Search

Nucleic acid-mediated interferon signaling plays a pivotal role in defense against microorganisms, especially during viral infection. Receptors sensing exogenous nucleic acid molecules are localized in the cytosol and endosomes. Cytosolic sensors, including cGAS, RIG-I, and MDA5, and endosome-anchored receptors are toll-like receptors (TLR3, TLR7, TLR8, and TLR9). These TLRs share the same domain architecture and have similar structures, facing the interior of endosomes so their binding to nucleic acids of invading pathogens via endocytosis is possible. The correct function of these receptors is crucial for cell homeostasis and effective response against pathogen invasion. A variety of endogenous mechanisms modulates their activities. Nevertheless, naturally occurring mutations lead to aberrant TLR-mediated interferon (IFN) signaling. Furthermore, certain pathogens require a more robust defense against control. Thus, manipulating these TLR activities has a profound impact. High-throughput virtual screening followed by experimental validation led to the discovery of numerous chemicals that can change these TLR-mediated IFN signaling activities. Many of them are unique in selectivity, while others regulate more than one TLR due to commonalities in these receptors. We summarized these nucleic acid-sensing TLR-mediated IFN signaling pathways and the corresponding chemicals activating or deactivating their signaling., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Toll-like receptor (TLR) activation in macrophages plays a critical role in the pathogenesis of acute lung injury (ALI). While TLR inhibition is a promising strategy to control the overwhelming inflammation in ALI, there still lacks effective TLR inhibitors for clinical uses to date. A unique class of peptide-coated gold nanoparticles (GNPs) is previously discovered, which effectively inhibited TLR signaling and protected mice from lipopolysaccharide (LPS)-induced ALI. To fast translate such a discovery into potential clinical applicable nanotherapeutics, herein an elegant strategy of "nano-enabled drug repurposing" with "nano-targeting" is introduced to empower the existing drugs for new uses. Combining transcriptome sequencing with Connectivity Map analysis, it is identified that the proton pump inhibitors (PPIs) share similar mechanisms of action to the discovered GNP-based TLR inhibitor. It is confirmed that PPIs (including omeprazole) do inhibit endosomal TLR signaling and inflammatory responses in macrophages and human peripheral blood mononuclear cells, and exhibits anti-inflammatory activity in an LPS-induced ALI mouse model. The omeprazole is then formulated into a nanoform with liposomes to enhance its macrophage targeting ability and the therapeutic efficacy in vivo. This research provides a new translational strategy of nano-enabled drug repurposing to translate bioactive nanoparticles into clinically used drugs and targeted nano-therapeutics for ALI., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Toll-like receptors (TLRs) are crucial transmembrane receptors that form part of the innate immune response. They play a role in the recognition of various microorganisms and their elimination from the host. TLRs have been proposed as vital immunomodulators in the regulation of multiple neonatal stressors that extend beyond infection such as oxidative stress and pain. The immune system is immature at birth and takes some time to become fully established. As such, babies are especially vulnerable to sepsis at this early stage of life. Findings suggest a gestational age-dependent increase in TLR expression. TLRs engage with accessory and adaptor proteins to facilitate recognition of pathogens and their activation of the receptor. TLRs are generally upregulated during infection and promote the transcription and release of proinflammatory cytokines. Several studies report that TLRs are epigenetically modulated by chromatin changes and promoter methylation upon bacterial infection that have long-term influences on immune responses. TLR activation is reported to modulate cardiorespiratory responses during infection and may play a key role in driving homeostatic instability observed during sepsis. Although complex, TLR signaling and downstream pathways are potential therapeutic targets in the treatment of neonatal diseases. By reviewing the expression and function of key Toll-like receptors, we aim to provide an important framework to understand the functional role of these receptors in response to stress and infection in premature infants.

A therapeutic strategy for prostate cancer (PCa) involves the use of 9-cis-retinoic acid (9cRA) to induce cancer stem cells (CSCs) differentiation and apoptosis. Polyinosinic:polycytidylic acid (PIC) is a Toll-like receptor 3 (TLR3) agonist that induces tumor cells apoptosis after activation. PIC+9cRA combination activates retinoic acid receptor β (RARβ) re-expression, leading to CSC differentiation and growth arrest. Since inorganic arsenic (iAs) targets prostatic stem cells (SCs), we hypothesized that arsenic-transformed SCs (As-CSCs) show an impaired TLR3-associated anti-tumor pathway and, therefore, are unresponsive to PIC activation. We evaluated TLR3-mediated activation of anti-tumor pathway based in RARβ expression, on As-CSC and iAs-transformed epithelial cells (CAsE-PE). As-CSCs and CAsE-PE showed lower TLR3 and RARβ basal expression compared to their respective isogenic controls WPE-Stem and RWPE-1. Also, iAs transformants showed reduced expression of mediators in TLR3 pathway. Importantly, As-CSCs were irresponsive to PIC+9cRA in terms of increased RARβ and decreased SC-markers expression, while CAsE-PE, a heterogeneous cell line having a small SC population, were partially responsive. These observations indicate that iAs can impair TLR3 expression and anti-tumor pathway activated by PIC+9cRA in SCs and prostatic epithelial cells. These findings suggest that TLR3-activation based therapy may be an ineffective therapeutic alternative for iAs-associated PCa., Competing Interests: Declaration of Competing Interest The authors declare that they have no knowledge of external financial or personal situations that have an influence on the work reported in this article., (Copyright © 2021 Elsevier B.V. All rights reserved.)

COVID-19 is both a viral illness and a disease of immunopathology. Proximal events within the innate immune system drive the balance between deleterious inflammation and viral clearance. We hypothesize that a divergence between the generation of excessive inflammation through over activation of the TLR associated myeloid differentiation primary response (MyD88) pathway relative to the TIR-domain-containing adaptor-inducing IFN-β (TRIF) pathway plays a key role in COVID-19 severity. Both viral elements and damage associated host molecules act as TLR ligands in this process. In this review, we detail the mechanism for this imbalance in COVID-19 based on available evidence, and we discuss how modulation of critical elements may be important in reducing severity of disease.

