Toll-like Receptor 4 Modulation Research Articles

Shedding light on the molecular and regulatory mechanisms of TLR4 signaling in endothelial cells under physiological and inflamed conditions.

Toll-like receptor 4 (TLR4) are part of the innate immune system. They are capable of recognizing pathogen-associated molecular patterns (PAMPS) of microbes, and damage-associated molecular patterns (DAMPs) of damaged tissues. Activation of TLR4 initiates downstream signaling pathways that trigger the secretion of cytokines, type I interferons, and other pro-inflammatory mediators that are necessary for an immediate immune response. However, the systemic release of pro-inflammatory proteins is a powerful driver of acute and chronic inflammatory responses. Over the past decades, immense progress has been made in clarifying the molecular and regulatory mechanisms of TLR4 signaling in inflammation. However, the most common strategies used to study TLR4 signaling rely on genetic manipulation of the TLR4 or the treatment with agonists such as lipopolysaccharide (LPS) derived from the outer membrane of Gram-negative bacteria, which are often associated with the generation of irreversible phenotypes in the target cells or unintended cytotoxicity and signaling crosstalk due to off-target or pleiotropic effects. Here, optogenetics offers an alternative strategy to control and monitor cellular signaling in an unprecedented spatiotemporally precise, dose-dependent, and non-invasive manner. This review provides an overview of the structure, function and signaling pathways of the TLR4 and its fundamental role in endothelial cells under physiological and inflammatory conditions, as well as the advances in TLR4 modulation strategies.

SPA4 peptide suppresses the lipopolysaccharide (LPS)-stimulated lactate secretion in lung epithelial cells through modulation of Toll-like receptor-4 (TLR4)

Abstract Lung infection and inflammation can cause severe injury leading to acute respiratory distress syndrome. An increased expression and activity of the TLR4 is associated with inflammatory response. Simultaneous elevated level of lactate is used as the marker of disease severity and is associated with poor outcomes in critically ill patients. Therapeutic treatment with TLR4-interacting SPA4 peptide (amino acid sequence: GDFRYSDGTPVNYTNWYRGE) suppresses inflammation in immune cells and in mouse models of lung infection and inflammation. In this study, we evaluated the effect of SPA4 peptide on lactate production by H441 human lung epithelial cells against Pseudomonas aeruginosa-derived LPS. Briefly, the H441 cells were challenged with LPS (1 μg/ml) and treated with SPA4 peptide (100 μM) after 1 h of LPS challenge. Next day, the cell-free supernatants and cell lysates were harvested. We also transfected the cells with TLR4 siRNA to achieve suppression of TLR4. Levels of protein and lactate were quantitated in cell lysates and in filtered supernatants using biochemical assays. The lactate levels were normalized with cell protein. The unchallenged, vehicle-treated cells served as control. Our results demonstrated reduced secretion of lactate by SPA4 peptide-treated cells (187.5% of control) versus vehicle-treated cells (324.7% of control) against LPS stimuli (p<0.05). The immunostaining and confocal imaging showed knockdown of TLR4 in up to 50% cells. The LPS-stimulated lactate secretion was reduced and SPA4 peptide treatment did not further affect the lactate levels in TLR4 siRNA transfected cells. Together, our results suggest that the SPA4 peptide suppresses lactate secretion through modulation of TLR4 in lung epithelial cells. Supported by grant from NIH (R01HL136325) and 2021 American Association of Immunologists Careers in Immunology Fellowship Program.

Small Molecules as Toll-like Receptor 4 Modulators Drug and In-House Computational Repurposing.

The innate immunity toll-like receptor 4 (TLR4) system is a receptor of paramount importance as a therapeutic target. Virtual screening following a “computer-aided drug repurposing” approach was applied to the discovery of novel TLR4 modulators with a non-lipopolysaccharide-like structure. We screened almost 29,000 approved drugs and drug-like molecules from commercial, public, and in-house academia chemical libraries and, after biological assays, identified several compounds with TLR4 antagonist activity. Our computational protocol showed to be a robust approach for the identification of hits with drug-like scaffolds as possible inhibitors of the TLR4 innate immune pathways. Our collaborative work broadens the chemical diversity for inspiration of new classes of TLR4 modulators.

Castration immunoregulates toll-like receptor-4 in male bladder cancer.

