Ablation Of Toll-like Receptor 4 Research Articles

Hepatocyte Toll-Like Receptor 4 Mediates Alcohol-Induced Insulin Resistance in Mice.

Accumulating evidence has demonstrated the association between alcohol overconsumption and the development of insulin resistance. However, the underlying mechanisms are not completely understood. To investigate the requirement and sufficiency of hepatocyte toll-like receptor 4 (TLR4) in alcohol-induced insulin resistance, we used two mouse models (Tlr4fl/fl and Tlr4LoxTB) that allow ablation of TLR4 only in hepatocytes (Tlr4LKO) and restoration of endogenous TLR4 expression in hepatocytes on a TLR4-null background (Tlr4LoxTB × Alb-Cre), respectively. A Lieber-DeCarli feeding model was used to induce glucose intolerance and insulin resistance in mice. Glucose tolerance test, insulin tolerance test, and insulin signaling experiments were performed to examine systemic and tissue-specific insulin sensitivity. We found that alcohol-fed hepatocyte TLR4 deficient mice (Tlr4LKO) had lower blood glucose levels in response to intraperitoneal injection of insulin. Moreover, increased phosphorylation of glycogen synthase kinase-3β (GSK3β) was observed in the liver of Tlr4LKO mice after chronic alcohol intake. In contrast, when hepatic TLR4 was reactivated in mice (Tlr4LoxTB × Alb-Cre), alcohol feeding caused glucose intolerance in these mice compared with littermate controls (Tlr4LoxTB). In addition, AKT phosphorylation was dramatically reduced in the liver and epididymal white adipose tissue (eWAT) of alcohol-fed Tlr4LoxTB × Alb-Cre mice, which was similar to that of mice with whole-body TLR4 reactivation (Tlr4LoxTB × Zp3-Cre). Collectively, these findings suggest that hepatocyte TLR4 is both required and sufficient in the development of insulin resistance induced by alcohol overconsumption.

Role of toll-like receptor 4 and sex in 6-hydroxydopamine–induced behavioral impairments and neurodegeneration in mice

Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive loss of the nigrostriatal dopaminergic neurons that are associated with motor alterations and non-motor manifestations (such as depression). Neuroinflammation is a process with a critical role in the pathogenesis of PD. In this regard, toll-like receptor 4 (TLR4) is a central mediator of immune response in PD. Moreover, there are gender-related differences in the incidence, prevalence, and clinical features of PD. Therefore, we aimed to elucidate the role of TLR4 in the sex-dependent response to dopaminergic denervation induced by 6-hydroxydopamine (6-OHDA) in mice. Female and male adult wildtype (WT) and TLR4 knockout (TLR4−/−) mice were administered with unilateral injection of 6-OHDA in the dorsal striatum, and non-motor and motor impairments were evaluated for 30 days, followed by biochemistry analysis in the substantia nigra pars compacta (SNc), dorsal striatum, and dorsoventral cortex. Early non-motor impairments (i.e., depressive-like behavior and spatial learning deficits) induced by 6-OHDA were observed in the male WT mice but not in male TLR4−/− or female mice. Motor alterations were observed after administration of 6-OHDA in both strains, and the lack of TLR4 was also related to motor commitment. Moreover, ablation of TLR4 prevented 6-OHDA–induced dopaminergic denervation and microgliosis in the SNc, selectively in female mice. These results reinforced the existence of sex-biased alterations in PD and indicated TLR4 as a promising therapeutic target for the motor and non-motor symptoms of PD, which will help counteract the neuroinflammatory and neurodegenerative processes.

TLR4-Endothelin Axis Controls Syncytiotrophoblast Motility and Confers Fetal Protection in Placental Malaria.

Pregnancy-associated malaria is often associated with adverse pregnancy outcomes. Placental circulatory impairments are an intriguing and unsolved component of malaria pathophysiology. Here, we uncovered a Toll-like receptor 4 (TLR4)-TRIF-endothelin axis that controls trophoblast motility and is linked to fetal protection during Plasmodium infection. In a cohort of 401 pregnancies from northern Brazil, we found that infection during pregnancy reduced expression of endothelin receptor B in syncytiotrophoblasts, while endothelin expression was only affected during acute infection. We further show that quantitative expression of placental endothelin and endothelin receptor B proteins are differentially controlled by maternal and fetal TLR4 alleles. Using murine malaria models, we identified placental autonomous responses to malaria infection mediated by fetally encoded TLR4 that not only controlled placental endothelin gene expression but also correlated with fetal viability protection. In vitro assays showed that control of endothelin expression in fetal syncytiotrophoblasts exposed to Plasmodium-infected erythrocytes was dependent on TLR4 via the TRIF pathway but not MyD88 signaling. Time-lapse microscopy in syncytiotrophoblast primary cultures and cell invasion assays demonstrated that ablation of TLR4 or endothelin receptor blockade abrogates trophoblast collective motility and cell migration responses to infected erythrocytes. These results cohesively substantiate the hypothesis that fetal innate immune sensing, namely, the TRL4-TRIF pathway, exerts a fetal protective role during malaria infection by mediating syncytiotrophoblast vasoregulatory responses that counteract placental insufficiency.

