TLR4 Signal Transduction Research Articles

Palmitate Stimulates Expression of the von Willebrand Factor and Modulates Toll-like Receptors Level and Activity in Human Umbilical Vein Endothelial Cells (HUVECs).

An increased concentration of palmitate in circulation is one of the most harmful factors in obesity. The von Willebrand factor (vWF), a protein involved in haemostasis, is produced and secreted by the vascular endothelium. An increased level of vWF in obese patients is associated with thrombosis and cardiovascular disease. The aim of this study was to investigate a palmitate effect on vWF in endothelial cells and understand the mechanisms of palmitate-activated signalling. Human umbilical vein endothelial cells (HUVECs) incubated in the presence of palmitate, exhibited an increased VWF gene expression, vWF protein maturation, and stimulated vWF secretion. Cardamonin, a Nuclear Factor kappa B (NF-κB) inhibitor, abolished the palmitate effect on VWF expression. The inhibition of Toll-like receptor (TLR) 2 with C29 resulted in the TLR4 overactivation in palmitate-treated cells. Palmitate, in the presence of TLR4 inhibitor TAK-242, leads to a higher expression of TLR6, CD36, and TIRAP. The silencing of TLR4 resulted in an increase in TLR2 level and vice versa. The obtained results indicate a potential mechanism of obesity-induced thrombotic complication caused by fatty acid activation of NF-κB signalling and vWF upregulation and help to identify various compensatory mechanisms related to TLR4 signal transduction.

Dual Functioned Hexapeptide-Coated Lipid-Core Nanomicelles Suppress Toll-Like Receptor-Mediated Inflammatory Responses through Endotoxin Scavenging and Endosomal pH Modulation.

Excessive activation ofToll-like receptor (TLR) signaling pathways and the circulating endotoxin are key players in the pathogenesis of many acute and chronic inflammatory diseases. Regulation of TLR-mediated inflammatory responses by bioactive nanodevices represents a promising strategy for treating these diseases. In searching for novel, clinically applicable nanodevices with potent TLR inhibitory activities, three types of hexapeptide-modified nano-hybrids with different cores of phospholipid nanomicelles, liposomes, and poly(lactic-co-glycolic acid) nanoparticles are constructed. Interestingly, only the peptide-modified lipid-core nanomicelles (M-P12) display potent TLR inhibitory activities. Further mechanistic studies disclose that lipid-core nanomicelles have a generic property to bind to and scavenge lipophilic TLR ligands including lipopolysaccharide to block the ligand-receptor interaction and down-regulate the TLR signaling extracellularly. In addition, the peptide modification enables M-P12 a unique capability to modulate endosomal acidification upon being endocytosed into macrophages, which subsequently regulates the endosomal TLR signal transduction. In an acute lung injury mouse model, intratracheal administration of M-P12 can effectively target lung macrophages and reduce lung inflammation and injuries. This work defines a dual mechanism of action of the peptide-modified lipid-core nanomicelles in regulating TLR signaling, and provides new strategies for the development of therapeutic nanodevices for treating inflammatory diseases.

Innate immune receptors co-recognition of polysaccharides initiates multi-pathway synergistic immune response

The law and mechanism of the interaction between polysaccharides and pattern recognition receptors (PRRs) has been unclear. Herein, three glucomannans with different structures were selected to explore the universal mechanism for PRRs to recognize glucomannans. Screening results showed that the silence of TLR4 but not TLR2 severely blocked the production of inflammatory cytokines and the transduction of signal pathways. In-depth results revealed that the participation of myeloid differentiation protein 2 (MD2) and CD14 and the dimerization of the TLR4-MD2 complex were required for glucomannan-activated TLR4 signal transduction. Mannose receptor (MR) was also engaged in glucomannan-induced respiratory burst, endocytosis, and inflammatory signaling pathways in a spleen tyrosine kinase-dependent manner. The internalization of glucomannans into the cytoplasm by MR directly initiated complex intracellular signaling cascades. Finally, molecular docking characterized the binding energy and binding sites between glucomannans and multiple receptors from other perspectives. The essence of glucomannans recognized by PRRs was the non-covalent interaction of multiple receptors and the subsequent transmission of the signal cascade was triggered in a multi-channel and cooperative manner. As a result, the hypothesis that “Innate immune receptors co-recognition of polysaccharides initiates multi-pathway synergistic immune response” was proposed to outline these meaningful phenomena.

