Propolis effects on pro-inflammatory cytokine production and Toll-like receptor 2 and 4 expression in stressed mice

IntroductionPropolis is a beehive product and its immunomodulatory action has been well documented; however, little is known concerning its activity on the immune system of stressed mice. This work investigated a possible role of propolis against the immunosuppressive effects induced by stress in mice, assessing the pro-inflammatory cytokine (IL-1β and IL-6) production and Toll-like receptor (TLR-2 and TLR-4) expression by spleen cells.MethodsBALB/c mice were divided into 3 groups: G1 was considered control; G2 was submitted to restraint stress for 3 days, and G3 was treated with propolis and immediately submitted to stress. After sacrifice, spleens were removed and TLR-2 and TLR-4 gene expression was analyzed, as well as the pro-inflammatory cytokine production. Serum corticosterone levels were determined by radioimmunoassay as a stress indicator.ResultsStressed mice, treated or not with propolis, produced higher corticosterone levels, whereas IL-1β and IL-6 production was inhibited. TLR-2 and TLR-4 expression was inhibited in stressed mice, while propolis exerted an immunorestorative role in TLR-4 expression. The immunosuppressive effects on IL-1β and IL-6 production and on TLR expression by stressed mice might have occurred due to a higher corticosterone production during stress.ConclusionPropolis treatment did not antagonize the inhibitory effects on pro-inflammatory cytokine production, however it restored at least partially TLR2 mRNA expression and counteracted the inhibition on TLR-4 expression in stressed animals, contributing to the recognition of microorganisms during stressful conditions.     

Toll-like receptor priming sensitizes macrophages to proinflammatory cytokine gene induction by deoxynivalenol and other toxicants.

Activation of the innate immune system might predispose a host to toxicant-induced inflammation. In vitro macrophage models were employed to investigate the effects of preexposure to Toll-like receptor (TLR) agonists on induction of proinflammatory cytokine gene expression by the trichothecene mycotoxin deoxynivalenol (DON) and other toxicants. Priming of the murine RAW 264.7 macrophage line or peritoneal murine macrophages with the TLR4 agonist lipopolysaccharide (LPS) at 100 ng/ml for 4, 8, and 16 h significantly increased DON-induced IL-1beta, IL-6, and TNF-alpha mRNA expression as compared to LPS or DON alone. The minimum LPS concentration for sensitization of both cell types was 1 ng/ml. LPS priming also potentiated IL-1beta mRNA induction by DON in human whole-blood cultures, suggesting the relevance of the murine findings. As observed for LPS, preexposure to TLR agonists including zymosan (TLR2), poly (I:C) (TLR3), flagellin (TLR5), R848 (TLR7/8), and ODN1826 (TLR9) sensitized RAW 267.4 cells to DON-induced proinflammatory gene expression. Amplified proinflammatory mRNA expression was similarly demonstrated in LPS-sensitized RAW 264.7 cells exposed to the microbial toxins satratoxin G, Shiga toxin, and zearalenone as well as the anthropogenic toxicants nickel chloride, triphenyltin, 2,4-dinitrochlorobenzene, and 2,3,7,8-tetrachlorodibenzodioxin. The results suggest that prior TLR activation might render macrophages highly sensitive to subsequent induction of proinflammatory gene expression by xenobiotics with diverse mechanisms of action.     

Selenium suppresses the activation of transcription factor NF-kappa B and IRF3 induced by TLR3 or TLR4 agonists

Toll-like receptors (TLRs) play an important role in recognition of microbial components and induce innate immune responses by recognizing invading microbial pathogens leading to the activation of the adaptive immune responses. The microbial components trigger the activation of two downstream signaling pathways of TLRs; MyD88- and TRIF-dependent pathways leading to the expression of pro-inflammatory cytokines and type I interferons (IFNs). The MyD88- and TRIF-dependent pathways lead to the activation of NF-kappa B and IRF3 through the activation of IKK-beta and TBK1, respectively. Selenium is an essential trace element nutrient possessing anticarcinogenic properties. Here, we attempted to identify the molecular targets of selenium in TLR signaling pathways. Selenium inhibited NF-kappaB activation induced by poly[I:C] (TLR3 agonist), LPS (TLR4 agonist) or overexpression of MyD88 or IKK-beta which is the key kinase of MyD88-dependent signaling pathway. Selenium inhibited IRF3 activation induced by poly[I:C], LPS or the overexpression of TRIF or TBK1. Selenium also suppressed the expression of COX-2 and iNOS and the endogenous IFN beta mRNA induced by poly[I:C] or LPS. Therefore, our results suggest that selenium can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs leading to decreased inflammatory gene expression.     

