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receptor potential canonical (TRPC) channels are presently an emerging target for airway disorders. Recent evidence has indicated that TRPC6 as a member of the TRPC family plays an important role in airway inflammation, but its precise function in bronchial epithelial cells remains unclear. The aim of this study was to investigate the role of TRPC6 in Toll-like receptor 4 (TLR4)-mediated inflammation in human bronchial epithelial cells stimulated by endotoxin [lipopolysaccharide (LPS)]. Hyp9 is a simplified phloroglucinol derivative of hyperforin that highly selectively activates TRPC6 channels. The results show that the activation of TRPC6 by Hyp9 induced the production of interleukin (IL)-8 and IL-6. LPS was also able to induce the release of IL-8 and IL-6, which was significantly aggravated by Hyp9 and reduced by knockdown of TRPC6. Treatment with LPS not only chronically induced the expression of TRPC6 mRNA and protein in a TLR4-dependent manner but also acutely increased Ca 2+ influx through TRPC6 channels. In addition, LPS-induced overexpression of TRPC6 and Ca 2+ influx were associated with the phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt. Importantly, TRPC6 was required for the activation of ERK1/2, p38, and NF-κB. In conclusion, these data reveal that LPS induced the overexpression of TRPC6 and TRPC6-dependent Ca 2+ influx via the TLR4/PI3K/Akt pathway resulting in Ca 2+ mobilization, which subsequently promoted the activation of ERK1/2, p38, and NF-κB and the inflammatory response in bronchial epithelial cells.

Neonates and infants are more vulnerable to infections and show reduced responses to vaccination. Consequently, repeated immunizations are required to induce protection and early life vaccines against major pathogens such as influenza are yet unavailable. Formulating antigens with potent adjuvants, including immunostimulators and delivery systems, is a demonstrated approach to enhance vaccine efficacy. Yet, adjuvants effective in adults may not meet the specific requirements for activating the early life immune system. Here, we assessed the neonatal adjuvanticity of three novel adjuvants including TLR4 (glucopyranosyl lipid adjuvant-squalene emulsion), TLR9 (IC31 ® ), and Mincle (CAF01) agonists, which all induce germinal centers (GCs) and potent antibody responses to influenza hemagglutinin (HA) in adult mice. In neonates, a single dose of HA formulated into each adjuvant induced T follicular helper (T FH ) cells. However, only HA/CAF01 elicited significantly higher and sustained antibody responses, engaging neonatal B cells to differentiate into GCs already after a single dose. Although antibody titers remained lower than in adults, HA-specific responses induced by a single neonatal dose of HA/CAF01 were sufficient to confer protection against influenza viral challenge. Postulating that the neonatal adjuvanticity of CAF01 may result from the functionality of the C-type lectin receptor (CLR) Mincle in early life we asked whether other C-type lectin agonists would show a similar neonatal adjuvanticity. Replacing the Mincle agonist trehalose 6,6'-dibehenate by Curdlan, which binds to Dectin-1, enhanced antibody responses through the induction of similar levels of T FH , GCs and bone marrow high-affinity plasma cells. Thus, specific requirements of early life B cells may already be met after a single vaccine dose using CLR-activating agonists, identified here as promising B cell immunostimulators for early life vaccines when included into cationic liposomes.

Systems biology approaches have recently provided new insights into the mechanisms of action of human vaccines and adjuvants. Here, we investigated early transcriptional signatures induced in whole blood of healthy subjects following vaccination with a recombinant HIV-1 envelope glycoprotein subunit CN54gp140 adjuvanted with the TLR4 agonist glucopyranosyl lipid adjuvant-aqueous formulation (GLA-AF) and correlated signatures to CN54gp140-specific serum antibody responses. Fourteen healthy volunteers aged 18-45 years were immunized intramuscularly three times at 1-month intervals and whole blood samples were collected at baseline, 6 h, and 1, 3, and 7 days post first immunization. Subtle changes in the transcriptomic profiles were observed following immunization, ranging from over 300 differentially expressed genes (DEGs) at day 1 to nearly 100 DEGs at day 7 following immunization. Functional pathway analysis revealed blood transcription modules (BTMs) related to general cell cycle activation, and innate immune cell activation at early time points, as well as BTMs related to T cells and B cell activation at the later time points post-immunization. Diverse CN54gp140-specific serum antibody responses of the subjects enabled their categorization into high or low responders, at early (<1 month) and late (up to 6 months) time points post vaccination. BTM analyses revealed repression of modules enriched in NK cells, and the mitochondrial electron chain, in individuals with high or sustained antigen-specific antibody responses. However, low responders showed an enhancement of BTMs associated with enrichment in myeloid cells and monocytes as well as integrin cell surface interactions. Flow cytometry analysis of peripheral blood mononuclear cells obtained from the subjects revealed an enhanced frequency of CD56 dim NK cells in the majority of vaccines 14 days after vaccination as compared with the baseline. These results emphasize the utility of a systems biology approach to enhance our understanding on the mechanisms of action of TLR4 adjuvanted human vaccines.

Elderly people are at high risk for influenza-related morbidity and mortality due to progressive immunosenescence. While toll-like receptor (TLR) agonist containing adjuvants, and other adjuvants, have been shown to enhance influenza vaccine-induced protective responses, the mechanisms underlying how these adjuvanted vaccines could benefit the elderly remain elusive. Here, we show that a split H1N1 influenza vaccine (sH1N1) combined with a TLR4 agonist, glucopyranosyl lipid adjuvant formulated in a stable oil-in-water emulsion (GLA-SE), boosts IgG2c:IgG1 ratios, enhances hemagglutination inhibition (HAI) titers, and increases protection in aged mice. We find that all adjuvanted sH1N1 vaccines tested were able to protect both young and aged mice from lethal A/H1N1/California/4/2009 virus challenge after two immunizations compared to vaccine alone. We show that GLA-SE combined with sH1N1, however, also provides enhanced protection from morbidity in aged mice given one immunization (based on change in weight percentage). While the GLA-SE-adjuvanted sH1N1 vaccine promotes the generation of cytokine-producing T helper 1 cells, germinal center B cells, and long-lived bone marrow plasma cells in young mice, these responses were muted in aged mice. Differential in vitro responses, dependent on age, were also observed from mouse-derived bone marrow-derived dendritic cells and lung homogenates following stimulation with adjuvants, including GLA-SE. Besides enhanced HAI titers, additional protective factors elicited with sH1N1 + GLA-SE in young mice were observed, including (a) rapid reduction of viral titers in the lung, (b) prevention of excessive lung inflammation, and (c) homeostatic maintenance of alveolar macrophages (AMs) following H1N1 infection. Collectively, our results provide insight into mechanisms of adjuvant-mediated immune protection in the young and elderly.

Background: Porphyromonas gingivalis lipopolysaccharide (P. gingivalis-LPS) is one of the major pathogenic factors of chronic periodontitis (CP). Few reports on the correlation between P. gingivalis-LPS and cognitive function exist. Thus, the present study aimed to investigate the effects of P. gingivalis-LPS on cognitive function and the associated underlying mechanism in C57BL/6 mice., Methods: The C57BL/6 mice were injected with P. gingivalis-LPS (5 mg kg -1 ) either with or without Toll-like receptor 4 (TLR4) inhibitor (TAK-242, 5 mg kg -1 ). After 7 days, behavioral alterations were assessed with the open field test (OFT), Morris water maze (MWM) test, and passive avoidance test (PAT). The activation of astrocytes and microglia in the cerebral cortex and hippocampus of mice was observed by immunohistochemistry. The expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-8), TLRs (TLR2, TLR3, and TLR4), and CD14 and the activation of the NF-κB signaling pathway (IRAK1, p65, and p-p65) in the cerebral cortex of the mice were evaluated by RT-PCR, ELISA, and western blot., Results: The OFT showed that P. gingivalis-LPS did not affect the initiative and activity of mice. Administration of P. gingivalis-LPS significantly impaired spatial learning and memory during the MWM test and attenuated the ability of passive avoidance learning during the PAT. Both astrocytes and microglia were activated in the cortex and hippocampus. The messenger RNA (mRNA) and protein expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-8) was upregulated by P. gingivalis-LPS in the cortex. In addition, the TLR4/NF-κB signaling pathway was activated (TLR4, CD14, IRAK1, and p-p65). These effects were effectively alleviated by TAK-242., Conclusions: Administration of P. gingivalis-LPS can lead to learning and memory impairment in C57BL/6 mice. This impairment is mediated by activation of the TLR4 signaling pathway. Our study suggests that P. gingivalis-LPS-induced neuroinflammation plays an important role in cognitive impairment. It also reveals that endotoxins of periodontal pathogens could represent a risk factor for cognitive disorders.

Activation or inhibition of TLR4 by small molecules will provide in the next few years a new generation of therapeutics. TLR4 stimulation (agonism) by high-affinity ligands mimicking lipid A gave vaccine adjuvants with improved specificity and efficacy that have been licensed and entered into the market. TLR4 inhibition (antagonism) prevents cytokine production at a very early stage; this is in principle a more efficient method to block inflammatory diseases compared to cytokines neutralization by antibodies. Advances in TLR4 modulation by drug-like small molecules achieved in the last years are reviewed. Recently discovered TLR4 agonists and antagonists of natural and synthetic origin are presented, and their mechanism of action and structure-activity relationship are discussed.

