Deletion Of Toll-like Receptor 4 Research Articles

Deletion of Extra Domain A of Fibronectin Reduces Acute Myocardial Ischaemia/Reperfusion Injury in Hyperlipidaemic Mice by Limiting Thrombo-Inflammation

Fibronectin splicing variant containing extra domain A (Fn-EDA), which is an endogenous ligand for Toll-like receptor 4 (TLR4), is present in negligible amounts in the plasma of healthy humans, but markedly elevated in patients with co-morbid conditions including diabetes and hyperlipidaemia, which are risk factors for myocardial infarction (MI). Very little is known about the role of Fn-EDA in the pathophysiology of acute MI under these co-morbid conditions. We determined the role of Fn-EDA in myocardial ischaemia/reperfusion (I/R) injury in the hyperlipidaemic apolipoprotein E-deficient (ApoE-/-) mice. Infarct size, plasma cardiac troponin I (cTnI) levels, intravascular thrombosis (CD41-positive), neutrophil infiltration (Ly6 B.2-positive), neutrophil extracellular traps (citrullinated H3-positive) and myocyte apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling-positive) were assessed in myocardial I/R injury model (1-hour ischaemia/23 hours of reperfusion). Irrespective of gender, Fn-EDA-/-ApoE-/- mice exhibited smaller infarct size and decreased cTnI levels concomitant with reduced post-ischaemic intra-vascular thrombi, neutrophils influx, neutrophil extracellular traps and myocyte apoptosis (p < 0.05 vs. ApoE-/- mice). Genetic deletion of TLR4 attenuated myocardial I/R injury in ApoE-/- mice (p < 0.05 vs. ApoE-/- mice), but did not further reduce in Fn-EDA-/- ApoE-/- mice suggesting that Fn-EDA requires TLR4 to mediate myocardial I/R injury. Bone marrow transplantation experiments revealed that Fn-EDA exacerbates myocardial I/R injury through TLR4 expressed on the haematopoietic cells. Infusion of a specific inhibitor of Fn-EDA, 15 minutes post-reperfusion, into ApoE-/- mice attenuated myocardial I/R injury. Fn-EDA exacerbates TLR4-dependent myocardial I/R injury by promoting post-ischaemic thrombo-inflammatory response. Targeting Fn-EDA may reduce cardiac damage following coronary artery re-canalization after acute MI.

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.

Fibronectin Containing Extra Domain A Induces Plaque Destabilization in the Innominate Artery of Aged Apolipoprotein E-Deficient Mice.

Fibronectin containing extra domain A (Fn-EDA) is an endogenous ligand of TLR4 (toll-like receptor 4) and is abundant in the extracellular matrix of advanced atherosclerotic lesions in human and mice. Irrespective of sex, deletion of Fn-EDA reduces early atherosclerosis in apolipoprotein E-deficient (Apoe-/-) mice. However, the contribution of Fn-EDA in advanced atherosclerosis remains poorly characterized. We determined the contribution of Fn-EDA in advanced atherosclerotic lesions of aged (1-year-old) Apoe-/- mice. Plaque composition was determined in the innominate artery, a plaque instability site that is known to mimic several histological features of vulnerable human plaques. Female Apoe-/-, Fn-EDA-/-Apoe-/-, TLR4-/-Apoe-/-, and Fn-EDA-/-TLR4-/-Apoe-/- mice were fed a high-fat Western diet for 44 weeks. Fn-EDA-/-Apoe-/- mice exhibited reduced plaque size characterized by smaller necrotic cores, thick fibrous caps containing abundant vascular smooth muscle cells and collagen, reduced CD68/MMP9 (matrix metalloproteinase 9)-positive content, less accumulation of MMP-cleaved extracellular matrix aggrecan, and decreased vascular smooth muscle cell and macrophage apoptosis (P<0.05 versus Apoe-/- mice). Together these findings suggest that Fn-EDA induces plaque destabilization. Deletion of TLR4 reduced histological features of plaque instability in Apoe-/- mice but did not further reduce features of plaque destabilization in Fn-EDA-/-Apoe-/- mice, suggesting that TLR4 may contribute to Fn-EDA-induced plaque destabilization. Fn-EDA potentiated TLR4-dependent MMP9 expression in bone marrow-derived macrophages, suggesting that macrophage TLR4 may contribute to Fn-EDA-mediated plaque instability. Fn-EDA induces histological features of plaque instability in established lesions of aged Apoe-/- mice. The abundance of Fn-EDA in advanced atherosclerotic lesions may increase the risk of plaque destabilization.

