did not affect the binding of LPS to the cell surface.

Significance

Our results indicated that pyroGlu-Leu inhibits LPS-induced inflammatory response via the blocking of NF-κB and MAPK pathways in RAW 264.7 macrophages.     

Melanocortin receptor signaling in RAW264.7 macrophage cell line

Melanocortin peptides modulate cytokine release and adhesion molecule expression. Here we have investigated the early cell-signaling pathway responsible for the induction of interleukin-10 (IL-10) in RAW264.7 cells. Cell incubation with ACTH(1-39) or MTII (melanotan II) did not alter ERK1/2 and JNK phosphorylation, while p38 phosphorylation and intracellular cAMP accumulation occurred within minutes. ACTH(1-39) and MTII provoked a time-dependent accumulation of IL-10 that was abrogated by the PKA inhibitor H-89 and only partially blocked by the p38 MAPK inhibitor SB203580. Thus, in RAW264.7 cells, IL-10 induction by the melanocortins is via the PKA pathway, and this mechanism could contribute to their anti-inflammatory profile.