Deletion Of Toll-like Receptor 4 Research Articles

SAT485 Knockout Of TLR4 Alters Reproductive And Metabolic Outcomes In Mouse Model Of Polycystic Ovary Syndrome

Abstract Disclosure: K.D. Wiggins: None. G. De Robles: None. J. De La Torre: None. D. Nicholas: None. Title: Knockout of TLR4 Alters Reproductive and Metabolic Outcomes in Mouse Model of Polycystic Ovary Syndrome. Polycystic Ovarian Syndrome (PCOS) is a heterogeneous endocrine disorder often characterized by reproductive and metabolic dysfunction. Prior literature has found a correlation between PCOS and chronic low-grade inflammation. However, it remains unknown if inflammation plays a causal role in the pathology of PCOS. In women with PCOS, serum levels of Lipopolysaccharides (LPS), a ligand for Toll-like receptor 4 (TLR4), are elevated. The binding of LPS to TLR4 leads to the production of pro-inflammatory cytokines, which can result in low-grade inflammation and metabolic syndrome, a prominent feature of PCOS. Given the role of TLR4, we hypothesize that the induced inflammatory environment mediates the development and pathogenesis of PCOS. To test this hypothesis, we subcutaneously implanted a nonsteroidal aromatase inhibitor, Letrozole, in TLR4 knockout mice (TLR4KO) and C57BL/6J (wild type) mice. Following five weeks of letrozole treatment, wild type mice failed to cycle, whereas TLR4KO mice were able to enter each estrous cycle phase. Furthermore, in contrast to wild type mice, TLR4KO mice display normalized Luteinizing Hormone (LH) levels with increased levels of Follicle Stimulating Hormone (FSH). We also characterized how inflammation influences the metabolic phenotypes of PCOS. Our results reveal that the deletion of TLR4 reduces weight gain and improves glucose intolerance. Moreover, we show that the development of insulin resistance in letrozole-treated TLR4KO mice was independent of other metabolic outcomes. Our study demonstrates that disruption of this pro-inflammatory pathway mediates the development of reproductive and metabolic phenotypes. Thus, research on the modulation of chronic low-grade inflammation may lead to the development of new therapeutics for PCOS patients. Keywords: Reproduction, Inflammation, Polycystic Ovary Syndrome, Presentation Date: Saturday, June 17, 2023

A positive feedback cycle between the alarmin S100A8/A9 and NLRP3 inflammasome-GSDMD signalling reinforces the innate immune response in Candida albicans keratitis.

Fungal keratitis is a severe sight-threatening ocular infection, without effective treatment strategies available now. Calprotectin S100A8/A9 has recently attracted great attention as a critical alarmin modulating the innate immune response againstmicrobialchallenges. However, the unique role of S100A8/A9 in fungal keratitis is poorly understood. Experimental fungal keratitis was established in wild-type and gene knockout (TLR4-/- and GSDMD-/-) mice by infecting mouse corneas with Candida albicans. The degree of mouse cornea injuries was evaluated by clinical scoring. To interrogate the molecular mechanism in vitro, macrophage RAW264.7 cell line was challenged with Candida albicans or recombinant S100A8/A9 protein. Label-free quantitative proteomics, quantitative real-time PCR, Western blotting, and immunohistochemistry were conducted in this research. Herein, we characterized the proteome of mouse corneas infected with Candida albicans and found that S100A8/A9 was robustly expressed at the early stage of the disease. S100A8/A9 significantly enhanced disease progression by promoting NLRP3 inflammasome activation and Caspase-1 maturation, accompanied by increased accumulation of macrophages in infected corneas. In response to Candida albicans infection, toll-like receptor 4 (TLR4) sensed extracellular S100A8/A9 and acted as a bridge between S100A8/A9 and NLRP3 inflammasome activation in mouse corneas. Furthermore, the deletion of TLR4 resulted in noticeable improvement in fungal keratitis. Remarkably, NLRP3/GSDMD-mediated macrophage pyroptosis in turn facilitates S100A8/A9 secretion during Candida albicans keratitis, thus forming a positive feedback cycle that amplifies the proinflammatory response in corneas. The present study is the first to reveal the critical roles of the alarmin S100A8/A9 in the immunopathology of Candida albicans keratitis, highlighting a promising approach for therapeutic intervention in the future.

