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.

Toll-like receptor-guided therapeutic intervention of human cancers: molecular and immunological perspectives.

Toll-like receptors (TLRs) serve as the body's first line of defense, recognizing both pathogen-expressed molecules and host-derived molecules released from damaged or dying cells. The wide distribution of different cell types, ranging from epithelial to immune cells, highlights the crucial roles of TLRs in linking innate and adaptive immunity. Upon stimulation, TLRs binding mediates the expression of several adapter proteins and downstream kinases, that lead to the induction of several other signaling molecules such as key pro-inflammatory mediators. Indeed, extraordinary progress in immunobiological research has suggested that TLRs could represent promising targets for the therapeutic intervention of inflammation-associated diseases, autoimmune diseases, microbial infections as well as human cancers. So far, for the prevention and possible treatment of inflammatory diseases, various TLR antagonists/inhibitors have shown to be efficacious at several stages from pre-clinical evaluation to clinical trials. Therefore, the fascinating role of TLRs in modulating the human immune responses at innate as well as adaptive levels directed the scientists to opt for these immune sensor proteins as suitable targets for developing chemotherapeutics and immunotherapeutics against cancer. Hitherto, several TLR-targeting small molecules (e.g., Pam3CSK4, Poly (I:C), Poly (A:U)), chemical compounds, phytocompounds (e.g., Curcumin), peptides, and antibodies have been found to confer protection against several types of cancers. However, administration of inappropriate doses of such TLR-modulating therapeutics or a wrong infusion administration is reported to induce detrimental outcomes. This review summarizes the current findings on the molecular and structural biology of TLRs and gives an overview of the potency and promises of TLR-directed therapeutic strategies against cancers by discussing the findings from established and pipeline discoveries.

Electroacupuncture stimulating Zhongji (CV3), Guanyuan (CV4), and bilateral Dahe (KI12) attenuates inflammation in rats with chronic nonbacterial prostatitis induced by estradiol through inhibiting toll-like receptor 4 pathway.

OBJECTIVE: To investigate the anti-inflammatory effect of electroacupuncture (EA) on chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), also known as chronic nonbacterial prostatitis (CNP), and explore its underlying mechanism. METHODS: A CNP rat was established by surgical castration combined with 17-ß estradiol injection in male Sprague-Dawley rats for thirty consecutive days. The CNP rats received EA treatment once a day for eight days. Chronic pelvic pain was evaluated by mechanical withdrawal threshold measurement. The histological change was assessed by hematoxylin-eosin staining. The inflammatory cytokines in prostates were determined by enzyme-linked immunosorbent assays. The expressions of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), inhibitors of kappa-B alpha (IκBα), and nuclear factor-kappa B (NF-κB) were detected by Western blotting. The nuclear translocation of NF-κB and the location of TLR4 were observed with immunofluorescence staining. RESULTS: The results showed that EA decreased the prostate index, upregulated the mechanical withdrawal threshold, restored the histomorphology of the prostate, reduced the inflammatory factor levels, inhibited NF-κB p65 nuclear translocation, and downregulated the expression levels of critical proteins involved in the TLR4/NF-κB signaling pathway in prostates. CONCLUSIONS: Our findings suggested that EA could relieve pelvic pain and attenuate prostatic inflammation in estradiol-induced CNP rats. The underlying mechanism may be related to the inhibition of the TLR4/NF-κB signaling pathway.

A novel therapeutic strategy for non-muscle invasive bladder cancer: OncoTherad® immunotherapy associated with platelet-rich plasma.