Objective: Current treatment and prognosis of Parkinson's disease (PD) are not ideal. This study explored the mechanism of long non-coding RNA (lncRNA) rhabdomyosarcoma 2-associated transcript (RMST) in dopaminergic (DA) neuron damage in PD rats., Methods: PD rats were modeled and injected with RMST silence or overexpression vectors to figure out its roles in oxidative stress, the apoptosis of DA neurons in brain substantia nigra (SN), and neurobehavioral activities of PD rats. Tyrosine hydroxylase (TH), synaptophysin (SYN), glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule (Iba-1) in SN were detected. RMST and Toll-like receptor (TLR)/nuclear factor kappa B (NF-κB) pathway-related factors were detected., Results: RMST expression in brain SN of rats, TLR2, TLR4 expression in neurons and NF-κB expression in cell nucleus were increased. Silenced RMST improved the neurobehavioral activities, depressed oxidative stress and neuronal apoptosis, increased TH and SYN expression, and reduced the activation degree of glial cells in SN and the inflammatory response via reducing GFAP and Iba-1. Moreover, reduced RMST reduced TLR2 and TLR4 expression in neurons and NF-κB expression in cell nucleus in PD rats., Conclusion: Inhibited RMST attenuates DA neuron damage in PD rats, which may be implicated with TLR/NF-κB signaling pathway., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory disease of the airway that represents a large global disease burden. Inflammation is a prominent feature of COPD and represents an important target for treatment. Toll-like receptors (TLRs) are pattern recognition receptors that detect invading microorganisms and nonmicrobial endogenous molecules to trigger inflammatory responses during host defense and tissue repair. The TLR signaling pathway is closely linked to the pathogenesis of COPD. Sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables, is well known for its anti-inflammatory activities. However, the molecular function of SFN in inhibition of COPD inflammation has yet to be fully elucidated. In this study, we investigated the effects of SFN on lipopolysaccharide (LPS)- or Pam3CysSerLys4 (Pam3CSK4)-induced inflammation in monocyte-derived macrophages (MDMs) from patients with COPD. MDMs from patients with COPD showed higher expression levels of TLR2, TLR4 and downstream myeloid differentiation factor 88 (MyD88) than healthy controls, along with increased secretion of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) (P < 0.05). Stimulation with TLR ligands (Pam3CSK4 and LPS) up-regulated the levels of TLR2, TLR4 and MyD88 in MDMs from patients with COPD and induced the release of IL-6 and TNF-α (P < 0.05). Pretreatment of MDMs from patients with COPD with SFN significantly suppressed Pam3CSK4- or LPS-induced TLR2, TLR4 and MyD88 expression, along with a reduction in the production of IL-6 and TNF-α (P < 0.05). Collectively, these data indicate that SFN exerts its anti-inflammatory activity in COPD by modulating the TLR pathway. SFN may represent a potential therapeutic agent for the treatment of COPD., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The objective of this study was to assess protective efficacy of vaccination using CPD-attenuated chimeric PRRSV and Toll like receptor (TLR) agonists (HSP70 c-terminal domain and HSPX) as adjuvants through different inoculation routes. In this study, a chimeric PRRSV composed of two field isolates was synthesized and attenuated by CPD in NSP1 as described in the previous study. The infection of the CPD-attenuated chimeric PRRSV to pigs of 3 weeks-old showed no clinical signs without pathological lesions in necropsy, while it induced improved cross immunity between its parent strains. The TLR agonists were expressed in E. coli and purified to be used. In challenge experiment, pigs of 3 weeks-old were vaccinated using the CPD-attenuated chimeric virus with the prepared TLR agonists through intramuscular or intradermal route, following heterologous challenge after 4 weeks of vaccination. In results, intramuscular or intradermal inoculation of the CPD-attenuated chimeric virus demonstrated excellent protective efficacy against heterologous challenges. Importantly, intradermal inoculation with the TLR agonists enhanced protective effects as shown in the significantly increased level of PRRSV-specific IFN-γ-SCs and cytokines in sera, and the significant reduction of pathological lesion and viral load in lung. This study suggested that the intradermal inoculation of CPD-attenuated chimeric PRRSV plus TLR agonists should be more effective for protection of pigs against diverse PRRS field viruses., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Morus alba L. bark has been widely used in traditional medicine for treating several inflammatory diseases, such as hypertension, diabetes mellitus and coughing; however, the molecular mechanisms underlying its anti-inflammatory effects are not well understood., Methods: We examined the effects of an extract of Morus alba L. bark (MabE) on Toll-like receptor (TLR) ligand-induced activation of RAW264.7 macrophages using a luciferase reporter assay and immunoassays. For the in vivo experiment, we used an imiquimod-induced ear edema model to examine the anti-inflammatory effects of MabE., Results: MabE inhibited the TLR ligand-induced activation of NF-κB in RAW264.7 cells without affecting their viability. Consistent with the inhibition of NF-κB activation, MabE also inhibited the production of IL-6 and IL-1β from TLR ligand-treated RAW264.7 cells. In vivo MabE treatment inhibited the ear swelling of IMQ-treated mice, in addition to the mRNA expression of IL-17A, IL-1β and COX-2. The increases in splenic γδT cells in IMQ-treated mice and the production of IL-17A from splenocytes were significantly inhibited by MabE treatment., Conclusion: Our study suggests that the anti-inflammatory effects of MabE on the activation of the macrophage cell line RAW246.7 by TLRs and IMQ-induced ear edema are through the inhibition of NF-κB activation and IL-17A-producing γδT cells, respectively.

Toxoplasma gondii is a prevalent parasite of medical and veterinary importance. Tachyzoïtes and bradyzoïtes are responsible for acute and chronic toxoplasmosis (AT and CT), respectively. In immunocompetent hosts, AT evolves into a persistent CT, which can reactivate in immunocompromised patients with dire consequences. Imiquimod is an efficient immunomodulatory drug against certain viral and parasitic infections. In vivo , treatment with Imiquimod, throughout AT, reduces the number of brain cysts while rendering the remaining cysts un-infectious. Post-establishment of CT, Imiquimod significantly reduces the number of brain cysts, leading to a delay or abortion of reactivation. At the molecular level, Imiquimod upregulates the expression of Toll-like receptors 7, 11, and 12, following interconversion from bradyzoïtes to tachyzoïtes. Consequently, MyD88 pathway is activated, resulting in the induction of the immune response to control reactivated Toxoplasma foci. This study positions Imiquimod as a potent drug against toxoplasmosis and elucidates its mechanism of action particularly against chronic toxoplasmosis, which is the most prevalent form of the disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hamie, Najm, Deleuze-Masquefa, Bonnet, Dubremetz, El Sabban and El Hajj.)

Background: Crohn's disease (CD) is characterized by chronic inflammation of the gastrointestinal tract with alternating periods of exacerbation and remission. The aim of this study was to determine the time-dependent effects of dietary oat beta-glucans on colon apoptosis and autophagy in the CD rat model., Methods: A total of 150 Sprague-Dawley rats were divided into two main groups: healthy control (H) and a TNBS (2,4,6-trinitrobenzosulfonic acid)-induced colitis (C) group, both including subgroups fed with feed without beta-glucans (βG-) or feed supplemented with low- (βGl) or high-molar-mass oat beta-glucans (βGh) for 3, 7, or 21 days. The expression of autophagy (LC3B) and apoptosis (Caspase-3) markers, as well as Toll-like (TLRs) and Dectin-1 receptors, in the colon epithelial cells, was determined using immunohistochemistry and Western blot., Results: The results showed that in rats with colitis, after 3 days of induction of inflammation, the expression of Caspase-3 and LC3B in intestinal epithelial cells did not change, while that of TLR 4 and Dectin-1 decreased. Beta-glucan supplementation caused an increase in the expression of TLR 5 and Dectin-1 with no changes in the expression of Caspase-3 and LC3B. After 7 days, a high expression of Caspase-3 was observed in the colitis-induced animals without any changes in the expression of LC3B and TLRs, and simultaneously, a decrease in Dectin-1 expression was observed. The consumption of feed with βGl or βGh resulted in a decrease in Caspase-3 expression and an increase in TLR 5 expression in the CβGl group, with no change in the expression of LC3B and TLR 4. After 21 days, the expression of Caspase-3 and TLRs was not changed by colitis, while that of LC3B and Dectin-1 was decreased. Feed supplementation with βGh resulted in an increase in the expression of both Caspase-3 and LC3B, while the consumption of feed with βGh and βGl increased Dectin-1 expression. However, regardless of the type of nutritional intervention, the expression of TLRs did not change after 21 days., Conclusions: Dietary intake of βGl and βGh significantly reduced colitis by time-dependent modification of autophagy and apoptosis, with βGI exhibiting a stronger effect on apoptosis and βGh on autophagy. The mechanism of this action may be based on the activation of TLRs and Dectin-1 receptor and depends on the period of exacerbation or remission of CD., Competing Interests: The authors declare no conflict of interest.

Rationale: Acute pancreatitis (AP) is a serious acute condition affecting the abdomen and shows high morbidity and mortality rates. Its global incidence has increased in recent years. Inflammation and oxidative stress are potential therapeutic targets for AP. This study was conducted to investigate the intrinsic anti-oxidative and anti-inflammatory effects of Prussian blue nanozyme (PBzyme) on AP, along with its underlying mechanism. Methods: Prussian blue nanozymes were prepared by polyvinylpyrrolidone modification method. The effect of PBzyme on inhibiting inflammation and scavenging reactive oxygen species was verified at the cellular level. The efficacy and mechanism of PBzyme for prophylactically treating AP were evaluated using the following methods: serum testing in vivo , histological scoring following hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling fluorescence staining, polymerase chain reaction array, Kyoto Encyclopedia of Genes and Genomes analysis and Western blotting analysis. Results: The synthetic PBzyme showed potent anti-oxidative and anti-inflammatory effects in reducing oxidative stress and alleviating inflammation both in vitro and in vivo in the prophylactic treatment of AP. The prophylactic therapeutic efficacy of PBzyme on AP may involve inhibition of the toll-like receptor/nuclear factor-κB signaling pathway and reactive oxygen species scavenging. Conclusion: The single-component, gram-level mass production, stable intrinsic biological activity, biosafety, and good therapeutic efficacy suggest the potential of PBzyme in the preventive treatment of AP. This study provides a foundation for the clinical application of PBzyme., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Nasopharyngeal carcinoma (NPC) originates in the nasopharyngeal epithelium. The most common treatments for NPC rT1-4 are radiotherapy and surgery. The pathogenesis of radiation-induced cognitive impairment is complex and includes oxidative stress, mitochondrial dysfunction, neuro-inflammation, and even apoptosis and cell death. Principally, toll-like receptors (TLRs) could regulate the inflammatory/anti-inflammatory balance in patients with radiation-induced brain injury. Vinpocetine has an anti-inflammatory effect as shown in both animal and in vitro studies. Also, dexamethasone is a widely used anti-inflammatory drug. Thus, it is important to test whether addition of vinpocetine could improve the anti-inflammatory properties of dexamethasone for the treatment of NPC patients with radiation-induced brain injuries., Methods: A total of 60 NPC patients with radiation-related brain injury were recruited for this study. All subjects were randomly and blindly assigned to the following groups: the dexamethasone group (D group, n = 30) and the vinpocetine and dexamethasone group (VD group, n = 30). Both medicine treatments were uninterrupted for 14 days of administration., Results: Combined administration of vinpocetine and dexamethasone lowered the expression levels of serum inflammatory cytokines, including TLR2, TLR4, interleukin (IL)-20, IL-8, tumor necrosis factor-α, interferon-γ, monocyte chemoattractant protein 2, and interferon-induced protein 20, when compared to dexamethasone monotherapy. Notably, combination therapy increased antioxidants (superoxide dismutase, glutathione, glutathione peroxidase, and glutathione reductase) and decreased oxidants (thiobarbituric acid reactive substances). Furthermore, combination therapy significantly increased the Mini Mental State Examination score, when compared to dexamethasone monotherapy., Conclusion: Administration of a combination of vinpocetine and dexamethasone may enhance the anti-inflammatory and anti-oxidative effects when compared to dexamethasone monotherapy, which leads to alleviated cognitive impairment in NPC patients with radiation injury., (© 2020 S. Karger AG, Basel.)

