GEF-H1-RhoA signaling pathway mediates LPS-induced NF-κB transactivation and IL-8 synthesis in endothelial cells

Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the roles of GEF-H1 (guanine-nucleotide exchange factor-H1)-RhoA signaling in LPS-induced interleukin-8 (IL-8, CXCL8) production in endothelial cells. First, we observed that GEF-H1 expression was upregulated in a dose- and time-dependent manner as consistent with TLR4 (Toll-like receptor 4) expression after LPS stimulation. Afterwards, Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activities, reduced LPS-induced NF-κB phosphorylation. Inhibition of GEF-H1 and RhoA expression reduced LPS-induced NF-κB and p38 phosphorylation. TLR4 knockout blocked LPS-induced activity of RhoA, however, MyD88 knockout did not impair the LPS-induced activity of RhoA. Nevertheless, TLR4 and MyD88 knockout both significantly inhibited transactivation of NF-κB. GEF-H1-RhoA and MyD88 both induced significant changes in NF-κB transactivation and IL-8 synthesis. Co-inhibition of GEF-H1-RhoA and p38 expression produced similar inhibitory effects on LPS-induced NF-κB transactivation and IL-8 synthesis as inhibition of p38 expression alone, thus confirming that activation of p38 was essential for the GEF-H1-RhoA signaling pathway to induce NF-κB transactivation and IL-8 synthesis. Taken together, these results demonstrate that LPS-induced NF-κB activation and IL-8 synthesis in endothelial cells are regulated by the MyD88 pathway and GEF-H1-RhoA pathway.     

黄芪总皂苷对脂多糖诱导的BV2小胶质细胞的抗炎机制

目的:探讨黄芪总皂苷(TAS)对脂多糖(LPS)诱导的BV2小胶质细胞炎症损伤的抗炎作用机制.方法:用CCK-8法筛选出对细胞活力无抑制的药物浓度;用浓度为1 mg/L的LPS刺激BV2细胞24 h,建立细胞炎症模型;实验分为正常组、LPS组、高剂量(75 mg/L)TAS组和低剂量(50 mg/L)TAS组;应用流式...     

Effect of chlorogenic acid on LPS-induced proinflammatory signaling in hepatic stellate cells

Objective and design

This study was aimed at investigating the effect of chlorogenic acid (CGA) on lipopolysaccharide (LPS)-induced proinflammatory signaling in hepatic stellate cells (HSCs).

Methods

An immortalized rat HSC line was cultured in vitro and treated with LPS in the absence or presence of CGA. Reactive oxygen species (ROS) production in the HSCs was monitored by flow cytometer using DCFH-DA. The protein expression levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor-κB (NF-κB), and p-IκB-α were determined by Western blot. The mRNA expression levels of TLR4, MyD88, monocyte chemotactic protein 1(MCP-1), and interleukin 6 (IL-6) were detected by RT-PCR. The levels of MCP-1 and IL-6 in the culture supernatant of HSCs were measured by ELISA.

Results

CGA had no effect on expression of TLR4 and MyD88. However, the treatment of CGA can inhibit LPS-induced production of ROS in HSCs. Meanwhile, CGA can inhibit LPS-induced nuclear translocation of NF-κB and IκB-α phosphorylation in HSCs, as well as NAC (a ROS scavenger). The mRNA expression and the levels of MCP-1 and IL-6 in the culture supernatant of the HSCs in this study were elevated by LPS stimulation and inhibited by CGA treatment, as well as NAC and PDTC (a NF-κB inhibitor).

Conclusion

Our results indicate that CGA can efficiently inhibit LPS-induced proinflammatory responses in HSCs and the anti-inflammatory effect may be due to the inhibition of LPS/ROS/NF-κB signaling pathway.     