Patients with non-muscle invasive bladder cancer (NMIBC) that are unresponsive to Bacillus Calmette-Guérin (BCG) have historically had limited treatment options. A new perspective is represented by OncoTherad® (MRB-CFI-1) immunotherapy, a nanostructured inorganic phosphate complex associated with glycosidic protein, developed by the University of Campinas in Brazil. Previous studies have shown that Platelet-Rich Plasma (PRP) also acts on immune activation and exerts antitumor effects. This study characterized the effects of the OncoTherad® associated with PRP in the treatment of NMIBC chemically induced in mice. When treated intravesically with PRP only, mice showed 28.6% of tumor progression inhibition rate; with OncoTherad® 85.7%; and with OncoTherad®+PRP 71.4%. Intravesical treatments led to distinct activation of Toll-like Receptors (TLRs) 2 and 4-mediated innate immune system in the interleukins (canonical) and interferons (non-canonical) signaling pathways. OncoTherad® isolated or associated with PRP upregulated TLR4 and its downstream cascade mediators as well as increased interleukins 6 (IL-6) and 1ß (IL-1ß), and interferon-γ (IFN-γ). In this way, the NMIBC microenvironment was modulated to a cytotoxic profile correlated with the IL-1ß increase by stimulating immune pathways for IFN-γ production and consequent cytotoxic T lymphocytes (as CD8+ T-cells) activation and regulatory T-cells (Tregs) reduction. In addition, PRP did not trigger carcinogenic effects through the biomarkers evaluated. Considering the possibility of personalizing the treatment with the PRP use as well as the antitumor properties of OncoTherad®, we highlight this association as a potential new therapeutic strategy for NMIBC, mainly in cases of relapse and/or resistance to BCG.

Intestinal microbiota regulates colonic inflammation in fluorosis mice by TLR/NF-κB pathway through short-chain fatty acids.

Intestinal inflammation and microbial dysbiosis are found simultaneously in patients with fluorosis. However, whether the inflammation derived from fluoride exposure only or intestinal microbial disorders has not been clarified. In this study, 100 mg/L NaF exposure for 90 days significantly elevated the expressions of inflammatory factors (TNF-α, IL-1ß, IL-6, IFN-γ, TGF-ß, and IL-10), and the levels of TLR4, TRAF6, Myd88, IKKß, and NF-κB P65 in mouse colon, while the above factors were reduced in pseudo germ-free mice with fluorosis, hinting that disordered microbiota might play a more direct role in the development of colonic inflammation than fluoride. Fecal microbiota transplantation (FMT) lowered the levels of inflammatory factors and inactivated the TLR/NF-κB pathway in fluoride-exposed mice. In addition, supplementing short-chain fatty acids (SCFAs) exhibited the identical effects to the model of FMT. In summary, intestinal microbiota may alleviate the colonic inflammatory of mice with fluorosis by regulating TLR/NF-κB pathway through SCFAs.

Tea polyphenols, astaxanthin, and melittin can significantly enhance the immune response of juvenile spotted knifejaw (Oplegnathus punctatus).

The frequent occurrence of diseases seriously hampers the sustainable development of the spotted knifejaw (Oplegnathus punctatus) breeding industry. Our previous genome-wide scan and cross-species comparative genomic analysis revealed that the immune gene family (Toll-like receptors, TLR) members of O. punctatus underwent a significant contraction event (tlr1, tlr2, tlr14, tlr5, and tlr23). To address immune genetic contraction may result in reduced immunity, we investigated whether adding different doses (0, 200, 400, 600, and 800 mg/kg) of immune enhancers (tea polyphenols, astaxanthin, and melittin) to the bait after 30 days of continuous feeding could stimulate the immune response of O. punctatus. We found that the expression of tlr1, tlr14, tlr23 genes in immune organs (spleen and head kidney) was stimulated when tea polyphenols were added at 600 mg/kg. The tlr2 (400 mg/kg), tlr14 (200 mg/kg), tlr5 (200 mg/kg), and tlr23 (200 mg/kg) genes expression of intestine were elevated in the tea polyphenol group. When the addition of astaxanthin is 600 mg/kg, it can effectively stimulate the expression of tlr14 gene in immune organs (liver, spleen and head kidney). In the astaxanthin group, the expression of the genes tlr1 (400 mg/kg), tlr14 (600 mg/kg), tlr5 (400 mg/kg) and tlr23 (400 mg/kg) reached their highest expression in the intestine. Besides, the addition of 400 mg/kg of melittin can effectively induce the expression of tlr genes in the liver, spleen and head kidney, except the tlr5 gene. The tlr-related genes expression in the intestine was not significantly elevated in the melittin group. We hypothesize that the immune enhancers could enhance the immunity of O. punctatus by increasing the expression of tlr genes, and thereby leading to increased resistance to diseases. Meanwhile, our findings further demonstrated that significant increases in weight gain rate (WGR), visceral index (VSI), and feed conversion rate (FCR) were observed at 400 mg/kg, 200 mg/kg and 200 mg/kg of tea polyphenols, astaxanthin and melittin in the diet, respectively. Overall, our study provided valuable insights for future immunity enhancement and viral infection prevention in O. punctatus, as well as offered guidance for the healthy development of the O. punctatus breeding industry.