Inhibitory Effects of Toll-Like Receptor 4, NLRP3 Inflammasome, and Interleukin-1β on White Adipocyte Browning.

Adipose tissue expansion is accompanied by infiltration and accumulation of pro-inflammatory macrophages, which links obesity to pathologic conditions such as type 2 diabetes. However, little is known regarding the role of pro-inflammatory adipose tissue remodeling in the thermogenic activation of brown/beige fat. Here, we investigated the effect of pattern recognition receptors (PRR) activation in macrophages, especially the toll-like receptor 4 (TLR4) and Nod-like receptor 3 (NLRP3), on white adipocyte browning. We report that TLR4 activation by lipopolysaccharide repressed white adipocyte browning in response to β3-adrenergic receptor activation and caused ROS production and mitochondrial dysfunction, while genetic deletion of TLR4 protected mitochondrial function and thermogenesis. In addition, activation of NLRP3 inflammasome in macrophages attenuated UCP1 induction and mitochondrial respiration in cultures of primary adipocytes, while the absence of NLRP3 protected UCP1 in adipocytes. The effect of NLRP3 inflammasome activation on browning was mediated by IL-1β signaling, as blocking IL-1 receptor in adipocytes protected thermogenesis. We also report that IL-1β interferes with thermogenesis via oxidative stress stimulation and mitochondrial dysfunction as we observed a statistically significant increase in ROS production, decrease in SOD enzyme activity, and increase in mitochondrial depolarization in adipocytes treated with IL-1β. Collectively, we demonstrated that inflammatory response to obesity, such as TLR4 and NLRP3 inflammasome activation as well as IL-1β secretion, attenuates β3-adrenoreceptor-induced beige adipocyte formation via oxidative stress and mitochondrial dysfunction. Our findings provide insights into targeting innate inflammatory system for enhancement of the adaptive thermogenesis against obesity.

Systemic Sympathoexcitation Was Associated with Paraventricular Hypothalamic Phosphorylation of Synaptic CaMKIIα and MAPK/ErK.

Systemic administration of adrenergic agonist (Isoproterenol; ISOP) is known to facilitate cardiovascular changes associated with heart failure through an upregulation of cardiac toll-like receptor 4 (TLR4). Furthermore, previous studies have shown that cardiac tissue-specific deletion of TLR4 protects the heart against such damage. Since the autonomic regulation of systemic cardiovascular function originates from pre-autonomic sympathetic centers in the brain, it is unclear how a systemically driven sympathetic change may affect the pre-autonomic paraventricular hypothalamic nuclei (PVN) TLR4 expression. Here, we examined how change in PVN TLR4 was associated with alterations in the neurochemical cytoarchitecture of the PVN in systemic adrenergic activation. After 48 h of intraperitoneal 150 mg/kg ISOP treatment, there was a change in PVN CaMKIIα and MAPK/ErK expression, and an increase in TLR4 in expression. This was seen as an increase in p-MAPK/ErK, and a decrease in synaptic CaMKIIα expression in the PVN (p < 0.01) of ISOP treated mice. Furthermore, there was an upregulation of vesicular glutamate transporter (VGLUT 2; p < 0.01) and a decreased expression of GABA in the PVN of Isoproterenol (ISOP) treated WT mice (p < 0.01). However, after a PVN-specific knockdown of TLR4, the effect of systemic administration of ISOP was attenuated, as indicated by a decrease in p-MAPK/ErK (p < 0.01) and upregulation of CaMKIIα (p < 0.05). Additionally, loss of inhibitory function was averted while VGLUT2 expression decreased when compared with the ISOP treated wild type mice and the control. Taken together, the outcome of this study showed that systemic adrenergic activation may alter the expression, and phosphorylation of preautonomic MAPK/ErK and CaMKIIα downstream of TLR4. As such, by outlining the roles of these kinases in synaptic function, we have identified the significance of neural TLR4 in the progression, and attenuation of synaptic changes in the pre-autonomic sympathetic centers.

Short-Term Versus Long-Term Effects of Adipocyte Toll-Like Receptor 4 Activation on Insulin Resistance in Male Mice.