A small but significant proportion of mild traumatic brain injury (mTBI) sufferers will report persistent symptoms, including depression, anxiety and cognitive deficits, in the months, or even years, following the initial event. This is known as post-concussion syndrome and its pathogenesis is not yet known. This study sought to investigate the role of a peripheral inflammatory insult in the development of ongoing behavioral symptoms following a mTBI. To investigate, male Sprague-Dawley rats were administered a single mTBI using the diffuse impact-acceleration model to generate ∼100G of force. Sham animals underwent surgery only. At 5days following surgery, rats were given either the TLR4 agonist, lipopolysaccharide (LPS, 0.1mg/kg), or saline via an intraperitoneal injection. mTBI animals showed an exaggerated response to LPS, with an increase in the expression of pro-inflammatory cytokines within the hippocampus at 24h post-dose, an effect not seen in sham animals. This was associated with the development of persistent behavioral deficits in the mTBI:LPS animals at 3 months post-injury. These behavioral deficits consisted of increased time spent immobile on the forced swim-test, indicative of depressive like behavior, impaired cognitive performance on the Barnes Maze and decreased anxiety on the Elevated Plus Maze. In contrast, animals administered mTBI alone had no deficits. This study provides evidence that a peripheral inflammatory stimulus can facilitate ongoing symptoms following a mTBI. As such this provides a basis for further exploration of exogenous factors which promote immune system activation as potential targets for intervention to allow the resolution of symptoms following a mTBI., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

In order to identify novel lead structures for human toll-like receptor 4 ( h TLR4) modulation virtual high throughput screening by a peta-flops-scale supercomputer has been performed. Based on the in silico studies, a series of 12 compounds related to tryptamine was rationally designed to retain suitable molecular geometry for interaction with the h TLR4 binding site as well as to satisfy general principles of drug-likeness. The proposed compounds were synthesized, and tested by in vitro and ex vivo experiments, which revealed that several of them are capable to stimulate h TLR4 in vitro up to 25% activity of Monophosphoryl lipid A. The specific affinity of the in vitro most potent substance was confirmed by surface plasmon resonance direct-binding experiments. Moreover, two compounds from the series show also significant ability to elicit production of interleukin 6., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Proximal tubule protein take-up is interceded by 2 receptors, megalin and cubilin. These receptors rescue an assortment of filtered ligands including fundamental vitamins and hormones. The objective of this study was to investigate the potential relation of megalin receptor injury with nuclear factor-kappa B (NF-κB) upregulation in acute kidney injury rat model. Twenty four rats were allocated into two groups: control group received saline, while the second group was intoxicated with cadmium chloride (2.4 mg Cd/kg/day i.p) for 30 days. Blood urea nitrogen, serum creatinine, tissue oxidant-antioxidant parameters (malondialdehyde [MDA] and reduced glutathione [GSH]) and expression levels for NF-κB, toll like receptor-2 (TLR2), toll like receptor-4 (TLR4), and megalin receptor were estimated. Noticeable downregulation of megalin receptor versus upregulation of NF-κB, TLR2, and TLR4 were observed in AKI rat model together with significant elevation in MDA as well as significant reduction in GSH. The study concluded that the oxidative stress in kidney tissue leads to megalin receptor damage, which indeed motivates upregulation of NF-κB through TLRs 2 and 4 pathways., (© 2017 Wiley Periodicals, Inc.)

Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by loss of dopaminergic neurons associated with neuroinflammation. Toll-like receptors (TLRs) are expressed in peripheral blood leukocytes and also in neurons and glial cells mediating inflammation. This study aimed to investigate the peripheral blood leukocyte response to TLR2 and TLR4 agonists in young and elderly PD patients. Two groups of patients with PD were evaluated (≤ 55 years old and ≥ 65 years old), age-matched with healthy controls (n = 26). Severity of PD was evaluated by Unified Parkinson's Disease Rating Scale (UPDRS). Whole blood cultures were stimulated with lipopolysaccharide (LPS), a TLR4 agonist or Pam 3 Cys (Pam), a TLR2 agonist. Tumor necrosis factor alpha (TNFα) and interleukin 10 (IL-10) were measured by immunoenzimatic assay. 6 h-TNFα production was increased after TLR4 stimulation, mainly in young PD patients, whereas TLR2-induced TNFα and IL-10 levels were decreased in PD patients independent of age (p < 0.05). A reverse correlation between LPS-induced TNFα production and age was observed in PD patients and controls, but TNFα induced by TLR2 agonist was not associated with age of PD patients or controls. TNFα production induced by TLR4 but not by TLR2 was reversely associated with the age at PD onset and disease duration. No associations between UPDRS scores and cytokine levels were detected. In conclusion, TLR4 and TLR2 responses seem to be differentially affected during PD. Data suggest that TLR2 deficiency in periphery is independent of age of the patients, age at PD onset, or PD duration.

Background: Most current prophylactic vaccines confer protection primarily through humoral immunity. Indeed, aluminum salts which have been widely used as adjuvants in vaccines primarily enhance Th2-driven antibody responses. Therefore, new vaccines formulation is moving toward a careful selection of adjuvants that also elicit significant Th1 or Tc1 responses. Several TLR agonists have been tested as potential new adjuvants in clinical and preclinical studies with some efficacy. These studies suggest that combining more than one of TLR ligands enhances the magnitude of immune responses to cancer and infectious disease., Objectives: In order to evaluate the synergistic effect of TLR agonists for effective induction of cellular immunity, we investigated the effects of single and/or combined TLR agonists on monocyte-derived DC maturation, DC-NK crosstalk and ultimately naïve T cells polarization into effector T cells., Results: Among the adjuvants tested, we found that TLR3, TLR4, TLR7/8 and TLR8 agonists were the most effective adjuvants to increase the expression levels of antigen-presenting, co-stimulatory molecules and production of cytokines by maturing DCs. When combined, TLR3+8 and TLR4+8 synergistically optimized DC maturation and IFN-γ secretion from NK cells co-cultured with DCs. Interestingly, co-culture of DC-NK-T treated with aluminum salt produced the highest percentage of effector memory CFSE-CCR7- Th1 cells whereas TLR3+8 and TLR4+8 treated co-cultures produced the highest percentage of effector memory CFSE-CCR7- Tc1 cells producing IFN-γ. Finally, while both TLR3+8 or TLR4+8 treated co-cultures generated similar frequency of Th1 and Tc1 effector cells, the effector cells from the latter co-culture produced quantitatively more IFN-γ in the supernatant., Conclusion: Our data indicate that if in need of an enhanced DC-NK mediated cellular immunity one may select TLR agonists with defined synergistic effects., (Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Iron is essential for basic cellular functions but in excess is highly toxic. For this reason, free iron and iron storage are controlled in the periphery by elaborate regulatory mechanisms. In contrast, iron regulation in the central nervous system (CNS) is not well defined. Given that excess iron is present after trauma, hemorrhagic stroke and neurodegeneration, understanding normal iron regulation and promoting iron uptake in CNS pathology is crucial. Peripherally, toll-like receptor 4 (TLR4) activation promotes iron sequestration by macrophages. Notably, iron-rich sites of CNS pathology typically contain TLR4 agonists, which may promote iron uptake. Indeed, our recent work showed impaired iron storage after acute spinal cord injury in mice with TLR4 deficiency. Here we used a reductionist model to ask if TLR4 activation in the CNS stimulates iron uptake and promotes neuroprotection from iron-induced toxicity. For this, we measured the ability of microglia/macrophages to sequester exogenous iron and prevent pathology with and without concomitant intraspinal TLR4 activation. Results show that, similar to the periphery, activating intraspinal TLR4 via focal LPS injection increased mRNA encoding iron uptake and storage proteins and promoted iron sequestration into ferritin-expressing macrophages. However, this did not prevent oligodendrocyte and neuron loss. Moreover, replacement of oligodendrocytes by progenitor cells - a normally robust response to in vivo macrophage TLR4 activation - was significantly reduced if iron was present concomitant with TLR4 activation. Thus, while TLR4 signaling promotes CNS iron uptake, future work needs to determine ways to enhance iron removal without blocking the reparative effects of innate immune receptor signaling., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Graft rejection due to immune incompatibility is a common occurrence in pearl culture, which often cause death to the host oyster. To improve cultured pearl production, host mortality and bead rejection rates must be reduced. Toll-like receptor 4 (TLR4) plays an important role in innate immunity, and may be related to allograft rejection. Here, we cloned the TLR4 cDNA from the pearl oyster Pinctada fucata martensii (PmTLR4). PmTLR4 cDNA was 3138bp, including a 2625bp open reading frame encoding 874 amino acids. The predicted PmTLR4 protein was structurally typical of the TLR family. PmTLR4 had relatively high sequence similarity and identity to the TLR4 of the Cyclina sinensis (48.1% and 27.6%, respectively). Multiple alignment of TLR4 sequences across species indicated that the Toll/interleukin-1 (IL-1) receptor domain was conserved among species. PmTLR4 mRNA was expressed in all tissues tested, with the most abundant mRNA expression in hepatopancreas and gill in P. fucata martensii. After being stressed by either lipopolysaccharide (LPS) exposure or the nucleus insertion operation, PmTLR4 mRNA expression increased significantly in the hemocytes as compared to controls. Peak level of PmTLR4 mRNA was observed 6h after the LPS injection, and 2d after the nucleus insertion operation. These data suggest that PmTLR4 may play a vital role in the induction of innate immunity and is therefore associated with allograft immunity in the pearl oyster P. fucata martensii., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Adjuvants are indispensable for achieving a sufficient immune response from vaccinations. From a functional viewpoint, adjuvants are classified into two categories: "physical adjuvants" increase the efficacy of antigen presentation by antigen-presenting cells (APC) and "signal adjuvants" induce the maturation of APC. Our previous study has demonstrated that a physical adjuvant can be encrypted into proteinous antigens by creating artificial proteins from combinatorial assemblages of epitope peptides and those peptide sequences having propensities to form certain protein structures (motif programming). However, the artificial antigens still require a signal adjuvant to maturate the APC; for example, co-administration of the Toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) was required to induce an in vivo immunoreaction. In this study, we further modified the previous artificial antigens by appending the peptide motifs, which have been reported to have agonistic activity for TLR4, to create "adjuvant-free" antigens. The created antigens with triple TLR4 agonistic motifs in their C-terminus have activated NF-κB signaling pathways through TLR4. These proteins also induced the production of the inflammatory cytokine TNF-α, and the expression of the co-stimulatory molecule CD40 in APC, supporting the maturation of APC in vitro. Unexpectedly, these signal adjuvant-encrypted proteins have lost their ability to be physical adjuvants because they did not induce cytotoxic T lymphocytes (CTL) in vivo, while the parental proteins induced CTL. These results confirmed that the manifestation of a motif's function is context-dependent and simple addition does not always work for motif-programing. Further optimization of the molecular context of the TLR4 agonistic motifs in antigens should be required to create adjuvant-free antigens.