猪苓多糖通过Toll样受体4对小鼠腹腔巨噬细胞的活化作用

目的观察猪苓多糖(PPS)对小鼠腹腔巨噬细胞的活化作用,并研究Toll样受体4(TLR4)在其中的作用。方法用PPS12.5,25,50及100mg·L-1刺激C3H/HeN小鼠和TLR4缺陷的C3H/HeJ小鼠脾细胞72h或腹腔巨噬细胞24h,以[3H]TdR掺入法检测脾细胞增殖反应;以Griess法测定腹腔巨噬细胞培养上清一氧化氮(NO)水平,ELISA检测白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)含量;以溴化氰活化多糖的方法制备荧光胺(Flu)标记的PPS(Flu-PPS)及Flu-葡聚糖,应用流式细胞仪及激光共聚焦显微镜检测Flu-PPS与小鼠腹腔巨噬细胞的结合。结果 PPS可明显促进C3H/HeN小鼠脾细胞增殖并诱导腹腔巨噬细胞分泌NO,IL-1β和TNF-α,且具有浓度依赖性(r=0.999,P<0.01)。用抗TLR4单抗20mg·L-1与C3H/HeN小鼠腹腔巨噬细胞预孵育1h后加入PPS50mg·L-1,与单独PPS50mg·L-1孵育比较,巨噬细胞培养上清IL-1β和TNF-α浓度分别由(0.38±0.06)μg·L-1和(0.29±0.05)μg·L-1降至(0.18±0.01)μg·L-1和(0.12±0.02)μg·L-1,降幅达52.6%和58.6%(P<0.01)。PPS对C3H/HeN小鼠的脾细胞增殖、腹腔巨噬细胞产生IL-1β和TNF-α的作用明显强于C3H/HeJ小鼠:PPS12.5～100mg·L-1组脾细胞增殖分别增加了2.4,1.7,1.5和22.2倍,IL-1β增加了0.9,1.3,1.2和1.1倍,TNF-α增加了1.3,0.9,0.6和0.6倍,差异均有统计学意义(P<0.01)。流式细胞仪及激光共聚焦显微镜分析结果表明,Flu-PPS1mg·L-1与小鼠腹腔巨噬细胞结合的荧光强度显著高于Flu-葡聚糖,增加Flu-PPS浓度至5mg·L-1,荧光强度并未相应增强,表明Flu-PPS与小鼠腹腔巨噬细胞的结合具有饱和性;未标记的PPS100mg·L-1及抗TLR4单抗200mg·L-1可明显阻断Flu-PPS与巨噬细胞的结合。结论 PPS可能通过TLR4活化小鼠腹腔巨噬细胞。     

Juzentaihoto, a Kampo medicine, enhances IL-12 production by modulating Toll-like receptor 4 signaling pathways in murine peritoneal exudate macrophages

Juzentaihoto (TJ-48), a Kampo medicine, has been reported to affect the immune system. Although toll-like receptors (TLRs) have been identified as receptors of innate immunity, the effects of TJ-48 on TLR signaling pathways have not been thoroughly investigated. Here we evaluated the effects of TJ-48 on TLR4 signaling pathways. Peritoneal exudate macrophages (PEMs) isolated from mice orally administered TJ-48 for 11 days were stimulated with lipopolysaccharide (LPS), a ligand of TLR4, in vitro. Production of IL-12 p40 was significantly augmented in TJ-48-treated PEMs compared with that in vehicle PEMs, without affecting the surface expression of TLR4. Treatment with chemical inhibitors of NF-kappa B and p38 mitogen-activated protein kinases (MAPKs) in vitro inhibited LPS-induced IL-12 production, whereas JNK and ERK inhibitors increased IL-12 production. Immunoblotting with phosphorylation-state specific antibodies demonstrated that TJ-48 differentially affected LPS-induced phosphorylation of NF-kappa B and MAPKs. In PEMs treated with TJ-48, LPS-induced phosphorylation of p65 NF-kappa B and p38 MAPK was augmented, while that of JNK and ERK was attenuated compared with those in vehicle PEMs. These results suggest that selective modulation of the TLR4 signaling pathways by TJ-48 is involved in enhanced production of IL-12 in PEMs.     

