Blocking Toll-like Receptor 4 Signaling Research Articles

Grape seed-derived procyanidins decreases neuropathic pain and nerve regeneration by suppression of toll-like receptor 4-myeloid differentiation factor-88 signaling.

Background: Recent studies have shown that peripheral nerve regeneration process is closely related to neuropathic pain. Toll-like receptor 4 (TLR4) signaling was involved in different types of pain and nerve regeneration. TLR4 induced the recruitment of myeloid differentiation factor-88 adaptor protein (MyD88) and NF-κB-depended transcriptional process in sensory neurons and glial cells, which produced multiple cytokines and promoted the induction and persistence of pain. Our study aimed to investigate procyanidins's effect on pain and nerve regeneration via TLR4-Myd88 signaling. Methods: Spinal nerve ligation (SNL) model was established to measure the analgesic effect of procyanidins. Anatomical measurement of peripheral nerve regeneration was measured by microscopy and growth associated protein 43 (GAP43) staining. Western blotting and/or immunofluorescent staining were utilized to detect TLR4, myeloid differentiation factor-88 adaptor protein (MyD88), ionized calcium-binding adapter molecule 1 (IBA1) and nuclear factor kappa-B-p65 (NF-κB-p65) expression, as well as the activation of astrocyte and microglia. The antagonist of TLR4 (LPS-RS-Ultra, LRU) were intrathecally administrated to assess the behavioral effects of blocking TLR4 signaling on pain and nerve regeneration. Result: Procyanidins reduced mechanical allodynia, thermal hyperalgesia and significantly suppressed the number of nerve fibers regenerated and the degree of myelination in SNL model. Compared with sham group, TLR4, MyD88, IBA1 and phosphorylation of NF-κB-p65 were upregulated in SNL rats which were reversed by procyanidins administration. Additionally, procyanidins also suppressed activation of spinal astrocytes and glial cells. Conclusion: Suppression of TLR4-MyD88 signaling contributes to the alleviation of neuropathic pain and reduction of nerve regeneration by procyanidins.

Myeloid Differentiation Factor 2 Inhibition Protects Against Doxorubicin‐induced Microglial Activation and Senescence

AbstractBackgroundDoxorubicin, an anti‐tumor drug, generates neuroinflammation that leads to microglial senescence and chemo‐brain (1, 2). Toll‐like receptor 4 (TLR4) signaling pathway is mainly expressed in microglia and it plays a key role in developing doxorubicin‐induced neuroinflammation, microglial activation and chemo‐brain (3). Myeloid differentiation factor 2 (MD‐2) is known as an essential protein in activation of TLR4 signaling pathway (1). However, the effect of blocking TLR4 signaling via targeting MD‐2 on doxorubicin‐induced microglial activation and senescence has never been investigated.MethodThe human microglial clone 3 cell line (HMC3) was treated with three different doses of MD‐2 inhibitor (L6H21: 5μM, 10μM, 20μM) for 30 minutes, then with doxorubicin hydrochloride (DOX) (50nM) for 24 hours. After 24 hours treatment with L6H21 and DOX, the microglial cells were assessed for the TLR4 signaling, microglial activation, cell death and cellular senescence pathways.ResultDOX treatment activated TLR4 signaling pathway as well as microglia, as evidenced by significantly increased protein expressions of TLR4, MD‐2, tumor necrosis factor alpha and ionized calcium‐binding adapter molecule 1. In addition, DOX treatment induced microglial cell death and senescence, as shown by significantly increased protein expressions of Bcl‐2 associated X‐protein to B‐cell lymphoma 2 ratio and p16Ink4a. Treatment with L6H21 (5μM) attenuated not only TLR4 signaling along with microglial activation, but also apoptosis and cellular senescence in DOX‐treated microglia. L6H21 (10μM and 20μM) protected against TLR4 signaling along with microglial activation, apoptosis as well as cellular senescence in DOX‐treated microglia.ConclusionThese findings imply that MD‐2 inhibition could be the potential therapeutic strategy for attenuating DOX‐induced microglial activation and senescence.

Toll-like receptor 4 and Syk kinase shape dendritic cell-induced immune activation to major house dust mite allergens.