Chronic exposure to high-saturated fat diets (HFDs) increases the prevalence of obesity and contributes to the development of low-grade inflammation and insulin resistance. A possible mediator accounting for obesity-associated inflammation and insulin resistance is Toll-like receptor 4 (TLR4). We investigated the role of adipocyte TLR4 in lipid and glucose homeostasis through an inducible, adipocyte-specific deletion of TLR4 in a mouse model that is referred to as the "Tadipo" mouse. Consistent with a critical role for inflammation as a positive force for healthy adipose tissue expansion, chronic HFD exposure results in exacerbated whole-body and muscle insulin resistance in the absence of TLR4 in the adipocyte. Elimination of TLR4 in adipocytes affects TLR4 expression in other tissues, with reduced TLR4 expression in peritoneal macrophages and in the liver. In contrast, TLR4 deletion from adipocytes protects whole-body insulin sensitivity after an acute lipid challenge during a hyperinsulinemic euglycemic clamp. Our results therefore demonstrate dichotomous effects of TLR4 on adipose tissue functionality, with an important positive role of TLR4 during a chronic HFD challenge due to the lack of adipose tissue remodeling and a negative role of TLR4 as a mediator of insulin resistance in the adipocyte during an acute challenge with saturated fatty acids.

Hyaluronan and TLR4 promote surfactant-protein-C-positive alveolar progenitor cell renewal and prevent severe pulmonary fibrosis in mice.

Successful recovery from lung injury requires the repair and regeneration of alveolar epithelial cells to restore the integrity of gas-exchanging regions within the lung and preserve organ function. Improper regeneration of the alveolar epithelium is often associated with severe pulmonary fibrosis, the latter of which involves the recruitment and activation of fibroblasts, as well as matrix accumulation. Type 2 alveolar epithelial cells (AEC2s) are stem cells in the adult lung that contribute to the lung repair process. The mechanisms that regulate AEC2 renewal are incompletely understood. We provide evidence that expression of the innate immune receptor Toll-like receptor 4 (TLR4) and the extracellular matrix glycosaminoglycan hyaluronan (HA) on AEC2s are important for AEC2 renewal, repair of lung injury and limiting the extent of fibrosis. Either deletion of TLR4 or HA synthase 2 in surfactant-protein-C-positive AEC2s leads to impaired renewal capacity, severe fibrosis and mortality. Furthermore, AEC2s from patients with severe pulmonary fibrosis have reduced cell surface HA and impaired renewal capacity, suggesting that HA and TLR4 are key contributors to lung stem cell renewal and that severe pulmonary fibrosis is the result of distal epithelial stem cell failure.

Pharmacological Inhibition of TLR4 Reduces Mast Cell Activation, Neuroinflammation and Hyperalgesia in Sickle Mice

