Chemical reagents modulate nucleic acid-activated toll-like receptors.

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.

Nano-Enabled Reposition of Proton Pump Inhibitors for TLR Inhibition: Toward A New Targeted Nanotherapy for Acute Lung Injury.

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.

TLRs in COVID-19: How they drive immunopathology and the rationale for modulation.

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-ß (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.

Targeting the Toll-like receptor pathway as a therapeutic strategy for neonatal infection.

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.

Human prostate epithelial cells and prostate-derived stem cells malignantly transformed in vitro with sodium arsenite show impaired Toll like receptor -3 (TLR3)-associated anti-tumor pathway.

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 ß (RARß) 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 RARß expression, on As-CSC and iAs-transformed epithelial cells (CAsE-PE). As-CSCs and CAsE-PE showed lower TLR3 and RARß 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 RARß 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.

Down-regulated long non-coding RNA RMST ameliorates dopaminergic neuron damage in Parkinson's disease rats via regulation of TLR/NF-κB signaling pathway.

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.

Intradermal co-inoculation of codon pair deoptimization (CPD)-attenuated chimeric porcine reproductive and respiratory syndrome virus (PRRSV) with Toll like receptor (TLR) agonists enhanced the protective effects in pigs against heterologous challenge.

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.

Anti-inflammatory effects of Morus alba Linne bark on the activation of toll-like receptors and imiquimod-induced ear edema in mice.

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-1ß 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-1ß 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.

Imiquimod Targets Toxoplasmosis Through Modulating Host Toll-Like Receptor-MyD88 Signaling.

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.

Sulforaphane suppresses lipopolysaccharide- and Pam3CysSerLys4-mediated inflammation in chronic obstructive pulmonary disease via toll-like receptors.

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.

Prussian blue nanozyme-mediated nanoscavenger ameliorates acute pancreatitis via inhibiting TLRs/NF-κB signaling pathway.

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.

Involvement of Pattern Recognition Receptors in the Direct Influence of Bacterial Components and Standard Antiacne Compounds on Human Sebaceous Gland Cells.

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.

Effects of Dietary Oat Beta-Glucans on Colon Apoptosis and Autophagy through TLRs and Dectin-1 Signaling Pathways-Crohn's Disease Model Study.

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 (ßG-) or feed supplemented with low- (ßGl) or high-molar-mass oat beta-glucans (ßGh) 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 ßGl or ßGh resulted in a decrease in Caspase-3 expression and an increase in TLR 5 expression in the CßGl 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 ßGh resulted in an increase in the expression of both Caspase-3 and LC3B, while the consumption of feed with ßGh and ßGl 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 ßGl and ßGh significantly reduced colitis by time-dependent modification of autophagy and apoptosis, with ßGI exhibiting a stronger effect on apoptosis and ßGh 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.

Combination of Vinpocetine and Dexamethasone Alleviates Cognitive Impairment in Nasopharyngeal Carcinoma Patients following Radiation Injury.

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.

Novel Molecular Mechanism of Aspirin and Celecoxib Targeting Mammalian Neuraminidase-1 Impedes Epidermal Growth Factor Receptor Signaling Axis and Induces Apoptosis in Pancreatic Cancer Cells.

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.

Methyl gallate attenuates inflammation induced by Toll-like receptor ligands by inhibiting MAPK and NF-Κb signaling pathways.

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 Pam3CSK4 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, Pam3CSK4 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.

Myelosuppression in Patients Treated with the Telomerase Inhibitor Imetelstat Is Not Mediated through Activation of Toll-Like Receptors.

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.

Advances in the use of chloroquine and hydroxychloroquine for the treatment of COVID-19.

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.

Different Mechanisms of Action of Regorafenib and Lenvatinib on Toll-Like Receptor-Signaling Pathways in Human Hepatoma Cell Lines.

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.

Neuroprotective effects of isoflurane against lipopolysaccharide-induced neuroinflammation in BV2 microglial cells by regulating HMGB1/TLRs pathway.

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.