House dust mite (HDM) is a major cause of respiratory allergic diseases. Dendritic cells (DCs) play a central role in orchestrating adaptive allergic immune responses. However, it remains unclear how DCs become activated by HDM. Biochemical functions of the major HDM allergens Der p 1 (cysteine protease) and Der p 2 (MD2-mimick) have been implicated to contribute to DC activation. We investigated the immune activating potential of HDM extract and its major allergens Der p 1 and Der p 2 using monocyte-derived DCs (moDCs). Maturation and activation markers were monitored by flow cytometry and cytokine production by ELISA. Allergen depletion and proteinase K digestion were used to investigate the involvement of proteins, and in particular of the major allergens. Inhibitors of spleen tyrosine kinase (Syk), Toll-like receptor 4 (TLR4) and of C-type lectin receptors (CLRs) were used to identify the involved receptors. The contribution of endotoxins in moDC activation was assessed by their removal from HDM extract. HDM extract induced DC maturation and cytokine responses in contrast to the natural purified major allergens Der p 1 and Der p 2. Proteinase K digestion and removal of Der p 1 or Der p 2 did not alter the immune stimulatory capacity of HDM extract. Antibodies against the CLRs Dectin-1, Dectin-2, and DC-SIGN did not affect cytokine responses. In contrast, Syk inhibition partially reduced IL-6, IL-12 and completely blocked IL-10. Blocking TLR4 signaling reduced the HDM-induced IL-10 and IL-12p70 induction, but not IL-6, while endotoxin removal potently abolished the induced cytokine response. Our data strongly suggest that HDM-induced DC activation is neither dependent on Der p 1 nor Der p 2, but depend on Syk and TLR4 activation, which might suggest a crosstalk between Syk and TLR4 pathways. Our data highlight that endotoxins play a potent role in immune responses targeting HDM.

Low-grade elevation of palmitate and lipopolysaccharide synergistically induced β-cell damage via inhibition of neutral ceramidase

High concentrations of free fatty acids (FFAs) or lipopolysaccharide (LPS) could lead to β-cell apoptosis and dysfunction, while low-grade elevation of FFAs or LPS, which are more common in people with type 2 diabetes mellitus (T2DM) or obesity, have no obvious toxic effect on β-cells. Palmitate is a component closely related to metabolic disorders in FFAs. Recent studies have found that low-grade elevation of palmitate and LPS synergistically affects the sphingolipid signaling pathway by activating Toll-like receptor 4 (TLR4) and further enhances the expression of inflammatory cytokines in immune cells. Previous studies demonstrated that sphingolipids also played an important role in the occurrence and development of T2DM. This study aimed to investigate the synergistic effects of low-grade elevation of palmitate and LPS on viability, apoptosis and insulin secretion in the rat pancreatic β-cell line INS-1 or islets and the role of sphingolipids in this process. We showed that low-grade elevation of palmitate or LPS alone did not affect the viability, apoptosis, glucose-stimulated insulin secretion (GSIS) or intracellular insulin content of INS-1 cells or islets, while the combination of the two synergistically inhibited cell viability, induced apoptosis and decreased basal insulin secretion in INS-1 cells or islets. Treatment with palmitate and LPS markedly upregulated TLR4 protein expression and downregulated neutral ceramidase (NCDase) activity and protein expression. Additionally, low-grade elevation of palmitate and LPS synergistically induced a significant increase in ceramide and a decrease in sphingosine-1-phosphate. Blocking TLR4 signaling or overexpressing NCDase remarkably attenuated INS-1 cell injury induced by the combination of palmitate and LPS. However, inhibition of ceramide synthase did not ameliorate injury induced by palmitate and LPS. Overall, we show for the first time that low-grade elevation of palmitate and LPS synergistically induced β-cell damage by activating TLR4 signaling, inhibiting NCDase activity, and further modulating sphingolipid metabolism, which was different from a high concentration of palmitate-induced β-cell injury by promoting ceramide synthesis.

Suppression of TLR4-MyD88 signaling pathway attenuated chronic mechanical pain in a rat model of endometriosis

