Cell‐line‐based assay for the toxicity/benefit analysis of lipopolysaccharides in plants

There are many immune‐boosting medicinal plants that can potently activate innate immune cells. Recent studies indicate that the active factors of some immune‐boosting plants are lipopolysaccharides (LPSs) of plant‐associated bacteria. However, little is currently known about the potential risk and benefit of LPSs in medicinal plants. To facilitate their characterization, we established a simple cell‐line‐based assay that can be used to screen the toxicity and benefit of LPSs in medicinal plants. The assay can distinguish endotoxic diphosphoryl lipid A (DPL) from beneficial monophosphoryl lipid A (MPL), which is a clinically used immunological adjuvant for vaccines. The established assay was used to characterize commercial supplements of Ashwagandha, which was shown to contain immunostimulatory LPSs. The study revealed that Ashwagandha activates macrophages in a manner similar to MPL. The current finding underscores the importance of further studies to characterize the LPSs in immune‐boosting medicinal plants.     

益气活血复方对内皮细胞TLR4及其下游MyD88、TRAF-6、TRAM、TRIF mRNA表达的影响

目的研究益气活血复方对脂多糖(LPS)诱导的人脐静脉内皮细胞(HUVECs)Toll样受体4(TLR4)及其下游信号转导通路元件髓样分化因子88(MyD88)、肿瘤坏死因子受体相关因子-6(TRAF-6)、Toll样受体相关分子(TRAM)、Toll样受体相关的干扰素活化子(TRIF)表达的影响,探讨益气活血复方含药血清防治动脉粥样硬化(AS)的机制。方法选择新西兰大耳白兔20只,随机分为4组,即正常组、中药高浓度组、中药中浓度组、中药低浓度组,每组5只。以上各组白兔分别以生理盐水和高、中、低浓度益气活血复方连续灌胃7d。末次灌胃给药2h后,心脏采血,离心后分离血清。体外培养人脐静脉内皮细胞,用LPS刺激后,分别加入高、中、低浓度益气活血复方含药血清干预24h,收集细胞,用Real ti me PCR方法测定TLR4、MyD88、TRAF-6、TRAM及TRIF mRNA的表达。结果用LPS刺激人脐静脉内皮细胞后,引起TLR4、MyD88、TRAF-6、TRAM及TRIF mRNA的高表达(与空白对照组比较,P0.01),用益气活血复方含药血清干预以后显著抑制TLR4、MyD88及TRAF-6 mRNA的高表达(与模型组比较P0.05或P0.01),对TRAM及TRIF作用不明显。结论益气活血复方可阻断TLR4高表达,同时阻断TLR4胞内信号转导的MyD88依赖性途径,而对MyD88非依赖性途径作用不明显,因此益气活血复方主要是通过阻断MyD88依赖性途径来发挥其抗动脉粥样硬化的作用。     

Total coumarins from Urtica dentata Hand prevent murine autoimmune diabetes via suppression of the TLR4-signaling pathways

Ethnopharmacological relevance

Urtica dentata Hand (UDH), the root of Laportea bulbifera (Sieb. et. Zucc.) Wedd, has been traditionally used in traditional Chinese medicine as an anti-inflammatory and immuno-regulatory agent for rheumatoid arthritis and some other autoimmune diseases treatment. And the coumarins are the major components of UDH.

Aim of the study

To investigate the effect of total coumarins (TC) isolated from UDH on the development of autoimmune diabetes.

Materials and methods

Eight-week-old non-obese diabetic (NOD) mice were randomly divided into four groups: control group, low-dose (37.5 mg/kg), middle-dose (75 mg/kg), and high-dose (150 mg/kg) TC-treatment groups. NOD mice were then given with a suspension of TC or saline by intragastric (i.g.) administration every other day. After 4 weeks of treatment, 8 mice at 12-weeks of age per group were randomly selected to be sacrificed to perform intraperitoneal glucose tolerance test, examine histopathological insulitis, spleen T lymphocyte proliferation, the percentage of CD4+CD25+Foxp3+ T regulatory cell (Treg), dendritic cell (DC) surface molecules, toll-like receptor (TLR)4 expression and signal pathways involved. The remaining 10 mice per group were kept until 26 weeks of age to assess the incidence of diabetes. We also studied the direct effect of TC on DC and CD4+CD25+ Tregs in vitro.