Dimethyl fumarate protects against intestine damage in necrotizing enterocolitis by inhibiting the Toll-like receptor (TLR) inflammatory signaling pathway

ObjectiveNecrotizing enterocolitis (NEC) is a severe disease in newborns, this study aimed to investigate the protective effect of dimethyl fumarate (DMF) on NEC and its possible mechanism. MethodsIn vivo, the mice were divided into the control, NEC, and NEC+DMF group. The NEC model was established by artificial feeding, hypoxic for 4 days, and lipopolysaccharide (LPS) stimulation on day 2 and day 3. DMF (25 mg/kg/d) was administered to NEC mice on day 1 and day 3. On the 11th day, the blood and intestinal tissues of mice were taken for enzyme-linked immunosorbent assay (ELISA), pathological examination, quantitative real-time PCR (RT-qPCR), Western blot, and immunohistochemical (IHC) detection. In vitro, human colorectal cells (FHC) were induced by LPS (100 ng/mL) and was divided into the control, LPS, and LPS+DMF group. The effect of DMF (20 μM) on cell viability and TLR4 signal transduction was detected by MTT and RT-qPCR, respectively. ResultsCompared to the NEC mice, DMF attenuated NEC-induced weight loss and abdominal distension diarrhea in mice, and alleviated NEC-induced intestinal pathological injuries. In addition, DMF reduced the expression of IL-6, IL-1β, TNF-α, NF-κB, and TLR4 in NEC mice intestinal tissues. Furthermore, DMF inhibited NEC-induced intestinal cell apoptosis as well as the protein expression of BCL2-Associated X (BAX), caspase-3, caspase-9, and increased Bcl-2 (B-cell lymphoma-2) expression. In vitro, DMF improved cell viability, and restrained NF-κB and TLR4 expression in LPS-induced NEC cells. ConclusionDMF has a protective effect against intestine damage of NEC, which is related to the inhibition of the TLR signaling pathway, alleviating the inflammatory response.

Comparison between Lactobacillus rhamnosus GG and LuxS-deficient strain in regulating gut barrier function and inflammation in early-weaned piglets.

Early weaning-induced stress impairs the intestinal barrier function and adversely affects the health of piglet. Probiotics can be used to prevent and treat various intestinal diseases. Lactobacillus rhamnosus GG (LGG) has an LuxS/AI-2 quorum sensing (QS) system that senses environmental changes through chemical signaling molecules. The aim of the study was to explore whether luxS mutant affects the protective role of LGG in the gut barrier of weaned piglets by comparing the luxS mutant (ΔluxS) with its wild-type (WT). Newborn piglets were orally administered with WT and ΔluxS at dosage of 109 CFU, respectively. Accordingly, newborn piglets in the Con group were orally administered with PBS. Piglets were weaned on day 21 and euthanized on day 24, three days following weaning. Supplementation of ΔluxS in advance significantly boosted the relative abundances of healthy microbes (including Catenibacterium, Eubacterium, Lachnospiraceae and Bifidobacterium). WT and ΔluxS maintain intestinal barrier function mainly by promoting intestinal villus to crypt ratio (VCR), occludin protein expression and mucus secretion (P<0.05). Furthermore, LGG reduces pro-inflammatory mediators by inhibiting TLR4 and MAPK signal transduction (P<0.05). Both WT and ΔluxS were shown to resist weaning stress by enhancing the intestinal barrier function of piglets. It has to be said that the ability of ΔluxS to maintain intestinal tissue morphology and promote mucus secretion significantly decreased compared with that of WT.