BackgroundAs a classic innate immunity pathway, Toll-like receptor 4 (TLR4) signaling has been intensively investigated for its function of pathogen recognition. The receptor is located not only on immune cells but also on sensory neurons and spinal glia. Recent studies revealed the involvement of neuronal TLR4 in different types of pain. However, the specific role of TLR4 signaling in the pain symptom of endometriosis (EM) remains obscure.MethodsThe rat endometriosis model was established by transplanting uterine horn tissue into gastrocnemius. Western blotting and/or immunofluorescent staining were applied to detect high mobility group box 1 (HMGB1), TLR4, myeloid differentiation factor-88 adaptor protein (MyD88), and nuclear factor kappa-B-p65 (NF-κB-p65) expression, as well as the activation of astrocyte and microglia. The antagonist of TLR4 (LPS-RS-Ultra, LRU) and MyD88 homodimerization inhibitory peptide (MIP) were intrathecally administrated to assess the behavioral effects of blocking TLR4 signaling on endometriosis-related pain.ResultsMechanical hyperalgesia was observed at the graft site, while HMGB1 was upregulated in the implanted uterine tissue, dorsal root ganglion (DRG), and spinal dorsal horn (SDH). Compared with sham group, upregulated TLR4, MyD88, and phosphorylated NF-κB-p65 were detected in the DRG and SDH in EM rats. The activation of astrocytes and microglia in the SDH was also confirmed in EM rats. Intrathecal application of LRU and MIP alleviated mechanical pain on the graft site of EM rats, with decreased phosphorylation of NF-κB-p65 in the DRG and reduced activation of glia in the SDH.ConclusionsHMGB1-TLR4-MyD88 signaling pathway in the DRG and SDH may involve in endometriosis-related hyperpathia. Blockade of TLR4 and MyD88 might serve as a potential treatment for pain in endometriosis.

Toll-like Receptor 4 Signaling in Neurons Enhances Calcium-Permeable α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Receptor Currents and Drives Post-Traumatic Epileptogenesis.

Traumatic brain injury is a major risk factor for acquired epilepsies, and understanding the mechanisms underlying the early pathophysiology could yield viable therapeutic targets. Growing evidence indicates a role for inflammatory signaling in modifying neuronal excitability and promoting epileptogenesis. Here we examined the effect of innate immune receptor Toll-like receptor 4 (TLR4) on excitability of the hippocampal dentate gyrus and epileptogenesis after brain injury. Slice and in vivo electrophysiology and Western blots were conducted in rats subject to fluid percussion brain injury or sham injury. The studies identify that TLR4 signaling in neurons augments dentate granule cell calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (CP-AMPAR) currents after brain injury. Blocking TLR4 signaling in vivo shortly after brain injury reduced dentate network excitability and seizure susceptibility. When blocking of TLR4 signaling after injury was delayed, however, this treatment failed to reduce postinjury seizure susceptibility. Furthermore, TLR4 signal blocking was less efficacious in limiting seizure susceptibility when AMPAR currents, downstream targets of TLR4 signaling, were transiently enhanced. Paradoxically, blocking TLR4 signaling augmented both network excitability and seizure susceptibility in uninjured controls. Despite the differential effect on seizure susceptibility, TLR4 antagonism suppressed cellular inflammatory responses after injury without impacting sham controls. These findings demonstrate that independently of glia, the immune receptor TLR4 directly regulates post-traumatic neuronal excitability. Moreover, the TLR4-dependent early increase in dentate excitability is causally associated with epileptogenesis. Identification and selective targeting of the mechanisms underlying the aberrant TLR4-mediated increase in CP-AMPAR signaling after injury may prevent epileptogenesis after brain trauma. ANN NEUROL 2020;87:497-515.

The effect of fenofibrate, a PPARα activator on toll-like receptor-4 signal transduction in melanoma both in vitro and in vivo.

The anti-cancer effect of peroxisome proliferator-activated receptor (PPAR) α ligands on growth and metastatic potential of melanoma cells has been shown previously. However, themechanismunderlying these effects remains to be elucidated. Here, we investigated the effects of fenofibrate (PPAR ligand) on Toll-like receptor-4 (TLR-4) signaling in mice melanoma. Mice melanoma cells (B16F10) were treated with fenofibrate or LPS or LPS + fenofibrate or pre-treated with CLI-095 (a TLR4 inhibitor), followed by fenofibrate. In in vivo model, C57BL/6 mice were subcutaneously injected with B16F10 cells (with/without LPS pre-treatment), and fenofibrate was administrated after development of palpable tumors. Cell proliferation, the expression level of Tlr4, Myd88, Nf-κb1 genes, TLR-4 protein expression, TNF-α levels, and tumor volume were measured. Our results indicated that fenofibrate significantly inhibited the Tlr-4, Myd-88, and Nf-kb1 mRNA expression and TNF-α concentration in B16F10 LPS-stimulated cells. In addition, blocking TLR-4 signaling increased the anti-inflammatory potential of fenofibrate. Also fenofibrate can reduce LPS-induced tumor volume, Tlr-4, Myd-88, Nf-kb1 mRNA, and TLR-4 protein expression in tumor tissue and also TNF-α level in tumor tissue lysate. Our data indicate that fenofibrate may exert its anti-melanoma effects via interaction with TLR4-dependent signaling pathway (TLR-4/MyD-88/ NF-kB).

