Iron Oxide Nanoparticles Inhibit Tumor Progression and Suppress Lung Metastases in Mouse Models of Breast Cancer.

Systemic exposure to starch-coated iron oxide nanoparticles (IONPs) can stimulate antitumor T cell responses, even when little IONP is retained within the tumor. Here, we demonstrate in mouse models of metastatic breast cancer that IONPs can alter the host immune landscape, leading to systemic immune-mediated disease suppression. We report that a single intravenous injection of IONPs can inhibit primary tumor growth, suppress metastases, and extend survival. Gene expression analysis revealed the activation of Toll-like receptor (TLR) pathways involving signaling via Toll/Interleukin-1 receptor domain-containing adaptor-inducing IFN-ß (TRIF), a TLR pathway adaptor protein. Requisite participation of TRIF in suppressing tumor progression was demonstrated with histopathologic evidence of upregulated IFN-regulatory factor 3 (IRF3), a downstream protein, and confirmed in a TRIF knockout syngeneic mouse model of metastatic breast cancer. Neither starch-coated polystyrene nanoparticles lacking iron, nor iron-containing dextran-coated parenteral iron replacement agent, induced significant antitumor effects, suggesting a dependence on the type of IONP formulation. Analysis of multiple independent clinical databases supports a hypothesis that upregulation of TLR3 and IRF3 correlates with increased overall survival among breast cancer patients. Taken together, these data support a compelling rationale to re-examine IONP formulations as harboring anticancer immune (nano)adjuvant properties to generate a therapeutic benefit without requiring uptake by cancer cells.

[Anti-herpes simplex virus type â of tectorigenin derivative and effect on Toll-like receptors in vitro].

The study investigated the inhibitory effect and mechanism of tectorigenin derivative(SGY) against herpes simplex virus type Ⅰ(HSV-1) by in vitro experiments. The cytotoxicity of SGY and positive drug acyclovir(ACV) on African green monkey kidney(Vero) cells and mouse microglia(BV-2) cells was detected by cell counting kit-8(CCK-8) method, and the maximum non-toxic concentration and median toxic concentration(TC_(50)) of the drugs were calculated. After Vero cells were infected with HSV-1, the virulence was determined by cytopathologic effects(CPE) to calculate viral titers. The inhibitory effect of the tested drugs on HSV-1-induced cytopathy in Vero cells was measured, and their modes of action were initially explored by virus adsorption, replication and inactivation. The effects of the drugs on viral load of BV-2 cells 24 h after HSV-1 infection and the Toll-like receptor(TLR) mRNA expression were detected by real-time fluorescence quantitative PCR(RT-qPCR). The maximum non-toxic concentrations of SGY against Vero and BV-2 cells were 382.804 µg·mL~(-1) and 251.78 µg·mL~(-1), respectively, and TC_(50) was 1 749.98 µg·mL~(-1) and 2 977.50 µg·mL~(-1), respectively. In Vero cell model, the half maximal inhibitory concentration(IC_(50)) of SGY against HSV-1 was 54.49 µg·mL~(-1), and the selection index(SI) was 32.12, with the mode of action of significantly inhibiting replication and directly inactivating HSV-1. RT-qPCR results showed that SGY markedly reduced the viral load in cells. The virus model group had significantly increased relative expression of TLR2, TLR3 and tumor necrosis factor receptor-associated factor 3(TRAF3) and reduced relative expression of TLR9 as compared with normal group, and after SGY intervention, the expression of TLR2, TLR3 and TRAF3 was decreased to different degrees and that of TLR9 was enhanced. The expression of inflammatory factors inducible nitric oxide synthase(iNOS), tumor necrosis factor-α(TNF-α), and interleukin-1ß(IL-1ß) was remarkably increased in virus model group as compared with that in normal group, and the levels of these inflammatory factors dropped after SGY intervention. In conclusion, SGY significantly inhibited and directly inactivated HSV-1 in vitro. In addition, it modulated the expression of TLR2, TLR3 and TLR9 related pathways, and suppressed the increase of inflammatory factor levels.