Among diverse Pattern Recognition Receptors (PRRs), Toll-like receptor-4 (TLR-4) is a key urothelial trigger for innate immune response impacting urothelial bladder carcinoma (BC). Androgen activation promotes immunotolerance, playing an immunoregulatory role by unknown mechanisms. We explored the castration impact on urothelial TLR-4 modulation in carcinogenesis and immunotherapeutic scenario. Intact (SHAM) versus castrated male Fisher-344 rats were evaluated in 2 scenarios: (A) Carcinogenesis: After randomization to SHAM (n = 5) and Castration (n = 5), carcinogenesis was induced by four intravesical doses of 1.5mg/kg n-methyl-n-nitrosourea (MNU) every 15days. (B) Treatment: After ultrasonographic confirmed MNU-induced papillary BC on week 8, rats were randomized to SHAM (n = 5) and Castration (n = 5) and offered 6 weekly intravesical treatment of 106CFU of bacillus Calmette Guerin (BCG) in 0.2ml saline. After 15weeks the urinary bladders underwent histopathology. Urothelial cell proliferation was measured by Ki-67 immunohistochemistry (IHC), and TLR-4 expression was quantified by IHC and WB. Castration induced higher TLR-4 urothelial expression (p = 0.007) and anticarcinogenic effect with fewer urothelial tumors (60 vs. 80%) and lower urothelial cell proliferation compared to intact animals (p = 0.008). In the intravesical BCG treatment setting, castration has potentialized the BCG activation of TLR-4 (p = 0.007) with no residual in situ carcinoma compared to intact animals, suggesting the potential to amplify the BCG immune response. To our knowledge, this is the first description of TLR-4 urothelial expression hormonal modulation. The described castration-mediated immunomodulation will help to improve the knowledge of urothelial cancer gender diversities and PRRs modulations with treatment implications.

Cyanidin and delphinidin restore colon physiology in high fat diet-fed mice: Involvement of TLR-4 and redox-regulated signaling

Consumption of high fat diets (HFD) mimics a modern or “Western style” diet pattern and can impair intestinal barrier integrity, leading to endotoxemia and associated unhealthy conditions. This study investigated if supplementation with an anthocyanin (cyanidin and delphinidin glucosides)-rich extract (CDRE) could revert or mitigate HFD-induced alterations of colonic physiology in part through the regulation of Toll-Like Receptor 4 (TLR-4)- and redox-regulated signaling. C57BL/6J male mice were fed for 4 weeks with a control or an HFD. Then, mice were divided in four groups fed either control or HFD, or these diets supplemented with CDRE for the subsequent 4 weeks. After 8 weeks on the HFD we observed in the colon: i) disruption of tight junction structure and function; ii) increased TLR-4 expression; iii) increased NADPH oxidase NOX1 expression, and iv) activation of redox-sensitive and TLR-4-triggered pathways, i.e. NF-κB, ERK1/2, JNK1/2, PI3K/Akt. All these events were prevented or reverted by CDRE supplementation. Supporting the relevance of CDRE-mediated downregulation of TLR-4 on its colon beneficial effect; in vitro (Caco-2 cell monolayers), cyanidin, delphinidin and their metabolites protocatechuic and gallic acid, mitigated lipopolysaccharide (LPS)-induced monolayer permeabilization by restoring tight junction structure and dynamics and preventing lipid/protein oxidation. The CDRE also mitigated HFD-mediated alterations in parameters of goblet cell differentiation and function, including the downregulation of markers of goblet cell differentiation (Klf4), and intestinal mucosa healing (Tff3). Results show that a short-term supplementation with cyanidin and delphinidin, protect from HFD-induced alterations in colon physiology in part through the modulation of TLR-4- and redox-regulated signaling.

Angiotensin 1–7 mitigates rhabdomyolysis induced renal injury in rats via modulation of TLR-4/NF-kB/iNOS and Nrf-2/heme‑oxygenase-1 signaling pathways