Sickle cell disease (SCD) is characterized by chronic hemolysis, inflammation, vascular dysfunction, and pain. Earlier we showed that mast cell activation contributes to neuroinflammation and pain and is accompanied by increased toll-like receptor 4 (TLR4) expression on mast cells (Vincent et al., Blood 2013), and that genetic deletion of TLR4 ameliorates neurogenic inflammation and hyperalgesia in HbSS-BERK sickle mice. Several other studies have shown increased TLR4 expression in peripheral system and its involvement in sickle pathobiology. We propose that free heme, due to hemolysis, activates TLR4 in the central nervous system in addition to peripheral activation, which further exacerbates neuroinflammation and hyperalgesia. Spinal cords of HbSS-BERK sickle mice show 3-fold mRNA transcripts for TLR4 and a 2-fold increase in hemin as compared to the spinal cords of HbAA-BERK control mice. Therefore, targeting TLR4 with pharmacological inhibitors may provide a therapeutic approach to attenuate peripheral and central inflammation and hyperalgesia. In the present study we examined the potential of pharmacological inhibition of mast cell activation, neuroinflammation and hyperalgesia in HbSS-BERK sickle mice with TLR4 inhibitor, TAK242. Sickle mice were administrated intravenously with TLR4 inhibitor TAK242 (1 mg/kg body weight/day) for 5 days. Sensory testing was performed at baseline at recruitment and periodically during the 5-day treatment and for another 8 days after concluding the treatment to evaluate mechanical hyperalgesia with von Frey filaments, thermal hyperalgesia in response to heat/cold and grip force for musculoskeletal/deep tissue hyperalgesia. Following the 5-day treatment with TAK242, release of cytokines, tryptase (marker of mast cell activation) and substance P released from skin biopsies and spinal cords were analyzed by ELISA. TAK242 significantly decreased the release of tryptase (TAK242: 5.178 ± 0.7613 pg/ml vs vehicle: 8.801 ± 0.9403 pg/ml, p = 0.0181), substance P (TAK242: 11.56 ± 1.945 pg/ml vs vehicle: 25.51 ± 4.283 pg/ml, p = 0.018), and IL-6 (TAK242: 15.59 ± 0.4541 pg/ml vs vehicle: 29.74 ± 0.8249 pg/ml, p = 0.0045) from skin biopsies, suggesting that TAK242 reduced SCD-induced mast cell activation and inflammation. TAK242 also significantly decreased substance P (TAK242: 0.7198 ± 0.0587 pg/mg vs vehicle: 0.931 ± 0.0676 pg/mg, p = 0.0462) and phosphorylation of p38/MAPK (p = 0.0184) in the spinal cord, as well as dorsal cutaneous blood flow (TAK242: 6.392 ± 0.3857 PU vs vehicle: 12.32 ± 0.5575 PU, p < 0.0001), indicating that TAK242 ameliorated SCD-evoked central and peripheral activation of inflammation and nociceptive mechanisms. Furthermore, TAK242 administration gradually reduced the mechanical, deep tissue, and thermal hyperalgesia upto 5-day treatment (p < 0.01, vs vehicle HbSS). However, discontinuation of treatment led to a gradual increase in hyperalgesia observed upto day-8 post-treatment. TAK242 also significantly decreased acute pain induced by hypoxia/reoxygenation and accelerated recovery from injury of hypoxia/reoxygenation. These data reveal the significant therapeutic effect of pharmacological inhibition of TLR4 on inflammation and hyperalgesia in sickle mice. Therapies targeting TLR4 inhibition may be potentially beneficial in ameliorating sickle pathobiology and pain. DisclosuresNo relevant conflicts of interest to declare.

Toll-like Receptor 4 Signaling on Dendritic Cells Suppresses Polymorphonuclear Leukocyte CXCR2 Expression and Trafficking via Interleukin 10 During Intra-abdominal Sepsis.

Toll-like receptor 4 (TLR4) is a critical receptor involved in the sensing of gram-negative bacterial infection. However, the roles of TLR4 in sepsis are cell-type specific. Dendritic cells (DCs) are known to play a central role in microbial detection, alerting the immune system to the presence of infection and coordinating adaptive immune response. The goal of this study was to investigate the impact of DC-specific TLR4 signaling on host defense against intra-abdominal polymicrobial sepsis. C57BL/6, global Tlr4 knockout, cell-specific knockout control, and CD11c-specific Tlr4(-/-) mice underwent cecal ligation and puncture (CLP). Specific deletion of TLR4 on DCs in mice improved survival and enhanced bacterial clearance. Deletion of TLR4 on DCs was associated with lower levels of circulating interleukin 10 (IL-10), higher polymorphonuclear leukocyte (PMN) accumulation in the peritoneal cavity, and higher expression of chemokine (C-X-C motif) receptor 2 (CXCR2) on PMNs after CLP. In vitro studies of DC and neutrophil cocultures confirmed that TLR4-dependent secretion of IL-10 from DCs regulated neutrophil CXCR2 expression. Our data shed light on a previously unrecognized role for TLR4 signaling on DCs in driving IL-10 secretion during sepsis and, through this pathway, regulates PMN recruitment via suppression of CXCR2 expression.

HMGB1-Driven Inflammation and Intimal Hyperplasia After Arterial Injury Involves Cell-Specific Actions Mediated by TLR4