Identification, functional characterization and expression pattern of myeloid differentiation factor 88 (MyD88) in Nibea albiflora

Myeloid differentiation factor 88 (MyD88), composed of an N-terminal death domain and a C-terminal Toll/interleukin (IL)-IR homology domain, is a key connector protein in the TLR signal transduction pathway. In this study a novel isoform of MyD88 in Nibea albiflora (named as NaMyD88) was identified and functionally characterized (GenBank accession no. MN384261.1). Its complete cDNA sequence was 1672 bp and contained an open reading frame of 879 bp encoding 292 amino acid residues, which was similar to its teleost fish counterparts in the length. The theoretical molecular mass was 33.63 kDa and the isoelectric point was 5.24. BLASTp analysis suggested that the deduced amino acids sequence of NaMyD88 shared high identity to the known MyD88, for instance, 94.77% identity with Collichthys lucidus. Sequence analysis showed that NaMyD88 protein was consistent with MyD88 protein of other species at three conserved domains, N-terminal DD, short middle domain and C-terminal TIR, and the TIR domain contained three highly conserved motifs: Box1, Box2, and Box3. NaMyD88 and red fluorescent protein (Dsred) were fused and expressed in the cytoplasm of the epithelioma papulosum cyprini (EPC cells). The NaTLR9-TIR-EGFP fusion protein, which was obtained in our previous studies, showed green fluorescence and mainly distributed in the cytoplasm. After co-transfection, NaMyD88-Dsred and NaTLR9-TIR-EGFP obviously overlapped and displayed orange-yellow color. The results showed that the homologous MyD88-Dsred could interact with NaTLR9-TIR-EGFP. Based on this result pcMV-NaMyD88-TIR-Myc plasmids and the pcDNA3.1-NaTLR9-TIR-flag were constructed and co-transfected into 293T cells for the immunoprecipitation test. According to Western blot, the protein eluted by Flag-beads could be detected by anti-Flag-tag antibody and anti-Myc tag antibody respectively, while the protein without NaTLR9-TIR could not be found, which further proved that TLR and MyD88 could interact each other. The prokaryotic plasmid of MyD88-TIR domain was constructed, expressed in BL21 (DE3) and purified by Ni-NAT super flow resin conforming to the expected molecular weight of 27 kDa with the corresponding active sites for its conferring protein-protein interaction functions. Real-time fluorescence quantitative PCR showed that NaMyD88 could be expressed in intestine, stomach, liver, kidney, gill, heart and spleen, with the highest in the kidney, and it was up-regulated after being infected with Polyinosinic:polycytidylic acid - Poly (I:C) and Pseudomonas plecoglossicida, which showed that NaMyD88 was involved in the immune response of N.albiflora. These data afforded a basis for understanding the role of NaMyD88 in the TLR signaling pathway of N.albiflora.

More Prominent Inflammatory Response to Pachyman than to Whole-Glucan Particle and Oat-β-Glucans in Dextran Sulfate-Induced Mucositis Mice and Mouse Injection through Proinflammatory Macrophages.

(1→3)-β-D-glucans (BG) (the glucose polymers) are recognized as pathogen motifs, and different forms of BGs are reported to have various effects. Here, different BGs, including Pachyman (BG with very few (1→6)-linkages), whole-glucan particles (BG with many (1→6)-glycosidic bonds), and Oat-BG (BG with (1→4)-linkages), were tested. In comparison with dextran sulfate solution (DSS) alone in mice, DSS with each of these BGs did not alter the weight loss, stool consistency, colon injury (histology and cytokines), endotoxemia, serum BG, and fecal microbiome but Pachyman–DSS-treated mice demonstrated the highest serum cytokine elicitation (TNF-α and IL-6). Likewise, a tail vein injection of Pachyman together with intraperitoneal lipopolysaccharide (LPS) induced the highest levels of these cytokines at 3 h post-injection than LPS alone or LPS with other BGs. With bone marrow-derived macrophages, BG induced only TNF-α (most prominent with Pachyman), while LPS with BG additively increased several cytokines (TNF-α, IL-6, and IL-10); inflammatory genes (iNOS, IL-1β, Syk, and NF-κB); and cell energy alterations (extracellular flux analysis). In conclusion, Pachyman induced the highest LPS proinflammatory synergistic effect on macrophages, followed by WGP, possibly through Syk-associated interactions between the Dectin-1 and TLR-4 signal transduction pathways. Selection of the proper form of BGs for specific clinical conditions might be beneficial.