Mercuric chloride induces the production of leukotriene B4 by rabbit alveolar macrophages

The effects of HgCl₂ on the production of leukotriene B₄ (LTB₄) by rabbit alveolar macrophages were investigated. Alveolar macrophages synthesized only a small amount of LTB₄ when incubated in Hanks’ balanced salt solution (HBSS) in the absence of stimuli or chemical reagents (0.2±0.1 ng/10⁶ cells). The amount of LTB₄ synthesized was greatly enhanced when macrophages were incubated with 5×10^?6 M HgCl₂ (5.3±0.2 ng/10⁶ cells). Increases in levels of free arachidonic acid (20∶4), a precursor of eicosanoids, were also observed on treatment of [³H]20∶4-labeled cells with HgCl₂ in a concentration-dependent manner. We confirmed that [³H]20∶4 was mainly liberated from the alkenyl subclass of ethanolamine glycerophospholipids (EGP). Degradation of alkenyl-EGP was also observed when HgCl₂ and [³H]20∶4-labeled lipids were incubated in HBSS, resulting in an increase in the level of [³H]20∶4-labeled lysoEGP. When a microsomal fraction prepared from macrophages was added to the same mixture, [³H]20∶4 was liberated from [³H]20∶4-containing lysoEGP. These results suggest that the alkenyl linkage of EGP was degraded in the presence of HgCl₂ with resultant formation of lysoEGP which was subsequently hydrolyzed by lysophospholipase. As a consequence, the level of free 20∶4 in and the rate of production of LTB₄ by alveolar macrophages were elevated.     

Inhibition of LPS-induced NO and PGE2 production by asiatic acid via NF-kappa B inactivation in RAW 264.7 macrophages: possible involvement of the IKK and MAPK pathways

In the present study, we investigated the effect of asiatic acid (the aglycon of asiaticoside) and asiaticoside isolated from the leaves of Centella asiatica (Umbelliferae) on LPS-induced NO and PGE(2) production in RAW 264.7 macrophage cells. Asiatic acid more potently inhibited LPS-induced NO and PGE(2) production than asiaticoside. Consistent with these observations, the protein and mRNA expression levels of inducible iNOS and COX-2 enzymes were inhibited by asiatic acid in a concentration-dependent manner. In addition, asiatic acid dose-dependently reduced the production of IL-6, IL-1 beta and TNF-alpha in LPS-stimulated RAW 264.7 macrophage cells. Furthermore, asiatic acid inhibited the NF-kappaB activation induced by LPS, and this was associated with the abrogation of I kappa B-alpha degradation and with subsequent decreases in nuclear p65 and p50 protein levels. Moreover, the phosphorylations of IKK, p38, ERK1/2, and JNK in LPS-stimulated RAW 264.7 cells were suppressed by asiatic acid in a dose-dependent manner. These results suggest that the anti-inflammatory properties of asiatic acid might be the results from the inhibition of iNOS, COX-2, IL-6, IL-1 beta, and TNF-alpha expressions through the down-regulation of NF-kappaB activation via suppression of IKK and MAP kinase (p38, ERK1/2, and JNK) phosphorylation in RAW 264.7 cells.     

4-Methoxylonchocarpin attenuates inflammation by inhibiting lipopolysaccharide binding to Toll-like receptor of macrophages and M1 macrophage polarization

The roots of Abrus precatorius (AP, Fabaceae) have traditionally been used in Vietnam and China for the treatment of inflammatory diseases such as stomatitis, asthma, bronchitis, and hepatitis. Therefore, in this study, we isolated 4-methoxylonchocarpin (ML), an anti-inflammatory compound present in AP, and studied its anti-inflammatory effects in mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. In lipopolysaccharide (LPS)-stimulated macrophages, ML was found to inhibit nuclear factor (NF)-κB activation and tumor necrosis factor (TNF) and interleukin (IL)-6 expression by inhibiting LPS binding to Toll-like receptor 4 (TLR4) in vitro. Oral administration of ML in mice with TNBS-induced colitis suppressed colon shortening and colonic myeloperoxidase activity. ML treatment significantly inhibited the activation of nuclear factor (NF)-κB and phosphorylation of transforming growth factor β-activated kinase 1 in the colon. Treatment with ML also inhibited TNBS-induced expression of IL-1β, IL-17A, and TNF. While ML reduced the TNBS-induced expression of M1 macrophage markers such as arginase-2 and TNF, it was found to increase the expression of M2 macrophage markers such as arginase-1 and IL-10. In conclusion, oral administration of ML attenuated colitis in mice by inhibiting the binding of LPS to TLR4 on immune cells and increasing the polarization of M1 macrophages to M2 macrophages.     