TLR4-MyD88-TRAF6-TAK1 Complex-Mediated NF-κB Activation Contribute to the Anti-Inflammatory Effect of V8 in LPS-Induced Human Cervical Cancer SiHa Cells

The synthetic compound 7-4-[Bis-(2-hydroxyethyl)-amino]-butoxy-5-hydroxy-8-methoxy-2-phenylchromen-4-one (V8) is a novel flavonoid-derived compound. In this study, we investigated the effects of V8 on Toll-like receptor 4 (TLR4)-mediated inflammatory reaction in human cervical cancer SiHa cells and lipopolysaccharide (LPS)-induced TLR4 activity in cervical cancer SiHa (HPV16+) cells, but not in HeLa (HPV18+) and C33A (HPV?) cells. In addition, V8 inhibited LPS-induced expression of TLR4, MyD88, TRAF6 and phosphorylation of TAK1, and their interaction with TLR4 in SiHa cells, resulting in an inhibition of TLR4-MyD88-TRAF6-TAK1 complex. Moreover, V8 blocked LPS-induced phosphorylation of IκB and IKK, resulting in inhibition of the nuclear translocation of P65-NF-κB in SiHa cells. We also found that V8 reduced the expression of NF-κB target genes, such as those for COX-2, iNOS, IL-6, IL-8, CCL-2, and TNF-α in LPS-stimulated SiHa cells. These results suggested that V8 exerted an anti-inflammatory effect on SiHa cells by inhibiting the TLR4-MyD88-TRAF6-TAK1 complex-mediated NF-κB activation.     

Treponema pallidum flagellin FlaA2 induces IL-6 secretion in THP-1 cells via the Toll-like receptor 2 signaling pathway

Treponema pallidum subsp. pallidum membrane proteins are considered as potent inducers in the initiation and development of inflammation. In the present study, the mechanism that leads to the production of interleukin 6 (IL-6), one of the key proinflammatory cytokines, by human monocytic THP-1 cells when these cells are treated with T. pallidum flagellin FlaA2 was investigated. Stimulation with flagellin FlaA2 can induce IL-6 expression in human monocytes and augment the phosphorylation of ERK, p38, and NF-κB, but has no effect on the phosphorylation of JNK. Likewise, FlaA2-induced IL-6 production was found to be attenuated by inhibitors for ERK, p38, and NF-κB, but not by JNK inhibitor. Immunofluorescence analysis showed that flagellin FlaA2 could stimulate the translocation of IκBα from the cytosol to the nucleus, and this phenomenon could be inhibited by the specific inhibitor BAY11-7082. FlaA2–induced IL-6 expression was also proved to be abrogated by transfection with dominant negative (DN) plasmid of MyD88. We further demonstrated that transfection with DN-TLR2 was sufficient to attenuate IL-6 expression and the phosphorylation of ERK, p38, and IκBα. These results suggest that flagellin FlaA2 induces IL-6 production via signaling pathways involving TLR2, MyD88, ERK, p38, and NF-κB in monocytes, which could contribute to the pathogenesis of T. pallidum.     

脂多糖预致敏的人骨髓间充质干细胞促炎机制研究

目的探索脂多糖（LPS）预致敏的人骨髓间充质干细胞（MSC）产生促炎功能的免疫调节机制。方法采用Real-time PCR和免疫荧光法检测MSC被预致敏前后TLR4信号通路相关分子（如TLR4、MyD88、TRAF6等）的表达水平,以及NF-κB的入核情况。通过Real-time PCR比较MSC被致敏前后促炎因子（IL-1β、IL-6、MIP-2、TNF-α）和Th1/Th2型细胞因子及其受体的表达差异。结果与未致敏的MSC相比,LPS预致敏的MSC中TLR4表达升高,NF-κB入核增加,促炎性因子IL-1β、IL-6、MIP-2、TNF-α表达升高,提示LPS预致敏可以激活MSC中的TLR4信号通路,并且诱导MSC中Th1型细胞因子及其受体表达升高,而Th2型细胞因子及其受体表达无变化或减少。结论MSC被LPS预致敏后TLR4信号通路激活,Th1型细胞因子及受体表达上调,从而诱导MSC分化成促炎表型。     

MD-2 regulates LPS-induced NLRP3 inflammasome activation and IL-1beta secretion by a MyD88/NF-κB-dependent pathway in alveolar macrophages cell line