Neuroprotective Effects of Resveratrol in Ischemic Brain Injury

Cerebral ischemia represents the third cause of death and the first cause of disability in adults. This process results from decreasing cerebral blood flow levels as a result of the occlusion of a major cerebral artery. This restriction in blood supply generates low levels of oxygen and glucose, which leads to a decrease in the energy metabolism of the cell, producing inflammation, and finally, neurological deterioration. Currently, blood restoration of flow is the only effective approach as a therapy in terms of ischemic stroke. However, a significant number of patients still have a poor prognosis, probably owing to the increase in the generation of reactive oxygen species (ROS) during the reperfusion of damaged tissue. Oxidative stress and inflammation can be avoided by modulating mitochondrial function and have been identified as potential targets for the treatment of cerebral ischemia. In recent years, the beneficial actions of flavonoids and polyphenols against cerebrovascular diseases have been extensively investigated. The use of resveratrol (RSV) has been shown to markedly decrease brain damage caused by ischemia in numerous studies. According to in vitro and in vivo experiments, there is growing evidence that RSV is involved in several pathways, including cAMP/AMPK/SIRT1 regulation, JAK/ERK/STAT signaling pathway modulation, TLR4 signal transduction regulation, gut/brain axis modulation, GLUT3 up-regulation inhibition, neuronal autophagy activation, and de novo SUR1 expression inhibition. In this review, we summarize the recent outcomes based on the neuroprotective effect of RSV itself and RSV-loaded nanoparticles in vitro and in vivo models focusing on such mechanisms of action as well as describing the potential therapeutic strategies in which RSV plays an active role in cases of ischemic brain injury.

Chrysin Derivative CM1 and Exhibited Anti-Inflammatory Action by Upregulating Toll-Interacting Protein Expression in Lipopolysaccharide-Stimulated RAW264.7 Macrophage Cells

Although our previous study revealed that gamma-irradiated chrysin enhanced anti-inflammatory activity compared to intact chrysin, it remains unclear whether the chrysin derivative, CM1, produced by gamma irradiation, negatively regulates toll-like receptor (TLR) signaling. In this study, we investigated the molecular basis for the downregulation of TLR4 signal transduction by CM1 in macrophages. We initially determined the appropriate concentration of CM1 and found no cellular toxicity below 2 μg/mL. Upon stimulation with lipopolysaccharide (LPS), CM1 modulated LPS-stimulated inflammatory action by suppressing the release of proinflammatory mediators (cytokines TNF-α and IL-6) and nitric oxide (NO) and downregulated the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. Furthermore, CM1 markedly elevated the expression of the TLR negative regulator toll-interacting protein (Tollip) in dose- and time-dependent manners. LPS-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II), proinflammatory cytokines (TNF-α and IL-6), COX-2, and iNOS-mediated NO were inhibited by CM1; these effects were prevented by the knockdown of Tollip expression. Additionally, CM1 did not affect the downregulation of LPS-induced expression of MAPKs and NF-κB signaling in Tollip-downregulated cells. These findings provide insight into effective therapeutic intervention of inflammatory disease by increasing the understanding of the negative regulation of TLR signaling induced by CM1.