AimsRhabdomyolysis (RM) is a critical condition with a high mortality rate, but effective management is still deficient. Till date, there are no studies that have addressed the effect of angiotensin 1–7 in this condition, hence, the rationale of this study was to evaluate the potential protective effect of Angiotensin 1–7 (Ang1–7), on rhabdomyolysis (RM) induced kidney injury in rats and detecting the underlying mechanistic insights. Main methodsForty adult male albino rats were divided into groups; the control group, RM group, RM+Ang1-7 group, and RM+Ang1-7+ A779 group. Sera and urine samples were collected for analysis of renal and muscle injury markers. Kidney tissues were taken for estimation of oxidative, inflammatory, and apoptotic markers as well as angiotensin-II (Ang II) and Ang1–7. Renal histology and expression of inducible nitric oxide synthase-1 (iNOS), real-time PCR for angiotensin-converting enzyme-2 (ACE-2), nuclear erythroid factor-2 (Nrf-2), Toll like receptor 4 (TLR-4) and NF-kB in kidney tissues were also measured. Key findingsInduction of RM caused renal oxidative stress injury, inflammation, apoptosis and marked deterioration in kidney functions as well as reduction of Ang1–7 and raised Angiotensin-II level in kidney tissues. Administration of Ang1–7 to the RM group reversed all the affected parameters which were blocked by A779 administration (Mas receptor blocker). SignificanceWe concluded that Ang1–7 could be a potential therapeutic agent that could mitigate RM-induced renal injury. The underlying mechanisms may involve Stimulation of the ACE-2/Ang1–7/MasR axis and modulation of TLR-4/NF-kB/iNOS and Nrf-2/heme‑oxygenase -1 pathways.

Improved cognition impairment by activating cannabinoid receptor type 2: Modulating CREB/BDNF expression and impeding TLR-4/NFκBp65/M1 microglia signaling pathway in D-galactose-injected ovariectomized rats.

Alzheimer’s disease (AD) is characterized by an active inflammatory response induced by the brain’s deposition and accumulation of amyloid-beta (Aβ). Cannabinoid receptor type 2 (CB2R) is expressed in specific brain areas, modulating functions, and pathophysiologies in CNS. Herein, we aimed to evaluate whether activation of CB2R can improve the cognitive impairment in the experimental AD-like model and determine the involved intracellular signaling pathway. Injection of D-galactose (150 mg/kg, i.p.) was performed to urge AD-like features in bilaterally ovariectomized female rats (OVC/D-gal rats) for 8-weeks. Then, AM1241, a CB2R-agonist (3 and 6 mg/kg), was injected intraperitoneally starting from the 6th week. Treatment with AM1241, significantly down-regulated; Toll-like receptor4 (TLR4), Myd88 (TLR4-adaptor protein) genes expression, and the pro-inflammatory cytokines (NFκB p65, TNF-α, IL-6, and IL-12). In contrast, it enhanced BDNF (the brain-derived neurotrophic factor) and CREB (the cyclic AMP response element-binding protein) as well as the immune-modulatory cytokines (IL-4 and IL-10) levels. Moreover, AM1241 lessened the immune-expression of GFAP, CD68, caspase-3, and NFκB p65 markers and mended the histopathological damage observed in OVC/D-gal rats by decreasing the deposition of amyloid plaques and degenerative neuronal lesions, as well as improving their recognition and learning memory in both novel object recognition and Morris water maze tests. In conclusion, activating CB2R by the selective agonist AM1241 can overrun cognitive deficits in OVC/D-gal rats through modulation of TLR4/ NFκB p65 signaling, mediated by modulating CREB/BDNF pathway, thereby can be applied as a potential therapeutic strategy in AD treatment.

Gamma-Oryzanol-Rich Fraction from Purple Rice Extract Attenuates Lipopolysaccharide-Stimulated Inflammatory Responses, Migration and VEGFA Production in SW480 Cells via Modulation of TLR4 and NF-κB Pathways

Inflammatory response facilitating colorectal cancer (CRC) progression is a serious event following operative infection, which can occur in CRC patients. This event is mainly mediated by bacterial lipopolysaccharide (LPS), via a toll like receptor 4 (TLR4) and NF-κB. Hexane soluble fraction (HSF) from purple rice extract (PRE) has been identified as a γ-oryzanol (OR)-rich fraction. Recently, HSF possessed inhibitory effect of LPS-stimulated metastasis of human colon cancer SW480 cells, however the related mechanism was unknown. Thus, this study aimed to investigate the effect of HSF on inflammatory response-associated cancer progression of LPS-stimulated SW480 cells. The various inflammatory mediators, vascular endothelial growth factor-A (VEGFA) and related pathways were evaluated by Western blot and ELISA. Furthermore, cell migration was also determined by migration assays. Of all, HSF seemed to be stronger than OR to attenuate the responsiveness of LPS on various inflammatory mediators, which was related to an obvious reduction of cancer cell migration as well as indistinct disruption on VEGFA production in SW480 cells, via downregulation of TLR4 and NF-κB. Therefore, OR-rich fraction from PRE, against the subsequent inflammatory response and CRC progression following surgery, which could be combined with conventional treatments to increase the survival rate.

