Blockade Of Toll-like Receptor 4 Research Articles

Abstract P111: Systemic TLR4 Blockade Ameliorates Neuroinflammation & Blood-Brain Barrier Disruption In Hypertensive Rats

Hypertension is linked to blood-brain barrier (BBB) disruption and neuroinflammation in areas such as the hypothalamic paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM). We have shown that Angiotensin II (AngII) induces BBB breakdown via type 1 receptors (AT1r) and activates hypothalamic microglia ex vivo through AT1r and Toll-like receptor 4 (TLR4). We hypothesized that TLR4 activation is a key contributor to AngII-mediated BBB disruption, neuroinflammation, and autonomic dysfunction. Spontaneously hypertensive rats (SHR) were treated with Losartan (AT1r blocker; 20 mg/kg; oral; 4 weeks; SHR-Los) or TAK-242 (TLR4 blocker; 2 mg/kg; i.p. ; 2 weeks; SHR-TAK), and age-matched to control SHR and Wistar Kyoto rats (WKY). Indirect tail-cuff mean arterial pressure (MAP) was normalized in SHR-Los (104 vs 100 mmHg in WKY) and reduced in SHR-TAK (129 vs 154 mmHg in SHR). During conscious baroreflex assessment, TAK-242 rescued the responsiveness of SHR baroreflex gain (ΔHR/ΔMAP; phenylephrine: WKY: -2.56; SHR: -1.47; SHR-TAK: -2.20; nitroprusside: WKY: -2.39; SHR: -0.91; SHR-TAK: -2.06). Sympathetic nervous system activity (indirect; hexamethonium) was elevated in SHR (-65 ΔMAP) versus WKY (-47 ΔMAP) and SHR-TAK (-43 ΔMAP). Microglial activation, indexed by morphological analysis of IBA1 immunofluorescence (IF), was enhanced in SHR ( PVN: end-points [EP]: -36.1%, branch length [BL]: -26.8%; RVLM: EP: -41.6%, BL: -33.4% vs WKY) and normalized in SHR-Los and SHR-TAK. Elevated TNF-α IF densities in SHR (PVN: +57.9; RVLM: +58.0% vs WKY) were abolished with AT1r or TLR4 blockade. Both treatments normalized PVN IL-6 IF (SHR: +46.6% vs WKY) and reduced RVLM IL-6 (SHR: +77.4%; SHR-Los: +32.4%; SHR-TAK: +33.9% vs WKY). Increased BBB permeability in SHR, quantified by leakage of a low MW dextran-conjugated dye, was apparent in the PVN (3.619% area), RVLM (3.700% area), and nucleus tractus solitarius (NTS; 3.588% area) compared to WKY (PVN: 1.558%, RVLM: 1.572%, NTS: 1.800% area), while TAK-242 restored the barrier’s integrity in each region. Taken together, these data support a significant contribution of TLR4 activation to AngII-driven autonomic dysfunction, neuroinflammation, and BBB disruption in hypertension.

Toll-like receptor 4 (TLR4) signaling in the trigeminal ganglion mediates facial mechanical and thermal hyperalgesia in rats

There is increasing evidence that the toll-like receptor 4 (TLR4) signaling pathway contribute to development of hyperalgesia in the trigeminal system. The aim of the present study was to investigate the role of TLR4 in the trigeminal ganglion (TG) in facial hyperalgesia induced by injection of Lipopolysaccharide (LPS) or intraoral mucosal incision, which is an orofacial postoperative pain model, in male Wistar rats. The TLR4 antagonist (LPS-RS, 20 µg/10 µL) was administrated 30 min before LPS injection into the TG (10 µg/10 µL) or oral mucosa (10 µg/50 µL). In the postoperative pain model, rats were treated with LPS-RS (20 µg/10 µL) into the TG for three consecutive days after the incision. Facial heat and mechanical hyperalgesia were assessed hourly after LPS injection or intraoral incision. In addition, expression of NFκB was assessed in the TG on day 3 after intraoral incision. Our results showed that blockade of TLR4 in the TG attenuated facial heat and mechanical hyperalgesia induced by LPS or by mucosal incision, and that both conditions are associated to increase of phosphorylated NFκB in the TG. In conclusion, the present study suggests that activation of TLR4-NFκB signaling pathway in the TG contributes to the development of facial heat and mechanical hyperalgesia and may contribute to pain in inflammatory oral conditions.

Toll-Like Receptor 4 Inhibitor TAK-242 Augments Acetylcholine-Induced Relaxation in Superior Mesenteric Arteries of the Streptozotocin-Induced Diabetic Rat.