[Mechanism of electroacupuncture penetration needling for relieving synovial inflammation of knee osteoarthritis through TLR4/MyD88/NF-κB signal pathway].

OBJECTIVE: To observe the effects of electroacupuncture (EA) penetration needling on Toll-like receptors 4/myeloid differentiation factor 88/nuclear factor-kappa B (TLR4/MyD88/NF-κB) signaling pathway in rat synovium and the serum-related inflammatory factors, so as to explore the mechanism of EA penetration needling on synovial inflammation in rats with knee osteoarthritis (KOA). METHODS: SD male rats were randomly divided into sham-operation group, model group, EA+penetration needling group, and conventional EA group, with 16 rats in each group. The rats model was prepared by anterior cruciate ligment transection and these rats were forced to exercise for 8 weeks after operation. After successful modeling, in the EA+penetration needling group, the needles were inserted at "Dubi" (ST35) "Neixiyan" (EX-LE4), and at "Xuehai"(SP10) "Liangqiu"(ST34) on the right hind limb, towards each other, 5-8 mm in depth, respectively. In the conventional EA group, the needles were inserted at ST35 and EX-LE4 on the right hind limb, obliquely, at 30° angle to the skin, 3-5 mm in depth; and were inserted at SP10 and ST34 on the right hind limb perpendicularly, 3-5 mm in depth. In these two groups, electric stimulation was operated with dense-disperse wave, 2 Hz/10 Hz in frequency and 0.5-1.5 mA in intensity, retained for 20 min in each treatment. The treatment was given once daily, 10 days as 1 course of treatment, and 2 courses were required at the interval of 2 days. After the intervention, the knee joint effusion was observed by musculoskeletal ultrasound; the contents of IL-1ß, IL-6 and TNF-α in serum were determined by ELISA; the morphological changes in the synovium were observed after H.E. staining; the positive expression of NF-κB p65 in the synovial membrane was detected by immunohistochemical method; the expression levels of TLR4, MyD88, TRAF-6 and NF-κB p65 proteins in the synovial membrane were determined by Western blot. RESULTS: Compared with the sham-operation group, in the model group, the knee joint effusion was obviously increased, the synovial lining cells were distributed irregularly, the cells were disarranged, the pannus was formed largely, and a great number of the inflammatory cells were infiltrated; the contents of serum IL-1ß, IL-6 and TNF-α, the positive expression of NF-κB p65, the protein expression levels of TLR4, MyD88, TRAF-6 and NF-κB p65 in the synovial tissue were increased (P<0.05). Compared with the model group, the knee joint effusion was reduced, the synovial lining cells were proliferated, a small number of the inflammatory cells were infiltrated, and the pannus was formed lightly; the contents of serum IL-1ß, IL-6 and TNF-α, the positive expression of NF-κB p65, the protein expression levels of TLR4, MyD88, TRAF-6 and NF-κB p65 in the synovial tissue were lower (P<0.05) in the EA+penetration needling group and the conventional EA group. In the conventional EA group, the knee joint effusion was increased, the synovial lining cells were proliferated, the inflammatory cells were infiltrated largely, and the pannus was formed increasingly; the contents of serum IL-1ß, IL-6 and TNF-α, and the protein expression levels of TLR4, MyD88 and NF-κB p65 in the synovial tissue were increased when compared with the EA+penetration needling group (P<0.05). CONCLUSION: The EA+penetration needling can significantly relieve the synovial inflammatory reaction and the knee joint effusion in KOA rats. The mechanism is probably related to down-regulating the downstream inflammatory cascade through inhibiting the transduction of TLR4/MyD88/NF-κB signaling pathway.

Mechanisms of interactions in pattern-recognition of common glycostructures across pectin-derived heteropolysaccharides by Toll-like receptor 4.