Cover Feature: Full‐Atom Model of the Agonist LPS‐Bound Toll‐like Receptor 4 Dimer in a Membrane Environment (Chem. Eur. J. 62/2021)

Computational full-atom models of the agonist lipopolysaccharide (LPS)-bound Toll-like receptor 4 (TLR4) embedded into a realistic plasma membrane reveal dynamic functional and structural insights of the TLR4 activation and signaling across the membrane, the ectodomain bouncing movement, and dimerization patterns (symmetric and asymmetric) of the intracellular Toll/interleukin-1 receptor (TIR) domain. These studies unveil molecular aspects of the TLR4, main actor in the innate immune pathways, and will promote the discovery of new TLR4 modulators. More information can be found in the Full Paper by S. Martin-Santamaria et al. (DOI: 10.1002/chem.202102995).

Full-Atom Model of the Agonist LPS-Bound Toll-like Receptor 4 Dimer in a Membrane Environment.

The Toll‐like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD‐2) innate immunity system is a membrane receptor of paramount importance as therapeutic target. Its assembly, upon binding of Gram‐negative bacteria lipopolysaccharide (LPS), and also dependent on the membrane composition, finally triggers the immune response cascade. We have combined ab‐initio calculations, molecular docking, all‐atom molecular dynamics simulations, and thermodynamics calculations to provide the most realistic and complete 3D models of the active full TLR4 complex embedded into a realistic membrane to date. Our studies give functional and structural insights into the transmembrane domain behavior in different membrane environments, the ectodomain bouncing movement, and the dimerization patterns of the intracellular Toll/Interleukin‐1 receptor domain. Our work provides TLR4 models as reasonable 3D structures for the (TLR4/MD‐2/LPS)2 architecture accounting for the active (agonist) state of the TLR4, and pointing to a signal transduction mechanism across cell membrane. These observations unveil relevant molecular aspects involved in the TLR4 innate immune pathways and will promote the discovery of new TLR4 modulators.

Neuroprotective role of camphor against ciprofloxacin induced depression in rats: modulation of Nrf-2 and TLR4

Background Depression affects people feeling to be anxious, worried, and restless. They also lose interest in activities, concentrating and appetite, they finally may attempt suicide. Depression is the second chronic disease, as a source of the global burden of disease, after heart disease. Its prevalence elevated seven times during the COVID-19. Aim The current study was designed to evaluate camphor neuroprotective role against rats’ ciprofloxacin-induced depression. Materials and methods Depression was induced by administration of ciprofloxacin (50 mg/kg; orally) for 21 days. Wister albino male rats were divided into five groups. Group I (normal control): rats were given normal saline. Group II: rats received camphor (10 mg/kg; i.p.) for 21 days. Group III (depression control): rats received ciprofloxacin only. Groups IV and V: rats received camphor (5 and 10 mg/kg; i.p.) for 21 days concurrent with ciprofloxacin. Behavior tests as forced swimming test, activity cage, and rotarod were estimated. Oxidative stress and antioxidant biomarkers as malondialdehyde (MDA), nitric oxide (NO), catalase, and nuclear factor erythroid 2-related factor 2 (Nrf-2) besides inflammatory biomarkers as Toll-like receptor 4 (TLR4) and tumor necrosis factor alpha (TNF-α) as well as neurotransmitters were determined. Finally, histopathological examination was done. Results Camphor increased catalase and Nrf-2 activities, decreased NO, MDA, TNF-α, TLR4 serum levels, and elevating brain contents of serotonin, dopamine, gamma-amino butyric acid (GABA) and P190-RHO GTP protein with normal neuronal cells of the frontal cortex. Conclusion Camphor has neuroprotective effect via modulation of Nrf-2 and TLR4 against ciprofloxacin-induced depression in rats.

Increasing the Chemical Variety of Small-Molecule-Based TLR4 Modulators: An Overview.