Monosodium urate crystal interleukin-1β release is dependent on Toll-like receptor 4 and transient receptor potential V1 activation.

Objective The present study aimed to elucidate the mechanisms involved in MSU-induced IL-1β release in a rodent animal model of acute gout arthritis. Methods Painful (mechanical and thermal hypersensitivity, ongoing pain and arthritis score) and inflammatory (oedema, plasma extravasation, cell infiltration and IL-1β release) parameters were assessed several hours after intra-articular injection of MSU (100 µg/articulation) in wild-type or knockout mice for Toll-like receptor 4 (TLR4), inducible nitric oxide synthase (iNOS), transient receptor potential (TRP) V1 and the IL-1 receptor (IL-1R). Also, wild-type animals were treated with clodronate, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) (TLR4 antagonist), spleen tyrosine kinase (SYK) inhibitor (iSYK), aminoguanidine (AMG, an iNOS inhibitor) or SB366791 (TRPV1 antagonist). Nitrite/nitrate and IL-1β levels were measured on the synovial fluid of wild-type mice, 2 h after intra-articular MSU injections, or medium from macrophages stimulated for MSU (1000 μg) for 2 h. Results Intra-articular MSU injection caused robust nociception and severe inflammation from 2 up to 6 h after injection, which were prevented by the pre-treatment with clodronate, LPS-RS, iSYK, AMG and SB366791, or the genetic ablation of TLR4, iNOS, TRPV1 or IL-1R. MSU also increased nitrite/nitrate and IL-1β levels in the synovial fluid, which was prevented by clodronate, LPS-RS, iSYK and AMG, but not by SB366791. Similarly, MSU-stimulated peritoneal macrophages released nitric oxide, which was prevented by LPS-RS, iSYK and AMG, but not by SB366791, and released IL-1β, which was prevented by LPS-RS, iSYK, AMG and SB366791. Conclusion Our data indicate that MSU may activate TLR4, SYK, iNOS and TRPV1 to induce the release of IL-1β by macrophages, triggering nociception and inflammation during acute gout attack.

Monosodium urate crystal interleukin-1β release is dependent on Toll-like receptor 4 and transient receptor potential V1 activation.

The present study aimed to elucidate the mechanisms involved in MSU-induced IL-1β release in a rodent animal model of acute gout arthritis. Painful (mechanical and thermal hypersensitivity, ongoing pain and arthritis score) and inflammatory (oedema, plasma extravasation, cell infiltration and IL-1β release) parameters were assessed several hours after intra-articular injection of MSU (100 µg/articulation) in wild-type or knockout mice for Toll-like receptor 4 (TLR4), inducible nitric oxide synthase (iNOS), transient receptor potential (TRP) V1 and the IL-1 receptor (IL-1R). Also, wild-type animals were treated with clodronate, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) (TLR4 antagonist), spleen tyrosine kinase (SYK) inhibitor (iSYK), aminoguanidine (AMG, an iNOS inhibitor) or SB366791 (TRPV1 antagonist). Nitrite/nitrate and IL-1β levels were measured on the synovial fluid of wild-type mice, 2 h after intra-articular MSU injections, or medium from macrophages stimulated for MSU (1000 μg) for 2 h. Intra-articular MSU injection caused robust nociception and severe inflammation from 2 up to 6 h after injection, which were prevented by the pre-treatment with clodronate, LPS-RS, iSYK, AMG and SB366791, or the genetic ablation of TLR4, iNOS, TRPV1 or IL-1R. MSU also increased nitrite/nitrate and IL-1β levels in the synovial fluid, which was prevented by clodronate, LPS-RS, iSYK and AMG, but not by SB366791. Similarly, MSU-stimulated peritoneal macrophages released nitric oxide, which was prevented by LPS-RS, iSYK and AMG, but not by SB366791, and released IL-1β, which was prevented by LPS-RS, iSYK, AMG and SB366791. Our data indicate that MSU may activate TLR4, SYK, iNOS and TRPV1 to induce the release of IL-1β by macrophages, triggering nociception and inflammation during acute gout attack.

TLR4-dependent upregulation of the platelet NLRP3 inflammasome promotes platelet aggregation in a murine model of hindlimb ischemia

Platelets play a critical role in the pathophysiology of peripheral arterial disease (PAD). The mechanisms by which muscle ischemia regulates aggregation of platelets are poorly understood. We have recently identified the Nod-like receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) expressed by platelets as a critical regulator of platelet activation and aggregation, which may be triggered by activation of toll-like receptor 4 (TLR4). In this study, we performed femoral artery ligation (FAL) in transgenic mice with platelet-specific ablation of TLR4 (TLR4 PF4) and in NLRP3 knockout (NLRP3−/−) mice. NLRP3 inflammasome activity of circulating platelets, as monitored by activation of caspase-1 and cleavage of interleukin-1β (IL-1β), was upregulated in mice subjected to FAL. Genetic ablation of TLR4 in platelets led to decreased platelet caspase 1 activation and platelet aggregation, which was reversed by the NLRP3 activator Nigericin. Two weeks after the induction of FAL, ischemic limb perfusion was increased in TLR4 PF4 and NLRP3−/− mice as compared to control mice. Hence, activation of platelet TLR4/NLRP3 signaling plays a critical role in upregulating platelet aggregation and interfering with perfusion recovery in muscle ischemia and may represent a therapeutic target to improve limb salvage.