Although vascular dysfunction is a key event in the development of diabetic complications, and abnormal toll-like receptor 4 (TLR4) may contribute to the pathophysiology of vascular diseases, the direct relationships between TLR4 and vascular function in diabetic arteries are still poorly understood. Thus, to investigate whether pharmacological blockade of TLR4 affects vascular function in the superior mesenteric artery (SMA) of streptozotocin (STZ)-induced diabetic rats, the SMA was isolated from male Wistar rat injected once with STZ (65 mg/kg, 27-34 weeks) which was treated with TAK-242 (10-6 M), a TLR4 inhibitor, for approximately 1 d using organ culture techniques. After incubation, functional and biochemical studies were performed. In the functional study, treatment with TAK-242 increased acetylcholine (ACh)-induced relaxation of the diabetic SMA in the intact condition. Sodium nitroprusside (SNP)-induced relaxation was also increased in the TAK-242-treated group compared with the vehicle-treated group. Under cyclooxygenase (COX) blockade by indomethacin (10-5 M), ACh-induced relaxation was similar in the vehicle- and TAK-242-treated groups. In addition, ACh-induced relaxation in the combined presence of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (L-NNA) (10-4 M), and indomethacin (10-5 M) was similar in the vehicle- and TAK-242-treated groups. The productions of thromboxane (TX) B2 in cultured medium in the presence of ACh (10-5 M) were lower in the TAK-242-treated group than in the vehicle-treated group. These data suggested that TAK-242 could augment endothelium-dependent relaxation by partly suppressing vasoconstrictor TXA2 or increasing NO signaling. TLR4 inhibition may be a novel therapeutic strategy to assist in the management of diabetes-associated vascular complications.

Saturated fatty acids activate the inflammatory signalling pathway in Schwann cells: Implication in sciatic nerve injury.

Sciatic nerve injury affects quality of life. Many immune cells and inflammatory cytokines have been reported to be involved in sciatic nerve injury, but little is known about the ligands and receptors that trigger inflammatory responses. By using a modified sciatic nerve clamp injury method, we found that the recruitment of Schwann cells and the inflammatory response were enhanced after sciatic nerve injury. Toll-like receptor 4 (TLR4), one of the major members of the TLR family, is highly expressed in Schwann cells. Under certain conditions, myeloid differentiation protein 2 (MD2) binds to TLR4 on the membrane and plays important roles in the inflammatory response. The reductions in the recruitment of Schwann cells and the inflammatory response induced by the blockade of TLR4 or MD2 suggest that TLR4 and MD2 are involved in sciatic nerve injury. What are the endogenous signals that activate the inflammatory response? A large number of free saturated fatty acids (SFAs) are released from Schwann cells, adipocytes and the blood after sciatic nerve injury. Liang et al reported that Schwann cells can be stimulated by palmitic acid (PA). Here, we found that the expression and secretion of TNF-α and IL-6 were enhanced by PA treatment. Moreover, PA activated TLR4 signalling pathway-related proteins and stimulated a strong association between TLR4 and MD2. Blocking TLR4 or MD2 reversed the PA-induced inflammatory response and TLR4 downstream signalling pathway. Thus, we speculated that SFAs act as endogenous ligands that activate TLR4/MD2, thus triggering Schwann cell inflammation during sciatic nerve injury.

640 CrispR/Cas9 deletion of TLR4 impacts the UV-induced stress response in human keratinocytes

Damage incurred by exposure to ultraviolet (UV) radiation stimulates multiple stress-responsive signaling pathways in the skin. There is mounting evidence that Toll-like receptor 4 (TLR4) mediates key inflammatory responses in skin cells, including keratinocytes. Pharmacological blockade of TLR4 using resatorvid (TAK-242) inhibits not only acute UV-induced signaling in vitro and in vivo, but blocks UV-induced skin carcinogenesis in mouse models. In order to better characterize the UV-induced responses linked to TLR4, we have recently utilized CRISPR/Cas9 techniques to delete TLR4 from the genome of human HaCaT keratinocyte cells in culture.

1735-P: Effect of TLR4 Inhibition in Fat-Induced Insulin Resistance in Human Subjects