Ebolaviruses Associated with Differential Pathogenicity Induce Distinct Host Responses in Human Macrophages.

Ebola virus (EBOV) and Reston virus (RESTV) are members of the Ebolavirus genus which greatly differ in their pathogenicity. While EBOV causes a severe disease in humans characterized by a dysregulated inflammatory response and elevated cytokine and chemokine production, there are no reported disease-associated human cases of RESTV infection, suggesting that RESTV is nonpathogenic for humans. The underlying mechanisms determining the pathogenicity of different ebolavirus species are not yet known. In this study, we dissected the host response to EBOV and RESTV infection in primary human monocyte-derived macrophages (MDMs). As expected, EBOV infection led to a profound proinflammatory response, including strong induction of type I and type III interferons (IFNs). In contrast, RESTV-infected macrophages remained surprisingly silent. Early activation of IFN regulatory factor 3 (IRF3) and NF-κB was observed in EBOV-infected, but not in RESTV-infected, MDMs. In concordance with previous results, MDMs treated with inactivated EBOV and Ebola virus-like particles (VLPs) induced NF-κB activation mediated by Toll-like receptor 4 (TLR4) in a glycoprotein (GP)-dependent manner. This was not the case in cells exposed to live RESTV, inactivated RESTV, or VLPs containing RESTV GP, indicating that RESTV GP does not trigger TLR4 signaling. Our results suggest that the lack of immune activation in RESTV-infected MDMs contributes to lower pathogenicity by preventing the cytokine storm observed in EBOV infection. We further demonstrate that inhibition of TLR4 signaling abolishes EBOV GP-mediated NF-κB activation. This finding indicates that limiting the excessive TLR4-mediated proinflammatory response in EBOV infection should be considered as a potential supportive treatment option for EBOV disease.IMPORTANCE Emerging infectious diseases are a major public health concern, as exemplified by the recent devastating Ebola virus (EBOV) outbreak. Different ebolavirus species are associated with widely varying pathogenicity in humans, ranging from asymptomatic infections for Reston virus (RESTV) to severe disease with fatal outcomes for EBOV. In this comparative study of EBOV- and RESTV-infected human macrophages, we identified key differences in host cell responses. Consistent with previous data, EBOV infection is associated with a proinflammatory signature triggered by the surface glycoprotein (GP), which can be inhibited by blocking TLR4 signaling. In contrast, infection with RESTV failed to stimulate a strong host response in infected macrophages due to the inability of RESTV GP to stimulate TLR4. We propose that disparate proinflammatory host signatures contribute to the differences in pathogenicity reported for ebolavirus species and suggest that proinflammatory pathways represent an intriguing target for the development of novel therapeutics.

Cell-Permeable Peptide Blocks TLR4 Signaling and Improves Islet Allograft Survival.

Toll-like receptor 4 (TLR4) activation in pancreatic β cells activates aberrant islet graft cellular pathways and contributes to immune rejection in allogeneic islet transplantation. As an approach to overcoming this problem, we determined the capacity of a 33-amino acid peptide consisting of a protein transduction domain (PTD) from the Hph-1 virus and a fragment of the intracellular domain of TLR4 from the C3H mice (PTD-dnTLR4) to block TLR4 signaling and improve allogeneic islet survival in vitro and after transplantation. The efficacy of PTD-dnTLR4 in blocking TLR4 signaling was assessed in the Raw264.7 macrophage line, in the islets, and the βTC3 cell line. In Raw264.7 cells, preculture with the peptide reduced LPS-induced NF-κB activation and production of proinflammatory cytokines (IL-1β, TNF-α, iNOS, and IL-6). In islets and β cells, preincubation with PTD-dnTLR4 suppressed LPS-induced TNF-α expression via inhibition of NF-κB activation and protected them from stress-induced cell death. In vivo, preincubation of BALB/c (H-2(d)) islets with PTD-dnTLR4 resulted in significantly longer survival than control islets in a streptozotocin-induced diabetes model (two of seven grafts survived long term >100 days). PTD-dnTLR4-treated grafts exhibited reduced expression of TNF-α and iNOS and reduced macrophage infiltration posttransplant. The data indicate that PTD-dnTLR4 blocked TLR4 signaling in both macrophages and β cells, and prolonged allograft survival at least in part by suppressing inflammation and macrophage infiltration. This strategy for blocking TLR4 activity has potential utilization in the treatment of diseases where excessive TLR4 activation contributes to the pathologic cellular pathways such as islet transplantation.