Profiling of extracellular vesicles of metastatic urothelial cancer patients to discover protein signatures related to treatment outcome.

The prognosis of metastatic urothelial carcinoma (mUC) patients is poor, and early prediction of systemic therapy response would be valuable to improve outcome. In this exploratory study, we investigated protein profiles in sequential plasma-isolated extracellular vesicles (EVs) from a subset of mUC patients treated within a Phase I trial with vinflunine combined with sorafenib. The isolated EVs were of exosome size and expressed exosome markers CD9, TSG101 and SYND-1. We found, no association between EVs/ml plasma at baseline and progression-free survival (PFS). Protein profiling of EVs, using an antibody-based 92-plex Proximity Extension Assay on the Oncology II® platform, revealed a heterogeneous protein expression pattern. Qlucore bioinformatic analyses put forward a protein signature comprising of SYND-1, TNFSF13, FGF-BP1, TFPI-2, GZMH, ABL1 and ERBB3 to be putatively associated with PFS. Similarly, a protein signature from EVs that related to best treatment response was found, which included FR-alpha, TLR 3, TRAIL and FASLG. Several of the markers in the PFS or best treatment response signatures were also identified by a machine learning classification algorithm. In conclusion, protein profiling of EVs isolated from plasma of mUC patients shows a potential to identify protein signatures that may associate with PFS and/or treatment response.

Bovine colostrum promoted ileal health in newborn lambs at 24 h after birth: insight from intestinal morphology and innate immunity.

The contribution of colostrum to passive immunity transfer and intestinal protection in newborn ruminants is well known; however, it is currently unclear how colostrum intake affects intestinal innate immunity. We investigated the effects of bovine colostrum intake on ileal morphology, expression of genes involved in intestinal innate immunity, and serum concentrations of inflammatory cytokines in newborn lambs. Twenty-seven newborn male Hu lambs were used, of which 18 were bottle-fed either bovine colostrum (C24h; n = 9) or bovine mature milk (M24h; n = 9) within the first 2 h after birth at an intake of approximately 8% of BW; the remaining nine lambs did not receive any feeding (N24h). Blood and ileal tissue samples were collected after the lambs were slaughtered at 24 h after birth. Ileal villus height and villus height-to-crypt depth ratio were significantly higher in C24h than those in N24h and M24h lambs (P < 0.01). Messenger RNA (mRNA) abundance of toll-like receptor (TLR)-2, TLR3, TLR4, TLR6, TLR7, TLR8 and tumour necrosis factor alpha in the ileum was lower in C24h than that in N24h lambs (P < 0.05). Moreover, C24h lambs had a lower TLR3 mRNA abundance (P < 0.01) and a trend of lower TLR6 (P = 0.06) and interleukin 1 beta (P = 0.08) expression compared with those in M24h lambs. We also observed strong positive correlations of tumour necrosis factor alpha expression with that of TLR2 (r = 0.71; P < 0.001), TLR4 (r = 0.88; P < 0.001) and TLR8 (r = 0.83; P < 0.001). Interestingly, the expression of barrier-related molecules, including mucin-13, lysozyme, claudin (CLDN)-1, CLDN2, CLDN4, CLDN7, CLDN12, occludin, zonula occluden-1 and junctional adhesion molecule-1, was consistently lower in C24h lambs than that in N24h and M24h lambs (P < 0.05). These results indicated that the beneficial roles of colostrum intake on intestinal protection in newborn lambs were associated with low TLR expression, which was reflected by improved intestinal development and reduced inflammatory response. Further studies using fluorescence in situ hybridisation and immunohistochemical methods are needed to further explore the mechanisms underlying the lower expression of intestinal barrier-related molecules due to colostrum feeding.

A long non-coding RNA H19/microRNA-138/TLR3 network is involved in high phosphorus-mediated vascular calcification and chronic kidney disease.