Introduction: Pattern recognition receptors are involved in innate and adaptive immunity by detecting microbial components. Bacteria have been accused to play a role in inflammatory acne. We investigated the potential involvement of Toll-like receptor (TLR)2, TLR4, TLR6, and CD14 in the direct influence of bacterial components and standard antiacne compounds on human sebocytes., Methods: mRNA and protein expression of TLR2, TLR4, TLR6, and CD14 in SZ95 sebocytes was evaluated by real-time qRT-PCR and immunocytochemistry. The effects of lipopolysaccharides (LPS) and lipoteichoic acid on TLR2, TLR4, and CD14 expression and of cytokine/chemokine secretion by 13-cis-retinoic acid, all-trans-retinoic acid, retinol, and hydrocortisone at the mRNA and protein levels were assessed by real-time qRT-PCR and ELISA and verified by cocultivation with neutralizing antibodies., Results: The constitutive expression of TLR2, TLR4, and CD14 in SZ95 sebocytes was augmented by exposure to LPS. Hydrocortisone induced TLR2, but markedly reduced TLR4 expression. 13-cis-retinoic acid and all-trans-retinoic acid regulated IL-6 release. LPS enhanced and hydrocortisone reduced cytokine and chemokine release. Anti-TLR4 and anti-CD14 mAb blocked LPS-induced IL-8 and IL-6 release., Conclusions: Microbial components use pattern recognition receptors to directly activate sebocytes to express a wide range of proinflammatory molecules and especially IL-8 and IL-6 in a TLR4- and CD14-specific manner. Retinoids, but mostly corticosteroids, also use this pathway to exhibit anti-inflammatory effects., (© 2021 S. Karger AG, Basel.)

Objective and Design: Methyl gallate (MG) is a prevalent polyphenol in the plant kingdom, which may be related to the effects of several medicinal plants. Although it is widely reported that polyphenols have therapeutic effects, there are few studies demonstrating that MG has anti-inflammatory action. This study aimed to investigate the molecular mechanism behind the anti-inflammatory activity of MG and its effect on hyperalgesia., Methods: Swiss mice were pretreated orally with different doses of MG and subjected to i.pl. injection of zymosan to induce paw edema. RAW264.7 macrophages and BMDMs stimulated with different TLR agonists such as zymosan, LPS, or Pam3CSK 4 were used to investigate the molecular mechanisms of MG RESULTS: MG inhibits zymosan-induced paw edema and hyperalgesia and modulates molecular pathways crucial for inflammation development. Pretreatment with MG inhibited cytokines production and NF-κB activity by RAW 264.7 cells stimulated with zymosan, Pam3CSK 4 or LPS, but not with PMA. Moreover, pretreatment with MG decreased IκB degradation, nuclear translocation of NF-κBp65, c-jun and c-fos and ERK1/2, p38 and JNK phosphorylation., Conclusion: Thus, the results of this study demonstrate that MG has a promising anti-inflammatory effect and suggests an explanation of its mechanism of action through the inhibition of NF-κB signaling and the MAPK pathway.

Background: Aspirin (acetylsalicylic acid) and celecoxib have been used as potential anti-cancer therapies. Aspirin exerts its therapeutic effect in both cyclooxygenase (COX)-dependent and -independent pathways to reduce tumor growth and disable tumorigenesis. Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, reduces factors that cause inflammation and pain. The question is whether aspirin and celecoxib have other molecular targets of equal or more therapeutic efficacy with significant anti-cancer preventive benefits., Aim: Here, we propose that aspirin and celecoxib exert their anti-cancer effects by targeting and inhibiting mammalian neuraminidase-1 (Neu-1). Neu-1 has been reported to regulate the activation of several receptor tyrosine kinases (RTKs) and TOLL-like receptors and their downstream signaling pathways. Neu-1 in complex with matrix metalloproteinase-9 (MMP-9) and G protein-coupled receptors (GPCRs) has been reported to be tethered to RTKs at the ectodomain., Materials and Methods: The WST-1 cell viability assay, Caspase 3/7 assay, and Annexin V assay were used to evaluate the cell viability and detect apoptotic and necrotic cells following treatment in MiaPaCa-2, PANC-1 and the gemcitabine-resistant PANC-1 variant (PANC-1 GemR) cells. Microscopic imaging, lectin cytochemistry, and flow cytometry were used to detect levels of α-2,3 sialic acid. Epidermal growth factor (EGF)-stimulated live cell sialidase assays and neuraminidase assays were used to detect Neu-1 activity. Immunocytochemistry was used to detect levels of EGFR and phosphorylated EGFR (pEGFR) following treatment., Results: For the first time, aspirin and celecoxib were shown to significantly inhibit Neu-1 sialidase activity in a dose- and time-dependent manner following stimulation with EGF. Aspirin blocked Neu-1 desialylation of α-2,3-sialic acid expression following 30 min stimulation with EGF. Aspirin and celecoxib significantly and dose-dependently inhibited isolated neuraminidase ( Clostridium perfringens ) activity on fluorogenic substrate 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MUNANA). Aspirin inhibited phosphorylation of the EGFR in EGF-stimulated cells. Aspirin dose- and time-dependently induced CellEvent caspase-3/7 + cells as well as apoptosis and necrosis on PANC-1 cells., Conclusion: These findings signify a novel multimodality mechanism(s) of action for aspirin and celecoxib, specifically targeting and inhibiting Neu-1 activity, regulating EGF-induced growth receptor activation and inducing apoptosis and necrosis in a dose- and time-dependent manner. Repurposing aspirin and celecoxib as anti-cancer agents may also upend other critical targets involved in multistage tumorigenesis regulated by mammalian neuraminidase-1., Significance: These findings may be the missing link connecting the anti-cancer efficacy of NSAIDs to the role of glycosylation in inflammation and tumorigenesis., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Qorri et al.)

Imetelstat sodium (GRN163L; hereafter, imetelstat) is a first-in-class telomerase inhibitor that has demonstrated activity in patients with myeloproliferative neoplasms (MPNs). Treatment with imetelstat has been associated with thrombocytopenia and other hematologic adverse effects that were manageable and reversible. Toll-like receptors (TLRs) are proteins that recognize pathogen-associated molecular patterns and stimulate innate immune and pro-apoptotic responses. Because imetelstat is an oligonucleotide, and some oligonucleotides can activate TLRs, we conducted an in vitro study to rule out the possibility of imetelstat-associated thrombocytopenia by off-target effects through activation of TLRs. We used HEK293 cell lines stably co-expressing a human TLR gene and an NFκB-inducible reporter to investigate whether imetelstat can activate TLR signaling. We treated the cells with imetelstat or control oligonucleotides for 20 h, and used absorbance of the culture media to calculate the reporter activity. Treatment with imetelstat within or beyond the clinically relevant concentrations had no stimulatory effect on TLR2, TLR3, TLR4, TLR5, TLR7, or TLR9. This result was not surprising since the structure of imetelstat does not meet the reported minimal structural requirements for TLR9 activation. Furthermore, imetelstat treatment of the MPN cell line HEL did not impact the expression of TLR signaling pathway target genes that are commonly induced by activation of different TLRs, whereas it significantly reduced its target gene hTERT , human telomerase reverse transcriptase, in a dose- and time-dependent manner. Hence, cytopenias, especially thrombocytopenia observed in some patients treated with imetelstat, are not mediated by off-target interactions with TLRs.

Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is spreading worldwide. Antiviral therapy is the most important treatment for COVID-19. Among the drugs under investigation, anti-malarials, chloroquine (CQ) and hydroxychloroquine (HCQ), are being repurposed as treatment for COVID-19. CQ/HCQ were shown to prevent receptor recognition by coronaviruses, inhibit endosome acidification, which interferes with membrane fusion, and exhibit immunomodulatory activity. These multiple mechanisms may work together to exert a therapeutic effect on COVID-19. A number of in vitro studies revealed inhibitory effects of CQ/HCQ on various coronaviruses, including SARS-CoV-2 although conflicting results exist. Several clinical studies showed that CQ/HCQ alone or in combination with a macrolide may alleviate the clinical symptoms of COVID-19, promote viral conversion, and delay disease progression, with less serious adverse effects. However, recent studies indicated that the use of CQ/HCQ, alone or in combination with a macrolide, did not show any favorable effect on patients with COVID-19. Adverse effects, including prolonged QT interval after taking CQ/HCQ, may develop in COVID-19 patients. Therefore, current data are not sufficient enough to support the use of CQ/HCQ as therapies for COVID-19 and increasing caution should be taken about the application of CQ/HCQ in COVID-19 before conclusive findings are obtained by well-designed, multi-center, randomized, controlled studies.

Multiple kinase inhibitors are available for patients with advanced hepatocellular carcinoma (HCC). It is largely unknown whether regorafenib or lenvatinib modulates innate immunity including Toll-like receptor (TLR)-signaling pathways in HCC. We performed real-time RT-PCR to investigate 84 TLR-associated gene expression levels and compared these gene expression levels in each hepatoma cells treated with or without regorafenib or lenvatinib. In response to regorafenib, nine and 10 genes were upregulated in Huh7 and HepG2 cells, respectively, and only C-X-C motif chemokine ligand 10 was upregulated in both cell lines. A total of 14 and 12 genes were downregulated in Huh7 and HepG2 cells, respectively, and two genes (Fos proto-oncogene, AP-1 transcription factor subunit, and ubiquitin conjugating enzyme E2 N) were downregulated in both cell lines. In response to lenvatinib, four and 16 genes were upregulated in Huh7 and HepG2 cells, respectively, and two genes (interleukin 1 alpha and TLR4) were upregulated in both cells. Six and one genes were downregulated in Huh7 and HepG2, respectively, and no genes were downregulated in both cell lines. In summary, regorafenib and lenvatinib affect TLR signaling pathways in human hepatoma cell lines. Modulation of TLR signaling pathway may improve the treatment of HCC patients with refractory disease.

Chronic lymphocytic leukaemia (CLL) cells are strongly influenced by microenvironmental signals through the activation of distinct membrane receptors including the B-cell receptor and toll-like receptors (TLR). Recapitulating TLR stimulation in vitro by treating CLL cells with the TLR9 ligand CpG can induce metabolic activation and protection from apoptosis. We hypothesized that interfering with TLR signalling may be beneficial for treating CLL, and we tested in preclinical studies the effect of a specific interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitory small molecule on primary leukaemic cells isolated from the peripheral blood of patients. We observed that IRAK4, an upstream kinase of the TLR pathway, is expressed in patients with CLL, and lower IRAK4 mRNA levels associate with a better outcome. The specific IRAK4 inhibitor disrupted TLR signalling as assessed by reduction of the specific biomarkers NFKBIZ and interleukin-6 mRNAs, and restrained the protective effect of in vitro TLR stimulation on cell viability. To note, IRAK4 inhibitor induced p53 and triggered apoptosis. Co-treatment of CLL cells with increasing concentrations of IRAK4i and the Bruton tyrosine kinase inhibitor ibrutinib demonstrated a synergistic effect. Our results suggest that targetting IRAK4 may represent a novel approach in CLL and may be combined with other signalling inhibitors., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Microglia, as the first line of defence of the central nervous system (CNS), has a major role in inflammatory response. It was reported that isoflurane has a neuroprotective role in the pathological process of CNS by interfering with inflammatory response. While the mechanism and function of isoflurane in microglia-mediated inflammation are still not clearly articulated. In our study, the inflammation model was established by the activation of lipopolysaccharide (LPS) in BV2 cells in vitro. The results demonstrated that isoflurane inhibited the release of nitric oxide (NO) and enhanced the survival of BV2 cells, meanwhile, isoflurane reduced the levels of inflammatory factors and downregulated the expressions of inflammation-related genes and proteins in LPS-mediated BV2 cells. Furthermore, we demonstrated that overexpression of high-mobility group box 1 protein (HMGB1) could reverse the reduction in NO concentration, enhancement of cell BV2 viability and inhibition of inflammatory response, which were mediated by isoflurane in LPS-induced BV2 cells. Therefore, we suggested that isoflurane inhibits the activation of LPS-induced neuro microglia and reduces the release of inflammatory factors by regulating HMGB1, suggesting that isoflurane might play a protective role in LPS-induced neuroinflammation through the HMGB1 pathway.

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediated by the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 3% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease. Here, we studied the effects of TLR activation and the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL. Our in vitro studies demonstrated that ND2158 preferentially killed CLL cells in a dose-dependent manner. We further observed a decrease in NF-κB and STAT3 signaling, cytokine secretion, proliferation and migration of primary CLL cells from MYD88-mutated and -unmutated cases. In the Eµ-TCL1 adoptive transfer mouse model of CLL, ND2158 delayed tumor progression and modulated the activity of myeloid and T cells. Our findings show the importance of TLR signaling in CLL development and suggest IRAK4 as a therapeutic target for this disease.

Emerging evidence shows that rheumatoid arthritis (RA) progression can be induced by the activation of Toll-like receptor (TLR) signaling pathway. Regulator of G-protein signaling 1 (RGS1) is observed to be a candidate biomarker for arthritis. Accordingly, the present study aims to determine the potential effects of RGS1 mediating TLR on RA. A rat model of collagen-induced arthritis (CIA) was established to mimic the features of RA by injection of bovine type II collagen. The rats with CIA were treated with short hairpin RNA (shRNA) against RGS1 or TLR pathway activator Poly I:C to elucidate the role of RGS1 in RA progression. The inflammatory factors were measured, and the thoracic gland and spleen indexes as well as the vascular density were determined. The expression levels of RGS1, TLR3, vascular endothelial growth factor (VEGF), metalloproteinase-2 (MMP-2), MMP-9, and interleukin 1 receptor-associated kinase-4 (IRAK4) were determined. RGS1 was robustly increased in RA. The TLR signaling pathway was suppressed by RGS1 silencing. shRNA-mediated depletion of RGS1 was shown to significantly enhance thoracic gland index and inhibit the serum levels of TNF-α, IL-1β, and IL-17, spleen index, vascular density, and the expression levels of TLR3, VEGF, MMP-2, MMP-9, and IRAK4. However, when the rats with CIA were treated with Poly I:C, the trend of effects was opposite. These findings highlight that functional suppression of RGS1 inhibits the inflammatory response and angiogenesis by inactivating the TLR signaling pathway in rats with CIA, thereby providing a novel therapeutic target for RA treatment., (© 2019 Wiley Periodicals, Inc.)

This study aims to investigate the role of Toll-like receptors (TLRs) on fine particulate matter (PM 2.5 )-induced inflammatory responses of vascular endothelial cells. Inflammatory factors and TLRs were examined in the aorta of mice after nonsurgical intratracheal instillation of PM 2.5 as well as in the human umbilical vein endothelial cells (HUVECs) treated with PM 2.5 . In addition, the effects of TLR2 and TLR4 inhibitors in the secretion of interleukin 6 (IL-6) and IL-1β and the expression of TLRs were determined in the HUVECs. The results showed that PM 2.5 could increase the expression of IL-1β, IL-6, TLR2, and TLR4 in vitro and in vivo . Anti-TLR2 IgG or TAK242, an inhibitor of TLR4, decreased the secretion of IL-1β and IL-6 by HUVECs and reduced the expression of corresponding TLRs. In conclusion, we demonstrate that both TLR2 and TLR4 are involved in PM 2.5 -induced inflammatory responses of vascular endothelial cells. Inhibition of TLR2 and TLR4 expression has the potential to prevent PM 2.5 -induced cardiovascular diseases.