Antimalarial artesunate protects sepsis model mice against heat-killed Escherichia coli challenge by decreasing TLR4, TLR9 mRNA expressions and transcription factor NF-kappa B activation

Bacterial DNA (bDNA) and lipopolysaccharide (LPS) are potent activators of immune cells such as monocytes and macrophages, which contribute to systemic inflammatory response syndrome (SIRS) and sepsis. Unfortunately, many experimental inflammatory antagonist-based therapies have failed in sepsis trials, and currently there is only one adjuvant therapy in clinical use, e.g. activated protein C. Artesunate (AS), a water-soluble derivative of dihydroartemisinin, has recently been demonstrated to protect against LPS-induced human umbilical vein endothelial cell (HUVEC) activation and injury by inhibiting tumor necrosis factor-alpha (TNF-alpha) mRNA expression. In the present study, heat-killed Escherichia coli was used to induce sepsis in the animal models. We observed that AS could protect mice against a lethal challenge with heat-killed E. coli in a dose-dependent manner. This protection was associated with reductions in serum TNF-alpha and measurable endotoxin levels. In addition, the treatment of murine peritoneal macrophage cells with AS strongly inhibited the release of TNF-alpha and IL-6 induced by CpG oligodeoxynucleotide (CpG ODN), LPS, or heat-killed E. coli in a dose-dependent manner. Experiments using affinity sensor technology revealed that AS could not directly bind to CpG ODN or LPS. Moreover, AS could not neutralize LPS in vitro. Further, flow cytometry revealed that AS could not alter the binding of CpG ODN to cell surfaces but could promote CpG ODN accumulation within RAW264.7 cells. Furthermore, AS reduced the expressions of TLR4 and TLR9 mRNA that were stimulated by LPS, CpG ODN, or heat-killed E. coli and inhibited heat killed E. coli-induced NF-kappaB activation. In conclusion, our results demonstrated that AS-mediated protection against a lethal heat-killed E. coli challenge was associated with a reduction in proinflammatory cytokine release and endotoxin levels via a mechanism involving a decrease in TLR4, TLR9 mRNA expression and NF-kappaB activation.     

Sodium methyldithiocarbamate inhibits MAP kinase activation through toll-like receptor 4, alters cytokine production by mouse peritoneal macrophages, and suppresses innate immunity.

Sodium methyldithiocarbamate (SMD; trade name, Metam Sodium) is an abundantly used soil fumigant that can cause adverse health effects in humans, including some immunological manifestations. The mechanisms by which SMD acts, and its targets within the immune system are not fully understood. Initial experiments demonstrated that SMD administered by oral gavage substantially decreased IL-12 production and increased IL-10 production induced by lipopolysaccharide in mice. The present study was conducted to further characterize these effects and to evaluate our working hypothesis that the mechanism for these effects involves alteration in signaling through toll-like receptor 4 and that this would suppress innate immunity to infection. SMD decreased the activation of MAP kinases and AP-1 but not NF-kappaB in peritoneal macrophages. The expression of mRNA for IL-1alpha, IL-1beta, IL-18, IFN-gamma, IL-12 p35, IL-12 p40, and macrophage migration inhibitory factor (MIF) was inhibited by SMD, whereas mRNA for IL-10 was increased. SMD increased the IL-10 concentration in the peritoneal cavity and serum and decreased the concentration of IL-12 p40 in the serum, peritoneal cavity, and intracellularly in peritoneal cells (which are >80% macrophages). Similar effects on LPS-induced cytokine production were observed following dermal administration of SMD. The major breakdown product of SMD, methylisothiocyanate (MITC), caused similar effects on cytokine production at dosages as low as 17 mg/kg, a dosage relevant to human exposure levels associated with agricultural use of SMD. Treatment of mice with SMD decreased survival following challenge with non-pathogenic Escherichia coli within 24-48 h, demonstrating suppression of innate immunity.     