There is no effective vaccine against human leishmaniasis. Achieving successful vaccines seems to need powerful adjuvants. Separate or combined use of toll like receptor (TLR) agonists as adjuvant is a promising approach in Leishmania vaccine research. In present study, we evaluated adjuvant effect of separate or combined use of a TLR7/8 agonist, R848 and a TLR4 agonist, monophosphoryl lipid A (MPL) beside soluble Leishmania antigen (SLA) in BALB/c mice. Mice were vaccinated three times by SLA with separate or combined TLR7/8 and TLR4 agonists and were then challenged by Leishmania major. Delay type hypersensitivity, lesion development, parasite load, and cytokines (interferon gamma, and interleukin-10) response were assessed. Results showed: 1) MPL can slightly assist SLA in parasite load reduction, but it is not able to increase SLA ability in evoking DTH and cytokine responses or decreasing lesion diameter. 2) R848 does not affect the DTH response and parasite load of mice vaccinated with SLA, but it decreases/inhibits cytokine responses induced by SLA, leading to increase lesion diameter. 3) MPL neutralized inhibitory effect of R848. In overall, these data emphasize that MPL slightly assists SLA to make a more potent vaccine, but R848 is not a good adjuvant to induce T cell-dependent immune response in BALB/c mice, and therefore combination of these TLR agonists in the current formulation, is not recommended for making a more powerful adjuvant., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Dendritic cells (DCs) are specialized antigen‑presenting cells which are important in immune diseases, in particular atherosclerosis, a chronic inflammatory disease, however their role in atherosclerosis‑associated immunity is unclear. To evaluate the role of DCs in atherosclerosis, exogenous bone marrow‑derived DCs were transferred into ApoE‑/‑ mice in the present study. The extent of disease was measured in the aorta and was compared with mice treated with phosphate‑buffered saline (PBS) or left untreated and fed a western diet. Mice receiving exogenous DCs demonstrated significantly larger atherosclerotic lesions compared with the mice treated with PBS, with increasing numbers of mature DCs in circulation and enhanced DC infiltration into plaque lesions, in addition to activation of circulating inflammatory components and atherosclerotic lesions. Furthermore, it was demonstrated that exogenous DCs upregulated the expression of Toll‑like receptor 4 (TLR4) on DCs, which may be an important mechanism to activate DCs and aggravate atherosclerosis. Therefore the present study concluded that exogenous DCs may induce maturation of endogenous DCs via upregulation of TLR4, further increasing the inflammatory response and accelerating atherosclerosis.

Cathelicidin are innate antimicrobial peptides with broad immunomodulatory functions; however, their role in regulating intestinal defenses is not well characterized. This study aimed to investigate the role of cathelicidin modulating expression of Toll-like receptors (TLRs) 4 and 9 in colonic epithelium in response to bacterial patterns. We demonstrated herein that intestinal epithelial cells, when primed by bacterial lipopolysaccharide (LPS), responded to cathelicidin by increased transcription and protein synthesis of TLR4. This cathelicidin-induced response required the interaction of LPS-TLR4 and activation of MAPK signalling pathways. However, cathelicidin blocked TLR9 responses induced by TLR9 ligand CpG oligodeoxynucleotide (CpG ODN) in these colonic epithelial cells. Modulations of TLRs triggered by cathelicidin in intestinal epithelium occurred mainly in the apical compartment of intestinal cells. Activation of TLR4 by ligands in combination with cathelicidin promoted CXCL8 chemokine secretion and epithelial antimicrobial defenses against Escherichia coli. We concluded that cathelicidin selectively modulated synthesis of TLR4 and 9 in intestinal epithelium, but only when cells were exposed to virulence factors, mostly from apical surfaces. Enhanced TLR4 expression promoted by cathelicidin in intestinal epithelium may be crucial for controlling enteric infectious diseases., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Bacterial lipopolysaccharide (LPS) is a known ligand of Toll-like receptor 4 (TLR4) which is expressed in cardiac fibroblasts (CF). Differentiation of CF to cardiac myofibroblasts (CMF) is induced by transforming growth factor-β1 (TGF-β1), increasing alpha-smooth muscle actin (α-SMA) expression. In endothelial cells, an antagonist effect between LPS-induced signaling and canonical TGF-β1 signaling was described; however, it has not been studied whether in CF and CMF the expression of α-SMA induced by TGF-β1 is antagonized by LPS and the mechanism involved. In adult rat CF and CMF, α-SMA, ERK1/2, Akt, NF-κβ, Smad3, and Smad7 protein levels were determined by western blot, TGF-β isoforms by ELISA, and α-SMA stress fibers by immunocytochemistry. CF and CMF secrete the three TGF-β isoforms, and the secretion levels of TGF-β2 was affected by LPS treatment. In CF, LPS treatment decreased the protein levels of α-SMA, and this effect was prevented by TAK-242 (TLR4 inhibitor) and LY294002 (Akt inhibitor), but not by BAY 11-7082 (NF-κβ inhibitor) and PD98059 (ERK1/2 inhibitor). TGF-β1 increased α-SMA protein levels in CF, and LPS prevented partially this effect. In addition, in CMF α-SMA protein levels were decreased by LPS treatment, which was abolished by TAK-242. Finally, in CF LPS decreased the p-Smad3 phosphorylation and increased the Smad7 protein levels. LPS treatment prevents the CF-to-CMF differentiation and reverses the CMF phenotype induced by TGF-β1, through decreasing p-Smad3 and increasing Smad7 protein levels.