Complex pectin, originating from terrestrial plant cell walls has been attracting research attention as a promising source of a new innate immune modulator. Numerous bioactive polysaccharides associated with pectin are newly reported every year, but the general mechanism of their immunological action remains unclear owing to the complexity and heterogeneity of pectin. Herein, we systematically investigated the interactions in pattern-recognition for common glycostructures of pectic heteropolysaccharides (HPSs) by Toll-like receptors (TLRs). The compositional similarity of glycosyl residues derived from pectic HPS was confirmed by conducting systematic reviews, leading to molecular modeling of representative pectic segments. Via structural investigation, the inner concavity of leucine-rich repeats of TLR4 was predicted to act as a binding motif for carbohydrate recognition, and subsequent simulations predicted the binding modes and conformations. We experimentally demonstrated that pectic HPS exhibits the non-canonical and multivalent binding aspects for TLR4 resulting in receptor activation. Furthermore, we showed that pectic HPSs were selectively clustered with TLR4 during endocytosis, inducing downstream signals to cause phenotypic activation of macrophages. Overall, we have presented a better explanation for the pattern recognition of pectic HPS and further proposed an approach to understand the interaction between complex carbohydrates and proteins.

TLR 2/1 interaction of pectin depends on its chemical structure and conformation.

Citrus pectins have demonstrated health benefits through direct interaction with Toll-like receptor 2. Methyl-ester distribution patterns over the homogalacturonan were found to contribute to such immunomodulatory activity, therefore molecular interactions with TLR2 were studied. Molecular-docking analysis was performed using four GalA-heptamers, GalA7Me0, GalA7Me1,6, GalA7Me1,7 and GalA7Me2,5. The molecular relations were measured in various possible conformations. Furthermore, commercial citrus pectins were characterized by enzymatic fingerprinting using polygalacturonase and pectin-lyase to determine their methyl-ester distribution patterns. The response of 12 structurally different pectic polymers on TLR2 binding and the molecular docking with four pectic oligomers clearly demonstrated interactions with human-TLR2 in a structure-dependent way, where blocks of (non)methyl-esterified GalA were shown to inhibit TLR2/1 dimerization. Our results may be used to understand the immunomodulatory effects of certain pectins via TLR2. Knowledge of how pectins with certain methyl-ester distribution patterns bind to TLRs may lead to tailored pectins to prevent inflammation.

Nimbolide attenuates complete Freund's adjuvant induced arthritis through expression regulation of toll-like receptors signaling pathway.

Nimbolide is an active constituent of Azadirachta indica and is known for its anti-inflammatory, anti-oxidant, immune-modulatory, and anti-cancer effects. Few studies suggest that nimbolide treatment influences the responses to rheumatoid arthritis, but the underlying molecular mechanisms involved are not yet well established. Therefore, the present study was designed to determine the effect of nimbolide on expression regulation of toll-like receptors to attenuate rheumatoid arthritis. The rheumatoid arthritis model was established by injecting complete Freund's adjuvant (CFA) intra-dermally into the sub-plantar region of the left hind paw of rats. Nimbolide (20 mg/kg) and piroxicam (10 mg/kg) were given to arthritic rats. Rats treated with nimbolide showed a significant reduction in inflammatory cells, rheumatoid factor, ESR, and improved the body weight. The results indicated that nimbolide possesses the capacity to attenuate rheumatoid arthritis by downregulating toll-like receptors, IL-17, IL-23, HSP70, and IFN-γ expression levels. Nimbolide treatment showed significant reduction in the severity of inflammation and destruction of joints and showed comparable effects to piroxicam, which is a standard non-steroidal anti-inflammatory drug used for the treatment of rheumatoid arthritis. It can be concluded that nimbolide can be considered as a potential candidate for therapeutic targeting of the toll-like receptors pathway in rheumatoid arthritis.

Vitamin D status can affect COVID-19 outcomes also in pediatric population.