Vascular calcification, characterized by the accumulation of calcium-phosphate crystals in blood vessels, is a major cause of cardiovascular complications and chronic kidney disease (CKD)-related death. This work focuses on the molecules involved in high-phosphorus-mediated vascular calcification in CKD. A rat model of CKD was established by 5/6 nephrectomy, and the rats were given normal phosphorus diet (NPD) or high phosphorus diet (HPD). HPD decreased kidney function, increased the concentration of calcium ion and damaged vascular structure in the thoracic aorta of diseased rats. A high phosphorus condition enhanced calcium deposition in vascular smooth muscle cells (VSMCs). High phosphorus also increased the expression of RUNX2 whereas reduced the expression of α-SM actin in the aortic tissues and VSMCs. Long non-coding RNA (lncRNA) H19 was upregulated in the aortic tissues after HPD treatment. H19 bound to microRNA (miR)-138 to block its inhibitory effect on TLR3 mRNA and activated the NF-κB signaling pathway. Downregulation of H19 or TLR3 alleviated, whereas downregulation of miR-138 aggravated the calcification and vascular damage in model rats and VSMCs. In conclusion, this study demonstrates that the H19/miR-138/TLR3 axis is involved in high phosphorus-mediated vascular calcification in rats with CKD.

Gender-specific development of experimental autoimmune cholangitis induced by double-stranded RNA.

Here we investigated the gender difference in murine cholangitis resembling human primary biliary cholangitis (PBC) caused by synthetic double-stranded RNA, and underlying hepatic innate immune responses. Female C57Bl/6 mice given repeated injections of polyinosinic-polycytidylic acid (poly I:C) for 24 weeks developed overt cholangitis with positive serum anti-mitochondria-M2 antibody, whereas male mice showed minimal pathological changes without induction in autoantibody. Poly I:C induced hepatic inflammatory cytokines and type-I interferons predominantly in females. Hepatic expression levels of toll-like receptor (TLR) 3 and melanoma differentiation-associated protein (MDA) 5 were equivalent in both genders; however, both mRNA and protein levels of retinoic acid-inducible gene (RIG)-I were nearly doubled in female livers. Following 4-week injections of poly I:C, not only hepatic RIG-I, but also TLR3 and MDA5 showed female-predominance. Moreover, hepatic RIG-I levels were 25% lower in ovariectomized mice, whereas supplementation of 17 ß-estradiol enhanced hepatic RIG-I expression, as well as cytokine induction. These results clearly indicate that hepatic RIG-I expression is potentiated by estrogen, and triggers gender-dependent hepatic innate immune response against double-stranded RNA, which most likely play a pivotal role in the pathogenesis of autoimmune cholangiopathies including PBC.

The antiviral mechanism of the crude extract from the flowers of Trollius chinensis based on TLR 3 signaling pathway.

The effects of crude extract from the flowers of Trollius chinensis on expressions of mRNA and proteins related to vital genes (TLR 3, TBK 1, IRF 3 and IFN ß) in TLR 3 signaling pathway were investigated in the presence/absence of Polyinosinic acid-polycytidylic acid (PolyI: C) to ascertain the antiviral mechanism of these flowers. Real-time PCR and western blot were applied to determine the expressions of mRNA and proteins, respectively, and immunofluorescence assay was employed to study the effect on IRF 3 distribution between nuclei and cytoplasma. In the absence of PolyI:C, the crude extract reduced the mRNA expression of TLR 3, IRF 3 and IFN ß and the protein expression of TLR 3, and increased the protein expression of IRF 3 and the distribution of IRF 3 in nuclei. In the presence of PolyI:C, the extract reduced the mRNA and protein expressions of TLR 3 and the mRNA expression of IFN ß, meanwhile inhibited the translocation of IRF 3 into nuclei. The antiviral mechanism of the crude extract from the flowers of T. chinensis is to protect the host from inflammatory damage through intervening the TLR 3 signaling pathway and reducing the secretion of inflammatory factors.