Hepatitis C virus (Flaviviridae: Hepacivirus: Hepacivirus C): regulation of signaling reactions of innate immunity

Studying the regulation of signaling reactions of innate immunity by the hepatitis C virus (HCV) will help to reveal the causes of the transition of the acute form of the disease to a chronic course. The molecular mechanisms of activation by HCV RNA of innate immunity receptors TLR and RLR and signal transduction processes leading to the synthesis of IFN and inflammatory cytokines are considered. The inhibitory effects of non-structural and structural HCV proteins on immune signaling reactions are analyzed in detail. The information presented is the result of an analysis of literature data published in international databases mainly over the past 5 years. In conclusion, signaling receptors are proposed as targets for the development of new antiviral drugs with immunotherapeutic activity.

Schisandrin B improves cerebral ischemia and reduces reperfusion injury in rats through TLR4/NF-κB signaling pathway inhibition

ABSTRACT It has been established that poor outcomes in ischemic stroke patients are associated with the post-reperfusion inflammatory response and up-regulation of TLR4. Therefore, suppression of the TLR4 signaling pathway constitutes a potential neuroprotective therapeutic strategy. Schisandrin B, a compound extracted from Schisandra chinensis, has been shown to possess anti-inflammatory and neuroprotective properties. However, the mechanism remains unclear. In the present study, the therapeutic effect of schisandrin B was assessed following cerebral ischemia and reperfusion (I/R) injury in a model of middle cerebral artery occlusion and reperfusion (MCAO/R) in rats. The effects of schisandrin B were investigated with particular emphasis on TLR4 signal transduction and on the inflammatory response. Schisandrin B treatment conferred significant protection against MCAO/R injury, as evidenced by decreases in infarct volume, neurological score, and the number of apoptotic neurons and inflammatory signaling molecules. Abbreviations I/R: schemia/reperfusion; IL: interleukin; MCAO/R: middle cerebral artery occlusion and reperfusion; NF-κB: nuclear; TLR4: Toll-like receptor 4; TNF-α: tumor necrosis factor-α

Cloning, Expression, and Purification of a Pathogenesis-Related Protein from &lt;i&gt;Oenanthe javanica&lt;/i&gt; and Its Biological Properties

Pathogenesis-related (PR) proteins are inducible and accumulated in plants upon pathogen challenge for survival. Interest in these proteins has arisen in many fields of research, including areas of protein defense mechanisms and plant-derived allergens. In this study, we cloned a PR protein gene (OJPR) from Oenanthe javanica, which consisted of 465 bp with an approximate molecular mass of 16 kDa. The DNA and deduced amino acid sequences of OJPR were 87% similar to Pimpinella brachycarpa PR-1 together with a glycine-rich loop which is a signature motif of PR-10. In microarray analysis, OJPR-transfected Raw264.7 (OJPR+) upregulated high mobility group box 1 and protein kinase Cα, and downregulated chemokine ligand 3 and interleukin 1β which are all related to toll-like receptor 4 (TLR4) and inflammation. TAK-242 and PD98059 inhibited the activation by OJPR, suggesting that OJPR transduce TLR4-mediated signaling. Interestingly, OJPR increased anti-viral repertoires, including interferon (IFN)α, IFNγ, OAS1, and Mx1 in CD4+ primary T cells. Taken together, we concluded that OJPR may play a role in modulating host defense responses via TLR signal transduction and provide new insights into the therapeutic and diagnostic advantages as a potential bioactive protein.

Low Cellular NAD+ Compromises Lipopolysaccharide-Induced Inflammatory Responses via Inhibiting TLR4 Signal Transduction in Human Monocytes.