The genus Acanthamoeba, which may cause different infections in humans, occurs widely in the environment. Lung inflammation caused by these parasites induces pulmonary pathological changes such as pulmonary necrosis, peribronchial plasma cell infiltration, moderate desquamation of alveolar cells and partial destruction of bronchial epithelial cells, and presence of numerous trophozoites and cysts among inflammatory cells. The aim of this study was to assess the influence of plant extracts from Artemisia annua L. on expression of the toll-like receptors TLR2 and TLR4 in lungs of mice with acanthamoebiasis. A. annua, which belongs to the family Asteraceae, is an annual plant that grows wild in Asia. In this study, statistically significant changes of expression of TLR2 and TLR4 were demonstrated. In the lungs of infected mice after application of extract from A. annua the expression of TLRs was observed mainly in bronchial epithelial cells, pneumocytes (to a lesser extent during the outbreak of infection), and in the course of high general TLR expression. TLR4 in particular was also visible in stromal cells of lung parenchyma. In conclusion, we confirmed that a plant extract of A. annua has a modulatory effect on components of the immune system such as TLR2 and TLR4., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objectives: To investigate the protective effects and potential mechanisms of Shenhua Tablet (, SHT) on the toll-like receptors (TLRs)-mediated signaling pathways in a rat model of kidney ischemia-reperfusion injury (IRI)., Methods: Sixty male Wistar rats were randomly divided into 5 groups: sham surgery, model control, astragaloside (150 mg•kg -1 •d -1 ), low- and high-dose SHT (1.5 and 3.0 g•kg -1 •d -1 , repectively) groups. One week after drug treatment, rats underwent surgery to establish the IRI models. At 24 h and 72 h after the modeling, serum creatinine (Scr) and blood urea nitrogen (BUN) were analyzed; pathological damage were scored after periodic acid-Schiffstaining. TLR2, TLR4 and myeloid differentiation factor 88 (MyD88) protein and mRNA expressions were detected by inmmunohistochemistry, Western blot and qPCR. Tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) protein expressions were detected by enzyme linked immunosorbent assay., Results: Compared with the sham group, the model group exhibited severe change in renal function (Scr: 189.42±21.50, P<0.05), pathological damage (damage score: 4.50±0.55, P<0.05), and the expression levels of TLR2, TLR4, MyD88, TNF-α, IL-6 were significantly higher than other groups. Meanwhile, the levels of TLRs in model group showed upward tendency from 24 to 72 h, unparalleled with pathological and functional changes. The aforementioned parameters were alleviated to a certain extent, and, in addition to TLRs, presented the obvious downward trending from the 24 to 72 h after the intervention in the SHT and astragaloside groups relative to the model (P<0.05); in particular, the most significant mitigation of these changes was observed in the SHT-H group (P<0.05)., Conclusion: TLRs may be an important spot to treat and research in acute kidney injury. SHT could effectively mitigate renal injuries and promote recovery of IRI injuries through suppression of degeneration induced by up-regulation of TLR2 and TLR4 expression levels in the MyD88-dependent signaling pathway and exhibit some dose dependence.

The antiviral and immunomodulatory role of vitamin D has been shown in various viral infections. However, there is scanty literature available about the effect of vitamin D supplementation in herpes simplex virus-1 (HSV-1) infection. Therefore, the present study aimed to evaluate the role of two different forms of vitamin D: 25-hydroxyvitamin D3 (25D3) and 1,25-dihydroxyvitamin D3 (1,25D3) against HSV-1 in HeLa cells. The HeLa cells were supplemented with either 25D3 or 1,25D3 before HSV-1 infection and were studied after 6, 12, and 24 h postinfection (p.i.). The mRNA levels of toll-like receptors (TLRs), (2, 3, 4, 7, and 9), vitamin D signaling genes, and HSV-1 were studied using real-time PCR. The HSV-1 DNA load was estimated in culture supernatant. The supplementation of 25D3 and 1,25D3 significantly downregulated the mRNA levels of TLR2 (p < 0.0001) at 12 h p.i. The mRNA levels of TLR9 were found to be significantly downregulated in 1,25D3-supplemented cells at 12 h p.i. Furthermore, the significant downregulation was observed in HSV-1 titer in both 25D3- and 1,25D3-supplemented cells at 24 h p.i.(p < 0.0001). However, the effect of 25D3 supplementation persisted till 24 h p.i. with significant downregulation of TLR2 (p < 0.05) mRNA levels. The supplementation of both 25D3 and 1,25D3 before HSV-1 infection was found to downregulate the viral titer and TLR2 mRNA during the intial phase of infection. However, the effect of 25D3 supplementation was found to last for a longer duration compared with 1,25D3.

Multipotent mesenchymal stromal cells (MSCs) support wound healing processes. These cells express toll-like receptors (TLRs). TLRs perform important key functions when the immune system is confronted with danger signals. TLR ligation by lipopolysaccharides (LPS) activates MSCs and induces intracellular signaling cascades, which affect their differentiation profile, increase the release of inflammatory cytokines and the production of reactive oxygen species. Continuing exposure to LPS triggers prolonged inflammatory reactions, which may lead to deleterious conditions, e.g. non-healing wounds. Cannabidiol (CBD) exerts anti-inflammatory processes through cannabinoid receptor dependent and independent mechanisms. In the present study, we examined whether CBD could influence the inflammatory MSC phenotype. Exposure to LPS increased the release of IL-6, as well as other soluble factors, and elevated levels of oxidized macromolecules found in cell homogenisates. While the amount of IL-6 was unaffected, co-treatment with CBD reduced the oxidative stress acting on the cells. LPS inhibited adipogenic as well as chondrogenic differentiation, which was attenuated by CBD treatment. In the case of adipogenesis, the disinhibitory effect probably depended on CBD interaction with the peroxisome proliferator-activated receptor-γ. CBD could exert mild immunosuppressive properties on MSCs, while it most effectively acted anti-oxidatively and by restoring the differentiation capacity upon LPS treatment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Receptor for advanced glycation end-products (RAGE) and toll-like receptors (TLRs) are the key factors indicating a danger to the organism. They recognize the microbial origin pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). The primary response induced by PAMPs or DAMPs is inflammation. Excessive stimulation of the innate immune system occurs in arterial wall with the participation of effector cells. Persistent adaptive responses can also cause tissue damage and disease. However, inflammation mediated by the molecules innate responses is an important way in which the adaptive immune system protects us from infection. The specific detection of PAMPs and DAMPs by host receptors drives a cascade of signaling that converges at nuclear factor- κ B (NF- κ B) and interferon regulatory factors (IRFs) and induces the secretion of proinflammatory cytokines, type I interferon (IFN), and chemokines, which promote direct killing of the pathogen. Therefore, signaling of these receptors' pathways also appear to present new avenue for the modulation of inflammatory responses and to serve as potential novel therapeutic targets for antiatherosclerotic therapy.