Abstract 369: Muscle Ischemia Induces Nlrp3 Inflammasome Activation in Platelets via Tlr4, Promoting Platelet Aggregation and Interfering With Perfusion Recovery

Introduction: Angiogenesis is an adaptive response to chronic ischemia, but is deficient in peripheral arterial disease (PAD). We have recently shown that the pattern recognition receptors toll-like receptor 4 (TLR4) and nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) expressed by platelets control aggregation and thrombosis. The role of platelet TLR4 and NLRP3 in PAD is unexplored. Methods: Unilateral femoral artery ligation (FAL) was performed in transgenic mice with platelet-specific ablation of TLR4 (TLR4 PF4) and in global NLRP3 knockout (NLRP3 KO) mice. Platelet NLRP3 inflammasome activation was monitored by caspase-1 activation (fluorescent labeled inhibitor of caspase-1, FLICA) and cleavage of IL1β (Western blot). Platelet aggregation was evaluated with impedance aggregometry. Laser Doppler perfusion imaging (LDPI) verified perfusion in the ischemic (right) and non-ischemic (left) limb over time. Angiogenesis and myoblast regeneration were measured histologically. Results: Platelet NLRP3 inflammasome activity was significantly upregulated following FAL (p<0.001), and reversed with TAK242, a TLR4 inhibitor. FAL significantly increased aggregation of circulating platelets, which was significantly suppressed in TLR4 PF4 (p<0.01) and NLRP3 KO mice (p<0.001). Down-regulation of platelet aggregation and caspase-1 activity in TLR4 PF4 mice was nearly completely reversed by nigericin, a NLRP3 activator. Ischemic limb perfusion (Fig 1A) was significantly higher in TLR4 PF4 (Fig1B, p<0.05) and NLRP3 KO mice (Fig1C, p<0.001) than in controls 14d after FAL. Angiogenesis and regeneration were significantly improved in TLR4 PF4 mice (p<0.05). Conclusion: We show that the platelet NLRP3 inflammasome is activated in muscle ischemia via platelet TLR4, which upregulates platelet aggregation and deters recovery from FAL. Thus, platelet TRL4/NLRP3 activation may be a therapeutic target to improve limb salvage in PAD.

Abstract 509: Fibronectin Containing Extra Domain a Modulates TLR4-Dependent Smooth Muscle Cell Proliferation and Neointimal Hyperplasia in Apolipoprotein E-Deficient Mice

Background: Fibronectin-splice variant containing extra domain A (Fn-EDA), an endogenous ligand of toll-like-receptor 4 (TLR4), is a characteristic feature of proliferating vascular smooth muscle cells (VSMCs) that contributes to intimal thickening and atherosclerosis. Very little is known about the role of Fn-EDA in VSMC proliferation or its clinical implication in neointima formation after vascular injury in the comorbid condition of hyperlipidemia. Methods: To explore the mechanism and therapeutic potential of targeting Fn-EDA in vascular diseases, we used carotid wire-injury model to induce neointimal hyperplasia (28 days) in hyperlipidemic Apoe -/- , Fn-EDA -/- Apoe -/- , TLR4 -/- Apoe -/- , Fn-EDA -/- TLR4 -/- Apoe -/- and SMC- specific Fn-EDA -/- mice (Fn-EDA fl/fl αSMACre + Apoe -/- ) (male and female; 10 weeks old). Furthermore, VSMCs were isolated from different genotypes and stimulated with PDGF to dissect the mechanisms. Results: Using immunostaining, we found increased expression of Fn-EDA in human atheroma, injured carotid artery and proliferating VSMCs of Apoe -/- mice (P<0.05 vs. control). Irrespective of gender, genetic deletion of Fn-EDA in Apoe -/- mice reduced neointimal area after vascular injury (P<0.05 vs. Apoe -/- ). Aortic VSMCs isolated from Fn-EDA -/- Apoe -/- mice exhibited reduced cell proliferation (BrdU assay, G1/S phase transition) and migration (wound healing assay) that was concomitant with elevated pAKT2 and pFOXO4 expression and reduction in pAKT1, pmTOR, pNFκB, TNF-α and IL-1β secretion levels (P<0.05 vs. Apoe -/- ). Genetic ablation of TLR4 in Apoe -/- mice reduced neointimal hyperplasia concomitant with decreased VSMCs proliferation and migration (P<0.05 vs. Apoe -/- ), but had no additive effect in Fn-EDA -/- Apoe -/- mice. Purified cellular Fn, which contains EDA, attenuated pAKT2 and pFOXO4 and potentiated pAKT1, pmTOR, pNFκB, TNF-α and IL-1β secretion in SMCs from Fn-EDA -/- Apoe -/- mice (P<0.05 vs. plasma Fn that lacks Fn-EDA) but not from Fn-EDA -/- TLR4 -/- Apoe -/- mice. Finally, SMC-specific Fn-EDA -/- mice (Fn-EDA fl/fl αSMACre + Apoe -/- ) exhibited reduced neointimal hyperplasia. Conclusion: Fn-EDA modulates TLR4-dependent VSMCs proliferation and intimal hyperplasia after vascular injury in the comorbid condition of hyperlipidemia.