Background: vitamin D influences the immune system and the inflammatory response. It is known that vitamin D supplementation reduces the risk of acute respiratory tract infection. In the last two years, many researchers have investigated vitamin D's role in the pathophysiology of COVID-19 disease. Results: the findings obtained from clinical trials and systematic reviews highlight that most patients with COVID-19 have decreased vitamin D levels and low levels of vitamin D increase the risk of severe disease. This evidence seems to be also confirmed in the pediatric population. Conclusions: further studies (systematic review and meta-analysis) conducted on children are needed to confirm that vitamin D affects COVID-19 outcomes and to determine the effectiveness of supplementation and the appropriate dose, duration and mode of administration.

Immunomodulatory effects of new phytotherapy on human macrophages and TLR4- and TLR7/8-mediated viral-like inflammation in mice.

Background: While all efforts have been undertaken to propagate the vaccination and develop remedies against SARS-CoV-2, no satisfactory management of this infection is available yet. Moreover, poor availability of any preventive and treatment measures of SARS-CoV-2 in economically disadvantageous communities aggravates the course of the pandemic. Here, we studied a new immunomodulatory phytotherapy (IP), an extract of blackberry, chamomile, garlic, cloves, and elderberry as a potential low-cost solution for these problems given the reported efficacy of herbal medicine during the previous SARS virus outbreak. Methods: The key feature of SARS-CoV-2 infection, excessive inflammation, was studied in in vitro and in vivo assays under the application of the IP. First, changes in tumor-necrosis factor (TNF) and lnteurleukin-1 beta (IL-1ß) concentrations were measured in a culture of human macrophages following the lipopolysaccharide (LPS) challenge and treatment with IP or prednisolone. Second, chronically IP-pre-treated CD-1 mice received an agonist of Toll-like receptors (TLR)-7/8 resiquimod and were examined for lung and spleen expression of pro-inflammatory cytokines and blood formula. Finally, chronically IP-pre-treated mice challenged with LPS injection were studied for "sickness" behavior. Additionally, the IP was analyzed using high-potency-liquid chromatography (HPLC)-high-resolution-mass-spectrometry (HRMS). Results: LPS-induced in vitro release of TNF and IL-1ß was reduced by both treatments. The IP-treated mice displayed blunted over-expression of SAA-2, ACE-2, CXCL1, and CXCL10 and decreased changes in blood formula in response to an injection with resiquimod. The IP-treated mice injected with LPS showed normalized locomotion, anxiety, and exploration behaviors but not abnormal forced swimming. Isoquercitrin, choline, leucine, chlorogenic acid, and other constituents were identified by HPLC-HRMS and likely underlie the IP immunomodulatory effects. Conclusions: Herbal IP-therapy decreases inflammation and, partly, "sickness behavior," suggesting its potency to combat SARS-CoV-2 infection first of all via its preventive effects.

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.

Isolation, Identification, and Immunomodulatory Mechanism of Peptides from Lepidium meyenii (Maca) Protein Hydrolysate.

Maca is a protein-enriched edible plant with immunomodulatory activity. However, the role of proteins in the immunomodulatory activity of maca is unclear. In this study, peptide products of maca proteins obtained through in vitro gastrointestinal digestion were isolated and purified, and the immunomodulatory activities of these peptides were assessed in macrophages (RAW 264.7 cells). The results show that the maca protein hydrolysate enhanced the phagocytic capacity and NO, TNF-α, and IL-6 secretion of RAW 264.7 cells. Forty-five peptides from known proteins of maca or the cruciferous family were identified by ultraperformance liquid chromatography-tandem mass spectrometry in the hydrolysate, and the peptide RNPFLP exhibited the strongest immunomodulatory activity. Antibody blocking, siRNA, pathway inhibitors, and western blot assays showed that RNPFLP-activated RAW 264.7 cells through the NF-κB and MAPK signaling pathways mediated by TLR2 and TLR4 receptors. An analysis of the structure-activity relationship showed that the N9-H60 active site in arginine plays an important role in the immunomodulatory activity of RNPFLP. This study provides a new understanding of the immunomodulatory activity of maca.

Herbal extracts that induce type I interferons through Toll-like receptor 4 signaling.

