The virus-induced signaling adaptor molecule enhances DNA-raised immune protection against H5N1 influenza virus infection in mice

As an adaptor molecule in the retinoic acid-inducible gene-I (RIG-I) signaling pathway, the virus-induced signaling adaptor (VISA) molecule activates NF-κB and IRF3 and thereby leads to the production of type I interferons (IFNs). To explore the potential of VISA as a genetic adjuvant for DNA vaccines, a eukaryotic expression plasmid, pVISA, was generated by cloning the VISA gene into the pVAX1vector. For comparison, the pTRIF plasmid was similarly constructed, encoding the known genetic adjuvant TRIF (TIR-domain-containing adapter-inducing interferon-β), an adapter in the Toll-like receptor (TLR) signaling pathway. Mice were immunized with the chimeric DNA vaccine pHA/NP_147-155, which encodes the HA (hemagglutinin) fused with NP (nucleoprotein) CTL epitope (NP_147-155) of H5N1 influenza virus, either alone or in combination with pVISA or pTRIF. Antigen-specific immune responses were examined in immunized mice. Our results demonstrate that co-immunization of the pHA/NP_147-155 plasmid with the VISA adjuvant augmented DNA-raised cellular immune responses and provided protection against H5N1 influenza virus challenge in mice. In addition, our data suggest that VISA acts as a stronger adjuvant for DNA immunization than TRIF. We conclude that co-inoculation with a vector expressing the adaptor molecule VISA enhanced the protective immunity against H5N1 infection induced by pHA/NP_147-155 and that VISA could be developed as a novel genetic adjuvant for DNA vaccines.     

Structures and interface mapping of the TIR domain-containing adaptor molecules involved in interferon signaling

Homotypic and heterotypic interactions between Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors (TLRs) and downstream adaptors are essential to evoke innate immune responses. However, such oligomerization properties present intrinsic difficulties in structural studies of TIR domains. Here, using BB-loop mutations that disrupt homotypic interactions, we determined the structures of the monomeric TIR domain-containing adaptor molecule (TICAM)-1 and TICAM-2 TIR domains. Docking of the monomeric structures, together with yeast two hybrid-based mutagenesis assays, reveals that the homotypic interaction between TICAM-2 TIR is indispensable to present a scaffold for recruiting the monomeric moiety of the TICAM-1 TIR dimer. This result proposes a unique idea that oligomerization of upstream TIR domains is crucial for binding of downstream TIR domains. Furthermore, the bivalent nature of each TIR domain dimer can generate a large signaling complex under the activated TLRs, which would recruit downstream signaling molecules efficiently. This model is consistent with previous reports that BB-loop mutants completely abrogate downstream signaling.The extracellular domain of toll-like receptor 4 (TLR4) specifically binds lipopolysaccharides (LPSs) from Gram-negative bacteria, inducing dimerization and leading to the dimerization of cytosolic Toll/interleukin-1 receptor (TIR) domains. This activated conformation of TLR4 recruits the TIR domain of a downstream adaptor molecule, TIR domain-containing adaptor molecule-2 (TICAM-2) [also known as TRIF-related adaptor molecule (TRAM)], that subsequently recruits the TIR domain of another adaptor molecule, TIR domain-containing adaptor molecule-1 (TICAM-1) [also known as TIR domain-containing adaptor inducing IFN-β (TRIF)] (1–3) at endosomes. Eventually this process activates IFN response factors and generates type-I interferons (IFNs) (4–7). Elucidation of the homotypic and heterotypic interactions between TICAM-1 and TICAM-2 is essential for understanding of TLR4-mediated type-I IFN generation (8).A large number of TIR domain structures, including receptors and adaptors, have been determined by X-ray crystallography and NMR. The receptors include TLR1 (9), TLR2 (10), and IL-1R accessory protein-like (IL-1RAPL) (11). Adaptors include myeloid differentiation factor 88 (MyD88) (12) and MyD88 adaptor-like (Mal) (13, 14). In addition, AtTIR (15, 16) derived from Arabidopsis thaliana and PdTIR (17) from bacteria have been solved. Each of these TIR domain structures has a ferredoxin fold with five β-strands (βA–βE), five α-helices (αA–αE), and loops connecting β-strands and α-helices (9). Although homotypic interactions of the TIR domains have been proposed based on the crystal structures, most proposed models have small interacting surfaces, possibly due to crystal contacts. Recently, however, a crystal structure of the TLR10 TIR domain was reported that forms a homotypic dimer mediated by the loop connecting βB and αB (designated “BB-loop”) (18). Interestingly, BB-loop mutations in TLR4 were reported to be dominant-negative and abrogated downstream signaling (19). TICAM-1 and TICAM-2 harboring BB-loop mutations are also dominant-negative and unable to form homotypic interactions (1, 2), reinforcing the importance of BB-loop–mediated homotypic dimer formation in signal propagation.Despite extensive structural studies, it is not known why homotypic interactions are essential for downstream signaling (20–27). To address this issue, it is necessary to discriminate residues required for homotypic and those required for heterotypic interactions. Here, we first determine the structures of the monomeric BB-loop mutants of the TICAM-1 and TICAM-2 TIR domains using NMR. Then, based on the solution structures of the BB-loop mutants, coupled mutagenesis/yeast two-hybrid experiments, and restrained docking calculations, we show that the homotypic interaction of TICAM-2 TIR is essential to form a scaffold for recruiting the TICAM-1 TIR domain.     