Obese subjects have increased levels of free fatty acids (FFA) in plasma that may mediate the inflammation and insulin resistance seen in these individuals. Data generated mostly in cells and rodents suggest that toll like receptor 4 (TLR4) mediates the inflammatory and insulin resistant states induced by FFA. In the present study, we tested the hypothesis that pharmacologic blockade of TLR4 with eritoran would prevent lipid-induced insulin resistance. We recruited 10 lean, healthy subjects (Age: 51 ± 1 y, Sex: 6M/4F, BMI: 23.8 ± 0.7 kg/m2, Fasting plasma glucose (FPG): 5.5 ± 0.1 mmol/l). They were randomized to receive the following 72 h long i.v. treatments on separate occasions: saline (30 ml/h)+vehicle (12 mg every 12 h); Intralipid (30 ml/h)+vehicle; Intralipid (30 ml/h)+eritoran (12 mg every 12 h). After these infusions, insulin sensitivity was measured with an hyperinsulinemic clamp. Infusion of Intralipid significantly decreased insulin sensitivity (M value) by 14%. FPG and fasting plasma insulin concentrations increased with Intralipid infusion by 7% and 22%, respectively. Intralipid also caused a low-grade inflammatory state, evidenced by increases in plasma levels of TNFa (32%), lipopolysaccharide (14%), LPS binding protein (21%), and blood monocytes counts (15%). However, metabolic and inflammatory outcomes were not different between the Intralipid+vehicle and the Intralipid+eritoran groups. We conclude that short-term TLR4 inhibition with eritoran fails to prevent lipid-induced inflammation and insulin resistance. Studies with longer acting TLR4 inhibitors may be needed to clarify the role of TLR4 on the pro-inflammatory and insulin resistant states seen obesity and type 2 diabetes. Disclosure H. Liang: None. N. Sathavarodom: None. C.V. Colmenares: None. V. Ganapathy: None. N. Musi: None. Funding American Diabetes Association (7-13-GSK-01 to N.M.); National Institutes of Health (R01DK080157)

Blockade of Toll-like receptor 4 (TLR4) reduces oxidative stress and restores phospho-ERK1/2 levels in Leydig cells exposed to high glucose

AimsHyperglycemia in combination with oxidative stress plays a significant pathophysiological role in diabetic testicular dysfunction, often leading to infertility. Activation of Toll-like receptor 4 (TLR4) has been reported to mediate oxidative stress during diabetes. However, engagement of the TLR4 signaling pathway in diabetic testicular dysfunction has not been previously explored. Herein, we investigated the role of TLR4 in reactive oxygen species (ROS) production and in the phosphorylation status of ERK1/2 in primary Leydig cells exposed to high glucose and in testis isolated from diabetic rats. Main methodsTesticular levels of TLR4 and phospho-ERK1/2 were determined by Western blotting. ROS production was detected with a fluorescent probe. Additionally, primary Leydig cells were exposed to normal (5.5 mmol/l) or elevated (33 mmol/l) glucose concentrations and treated with or without a TLR4 inhibitor, CLI095 (10−5 mol/l) for 24 h, followed by evaluation of TLR4 and phospho-ERK1/2 expression levels by Western blotting and immunofluorescence staining, respectively. Key findingsWe show that high glucose induces the expression of TLR4 in Leydig cells. Additionally, we demonstrate that blockade of this receptor in this cell population reduces oxidative stress and restores the levels of phospho-ERK1/2. SignificanceOur findings provide new insight into TLR4 interaction with ROS and MEK/ERK pathway in Leydig cells exposed to high glucose and present a rationale for the development of new therapeutics for diabetic testicular dysfunction.

A (1→6)-Branched (1→4)-β-d-Glucan from Grifola frondosa Inhibits Lipopolysaccharide-Induced Cytokine Production in RAW264.7 Macrophages by Binding to TLR2 Rather than Dectin-1 or CR3 Receptors.

Mushroom polysaccharides including β-glucans possess various health-promoting properties and are known to be the major bioactive constituents of Grifola frondosa (GF), which is a popular edible and medicinal mushroom. Dectin-1, a pattern-recognition receptor, is responsible for recognizing β-glucans. In this study, parental RAW264.7 macrophages and Dectin-1-expressing RAW264.7 macrophages were used to investigate the anti-inflammatory activity and receptor involvement of the water-soluble polysaccharides from GF. Results indicated that the high molecular weight fraction of GF (GF70-F1; 1260 kDa) inhibited TNF-α and IL-6 production as well as NF-κB activation in lipopolysaccharide-induced macrophages. Chemical and enzymatic linkage analyses indicated that GF70-F1 mainly contained the known (1→3),(1→6)-β-d-glucan and a polysaccharide not previously isolated from GF, a nondigestible glucan with a β-(1→4)-linked backbone and β-(1→6)-linked branches. The ability of GF70-F1 to inhibit cytokine production was not affected by the expression level of Dectin-1 in cells, and a similar inhibitory activity was observed after removing the (1→3),(1→6)-β-d-glucan from GF70-F1. Blockade of Toll-like receptor 2 (TLR2) but not Dectin-1 or complement receptor 3 (CR3) attenuated the inhibitory activity of GF70-F1. The nondigestible (1→6)-branched (1→4)-β-d-glucan in GF70-F1 may contribute to the anti-inflammatory activity via interacting with TLR2 rather than Dectin-1 or CR3 receptors.