Myeloid differentiation protein 2 (MD-2) is required in the recognition of lipopolysaccharide (LPS) by toll-like receptor 4 (TLR4), and participates in LPS-induced alveolar macrophage (AM) inflammation during acute lung injury (ALI). Activation of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome aggravates inflammation in LPS-induced ALI. However, there is currently little known about the relationship between MD-2 signaling and the NLRP3 inflammasome. This study showed that NLRP3 expression, IL-1beta (IL-1β) secretion, and pyroptosis were up-regulated after LPS stimulation in the NR8383 AM cell-line. MD-2 gene knock-down reduced LPS-induced mRNA and protein expression of NLRP3 and IL-1β secretion in NR8383 cells, and inhibited the MyD88/NF-κB signaling pathway. Conversely, over-expression of MD-2 not only heightened NLRP3, MyD88, and NF-κB p65 protein expression, it also aggravated the LPS-induced inflammatory response. Furthermore, the NF-κB inhibitor SN50 had a beneficial role in decreasing NLRP3 and caspase-1 mRNA and protein expression. The observations suggest that MD-2 helps to regulate LPS-induced NLRP3 inflammasome activation and the inflammatory response in NR8383 cells, and likely does so by affecting MyD88/NF-κB signaling.     

CD103−CD11b+ dendritic cells regulate the sensitivity of CD4 T-cell responses to bacterial flagellin

Toll-like receptor 5 (TLR5) has been widely studied in an inflammatory context, but the effect of TLR5 on the adaptive response to bacterial flagellin has received considerably less attention. Here, we demonstrate that TLR5 expression by dendritic cells (DCs) allows a 1,000-fold enhancement of T-cell sensitivity to flagellin, and this enhancement did not require the expression of NLRC4 or Myd88. The effect of TLR5 on CD4 T-cell sensitivity was independent of the adjuvant effect of flagellin and TLR5 ligation did not alter the sensitivity of ovalbumin (OVA)-specific T cells to OVA. In the spleen, the exquisite T-cell sensitivity to flagellin was regulated by CD4−CD8α− DCs and was blocked by a monoclonal antibody to TLR5. In the mesenteric lymph nodes, flagellin-specific T-cell activation was regulated by a population of CD103−CD11b+ DCs. Thus, TLR5 expression by mucosal and systemic DC subsets controls the sensitivity of the adaptive immune response to flagellated pathogens.     

Toll样受体4信号转导研究进展

Toll样受体（Toll-like-receptors,TLRs）是一个主要分布于炎症细胞的识别病源分子的受体超家族,其中TLR4主要识别革兰阴性细菌细胞壁成分脂多糖（lipopolysaccharide,LPS）。LPS与TLR4结合后活化髓样分化因子88 (myeloid differentiation factor 88, MyD88)依赖性和非依赖性两条信号途径;前者活化丝裂原激活的蛋白激酶（mitogen-activated protein kinase,MAPK）和核因子-κB（nuclear factor kappa B,NF-κB）信号通路,后者活化NF-κB和干扰素调节因子-3(IFN-regulated factor-3,IRF3)信号通路。通过这些信号途径TLR4诱导炎症细胞释放炎症因子介导炎症反应;同时TLR4通过活化树突状细胞促进抗原递呈,介导先天性免疫向获得性免疫的转化。此外,TLR4能诱导磷脂酰肌醇-3激酶-蛋白激酶B（PI3K-AKT）的信号转导,LPS介导的细胞存活和增殖与TLR4活化 PI3K-AKT途径有关。     

Role of myeloid differentiation factor 88 in HSP60 signal transduction in dendritic cells