Toll-Like Receptor 4 (TLR4) is one of the receptors of innate immunity. It is activated by Pathogen- and Damage-Associated Molecular Patterns (PAMPs and DAMPs) and triggers pro-inflammatory responses that belong to the repertoire of innate immune responses, consequently protecting against infectious challenges and boosting adaptive immunity. Mild TLR4 stimulation by non-toxic molecules resembling its natural agonist (lipid A) provided efficient vaccine adjuvants. The non-toxic TLR4 agonist monophosphoryl lipid A (MPLA) has been approved for clinical use. This suggests the development of other TLR4 agonists as adjuvants or drugs for cancer immunotherapy. TLR4 excessive activation by a Gram-negative bacteria lipopolysaccharide (LPS) leads to sepsis, while TLR4 stimulation by DAMPs is a common mechanism in several inflammatory and autoimmune diseases. TLR4 inhibition by small molecules and antibodies could therefore provide access to innovative therapeutics targeting sepsis as well as acute and chronic inflammations. The potential use of TLR4 antagonists as anti-inflammatory drugs with unique selectivity and a new mechanism of action compared to corticosteroids or other non-steroid anti-inflammatory drugs fueled the search for compounds of natural or synthetic origin able to block or inhibit TLR4 activation and signaling. The wide spectrum of clinical settings to which TLR4 inhibitors can be applied include autoimmune diseases (rheumatoid arthritis, inflammatory bowel diseases), vascular inflammation, neuroinflammations, and neurodegenerative diseases. The last advances (from 2017) in TLR4 activation or inhibition by small molecules (molecular weight <2 kDa) are reviewed here. Studies on pre-clinical validation of new chemical entities (drug hits) on cellular or animal models as well as new clinical studies on previously developed TLR4 modulators are reported. Innovative TLR4 modulators discovered by computer-assisted drug design and an artificial intelligence approach are described. Some “old” TLR4 agonists or antagonists such as MPLA or Eritoran are under study for repositioning in different pharmacological contexts. The mechanism of action of the molecules and the level of TLR4 involvement in their biological activity are critically discussed.

Distinct cellular mediators drive the Janus faces of toll-like receptor 4 regulation of network excitability which impacts working memory performance after brain injury

The mechanisms by which the neurophysiological and inflammatory responses to brain injury contribute to memory impairments are not fully understood. Recently, we reported that the innate immune receptor, toll-like receptor 4 (TLR4) enhances AMPA receptor (AMPAR) currents and excitability in the dentate gyrus after fluid percussion brain injury (FPI) while limiting excitability in controls. Here, we examine the cellular mediators underlying TLR4 regulation of dentate excitability and its impact on memory performance. In ex vivo slices, astrocytic and microglial metabolic inhibitors selectively abolished TLR4 antagonist modulation of excitability in controls, but not in rats after FPI, demonstrating that glial signaling contributes to TLR4 regulation of excitability in controls. In glia-depleted neuronal cultures from naïve mice, TLR4 ligands bidirectionally modulated AMPAR charge transfer consistent with neuronal TLR4 regulation of excitability, as observed after brain injury. In vivo TLR4 antagonism reduced early post-injury increases in mediators of MyD88-dependent and independent TLR4 signaling without altering expression in controls. Blocking TNFα, a downstream effector of TLR4, mimicked effects of TLR4 antagonist and occluded TLR4 agonist modulation of excitability in slices from both control and FPI rats. Functionally, transiently blocking TLR4 in vivo improved impairments in working memory observed one week and one month after FPI, while the same treatment impaired memory function in uninjured controls. Together these data identify that distinct cellular signaling mechanisms converge on TNFα to mediate TLR4 modulation of network excitability in the uninjured and injured brain and demonstrate a role for TLR4 in regulation of working memory function.

Deletion of TLR4 in Townes SS Mice Prevents Microvascular Stasis in Response to Hemin, LPS and Hypoxia/Reoxygenation: Role of Inflammation