Blockade of TLR4 using TAK-242 (resatorvid) enhances anti-cancer effects of chemotherapeutic agents: a novel synergistic approach for breast and ovarian cancers.

It is believed that pathways of the immune system are responsible for eradicating cancer cells; however, their over-activation and also their ectopic expression in tumor cells and microenvironment are major contributors to tumor growth and chemoresistance. Toll-like receptor 4 (TLR4) pathway is an innate immune-related pathway which is usually overexpressed in tumor cells that leads to excessive pro-inflammatory cytokines and eventually results in tumor survival, drug resistance, and metastasis. In this study, we investigated whether TLR4 expression is affected upon the treatment of breast and ovarian cancer cells with common chemotherapeutics (paclitaxel, cisplatin, doxorubicin, and arsenic trioxide) and if TLR4 inhibition using its specific inhibitor TAK-242 could enhance cancer cells' response to the drugs. Both breast (MCF7) and ovarian (2008C13) cancer cells experienced an elevated expression of TLR4 after treatment with the drugs. The expression of this receptor was also upregulated in cisplatin-resistant 2008C13 cells; however, it was significantly higher upon short-term treatment with cisplatin. More importantly, the combination treatment of the drugs with TAK-242 intensified the chemosensitivity of six different breast and ovarian cancer cells to chemotherapeutic drugs. It was also identified that co-treatment of paclitaxel and TAK-242 not only led to enhanced G2/M arrest and apoptosis but also satisfactorily decreased the expression of TLR4 and different interleukins in these cells. Taken together, the results of the present study emphasize that chemotherapy may lead to chemoresistance through inducing TLR4 expression, and therefore inhibiting this receptor using TAK-242 could be a promising approach to improve the outcome of chemotherapy in foreseeable future.

EFFECT OF TLR4 INHIBITION IN FAT-INDUCED INSULIN RESISTANCE IN HUMAN SUBJECTS

Older and obese subjects have increased levels of free fatty acids (FFA) in plasma that may mediate the inflammation and insulin resistance seen in these individuals. Data generated mostly in cells and rodents suggest that toll like receptor 4 (TLR4) mediates the inflammatory and insulin resistant states induced by FFA. In the present study, we tested the hypothesis that pharmacologic blockade of TLR4 would prevent lipid-induced insulin resistance. We recruited 10 lean, healthy subjects (Age: 51 ± 1 y, Sex: 6M/4F, BMI: 23.8 ± 0.7 kg/m2, Fasting plasma glucose (FPG): 5.5 ± 0.1 mmol/l). They were randomized to receive the following 72 h long i.v. treatments on separate occasions: saline (30 ml/h)+placebo (12 mg every 12 h); Intralipid (30 ml/h)+placebo; Intralipid (30 ml/h)+eritoran (12 mg every 12 h). After these infusions, insulin sensitivity was measured with an hyperinsulinemic clamp. Infusion of Intralipid significantly decreased insulin sensitivity (M value) by 14%. FPG and fasting plasma insulin concentrations increased with Intralipid infusion by 7% and 22%, respectively. Intralipid also caused a low-grade inflammatory state, evidenced by increases in plasma levels of TNFa (32%), lipopolysaccharide (14%), LPS binding protein (21%), and blood monocytes counts (15%). However, metabolic and inflammatory outcomes were not different between the Intralipid+placebo and the Intralipid+eritoran groups. We conclude that short-term TLR4 inhibition with eritoran fails to prevent lipid-induced inflammation and insulin resistance. Studies with longer acting TLR4 inhibitors may be needed to clarify the role of TLR4 on the pro-inflammatory and insulin resistant states seen with aging and obesity.

P4691Chronic blockade of toll-like receptor 9 ameliorated pulmonary arterial hypertension by reducing perivascular inflammation in rats