MiR-223 Inhibits Lipid Deposition and Inflammation by Suppressing Toll-Like Receptor 4 Signaling in Macrophages.

Atherosclerosis and its complications rank as the leading cause of death with the hallmarks of lipid deposition and inflammatory response. MicroRNAs (miRNAs) have recently garnered increasing interests in cardiovascular disease. In this study, we investigated the function of miR-223 and the underlying mechanism in atherosclerosis. In the atherosclerotic ApoE−/− mice models, an obvious increase of miR-223 was observed in aortic atherosclerotic lesions. In lipopolysaccharide (LPS) activated macrophages, its expression was decreased. The miR-223 overexpression significantly attenuated macrophage foam cell formation, lipid accumulation and pro-inflammatory cytokine production, which were reversed by anti-miR-223 inhibitor transfection. Mechanism assay corroborated that miR-223 negatively regulated the activation of the toll-like receptor 4 (TLR4)-nuclear factor-κB (NF-κB) pathway. Pretreatment with a specific inhibitor of NF-κB (pyrrolidinedithiocarbamate, PDTC) strikingly abrogated miR-223 silence-induced lipid deposition and inflammatory cytokine production. Furthermore, PI3K/AKT was activated by miR-223 up-regulation. Pretreatment with PI3K/AKT inhibitor LY294002 strikingly ameliorated the inhibitory effects of miR-223 on the activation of TLR4 and p65, concomitant with the increase in lipid deposition and inflammatory cytokine production. Together, these data indicate that miR-223 up-regulation might abrogate the development of atherosclerosis by blocking TLR4 signaling through activation of the PI3K/AKT pathway, and provides a promising therapeutic avenue for the treatment of atherosclerosis.

The Therapeutic Potential of Toll‐like Receptor 4 Antagonists in Preventing Liver Injury Induced by Biliary Obstruction and Subsequent Endotoxemia

ObjectiveThe objective of this study was to elucidate the efficacy of toll‐like receptor 4 (TLR4) antagonists in preventing liver injury induced by biliary obstruction and subsequent intra‐portal lipopolysaccharide (LPS) infusion in rats.BackgroundBiliary obstruction often leads to the development of bacterial translocation and subsequent endotoxemia. Both biliary obstruction and endotoxemia lead to a severe liver injury.Materials and MethodsRats were subjected to either a sham operation (Sham group) or bile duct ligation for 7 days (BDL group). Seven days after each operation, LPS (0.5 μg) was injected through the ileocecal vein. In other experiments, rats that had undergone BDL were pretreated, prior to LPS challenge, with intravenous TAK‐242, a TLR4 inhibitor.ResultsThe expression of the TLR4 protein as well as the number of Kupffer cells in the liver were significantly increased in the BDL group compared with the Sham group. The expression of TLR4 co‐localized with Kupffer cells, which was confirmed by double immunofluorescence staining. Serum levels of liver enzymes and proinflammatory cytokines after intra‐portal LPS injection were significantly higher in the BDL group than in the Sham group. However, pretreatment with TAK‐242 strongly attenuated these changes.ConclusionsThese results imply that blocking TLR4 signaling effectively attenuates liver damage in rats with BDL and subsequent intra‐portal LPS infusion. TAK‐242 treatment may be used for patients who are susceptible to liver damage by biliary obstruction and endotoxemia.