Abstract 374: Lack of Fibronectin Containing Extra Domain a Attenuates Acute Myocardial Ischemia/Reperfusion Injury in Hyperlipidemic Mice by Limiting Postischemic Thrombo-inflammation

Background: Reperfusion therapy is standard care for patients following acute myocardial infarction. Fibronectin-splicing variant containing extra domain A (Fn-EDA), which is an endogenous ligand for toll-like-receptor 4 (TLR4), is upregulated during vascular hypertension, myocardial injury, and atherosclerosis. An earlier study done in rabbit showed that Fn-EDA accumulates more rapidly (within 24 hrs) in the reperfused hearts. Very little is known about the mechanistic role of Fn-EDA in the pathophysiology of myocardial infarction (MI) in the comorbid condition of hyperlipidemia. Methods: Susceptibility to MI outcome was evaluated in hyperlipidemic Apoe -/- , Fn-EDA -/- Apoe -/- , TLR4 -/- Apoe -/- and Fn-EDA -/- TLR4 -/- Apoe -/- mice (male and female; 8-10 weeks old) by transient 1-hour ischemia/23 hours of reperfusion (I/R). Myocardial I/R injury outcome was evaluated by measuring infarct area, cardiac Troponin I (cTnI) levels in plasma, postischemic thrombo-inflammation (thrombi and neutrophil influx) and myocyte apoptosis. Results: Irrespective of gender, Fn-EDA -/- Apoe -/- mice (Apoe -/- mice expressing Fn deficient in EDA) exhibited smaller infarcts and decreased cTnI levels concomitant with reduced postischemic thrombosis, inflammation and myocyte apoptosis ( P <0.05 vs. Apoe -/ - mice). Genetic ablation of TLR4 attenuated myocardial I/R injury outcome in Apoe -/- mice ( P <0.05 vs. Apoe -/- mice), but did not further reduce in Fn-EDA -/- Apoe -/- mice, suggesting Fn-EDA modulates TLR4-dependent MI exacerbation. Bone marrow transplantation experiments revealed that nonhematopoietic cells-derived Fn-EDA exacerbates myocardial I/R injury through TLR4 expressed on the hematopoietic cell. Infusion of a specific inhibitor of Fn-EDA into Apoe -/- mice significantly reduced myocardial I/R injury. Conclusions: Hyperlipidemic mice deficient in Fn-EDA exhibit TLR4-dependent reduced myocardial I/R injury that was associated with decreased thrombo-inflammatory response. These findings suggest that targeting Fn-EDA combined with thrombolytic agents could be an effective therapeutic strategy to inhibit myocardial I/R injury in patients with hyperlipidemia.

Ablation of toll-like receptor 4 attenuates aging-induced myocardial remodeling and contractile dysfunction through NCoRI-HDAC1-mediated regulation of autophagy

Aging is usually accompanied with overt structural and functional changes as well as suppressed autophagy in the heart although the precise regulatory mechanisms are somewhat unknown. Here we evaluated the role of the innate proinflammatory mediator toll-like receptor 4 (TLR4) in cardiac aging and the underlying mechanism with a focus on autophagy. Cardiac geometry and function were monitored in young or old wild-type (WT) and TLR4 knockout (TLR4−/−) mice using echocardiography, IonOptix® edge-detection and fura-2 techniques. Levels of autophagy and mitophagy, nuclear receptor corepressor 1 (NCoR1) and histone deacetylase I (HDAC1) were examined using western blot. Transmission electronic microscopy (TEM) was employed to monitor myocardial ultrastructure. Our results revealed that TLR4 ablation alleviated advanced aging (24 months)-induced changes in myocardial remodeling (increased heart weight, chamber size, cardiomyocyte cross-sectional area), contractile function and intracellular Ca2+ handling as well as autophagy and mitophagy [Beclin-1, Atg5, LC3B, PTEN-induced putative kinase 1 (PINK1), Parkin and p62]. Aging downregulated levels of NCoR1 and HDAC1 as well as their interaction, the effects were significantly attenuated or negated by TLR4 ablation. Advanced aging disturbed myocardial ultrastructure as evidenced by loss of myofilament alignment and swollen mitochondria, which was obliterated by TLR4 ablation. Moreover, aging suppressed autophagy (GFP-LC3B puncta) in neonatal mouse cardiomyocytes, the effect of which was negated by the TLR4 inhibitor CLI-095. Inhibition of HDCA1 using apicidin cancelled off CLI095-induced beneficial response of GFP-LC3B puncta against aging. Our data collectively indicate a role for TLR4-mediated autophagy in cardiac remodeling and contractile dysfunction in aging through a HDAC1-NCoR1-dependent mechanism.