Abstract Background/Introduction Perivascular inflammation plays an important role in the pathogenesis of pulmonary arterial hypertension (PAH). Recent studies have demonstrated that damaged mitochondrial DNA induces sterile inflammation by activating toll-like receptor (TLR)9 in spontaneous hypertensive rats. However, it remains unclear whether TLR9 is involved in perivascular inflammation and subsequent development of PAH. Purpose The purpose of the present study is to investigate whether chronic inhibition of TLR9 can ameliorate monocrotaline (MCT)-induced PAH in rats. Methods Male Sprague-Dawley rats were injected with MCT (60 mg/kg). First, we conducted immunohistochemistory to examine which cell types express TLR9 in lungs of normal rats and MCT-exposed rats. Second, we extracted cell-free DNA from plasma of rats and amplified genes of COX2 by real-time PCR to detect circulating cell-free mitochondrial DNA, a ligand of TLR9. Third, the administration of a selective TLR9 inhibitor (E6446, 10mg/kg/day, drinking water) or non-selective TLR9 inhibitor (chloroquine: 50mg/kg/day, ip) started three days before MCT injection and sacrificed on day 21. We assessed hemodynamic data and histopathological analysis (EVG stain for medial wall thickness (MWT) in pulmonary arteries (outer diameter: 50 ∼ 100 μm) and CD68 for macrophage accumulation around pulmonary arteries (outer diameter: <50 μm)), and measured the levels of interleukin-6 (IL-6) in lungs by real time PCR. Finally, we investigated survival rate in the reversal protocol, where we started the administration of E6446 on day 14. Results TLR9 was expressed dominantly in pulmonary endothelial cells and macrophages in the lungs of both normal rats and MCT-exposed rats. Compared with normal rats, MCT-exposed rats showed increased gene expression of COX2 (0.048±0.001 vs. 0.052±0.001 expressed by 1/Ct) in plasma on day 14. MCT-exposed rats also had increased right ventricular systolic pressure (RVSP: 21±1 vs. 60±2 mmHg), total pulmonary vascular resistance index (TPRI: 0.07±0.01 vs. 0.43±0.02 mmHg/min/mL/kg), MWT (0.07±0.01 vs. 0.26±0,02) and accumulation of macrophages (1.6±0.3 vs. 20.0±1.7 cells/HPF) on day 21. In the prevention protocol, either E6446 or chloroquine significantly prevented the elevations of RVSP (49±4 or 48±3 mmHg) and TPRI (0.29±0.04 or 0.27±0.03 mmHg/min/mL/kg) with reducing MWT (0.18±0.01 or 0.18±0.01) and macrophage accumulation (9.7±1.3 or 9.8±2.5 cell/HPF) on day 21. In addition, these drugs significantly reduced the levels of IL-6 mRNA compared with MCT group (4.4±1.0 or 4.8±1.4 vs. 11.9±1.0). In the reversal protocol, the treatment of E6446 had significantly increased the survival rate (50 vs. 10%). Conclusions TLR9 largely contributes to the development of PAH by reducing perivascular inflammation. Inhibition of TLR9 could be a novel therapeutic target for PAH.

TLR4 blockade using TAK-242 suppresses ovarian and breast cancer cells invasion through the inhibition of extracellular matrix degradation and epithelial-mesenchymal transition

Numerous links exist between inflammation and tumor development. Toll-like receptor 4 (TLR4) expression by tumor cells can be a contributing factor that promotes tumor cell proliferation, survival, migration, and metastasis. In this study, we explored the impact of TLR4 inhibition using TAK-242, a specific inhibitor of TLR4, on the invasion properties of ovarian (A2780CP, 2008C13, SKOV3, and A2780S) and breast (MCF7, SKBR3, MDA-MB-231, and BT-474) cancer cell lines. Six out of eight cell lines expressed TLR4 and its downstream mediators (MyD88, NF-ĸB1, and RELB), indicating that these cell lines could be proper candidates for the TLR4 inhibition. TAK-242 induced a cytotoxic effect on all tested cell lines; however, a different cell sensitivity pattern was noticeable. Interestingly, in the TLR4-expressing cell lines, there was a significant correlation between the TLR4/MyD88 expressions and the cancer cell response to TAK-242: the higher the expression, the higher the IC50. To the best of our knowledge, no study has addressed the effects of TAK-242 on invasive abilities of cancer cells and our study suggests for the first time that TAK-242 could considerably decrease invasion properties of ovarian and breast cancer cell lines. We found that not only did TAK-242 reduce the enzymatic activity of MMP2 and MMP9, but also down-regulated gene expressions of epithelial-mesenchymal transition (EMT)-related genes. In sum, it seems that targeting TLR4 using TAK-242 possesses novel promising potential in cancer treatment strategies and may prevent invasion in patients suffering from ovarian and breast cancers, especially in those with over-expression of TLR4.

Nanoparticle incorporating Toll-like receptor 4 inhibitor attenuates myocardial ischaemia-reperfusion injury by inhibiting monocyte-mediated inflammation in mice.