Isobavachalcone attenuates lipopolysaccharide-induced ICAM-1 expression in brain endothelial cells through blockade of toll-like receptor 4 signaling pathways

Inflammation has been implicated in the pathogenesis of various cerebral diseases. Thus, control of brain inflammation is regarded as one of the important therapeutic strategies for the treatment of neurodegenerative diseases such as Alzheimer׳s disease and stroke. Isobavachalcone, a flavonoid from Psoralea corylifolia, is known to possess a wide spectrum of biological activities and is expected to be useful in preventing or treating neurodegenerative diseases. However, very little is known regarding its effects on cerebral inflammation. In this study, we examined the effect of isobavachalcone on leukocyte adhesion and intercellular adhesion molecule-1 (ICAM-1) expression in brain endothelial cells activated with lipopolysaccharide (LPS) and explored the possible mechanisms involved. Isobavachalcone significantly down-regulated LPS-induced ICAM-1 expression and leukocyte–endothelial cell adhesion and suppressed NF-κB activity which is implicated in the expression of ICAM-1. It attenuated ICAM-1 expression as well as NF-κB transcriptional activity induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or polyriboinosinic polyribocytidylic acid (poly[I:C]). Isobavachalcone also down-regulated LPS or poly[I:C]-induced expression of IFN-β, which can indirectly activate NF-κB. These data imply that isobavachalcone can modulate both MyD88-dependent and TRIF-dependent signaling of toll-like receptor 4 (TLR4). Taken together, our data suggest that isobavachalcone inhibits LPS-induced ICAM-1 expression and leukocyte adhesion to brain endothelial cell by blocking TLR4 signaling and thus, has the potential to ameliorate neuronal injury in brain diseases associated with inflammation.

Diallyl trisulfide exerts anti-inflammatory effects in lipopolysaccharide-stimulated RAW 264.7 macrophages by suppressing the Toll-like receptor 4/nuclear factor-κB pathway.

Diallyl trisulfide (DATS; di‑2‑propen‑1‑yl trisulfide) is an organic polysulfide compound found in garlic and other allium vegetables. Although certain studies have demonstrated that DATS possesses strong anti‑inflammatory activity, the underlying molecular mechanisms remain largely unresolved. In the present study, the anti‑inflammatory potential of DATS was investigated using the murine macrophage RAW 264.7 cell model. At non‑toxic concentrations, DATS inhibited the production of nitric oxide (NO) and prostaglandin E2 by inhibiting inducible NO synthase and cyclooxygenase‑2 expression at the transcriptional level in lipopolysaccharide (LPS)‑activated RAW 264.7 macrophages. DATS attenuated the release of the pro‑inflammatory cytokines, tumor necrosis factor‑α and interleukin‑1β, by inhibiting mRNA expression, respectively. DATS also suppressed LPS‑induced DNA‑binding activity of nuclear factor‑κB (NF‑κB), as well as the nuclear translocation of the NF‑κB p65, which correlated with the inhibitory effects of DATS on inhibitor κB (IκB) degradation. In addition, DATS was observed to significantly suppress LPS‑induced Toll‑like receptor 4 (TLR4) and myeloid differentiation factor 88 expression and the binding of LPS to macrophages, indicating the antagonistic effect of DATS against TLR4. Furthermore, blocking TLR4 signaling with the specific TLR4 signaling inhibitor, CLI‑095, increased the anti‑inflammatory potential of DATS in LPS‑stimulated RAW 264.7 macrophages. These data demonstrate that DATS may attenuate the initiation of LPS‑mediated intracellular signaling cascades by suppressing activation of NF‑κB and by inhibiting binding of LPS to TLR4 on macrophages.

Anti-inflammatory effects of genistein via suppression of the toll-like receptor 4-mediated signaling pathway in lipopolysaccharide-stimulated BV2 microglia

Genistein, a principal soy isoflavone, has received considerable attention as a protein kinase inhibitor. Although some studies have demonstrated that genistein possesses anti-inflammatory effects, the molecular mechanisms of genistein-mediated anti-inflammatory potential are unclear in microglial cells. In this study, we determined whether genistein attenuates pro-inflammatory responses in lipopolysaccharide (LPS)-stimulated BV2 microglia and attempted to establish the possible mechanisms. Our results indicated that genistein inhibited the production of nitric oxide (NO) and prostaglandin E2 at non-toxic concentrations by inhibiting inducible NO synthase and cyclooxygenase-2 expression. The increased release and expression of inflammatory cytokines, including interleukin-1β, tumor necrosis factor-α, by LPS, were markedly reduced by genistein. Genistein also attenuated LPS-induced reactive oxygen species generation and LPS-mediated nuclear translocation of nuclear factor-kappa B (NF-κB), associated with blocking degradation of the inhibitor of NF-κB-α. Furthermore, genistein potently suppressed binding of LPS to the microglial cell surface, indicating the antagonistic effect of genistein against toll like receptor 4 (TLR4), and inhibited LPS-induced TLR4 and myeloid differentiation factor 88 expression. In addition, blocking TLR4 signaling using the specific TLR4 signaling inhibitor CLI-095 increased the anti-inflammatory potential of genistein in BV2 microglia. Our data indicate that genistein may attenuate the initiation of intracellular signaling cascades by LPS through inhibiting NF-κB activation by inhibiting the binding of LPS to TLR-4 on microglial cells.