ω-3 fatty acid alleviates virus-induced myocardial injury by regulating TLR4 and TLR3 expression.

The specific pathogenesis of viral-induced myocardial injury is unclear. TLR regulation plays an important role in virus-induced myocardial injury. The therapeutic effect and possible mechanism of omega-3 fatty acids in patients with viral-induced myocardial injury must be investigated. The study population was randomly divided into three groups: a healthy control group (n = 50); general treatment group (n = 40); and general treatment with ω-3 polyunsaturated fatty acid group (n = 36). We detected the mRNA levels of TLR3 and TLR4, downstream signal pathway proteins, inflammatory factors, oxidative stress markers, and myocardial enzymes in patients and healthy controls. ω-3 fatty acid therapy in patients with virus-induced myocardial injury significantly regulates the expression of TLR3 and TLR4 and their downstream signal protein, increases antioxidant expression, reduces the secretion of inflammatory factors, alleviates myocardial injury, and improves cardiac function. This provides a new strategy to treat virus-induced myocardial injury.

Santalum album extract exhibits neuroprotective effect against the TLR3-mediated neuroinflammatory response in human SH-SY5Y neuroblastoma cells.

Neuroinflammation is an inflammatory response in the nervous system that is associated with various neurological diseases including Alzheimer's diseases and others. Many studies evaluated the anti-inflammatory potential of Santalum album (S. album) extract, but none of them analyzed its effects against neuroinflammatory response in vitro. In addition, the precise mechanism underlying the anti-inflammatory effect of the extract has not yet been elucidated. Therefore, in this study, we investigated the effect of S. album extract on modulation of toll-like receptor 3 (TLR3) agonist polyinosnic-polycytidylic acid (PolyI:C)-induced neuroinflammatory response in human neuroblastoma cells. The TLR3-mediated immune response was differentially modulated by S. album extract in SH-SY5Y cells. In addition, treatment of cells with the conditioned medium (CM) of S. album extract significantly increased the mRNA levels of IFN-ß, IFN-α, MxA and OAS-1 and decreased IL-6, CXCL8, CCL2 and IP-10. S. album extract has indirectly affected the expression of IFNs and inflammatory cytokines in SH-SY5Y cells. Furthermore, the extract was able to modulate PolyI:C-induced inflammatory response in Caco2 cells. Overall, S. album was capable to attenuate PolyI:C-induced neuroinflammatory effect through the induction of TLR2, TLR4 and the modulation of TLR negative regulators of the TRAF3, IRF3 and NF-κB pathways.

Protective effect of Chushizi (Fructus Broussonetiae) on acetaminophen-induced rat hepatitis by inhibiting the Toll-like receptor 3/c-Jun N-terminal kinase/c-jun/c-fos/janus protein tyrosine kinase/activators of transcription 3 pathway.

OBJECTIVE: To observe the intervention of Chushizi (Fructus Broussonetiae) (CSZ) on drug-induced liver injury (DILI) in rats, as well as indicators of liver function, serum levels of inflammatory cytokines, and expression of proteins and mRNA associated with toll-like receptor 3 (TLR3) and the signal transducer and activator of transcription 3 (STAT3) pathway in the liver [TLR3, janus protein tyrosine kinase 2 (JAK2), c-jun, c-fos, c-Jun N-terminal kinase 2 (JNK2), and STAT3]. METHODS: Forty specified pathogen free grade Sprague-Dawley rats were randomly divided into the control group, the model group, the silybin group and the CSZ group. Rats were given acetaminophen (APAP) to trigger DILI. Histopathology of the liver was observed by hematoxylin-eosin staining. The levels of alanine aminotransferase (ALT), aspartate transaminase (AST), direct bilirubin (DBIL), and total bilirubin (TBIL) in serum were detected by a semi-automatic biochemical instrument. Content of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-13, and IL-22 in serum were detected by the enzyme-linked immunosorbent assay, the expression of TLR3, phosphorylation of JAK2 (p-JAK2), while c-jun and c-fos proteins in the liver were determined by immunohistochemistry; expression of JNK2, and STAT3 in the liver were assayed by Western blot and real-time quantitative polymerase chain reaction. P-JNK2 and p-STAT3 in the liver were assayed by Western blot. RESULTS: After treatment, the activity of ALT, AST, and concentrations of TBIL, DBIL, TNF-α, IL-6, as well as IL-13 in serum, were lower than those in the model group, and expression of p-JAK2, TLR3, c-jun, c-fos, p-STAT3, and p-JNK2 could be downregulated. CONCLUSION: Our findings suggest that CSZ is a valid medicine to alleviate APAP-induced DILI, while its partial mechanism may regulate the TLR3/JNK/ c-jun/c-fos/JAK/STAT3 pathway.