BACKGROUND: A mixture of five herbal extracts called internatural (INT), which is prepared from pumpkin seeds, purple turmeric, pearl barley, corn pistil, and cinnamon, is widely used by people in Japan and elsewhere for its immunity-enhancing effects and general health. Although anecdotal evidence indicates its efficacy, the mechanisms by which INT boosts immunity have remained unknown. OBJECTIVE: The aim of this study was to investigate whether INT induces type I interferons (IFNs) in murine bone marrow-derived macrophages (BMDMs) and by what mechanism. DESIGN: We measured induction of type I IFNs (IFNß and IFNα) in BMDMs treated with INT or other Toll-like receptor ligands: bacterial lipopolysaccharides (LPS), dsRNA, poly(I:C), and CpG oligonucleotides. To investigate whether INT signals through Toll-like receptor 4 (TLR4), we tested TLR4-specific inhibitor. We also tested if INT utilizes TLR4 adaptors, toll/IL-1 receptor (TIR) domain-containing adaptor (TRIF), or myeloid differentiation factor 88 (MyD88), we examined INT induction of IFNß in TRIF-KO and MyD88-KO BMDMs. We then investigated whether INT provides an antiviral effect upon fibroblasts either directly or indirectly using the encephalomyocarditis virus (EMCV) model. RESULTS: We first observed that INT, when added to BMDMs, potently induces type I IFNs (IFNß and IFNα) within 2 h. INT induction of IFN expression was mediated by TLR4, which signaled through the TRIF/MyD88 adaptors, similar to LPS. A high-molecular-weight fraction (MW > 10,000) of INT extracts contained IFN-inducing activity. Supernatants from INT-treated BMDMs protected untreated fibroblast from EMCV infection as reduced viral titers. CONCLUSIONS: INT induced type I IFN mRNA and proteins in BMDMs and other cell types. This induction was mediated by TLR4, which transduces signals using the TRIF/MyD88 pathway. The high-MW component of INT contained type I IFN inducing activity. The supernatants from INT-treated cells displayed antiviral activity and protected cells from EMCV infection. These findings indicate that INT is a novel natural IFN inducer that strengthens host's innate immunity.

Inhibitory effect of main phenolic acid components of Jacobaea cannabifolia (Less.) on inflammation caused by PM2.5.

PM2.5 is an important environmental problem threatening human health at present, which poses serious harm to human body after inhalation. J. cannabifolia is a traditional Chinese medicine which exhibits anti-inflammatory effect. This study aimed to investigate the inhibitory effect of main phenolic acid components of J. cannabifolia on inflammation caused by PM2.5. Effect of PM2.5 on cell activity and apoptosis were determined by MTT, flow cytometry and calcein AM/PI staining. PHBA, PHPAA, and mixture of PHBA and PHPAA of different concentrations were given to RAW264.7 cells pretreated with PM2.5. The effect of drugs on cellular inflammatory factors was detected by ELISA. The expressions of TLRs related signal pathway at protein and gene levels were detected by western blot and qRT-PCR. The results showed that PM2.5 had no effect on cell activity and apoptosis within the determined concentration range. PHBA and PHPAA could markly inhibit the level of IL-1ß, IL-6, and TNF-α in RAW264.7 cells. Furthermore, the expressions of TLR2, TLR4, MyD88, IRAK1, TRAF6, TAK1, IKKß, and NF-κB induced by PM2.5 were markedly inhibited by PHBA and PHPAA at protein and gene levels. This study demonstrated that PHBA and PHPAA could attenuated inflammation caused by PM2.5 through suppressing TLRs related signal pathway.

Early redox homeostasis disruption contributes to the differential cytotoxicity of imiquimod on transformed and normal endothelial cells.