We have previously shown that heme, a damage-associated molecular pattern (DAMP), released from sickle RBCs interacts with the innate immune toll-like receptor 4 (TLR4) on endothelium and blood cells to activate cell signaling. This activates the pro-inflammatory transcription factor NF-κB leading to the production of cytokines and adhesion molecules that promote inflammation, coagulation, and vaso-occlusion (VO). Previous studies in our lab have demonstrated that inhibition of TLR4 in Townes-SS mice with TAK242 reduces microvascular stasis in response to hemin, hypoxia/reoxygenation and LPS. To further delineate the role of TLR4 in sickle cell disease (SCD), we bred a global Tlr4-/- deficiency state into Townes-AA mice expressing normal human adult hemoglobin A and Townes-SS mice expressing sickle hemoglobin S. These Tlr-/- Townes mice were backcrossed 10 generations to homogenize their genetic background with our Tlr4+/+ Townes colony. Townes-SS Tlr4-/- mice developed less microvascular stasis than Townes-SS Tlr4+/+ mice in response to challenges with heme infusion (4% vs 30% at 1 hr, p&amp;lt;0.05, Fig 1A); LPS infusion (13% vs 36% at 1 hr, p&amp;lt;0.05, Fig 1B); and hypoxia/reoxygenation (3% vs 20% at 1 hr, p&amp;lt;0.05, Fig 1C). We next measured the complete blood counts, serum chemistries, and urine analytes in the Tlr4+/+ and -/- mice. We also quantified mRNA levels via qPCR for liver and kidney cytokines, adhesion molecule, heme oxygenase 1 (HO-1) and coagulation factors in all four genotypes (AA Tlr4+/+; AA Tlr4-/-; SS Tlr4+/+; SS Tlr4-/-). Hemoglobin levels in SS mice were lower than AA mice, but there were no differences between Tlr4+/+ and Tlr4-/- mice. WBC counts were greater in SS than AA mice, but there were no differences between Tlr4+/+ and Tlr4-/- mice. Bilirubin and liver function tests (LFTs) were higher in SS than AA, but there were no differences between Tlr4+/+ and Tlr4-/- mice. In the liver, adhesion molecules and HO-1 were higher in SS than AA, but there were no differences between Tlr4+/+ and Tlr4-/- mice. To define a potential mechanism for decreased microvascular stasis in response to hemin infusion in SS Tlr4-/-, we measured cytokines and adhesion molecule expression after heme challenge. When SS Tlr4+/+ mice were infused with hemin, we found increased expression of pro-inflammatory cytokines and cell adhesion molecules. SS Tlr4-/- mice infused with hemin had reduced expression of cytokines and cell adhesion molecules compared to SS Tlr4+/+ mice; most notably in IL-6 in the liver, adhesion molecules VCAM-1 and ICAM-1 in the liver and kidneys (p&amp;lt;0.05), and P-selectin and von Willebrand factor in the lungs (Fig 2). Our data thus far indicate that TLR4 of the innate immune system plays a critical role in VO and inflammation in challenged SS mice. We speculate that modulation of TLR4 with targeted inhibitors would be beneficial for SCD patients. Disclosures Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.

Saxagliptin mitigates airway inflammation in a mouse model of acute asthma via modulation of NF-kB and TLR4

Saxagliptin (Saxa), a dipeptidyl dipeptidase-4 (DPP-4) inhibitor, is widely used for the treatment of type 2 diabetes mellitus. It has been documented to have immunomodulatory and anti-inflammatory actions. Our objective was to delineate the protective effect and the underlying mechanism of Saxa-in comparison with Dexamethasone (Dexa) – in airway inflammation induced by ovalbumin (OVA) in mice. MethodsMice were OVA-sensitized and challenged for the induction of acute asthma. Mice were orally administrated Saxa or Dexa. Total and differential cell counts, lactate dehydrogenase (LDH) and total protein concentrations were assessed in bronchoalveolar lavage fluid (BALF). The toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-kB), reduced glutathione (GSH), and total nitrate/nitrite products (NOx) levels as well as myeloperoxidase (MPO) activity in lung tissues were measured. Histopathological examination of the lung specimens was carried out using the hematoxylin and eosin (H & E) staining. ResultsHistopathological examination revealed that both Saxa and Dexa ameliorated OVA-induced inflammatory changes and significantly reduced total and differential leukocyte counts, LDH and total protein level in BALF upon comparison with OVA group. In addition, both treatments significantly mitigated OVA-induced oxidative stress as evidenced by diminished lung NOx level and MPO activity and elevated GSH level. The elevation of TLR4 and NF-kB levels in lung tissue were ameliorated by Saxa and Dexa administration. ConclusionSaxa had marked antiasthmatic effect in OVA-induced allergic asthma through modulation of TLR4 and NF-κB signaling. Also, Saxa may represent a promising therapeutic agent for acute allergic asthma.