166 TLR-4-Regulated Cerebrospinal Fluid Hypersecretion in Post-Hemorrhagic Hydrocephalus

Abstract INTRODUCTION Intraventricular hemorrhage (IVH) frequently causes post-hemorrhagic hydrocephalus (PHH) as a result of impaired cerebrospinal fluid (CSF) homeostasis. The most common treatment for PHH is surgical CSF shunting, a procedure fraught with complications. Historically, it is thought that PHH results from impaired reabsorption of CSF, however, little consideration has been given to the role of CSF hypersecretion. Recently, toll-like receptor-4 (TLR-4) has been implicated in the CNS by detecting “alarmins”, including IVH-derived metabolites, via recognition of damage-associated molecular patterns. We speculate that IVH triggers TLR-4-dependent inflammation at the choroid plexus epithelia (CPE), leading to CSF hypersecretion contributing to the development of PHH. METHODS In an established rat model of IVH, we assessed the rate of CSF secretion, CPE inflammation and the effect of genetic and pharmacological intervention on the development of PHH. We developed and implemented a novel surgical method in rats to measure the rate of CSF secretion. TLR-4 knockout rats, antisense oligodeoxynucleotide-mediated gene knockdown, and intracerebroventricular delivery of FDA-approved drugs were used to assess the role of specific inflammatory and ion transport targets on CSF secretion rate and ventriculomegaly following IVH. Immunoblot and immunohistochemistry were used to detect and quantify inflammatory markers. RESULTS >We show IVH triggers TLR4-NFkB-mediated inflammation at the CPE that is associated with a striking ∼3-fold increase in CSF secretion sufficient to cause PHH. CSF hypersecretion is dependent on the NFkB-regulated kinase SPAK, which interacts with and phosphorylates the bumetanide-sensitive NKCC1 at the CPE apical membrane. Genetic ablation of TLR-4 or SPAK, and pharmacology antagonizing TLR-4-NFkB signaling or the SPAK-NKCC1 complex normalizes CSF secretion rates and ventriculomegaly after IVH. CONCLUSION These data uncover a previously unrecognized role for CSF hypersecretion in the pathogenesis of PHH, and reveal a novel TLR-4-dependent regulatory pathway of CSF secretion that can be targeted with repurposed, FDA-approved drugs for the non-surgical treatment of PHH.

TLR4 knockout attenuated high fat diet-induced cardiac dysfunction via NF-κB/JNK-dependent activation of autophagy

Obesity is commonly associated with a low grade systemic inflammation, which may contribute to the onset and development of myocardial remodeling and contractile dysfunction. Toll-like receptor 4 (TLR4) plays an important role in innate immunity and inflammation although its role in high fat diet-induced obesity cardiac dysfunction remains elusive. This study was designed to examine the effect of TLR4 ablation on high fat diet intake-induced cardiac anomalies, if any, and underlying mechanism(s) involved. Wild-type (WT) and TLR4 knockout mice were fed normal or high fat (60% calorie from fat) diet for 12weeks prior to assessment of mechanical and intracellular Ca2+ properties. The inflammatory signaling proteins (TLR4, NF-κB, and JNK) and autophagic markers (Atg5, Atg12, LC3B and p62) were evaluated. Our results revealed that high fat diet intake promoted obesity, marked decrease in fractional shortening, and cardiomyocyte contractile capacity with dampened intracellular Ca2+ release and clearance, elevated ROS generation and oxidative stress as measured by aconitase activity, the effects of which were significantly attenuated by TLR4 knockout. In addition, high fat intake downregulated levels of Atg5, Atg12 and LC3B, while increasing p62 accumulation. TLR4 knockout itself did not affect Atg5, Atg12, LC3B and p62 levels while it reconciled high fat diet intake-induced changes in autophagy. In addition, TLR4 knockout alleviated high fat diet-induced phosphorylation of IKKβ, JNK and mTOR. In vitro study revealed that palmitic acid suppressed cardiomyocyte contractile function, the effect of which was inhibited the TLR4 inhibitor CLI-095, the JNK inhibitor AS601245 or the NF-κB inhibitor Celastrol. Taken together, these data showed that TLR4 knockout ameliorated high fat diet-induced cardiac contractile and intracellular Ca2+ anomalies through inhibition of inflammation and ROS, possibly through a NF-κB/JNK-dependent activation of autophagy. This article is part of a Special Issue entitled: Genetic and epigenetic control of heart failure - edited by Jun Ren & Megan Yingmei Zhang.

Toll-like receptor 4 ablation rescues against paraquat-triggered myocardial dysfunction: Role of ER stress and apoptosis.