Endoluminal vascular interventions such as angioplasty initiate a sterile inflammatory response resulting from local tissue damage. This response drives the development of intimal hyperplasia (IH) that, in turn, can lead to arterial occlusion. We hypothesized that the ubiquitous nuclear protein and damage-associated molecular pattern molecule, high-mobility group box 1 (HMGB1), is one of the endogenous mediators that activates processes leading to IH after endoluminal injury to the arterial wall. The aim of this study is to investigate whether approaches that reduce the levels of HMGB1 or inhibit its activity suppresses IH after arterial injury. Here, we show that HMGB1 regulates IH in a mouse carotid wire injury model. Induced genetic deletion or neutralization of HMGB1 prevents IH, monocyte recruitment, and smooth muscle cell growth factor production after endoluminal carotid artery injury. A specific inhibitor of HMGB1 myeloid differentiation factor 2-toll-like receptor 4 (TLR4) interaction, P5779, also significantly inhibits IH. HMGB1 deletion is mimicked in this model by global deletion of TLR4 and partially replicated by myeloid-specific deletion of TLR4 but not TLR2 or receptor for advanced glycation endproducts deletion. The specific HMGB1 isoform known to activate TLR4 signaling (disulfide HMGB1) stimulates smooth muscle cell to migrate and produce monocyte chemotactic protein 1/CCL2) via TLR4. Macrophages produce smooth muscle cell mitogens in response to disulfide HMGB1 also in a TLR4/myeloid differentiation primary response gene (88)/Trif-dependent manner. These findings place HMGB1 and its receptor, TLR4 as critical regulators of the events that drive the inflammation leading to IH after endoluminal arterial injury and identify this pathway as a possible therapeutic target to limit IH to attenuate damage-associated molecular pattern molecule-mediated vascular inflammatory responses.

Cellular fibronectin containing extra domain A promotes arterial thrombosis in mice through platelet Toll-like receptor 4

AbstractCellular fibronectin containing extra domain A (Fn-EDA+), which is produced in response to tissue injury in several disease states, has prothrombotic activity and is known to interact with Toll-like-receptor 4 (TLR4). The underlying mechanism and cell types involved in mediating the prothrombotic effect of Fn-EDA+ still remain unknown. Using intravital microscopy, we evaluated susceptibility to carotid artery thrombosis after FeCl3-induced injury in mice expressing Fn lacking EDA (Fn-EDA−/− mice) or Fn containing EDA (Fn-EDA+/+ mice). Fn-EDA−/− mice exhibited prolonged times to first thrombus formation and complete occlusion and a significant decrease in the rate of thrombus growth (P < .05 vs Fn-EDA+/+ mice). Genetic deletion of TLR4 reversed the accelerated thrombosis in Fn-EDA+/+ mice (P < .05) but had no effect in Fn-EDA−/− mice. Bone marrow transplantation experiments revealed that TLR4 expressed on hematopoietic cells contributes to accelerated thrombosis in Fn-EDA+/+ mice. In vitro studies showed that cellular Fn-EDA+ interacts with platelet TLR4 and promotes agonist-induced platelet aggregation. Finally, Fn-EDA+/+ mice specifically lacking platelet TLR4 exhibited prolonged times to first thrombus formation and complete occlusion (P < .05 vs Fn-EDA+/+ mice containing platelet TLR4). We conclude that platelet TLR4 contributes to the prothrombotic effect of cellular Fn-EDA+, suggesting another link between thrombosis and innate immunity.

Toll‐like Receptor 4 (TLR4) Knock‐out Mice Are Partially Protected from Diabetic Bladder Dysfunction

Bladder dysfunction affects a majority of patients with diabetes and therapeutical options for diabetic bladder dysfunction (DBD) are limited. The innate immune system is now thought to be implicated in the pathogenesis of diabetic complications via inflammation and oxidative stress. We have previously shown that activation of Toll‐like receptor 4 (TLR4), a component of the innate immune system, was increased in the bladder of streptozotocin (STZ)‐induced diabetic mice, and that inhibition of TLR4 in vitro normalized the augmented bladder contraction observed in these mice. Here we hypothesized that deletion of TLR4 would ameliorate diabetic bladder dysfunction. We evaluated the development of DBD in TLR4 knock‐out (TLR4KO) and C57Bl/6 wild type (WT) mice treated with vehicle (CON) or STZ (50 mg/kg/day i.p. for 5 days). After 4 weeks of STZ treatment, we observed the same levels of glycemia in TLR4KO and WT mice (mg/dl: WT STZ=349±41, TLR4KO STZ=403±11). Bladder hypertrophy was slightly attenuated in TLR4KO compared to WT mice (% of CON bladder weight/body weight: WT STZ=145±8 TLR4KO STZ=126±11). Contraction to carbacholine (CCh) and electrical field stimulation (EFS) was increased in urothelium‐denuded bladder strips from WT STZ compared to WT CON mice, however no difference was observed between TLR4KO CON and TLR4KO STZ (CCh Emax in mN: WT CON=7.87±0.45, WT STZ=13.07±2.47, TLR4KO CON=8.01±1.35, TLR4KO STZ=9.23±3.13; mN contraction at 32 Hz EFS: WT CON=13.4±0.45, WT STZ=25.51±4.45, TLR4KO CON=14.67±1.66, TLR4KO STZ=19.07±6.45). We conclude that TLR4 contributes to the development of DBD independently of glycemia levels and the mechanism is coupled to innate immune activation in DBD. Support: NIDDK‐13GHSU302.