Abstract #4312 Microglial repopulation attenuates age-associated dysfunction independent of neuroinflammation

The mechanisms by which the neurophysiological and inflammatory responses to brain injury contribute to memory impairments are not fully understood. Recently, we reported that the innate immune receptor, toll-like receptor 4 (TLR4) enhances AMPA receptor (AMPAR) currents and excitability in the dentate gyrus after fluid percussion brain injury (FPI) while limiting excitability in controls. Here, we examine the cellular mediators underlying TLR4 regulation of dentate excitability and its impact on memory performance. In ex vivo slices, astrocytic and microglial metabolic inhibitors selectively abolished TLR4 antagonist modulation of excitability in controls, but not in rats after FPI, demonstrating that glial signaling contributes to TLR4 regulation of excitability in controls. In glia-depleted neuronal cultures from naïve mice, TLR4 ligands bidirectionally modulated AMPAR charge transfer consistent with neuronal TLR4 regulation of excitability, as observed after brain injury. In vivo TLR4 antagonism reduced early post-injury increases in mediators of MyD88-dependent and independent TLR4 signaling without altering expression in controls. Blocking TNFα, a downstream effector of TLR4, mimicked effects of TLR4 antagonist and occluded TLR4 agonist modulation of excitability in slices from both control and FPI rats. Functionally, transiently blocking TLR4 in vivo improved impairments in working memory observed one week and one month after FPI, while the same treatment impaired memory function in uninjured controls. Together these data identify that distinct cellular signaling mechanisms converge on TNFα to mediate TLR4 modulation of network excitability in the uninjured and injured brain and demonstrate a role for TLR4 in regulation of working memory function.

Abstract # 3226 Demyelination and remyelination following a gliotoxic injury in the brainstem of XID mice

The mechanisms by which the neurophysiological and inflammatory responses to brain injury contribute to memory impairments are not fully understood. Recently, we reported that the innate immune receptor, toll-like receptor 4 (TLR4) enhances AMPA receptor (AMPAR) currents and excitability in the dentate gyrus after fluid percussion brain injury (FPI) while limiting excitability in controls. Here, we examine the cellular mediators underlying TLR4 regulation of dentate excitability and its impact on memory performance. In ex vivo slices, astrocytic and microglial metabolic inhibitors selectively abolished TLR4 antagonist modulation of excitability in controls, but not in rats after FPI, demonstrating that glial signaling contributes to TLR4 regulation of excitability in controls. In glia-depleted neuronal cultures from naïve mice, TLR4 ligands bidirectionally modulated AMPAR charge transfer consistent with neuronal TLR4 regulation of excitability, as observed after brain injury. In vivo TLR4 antagonism reduced early post-injury increases in mediators of MyD88-dependent and independent TLR4 signaling without altering expression in controls. Blocking TNFα, a downstream effector of TLR4, mimicked effects of TLR4 antagonist and occluded TLR4 agonist modulation of excitability in slices from both control and FPI rats. Functionally, transiently blocking TLR4 in vivo improved impairments in working memory observed one week and one month after FPI, while the same treatment impaired memory function in uninjured controls. Together these data identify that distinct cellular signaling mechanisms converge on TNFα to mediate TLR4 modulation of network excitability in the uninjured and injured brain and demonstrate a role for TLR4 in regulation of working memory function.

Abstract #4300 TLR-7 agonist imiquimod may trigger pro-inflammatory reaction by acting on brain cells directly

The mechanisms by which the neurophysiological and inflammatory responses to brain injury contribute to memory impairments are not fully understood. Recently, we reported that the innate immune receptor, toll-like receptor 4 (TLR4) enhances AMPA receptor (AMPAR) currents and excitability in the dentate gyrus after fluid percussion brain injury (FPI) while limiting excitability in controls. Here, we examine the cellular mediators underlying TLR4 regulation of dentate excitability and its impact on memory performance. In ex vivo slices, astrocytic and microglial metabolic inhibitors selectively abolished TLR4 antagonist modulation of excitability in controls, but not in rats after FPI, demonstrating that glial signaling contributes to TLR4 regulation of excitability in controls. In glia-depleted neuronal cultures from naïve mice, TLR4 ligands bidirectionally modulated AMPAR charge transfer consistent with neuronal TLR4 regulation of excitability, as observed after brain injury. In vivo TLR4 antagonism reduced early post-injury increases in mediators of MyD88-dependent and independent TLR4 signaling without altering expression in controls. Blocking TNFα, a downstream effector of TLR4, mimicked effects of TLR4 antagonist and occluded TLR4 agonist modulation of excitability in slices from both control and FPI rats. Functionally, transiently blocking TLR4 in vivo improved impairments in working memory observed one week and one month after FPI, while the same treatment impaired memory function in uninjured controls. Together these data identify that distinct cellular signaling mechanisms converge on TNFα to mediate TLR4 modulation of network excitability in the uninjured and injured brain and demonstrate a role for TLR4 in regulation of working memory function.