Double-Stranded Structure of the Polyinosinic-Polycytidylic Acid Molecule to Elicit TLR3 Signaling and Adjuvant Activity in Murine Intranasal A(H1N1)pdm09 Influenza Vaccination.

Polyinosinic-polycytidylic acid (PIC) is a potent double-stranded RNA (dsRNA) adjuvant useful in intranasal influenza vaccination. In mice, the intensity and duration of immune responses to PIC correlated with the double-stranded chain length. A rational method to avoid PIC chain extension in PIC production is to use multiple short poly(I) molecules and one long poly(C) molecule for PIC assembly. In this study, we elucidate that a newly developed uPIC100-400 molecule comprising multiple 0.1 kb poly(I) molecules and one 0.4 kb poly(C) molecule effectively enhanced the immune responses in mice, by preventing the challenged viral propagation and inducing hemagglutinin-specific IgA, after intranasal A(H1N1)pdm09 influenza vaccination. Reduced intraperitoneal toxicity of PIC prepared with multiple short poly(I) molecules in mice indicates the widened effective range of uPIC100-400 as an adjuvant. In contrast to uPIC100-400, the PIC molecule comprising multiple 0.05 kb poly(I) molecules failed to elicit mouse mucosal immunity. These results were consistent with TLR3 response but not retinoic acid inducible gene I (RIG-I)-like receptor response in the cell assays, which suggests that the adjuvant effect of PIC in mouse intranasal immunization depends on TLR3 signaling. In conclusion, the double-stranded PIC with reduced toxicity developed in this study would contribute to the development of PIC-adjuvanted vaccines.

FAAH inhibition attenuates TLR3-mediated hyperthermia, nociceptive- and anxiety-like behaviour in female rats.

Aberrant activation of toll-like receptor (TLR)s results in persistent and prolonged neuroinflammation and has been implicated in the pathogenesis and exacerbation of psychiatric and neurodegenerative disorders. TLR3 coordinates the innate immune response to viral infection and recent data have demonstrated that inhibiting fatty acid amide hydrolase (FAAH), the enzyme that primarily metabolizes anandamide, modulates TLR3-mediated neuroinflammation. However, the physiological and behavioural consequences of such modulation are unknown. The present study examined the effect of URB597, a selective FAAH inhibitor, on neuroinflammation, physiological and behavioural alterations following administration of the TLR3 agonist and viral mimetic poly I:C to female rats. URB597 attenuated TLR3-mediated fever, mechanical and cold allodynia, and anxiety-like behaviour in the elevated plus maze and open field arena. There was no effect of URB597 on TLR3-mediated decreases in body weight and no effect in the sucrose preference or forced swim tests. URB597 attenuated the TLR3-mediated increase in the expression of CD11b and CD68, markers of microglia/macrophage activation. In summary, these data demonstrate that enhancing FAAH substrate levels suppresses TLR3-mediated microglia/macrophage activation and associated changes in fever, nociceptive responding and anxiety-related behaviour. These data provide further support for FAAH as a novel therapeutic target for neuroinflammatory disorders.