Reduced Radiation Pulmonary Fibrosis in Toll-Like Receptor-4 (TLR4) Deletion Recombinant Negative Mice

The inflammatory mediator receptor, TLR4 has been demonstrated to be upregulated in the lungs of thoracic irradiated C57BL/6J/NHsd mice. We investigated whether pulmonary irradiation of TLR4 -/- mice (B6 background) led to reduced lung fibrosis (organizing alveolitis). Since lung fibrosis involves pulmonary migration of marrow-origin fibroblasts, we compared bone marrow hematopoiesis in TLR4 -/- mice compared to TLR4 +/+ C57BL/6NHsd mouse long term marrow cultures (LTBMCS) LTBMC were established from femur bone marrow of TLR4-/- or wild type C57BL/6J into T25 flasks and hematopoiesis was measured over 30 weeks. Bone marrow stromal and IL-3 dependent hematopoietic progenitor cells lines were established from each genotype. TLR4 -/- and wild type 6-8 week old C57BL/6J mice were thoracic irradiated to 20 Gy, and pulmonary fibrosis was quantitated at 200 days after irradiation. Homozygous deletion of TLR4 resulted in improvement of multiple parameters of hematopoiesis in LTBMC including: Increased formation of cobblestone islands from week 1 (297.8±80.6 vs 136.0±36.6 for wild type, p = 0.0107) and week 7 (2228.5±165.5 vs 1994.5±47.2, p = 0.0346). Non-adherent cell production, another indicator of stem cell activity was significantly higher in TLR4-/- LTBMCS compared to wild type LTBMCS from week 1 (35.7±2.0 x105 vs 21.4±3.2 x105, p = 0.0003) through week 17 (8.2±2.2 x105 vs 3.3±1.7 x105 p = 0.0194). LTBMC from TLR4-/- continued to produce cobblestone islands and non-adherent hematopoietic cells until week 31,while those from wild type mice ceased at week 21. Production of day 14 colony forming CFU-GEMM cells was higher in TLR4-/- LTBMCS from week 3 (349.7±15.2 vs 194.3±8.0, p = 0.0001) to week 20 (164.0±7.0 vs 11.7±2.1, p = <0.0001). Lungs from 20GY thoracic irradiated TLR4-/- mice showed reduced irradiation pulmonary fibrosis and collagen compared to those from wild type mice (1.0 +/- 0.5 and 20.83 +/- 3.4, p < 0.0001). Absence of TLR4 decreases irradiation pulmonary fibrosis in C57BL/6 mice and improves hematopoiesis in LTBMCS suggesting a common role of bone marrow stromal cells in both processes.

98. Consequences of toll-like receptor 4 deletion on behavior, immune responses to stress, and age-related pathology in mice

Pro-inflammatory responses are potentiated in response to stress and play a role in age-related pathology and cognitive decline. We have undertaken an extensive littermate-controlled mouse phenotyping study to characterize the consequences of toll-like receptor 4 (TLR4) deletion in young and aged mice. Our study includes behavioral tests, next-generation microbiome sequencing, complete blood counts, clinical chemistries, serum glucocorticoid and cytokine levels, histology of all tissues, and assessments of function in immune cells isolated from stressed and unstressed animals. Deletion of TLR4 improves memory in both young and old mice, alters social motivation, reduces anxiety-like behavior, and changes behavioral and immune responses to acute stress. TLR4 deletion also leads to chronic intermittent seizures in some animals. Basal and stress-induced changes in serum and splenocyte secreted cytokines associated with stress and depressive-like behavior (IL-6 and IL-10, specifically) differ between wild-type and TLR4 knockout mice. Correlations between serum cytokines and circulating leukocytes also differ. Abundance of specific gut microbes also differ significantly between wild-type and TLR4 KO littermates who have lived long-term in the same cage. TLR4 functions in different tissues may offer mechanistic insight to the differences observed. Ongoing studies are delineating the roles of specific cell types and downstream pathways underlying the observed phenotype. Our results highlight the need for interdisciplinary work that will help elucidate how animals integrate information across multiple systems to coordinate adaptive responses.

