Bushen recipe and its disassembled prescriptions inhibit inflammation of liver injury associated with Concanavalin A through Toll­like receptor 3/9 signaling pathway.

The aim of the present study was to explore the effect of Bushen recipe and its disassembled prescriptions on liver injury and chronic hepatitis B. Liver injury was induced in normal and hepatitis B virus (HBV)­transgenic mice through injection of Concanavalin A, followed by treatment with Bushen recipe and its disassembled prescriptions including the Bushen­yang, the Bushen­yin and the QingHua groups as well as the GanYanLing group (positive control). Subsequently, their liver function indexes were investigated by a microplate method and liver sections were blindly evaluated using an optical microscope by a pathologist. Subsequently, the activation state of Toll­like receptor (TLR)3/9 signaling pathway in liver tissues was analyzed by western blotting. Additionally, the inflammatory factors produced following liver injury in peripheral blood were detected via ELISA. Following intervention with the Bushen recipe and its disassembled prescriptions, the liver function indexe alanine aminotransferase had declined, whereas cholinesterase increased. The pathological alterations of liver tissue in HBV transgenic mice were reversed by Bushen recipe and its disassembled prescriptions. In addition, the TLR3/9 signaling pathway in liver tissues of HBV transgenic mice was inhibited and inflammatory factors such as interleukin (IL)­6, IL­1, tumor necrosis factor­α and interferon­Î³ were reduced significantly. In conclusion, the present study demonstrated that Bushen recipe and its disassembled prescriptions repaired liver injury induced by Concanavalin A through inhibition of TLR3/9 signaling pathway.

ROS-Inducing Micelles Sensitize Tumor-Associated Macrophages to TLR3 Stimulation for Potent Immunotherapy.

One approach to cancer immunotherapy is the repolarization of immunosuppressive tumor-associated macrophages (TAMs) to antitumor M1 macrophages. The present study developed galactose-functionalized zinc protoporphyrin IX (ZnPP) grafted poly(l-lysine)- b-poly(ethylene glycol) polypeptide micelles (ZnPP PM) for TAM-targeted immunopotentiator delivery, which aimed at in vivo repolarization of TAMs to antitumor M1 macrophages. The outcomes revealed that ROS-inducing ZnPP PM demonstrated specificity for the in vitro and in vivo targeting of macrophages, elevated the level of ROS, and lowered STAT3 expression in BM-TAMs. Poly I:C (PIC, a TLR3 agonist)-loaded ZnPP PM (ZnPP PM/PIC) efficiently repolarized TAMs to M1 macrophages, which were reliant on ROS generation. Further, ZnPP PM/PIC substantially elevated the activated NK cells and T lymphocytes in B16-F10 melanoma tumors, which caused vigorous tumor regression. Therefore, the TAM-targeted transport of an immunologic adjuvant with ZnPP-grafted nanovectors may be a potential strategy to repolarize TAMs to M1 macrophages in situ for effective cancer immunotherapy.

Radix isatidis Polysaccharides Inhibit Influenza a Virus and Influenza A Virus-Induced Inflammation via Suppression of Host TLR3 Signaling In Vitro.

Influenza remains one of the major epidemic diseases worldwide, and rapid virus replication and collateral lung tissue damage caused by excessive pro-inflammatory host immune cell responses lead to high mortality rates. Thus, novel therapeutic agents that control influenza A virus (IAV) propagation and attenuate excessive pro-inflammatory responses are needed. Polysaccharide extract from Radix isatidis, a traditional Chinese herbal medicine, exerted potent anti-IAV activity against human seasonal influenza viruses (H1N1 and H3N2) and avian influenza viruses (H6N2 and H9N2) in vitro. The polysaccharides also significantly reduced the expression of pro-inflammatory cytokines (IL-6) and chemokines (IP-10, MIG, and CCL-5) stimulated by A/PR/8/34 (H1N1) at a range of doses (7.5 mg/mL, 15 mg/mL, and 30 mg/mL); however, they were only effective against progeny virus at a high dose. Similar activity was detected against inflammation induced by avian influenza virus H9N2. The polysaccharides strongly inhibited the protein expression of TLR-3 induced by PR8, suggesting that they impair the upregulation of pro-inflammatory factors induced by IAV by inhibiting activation of the TLR-3 signaling pathway. The polysaccharide extract from Radix isatidis root therefore has the potential to be used as an adjunct to antiviral therapy for the treatment of IAV infection.

Nanoformulation of synergistic TLR ligands to enhance vaccination against Entamoeba histolytica.