Purpose: The toll-like receptors (TLRs) stand at the interface of innate immune activation. We hypothesize to decrease the response of innate immunity activated by TLR4 by a safe, short-term, systemic immunosuppression., Methods: Two silicone block implants were placed into two dorsal subcutaneous pockets in 32 rats that were subdivided into four groups: The two study groups were the IV DEX group (single intravenous injection of dexamethasone 1 h before surgery) and the IV DEX + IP DEX group (in addition to a single intravenous injection of dexamethasone 1 h before surgery, intraperitoneal dexamethasone was administered for 10 days after surgery), and the two control groups were the untreated control group and the saline-treated control group. After 10 weeks, all animals were killed to determine capsular thickness, inflammatory cell density, presence of pseudoepitheliomatous hyperplasia, edema, necrosis, vascularization, TLR4 expression and myofibroblast proliferation., Results: No significant difference was observed in any parameter between the untreated and saline-treated control groups (p > 0.05). Capsular thickness, myofibroblast proliferation, TLR4 expression density were statistically different among study groups compared to control (p < 0.05)., Conclusions: This study demonstrates the relationship between toll-like receptors and fibrous capsule after implant surgery. Decreasing the innate immunity by a safe, short-term perioperative systemic immunosuppression resulted in decreased TLR4 expression and myofibroblast differentiation which could be a new research field in profibrotic pathophysiology underlying breast capsule formation., No Level Assigned: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Previous studies have shown that isosteviol sodium (STVNa) protects against permanent cerebral ischemia injury by inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-mediated inflammatory responses. Overwhelming evidence shows that toll-like receptors (TLRs) are the upstream regulators of NF-κB. On the basis of the similarity of the pathology caused by traumatic brain injury (TBI) and stroke, we speculated that STVNa may have a therapeutic effect against TBI through regulation of the TLRs/NF-κB signaling-mediated inflammatory response. Thus, we studied the potential therapeutic effects of STVNa and the underlying mechanisms. Male rats, subjected to controlled cortical impact (CCI) injury, were injected intraperitoneally with STVNa (5, 10, 20, 40, and 80 mg/kg, daily for 3 or 7 days) after trauma. Neurobehavioral scores, relative numbers of cortical lesions, and histology were examined. We also measured the mRNA and protein expression levels of TLRs/NF-κB signaling pathway-related genes including TLR2, TLR4, and NF-κB by quantitative real-time-PCR and western blotting, respectively, and concentrations of tumor necrosis factor-α and interleukin-1β by an enzyme-linked immunosorbent assay. The results indicated that STVNa (20 mg/kg) showed significant neuroprotective effects 3 and 7 days after TBI, including the reduction of cortical lesions, improvement of the neurological severity score, significantly increased number of restored neurons, decreased number of astrocytes, and lower concentrations of tumor necrosis factor-α and interleukin-1β. Results from quantitative real-time-PCR and western blotting also show that the mRNA and protein expression levels of TLR2, TLR4, and NF-κB were significantly lower in STVNa-treated rats compared with the vehicle-treated rats. The administration of STVNa attenuates the TLR/NF-κB signaling pathway-mediated inflammatory responses in the injured rat brain, and this may be the mechanism by which STVNa improves the outcome following TBI.

Inflammatory injury and neuronal apoptosis participate in the period of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Suppression of inflammation has recently been shown to reduce neuronal death and neurobehavioral dysfunction post SAH. Biochanin A (BCA), a natural bioactive isoflavonoid, has been confirmed to emerge the anti-inflammatory pharmacological function. This original study was aimed at evaluating and identifying the neuroprotective role of BCA and the underlying molecular mechanism in an experimental Sprague-Dawley rat SAH model. Neurobehavioral function was evaluated via the modified water maze test and modified Garcia neurologic score system. Thus, we confirmed that BCA markedly decreased the activated level of TLRs/TIRAP/MyD88/NF- κ B pathway and the production of cytokines. BCA also significantly ameliorated neuronal apoptosis which correlated with the improvement of neurobehavioral dysfunction post SAH. These results indicated that BCA may provide neuroprotection against EBI through the inhibition of inflammatory injury and neuronal apoptosis partially via the TLRs/TIRAP/MyD88/NF- κ B signal pathway.

BACKGROUND Emerging evidence suggests the potential of Dendrobium officinale Kimura et Migo (DO) in treating the complications of diabetes mellitus (DM). We evaluated the therapeutic potential of DO in treating diabetic nephropathy (DN) by preventing insulin resistance. MATERIAL AND METHODS A DN model was established. Mean glomerular volume of rats was estimated by the method of Weibel-Gomez. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to determine the expression of mRNAs and we used Western blot assay to determine the expression of proteins. The levels of fasting insulin (FINS) and glucagon (GLU) were measured and we assessed the levels of high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor-a (TNF-a), and interleukin-6 (IL-6) using the enzyme-linked immunosorbent assay (ELISA). RESULTS Compared with the Normal rats, the levels of urinary glucose, albuminuria, Scr, albuminuria/Scr and BUN, and the expression levels of CaN, TLR-2, TLR-4, MyD88, hs-CRP, TNF-a, and IL-6, the level of FINS, GLU, and HOMAIR were increased in DN, DO 1.0, DO 2.0, and DMBG groups. Compared with the DN rats, in DO 1.0, DO 2.0, and DMBG groups the glomerular volume was smaller, the levels of urinary glucose, albuminuria, Scr, albuminuria/Scr, and BUN, the expression levels of CaN, TLR-2, TLR-4, MyD88, hs-CRP, TNF-a, and IL-6, the level of FINS, GLU, and HOMA-IR were decreased. CONCLUSIONS We found that DO prevents insulin resistance in rats with DN. This may be associated with reduction of TLRs and inflammatory response, which should be further verified by loss of DO effects on DN after treatment of inhibitors of TLRs.

The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system - such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1beta, chemokine IL-8 and enzyme caspase-1 - was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and beta-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1beta, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.

Toll-like receptor (TLR) ligands (TLR-Ls) are critical activators of immunity and are successfully being developed as vaccine adjuvants in both mammals and birds. In this study, we investigated the synergistic effect of co-stimulation of membrane and endosomal TLRs on the innate immune responses using chicken bone marrow-derived macrophages (BMMs), and studied the effect of age on the induction of innate responses. BMMs from 1 and 4-week-old birds were stimulated with Pam3Cys-SK4 (PCSK; TLR2), synthetic monophosphoryl lipid A (MPLA), Di[3-deoxy-d-manno-octulosonyl]-lipid A ammonium salt (KLA; TLR4), Gardiquimod, Resiquimod (R848; TLR7), CpG class B and C (TLR21). Nitric oxide (NO) production and mRNA levels of IL-1β, IL-10 and IL-12p40 showed macrophages from 4-week-old birds showed more sensitive responses compared to 1-week-old birds. The most potent TLR-Ls, PCSK, MPLA and CpG B were used to study the effect of co-stimulation on macrophages. Co-stimulation with TLR21 and TLR4 synergistically up-regulated inflammatory-related genes, as well as NO production. However, incubation of splenocytes with PCSK, MPLA and CpG B did not induce cell proliferation. Moreover, treatment with CpG B led to significant cell death., (Copyright © 2018 Elsevier B.V. All rights reserved.)