[Immunologic mechanisms of Yinhua Pinggan granule and San-ao decoction against influenza virus in vivo].

This paper aimed to investigate the effect of Yinhua Pinggan granule and San-ao decoction on the immunologic mechanisms of influenza viral pneumonia mice in vivo, in order to study the activity of the combined administration of different formulas on influenza A/H1N1 virus. The model of pneumonia was established in mice through nasal dropping influenza virus, and then divided randomly into five groups: normal control group, influenza virus model group, oseltamivir control group, Yinhua Pinggan granule group, and San-ao decoction group. The animals were put to death at the 5th day after gavage administration with the corresponding drugs. The contents in mice serum of TNF-α, IL-6 and IFN-γ were respectively measured by ELISA. The mRNA expressions of TLR3/7, MyD88, JNK, p38MAPK and NF-κB p65 in lung tissues were respectively detected by RT-PCR. The protein expressions of JNK, p38MAPK and NF-κB p65 in lung tissues were determined by immunohistochemical analysis, respectively. According to the results, Yinhua Pinggan granule and San-ao decoction could significantly decrease the levels of TNF-α and IL-6, increase the level of IFN-γ in mice serum of lung tissues, significantly reduce the gene expressions of TLR3/7, MyD88, JNK, p38MAPK and NF-κB p65 in influenza virus-infected mice lung tissues, and significantly reduce the protein expressions of JNK, p38MAPK and NF-κB p65 in lung tissues. Furthermore, the regulatory effect of Yinhua Pinggan granule was superior to that of San-ao decoction. In conclusion, Yinhua Pingan granule and San-ao decoction have the therapeutic effect on pneumonia mice infected by H1N1 virus in vivo. The anti-influenza mechanisms of Yinhua Pinggan granule and San-ao decoction may be the results of interactions by regulating the immunologic function of influenza virus-infected mice and TLR3/7 signaling pathway with multiple links of the gene and protein expressions. Moreover, the combined administration of warm-natured and cold-natured Yinhua Pinggan granule with the effects of detoxification and exhalation has a better effect than the single administration of warm-natured San-ao decoction.

Andrographolide suppresses TRIF-dependent signaling of toll-like receptors by targeting TBK1.

Toll-like receptors (TLRs) play a crucial role in danger recognition and induction of innate immune response against bacterial and viral infections. The TLR adaptor molecule, toll-interleukin-1 receptor domain-containing adapter inducing interferon-ß (TRIF), facilitates TLR3 and TLR4 signaling, leading to the activation of the transcription factor, NF-κB and interferon regulatory factor 3 (IRF3). Andrographolide, the active component of Andrographis paniculata, exerts anti-inflammatory effects; however, the principal molecular mechanisms remain unclear. The objective of this study was to investigate the role of andrographolide in TLR signaling pathways. Andrographolide suppressed NF-κB activation as well as COX-2 expression induced by TLR3 or TLR4 agonists. Andrographolide also suppressed the activation of IRF3 and the expression of interferon inducible protein-10 (IP-10) induced by TLR3 or TLR4 agonists. Andrographolide attenuated ligand-independent activation of IRF3 following overexpression of TRIF, TBK1, or IRF3. Furthermore, andrographolide inhibited TBK1 kinase activity in vitro. These results indicate that andrographolide modulates the TRIF-dependent pathway of TLRs by targeting TBK1 and represents a potential new anti-inflammatory candidate.

(-)-Epigallocatechin-3-gallate enhances poly I:C-induced interferon-λ1 production and inhibits hepatitis C virus replication in hepatocytes.