Genetic Deletion of Toll-Like Receptor 4 on Platelets Attenuates Experimental Pulmonary Hypertension

Recent studies demonstrate a role for toll-like receptor 4 (TLR4) in the pathogenesis of pulmonary hypertension (PH); however, the cell types involved in mediating the effects of TLR4 remain unknown. The objective of this study was to determine the contribution of TLR4 expressed on nonparenchymal cells to the pathogenesis of PH. TLR4 bone marrow chimeric mice revealed an equal contribution of TLR4 on nonparenchymal and parenchymal cells in the pathogenesis of PH as determined by measuring right ventricular (RV) systolic pressure and RV hypertrophy. However, the deletion of TLR4 from myeloid lineage cells had no effect on the development of PH because we found no difference in RV systolic pressure or RV hypertrophy in wild-type versus LysM-TLR4(-/-) mice. To explore the potential role of platelet TLR4 in the pathogenesis of PH, platelet-specific TLR4(-/-) mice were generated (PF4-TLR4(-/-) mice). TLR4(-/-) platelets from either global TLR4(-/-) or PF4-TLR4(-/-) mice were functional but failed to respond to lipopolysaccharide, demonstrating a lack of TLR4. PF4-TLR4(-/-) mice demonstrated significant protection from hypoxia-induced PH, including attenuated increases in RV systolic pressure and RV hypertrophy, decreased platelet activation, and less pulmonary vascular remodeling. The deletion of TLR4 from platelets attenuated serotonin release after chronic hypoxia, and lipopolysaccharide-stimulated platelets released serotonin and promoted pulmonary artery smooth muscle cell proliferation in a serotonin-dependent manner. Our data demonstrate that TLR4 on platelets contributes to the pathogenesis of PH and further highlights the role of platelets in PH.

Toll-like receptor 4 in bone marrow-derived cells contributes to the progression of diabetic retinopathy.

Diabetic retinopathy (DR) is a major microvascular complication in diabetics, and its mechanism is not fully understood. Toll-like receptor 4 (TLR4) plays a pivotal role in the maintenance of the inflammatory state during DR, and the deletion of TLR4 eventually alleviates the diabetic inflammatory state. To further elucidate the mechanism of DR, we used bone marrow transplantation to establish reciprocal chimeric animals of TLR4 mutant mice and TLR4 WT mice combined with diabetes mellitus (DM) induction by streptozotocin (STZ) treatment to identify the role of TLR4 in different cell types in the development of the proinflammatory state during DR. TLR4 mutation did not block the occurrence of high blood glucose after STZ injection compared with WT mice but did alleviate the progression of DR and alter the expression of the small vessel proliferation-related genes, vascular endothelial growth factor (VEGF), and hypoxia inducible factor-1α (HIF-1α). Grafting bone marrow-derived cells from TLR4 WT mice into TLR4 mutant mice increased the levels of TNF-α, IL-1β, and MIP-2 and increased the damage to the retina. Similarly, VEGF and HIF-1α expression were restored by the bone marrow transplantation. These findings identify an essential role for TLR4 in bone marrow-derived cells contributing to the progression of DR.

Impaired cavernosal relaxation in Angiotensin‐ II infused mice is improved by deletion of Toll like receptor 4 (TLR4)

TLRs are important contributors of the innate immune system. TLR‐mediated signaling is involved in inflammatory responses and TLR4 has been associated with cardiovascular diseases (CVD). Erectile dysfunction (ED) is an early marker for CVD. Upregulation of angiotensin II (Ang‐II) is linked with CDV and ED. Ang II is found in human corpus cavernosum (CC) and abnormal levels induce vasoconstriction and endothelial dysfunction. We hypothesize that deletion of TLR4 contributes to improve mice cavernosal relaxation impaired due to Ang‐II infusion. TLR4 knockout (KO) mice and wild type (WT) were infused with Ang II (42 ng/min) for 2 weeks. Functional responses of cavernosal tissue were performed. Cavernosal strips from Ang II WT mice exhibited increased contraction to phenylephrine (PE, 10−8 to 10−4M) and electrical field stimulation (EFS, 1–32 Hz) compared to WT control (CTL), which is attenuated around 25 ± 4.5 % in Ang II TLR4 KO compared to Ang II WT mice. Impaired endothelium‐dependent relaxation to acetylcholine (Ach, 10−9 to 10−5M) in CC from Ang II was improved almost 20 ± 3.7% compared to Ang II TLR KO mice. Furthermore, TLR4 deletion enhanced nitrergic‐induced relaxation in penile tissue from Ang‐II infused mice. In conclusion, these results suggest that TLR4 contributes to impaired penile relaxation and contraction in response to pathological stimulation such as upregulation of Ang‐ II.Financial support: NIH, AHA.