Inhibition of Toll-like receptor 4 suppresses liver injury induced by biliary obstruction and subsequent intraportal lipopolysaccharide injection

The objective of this study was to elucidate the role of Toll-like receptor 4 (TLR4) in liver injury induced by biliary obstruction and subsequent intraportal lipopolysaccharide (LPS) infusion in rats. Biliary obstruction often leads to the development of bacterial translocation. Rats were subjected to either a sham operation (Sham group) or bile duct ligation for 7 days (BDL group). Seven days after each operation, LPS (0.5 μg) was injected through the ileocecal vein. In other experiments, rats that had undergone BDL were pretreated, before LPS challenge, with internal biliary drainage (Drainage group); intravenous TAK-242, a TLR4 inhibitor (TAK group); or intravenous GdCl3, a Kupffer cell deactivator (GdCl3 group). The expression of the TLR4 protein and the number of Kupffer cells in the liver were significantly increased in the BDL group compared with the Sham group. These changes were normalized after biliary drainage. The expression of TLR4 colocalized with Kupffer cells, which was confirmed by double immunostaining. Serum levels of liver enzymes and proinflammatory cytokines after intraportal LPS injection were significantly higher in the BDL group than in the Sham group. However, pretreatment with TAK-242 or GdCl3 strongly attenuated these changes to levels similar to those seen with biliary drainage. These results imply that blocking TLR4 signaling effectively attenuates liver damage to the same level as that observed with biliary drainage in rats with BDL and subsequent intraportal LPS infusion. TAK-242 treatment may be used for patients who are susceptible to liver damage by biliary obstruction and endotoxemia.

A peptide antagonist of the TLR4-MD2 interaction.

Toll-like receptors are an integral part of innate immunity in the central nervous system (CNS); they orchestrate a robust defense in response to both exogenous and endogenous danger signals. Recently, toll-like receptor 4 (TLR4) has emerged as a therapeutic target for the treatment of CNS-related diseases such as sepsis and chronic pain. We herein report a chemical biology approach by using a rationally designed peptide inhibitor to disrupt the TLR4-MD2 association, thereby blocking TLR4 signaling.

Cellular and humoral factors associated with polyriboinosinic-polyribocytidylic acid (poly I:C) induced resistance to banzi virus encephalitis : D.C. Barnhart, E.P. Mayer, A. Ghaffar, and J.D. Gangemi, Department of Microbiology and Immunology, USC School of Medicine, Columbia, SC, 29208, USA

Inflammation has been implicated in the pathogenesis of various cerebral diseases. Thus, control of brain inflammation is regarded as one of the important therapeutic strategies for the treatment of neurodegenerative diseases such as Alzheimer׳s disease and stroke. Isobavachalcone, a flavonoid from Psoralea corylifolia, is known to possess a wide spectrum of biological activities and is expected to be useful in preventing or treating neurodegenerative diseases. However, very little is known regarding its effects on cerebral inflammation. In this study, we examined the effect of isobavachalcone on leukocyte adhesion and intercellular adhesion molecule-1 (ICAM-1) expression in brain endothelial cells activated with lipopolysaccharide (LPS) and explored the possible mechanisms involved. Isobavachalcone significantly down-regulated LPS-induced ICAM-1 expression and leukocyte–endothelial cell adhesion and suppressed NF-κB activity which is implicated in the expression of ICAM-1. It attenuated ICAM-1 expression as well as NF-κB transcriptional activity induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or polyriboinosinic polyribocytidylic acid (poly[I:C]). Isobavachalcone also down-regulated LPS or poly[I:C]-induced expression of IFN-β, which can indirectly activate NF-κB. These data imply that isobavachalcone can modulate both MyD88-dependent and TRIF-dependent signaling of toll-like receptor 4 (TLR4). Taken together, our data suggest that isobavachalcone inhibits LPS-induced ICAM-1 expression and leukocyte adhesion to brain endothelial cell by blocking TLR4 signaling and thus, has the potential to ameliorate neuronal injury in brain diseases associated with inflammation.