AIM: To investigate the effect of (-)-epigallocatechin-3-gallate (EGCG) on polyinosinic-polycytidylic acid (poly I:C)-triggered intracellular innate immunity against hepatitis C virus (HCV) in hepatocytes. METHODS: A cell culture model of HCV infection was generated by infecting a hepatoma cell line, Huh7, with HCV JFH-1 strain (JFH-1-Huh7). Poly I:C with a high molecular weight and EGCG were used to stimulate the JFH-1-Huh7 cells. Real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of intracellular mRNAs and of intracellular and extracellular HCV RNA. Enzyme-linked immunosorbent assay was used to evaluate the interferon (IFN)-λ1 protein level in the cell culture supernatant. Immunostaining was used to examine HCV core protein expression in Huh7 cells. RESULTS: Our recent study showed that HCV replication could impair poly I:C-triggered intracellular innate immune responses in hepatocytes. In the current study, we showed that EGCG treatment significantly increased the poly I:C-induced expression of Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and IFN-λ1 in JFH-1-Huh7 cells. In addition, supplementation with EGCG increased the poly I:C-mediated antiviral activity in JFH-1-Huh7 cells at the intracellular and extracellular HCV RNA and protein levels. Further investigation of the mechanisms showed that EGCG treatment significantly enhanced the poly I:C-induced expression of IFN-regulatory factor 9 and several antiviral IFN-stimulated genes, including ISG15, ISG56, myxovirus resistance A, and 2'-5'-oligoadenylate synthetase 1, which encode the key antiviral elements in the IFN signaling pathway. CONCLUSION: Our observations provide experimental evidence that EGCG has the ability to enhance poly I:C-induced intracellular antiviral innate immunity against HCV replication in hepatocytes.

The anti-HSV-1 effect of quercetin is dependent on the suppression of TLR-3 in Raw 264.7 cells.

Quercetin is a major component of the plant Glycyrrhiza uralensis, which is largely used as a traditional medicine in Asia. Quercetin has been reported to have several biological activities, which include anti-viral and anti-inflammatory effects. We explored the molecular mechanism linking anti-viral and anti-inflammatory activities using an in vitro herpes simplex virus-1 (HSV-1) infection model. Raw 264.7 cells were infected with HSV-1 in the presence or absence of different concentrations of quercetin and infected cell lysates were harvested 24 h later. HSV plaque reduction assays, western blotting (HSV-1gD, HSV-1 ICP0, TLR-2, 3, 9, NF-κB, IRF3), and real time PCR (HSV-1ICP0, HSV-1UL13, HSV-1UL52) were performed to elucidate the mechanism responsible for the anti-HSV-1 effect of quercetin. In addition, TNF-α level was measured. Quercetin significantly lowered HSV infectivity in Raw 264.7 cells and inhibited the expressions of HSV proteins (gD, ICP0) and genes (ICP0, UL13, UL52). Interestingly, quercetin specifically suppressed the expression of TLR-3, and this led to the inhibitions of inflammatory transcriptional factors (NF-κB and IRF3). These findings suggest that the anti-HSV-1 effects of quercetin are related to the suppression of TLR-3 dependent inflammatory responses in Raw 264.7 cells.

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.

MDA5 complements TLR3 in suppression of neuroblastoma.

Toll-like receptor3 (TLR3) has been confirmed to be differentially expressed in neuroblastoma (NB), and predicts a favorable prognosis with a high expression in tumor tissues. Treatment with TLR3 agonist--polyinosinic-polycytidylic acid [poly(I:C)] could induce significant but limited apoptosis in TLR3-expressing NB cells, suggesting that other viral RNA sensors, including melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I) in the cytosolic compartment might also be implicated in poly(I:C)-induced NB cell death. MDA5 and RIG-I were induced by poly(I:C) to express in two of six NB cell lines, SK-N-AS (AS) and SK-N-FI, which were associated with up-regulation of caspase9 and active caspase3. While knockdown of either MDA5 or RIG-I alone is ineffective to decrease caspase9 and active caspase3, simultaneously targeting MDA5 and TLR3 showed the best effect to rescue poly(I:C) induced up-regulation of mitochondrial antiviral signaling protein (MAVS), caspase9, active caspase3, and apoptosis in AS cells. Over-expression of MDA5 in FaDu cells resulted in significantly less colony formation and more poly(I:C)-induced cell death. Further studies in human NB tissue samples revealed that MDA5 expression in NB tissues predicted a favorable prognosis synergistically with TLR3. Our findings indicate that MDA5 may serve as a complementary role in the TLR3 activated suppression of NB.