NAD+ is an essential cofactor in reduction-oxidation metabolism with impact on metabolic and inflammatory diseases. However, data elucidating the effects of NAD+ on the proinflammatory features of human primary monocytes are scarce. In this study, we explored how NAD+ affects TLR4 and NOD-like receptor with a PYD-domain 3 (NLRP3) inflammasome activation, two key innate immune responses. Human primary monocytes were isolated from buffy coats obtained from healthy individuals. Intracellular NAD+ was manipulated by nicotinamide riboside and the NAMPT inhibitor FK866. Cells were primed with LPS with or without subsequent NLRP3 activation with ATP or cholesterol crystals to analyze the effects of NAD+ levels on TLR4-mediated NF-κB activation and NLRP3 activity, respectively. Cytokine release was quantified, and the downstream signal pathway of TLR4 was investigated with Western blot and proteomic analysis. The impact of sirtuin and PARP inhibition was also explored. Our main findings were: 1) elevated NAD+ enhanced IL-1β release in LPS-primed human monocytes exposed to ATP in vitro, 2) both NLRP3-dependent and -independent inflammatory responses in LPS-exposed monocytes were inhibited by NAD+ depletion with FK866, 3) the inhibition was not caused by suppression of sirtuins or PARP1, and 4) phosphorylation of several proteins TLR4 signal pathway was inhibited by FK866-mediated NAD+ depletion, specifically TAK1, IKKβ, IkBα, MEK 1/2, ERK 1/2, and p38. Hence, we suggest a novel mechanism in which NAD+ affects TLR4 signal transduction. Furthermore, our data challenge previous reports of the interaction between NAD+ and inflammation and question the use of nicotinamide riboside in the therapy of inflammatory disorders.

SIRT1-regulated HMGB1 release is partially involved in TLR4 signal transduction: A possible anti-neuroinflammatory mechanism of resveratrol in neonatal hypoxic-ischemic brain injury

Neonatal hypoxic-ischemic brain injury (HIBI) is a knotty disease that lacks appropriate treatment. Inflammation is an important contributor to brain damage, and microglia are responsible for eliciting early and pronounced inflammatory reactions in the immature brain after hypoxic-ischemic (HI) insult. Acetylated HMGB1 can be released from immune cells into the extracellular space, where it acts as a danger-associated molecular pattern molecule to activate TLR4 signalling-mediated inflammatory responses. Resveratrol has neuroprotective and anti-inflammatory effects against HIBI, but whether these effects involve the regulation of the TLR4 signalling pathway and whether HMGB1 participates in this process is still unclear. We investigated the anti-inflammatory effects of resveratrol in HIBI and the molecular mechanisms potentially involved in the effect. The in vivo and in vitro results indicated that the level of cytoplasmic HMGB1 in microglia increased after insult and that treating experimental animals or mouse BV2 microglial cells with resveratrol attenuated HI insult-induced neuroinflammation, which was characterized by improved behavioural defects, reduced microglial activation and TLR4/MyD88/NF-κB signalling, and attenuated primary neuronal damage; this was accompanied by the inhibition of HMGB1 nucleoplasmic transfer and extracellular release. EX527 pretreatment reversed these effects. In addition, co-immunoprecipitation confirmed that SIRT1 was directly involved in the HMGB1 acetylation process in BV2 cells after oxygen glucose deprivation. These data demonstrate that resveratrol plays a neuroprotective role in neonatal HIBI by activating SIRT1 to inhibit HMGB1/TLR4/MyD88/NF-κB signalling and subsequent neuroinflammatory responses.

Citric acid mitigates soybean meal induced inflammatory response and tight junction disruption by altering TLR signal transduction in the intestine of turbot, Scophthalmus maximus L