Toll样受体 2、Toll样受体 4 mRNA和蛋白在急性出血坏死性胰腺炎肝损伤中的表达及意义

目的 探讨Toll样受体2(TLR2)、TLR4信使核糖核酸(mRNA)和蛋白在急性出血坏死性胰腺炎(AHNP)肝损伤中的表达及意义.方法 2019年6月至12月选取健康SD大鼠(郑州大学实验动物中心)60只为研究对象,采用随机数字表法分为模型组和对照组,每组30只,同时模型组和对照组再随机分为3 h组、6 h组和12 h组各10只.模型组采用牛磺胆酸钠建立AHNP模型,对照组注射生理盐水,检测各组血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)和淀粉酶,qRT-PCR和Western blotting检测肝脏组织TLR2、TLR4 mRNA和蛋白表达,HE染色观察肝脏组织.结果 模型组造模后3 h组、6 h组和12 h组血清ALT[(170.02±32.21)U/L、(310.01±80.02)U/L、(720.11±112.28)U/L]、AST[(450.13±100.21)U/L、(980.12±132.21)U/L、(1100.32±145.03)U/L]和淀粉酶[(7099.27±1209.02)U/L、(11010.19±1200.01)U/L、(17003.21±2007.82)U/L],肝脏组织TLR2和TLR4 mRNA及蛋白相对表达量[TLR2 mRNA(0.962±0.201)、(1.201±0.223)、(1.501±0.212),TLR2蛋白(0.713±0.091)、(1.102±0.100)、(1.343±0.121);TLR4 mRNA(1.701±0.332)、(2.011±0.350)、(2.562±0.313),TLR4蛋白(1.210±0.170)、(1.446±0.182)、(1.788±0.190)]均逐渐增高(P<0.001),且均明显高于对照组[ALT(84.40±14.92)U/L、(85.50±15.20)U/L、(84.19±14.49)U/L;AST(170.02±20.01)U/L、(168.82±21.14)U/L、(171.14±20.11)U/L;淀粉酶(1102.00±183.29)U/L、(1154.49±190.04)U/L、(1160.03±186.27)U/L;TLR2 mRNA(0.030±0.010)、(0.031±0.011)、(0.029±0.009),TLR2蛋白(0.021±0.008)、(0.020±0.009)、(0.022±0.010);TLR4 mRNA(0.882±0.181)、(0.880±0.170)、(0.883±0.179),TLR4蛋白(0.961±0.192)、(0.967±0.181)、(0.966±0.180)](P<0.001).肝组织TLR2、TLR4 mRNA表达与血清ALT、AST和淀粉酶呈正相关(P<0.05),TLR2、TLR4蛋白表达与血清ALT、AST和淀粉酶呈正相关(P<0.05);模型组造模3 h后可见肝细胞气球样变性,6 h后炎性细胞浸润汇管区,小叶周边细胞发生嗜酸性变性,12 h后出现大片状出血坏死及灶性坏死.结论 TLR2、TLR4 mRNA和蛋白在AHNP肝损伤组织中的表达升高,可能在肝脏损伤过程中有重要作用.     

The effect of magnolol on the Toll-like receptor 4/nuclear factor kappa B signaling pathway in lipopolysaccharide-induced acute lung injury in mice

Magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis has been reported to have anti-inflammatory properties. The purpose of this study was to evaluate the effect of magnolol on acute lung injury induced by lipopolysaccharide in mice. Male BALB/c mice were pretreated with dexamethasone or magnolol 1 h before intranasal instillation of lipopolysaccharide (LPS). 7 h after LPS administration, the myeloperoxidase in lung tissues, lung wet/dry weight ratio and inflammatory cells in the bronchoalveolar lavage fluid were determined. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in the bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assay (ELISA). The extent of phosphorylation of nuclear factor of inhibitory kappa B alpha (IκB-α), nuclear factor kappa-B (NF-κB) p65 and the expression of Toll-like receptor-4 (TLR4) were detected by western blot. The results showed that magnolol markedly attenuated the histological alterations in the lung; reduced the number of total cells, neutrophils, and macrophages in the bronchoalveolar lavage fluid; decreased the wet/dry weight ratio of lungs in the bronchoalveolar lavage fluid; down-regulated the level of pro-inflammatory mediators, including TNF-α, IL-1β and IL-6; inhibited the phosphorylation of IκB-α, NF-κB p65 and the expression of TLR4, caused by LPS. Taken together, our results suggest that anti-inflammatory effects of magnolol against the LPS-induced acute lung injury may be due to its ability of inhibition TLR4 mediated NF-κB signaling pathways. Magnolol may be a promising potential therapeutic reagent for acute lung injury treatment.