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) complex is essential for LPS recognition and induces innate immune responses against Gram-negative bacteria. As activation of TLR4/MD-2 is also critical for the induction of adaptive immune responses, TLR4/MD-2 agonists have been developed as vaccine adjuvants, but their efficacy has not yet been ascertained. Here, we demonstrate that a funiculosin (FNC) variant, FNC-RED, and FNC-RED and FNC derivatives are agonists for both murine and human TLR4/MD-2. FNC-RED induced nuclear factor-κB (NF-κB) activation via murine TLR4/MD-2, whereas FNC had no TLR4/MD-2 stimulatory activity. Biacore analysis revealed that FNC-RED binds to murine TLR4/MD-2 but not murine radioprotective 105 (RP105)/myeloid differentiation factor-1 (MD-1), another LPS sensor. FNC-RED induced CD14-independent expressions of pro-inflammatory cytokines and co-stimulatory molecules in murine macrophages and dendritic cells. In contrast, FNC-RED stimulation was reduced in CD14-dependent LPS responses, including dimerization and internalization of TLR4/MD-2 and IFN-β expression. FNC-RED-induced IL-12p40 production from murine dendritic cells was dependent on NF-κB but not MAPK pathway. In addition, fetal bovine serum augmented lipid A-induced NF-κB activation but blocked FNC-RED-mediated responses. Two synthetic phosphate group-containing FNC-RED and FNC derivatives, FNC-RED-P01 and FNC-P01, respectively, activated human TLR4/MD-2, unlike FNC-RED. Finally, computational analysis revealed that this species-specific activation by FNC-RED and FNC-RED-P01 resulted from differences in electrostatic surface potentials between murine and human TLR4/MD-2. We conclude that FNC-RED and its synthetic derivative represent a novel category of murine and human TLR4/MD-2 agonist., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Glioblastoma is among the most aggressive brain tumors and has an exceedingly poor prognosis. Recently, the importance of the tumor microenvironment in glioblastoma cell growth and progression has been emphasized. Toll-like receptor 4 (TLR4) recognizes bacterial lipopolysaccharide (LPS) and endogenous ligands originating from dying cells or the extracellular matrix involved in host defense and in inflammation. G-protein coupled receptors (GPCRs) have gained interest in anti-tumor drug discovery due to the role that they directly or indirectly play by transactivating other receptors, causing cell migration and proliferation. A proteomic analysis showed that the nociceptin receptor (NOPr) is among the GPCRs significantly expressed in glioblastoma cells, including U87 cells. We describe a novel role of the peptide nociceptin (N/OFQ), the endogenous ligand of the NOPr that counteracts cell migration, proliferation and increase in IL-1β mRNA elicited by LPS via TLR4 in U87 glioblastoma cells. Signaling pathways through which N/OFQ inhibits LPS-mediated cell migration and elevation of [Ca 2+ ] i require β-arrestin 2 and are sensitive to TNFR-associated factor 6, c-Src and protein kinase C (PKC). LPS-induced cell proliferation and increase in IL-1β mRNA are counteracted by N/OFQ via β-arrestin 2, PKC and extracellular signal-regulated kinase 1/2; furthermore, the contributions of the transcription factors NF-kB and AP-1 were investigated. Independent of LPS, N/OFQ induces a significant increase in cell apoptosis. Contrary to what was observed in other cell models, a prolonged exposure to this endotoxin did not promote any tolerance of the cellular effects above described, including NOPr down-regulation while N/OFQ loses its inhibitory role., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS), produces pro-inflammatory cytokines and type I interferons, and associates with a trigger of endotoxin shock. TLR4 is interacted with a TIR domain-containing adaptor molecule-2 (TICAM-2)/TRAM [TRIF (TIR domain-containing adaptor-inducing interferon-β)-related adaptor molecule] via its Toll-interleukin-1 receptor homology (TIR) domain. TICAM-2 acts as a scaffold protein and activates TIR domain-containing adaptor molecule-1 (TICAM-1)/TRIF. According to the structural analysis by NMR, TICAM-2 interacts with TICAM-1 by the acidic amino acids motif, E87/D88/D89. The TIR domain of TICAM-2 couples with the dimer of TIR domain of TLR4 beneath the membrane, and TICAM-2 itself also forms dimer and constitutes a binding site with TICAM-1. Endosomal localization of TICAM-2 is essential for TLR4-mediated type I interferon-inducing signal from the endosome. N-terminal myristoylation allows TICAM-2 to anchor to the endosomal membrane. Additionally, we have identified two acidic amino acids, D91/E92, as a functional motif that cooperatively determines endosomal localization of TICAM-2. This structural information of TICAM-2 suggests that the specific structure is indispensable for the endosomal localization and type I interferon production of TICAM-2. Taken together with the knowledge on cytoplasmic sensors for LPS, TICAM-2/TICAM-1 may conform to a signal network on TLR4 to facilitate induction of cytokine disorders., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Age-related alterations in immunity have been linked to increased incidence of infections and decreased responses to vaccines in the aging population. Human peripheral blood monocytes are known to promote Ag presentation and antiviral activities; however, the impact of aging on monocyte functions remains an open question. We present an in-depth global analysis examining the impact of aging on classical (CD14 + CD16 - ), intermediate (CD14 + CD16 + ), and nonclassical (CD14 dim CD16 + ) monocytes. Monocytes sorted from nonfrail healthy adults (21-40 y) and old (≥65 y) individuals were analyzed after stimulation with TLR4, TLR7/8, and retinoic acid-inducible gene I agonists. Our data showed that under nonstimulated conditions, monocyte subsets did not reveal significant age-related alternations; however, agonist stimulated-monocytes from adults and old subjects did show differences at the transcriptional and functional levels. These alternations in many immune-related transcripts and biological processes resulted in reduced production of IFN-α, IFN-γ, IL-1β, CCL20, and CCL8, and higher expression of CX3CR1 in monocytes from old subjects. Our findings represent a comprehensive analysis of the influence of human aging on pattern recognition receptors signaling and monocyte functions, and have implications for strategies to enhance the immune response in the context of infection and immunization., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Most vaccine adjuvants directly stimulate and activate antigen presenting cells but do not sustain immunostimulation of these cells. A high throughput screening (HTS) strategy was designed to identify compounds that would sustain NF-κB activation by a stimulus from the Toll-like receptor (TLR)4 ligand, lipopolysaccharide (LPS). Several pilot studies optimized the parameters and conditions for a cell based NF-κB reporter assay in human monocytic THP-1 cells. The final assay evaluated prolongation of LPS induced NF-κB activation at 12 h. The dynamic range of the assay was confirmed in a pilot screen of 14 631 compounds and subsequently in a main extensive screen with 166 304 compounds. Hit compounds were identified using an enrichment strategy based on unsupervised chemoinformatic clustering, and also by a naı̈ve "Top X" approach. A total of 2011 compounds were then rescreened for levels of coactivation with LPS at 5 h and 12 h, which provided kinetic profiles. Of the 407 confirmed hits, compounds that showed correlation of the kinetic profiles with the structural similarities led to identification of four chemotypes: pyrimido[5,4-b]indoles, 4H-chromene-3-carbonitriles, benzo[d][1,3]dioxol-2-ylureas, and tetrahydrothieno[2,3-c]pyridines, which were segregated by 5 h and 12 h kinetic characteristics. Unlike the TLR4 agonistic pyrimidoindoles identified in previous studies, the revealed pyrimidoindoles in the present work did not intrinsically stimulate TLR4 nor induce NF-κB but rather prolonged NF-κB signaling induced by LPS. A 42-member combinatorial library was synthesized which led to identification of potent N3-alkyl substituted pyrimidoindoles that were not only active in vitro but also enhanced antibody responses in vivo when used as a coadjuvant. The novel HTS strategy led to identification of compounds that are intrinsically quiescent but functionally prolong stimulation by a TLR4 ligand and thereby potentiate vaccine efficacy.

Development of non-infectious subunit vaccines is hampered by a slow pipeline of new adjuvants to replace or enhance alum in part because expectations of safety are high. Transient vaccine side effects are not clinical priorities because they cause no lasting harm and vaccine development has appropriately been focused on avoidance of serious adverse events. As a result, surprisingly little is known about the extent to which side effects caused by a vaccine's reactogencicity are predictive of successful immunization outcomes. Recent clinical studies of pertussis and human papillomavirus vaccines adjuvanted with alum or the TLR4 agonist monophosphoryl lipid A can be used to advance understanding of the relationship between vaccine side effects and immunization outcomes., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Signaling pathways induced by the activation of renal toll-like receptor 4 (TLR4) play a pivotal role in chronic kidney disease (CKD). Some recent studies suggested that clarithromycin (CAM), a 14-membered ring macrolide, exerts renoprotective effects by suppressing proinflammatory chemokines. However, its beneficial effects on signaling pathways through renal TLR4 activation are unknown., Methods: Cultured human mesangial cells (MCs) were treated with lipopolysaccharide (LPS). Expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and interleukin-8 (IL-8/CXCL8) was analyzed by quantitative RT-PCR and enzyme-linked immunosorbent assay. Signaling pathways affected by CAM were determined by examining the activation of nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) by performing western blotting., Results: CAM inhibited both the mRNA and protein expression of MCP-1 without cell injury but did not affect those expressions of IL-8 in LPS-stimulated MCs. Interestingly, CAM decreased p38 MAPK activation by inhibiting phosphorylation but did not affect NF-κB activation., Conclusion: Our results indicated that CAM exerted renoprotective effects by suppression of p38 MAPK activity and by decreasing the expression of MCP-1 in LPS-stimulated MCs. Given the implication of TLR4 signaling in CKD, CAM may be a potential treatment of choice for CKD.

A history of repeated concussion has been linked to the later development of neurodegeneration, which is associated with the accumulation of hyperphosphorylated tau and the development of behavioral deficits. However, the role that exogenous factors, such as immune activation, may play in the development of neurodegeneration following repeated mild traumatic brain injury (rmTBI) has not yet been explored. To investigate, male Sprague-Dawley rats were administered three mTBIs 5days apart using the diffuse impact-acceleration model to generate ∼100G. Sham animals underwent surgery only. At 1 or 5days following the last injury rats were given the TLR4 agonist, lipopolysaccharide (LPS, 0.1mg/kg), or saline. TLR4 activation had differential effects following rmTBI depending on the timing of activation. When given at 1day post-injury, LPS acutely activated microglia, but decreased production of pro-inflammatory cytokines like IL-6. This was associated with a reduction in neuronal injury, both acutely, with a restoration of levels of myelin basic protein (MBP), and chronically, preventing a loss of both MBP and PSD-95. Furthermore, these animals did not develop behavioral deficits with no changes in locomotion, anxiety, depressive-like behavior or cognition at 3months post-injury. Conversely, when LPS was given at 5days post-injury, it was associated acutely with an increase in pro-inflammatory cytokine production, with an exacerbation of neuronal damage and increased levels of aggregated and phosphorylated tau. At 3months post-injury, there was a slight exacerbation of functional deficits, particularly in cognition and depressive-like behavior. This highlights the complexity of the immune response following rmTBI and the need to understand how a history of rmTBI interacts with environmental factors to influence the potential to develop later neurodegeneration., (Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.)