Paraquat is a nitrogen herbicide imposing severe organ toxicity in human leading to acute lung injury and heart failure. The present study was designed to examine the impact of ablation of the innate proinflammatory mediator toll-like receptor 4 (TLR4) in paraquat-induced cardiac contractile dysfunction and the underlying mechanisms involved with a focus on endoplasmic reticulum (ER) stress and apoptosis. Adult male wild-type (WT) and TLR4 knockout (TLR4-/- ) mice were challenged with paraquat (45 mg/kg, i.p.) for 48 h prior to the assessment of myocardial and cardiomyocyte sarcomere function, ER stress, apoptosis and inflammation. Acute paraquat challenge exerted myocardial functional and geometric alterations including enlarged left ventricular end systolic diameter (LVESD), reduced fractional shortening, decreased sarcomere shortening, maximal velocities of sarcomere shortening and relengthening associated with unchanged LV posterior wall thickness, septal thickness, LV end diastolic diameter (LVEDD), heart rate, sarcomere length, time-to-peak shortening and time-to-90% relengthening. Although TLR4 ablation did not affect mechanical properties in the heart, it significantly attenuated or ablated paraquat-induced cardiac contractile anomalies. Moreover, paraquat imposed overt ER stress, apoptosis and inflammation as evidenced by upregulation of Bip, CHOP, Caspase-3, -9, Bax, Bad, and IL-1β, phosphorylation of PERK, eIF2α and IΚB, as well as activation of the stress molecules ERK and p38, with unchanged Caspase-8, Bcl2, TNF-α, p53, HMGB1, MyD88 and phosphorylation of Akt, GSK3β and JNK, the effects of which were attenuated or negated by TLR4 knockout. Taken together, our results suggested that TLR4 ablation alleviated paraquat-induced myocardial contractile dysfunction possibly through attenuation of ER stress, apoptosis and inflammation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 656-668, 2017.

Toll-like receptor 4 knockout alleviates paraquat-induced cardiomyocyte contractile dysfunction through an autophagy-dependent mechanism.

Paraquat, a quarternary nitrogen herbicide, is a toxic prooxidant leading to multi-organ failure including the heart although the underlying mechanism remains poorly understood. This study was designed to examine the role of the innate proinflammatory mediator toll-like receptor 4 (TLR4) in paraquat-induced cardiac contractile anomalies and the underlying mechanisms involved with a focus on autophagy, a conservative machinery governing protein and organelle degradation and recycling for cardiac homeostasis. Wild-type (WT) and TLR4 knockout (TLR4(-/-)) mice were challenged with paraquat (45mg/kg, i.p.) for 48h. Paraquat challenge did not affect mRNA levels of TLR2, TLR4 and TLR9 in WT mice nor did paraquat treatment alter TREM-1 levels. Paraquat challenge elicited cardiac mechanical defects including compromised cardiomyocyte contractile function, intracellular Ca(2+) handling, and overt autophagy as manifested by increased LC3BII-to-LC3BI ratio, Atg5, Atg7 and p62 levels. Interestingly, TLR4 knockout significantly attenuated paraquat-induced cardiac contractile and intracellular Ca(2+) derangement as well as alterations of autophagy markers. Paraquat-elicited changes in cardiac autophagy markers (LC3BII, LC3BII-to-LC3BI ratio and p62) were augmented by lysosomal inhibition using bafilomycin A1 in WT mice. TLR4 knockout significantly attenuated or negated paraquat-elicited increase in LC3BII, LC3BII-to-LC3BI ratio and p62 levels in the presence of lysosomal inhibition. In addition, paraquat challenge promoted phosphorylation of AMPK while suppressing the phosphorylation of mTOR and ULK1 (the autophagy inhibitory Ser(757)), the effects of which were significantly attenuated by TLR4 ablation. In vitro study revealed that AMPK activation using AICAR or mTOR inhibition using rapamycin effectively negated the beneficial cardiomyocyte mechanical effects of TLR4 inhibition (CLI-095) against paraquat toxicity, supporting a permissive role for AMPK-mTOR in TLR4 inhibition-offered cardioprotection against paraquat. Our results suggested that TLR4 knockout alleviated paraquat-induced cardiac dysfunction possibly through regulation of AMPK-mediated cardiac autophagy.

Fibronectin Splicing Variants Containing Extra Domain A Promote Atherosclerosis in Mice Through Toll-Like Receptor 4