Diarrheal infectious diseases represent a major cause of global morbidity and mortality. There is an urgent need for vaccines against diarrheal pathogens, especially parasites. Modern subunit vaccines rely on combining a highly purified antigen with an adjuvant to increase their efficacy. In the present study, we evaluated the ability of a nanoliposome adjuvant system to trigger a strong mucosal immune response to the Entamoeba histolytica Gal/GalNAc lectin LecA antigen. CBA/J mice were immunized with alum, emulsion or liposome based formulations containing synthetic TLR agonists. A liposome formulation containing TLR4 and TLR7/8 agonists was selected based on its ability to generate intestinal IgA, plasma IgG2a/IgG1, IFN-γ and IL-17A. Immunization with a mucosal prime followed by a parenteral boost generated a high mucosal IgA response that inhibited adherence of parasites to mammalian cells. Inclusion of the immune potentiator all-trans retinoic acid in the regimen further improved the mucosal IgA response. Immunization protected from infection with up to 55% efficacy. Our results show that a nanoliposome delivery system containing TLR agonists is a promising prospect for the development of vaccines against enteric pathogens, especially when a multifaceted immune response is desired.

Application of Magnesium Pyrophosphate-Based Sponge-Like Microparticles to Enhance the Delivery Efficiency and Adjuvant Effects of Polyriboinosinic-Polyribocytidylic Acid in Immune Cells.

The magnesium pyrophosphate particle (MgPP) is a unique and safe carrier that is prepared by simply mixing magnesium chloride and sodium pyrophosphate. In this study, we investigated whether MgPP can be used to deliver nucleic acid-based adjuvants to immune cells. Polyriboinosinic-polyribocytidylic acid (polyI:C), a ligand for toll-like receptor 3, was selected as a model nucleic acid-based adjuvant. PolyI:C-loaded MgPP (polyI:C-MgPP) was prepared by adding polyI:C during the MgPP preparation process. Efficient loading of polyI:C into MgPP was confirmed by measuring the absorbance at 260 nm after disruption of polyI:C-MgPP by ethylenediaminetetraacetic acid. Scanning electron microscopy revealed that both MgPP and polyI:C-MgPP had a unique sponge-like shape with a diameter of approximately 1 µm. PolyI:C-MgPP was more efficiently taken up by toll-like receptor 3-positive RAW264.7 cells than naked polyI:C, and its uptake stimulated increased tumor necrosis factor-α production. When the presentation of ovalbumin (OVA), a model antigen, was evaluated after the addition of OVA along with naked polyI:C or polyI:C-MgPP to mouse dendritic DC2.4 cells, polyI:C-MgPP substantially increased OVA presentation. These results indicate that MgPP is a useful delivery vehicle for polyI:C and that polyI:C-MgPP is an effective immune cell adjuvant.

A novel rabies vaccine based-on toll-like receptor 3 (TLR3) agonist PIKA adjuvant exhibiting excellent safety and efficacy in animal studies.

Vaccination alone is not sufficiently effective to protect human from post-exposure rabies virus infection due to delayed generation of rabies virus neutralizing antibodies and weak cellular immunity. Therefore, it is vital to develop safer and more efficacious vaccine against rabies. PIKA, a stabilized chemical analog of double-stranded RNA that interacts with TLR3, was employed as adjuvant of rabies vaccine. The efficacy and safety of PIKA rabies vaccine were evaluated. The results showed that PIKA rabies vaccine enhanced both humoral and cellular immunity. After viral challenge, PIKA rabies vaccine protected 70-80% of animals, while the survival rate of non-adjuvant vaccine group (control) was 20-30%. According to the results of toxicity tests, PIKA and PIKA rabies vaccine are shown to be well tolerated in mice. Thus, this study indicates that PIKA rabies vaccine is an effective and safe vaccine which has the potential to develop next-generation rabies vaccine and encourage the start of clinical studies.

Particulate formulations for the delivery of poly(I:C) as vaccine adjuvant.