The biophysical parameters governing nanoparticle (NP)-cell interactions significantly affect biological responses, particularly in the application of NP-based immunotherapeutics. Modulation of the surface biophysical character of NPs can be achieved via introduction of amino acids, which offer the ability to fine tune a range of biophysical parameters of interest. We employed this approach using monodisperse silica NPs coated with numerous poly(amino acid)s (PAAs). The NPs were incubated with dendritic cells (DCs) in conjunction with TLR ligands and production of IL-1β from DCs and IFNγ from T cells primed by these DCs were measured. These key cytokines can prognosticate the efficacy of the NP platform as a potential vaccine or active cellular immunotherapy carrier. IL-1β production showed a correlation with both NP size and degree of hydrophobicity. High IFNγ secretion from T cells was shown to be correlated with both the hydrophobicity and charge of the NPs used to activate the DCs. Other cytokines were also screened in order to compare the immune responses. The results of this study highlight the importance of nanoparticle biophysical parameters and the selection of TLR ligands to the rational design of nanoparticle-based vaccines and immunotherapies., Statement of Significance: The manuscript describes a systematic investigation into the effects of biophysical parameters of nanoparticles (NPs) on immune cells. Modulation of the biophysical character of the NP surface can be achieved by introduction of amino acids on monodisperse silica NPs, introducing a range of tunable biophysical parameters of interest, i.e. distinct sizes, different surface charges and varying degrees of surface hydrophobicity. We examine internalization of the NP in dendritic cells (DCs) and measure a myriad of cytokines, including IL-1β and IFNγ, which prognosticate the efficacy of the NPs as a potential vaccine (IL-1β metric) or active cellular immunotherapy carrier (IFNγ metric). Two different TLR ligands (a viral TLR3 ligand and a bacterial TLR4 ligand) were used along with the PAA NPs to compare their costimulatory immunogenicity. We strongly believe that this study will provide crucial information to many readers of Acta Biomaterialia and further drive the use of nanoparticle platforms in modulating immune responses., (Copyright © 2017. Published by Elsevier Ltd.)

IL-27 bridges innate and adaptive immunity by modulating cytokine production from myeloid cells and regulating Th cell differentiation. During bacterial infection, TLR4 triggering by LPS induces IL-27 production by monocytes and macrophages. We have previously shown that IL-27 can prime monocytes for LPS responsiveness by enhancing TLR4 expression and intracellular signaling. If unregulated, this could result in damaging inflammation, whereas on the other hand, this may also provide greater responses by inflammatory processes induced in response to bacterial pathogens. A key process in fine-tuning inflammatory responses is activation of the inflammasome, which ultimately results in IL-1β production. Herein, we investigated the molecular mechanisms by which IL-27 modulates LPS-induced IL-1β secretion in monocytes and macrophages. We found that when delivered simultaneously with LPS, IL-27 augments activation of caspase-1 and subsequent release of IL-1β. Furthermore, we determined that IL-27 primes cells for enhanced IL-1β production by up-regulating surface expression of TLR4 and P2X purinoceptor 7 (P2X7) for enhanced LPS and ATP signaling, respectively. These findings provide new evidence that IL-27 plays an important role in the proinflammatory capacity of monocytes and macrophages via enhancing IL-1β secretion levels triggered by dual LPS-ATP stimulation., (© Society for Leukocyte Biology.)

Protein tyrosine phosphorylation guides many molecular interactions for cellular functions. SCIMP is a transmembrane adaptor protein (TRAP) family member that mediates selective proinflammatory cytokine responses generated by pathogen-activated Toll-like receptor (TLR) pathways in macrophages. TLR activation triggers SCIMP phosphorylation and selective phosphorylation of distinct tyrosine residues on this adaptor offers the potential for regulating or biasing inflammatory responses. To analyze site-specific phosphorylation events, we developed three probes based on the SH2 domains of known SCIMP effectors, and used them for pull-downs from macrophage extracts. CRISPR-mediated SCIMP-deficient RAW264.7 macrophage-like cells were reconstituted with various phosphorylation-deficient (Y58F, Y96F, Y120F) SCIMPs, and used to demonstrate the specificity of LPS/TLR4-induced, site-specific phosphorylation of SCIMP for the temporal recruitment of the effectors Grb2, Csk and SLP65. Our findings reveal potential for differential SCIMP phosphorylation and specific effectors to influence TLR signaling and inflammatory programs. Furthermore, the use of Csk-SH2 pull-downs to identify additional known and new Csk targets in LPS-activated macrophages reveals the wider utility of our SH2 probes.

Endotoxins, also referred to as lipopolysaccharides (LPS), are powerful immunostimulators involved in a number of severe diseases. Forsythoside A (FTA), a monomer of phenethyl alcohol glycosides extracted from Forsythia suspensa, has been shown to possess anti-bacterial and immunomodulatory properties. However, it is currently not known whether FTA can counter the adverse effects of endotoxins. We investigated the effect of FTA on LPS-stimulated RAW264.7 cells and primary lymphocytes to determine its molecular mechanism of action. RAW264.7 cells and primary lymphocytes were incubated with or without LPS (100 ng/ml) in the presence or absence of FTA or polymyxin B. We found that FTA increased the viability of LPS-treated RAW264.7 cells and primary lymphocytes suggesting that FTA effectively counters the adverse effects of endotoxins. FTA decreased the percentage of regulatory T cells (Tregs) and inhibited the TLR4/MyD88/NF-κB signaling pathway, downregulating Foxp3, IL-10 and TGF-β1, molecules involved in the immunosuppressive function of Tregs. These findings elucidate the molecular mechanism underlying the anti-endotoxin effects of FTA and suggest its use as a new treatment for LPS-induced diseases.

Ectromelia virus (ECTV) is an orthopoxvirus responsible for mousepox, a lethal disease of certain strains of mice that is similar to smallpox in humans, caused by variola virus (VARV). ECTV, similar to VARV, exhibits a narrow host range and has co-evolved with its natural host. Consequently, ECTV employs sophisticated and host-specific strategies to control the immune cells that are important for induction of antiviral immune response. In the present study we investigated the influence of ECTV infection on immune functions of murine GM-CSF-derived bone marrow cells (GM-BM), comprised of conventional dendritic cells (cDCs) and macrophages. Our results showed for the first time that ECTV is able to replicate productively in GM-BM and severely impaired their innate and adaptive immune functions. Infected GM-BM exhibited dramatic changes in morphology and increased apoptosis during the late stages of infection. Moreover, GM-BM cells were unable to uptake and process antigen, reach full maturity and mount a proinflammatory response. Inhibition of cytokine/chemokine response may result from the alteration of nuclear translocation of NF-κB, IRF3 and IRF7 transcription factors and down-regulation of many genes involved in TLR, RLR, NLR and type I IFN signaling pathways. Consequently, GM-BM show inability to stimulate proliferation of purified allogeneic CD4+ T cells in a primary mixed leukocyte reaction (MLR). Taken together, our data clearly indicate that ECTV induces immunosuppressive mechanisms in GM-BM leading to their functional paralysis, thus compromising their ability to initiate downstream T-cell activation events.