A 12-week feeding trial was conducted to investigate the effect of citric acid on the involvement of TLRs in the soybean meal induced inflammatory response and tight junction disruption in the distal intestine of juvenile turbot (Scophthalmus maximus L.). Four isonitrogenous and isolipidic practical diets were formulated: fish meal-based diet (FM); 40% fish meal protein in FM replaced with soybean meal protein (SBM); SBM + 1.5% citric acid and SBM + 3% citric acid. Compared to the FM, diet SBM significantly increased the gene expression of TLRs (TLR2, TLR3, TLR5b, TLR9, TLR21, TLR22) and MyD88, as well as TLR related molecules (NF-κB, IRF-3, p38 and JNK), which were remarkably reduced by dietary citric acid. Similarly, citric acid supplementation in SBM markedly depressed gene expression of pro-inflammatory cytokines (TNF-α and IFN-γ) and pore-forming tight junction protein Claudin-7, and enhanced gene expression of the anti-inflammatory cytokine TGF-β1 and TJ proteins related to the decrease in paracellular permeability (Claudin-3, Claudin-4, Occludin, Tricellulin and ZO-1). Compared to the SBM, the concentration of IgM and C4 in serum was significantly reduced by dietary citric acid. In brief, dietary citric acid could synchronously inhibit TLRs-dependent inflammatory response regulated by NF-κB and IRF3, as well as cause TLRs-dependent tight junction disruption modulated by p38 and JNK. Therefore, citric acid could function on mitigating soybean meal induced enteropathy in the distal intestine of juvenile turbot.

PO-01-009 Electric stimulation hyperthermia relieves inflammation through inhibiting TLR4 signal transduction in chronic prostatitis/chronic pelvic pain syndrome

PO-01-009 Electric stimulation hyperthermia relieves inflammation through inhibiting TLR4 signal transduction in chronic prostatitis/chronic pelvic pain syndrome

Molecular mechanism of action responsible for carrageenan-induced inflammatory response

Carrageenan–induced inflammation has long been used as an in vivo model of local inflammation. We developed an in vitro model of inflammation using primary blood cells to characterize gene induction following carrageenan (λ-CGN) stimulation and identify the signal transduction pathway(s) through which λ-CGN worked, using swine whole blood cultures from Yorkshire barrows. Blood samples were divided into stimulated and unstimulated groups. Unstimulated blood was a control for λ-CGN treated cultures to delineate treatment effects from time-in-culture effects. All cultures were collected and separated into two fractions; supernatant for ELISA analyses and white blood cells for mRNA expression. Lambda (λ)-CGN induced MCP-1 at the proteomic and the genomic levels. Lambda-CGN increased IL-8 protein production but had no impact on serum amyloid A protein levels. Alveolar Macrophage-Derived Neutrophil Chemotactic Factor-II (AMCF-II), a swine-specific member of the IL8/GRO family, showed increased gene expression. TNF-α and IL-6 protein levels were not induced by λ-CGN stimulation. Stimulation of HEK-293 cells co-transfected with a single pattern recognition receptor and the secreted embryonic alkaline phosphatase (SEAP) read-out system demonstrated that λ-CGN signals through the TLR-2 and TLR-4 signal transduction pathways. Using silencing RNA to inhibit TLR6 expression in TLR2 transfected HEK-293 cells indicated that λ-CGN works through the TLR2/6 pathway. Silencing TLR6 expression in TLR4 transfected HEK-293 cells showed that λ-CGN stimulation of this cell line worked through a TLR4/6 heterodimer, as lipopolysaccharide (LPS) induced SEAP production through a TLR4 homodimer. These results demonstrate that although carrageenan can stimulate through TLR4 signaling pathways, it initiates an inflammatory response in these cells that differs from a typical endotoxin effect such as LPS stimulation, in terms of the pathways and gene products altered, suggesting that activation of TLR2/6 and TLR4/6 are the predominant pathways through which carrageenan induces inflammatory responses.

Scientific validation of anti-arthritic effect of Kashayams – A polyherbal formulation in collagen induced arthritic rats