Results

Treatment with TC for 4 weeks significantly inhibited insulitis, increased pancreatic islet number, delayed the onset and decreased the development of diabetes by 26 weeks of age in NOD mice, compared with the untreated control mice. TC suppressed spleen T lymphocyte proliferation, induced Th2-biased cytokine response, the generation of CD4+CD25+Foxp3+ Tregs and Foxp3 mRNA expression. And TC-treated DCs were characterized as low expression of MHC class II and CD86 molecules. TLR4 gene and protein expressions in the spleen, thymus and pancreas were down-regulated in TC-treated groups. The key molecules in the downstream signaling cascades of TLR4, including myeloid differentiation factor (MyD)88, nuclear factor (NF)-κB, IL-1β, Toll–IL-1 receptor domain-containing adaptor inducing interferon-β(TRIF), TRIF-related adaptor molecule (TRAM), interferon regulatory factor (IRF)-3 and IFN-β, all decreased significantly in TC groups, suggesting that TC inhibits both MyD88-dependent and -independent pathways of TLR4. At the cellular level, however, TLR4 protein expression in DCs, but not in Tregs, was downregulated by TC. And TC strengthened the role of DC, not Treg, in negative immune regulation in vitro. In contrast, anti-TLR4 antibody could block the effect of TC on DCs immune function.

Conclusion

These results suggest that TC extracted from UDH prevent the development of autoimmune diabetes in NOD mice via suppression of the TLR4-signaling pathways. TC maintain the DCs in an immature tolerogenic state, at least in part, mediated by down-regulating TLR4-signaling pathways in DCs, then enhance Treg differentiation, shift toward Th2 and suppress T lymphocyte proliferation.     

Role of airway epithelial barrier dysfunction in pathogenesis of asthma

Bronchial asthma is characterized by persistent cough, increased sputum, and repeated wheezing. The pathophysiology underlying these symptoms is the hyper-responsiveness of the airway along with chronic airway inflammation. Repeated injury, repair, and regeneration of the airway epithelium following exposure to environmental factors and inflammation results in histological changes and functional abnormalities in the airway mucosal epithelium; such changes are believed to have a significant association with the pathophysiology of asthma. Damage to the barrier functions of the airway epithelium enhances mucosal permeability of foreign substances in the airway epithelium of patients with asthma. Thus, epithelial barrier fragility is closely involved in releasing epithelial cytokines (e.g., TSLP, IL-25, and IL-33) because of the activation of airway epithelial cells, dendritic cells, and innate group 2 innate lymphoid cells (ILC2). Functional abnormalities of the airway epithelial cells along with the activation of dendritic cells, Th2 cells, and ILC2 form a single immunopathological unit that is considered to cause allergic airway inflammation. Here we use the latest published literature to discuss the potential pathological mechanisms regarding the onset and progressive severity of asthma with regard to the disruption of the airway epithelial function.     

A presumed antagonistic LPS identifies distinct functional organization of TLR4 in mouse microglia