Cellular fibronectin containing extra domain A (EDA(+)-FN) is abundant in the arteries of patients with atherosclerosis. Several in vitro studies suggest that EDA(+)-FN interacts with Toll-like receptor 4 (TLR4). We tested the hypothesis that EDA(+)-FN exacerbates atherosclerosis through TLR4 in a clinically relevant model of atherosclerosis, the apolipoprotein E-deficient (Apoe(-/-)) mouse. The extent of atherosclerosis was evaluated in whole aortae and cross sections of the aortic sinus in male and female EDA(-/-)Apoe(-/-) mice (which lack EDA(+)-FN), EDA(fl/fl)Apoe(-/-) mice (which constitutively express EDA(+)-FN), and control Apoe(-/-) mice fed a high-fat Western diet for 14 weeks. Irrespective of sex, EDA(fl/fl)Apoe(-/-) mice exhibited a 2-fold increase in atherosclerotic lesions (aorta and aortic sinus) and macrophage content within plaques, whereas EDA(-/-)Apoe(-/-) mice exhibited reduced atherosclerotic lesions (P<0.05 versus Apoe(-/-), n=10-12 mice/group), although cholesterol and triglyceride levels and circulating leukocytes were similar. Genetic ablation of TLR4 partially reversed atherosclerosis exacerbation in EDA(fl/fl)Apoe(-/-) mice (P<0.05) but had no effect on atherosclerotic lesions in EDA(-/-)Apoe(-/-) mice. Purified cellular FN, which contains EDA, potentiated dose-dependent NFκB-mediated inflammation (increased phospho-NFκB p65/NFκB p65, tumor necrosis factor-α, and interleukin-1β) in bone marrow-derived macrophages from EDA(-/-)Apoe(-/-) mice but not from EDA(-/-)TLR4(-/-)Apoe(-/-) mice. Finally, using immunohistochemistry, we provide evidence for the first time that EDA(+)-FN colocalizes with macrophage TLR4 in murine aortic lesions and human coronary artery atherosclerotic plaques. Our findings reveal that TLR4 signaling contributes to EDA(+)-FN-mediated exacerbation of atherosclerosis. We suggest that EDA(+)-FN could be a therapeutic target in atherosclerosis.

Toll-like receptor 4 ablation in mdx mice reveals innate immunity as a therapeutic target in Duchenne muscular dystrophy.

Toll-like receptor 4 (TLR4) recognizes specific structural motifs associated with microbial pathogens and also responds to certain endogenous host molecules associated with tissue damage. In Duchenne muscular dystrophy (DMD), inflammation plays an important role in determining the ultimate fate of dystrophic muscle fibers. In this study, we used TLR4-deficient dystrophic mdx mice to assess the role of TLR4 in the pathogenesis of DMD. TLR4 expression was increased and showed enhanced activation following agonist stimulation in mdx diaphragm muscle. Genetic ablation of TLR4 led to significantly increased muscle force generation in dystrophic diaphragm muscle, which was associated with improved histopathology including decreased fibrosis, as well as reduced pro-inflammatory gene expression and macrophage infiltration. TLR4 ablation in mdx mice also altered the phenotype of muscle macrophages by inducing a shift toward a more anti-inflammatory (iNOS(neg) CD206(pos)) profile. In vitro experiments confirmed that lack of TLR4 is sufficient to influence macrophage activation status in response to classical polarizing stimuli such as IFN-gamma and IL-4. Finally, treatment of dystrophic mice with glycyrrhizin, an inhibitor of the endogenous TLR4 ligand, high mobility group box (HMGB1), also pointed to involvement of the HMGB1-TLR4 axis in promoting dystrophic diaphragm pathology. Taken together, our findings reveal TLR4 and the innate immune system as important players in the pathophysiology of DMD. Accordingly, targeting either TLR4 or its endogenous ligands may provide a new therapeutic strategy to slow disease progression.

Abstract 59: Fibronectin Extradomain A Modulates Atherosclerotic Lesion Progression in Hypercholesterolemic Mice Through Toll-Like Receptor 4

Background and Hypothesis: Fibronectin (FN) containing the alternatively-spliced extra domain A (EDA + -FN) is absent in the arteries or circulation of healthy humans and mice, but specifically expressed in the endothelium of atherosclerotic arteries and elevated in circulation during pathological settings including stroke and atherosclerosis. We tested the hypothesis that EDA + -FN exacerbates atherosclerosis. Model and Methods: We generated EDA hi/hi /ApoE -/- mice (over express EDA + -FN in plasma) and EDA -/- /ApoE -/- mice (EDA-deleted allele of FN). ApoE -/- mice, which express EDA + -FN in plasma (~3-5 μg/mL) similar to levels found in human pathological settings including stroke and atherosclerosis, were used as control. Male mice (n=14/group) were fed a high-fat Western diet beginning at 6 weeks until they were sacrificed at 5 and 8 months of age. Extent of atherosclerosis was evaluated in whole aortae (Oil Red O stain) and in the cross section area of the aortic sinus (Verhoeff-Van Gieson stain). Markers of inflammation were quantified by immunohistochemistry. Results: We report that atherosclerotic plaque formation in the aortae and aortic sinus of EDA hi/hi /ApoE -/- mice were increased by 2-fold, concomitant with increased plaque inflammatory content (neutrophils, macrophages, and NF-κB p65 expression; P&lt;0.01 vs. ApoE -/- ). Conversely, EDA -/- /ApoE -/- mice had smaller plaques (P&lt;0.01 vs. ApoE -/- mice), concordant with decreased inflammatory content within plaque. Because EDA + -FN activates toll-like receptor 4 (TLR4) in vitro , we investigated the role of TLR4 in the EDA + -FN-mediated accelerated atherosclerosis. Genetic ablation of TLR4 in EDA hi/hi /ApoE -/- mice partially reversed exacerbated atherosclerosis (P&lt;0.05 vs. EDA hi/hi /ApoE -/- mice), whereas TLR4 deficiency in EDA -/- /ApoE -/- had no effect. Total cholesterol and triglycerides levels were similar among groups. Finally, we report co-localization of EDA + -FN with TLR4 on macrophages in human coronary atherosclerotic plaque samples. Conclusions: Endogenous EDA + -FN exacerbates atherosclerosis via TLR4. Targeting EDA + -FN/TLR4 pathway may have the potential to reduce atherosclerosis and improve outcomes in patients at high risk for coronary artery disease.