Current research and development of antigens for vaccination often center on purified recombinant proteins, viral subunits, synthetic oligopeptides or oligosaccharides, most of them suffering from being poorly immunogenic and subject to degradation. Hence, they call for efficient delivery systems and potent immunostimulants, jointly denoted as adjuvants. Particulate delivery systems like emulsions, liposomes, nanoparticles and microspheres may provide protection from degradation and facilitate the co-formulation of both the antigen and the immunostimulant. Synthetic double-stranded (ds) RNA, such as polyriboinosinic acid-polyribocytidylic acid, poly(I:C), is a mimic of viral dsRNA and, as such, a promising immunostimulant candidate for vaccines directed against intracellular pathogens. Poly(I:C) signaling is primarily dependent on Toll-like receptor 3 (TLR3), and on melanoma differentiation-associated gene-5 (MDA-5), and strongly drives cell-mediated immunity and a potent type I interferon response. However, stability and toxicity issues so far prevented the clinical application of dsRNAs as they undergo rapid enzymatic degradation and bear the potential to trigger undue immune stimulation as well as autoimmune disorders. This review addresses these concerns and suggests strategies to improve the safety and efficacy of immunostimulatory dsRNA formulations. The focus is on technological means required to lower the necessary dosage of poly(I:C), to target surface-modified microspheres passively or actively to antigen-presenting cells (APCs), to control their interaction with non-professional phagocytes and to modulate the resulting cytokine secretion profile.

Triggering of the dsRNA sensors TLR3, MDA5, and RIG-I induces CD55 expression in synovial fibroblasts.

BACKGROUND: CD55 (decay-accelerating factor) is a complement-regulatory protein highly expressed on fibroblast-like synoviocytes (FLS). CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor abundantly present on leukocytes. Little is known regarding the regulation of CD55 expression in FLS. METHODS: FLS isolated from arthritis patients were stimulated with pro-inflammatory cytokines and Toll-like receptor (TLR) ligands. Transfection with polyinosinic-polycytidylic acid (poly(I:C)) and 5'-triphosphate RNA were used to activate the cytoplasmic double-stranded (ds)RNA sensors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I). CD55 expression, cell viability, and binding of CD97-loaded beads were quantified by flow cytometry. RESULTS: CD55 was expressed at equal levels on FLS isolated from patients with rheumatoid arthritis (RA), osteoarthritis, psoriatic arthritis and spondyloarthritis. CD55 expression in RA FLS was significantly induced by IL-1ß and especially by the TLR3 ligand poly(I:C). Activation of MDA5 and RIG-I also enhanced CD55 expression. Notably, activation of MDA5 dose-dependently induced cell death, while triggering of TLR3 or RIG-I had a minor effect on viability. Upregulation of CD55 enhanced the binding capacity of FLS to CD97-loaded beads, which could be blocked by antibodies against CD55. CONCLUSIONS: Activation of dsRNA sensors enhances the expression of CD55 in cultured FLS, which increases the binding to CD97. Our findings suggest that dsRNA promotes the interaction between FLS and CD97-expressing leukocytes.

Anticancer function of polyinosinic-polycytidylic acid.

Polyinosinic-polycytidylic acid (polyi:c) is a synthetic analog of double-stranded RNA and an agonist of toll-like receptor (TLR) 3 and retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I and melanoma differentiation-associated gene 5 (MDA5). The effect of polyi:c on tumor immunotherapy has been well explored for several decades. The accumulated evidence suggests that polyi:c could be used as a vaccine adjuvant to enhance innate and adaptive immune responses, and to alter the tumor microenvironment. Recent studies have also shown that activation of TLR3 and RLR signaling by polyi:c can directly trigger apoptosis in some cancer cells. This review focuses on polyi:c-induced signaling pathways and the applications of polyi:c in cancer treatment.

Suppression of the effector phase of inflammatory arthritis by double-stranded RNA is mediated by type I IFNs.

Innate immune receptors that recognize nucleic acids, such as TLRs and RNA helicases, are potent activators of innate immunity that have been implicated in the induction and exacerbation of autoimmunity and inflammatory arthritis. Polyriboinosine-polyribocytidylic acid sodium salt (poly(IC)) is a mimic of dsRNA and viral infection that activates TLR3 and the RNA helicases retinoic acid-induced gene-1 and melanoma differentiation-associated gene-5, and strongly induces type I IFN production. We analyzed the effects of systemic delivery of poly(IC) on the inflammatory effector phase of arthritis using the collagen Ab-induced and KRN TCR-transgenic mouse serum-induced models of immune complex-mediated experimental arthritis. Surprisingly, poly(IC) suppressed arthritis, and suppression was dependent on type I IFNs that inhibited synovial cell proliferation and inflammatory cytokine production. Administration of exogenous type I IFNs was sufficient to suppress arthritis. These results suggest a regulatory role for innate immune receptors for dsRNA in modulating inflammatory arthritis and provide additional support for an anti-inflammatory function of type I IFNs in arthritis that directly contrasts with a pathogenic role in promoting autoimmunity in systemic lupus.