The off-label use of imiquimod (IQ) for hemangioma treatment has shown clinical benefits. We have previously reported a selective direct IQ-cytotoxic effect on transformed (H5V) vs. normal (1G11) endothelial cells (EC). In the present study, we investigated the mechanism underlying this selective cytotoxicity in terms of TLR7/8 receptor expression, NF-κB signalling and time-dependent modifications of oxidative stress parameters (ROS: reactive oxygen species, catalase and superoxide dismutase activities, GSH/GSSG and lipid peroxidation). TLR7/8 level was extremely low in both cell lines, and IQ did not upregulate TLR7/8 expression or activate NF-κB signalling. IQ significantly induced ROS in H5V after 2 h and strongly affected antioxidant defenses. After 12 h, enzyme activities were restored to baseline levels but a robust drop in GSH/GSSG persisted together with increased lipid peroxidation levels and a marked mitochondrial dysfunction. Although in normal IQ-treated EC some oxidative stress parameters were affected after 4 h, mitochondrial health and GSH/GSSG ratio remained notably unaffected after 12 h. Therefore, the early alterations (0-2 h) in transformed EC breached redox homeostasis as strongly as to enhance their susceptibility to IQ. This interesting facet of IQ as redox disruptor could broaden its therapeutic potential for other skin malignancies, alone or in adjuvant schemes.

Achyranthes bidentata Polysaccharide Activates Nuclear Factor-Kappa B and Promotes Cytokine Production in J774A.1 Cells Through TLR4/MyD88 Signaling Pathway.

Achyranthes bidentata Blume, a traditional Chinese medicine, is widely acknowledged for its function of invigorating the liver and kidneys and as a stranguria-relieving diuretic and used in the treatment of edema, gonorrhea, and other diseases. Polysaccharide (ABPS), isolated from Achyranthes bidentata Blume, has been demonstrated to have multiple biological activities including immunomodulatory effects. However, the mechanisms underlying the effects of ABPS have not been fully investigated. The present study is conducted to explore the underlying mechanism of immunomodulatory activities of ABPS. Results showed that ABPS significantly increased the secretion of IL-1ß and TNF-α in J744 A.1 cells. Nitric oxide (NO) also significantly increased after ABPS treatment. The special antibodies (Toll-like receptor 4 (TLR4) antibody and CD14/TLR4 antibody) significantly decreased the activation, while the Toll-like receptor 2 (TLR2) antibody could not abolish this activation. Meanwhile, pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of NF-κB, remarkably inhibited the secretion of IL-1ß and TNF-α induced by ABPS in J744 A.1 cells. Western blotting (WB) and confocal laser scanning microscopy (CLSM) showed that ABPS promoted NF-κB translocation into the nucleus. Furthermore, the mRNA and protein expression of TLR4 and MyD88 were significantly increased after ABPS treatment. Taken together, these findings suggested that the immunomodulatory mechanism of ABPS was associated with the secretion of cytokines by stimulating the NF-κB pathway through TLR4/MyD88 signaling.

PolyI:C suppresses TGF-ß1-induced Akt phosphorylation and reduces the motility of A549 lung carcinoma cells.

BACKGROUNDS: Epithelial mesenchymal transition (EMT) is a critical process involved in the invasion and metastasis of cancer, including lung cancer (LC). Transforming growth factor (TGF)-ß is one of factors capable of inducing EMT. Polyinosinic-polycytidylic acid (polyI:C), a synthetic agonist for toll-like receptor (TLR) 3, can enhance immune responses and has been used as an adjuvant for cancer vaccines; however, it remains unclear whether it influences other process, such as EMT. In the present study, we examined the effects of polyI:C on TGF-ß-treated A549 human LC cells. METHODS AND RESULTS: By in vitro cell proliferation assay, polyI:C showed no effect on the growth of A549 cells treated with TGF-ß1 at the concentration range up to 10 µg/ml; however, it markedly suppressed the motility in a cell scratch and a cell invasion assay. By Western blotting, polyI:C dramatically decreased TGF-ß1-induced Ak strain transforming (Akt) phosphorylation and increased phosphatase and tensin homologue (PTEN) expression without affecting the Son of mothers against decapentaplegic (Smad) 3 phosphorylation or the expression level of E-cadherin, N-cadherin or Snail, indicating that polyI:C suppressed cell motility independently of the 'cadherin switching'. The Akt inhibitor perifosine inhibited TGF-ß1-induced cell invasion, and the PTEN-specific inhibitor VO-OHpic appeared to reverse the inhibitory effect of polyI:C. CONCLUSION: PolyI:C has a novel function to suppress the motility of LC cells undergoing EMT by targeting the phosphatidylinositol 3-kinase/Akt pathway partly via PTEN and may prevent or reduce the metastasis of LC cells.