The development of new approaches leading to fast and successful vascularization of tissue-engineered constructs is one of the most intensively studied subjects in tissue engineering and regenerative medicine. Recently, TLR4 activation and LPS stimulation of endothelial cells have been reported to promote angiogenesis in a variety of settings. In this study, we demonstrate that TLR4 activation by Ultrapure LPS Escherichia coli 0111:B4 (LPS-EB) significantly enhances microvessel formation in a co-culture system consisting of outgrowth endothelial cells (OECs) and primary human osteoblasts (pOBs). The precise modes of TLR4 action on the process of angiogenesis have also been investigated in this study. Using quantitative fluorescence microscopy in monocultures of OECs and pOBs, it was found that TLR4 activation through LPS-EB upregulates the expression level of TLR4/MYD88 and enhances both angiogenesis and osteogenesis. Furthermore, ELISA and qRT-PCR have shown that the level of two adhesion molecules (ICAM-1 and E-selectin), two cytokines (IL-6 and IL-8) and two growth factors (VEGF and PDGF-BB) related to angiogenesis increase significantly after LPS-EB treatment. This increased understanding of the role of TLR4 in angiogenesis could be of value in various settings related to tissue repair and tissue engineering. Moreover, since LPS and TLR4 agonists improve angiogenesis and osteogenesis, TLR4 agonists (endogenous or synthetic) could be used for angiogenesis intervention in vivo and therefore could be tested for their potential clinical applications in promoting angiogenesis in bone tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

IL-1β release is integral to the innate immune system. The release of mature IL-1β depends on 2 regulated events: the de novo induction of pro-IL-1β, generally via NF-κB-dependent transduction pathways; and the assembly and activation of the NLRP3 inflammasome. This latter step is reliant on active caspase-1, pannexin-1, and P2X 7 receptor activation. Pathogen-associated molecular patterns in gram-positive and gram-negative bacteria activate IL-1β release from immune cells via TLR2 and TLR4 receptors, respectively. We found that pro-IL-1β and mature IL-1β release from human monocytes is stimulated by the TLR2 agonists Pam 3 CSK4 or FSL-1, as well as the TLR4 agonist LPS in the absence of additional ATP. TLR2 agonists required pannexin-1 and P2X 7 receptor activation to stimulate IL-1β release. In contrast, IL-1β release stimulated by the TLR4 agonist LPS is independent of both pannexin-1 and P2X 7 activation. In the absence of exogenous ATP, P2X 7 activation requires endogenous ATP release, which occurs in some cells via pannexin-1. In line with this, we found that LPS-stimulated human monocytes released relatively low levels of ATP, whereas cells stimulated with TLR2 agonists released high levels of ATP. These findings suggest that in human monocytes, both TLR2 and TLR4 signaling induce pro-IL-1β expression, but the mechanism by which they activate caspase-1 diverges at the level of the pannexin-1/ATP/P2X 7 axis.-Parzych, K., Zetterqvist, A. V., Wright, W. R., Kirkby, N. S., Mitchell, J. A., Paul-Clark, M. J. Differential role of pannexin-1/ATP/P2X 7 axis in IL-1β release by human monocytes., (© FASEB.)

Oligodendrocyte progenitor cells (OPCs) are present throughout the adult brain and spinal cord and can replace oligodendrocytes lost to injury, aging, or disease. Their differentiation, however, is inhibited by myelin debris, making clearance of this debris an important step for cellular repair following demyelination. In models of peripheral nerve injury, TLR4 activation by lipopolysaccharide (LPS) promotes macrophage phagocytosis of debris. Here we tested whether the novel synthetic TLR4 agonist E6020, a Lipid A mimetic, promotes myelin debris clearance and remyelination in spinal cord white matter following lysolecithin-induced demyelination. In vitro, E6020 induced TLR4-dependent cytokine expression (TNFα, IL1β, IL-6) and NF-κB signaling, albeit at ∼10-fold reduced potency compared to LPS. Microinjection of E6020 into the intact rat spinal cord gray/white matter border induced macrophage activation, OPC proliferation, and robust oligodendrogenesis, similar to what we described previously using an intraspinal LPS microinjection model. Finally, a single co-injection of E6020 with lysolecithin into spinal cord white matter increased axon sparing, accelerated myelin debris clearance, enhanced Schwann cell infiltration into demyelinated lesions, and increased the number of remyelinated axons. In vitro assays confirmed that direct stimulation of macrophages by E6020 stimulates myelin phagocytosis. These data implicate TLR4 signaling in promoting repair after CNS demyelination, likely by stimulating phagocytic activity of macrophages, sparing axons, recruiting myelinating cells, and promoting remyelination. This work furthers our understanding of immune-myelin interactions and identifies a novel synthetic TLR4 agonist as a potential therapeutic avenue for white matter demyelinating conditions such as spinal cord injury and multiple sclerosis., (© 2017 Wiley Periodicals, Inc.)

Recently, we reported a novel function for C4b-binding protein (C4BP) in inhibiting the toll-like receptor (TLR)1/2 response by interacting with TLR2. TLRs share a common structure; hence, we examined the effect of C4BP on activation of other TLRs-TLR4 and TLR3. The results of immunoprecipitation assays suggest that C4BP interacts with TLR4/MD-2 but not TLR3. C4BP inhibits TLR4/MD-2-mediated, but not TLR3-mediated, proinflammatory cytokine production and nuclear factor (NF)-κB signaling. C4BP-deficient mice show increased interleukin (IL)-6 production in response to the TLR4/MD-2 ligand. A competition assay revealed that C4BP prevents an interaction between TLR4/MD-2 and its ligand. These findings indicate that C4BP binds to cell surface TLRs and inhibits the TLR-TLR ligand interaction, thereby inhibiting TLR activation., (© 2017 Federation of European Biochemical Societies.)

Sensing of pathogens by innate immune cells is essential for the initiation of appropriate immune responses. Toll-like receptors (TLRs), which are highly sensitive for various structurally and evolutionary conserved molecules derived from microbes have a prominent role in this process. TLR engagement results in the activation of the transcription factor NF-κB, which induces the expression of cytokines and other inflammatory mediators. The exquisite sensitivity of TLR signalling can be exploited for the detection of bacteria and microbial contaminants in tissue cultures and in protein preparations. Here we describe a cellular reporter system for the detection of TLR ligands in biological samples. The well-characterized human monocytic THP-1 cell line was chosen as host for an NF-ᴋB-inducible enhanced green fluorescent protein reporter gene. We studied the sensitivity of the resultant reporter cells for a variety of microbial components and observed a strong reactivity towards TLR1/2 and TLR2/6 ligands. Mycoplasma lipoproteins are potent TLR2/6 agonists and we demonstrate that our reporter cells can be used as reliable and robust detection system for mycoplasma contaminations in cell cultures. In addition, a TLR4-sensitive subline of our reporters was engineered, and probed with recombinant proteins expressed in different host systems. Bacterially expressed but not mammalian expressed proteins induced strong reporter activity. We also tested proteins expressed in an E. coli strain engineered to lack TLR4 agonists. Such preparations also induced reporter activation in THP-1 cells highlighting the importance of testing recombinant protein preparations for microbial contaminations beyond endotoxins. Our results demonstrate the usefulness of monocytic reporter cells for high-throughput screening for microbial contaminations in diverse biological samples, including tissue culture supernatants and recombinant protein preparations. Fluorescent reporter assays can be measured on standard flow cytometers and in contrast to established detection methods, like luciferase-based systems or Limulus Amebocyte Lysate tests, they do not require costly reagents.

Adjuvant properties of bacterial cell wall components like MPLA (monophosphoryl lipid A) are well described and have gained FDA approval for use in vaccines such as Cervarix. MPLA is the product of chemically modified lipooligosaccharide (LOS), altered to diminish toxic proinflammatory effects while retaining adequate immunogenicity. Despite the virtually unlimited number of potential sources among bacterial strains, the number of useable compounds within this promising class of adjuvants are few. We have developed bacterial enzymatic combinatorial chemistry (BECC) as a method to generate rationally designed, functionally diverse lipid A. BECC removes endogenous or introduces exogenous lipid A-modifying enzymes to bacteria, effectively reprogramming the lipid A biosynthetic pathway. In this study, BECC is applied within an avirulent strain of Yersinia pestis to develop structurally distinct LOS molecules that elicit differential Toll-like receptor 4 (TLR4) activation. Using reporter cell lines that measure NF-κB activation, BECC-derived molecules were screened for the ability to induce a lower proinflammatory response than Escherichia coli LOS. Their structures exhibit varied, dose-dependent, TLR4-driven NF-κB activation with both human and mouse TLR4 complexes. Additional cytokine secretion screening identified molecules that induce levels of tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8) comparable to the levels induced by phosphorylated hexa-acyl disaccharide (PHAD). The lead candidates demonstrated potent immunostimulation in mouse splenocytes, human primary blood mononuclear cells (PBMCs), and human monocyte-derived dendritic cells (DCs). This newly described system allows directed programming of lipid A synthesis and has the potential to generate a diverse array of TLR4 agonist candidates. IMPORTANCE There is an urgent need to develop effective vaccines against infectious diseases that continue to be major causes of morbidity and mortality worldwide. Making effective vaccines requires selecting an adjuvant to strengthen an appropriate and protective immune response. This work describes a practical method, bacterial enzymatic combinatorial chemistry (BECC), for generating functionally diverse molecules for adjuvant use. These molecules were analyzed in cell culture for their ability to initiate immune stimulatory activity. Several of the assays described herein show promising in vitro cytokine production and costimulatory molecule expression results, suggesting that the BECC molecules may be useful in future vaccine preparations., (Copyright © 2017 Gregg et al.)