Objective: To explore the role and mechanism of myeloid differentiation factor88 (MyD88) in HSP60 signal transduction in dendritic cells. Methods:Mouse DCs were cultured from murine bone marrow cells. The DC marker CD11c was detected by flow cytometry, then DCs were divided into control group, HSP60 groupand RNA interference group. Control group was cultured under normal condition, and HSP60 group was cultured with 10 μg/ml of HSP60. RNA interference group was first cultured with MyD88 siRNA forl2 hours and then HSP60 was added into the culture mixture. All groups were cultured for 48 hours. Immunochemistry was used to detect the concentration of MyD88 and NF- κB. Western blot was used to detect the concentration of MyD88. Flow cytometry and mixed lymphocyte reaction (MLR) were used to detect the phenotype and functional properties of DCs. ELISA was used to detect the concentration of TNF-α, IFN-γ and IL-12 in the supernatant. Results:The expression of CD11c in marine bone marrow DCs was 88.76%. HSP60 stimulation increased the expression of CD80, CD86, MHC-Ⅱ in DCs and TNF-α, IFN-γ, IL-12 secretion in the supematant. HSP60 stimulation also increased the level of MyD88 in the cytoplasm and promoted the shift of NF-κB to karyon and the proliferation of allogeneic T cells. MyD88 siRNA could decreaseMyD88 and inhibit these effects induced by HSP60. Conclusion:HSP60 activates DCs through MyD88-dependent pathway. MyD88 plays a critical role in HSP60 signal transduction. Inhibition of MyD88 may be a novel way for treating disease correlated with HSP60.     

Propofol Attenuates Airway Inflammation in a Mast Cell-Dependent Mouse Model of Allergic Asthma by Inhibiting the Toll-like Receptor 4/Reactive Oxygen Species/Nuclear Factor κB Signaling Pathway

Propofol, an intravenous anesthetic agent widely used in clinical practice, is the preferred anesthetic for asthmatic patients. This study was designed to determine the protective effect and underlying mechanisms of propofol on airway inflammation in a mast cell-dependent mouse model of allergic asthma. Mice were sensitized by ovalbumin (OVA) without alum and challenged with OVA three times. Propofol was given intraperitoneally 0.5 h prior to OVA challenge. The inflammatory cell count and production of cytokines in the bronchoalveolar lavage fluid (BALF) were detected. The changes of lung histology and key molecules of the toll-like receptor 4 (TLR4)/reactive oxygen species (ROS)/NF-κB signaling pathway were also measured. The results showed that propofol significantly decreased the number of eosinophils and the levels of IL-4, IL-5, IL-6, IL-13, and TNF-α in BALF. Furthermore, propofol significantly attenuated airway inflammation, as characterized by fewer infiltrating inflammatory cells and decreased mucus production and goblet cell hyperplasia. Meanwhile, the expression of TLR4, and its downstream signaling adaptor molecules—–myeloid differentiation factor 88 (MyD88) and NF-κB, were inhibited by propofol. The hydrogen peroxide and methane dicarboxylic aldehyde levels were decreased by propofol, and the superoxide dismutase activity was increased in propofol treatment group. These findings indicate that propofol may attenuate airway inflammation by inhibiting the TLR4/MyD88/ROS/NF-κB signaling pathway in a mast cell-dependent mouse model of allergic asthma.     

1-[4-Fluoro-2-(2-nitrovinyl)phenyl]pyrrolidine Suppresses Toll-Like Receptor 4 Dimerization Induced by Lipopolysaccharide

Toll-like receptor 4 (TLR4) recognizes LPS and triggers the activation of the myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter, inducing interferon-β (TRIF)-dependent major downstream signaling pathways. Previously, we presented biochemical evidence that 1-[4-Fluoro-2-(2-nitrovinyl)phenyl]pyrrolidine (FPP), which was synthesized in our laboratory, inhibits NF-κB activation induced by LPS. Here, we investigated whether FPP modulates the TLR4 downstream signaling pathways and what anti-inflammatory target in TLR4 signaling is regulated by FPP. FPP inhibited LPS-induced NF-κB activation by targeting TLR4 dimerization. These results suggest that FPP can modulate the TLR4 signaling pathway at the receptor level to decrease inflammatory gene expression.     