Microglia as principle innate immune cells of the central nervous system (CNS) are the first line of defense against invading pathogens. They are capable of sensing infections through diverse receptors, such as Toll‐like receptor 4 (TLR4). This receptor is best known for its ability to recognize bacterial lipopolysaccharide (LPS), a causative agent of gram‐negative sepsis and septic shock. A putative, naturally occurring antagonist of TLR4 derives from the photosynthetic bacterium Rhodobacter sphaeroides. However, the antagonistic potential of R. sphaeroides LPS (Rs‐LPS) is no universal feature, since several studies suggested agonistic rather than antagonistic actions of this molecule depending on the investigated mammalian species. Here we show the agonistic versus antagonistic potential of Rs‐LPS in primary mouse microglia. We demonstrate that Rs‐LPS efficiently induces the release of cytokines and chemokines, which depends on TLR4, MyD88, and TRIF, but not CD14. Furthermore, Rs‐LPS is able to regulate the phagocytic capacity of microglia as agonist, while it antagonizes Re‐LPS‐induced MHC I expression. Finally, to our knowledge, we are the first to provide in vivo evidence for an agonistic potential of Rs‐LPS, as it efficiently triggers the recruitment of peripheral immune cells to the endotoxin‐challenged CNS. Together, our results argue for a versatile and complex organization of the microglial TLR4 system, which specifically translates exogenous signals into cellular functions. Importantly, as demonstrated here for microglia, the antagonistic potential of Rs‐LPS needs to be considered with caution, as reactions to Rs‐LPS not only differ by cell type, but even by function within one cell type.     

Myrsine seguinii ethanolic extract and its active component quercetin inhibit macrophage activation and peritonitis induced by LPS by targeting to Syk/Src/IRAK-1

Ethnopharmacological relevance

Myrsine seguinii H. LÉVEILLÉ (syn. Rapanea neriifolia) (Myrsinaceae) is a medicinal plants traditionally used in Myanmar to treat infectious and inflammatory diseases. Since none of reports have systematically demonstrated the anti-inflammatory activity of this plant, we aimed to mechanistically understand the regulatory roles of the plant in inflammatory responses using the ethanolic extract of Myrsine seguinii (Ms-EE).

Materials and methods

Activated macrophages and peritonitis symptoms induced by lipopolysaccharide (LPS) were employed. HPLC analysis was used to identify active components. To characterize direct target enzymes, kinase assay was established.

Results

Ms-EE inhibited the production of nitric oxide (NO) and prostaglandin (PG)E₂ in RAW264.7 cells and peritoneal macrophages stimulated by LPS. This extract suppressed the mRNA expression of the inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 genes by down-regulating the activation of nuclear factor (NF)-κB and activator protein (AP-1). Interestingly, it was found that Ms-EE can directly suppress the enzyme activities of Syk, Src, and interleukin-1 receptor-associated kinase-1 (IRAK-1). Similarly, orally administered Ms-EE inhibited the phosphorylation of Src and Syk in peritoneal exudate-derived cells prepared from peritonitis. Finally, HPLC analysis clearly demonstrated that quercetin is a major active component with suppressing activity on the release of inflammatory mediators (NO and PGE₂), and the enzyme activities of Src, Syk, and IRAK-1.

Conclusion

Ms-EE containing quercetin negatively modulates macrophage-mediated in vitro inflammatory responses and LPS-induced peritonitis by blocking the Src/Syk/NF-κB and IRAK-1/AP-1 pathways, which contributes to its major ethnopharmacological use as an anti-inflammatory herbal medicine.     

IRF9 as a negative regulator involved in TRIF-mediated NF-κB pathway in a teleost fish,Miichthys miiuy

Proinflammatory cytokines and type I IFNs were produced by TLR signaling and these responses are crucial for host defensive responses against pathogens. In order to avoid harmful and inappropriate inflammatory responses, there are multiple mechanisms to negatively regulate TLR signaling. In this paper, we have firstly studied IRF9 functions as a negative regulator involved in TRIF-mediated NF-κB pathway. Moreover, we found inhibitory effect of IRF9 primary depends on DBD domain. Interestingly, we also demonstrated that else mutants of IRF9, except for IRF9-ΔDBD, have different inhibitory effects upon TRIF-mediated NF-κB pathway. This study provides a novel evidence about the negatively regulation of innate immune signaling pathway in teleost fish. In addition, this finding provides new insights into the regulatory mechanism in mammals.