Background: Ethanol (EtOH) consumption leads to an increase of proinflammatory signaling via activation of Toll-like receptors (TLRs) such as TLR3 and TLR4 that leads to kinase activation (ERK1/2, p38, TBK1), transcription factor activation (NFκB, IRF3), and increased transcription of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6. This immune signaling cascade is thought to play a role in neurodegeneration and alcohol use disorders. While microglia are considered to be the primary macrophage in brain, it is unclear what if any role neurons play in EtOH-induced proinflammatory signaling., Methods: Microglia-like BV2 and retinoic acid-differentiated neuron-like SH-SY5Y were treated with TLR3 agonist Poly(I:C), TLR4 agonist lipopolysaccharide (LPS), or EtOH for 10 or 30 minutes to examine proinflammatory immune signaling kinase and transcription factor activation using Western blot, and for 24 hours to examine induction of proinflammatory gene mRNA using RT-PCR., Results: In BV2, both LPS and Poly(I:C) increased p-ERK1/2, p-p38, and p-NFκB by 30 minutes, whereas EtOH decreased p-ERK1/2 and increased p-IRF3. LPS, Poly(I:C), and EtOH all increased TNF-α and IL-1β mRNA, and EtOH further increased TLR2, 7, 8, and MD-2 mRNA in BV2. In SH-SY5Y, LPS had no effect on kinase or proinflammatory gene expression. However, Poly(I:C) increased p-p38 and p-IRF3, and increased expression of TNF-α, IL-1β, and IL-6, while EtOH increased p-p38, p-IRF3, p-TBK1, and p-NFκB while decreasing p-ERK1/2 and increasing expression of TLR3, 7, 8, and RAGE mRNA. HMGB1, a TLR agonist, was induced by LPS in BV2 and by EtOH in both cell types. EtOH was more potent at inducing proinflammatory gene mRNA in SH-SY5Y compared with BV2., Conclusions: These results support a novel and unique mechanism of EtOH, TLR3, and TLR4 signaling in neuron-like SH-SY5Y and microglia-like BV2 that likely contributes to the complexity of brain neuroimmune signaling., (Copyright © 2017 The Authors Alcoholism: Clinical and Experimental Research published by Wiley Periodicals, Inc. on behalf of Research Society on Alcoholism.)

Aberrant activation of toll-like receptors (TLRs), key components of the innate immune system, has been proposed to underlie and exacerbate a range of central nervous system disorders. Increasing evidence supports a role for the endocannabinoid system in modulating inflammatory responses including those mediated by TLRs, and thus this system may provide an important treatment target for neuroinflammatory disorders. However, the effect of modulating endocannabinoid tone on TLR-induced neuroinflammation in vivo and associated behavioural changes is largely unknown. The present study examined the effect of inhibiting fatty acid amide hydrolyase (FAAH), the primary enzyme responsible for the metabolism of anandamide (AEA), in vivo on TLR4-induced neuroimmune and behavioural responses, and evaluated sites and mechanisms of action. Systemic administration of the FAAH inhibitor PF3845 increased levels of AEA, and related FAAH substrates N-oleoylethanolamide (OEA) and N-palmitoylethanolamide (PEA), in the frontal cortex and hippocampus of rats, an effect associated with an attenuation in the expression of pro- and anti-inflammatory cytokines and mediators measured 2hrs following systemic administration of the TLR4 agonist, lipopolysaccharide (LPS). These effects were mimicked by central i.c.v. administration of PF3845, but not systemic administration of the peripherally-restricted FAAH inhibitor URB937. Central antagonism of TRPV1 significantly attenuated the PF3845-induced decrease in IL-6 expression, effects not observed following antagonism of CB 1, CB 2 , PPARα, PPARγ or GPR55. LPS-induced a robust sickness-like behavioural response and increased the expression of markers of glial activity and pro-inflammatory cytokines over 24hrs. Systemic administration of PF3845 modulated the TLR4-induced expression of neuroimmune mediators and anhedonia without altering acute sickness behaviour. Overall, these findings support an important role for FAAH substrates directly within the brain in the regulation of TLR4-associated neuroinflammation and highlight a role for TRPV1 in partially mediating these effects., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Immunotherapy has become one of the fastest growing areas of cancer research. A promising in situ autologous cancer vaccine (inCVAX) uses a novel immune activator, N-dihydrogalactochitosan (GC), that possesses the ability to stimulate dendritic cells (DC). inCVAX is a combination treatment procedure involving treatment of the tumor with a thermal near-infrared laser to liberate whole cell tumor antigens, followed by injection of GC (a glucosamine polymer with galactose attached to the amino groups) into the treated tumor thereby inducing a systemic antitumor immune response. Regression of both the treated tumor and distant untreated metastases has been observed in both nonclinical and clinical settings following inCVAX. We studied the stimulatory action of GC on relatively immature DCs (DC2.4 cell line) in vitro. GC at 1 mg/mL was a potent stimulator for DC with limited toxicity, giving increased expression of major histocompatibility complex class 2, CD80, and CD11c. Confocal imaging also revealed qualitatively increased uptake of antigen (Texas red-labeled ovalbumin) by DCs after the introduction of GC. To visualize cellular uptake, GC was conjugated with FITC-fluorophore revealing its cellular internalization after 8 hours. In some cases GC was more effective than the toxic TLR4 agonist, lipopolysaccharide. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 963-972, 2017., (© 2017 Wiley Periodicals, Inc.)

Background: Generation of monocyte-derived dendritic cells (MDDC) is induced in the presence of GM-CSF and IL-4, and a maturation stimulus is added to the monocyte culture to obtain mature Dendritic Cells (DCs) suitable for therapy. TNF-α is the most common cytokine used for activating DCs and generating mature MDDC either alone or in combination with other cytokines., Objective: To compare effects of traditional cytokine cocktail (TNF-α + IL-1β) versus TLR4-agonist monophosphoryl lipid A on the viability, phenotype, cytokine profile and functionality of MDDC., Methods: The study included 32 individuals; twenty Acute Myeloid Leukaemia (AML) cases in complete remission and 12 healthy volunteers. They were divided into 3 groups: Group 1: control group: 12 subjects to measure the baseline levels of all markers in the monocytic preparation. Group 2: cytokine cocktail (TNF-α) group, which included 10 AML subjects. Group 3: MPLA group which included 10 AML subjects., Results: TNF-α group showed higher expression of CD83 than MPLA group indicating higher capacity to induce DC maturation but both were similar in CD86, CCR7 and IL-10 expression. Preparation of dendritic cells from AML cases in remission and loading them with tumor peptides was successful., Conclusion: MPLA effect in DC maturation is comparable with traditional DC maturation cocktail.

Cholesterol deposits and pro-inflammatory cytokines play an essential role in the pathogenesis of atherosclerosis, a predominant cause of cardiovascular disease (CVD). Epidemiological evidence has linked periodontal disease (PD) with atherosclerotic CVD. Accordingly, viable periodontal pathogens, including Porphyromonas gingivalis, have been found in atherosclerotic plaques in humans and mice. We aimed to determine whether cholesterol crystals (CHCs) and oral bacteria synergize in the stimulation of human monocytes. Incubation of human monocytes with CHCs induced secretion of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, and IL-8. Moreover, CHCs markedly enhanced secretion of IL-1β by monocytes stimulated with the toll-like receptor (TLR) 4 agonist Escherichia coli lipopolysaccharide (LPS), and the TLR2 agonist Staphylococcus aureus lipoteichoic acid. Notably, CHCs also enhanced IL-1β secretion induced by P. gingivalis LPS and IL-1β secretion induced by whole P. gingivalis bacteria. This enhancement was abrogated by the NLRP3 inflammasome inhibitors Z-YVAD-FMK and glibenclamide. CHCs had no effect on cytokine production induced by P. gingivalis gingipains. Taken together, our findings support that CHCs, via stimulation of NLRP3 inflammasomes, act in synergy with the periodontal pathogen P. gingivalis to promote monocyte secretion of pro-atherogenic cytokines.

Background: Multiple toll-like receptors (TLRs) are expressed in cells of the monocytic lineage, including microglia, which constitute the major reservoir for human immunodeficiency virus (HIV) infection in the brain. We hypothesized that TLR receptor mediated responses to inflammatory conditions by microglial cells in the central nervous system (CNS) are able to induce latent HIV proviruses, and contribute to the etiology of HIV-associated neurocognitive disorders., Results: Newly developed human microglial cell lines (hµglia), obtained by immortalizing human primary microglia with simian virus-40 (SV40) large T antigen and the human telomerase reverse transcriptase, were used to generate latently infected cells using a single-round HIV virus carrying a green fluorescence protein reporter (hµglia/HIV, clones HC01 and HC69). Treatment of these cells with a panel of TLR ligands showed surprisingly that two potent TLR3 agonists, poly (I:C) and bacterial ribosomal RNA potently reactivated HIV in hμglia/HIV cells. LPS (TLR4 agonist), flagellin (TLR5 agonist), and FSL-1 (TLR6 agonist) reactivated HIV to a lesser extent, while Pam3CSK4 (TLR2/1 agonist) and HKLM (TLR2 agonist) only weakly reversed HIV latency in these cells. While agonists for TLR2/1, 4, 5 and 6 reactivated HIV through transient NF-κB induction, poly (I:C), the TLR3 agonist, did not activate NF-κB, and instead induced the virus by a previously unreported mechanism mediated by IRF3. The selective induction of IRF3 by poly (I:C) was confirmed by chromatin immunoprecipitation (ChIP) analysis. In comparison, in latently infected rat-derived microglial cells (hT-CHME-5/HIV, clone HC14), poly (I:C), LPS and flagellin were only partially active. The TLR response profile in human microglial cells is also distinct from that shown by latently infected monocyte cell lines (THP-1/HIV, clone HA3, U937/HIV, clone HUC5, and SC/HIV, clone HSCC4), where TLR2/1, 4, 5, 6 or 8, but not for TLR3, 7 or 9, reactivated HIV., Conclusions: TLR signaling, in particular TLR3 activation, can efficiently reactivate HIV transcription in infected microglia, but not in monocytes or T cells. The unique response profile of microglial cells to TLR3 is fundamental to understanding how the virus responds to continuous microbial exposure, especially during inflammatory episodes, that characterizes HIV infection in the CNS.