One of the fundamental mechanisms whereby the innate immune system coordinates inflammatory signal transduction is through Toll-like receptors (TLRs), which function to protect and defend the host organism by initiating inflammatory signaling cascades in response to tissue damage or injury. TLRs are positioned at the neuroimmune interface, and accumulating evidence suggests that the inflammatory consequences of TLR activation on glia (including microglia and astrocytes), sensory neurons, and other cell types can influence nociceptive processing and lead to states of exaggerated and unresolved pain. In this review, we summarize our current understanding of how different TLRs and their accessory or adaptor molecules can contribute to the development and maintenance of persistent pain. The challenges and opportunities of targeting TLRs for new treatment strategies against chronic pain are discussed, including the therapeutic context of TLR-mediated signaling in opioid analgesia and chemotherapy-induced pain. Considering the prevalence of persistent pain and the insufficient efficacy and safety of current treatment options, a deeper understanding of Toll-like receptors holds the promise of novel therapies for managing pathological pain., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The treatment of acanthamoebiasis is a still a problem. Our previous studies showed that the application of extracts from Artemisia annua L. significantly prolonged the survival of mice infected by Acanthamoeba. This plant has medicinal properties in the treatment of human parasitic diseases. The aim of this study was to evaluate the effects of A. annua on expression of Toll-like receptors (TLRs) 2 and 4 in brain of mice with Acanthamoeba infection. Mice were infected with Acanthamoeba sp. strain Ac309 (KY203908) by intranasal inoculation without and after application of A. annua extract. The administration of extract from A. annua significantly reduced the level of expression of TLR2 and modified the level of expression of TLR4. A. annua extract is a natural substance that is well tolerated in animals and may be considered as a combination therapy in treatment of acanthamoebiasis. Our study suggested that A. annua extract may be used as an alternative therapeutic tool., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Lycium barbarum polysaccharides (LBPs) have multiple biological and pharmacological functions, including antioxidant, anti-inflammatory and anticancer activities. This research was conducted to evaluate whether LBPs could alleviate carbon tetrachloride (CCl 4 )-induced liver fibrosis and the underlying signaling pathway mechanism. Fifty male wistar rats were randomly allocated to five groups (n=10): control, CCl 4 and CCl 4 with 400, 800 or 1600mg/kg LBPs, respectively. Each wistar rat from each group was used for blood and tissue collections at the end of experiment. The results showed that CCl 4 induced liver fibrosis as demonstrated by increasing histopathological damage, α-smooth muscle actin expression, aspartate transaminase activities, alkaline phosphatase activities and alanine aminotransferase activities. LBPs supplementation alleviated CCl 4 -induced liver fibrosis as demonstrated by reversing the above parameters. In addition, CCl 4 treatment induced the oxidative injury, increased the mRNA levels of tumor necrosis factor-α, monocyte chemoattractant protein-1 and interleukin-1β, and up-regulated the protein expressions of toll-like receptor 4 (TLR4), TLR2, myeloid differentiation factor 88, nuclear factor-kappa B (NF-kB) and p-p65. LBPs supplementation alleviated CCl 4 -induced oxidative injury, inflammatory response and TLRs/NF-kB signaling pathway expression by reversing the above some parameters. These results suggest that the alleviating effects of LBPs on CCl 4 -induced liver fibrosis in wistar rats may be through inhibiting the TLRs/NF-kB signaling pathway expression., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Oral cancer accounts for 10% of head and neck tumors. Despite the recent advancements in surgical techniques, as well as in chemo- and radiotherapy therapeutic protocols, survival rates for oral cancer patients have not improved significantly in the last decades. Recently, toll like receptors (TLRs) have been described as promoters of cell proliferation, invasiveness, and angiogenesis in a variety of cancers. The aim of this review is to identify how TLRs participate in oral carcinogenesis in order to evaluate their biological relevance. Data revealed that some TLRs participate in oral carcinogenesis since TLR5 and TLR9 promote tumor growh, whereas TLR3 is closely involved to anti-cancer properties. They represent a promising alternative for oral cancer therapy. However, TLR2, TLR4 and TLR7 need further investigation, since current data are not sufficient to conclude about their role. Certainly, such data will contribute to new scientific knowledge, which will be incorporated to the preventive actions and therapeutic protocols for oral cancer patients suffering from this devasting disease., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objectives: Lipopolysaccharides (LPSs) and lipoteichoic acids (LTAs), or bacterial endotoxins, bind with Toll-like receptors (TLRs) that are expressed on host cells of the periodontium, thereby contributing to the periodontal pathogenicity of oral bacteria. Stannous fluoride (SnF2), an antibacterial fluoride that treats and controls gingivitis, has been shown to react with lipophilic domains/anionic charges in LPS and LTA. The effects of bacterial species and dental plaque on toll receptors can be studied using genetically engineered cell lines containing linked toll receptors on their surfaces. This randomized, examiner-blinded study examined the clinical effects of stabilized SnF2 dentifrice intervention on gingivitis and dental plaque virulence in populations exhibiting high and low levels of clinical gingivitis., Methods: Recruited populations were evaluated for gingival inflammation (MGI) and gingival bleeding (GBI) at baseline and assigned into two cohorts of 20 each, those with high (GBI > 20 sites) and low (GBI < 3 sites) levels of observed bleeding/gingivitis. Participants were sampled at baseline for both supra- and subgingival dental plaque at both healthy (no bleeding, PD = 2 mm), as well as clinically diseased sites (bleeding, PD = 3-4 mm), and then provided with an intervention hygiene product including a stabilized SnF2 dentifrice and a new soft bristle manual toothbrush. Following two and four weeks of assigned dentifrice use, participants returned for a re-evaluation of gingival inflammation and bleeding and repeat samplings of dental plaque. Plaque samples were analyzed by anaerobic culturing of gram negative anaerobes (GNA), as well as by incubation of subgingival sampled plaques with TLR4 transfected HEK293 cells, where gene expression was assessed by measurement of a SEAP alkaline phosphatase reporter as a marker of toll receptor activation., Results: Clinical assessments showed statistically significant reductions in MGI (24-26%) and GBI (42-53%) gingivitis in both diseased and healthy cohorts following four weeks of dentifrice intervention. For all clinical examinations, MGI and bleeding sites were statistically significantly different (lower) in the low bleeding versus the higher bleeding cohort. Supragingival and subgingival GNAs were significantly reduced (p < 0.05) in both high and low disease cohorts at bleeding and healthy sites following four weeks of stabilized SnF2 dentifrice use. TLR activation from subgingival sampled plaque was reduced following four weeks of stabilized SnF2 dentifrice use in both high and low disease cohorts and in both healthy, as well as diseased sites., Conclusions: Collectively, these results support the potential for stabilized SnF2 dentifrice to provide clinical gingivitis benefits via mechanisms beyond control of plaque mass, potentially directly decreasing the pathogenicity of plaque biofilms by blocking reactivity of LPS and LTA ligands with tissue receptors associated with inflammation. Importantly, benefits could be seen in both diseased sites, as well as sites not yet exhibiting symptoms of inflammation, supporting the activity of SnF2 not just in treating diseased sites, but in preventing disease development. These learnings may influence treatment planning for patients susceptible to gingivitis., Competing Interests: This study was supported by Procter & Gamble. All authors are employees of Procter & Gamble.

Background: Toll-like receptor (TLR)-mediated inflammation may contribute to neonatal sepsis, for which pentoxifylline (PTX), a phosphodiesterase inhibitor that raises intracellular cAMP, is a candidate adjunctive therapy. We characterized the anti-inflammatory effects of PTX toward TLR-mediated production of inflammatory (tumor necrosis factor (TNF) and interleukin (IL)-1β) and proresolution (IL-6 and IL-10) cytokines in human newborn and adult blood., Methods: Newborn cord and adult blood were treated with PTX (50-400 µmol/l) before, during or after stimulation with LPS (TLR4 agonist), R848 (TLR7/8 agonist) or LPS/ATP (inflammasome activation). Cytokines were measured by multiplex assay (supernatants), intracellular cytokines and signaling molecules by flow cytometry, and mRNA by quantitative real-time PCR., Results: Whether added 2 h pre-, simultaneously to, or 2 h post-TLR stimulation, PTX inhibited TLR-mediated cytokine production in a concentration-dependent manner, with greater efficacy and potency in newborn blood, decreasing intracellular TNF and IL-1β with relative preservation of IL-10 and IL-6. PTX decreased TLR-mediated TNF mRNA while increasing IL-10 mRNA. Neonatal plasma factors contributed to the anti-inflammatory effects of PTX in newborn blood that were independent of soluble TNF receptor concentrations, p38 MAPK phosphorylation and IĸB degradation., Conclusion: PTX is a potent and efficacious inhibitor of TLR-mediated inflammatory cytokines in newborn cord blood and a promising neonatal anti-inflammatory agent.

Introduction: The potential roles of toll-like receptors (TLRs) in immunopathogenesis of Ebola virus disease should be unraveled to provoke possible prophylactic or therapeutic implications of TLRs for EVD. Areas covered: The Ebola virus (EBOV) infection virtually paralyses all the main mechanisms responsible for induction of type I interferon (IFN-I) response. To summarize, EBOV infection interferes with: a) the TIR-domain-containing adapter-inducing interferon-β (TRIF) pathway that is mediated by TLR3 and TLR4 signaling; b) the interferon regulatory factor 7 (IRF7) pathway that is stimulated by TLR7 and TLR9; c) the intracellular signaling that is induced by retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs); and d) the autocrine/paracrine feedback loop that is mediated by the IFN-stimulated gene factor 3 (ISGF3) complex. Upon infection with EBOV infection, TLR4 plays a key role in production of proinflammatory mediators. Expert opinion: It is theoretically possible that use of TLRs 3, 4, 7, and 9 agonists would be beneficial to improve the IFN-I response, despite their systemic side effects. Also, antagonist of TLR4 can be utilized to prevent production of proinflammatory cytokines. Additionally, it is highly recommended to design future investigations aimed at determining if the utilization of IFN-I would be beneficial for prophylactic/therapeutic programs of Ebola.