BackgroundToll-like receptor-4 (TLR-4) mediates activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) resulting in induction of proinflammatory genes such as that encoding tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) which played a significant role in cartilage destruction of rheumatoid arthritis (RA). Low risk and better efficacy made herbal drugs more reliable than nonsteroid anti-inflammatory drugs (NSAIDS) in RA treatment. Gugguluthiktam Kashayam (GuK), Punarnavadi Kashayam (PuK) and Balaguluchiadi Kashayam (BgK) are ayurvedic polyherbal formulations prescribed in classical ayurvedic texts Sahasrayogam and Ashtangahridayam as medicines for the treatment of RA. ObjectiveThe objective of the present study was to elucidate the molecular mechanism of anti-arthritic effect of these Kashayams on TLR-4 signal transduction pathway in collagen induced arthritic rats. Material and methodsThe wistar rats grouped into group I - Normal, group II- Collagen induced arthritis (CIA), group III- CIA + BgK, group IV– CIA + PuK, group V- CIA + GuK, group VI - CIA + Indomethacin (3 mg/kg b.wt.). Treatment with Kashayam (2 ml/kg b.wt) started after 14 days of primary immunization with type II collagen and continued for a period of 45 days. ResultsArthritis index, C-reactive protein (CRP), rheumatoid factor (RF) and myeloperoxidase (MPO) in serum and protein level of TLR-4, myeloid differentiation factor 88 (MYD88), NF-κB, TNF-α, IL-1β, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 COX-2) and prostaglandin E-2 (PGE-2) in cartilage were significantly elevated in CIA rats. Further, treatment with Kashayams downregulated all these inflammatory mediators hitherto TLR-4-NF-kB signal transduction pathway except IL-10, an anti-inflammatory cytokine which showed a reverse effect. ConclusionThis molecular mechanism of the investigation confirmed the clinical efficacy of Kashayams in preventing the progression of RA and gave an intuition of the scientific validation of Kashayams, an Ayurvedic classical medicine.

Understanding early TLR signaling through the Myddosome.

TLRs are expressed on the plasma and endosomal membranes of innate immune cells acting as sensors of foreign and inherent danger signals that threaten the host. Upon activation, TLRs facilitate the assembly of large intracellular oligomeric signaling complexes, termed Myddosomes, which initiate key signal transduction pathways to elicit critical inflammatory immune responses. The formation of the Myddosome is integral for TLR signaling; however, the molecular mechanisms controlling its formation, disassembly, and the subsequent proximal signaling events remain to be clearly defined. In this review, we present a brief overview of TLR signal transduction pathways, summarize the current understanding of the Myddosome and the proteins that comprise its structure, including MyD88 and members of the IL-1 receptor-associated kinase (IRAK) family. Finally, we will discuss recent advances and open questions regarding early TLR signaling in the context of the Myddosome complex.

The innate immune response to ischemic injury: a multiscale modeling perspective

BackgroundCell death as a result of ischemic injury triggers powerful mechanisms regulated by germline-encoded Pattern Recognition Receptors (PRRs) with shared specificity that recognize invading pathogens and endogenous ligands released from dying cells, and as such are essential to human health. Alternatively, dysregulation of these mechanisms contributes to extreme inflammation, deleterious tissue damage and impaired healing in various diseases. The Toll-like receptors (TLRs) are a prototypical family of PRRs that may be powerful anti-inflammatory targets if agents can be designed that antagonize their harmful effects while preserving host defense functions. This requires an understanding of the complex interactions and consequences of targeting the TLR-mediated pathways as well as technologies to analyze and interpret these, which will then allow the simulation of perturbations targeting specific pathway components, predict potential outcomes and identify safe and effective therapeutic targets.ResultsWe constructed a multiscale mathematical model that spans the tissue and intracellular scales, and captures the consequences of targeting various regulatory components of injury-induced TLR4 signal transduction on potential pro-inflammatory or pro-healing outcomes. We applied known interactions to simulate how inactivation of specific regulatory nodes affects dynamics in the context of injury and to predict phenotypes of potential therapeutic interventions. We propose rules to link model behavior to qualitative estimates of pro-inflammatory signal activation, macrophage infiltration, production of reactive oxygen species and resolution. We tested the validity of the model by assessing its ability to reproduce published data not used in its construction.ConclusionsThese studies will enable us to form a conceptual framework focusing on TLR4-mediated ischemic repair to assess potential molecular targets that can be utilized therapeutically to improve efficacy and safety in treating ischemic/inflammatory injury.