Impact of toll-like receptor 4 on the severity of acute pancreatitis and pancreatitis-associated lung injury in mice

Background and Aims:Acute pancreatitis is an inflammatory disease involving acinar cell injury, and the rapid production and release of inflammatory cytokines, which play a dominant role in local pancreatic inflammation...

Deletion of toll-like receptor-4 downregulates protein kinase C-ζ and attenuates liver injury in experimental pancreatitis

Toll-like receptor-4 (TLR4) and protein kinase C-zeta (PKC-zeta) play a role in macrophage activation. We hypothesized that deletion of TLR4 downregulates PKC-zeta and attenuates liver cell apoptosis in experimental pancreatitis. Acute pancreatitis was induced by choline-deficient ethionine diet in C57/BL6 (TLR4+/+ and TLR4-/-) mice. During pancreatitis, staining for TLR4 and PKC-zeta, which colocalized in Kupffer cells but not in hepatocytes, increased in TLR4+/+ mice and decreased in TLR4-/- mice. In TLR4+/+ mice, pancreatitis increased TLR4 protein and mRNA and PKC-zeta protein and activity, nuclear factor (NF)-kappaB, ERK1/2, caspase-3 cleavage, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining; all P < .01 versus controls. In TLR4-/- mice with pancreatitis, PKC-zeta mRNA and activity were reduced, ERK1/2 and caspase-3 did not increase, and NF-kappaB and TUNEL (mostly in hepatocytes) increased mildly (all P < .01 vs control). PKC-zeta did not interact directly with NF-kappaB; however, during pancreatitis, coimmunoprecipitation of PKC-zeta with ERK1/2 was increased in TLR4+/+ mice and was attenuated in TLR4-/- mice (all P < .01 vs control), indicating that PKC-zeta interacts with ERK1/2. Acute pancreatitis upregulates TLR4, PKC-zeta, NF-kappaB, and ERK1/2, and increases apoptosis in mice livers. PKC-zeta induces nuclear translocation of NF-kappaB via ERK1/2-dependent mechanisms. Deletion of TLR4 downregulates PKC-zeta, NF-kappaB, and ERK1/2, and attenuates pancreatitis-induced liver cell apoptosis.

Inhibition or deletion of the lipopolysaccharide receptor Toll-like receptor-4 confers partial protection against lipid-induced insulin resistance in rodent skeletal muscle

A role for increased activity of the innate immune system in the pathogenesis of insulin resistance is supported by a number of studies. The current study assessed the potential role of the lipopolysaccharide receptor known as Toll-like receptor-4 (TLR-4), a component of the innate immune system, in mediating lipid-induced insulin resistance in skeletal muscle. The effects of TLR-4 inhibition/deletion on lipid-induced insulin resistance was determined in skeletal muscle of TLR-4 null mice in vivo and in rat L6 myotubes in vitro. In mice, acute hyperlipidaemia induced skeletal muscle insulin resistance, but a deletion of TLR-4 conferred significant protection against these effects. In L6 myotubes, inhibition of TLR-4 activity substantially reduced the capacity of the saturated fatty acid palmitate to induce insulin resistance. Importantly, palmitate activated the nuclear factor kappaB (NFkappaB) pathway in L6 myotubes and mouse skeletal muscle, and these effects were blocked by inhibition of TLR-4 in L6 myotubes and absence of TLR-4 in skeletal muscle. Furthermore, inhibition of the NFkappaB pathway downstream of TLR-4 in L6 myotubes also protected against the induction of insulin resistance by palmitate. Inhibition or absence of TLR-4 confers protection against the detrimental effects of lipids on skeletal muscle insulin action, and these effects are associated with a prevention of the activation of the NFkappaB pathway by lipids. Importantly, inhibition of the NFkappaB pathway in myotubes downstream of TLR-4 also protects against lipid-induced insulin resistance, suggesting a mechanism by which reduced TLR-4 activity confers beneficial effects on insulin action.