黄芪甲苷通过toll样受体4/核因子κB通路介导辐射所致肾损伤的防护作用

目的 探讨黄芪甲苷（AS-Ⅳ）是否通过toll样受体4（TLR4）/核因子κB（NF-κB）介导的信号通路对辐射诱导肾脏损伤起到保护作用。 方法 将小鼠分为正常对照组、DMSO溶剂组、辐射组(IR)、IR + AS-Ⅳ 20 mg/kg 组和IR + AS-Ⅳ 40 mg/kg 组。小鼠给予AS-Ⅳ腹腔注射1个月后,以8Gy的60Coγ进行全身辐射。测定血清肌酐（Cr）和尿酸（BUA）的含量,进行肾组织HE和免疫组织化学染色,并检测肾脏TLR4/NF-κB信号通路相关蛋白的表达情况。 结果 与对照组相比,辐射组小鼠血清中Cr和BUA含量明显升高（P<0.001）,肾小球萎缩,肾小管扩张,TLR4和髓样分化因子88（MyD88）阳性表达明显增多(P<0.001),且TLR4/NF-κB信号通路相关蛋白［TLR4、MyD88、NF-κB、白细胞介素1β（IL-1β）和肿瘤坏死因子α（TNF-α）］表达极显著升高（P<0.01）;AS-Ⅳ预处理能降低血清中Cr和BUA的含量（P<0.05, P<0.01或 P<0.001）,明显改善辐射所致的病理反应,降低TLR4/NF-κB 信号通路相关蛋白的表达（P<0.05或 P<0.01）。 结论 AS-Ⅳ可能通过TLR4/NF-κB信号通路下调炎症因子的释放,从而改善辐射诱导的小鼠肾损伤,发挥保护作用。     

Atractylenolide I inhibits lipopolysaccharide-induced inflammatory responses via mitogen-activated protein kinase pathways in RAW264.7 cells

Atractylenolide I (ATL-I) is a bioactive component of Rhizoma Atractylodis macrocephalae. Although increasing evidence shows that ATL-I has an anti-inflammatory effect, the anti-inflammatory molecular mechanism of ATL-I is still unknown. In this study, we investigated the effect of ATL-I on cell viability by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and the level of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) by enzyme-linked immunosorbent assay (ELISA) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Further, we examined the effect of ATL-I on the activation of nuclear factor-kappaB (NF-κB) and phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38) by Western blot. We also investigated the effect of ATL-I on the expression of myeloid differentiation protein-2 (MD-2), CD14, complement receptor 3 (CR3), scavenger receptor class A (SR-A), toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). We found that ATL-I showed no inhibitory effect on cell viability at concentrations ranging from 1?µM to 100?µM and markedly reduced the release of IL-6 and TNF-α at a concentrate-dependent manner. In addition, ATL-I suppressed the activity of nuclear NF-κB and the phosphorylation of ERK1/2 and p38 in LPS-treated RAW264.7 cells. Further analysis showed that ATL-I inhibited the expression of MD-2, CD14, SR-A, TLR4 and MyD88, but the expression of CR3 was unaffected. These data suggest that ATL-I shows an anti-inflammatory effect by inhibiting TNF-α and IL-6 production. The anti-inflammatory effects of ATL-I may be associated with the inhibition of the NF-κB, ERK1/2 and p38 signaling pathways.     

CD300a and CD300f differentially regulate the MyD88 and TRIF-mediated TLR signalling pathways through activation of SHP-1 and/or SHP-2 in human monocytic cell lines

CD300a, a membrane protein expressed on myeloid lineages and specific subsets of CD4(+) T cells, has been reported to have inhibitory activities in cellular activation. However, the role of CD300a in Toll-like receptor (TLR) -mediated macrophage activation has not been investigated. The human monocytic cell lines THP-1 and U937 were stimulated with various TLR ligands after triggering of CD300a with specific monoclonal antibody. Interestingly, CD300a blocked TLR4-mediated and TLR9-mediated expression of pro-inflammatory mediators without affecting TLR3-mediated events. In contrast, CD300f, another member of the CD300 family, blocked the activation of cells induced by all TLR ligands. A transient transfection assay using luciferase reporter gene under the regulation of nuclear factor-κB binding sites indicated that co-transfection of CD300f blocked reporter expression induced by over-expression of both myeloid differentiation factor 88 (MyD88) and toll-interleukin 1 receptor-domain-containing adapter-inducing interferon-β (TRIF), whereas CD300a blocked only MyD88-induced events. Synthetic peptides representing immunoreceptor tyrosine-based inhibitory motifs of CD300a or CD300f mimicked the differential inhibition patterns of their original molecules. The use of various signalling inhibitors and Western blotting analysis revealed that TLR9/MyD88-mediated signalling was regulated mainly by SH2-containing tyrosine phosphatase 1 (SHP-1), which could be activated by CD300a or CD300f. In contrast, regulation of the TLR3/TRIF-mediated pathway required the combined action of SHP-1 and SHP-2, which could be accomplished by CD300f but not CD300a. These data indicate that CD300a and CD300f regulate the MyD88 and TRIF-mediated TLR signalling pathways through differential activation of SHP-1 and SHP-2.     