Increased Thymic Cell Turnover under Boron Stress May Bypass TLR3/4 Pathway in African Ostrich.

Previous studies revealed that thymus is a targeted immune organ in malnutrition, and high-boron stress is harmful for immune organs. African ostrich is the living fossil of ancient birds and the food animals in modern life. There is no report about the effect of boron intake on thymus of ostrich. The purpose of present study was to evaluate the effect of excessive boron stress on ostrich thymus and the potential role of TLR3/4 signals in this process. Histological analysis demonstrated that long-term boron stress (640 mg/L for 90 days) did not disrupt ostrich thymic structure during postnatal development. However, the numbers of apoptotic cells showed an increased tendency, and the expression of autophagy and proliferation markers increased significantly in ostrich thymus after boron treatment. Next, we examined the expression of TLR3 and TLR4 with their downstream molecular in thymus under boron stress. Since ostrich genome was not available when we started the research, we first cloned ostrich TLR3 TLR4 cDNA from thymus. Ostrich TLR4 was close to white-throated Tinamou. Whole avian TLR4 codons were under purify selection during evolution, whereas 80 codons were under positive selection. TLR3 and TLR4 were expressed in ostrich thymus and bursa of fabricius as was revealed by quantitative real-time PCR (qRT-PCR). TLR4 expression increased with age but significantly decreased after boron treatment, whereas TLR3 expression showed the similar tendency. Their downstream molecular factors (IRF1, JNK, ERK, p38, IL-6 and IFN) did not change significantly in thymus, except that p100 was significantly increased under boron stress when analyzed by qRT-PCR or western blot. Taken together, these results suggest that ostrich thymus developed resistance against long-term excessive boron stress, possibly by accelerating intrathymic cell death and proliferation, which may bypass the TLR3/4 pathway. In addition, attenuated TLRs activity may explain the reduced inflammatory response to pathogens under boron stress.

Antiviral activity and possible mechanism of action of constituents identified in Paeonia lactiflora root toward human rhinoviruses.

Human rhinoviruses (HRVs) are responsible for more than half of all cases of the common cold and cost billions of USD annually in medical visits and missed school and work. An assessment was made of the antiviral activities and mechanisms of action of paeonol (PA) and 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose (PGG) from Paeonia lactiflora root toward HRV-2 and HRV-4 in MRC5 cells using a tetrazolium method and real-time quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Results were compared with those of a reference control ribavirin. Based on 50% inhibitory concentration values, PGG was 13.4 and 18.0 times more active toward HRV-2 (17.89 µM) and HRV-4 (17.33 µM) in MRC5 cells, respectively, than ribavirin. The constituents had relatively high selective index values (3.3->8.5). The 100 µg/mL PA and 20 µg/mL PGG did not interact with the HRV-4 particles. These constituents inhibited HRV-4 infection only when they were added during the virus inoculation (0 h), the adsorption period of HRVs, but not after 1 h or later. Moreover, the RNA replication levels of HRVs were remarkably reduced in the MRC5 cultures treated with these constituents. These findings suggest that PGG and PA may block or reduce the entry of the viruses into the cells to protect the cells from the virus destruction and abate virus replication, which may play an important role in interfering with expressions of rhinovirus receptors (intercellular adhesion molecule-1 and low-density lipoprotein receptor), inflammatory cytokines (interleukin (IL)-6, IL-8, tumor necrosis factor, interferon beta, and IL-1ß), and Toll-like receptor, which resulted in diminishing symptoms induced by HRV. Global efforts to reduce the level of synthetic drugs justify further studies on P. lactiflora root-derived materials as potential anti-HRV products or lead molecules for the prevention or treatment of HRV.