Myocardial ischaemia-reperfusion (IR) injury hampers the therapeutic effect of revascularization in patients with acute myocardial infarction (AMI). Innate immunity for damage-associated protein patterns promotes the process of IR injury; however, the blockade of Toll-like receptor 4 (TLR4) in myocardial IR injury has not been translated into clinical practice. Therefore, we aimed to examine whether the nanoparticle-mediated administration of TAK-242, a chemical inhibitor of TLR4, attenuates myocardial IR injury in a clinically feasible protocol in a mouse model. We have prepared poly-(lactic-co-glycolic acid) nanoparticles containing TAK-242 (TAK-242-NP). TAK-242-NP significantly enhanced the drug delivery to monocytes/macrophages in the spleen, blood, and the heart in mice. Intravenous administration of TAK-242-NP (containing 1.0 or 3.0 mg/kg TAK-242) at the time of reperfusion decreased the infarct size, but the TAK-242 solution did not even when administered at a dosage of 10.0 mg/kg. TAK-242-NP inhibited the recruitment of Ly-6Chigh monocytes to the heart, which was accompanied by decreased circulating HMGB1, and NF-κB activation and cytokine expressions in the heart. TAK-242-NP did not decrease the infarct size further in TLR4-deficient mice, confirming the TLR4-specific mechanism in the effects of TAK-242-NP. Furthermore, TAK-242-NP did not decrease the infarct size further in CCR2-deficient mice, suggesting that monocyte/macrophage-mediated inflammation is the primary therapeutic target of TAK-242-NP. The nanoparticle-mediated delivery of TAK-242-NP represent a novel and clinical feasible strategy in patients undergone coronary revascularization for AMI by regulating TLR4-dependent monocytes/macrophages-mediated inflammation.

Role of Toll-like receptor 4 signaling in mast cell-mediated migrainepain pathway.

Degranulation of meningeal mast cells leading to the sensitization of trigeminal vascular afferent processing is believed to be one of the mechanisms underlying the migraine pain pathway. Recent work suggests that Toll-like receptor 4 (TLR4) may be involved in signaling states of central sensitization. Using a murine model of light aversion produced by compound 48/80 (2 mg/kg, intraperitoneal) mast cell degranulation, employed as a surrogate marker for photophobia observed in migraineurs, we examined the role of TLR4 in migraine-like behavior and neuronal activation. Using a two-chambered light/dark box, we found that compound 48/80 administration in male and female C57Bl/6 mice produced light aversion lasting up to 2 h, and that pre-treatment with sumatriptan (1 mg/kg, i.p.) reliably prevented this effect. Genetic deletion and pharmacological blockade of TLR4 with TAK-242 (3 mg/kg, i.p.) reversed the light aversive effects of compound 48/80 in males but not in females. Assessing the downstream signaling pathway in mutant mice, we found that the TLR4-mediated, light aversion was dependent upon myeloid differentiation primary response gene 88 but not Toll-interleukin-1 receptor domain-containing adapter-inducing interferon-β signaling. In separate groups, male mice sacrificed at 10 min following compound 48/80 revealed a significant increase in the incidence of evoked p-extracellular signal–regulated kinases (+) neurons in the nucleus caudalis of wild type but not Tlr4−/− mice or in mice pre-treated with sumatriptan. This study thus provides the first evidence for involvement of TLR4 signaling through MyD88 in initiating and maintaining migraine-like behavior and nucleus caudalis neuronal activation in the mouse.

Trapping of Lipopolysaccharide to Promote Immunotherapy against Colorectal Cancer and Attenuate Liver Metastasis.

The development and progression of colorectal cancer (CRC) is closely related to gut microbiome. Here, the impact of lipopolysaccharide (LPS), one of the most prevalent products in the gut microbiome, on CRC immunotherapy is investigated. It is found that LPS is abundant in orthotopic CRC tissue and is associated with low responses to anti-PD-L1 mAb therapy, and clearance of Gram-negative bacteria from the gut using polymyxin B (PmB) or blockade of Toll-like receptor 4 using TAK-242 will both relieve the immunosuppressive microenvironment and boost T-cell infiltration into the CRC tumor. Further, an engineered LPS-targeting fusion protein is designed and its coding sequence is loaded into a lipid-protamine-DNA (LPD) nanoparticle system for selective expression of LPS trap protein and blocking LPS inside the tumor, and this nanotrapping system significantly relieves the immunosuppressive microenvironment and boosts anti-PD-L1 mAb therapy against CRC tumors. This LPS trap system even attenuates CRC liver metastasis when applied, suggesting the importance of blocking LPS in the gut-liver axis. The strategy applied here may provide a useful new way for treating CRC as well as other epithelial cancers that interact with mucosa microbiome.

Early TLR4 Blockade Attenuates Sterile Inflammation-mediated Stress in Islets During Isolation and Promotes Successful Transplant Outcomes.