Herbal medicines for the prevention of alcoholic liver disease: A review

Ethnopharmacological relevance

Long-term excess alcohol exposure leads to alcoholic liver disease (ALD)—a global health problem without effective therapeutic approach. ALD is increasingly considered as a complex and multifaceted pathological process, involving oxidative stress, inflammation and excessive fatty acid synthesis. Over the past decade, herbal medicines have attracted much attention as potential therapeutic agents in the prevention and treatment of ALD, due to their multiple targets and less toxic side effects. Several herbs, such as Cnidium monnieri (L.) Cusson (Apiaceae), Curcuma longa L. (Zingiberaceae) and Pueraria lobata (Willd.) Ohwi (Leguminosae), etc., have been shown to be quite effective and are being widely used in China today for the treatment of ALD when used alone or in combination.

Aim of the review

To review current available knowledge on herbal medicines used to prevent or treat ALD and their underlying mechanisms.

Materials and methods

We used the pre-set searching syntax and inclusion criteria to retrieve available published literature from PUBMED and Web of Science databases, all herbal medicines and their active compounds tested on ALD induced by both acute and chronic alcohol ingestion were included.

Results

A total of 40 experimental studies involving 34 herbal medicines and (or) active compounds were retrieved and reviewed. We found that all reported extracts and individual compounds from herbal medicines/natural plants could be beneficial to ALD, which might be attributed to regulate multiple critical targets involved in the pathways of oxidation, inflammation and lipid metabolism.

Conclusions

Screening chemical candidate from herbal medicine might be a promising approach to drug discovery for the prevention or treatment of ALD. However, further studies remain to be done on the systematic assessment of herbal medicines against ALD and the underlying mechanisms, as well as their quality control studies.     

Inhibition of homodimerization of Toll-like receptor 4 by curcumin

Toll-like receptors play a key role in sensing microbial components and inducing innate immune responses. Ligand-induced dimerization of TLR4 is required for the activation of downstream signaling pathways. Thus, the receptor dimerization may be one of the first lines of regulation in activating TLR-mediated signaling pathways and induction of subsequent immune responses. LPS induces the activation of NF-kappaB and IRF3 through MyD88- or TRIF-dependent pathways. Curcumin, a polyphenol found in the plant Curcuma longa, has been shown to suppress the activation of NF-kappaB induced by various pro-inflammatory stimuli by inhibiting IKKbeta kinase activity in MyD88-dependent pathway. Curcumin also inhibited LPS-induced IRF3 activation. These results imply that curcumin inhibits both MyD88- and TRIF-dependent pathways in LPS-induced TLR4 signaling. However, in TRIF-dependent pathway, curcumin did not inhibit IRF3 activation induced by overexpression of TRIF in 293T cells. These results suggest that TLR4 receptor complex is the molecular target of curcumin in addition to IKKbeta. Here, we report biochemical evidence that phytochemicals (curcumin and sesquiterpene lactone) inhibit both ligand-induced and ligand-independent dimerization of TLR4. Furthermore, these results demonstrate that small molecules with non-microbial origin can directly inhibit TLRs-mediated signaling pathways at the receptor level. These results imply that the activation of TLRs and subsequent immune/inflammatory responses induced by endogenous molecules or chronic infection can be modulated by certain dietary phytochemicals we consume daily.     