Abstract 127: Genetic Ablation of Alternatively-spliced Extra Domain A of Fibronectin Protects Hypercholesterolemic Mice from Acute Stroke Injury

Background and Hypothesis: Fibronectin containing the alternatively-spliced extra domain A (EDA + -FN) is absent in the arteries or circulation of healthy humans and mice; but is specifically expressed in the endothelium of atherosclerotic arteries, and elevated in circulation during pathological settings, including stroke. We tested the hypothesis that deletion of EDA in FN protects hypercholesterolemic apolipoprotein E-deficient (ApoE -/- ) mice from acute stroke injury. Methods: We generated EDA hi/hi /ApoE -/- mice (constitutively overexpress EDA + -FN in plasma) and EDA -/- /ApoE -/- mice (EDA-deleted allele of FN). ApoE -/- mice (express EDA + -FN in plasma (~3-5 μg/mL) similar to levels found in human pathological settings) were used as control. Susceptibility to thrombosis was assessed using FeCl 3 -induced carotid thrombosis model. Stroke outcome was evaluated in male mice following 60 min of ischemia/ 23 h of reperfusion injury by measuring infarct size, neurological deficit and markers of inflammation within the infarct and surrounding regions (immunohistochemistry). Results: Using intravital microscopy, we found that the time to stable occlusion of the carotid artery was accelerated in EDA hi/hi /ApoE -/- mice (P&lt;0.05 vs ApoE -/- mice), whereas EDA -/- /ApoE -/- mice showed opposite effect. EDA -/- /ApoE -/- mice had smaller infarcts and improved neurological outcome (P&lt;0.01 vs. ApoE -/- mice), concordant with decreased vascular inflammation (neutrophils, macrophages, and NF-κB p65 expression). Conversely, EDA hi/hi /ApoE -/- mice had larger infarcts and more severe neurological deficits, concordant with increased vascular inflammation (P&lt;0.01 vs. ApoE -/- mice). Because EDA + -FN activates toll-like receptor 4 (TLR4) in vitro , we investigated the role of TLR4 in the EDA + -FN-mediated aggravated stroke injury. Genetic ablation of TLR4 in EDA hi/hi /ApoE -/- mice completely reversed aggravated stroke injury (P&lt;0.05 vs. EDA hi/hi /ApoE -/- mice), whereas TLR4 deficiency in EDA -/- /ApoE -/- had no effect. Conclusions: EDA + -FN is pro-thrombotic and pro-inflammatory, and promotes acute stroke injury via a TLR4 -dependent mechanism. We suggest that monitoring plasma EDA + -FN levels may have prognostic value for patients at high risk for stroke.

TLR4 Deletion Ameliorates MSC –mediated Protection Effects in Liver Ischemia/Reperfusion Model

Liver ischemia-reperfusion (I/R) injury is common in liver transplantation, liver surgery, and hemorrhagic shock. Mesenchymal stem cell (MSC) is potential therapy to prevent liver injury. Toll-like receptor 4 (TLR4) is expressed by MSC and TLR4 ablation improves stem cell survival after hypoxia. However, it is unknown whether TLR4 deletion will enhance MSC function in liver I/R model.

Gut-Derived Lipopolysaccharide Promotes T-Cell–Mediated Hepatitis in Mice through Toll-Like Receptor 4

Robust clinical and epidemiologic data support the role of inflammation as a key player in hepatocellular carcinoma (HCC) development. Our previous data showed that gut-derived lipopolysaccharide (LPS) promote HCC development by activating Toll-like receptor 4 (TLR4) expressed on myeloid-derived cells. However, the effects of gut-derived LPS on other types of liver injury models are yet to be studied. The purpose of this study was to determine the importance of gut-derived LPS and TLR4 signaling in a T-cell-mediated hepatitis-Con A-induced hepatitis model, which mimic the viral hepatitis. Reduction of endotoxin using antibiotics regimen or genetic ablation of TLR4 in mice significantly alleviate Con A-induced liver injury by inhibiting the infiltration of T lymphocytes into the liver and the activation of CD4(+) T lymphocytes as well as the production of T helper 1 cytokines; in contrast, exogenous LPS can promote Con A-induced hepatitis and CD4(+) T cells activation in vivo and in vitro. Reconstitution of TLR4-expressing myeloid cells in TLR4-deficient mice restored Con A-induced liver injury and inflammation, indicating the major cell target of LPS. In addition, TLR4 may positively regulate the target hepatocellular apoptosis via the perforin/granzyme B pathway. These data suggest that gut-derived LPS and TLR4 play important positive roles in Con A-induced hepatitis and modulation of the gut microbiotia may represent a new avenue for therapeutic intervention to treat acute hepatitis induced by hepatitis virus infection, thus to prevent hepatocellular carcinoma.