During the isolation process, pancreatic islets are exposed to an environment of sterile inflammation resulting in an upregulated inflammatory state before transplantation. Toll-like receptor 4 (TLR4) has been identified as a major mediator of sterile inflammation. Therefore, we sought to determine whether early TLR4 blockade would be effective in reducing the inflammatory burden in islets pretransplant. Islets from C57BL/6 mice were treated with a TLR4 antagonist during the pancreatic ductal perfusion and digestion steps of the isolation process. Islets were then analyzed for inflammation by RT-PCR and Western blot, and for viability and function in vitro. A syngeneic transplant model using a marginal mass of islets transplanted intraportally into mice with streptozotocin-induced diabetes was used to study transplant outcomes after early TLR4 blockade. Diabetic mice receiving 150 islets treated with early TLR4 blockade achieved euglycemia at a higher rate than mice receiving untreated islets (75% vs 29%; P < 0.05) and had improved long-term function (P < 0.05). Serum markers for islet damage and inflammation were significantly reduced posttransplant (P < 0.05). Both the expression of key inflammatory genes and the activation of mitogen-activated protein kinases were reduced by early TLR4 blockade. Islet viability was improved (P < 0.05) while preserving islet insulin secretory capacity postisolation. Early TLR4 blockade protects islets from sterile inflammation-mediated stress sustained during isolation and promotes positive transplant outcomes. Our findings support the use of early TLR4 blockade during clinical islet isolation procedures to reduce pretransplant inflammation and improve transplant outcomes.

Blockade of Toll-Like Receptor 4 Attenuates Erectile Dysfunction in Diabetic Rats

IntroductionWhile increased toll-like receptor (TLR)4 activity may contribute to the pathophysiology of vascular diseases, the molecular mechanisms disrupted by this receptor in the vasculature are still poorly understood. Additionally, it is unknown if TLR4 mediates erectile dysfunction (ED) during diabetes. AimTo investigate whether pharmacological blockade of TLR4 affects erectile function in a murine model of diabetes. MethodsSprague Dawley rats (Charles River Laboratory, Wilmington, MA, USA) received a single streptozotocin injection (65 mg/kg, 28 days) and were treated with an anti-TLR4 antibody (1 μg/d, intraperitoneally) for the last 14 days of the treatment. Additionally, cavernosal strips were acutely incubated for 30 minutes with CLI-095 (10−5 mol/L), a TLR4 inhibitor. Functional studies, Western blotting, erectile function, immunohistochemistry, and biochemical analyses were performed. Main Outcome MeasuresOxidative stress, cyclic guanosine monophosphate (cGMP) levels, and functional studies were evaluated in treated and nontreated cavernosal strips from control and diabetic animals. Additionally, in vivo erectile function was assessed. ResultsEnhanced TLR4 expression was observed in corpus cavernosum from diabetic rats compared with control animals. Long-term blockade of TLR4 slightly improved diabetes–induced ED in rats due to attenuation of oxidative stress and increased cGMP levels in penile tissue, which ameliorated cavernosal relaxation. Functional experiments revealed that acute or chronic inhibition of TLR4 decreased hypercontractility in response to phenylephrine and improved nitrergic relaxation in corpus cavernosum from diabetic rats. Clinical ImplicationsTLR4 blockade may be a novel therapeutic strategy to assist in ED management. Strengths & LimitationsThe strength of this article stems from the fact that we showed that TLR4 blockade partly improves erectile function in vivo in diabetic rats. Its limitations mainly include that messenger RNA analysis for the nitric oxide/cGMP pathway were not performed. ConclusionIn summary, TLR4 participates in the mechanisms of diabetes-associated ED and blockade of this receptor positively affects penile vascular function.Nunes KP, de Oliveira AA, Szasz T, et al. Blockade of Toll-Like Receptor 4 Attenuates Erectile Dysfunction in Diabetic Rats. J Sex Med 2018;15:1235–1245.

Varying magnesium concentration elicits changes in inflammatory response in human umbilical vein endothelial cells (HUVECs).

The aims of this study were to determine whether low concentrations of magnesium in vitro exacerbated the human umbilical vein endothelial cell (HUVEC) response to inflammatory challenge, and whether expression of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) through the toll-like receptor 4 (TLR4) played a role in this process. HUVECs were incubated with different concentrations of Mg (low- 0.1mM, control- 1mM, high- 5mM) for 72 h before being stimulated with bacterial lipopolysaccharide (LPS) for 4 h. The response of cells to LPS was greater in cells cultured in low Mg, relative to control cells and suppressed in high Mg. Expression of NF-κB was increased in low-Mg and decreased with high Mg. Low Mg increased the expression of TLR4 mRNA, but only in the presence of LPS. Antibody blockade of TLR4 but not TLR2 blunted the response of cells to LPS in low Mg, such that they were similar to unblocked 1mM Mg cells. Associations of Mg with cardiovascular disease may therefore relate to inflammatory responses mediated through the TLR4/NF-κB pathway.