Role of toll-like receptor 4 in the regulation of the cell death pathway and neuroinflammation

Septic brain injury is a severe disease of the central nervous system and involves alteration of the cell death pathway due to sepsis. Increasing evidence indicates that Toll-like receptor 4 (TLR4) plays a key role in the development of sepsis. Cell death is also thought to contribute to septic brain injury. However, the mechanism that regulates cell death pathways in response to TLR4 in neurons exposed to septic brain injury is unclear. Here, we established a rat model of septic brain injury to evaluate the effect of TLR4 inhibition on cell death pathways. Furthermore, primary neurons with and those without TLR4 inhibition were exposed to lipopolysaccharide (LPS). We found that septic brain injury induction by cecum ligation and puncture evoked autophagy, induced high-mobility group box-1(HMGB1) translocation and increased TLR4 expression. Inhibition of TLR4 also enhanced autophagy and increased nuclear HMGB1 levels in the sepsis-injured rat brain cortex. After LPS treatment in vitro, HMGB1 was released from neurons. This release of HMGB1 could be inhibited using a TLR4 inhibitor. Collectively, these results indicate that modulation of TLR4 may result in the regulation of neuron cell death pathways by regulating autophagy in cortical tissues. Thus, it may represent a potential therapeutic strategy for neuronal protection in septic brain injury.

Polysaccharides from radix astragali exert immunostimulatory effects to attenuate the dampness stagnancy due to spleen deficiency syndrome

Pharmacognosy Magazine ,2019,15,63,500-506.DOI:10.4103/pm.pm_503_18Published:May 2019Type:Original Article Authors:Wenxiao Zhao, Lijun Chen, Ning Cui, Xuming Ji, Haiqiang Jiang, Tiantian Deng, and Shijun Wang Author(s) affiliations:Wenxiao Zhao1, Lijun Chen2, Ning Cui3, Xuming Ji4, Haiqiang Jiang5, Tiantian Deng3, Shijun Wang6 1School of Nursing, Shandong University of Traditional Chinese Medicine; Shandong Co-Innovation Center of Classic Traditional Chinese Medicine Formula, Jinan, Shandong, China 2School of Nursing, Shandong University of Traditional Chinese Medicine, Jinan, Shandong, China 3School of Traditional Chinese Medical, Shandong University of Traditional Chinese Medicine, Jinan, Shandong, China. 4College of Basic Medicine, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China 5Key Laboratory of Traditional Chinese Medicine Basic Research, Shandong University of Traditional Chinese Medicine, Jinan, Shandong, China 6Shandong Co-Innovation Center of Classic Traditional Chinese Medicine Formula; School of Traditional Chinese Medical, Shandong University of Traditional Chinese Medicine, Jinan, Shandong, China Abstract:Background: Radix Astragali is an herb with tonifying qi or adaptogenic effects and commonly used for spleen-qi deficiency in traditional Chinese medicine. Astragalus polysaccharides (APS) is one of the major bioactive ingredients, and little is known on APS in attenuating the dampness stagnancy due to spleen deficiency (DSSD). Objective: The objective of the study is to investigate the potential mechanism of action of APS underlying attenuating water and fluid retention of rats with the DSSD syndrome in order to provide novel insights into the application of APS on modulating the DSSD syndrome. Materials and Methods: Four-week-old Wistar rats were fed with one high-fat and low-protein diet and subjected to exhaustive swimming to induce the DSSD model for 8 weeks. APS (300, 600, and 1200 mg/kg) and Shen Ling Bai Zhu San (SLBZS) (2.5 g/kg) were administrated starting at 9 weeks for 2 weeks. Results: APS increased the body weight, decreased the water load index, and attenuated symptoms of the DSSD syndrome. APS also increased spleen and thymus indices, the percentage of CD3+, the percentage of CD3+ CD4+, and CD3+ CD4+ to CD3+ CD8+ ratio and decreased levels of serum interleukin (IL)-6, IL-10, and tumor necrosis factor-α in DSSD rats. Significant effects were seen for toll-like receptor 4 (TLR4) at both protein and gene expression levels with amelioration of expression levels in APS groups. Conclusion: These findings suggest that APS may be used to attenuate the fluid retention of the DSSD syndrome through the immunoregulatory effect, which is driven in part by the modulation of TLR4. Keywords:Astragalus polysaccharides, cytokine secretion, dampness stagnancy due to spleen deficiency, immunostimulatory effects, toll-like receptor 4View:PDF (1.85 MB)