Myeloid differentiation 2 as a therapeutic target of inflammatory disorders

Lipopolysaccharide (LPS), an endotoxin of Gram-negative bacteria, activates the innate immunity system through a receptor complex of myeloid differentiation 2 (MD-2) and toll-like receptor 4 (TLR4). MD-2 directly recognizes the lipid A domain of LPS, which triggers MD-2/TLR4-mediated cellular response aimed at eliminating the invaded pathogen. However, excess production of inflammatory mediators is harmful to host tissue and this can cause septic death in extreme cases. MD-2 represents an attractive therapeutic target of inflammatory and immune diseases in human. In particular, eritoran is a synthetic tetraacylated lipid A that binds directly to MD-2 and antagonizes LPS binding to the same site, and it ameliorates various inflammatory conditions due to infection or sterile organ injury. In this review, we outline the recent advances in the structure biology of ligand interaction with MD-2/TLR4, and highlight the MD-2-directed LPS antagonists, which are natural and synthetic chemicals, under development to treat inflammatory diseases.     

Toll-like receptor 3 agonist Poly I:C protects against simulated cerebral ischemia in vitro and in vivo

Aim:

To examine the neuroprotective effects of the Toll-like receptor 3 (TLR3) agonist Poly I:C in acute ischemic models in vitro and in vivo.

Methods:

Primary astrocyte cultures subjected to oxygen-glucose deprivation (OGD) were used as an in vitro simulated ischemic model. Poly I:C was administrated 2 h before OGD. Cell toxicity was measured using MTT assay and LDH leakage assay. The levels of TNFα, IL-6 and interferon-β (IFNβ) in the media were measured using ELISA. Toll/interleukin receptor domain-containing adaptor-inducing IFNβ (TRIF) protein levels were detected using Western blot analysis. A mouse middle cerebral artery occlusion (MCAO) model was u sed for in vivo study. The animals were administered Poly I:C (0.3 mg/kg, im) 2 h before MCAO, and examined with neurological deficit scoring and TTC staining. The levels of TNFα and IL-6 in ischemic brain were measured using ELISA.

Results:

Pretreatment with Poly I:C (10 and 20 μg/mL) markedly attenuated OGD-induced astrocyte injury, and significantly raised the cell viability and reduced the LDH leakage. Poly I:C significantly upregulated TRIF expression accompanied by increased downstream IFNβ production. Moreover, Poly I:C significantly suppressed the pro-inflammatory cytokines TNFα and IL-6 production. In mice subjected to MCAO, administration of Poly I:C significantly attenuated the neurological deficits, reduced infarction volume, and suppressed the increased levels of TNFα and IL-6 in the ischemic striatum and cortex.

Conclusion:

Poly I:C pretreatment exerts neuroprotective and anti-inflammatory effects in the simulated cerebral ischemia models, and the neuroprotection is at least in part due to the activation of the TLR3-TRIF pathway.     

桔梗汤总黄酮对慢性阻塞性肺病大鼠TLR4/TRIF/IRF3通路的影响＊

目的 考察桔梗汤总黄酮（FPG）对慢性阻塞性肺病（COPD）大鼠的治疗作用，研究其对TLR4/TRIF/IRF3和TLR4/MyD88通路的影响，探讨其治疗COPD可能的分子机制。方法 建立COPD大鼠模型，将60只SD大鼠分为空白组、模型组、阳性对照组及不同剂量的桔梗汤总黄酮治疗组，经过3周治疗后，通过分析大鼠肺功能指标、肺灌洗液中IL-6、IL-12、TNF-α的含量、血清中IFN-α、IFN-β的含量、肺组织病理切片及MyD88、TLR4、TRAM、TRIF、TRAF6、IRF3、P-IRF3蛋白表达量变化，探讨桔梗汤总黄酮防治COPD的分子机制。结果 各个剂量组的桔梗汤总黄酮均可明显缓解COPD模型大鼠的肺部通气功能，可显著抑制COPD大鼠肺灌洗液中IL-6、IL-12、TNF-α的含量，抑制其血清中IFN-α、IFN-β的含量，以上结果均呈现明显的剂量依赖性；Western blot实验结果表明，桔梗汤总黄酮对COPD大鼠肺组织中的MyD88无显著影响，但可显著降低其中TLR4、TRAM、TRIF、TRAF6、IRF3、P-IRF3蛋白表达，差异具有统计学意义。结论 桔梗汤总黄酮治疗COPD可能是通过抑制TLR4/TRIF/IRF3通路发挥作用，不是通过TLR4/MyD88经典通路实现的。