Deletion or pharmacological blockade of TLR4 confers protection against cyclophosphamide-induced mouse cystitis.

Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is a chronic inflammatory disease without consistently effective treatment. We investigate the role of toll-like receptor 4 (TLR4) on voiding dysfunction and inflammation in the cyclophosphamide (CYP)-induced mouse cystitis. Male C57BL/6 [wild-type, (WT)] and/or TLR4 knockout (TLR4-/-) mice were treated with an injection of CYP (300 mg/kg, 24 h) or saline (10 ml/kg). The pharmacological blockade of the TLR4 by resatorvid (10 mg/kg) was also performed 1 h prior CYP-injection in WT mice. Urodynamic profiles were assessed by voiding stain on filter paper and filling cystometry. Contractile responses to carbachol were measured in isolated bladders. In CYP-exposed WT mice, mRNA for TLR4, myeloid differentiation primary response 88, and TIR-domain-containing adapter-inducing interferon-β increased by 45%, 72%, and 38%, respectively ( P < 0.05). In free-moving mice, CYP-exposed mice exhibited a higher number of urinary spots and smaller urinary volumes. Increases of micturition frequency and nonvoiding contractions, concomitant with decreases of intercontraction intervals and capacity, were observed in the filling cystometry of WT mice ( P < 0.05). Carbachol-induced bladder contractions were significantly reduced in the CYP group, which was paralleled by reduced mRNA for M2 and M3 muscarinic receptors. These functional and molecular alterations induced by CYP were prevented in TLR4-/- and resatorvid-treated mice. Additionally, the increased levels of inflammatory markers induced by CYP exposure, myeloperoxidase activity, interleukin-6, and tumor necrosis factor-alpha were significantly reduced by resatorvid treatment. Our findings reveal a central role for the TLR4 signaling pathway in initiating CYP-induced bladder dysfunction and inflammation and thus emphasize that TLR4 receptor blockade may have clinical value for IC/BPS treatment.

High Mobility Group Box 1 Neutralization in the Brain Prevents Inflammation, Sympathoexcitation and Hypertension

High mobility group box 1 (HMGB1) is a nuclear protein, that once translocated to cytoplasm, can act as a chemoattractant and a proinflammatory mediator. Post‐translationally modified HMGB1 can interact with different receptors, such as toll‐like receptor 4 (TLR4) and chemokine receptor CXC receptor 4 (CXCR4), to induce acute and chronic inflammation. We have previously showed that central TLR4 blockade delayed progression of hypertension and improved cardiac function. However, the involvement of HMGB1 and its interaction with TLR4 in the pathogenesis of hypertension has not been fully explored. Therefore, in this study, we investigated the role of HMGB1 in mediating hypertension, inflammation and sympathoexcitation. We hypothesized that blockade of HMGB1 prevents the development of hypertension. Chronic angiotensin II (AngII) infusion (200 ng/kg/min, SC, 14 days) significantly increased mean arterial pressure (telemetry) in male Sprague‐Dawley rats compared to saline‐infused controls (163 ±5 vs. 105 ±4 mmHg, n=7, p&lt;0.01). AngII‐infusion significantly increased HMGB1 levels in the plasma (21 ±3 vs.15 ±2 ng/ml, p&lt;0.01) and cerebrospinal fluid (34±3 vs. 21±1 ng/ml, p&lt;0.01), and elevated HMGB1 protein expression (2.5 fold, n=6, p&lt;0.01 vs. Control) in the brain hypothalamic paraventricular nucleus (PVN). Intracerebroventricular infusion of neutralizing monoclonal anti‐HMGB1 antibody (10 μg/kg/day) attenuated AngII‐induced hypertension (120 ±2 mmHg, p&lt;0.01 vs. AngII). Furthermore, AngII‐infusion significantly increased expression of TNF, IL‐1, IL‐6, TLR4, CXCR4, and NF‐kB in the PVN, which was prevented by HMGB1 neutralization. AngII infusion resulted in sympathoexcitation, as indicated by increased plasma norepinephrine levels (1602 ±53 vs. 549 ±25 pg/ml, P&lt;0.01 vs. Control), but this increase was blunted by HMGB1 neutralization (720 ±25 pg/ml, P&lt;0.05 vs. AngII). Taken together, these data suggest that neutralization of HMGB1 in the brain reduces inflammation and decreases sympathoexcitation, thereby preventing the development of AngII‐induced hypertension.Support or Funding InformationThe American Heart Association, 15SDG25720021 (SS), 16GRNT30510012 (JF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.