As essential proteins of the innate immune system, Toll-like receptors (TLRs) are involved in a plethora of physiological pathologies and their modulation is an ongoing quest in the field of drug discovery. Although TLRs recognize an unusually broad range of different molecular patterns, only a few small-molecule TLR modulators have been reported to date. Recent advances in crystallography and in silico techniques provide promising opportunities for TLR investigations and drug design. Here, three application areas for computational approaches are considered: (i) exploration of TLR structure and activation; (ii) understanding TLR modulation; and (iii) TLR drug discovery. By providing an overview on state-of-the-art computational methods, we highlight the value of molecular modeling in mechanistically understanding TLR function and guiding drug design., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The aim of this study was to determine, using murine RAW 264.7 macrophages, the immunomodulatory effect of extracellular β-glucan isolated from Pleurotus eryngii (PEBG) and its sulfated derivative (PEBG-S) on signaling molecules implicated in host innate immunity. β-Glucan was extracted and purified from the mycelial culture using optimal medium concentrations. It was then chemically converted to its sulfated form. Monosaccharide composition of β-glucan was characterized with p-aminobenzoic acid ethyl ester-derivatized sugars through highperformance liquid chromatography analysis. Fourier transform infrared structural analysis showed an S=O bond at 1250 cm-1 and C-S-O binding at 815 cm-1 in PEBG-S. 13C nuclear magnetic resonance analysis showed 1,3-linked α-D-mannopyranosyl and 1,3-β-D-glucopyranosyl in PEBG-S. A concentration-dependent increase of nitric oxide production was noticed in RAW 264.7 cells treated with PEBG-S or PEBG; those treated with PEBG-S showed less cytotoxicity than those treated with PEBG. Cellular levels of tumor necrosis factor-α, interleukin-1β, and interleukin-6 were increased by PEBG and PEBG-S treatment, suggesting that they have immunomodulatory activity. Real-time polymerase chain reaction array revealed that the expression levels of nuclear factor-κB and Toll-like receptor signaling genes in cells were upregulated by PEBG and PEBG-S. Moreover, the expression of the β-glucan receptor dectin-2 was significantly upregulated by PEBG and PEBG-S treatment, reflecting immune activation through the dectin-2-Syk-(CARD9/Bcl-10/MALT1) pathway. Our results suggest that PEBG-S could be used as an effective adjuvant or immune enhancer that can be sustainably produced by recycling the by-product of mycelial culture.

The invading microbial pathogens are controlled by the rapid and effective innate immune responses sequentially formation of the long-lasting adaptive memories. Toll-like receptors (TLRs) play a vital role in innate and adaptive immune response by function as a bridge to modulate the immune response. Further, genetic studies in human or animals showed that regulation of TLR signaling contributes to the antibacterial efficacy, and developing novel reagent to modulate TLR related immune response becomes an interesting therapy method to against bacterial infections. Herein we review the recent developments of this area, focusing on the reagent of synthetic molecules, natural products and peptides (or proteins), as TLR-related antibacterial drugs.

Drugs of abuse cause persistent alterations in synaptic plasticity that may underlie addiction behaviors. Evidence suggests glial cells have an essential and underappreciated role in the development and maintenance of drug abuse by influencing neuronal and synaptic functions in multifaceted ways. Microglia and astrocytes perform critical functions in synapse formation and refinement in the developing brain, and there is growing evidence that disruptions in glial function may be implicated in numerous neurological disorders throughout the lifespan. Linking evidence of function in health and under pathological conditions, this review will outline the glial and neuroimmune mechanisms that may contribute to drug-abuse liability, exploring evidence from opioids, alcohol, and psychostimulants. Drugs of abuse can activate microglia and astrocytes through signaling at innate immune receptors, which in turn influence neuronal function not only through secretion of soluble factors (eg, cytokines and chemokines) but also potentially through direct remodeling of the synapses. In sum, this review will argue that neural-glial interactions represent an important avenue for advancing our understanding of substance abuse disorders.

Purpose: To apply quantitative Toll-like receptors (TLR) cell assays to compare lipopolysaccharides (LPSs) and lipoteichoic acids (LTAs) from different oral bacterial strains for potential pathogenicity in vitro., Methods: The potency of LPS and LTA from different bacteria on activation of TLR reporter genes in HEK-tlr cell lines was examined. P. gingivalis LPS mix, P. gingivalis 1690 LPS, P. gingivalis 1435/50 LPS, E. coli LPS (E. coli K12), B. subtilis LTA, S. aureus LTA, E. hirae LTA and S. pyogenes LTA were examined in both TLR2 and TLR4 HEK cell line reporter assays. Solutions of LPS and LTA from selected bacteria were applied in a dose response fashion to the TLR reporter cells under standard culture conditions for mammalian cells. Reporter gene secreted-embryonic-alkaline-phosphatase (SEAP) was measured, and half maximal effective concentration (EC50) was determined for each sample. Concentration dependent TLR activation was compared to similar responses to LPS and LTA for commercial BODIPY-TR-Cadaverine and LAL biochemical (non cell based) assays., Results: All LPS from P. gingivalis activated both TLR2 and TLR4 responses. E. coli LPS is a strong activator for TLR4 but not for TLR2 responses. In contrast, both B. subtilis and S. aureus LTA provoked responses only in TLR2, but not in the TLR4 assay. Interestingly, E. hirae LTA and S. pyogenes LTA did not stimulate strong TLR2 responses. Instead, both E. hirae LTA and S. pyogenes LTA mounted a reasonable response in TLR4 reporter gene assay. Both LPS and LTA showed deactivation of fluorescence in BODIPY-TR-Cadaverine while only LPS was active in LAL. As with biochemical assays, an EC50 could be determined for LPS and LTA from various bacterial strains. The EC50 is defined as a concentration of LPS or LTA that provokes a response halfway between the baseline and maximum responses. Lower EC50 means higher potency in promoting TLR responses, and in principle indicates greater toxicity to the host., Clinical Significance: InvivoGen TLR2 and TLR4 assays distinguish specific types of microbial products, such as LPS and LTA from different bacteria. Application of EC50 determinations creates a means for quantitative and comparisons of LPS and LTA virulence in a cellular-based assay and combinations of TLR reporter cell assays along with biochemical evaluation of LPS#47;LTA in BODIPY-TR-Cadaverine and LPS in LAL assays provides a means to quantitate virulence of plaque samples with respect to both LPS and LTA. These learnings have long-term implications for patient care in that understanding the virulence of patients' plaque provides important information to assess risk of oral diseases., Competing Interests: All authors are employees of Procter $ Gamble Co.

Purpose: To study the reactivity of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) with the cationically charged agents cetylpyridinium chloride, stannous fluoride, and the non-cationic agent triclosan. We also assessed the effect of these agents to inhibit LPS and LTA binding to cellular Toll-like Receptors (TLRs) in vitro., Methods: The ability of these antimicrobials to bind with LPS and/or LTA was assessed in both the Limulus amebocyte lysate and BODIPY-TR-cadaverine dye assays. Mass spectroscopy was then used to confirm that stannous fluoride directly binds with LPS and to determine stoichiometry. Lastly, we looked for possible inhibitory effects of these antimicrobial agents on the ability of fluorescently conjugated LPS to bind to TLR4 expressed on HEK 293 cells., Results: Cetylpyridinium chloride (CPC) and stannous salts including stannous fluoride interfered with LPS and LTA reactivity in both dye assays, while triclosan had no effect. Mass spectroscopy revealed direct binding of stannous fluoride with E. Coli LPS at 1:1 stoichiometric ratios. In the cellular assay, cetylpyridinium chloride and stannous fluoride, but not triclosan, inhibited LPS binding to TLR4., Clinical Significance: These results support a potential mechanism of action for stannous fluoride and CPC formulated in oral products in which these ingredients bind bacterial toxins and potentially render them less toxic to the host. These results may influence home care recommendations for patients at risk for plaque-related diseases., Competing Interests: All authors are employees of Procter $ Gamble Co.

Objective: Oral epithelial cells have currently been found to play an important role in inflammatory modulation in periodontitis. Mangiferin is a natural glucosylxanthone with anti-inflammatory activity. The aim of this study was to investigate the regulatory effect of mangiferin on lipopolysaccharide (LPS)-induced production of proinflammatory cytokine interleukin-6 (IL-6) in oral epithelial cells and the underlying mechanisms., Design: The levels of LPS-induced IL-6 production in OKF6/TERT-2 oral keratinocytes were detected using enzyme-linked immunosorbent assay (ELISA). The expression of Toll-like receptor (TLR) 2 and TLR4 was determined using western blot analysis. And the phosphorylation of TLR downstream nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) was examined using cell-based protein phosphorylation ELISA kits., Results: We found that mangiferin reduced LPS-upregulated IL-6 production in OKF6/TERT-2 cells. Additionally, mangiferin inhibited LPS-induced TLR2 and TLR4 overexpression, and suppressed the phosphorylation of NF-κB, p38 MAPK and JNK. Moreover, mangiferin repressed IL-6 production and TLR signaling activation in a dose-dependent manner after 24h treatment., Conclusions: Mangiferin decreases LPS-induced production of IL-6 in human oral epithelial cells by suppressing TLR signaling, and this glucosylxanthone may have potential for the treatment of periodontitis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)