In this review, we discuss small-molecule, carbohydrate-based immunostimulants that target Toll-like receptor 4 (TLR-4) and cluster of differentiation 1D (CD1d) receptors. The design and use of these molecules in immunotherapy as well as results from their use in clinical trials are described. How these molecules work and their utilization as vaccine adjuvants are also discussed. Future applications and extensions for the use of these analogues as therapeutic agents will be outlined., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The paucity of FDA approved adjuvants renders the synthesis, characterization, and use of new compounds as vaccine adjuvants, a necessity. For this purpose, a novel saccharide analog has been synthesized from glucosamine, pyruvylated galactose and 1,4-cyclohexanediol and its biological efficacy was determined in innate immune cells. More specifically, we assessed the production of pro-inflammatory cytokines from the murine monocyte cell line, Raw 264.7 and from C57 BL/6 mouse peritoneal macrophages following exposure to the saccharide analog. Our data conclude that the novel saccharide has immunostimulatory activity on mouse macrophages as indicated by the elevated levels of IL-6 and TNF-α in culture supernatants. This effect was TLR-4-dependent but TLR-2-independent. Our data, suggest TLR-4 agonism; a key feature of vaccine adjuvants., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: This study is to determine the effects of P. gingivalis LPS and CpG on B10 cell expansion and IL-10 competency in vitro., Material and Methods: Spleen B cells were isolated from C57BL/6J mice with or without formalin-fixed P. gingivalis immunization. B cells were cultured for 48 hours under the following conditions: CD40L, CD40L+LPS, CD40L+CpG, and CD40L+LPS+CpG in the presence or absence of fixed P. gingivalis. Percentages of CD1dhiCD5+ B cells were measured by flow cytometry. IL-10 mRNA expression and secreted IL-10 were measured by real-time quantitative PCR and by ELISA respectively., Results: P. gingivalis LPS plus CD40L significantly increased CD1dhiCD5+ B cell percentages and secreted IL-10 levels in both immunized and non-immunized mice B cells in the presence or absence of P. gingivalis, compared with control group. Secreted IL-10 levels were significantly increased in CD40L+LPS treated group compared with CD40L treatment group in the absence of P. gingivalis. CpG plus CD40L significantly decreased CD1dhiCD5+ B cell percentages, but greatly elevated secreted IL-10 levels in immunized and non-immunized mice B cells in the absence of P. gingivalis, compared with CD40L treatment group., Conclusions: P. gingivalis LPS and CpG differentially enhance IL-10 secretion and expansion of mouse B10 cells during innate and adaptive immune responses.

Background/aims: Vitamin A (VA) protects the intestinal epithelial barrier by improving cell migration and proliferation. Our previous studies demonstrated that VA deficiency (VAD) during pregnancy suppresses the systemic and mucosal immune responses in the intestines of offspring and that VA supplementation (VAS) during early life can increase immune cell counts. However, little is known about the mechanisms by which VA regulates intestinal epithelial barrier function., Methods: Caco-2 cells were treated with all-trans retinoic acid (ATRA) for 24 hours to determine the optimum ATRA concentration to which the cells in question respond. Caco-2 cells were infected with recombinant adenoviruses carrying retinoic acid receptor beta (Ad-RARβ) and small interfering RARβ(siRARβ) to assess the effects of RARβ signalling on the expression of specific proteins. A siTLR4 lentivirus was used to knockdown Toll-like receptor 4 (TLR4) in Caco-2 cells to determine its role in the protective effects of VA on the intestinal epithelial barrier, and experiments involving TLR4-knock-out mice were performed to verify the effect of TLR4. VA normal (VAN), VAD and VAS rat models were established to confirm that changes in RARβ, TLR4 and ZO-2 expression levels that occurred following decreases or increases in retinol concentrations in vivo, and the permeability of the Caco-2 cell monolayer, as well as that of the epithelial barrier of the rat intestine was detected by measuring transepithelial resistance (TER) or performing enzyme-linked immunosorbent assay (ELISA). Retinoic acid receptor (RAR), toll like receptor (TLR) and tight junction (TJ) mRNA and protein expression levels in Caco-2 cells and the colon monolayers in the rat and mouse models were measured by PCR and western blotting, respectively. Co-immunoprecipitation (co-IP) and immunofluorescence staining were performed to assess the interactions among RARβ, TLR4 and zonula occluden-2 (ZO-2) in Caco-2 cells, and chromatin immunoprecipitation (ChIP) assay was performed to assess the binding between RARβ and the TLR4 promoter sequence in Caco-2 cells., Results: In the present study, ATRA treatment not only increased the TER of the Caco-2 monolayer but also up-regulated the expression levels of RARβ, TLR4 and ZO-2 in Caco-2 cells. The expression levels of these three proteins were significantly decreased in the colonic epithelial monolayers of VAD rats compared with those of VAN rats and were significantly increased following VAS in the corresponding group compared with the control group. Furthermore, the above changes in TLR4 and ZO-2 expression levels were augmented or attenuated by Ad-RARβ or siRARβ infection, respectively, in Caco-2 cells. Interestingly, siTLR4 down-regulated ZO-2 expression but did not affect RARβ expression in Caco-2 cells, and in VAD mice the lack of TLR4 did not affect ZO-2 expression. We noted direct interactions between RARβ and TLR4, TLR4 and ZO-2 in Caco-2 cells, and ChIP assay showed that RARβ could bind to the TLR4 promoter but not the ZO-2 promoter in Caco-2 cells., Conclusion: Taken together, our results indicate that RARβ enhanced ZO-2 expression by regulating TLR4 to improve intestinal epithelial barrier function in Caco-2 cells, as well as in rat and mouse models, but not in humans., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Aim: Toll like receptors plays a significant anti-viral role in different infections. The aim of this study was to look into the role of toll like receptor 4 (TLR4) in hepatitis B virus (HBV) infection., Methods: Real time PCR was used to analyze the transcription of TLR4 signaling molecules, cell cycle regulators and HBV DNA viral load after triggering the HepG2.2.15 cells with TLR4 specific ligand. Nuclear factor (NF)-κB translocation on TLR4 activation was analyzed using microscopic techniques. Protein and cell cycle analysis was done using Western Blot and FACS respectively., Results: The present study shows that TLR4 activation represses HBV infection. As a result of HBV suppression, there are several changes in host factors which include partial release in G1/S cell cycle arrest and changes in host epigenetic marks. Finally, it was observed that anti-viral action of TLR4 takes place through the NF-κB pathway., Conclusion: The study shows that TLR4 activation in HBV infection brings about changes in hepatocyte microenvironment and can be used for developing a promising therapeutic target in future., Competing Interests: Conflict-of-interest statement: The authors declare no conflict of interest.

The contribution of B cells to immunity against many infectious diseases is unquestionably important and well characterized. Here, we sought to determine the role of B cells in the induction of T-helper 1 (T H 1) CD4 + T cells upon vaccination with a tuberculosis (TB) antigen combined with a TLR4 agonist. We used B-cell deficient mice (μMT -/- ), tetramer-positive CD4 + T cells, markers of memory "precursor" effector cells (MPECs), and T-cell adoptive transfers and demonstrated that the early antigen-specific cytokine-producing T H 1 responses are unaffected in the absence of B cells, however MPEC induction is strongly impaired resulting in a deficiency of the memory T H 1 response in μMT -/- mice. We further show that antigen-presentation by B cells is necessary for their role in MPEC generation using B-cell adoptive transfers from wt or MHC class II knock-out mice into μMT -/- mice. Our study challenges the view that B-cell deficiency exclusively alters the T H 1 response at memory time-points. Collectively, our results provide new insights on the multifaceted roles of B cells that will have a high impact on vaccine development against several pathogens including those requiring T H 1 cell-mediated immunity., Competing Interests: The authors declare no financial or commercial conflicts of interest., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Dendritic cells (DCs) have shown promise for use in cancer vaccine and cancer immunotherapy studies. However, we demonstrate that cancer cell lines can negatively interfere with DC generation in granulocyte-macrophage colony-stimulating factor (GM-CSF)-derived cultures, although cancer cells are able to enhance CD80 cell surface activation marker and cytokine secretion. Furthermore, in the presence of cancer cells, GM-CSF-derived DCs are unable to stimulate T-cells. Additional stimulation with toll-like receptor 4 cannot fully reverse the suppressive effect of cancer cells or supernatant. Hence, it is imperative to understand the immunosuppressive effects of cancer on DCs in order for DC-based cancer immunotherapy to be successful., (© The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)