TLR4／NF-κB信号通路在SiO2所致矽肺模型肺泡巨噬细胞脂质代谢调控中的作用#br#

目的 探讨Toll样受体4（Toll like receptor 4,TLR4）/核转录因子κB（nuclear factor-κB,NF-κB）通路在二氧化硅（SiO2）所致大鼠矽肺模型肺泡巨噬细胞的脂质代谢中的作用。 方法 按随机数字表法将36只SD大鼠分为正常组、模型组、抑制剂组。模型组、抑制剂组采用一次性气管内缓慢滴注1 mL SiO2悬浮液进行造模。造模成功后抑制剂组大鼠每天定时耳缘静脉注射TAK-242（TLR4/NF-κB信号特异性抑制剂）,剂量为0.5 mg/kg,正常组和模型组注射等剂量生理盐水。4周后处死大鼠收集支气管灌洗液（bronchial lavage fluid,BALF）,ELISA测定各组大鼠BALF中白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的含量;提取BALF中的肺泡巨噬细胞进行培养,油红O染色观察各组大鼠肺泡巨噬细胞的脂滴形成;Western blot检测各组肺泡巨噬细胞中TLR4、MyD88、NF-κB的表达水平。 结果 与正常组相比,模型组、抑制剂组大鼠BALF中IL-6和TNF-α的含量,巨噬细胞中油红O阳性比例,巨噬细胞中TLR4、MyD88、NF-κB的表达升高,差异具有统计学意义（均P<0.05）;与模型组相比,抑制剂组大鼠上述指标降低,差异具有统计学意义（均P<0.05）。 结论 在SiO2所致大鼠矽肺模型中,TLR4/NF-κB信号参与肺泡巨噬细胞的脂质代谢的调控,抑制该信号的表达,能明显抑制矽肺的病理损伤。     

SHPS-1 and a synthetic peptide representing its ITIM inhibit the MyD88, but not TRIF, pathway of TLR signaling through activation of SHP and PI3K in THP-1 cells

Background

Src homology 2 domain-containing protein tyrosine phosphatase substrate (SHPS)-1 is known to have regulatory effects on myeloid cells. However, its role in macrophage activation is not clearly understood.

Methods and results

In order to investigate the role of SHPS-1 in Toll-like receptor (TLR)-mediated activation, human monocytic cell lines were treated with anti-SHPS-1 monoclonal antibody. The triggering of SHPS-1 blocked the expression of IL-8 and TNF-α in cells treated with a TLR4 ligand that induces a signaling pathway involving myeloid differentiation factor 88 (MyD88) and Toll-interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-β (TRIF). Interestingly, SHPS-1 inhibited TLR9/MyD88-mediated, but not TLR3/TRIF-mediated, expression of IL-8. Accordingly, a synthetic peptide representing the immunoreceptor tyrosine-based inhibition motif (ITIM) of SHPS-1 suppressed only the MyD88 pathway. Utilization of specific inhibitors and Western blot analysis indicated that the inhibitory effects were mediated by Src homology 2 domain-containing phosphatases (SHPs) and phosphoinositide 3-kinase (PI3K).

Conclusion

SHPS-1 negatively regulates the MyD88-dependent TLR signaling pathway through the inhibition of NF-κB activation.