Subchronic exposure to 1,2-naphthoquinone induces adipose tissue inflammation and changes the energy homeostasis of mice, partially due to TNFR1 and TLR4

Air pollution affects energy homeostasis detrimentally. Yet, knowledge of how each isolated pollutant can impact energy metabolism remains incomplete. The present study was designed to investigate the distinct effects of 1,2-naphthoquinone (1,2-NQ) on energy metabolism since this pollutant increases at the same rate as diesel combustion. In particular, we aimed to determine in vivo effects of subchronic exposure to 1,2-NQ on metabolic and inflammatory parameters of wild-type mice (WT) and to explore the involvement of tumor necrosis factor receptor 1 (TNFR1) and toll-like receptor 4 (TLR4) in this process. Males WT, TNFR1KO, and TLR4KO mice at eight weeks of age received 1,2-NQ or vehicle via nebulization five days a week for 17 weeks. In WT mice, 1,2-NQ slightly decreased the body mass compared to vehicle-WT. This effect was likely due to a mild food intake reduction and increased energy expenditure (EE) observed after six weeks of exposure. After nine weeks of exposure, we observed higher fasting blood glucose and impaired glucose tolerance, whereas insulin sensitivity was slightly improved compared to vehicle-WT. After 17 weeks of 1,2-NQ exposure, WT mice displayed an increased percentage of M1 and a decreased (p = 0.057) percentage of M2 macrophages in adipose tissue. The deletion of TNFR1 and TLR4 abolished most of the metabolic impacts caused by 1,2-NQ exposure, except for the EE and insulin sensitivity, which remained high in these mice under 1,2-NQ exposure. Our study demonstrates for the first time that subchronic exposure to 1,2-NQ affects energy metabolism in vivo. Although 1,2-NQ increased EE and slightly reduced feeding and body mass, the WT mice displayed higher inflammation in adipose tissue and impaired fasting blood glucose and glucose tolerance. Thus, in vivo subchronic exposure to 1,2-NQ is harmful, and TNFR1 and TLR4 are partially involved in these outcomes.

Spinal alarmin HMGB1 and the activation of TLR4 lead to chronic stress-induced nociceptive hypersensitivity in rodents

Chronic stress affects millions of people around the world, and it can trigger different behavioral disorders like nociceptive hypersensitivity and anxiety, among others. However, the mechanisms underlaying these chronic stress-induced behavioral disorders have not been yet elucidated. This study was designed to understand the role of high-mobility group box-1 (HMGB1) and toll-like receptor 4 (TLR4) in chronic stress-induced nociceptive hypersensitivity. Chronic restraint stress induced bilateral tactile allodynia, anxiety-like behaviors, phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) and activation of spinal microglia. Moreover, chronic stress enhanced HMGB1 and TLR4 protein expression at the dorsal root ganglion, but not at the spinal cord. Intrathecal injection of HMGB1 or TLR4 antagonists reduced tactile allodynia and anxiety-like behaviors induced by chronic stress. Additionally, deletion of TLR4 diminished the establishment of chronic stress-induced tactile allodynia in male and female mice. Lastly, the antiallodynic effect of HMGB1 and TLR4 antagonists were similar in stressed male and female rats and mice. Our results suggest that chronic restraint stress induces nociceptive hypersensitivity, anxiety-like behaviors, and up-regulation of spinal HMGB1 and TLR4 expression. Blockade of HMGB1 and TLR4 reverses chronic restraint stress-induced nociceptive hypersensitivity and anxiety-like behaviors and restores altered HMGB1 and TLR4 expression. The antiallodynic effects of HMGB1 and TLR4 blockers in this model are sex independent. TLR4 could be a potential pharmacological target for the treatment of the nociceptive hypersensitivity associated with widespread chronic pain.

Saturated fatty acid-enriched small extracellular vesicles mediate a crosstalk inducing liver inflammation and hepatocyte insulin resistance

Background & AimsLipotoxicity triggers non-alcoholic fatty liver disease (NAFLD) progression owing to the accumulation of toxic lipids in hepatocytes including saturated fatty acids (SFAs), which activate pro-inflammatory pathways. We investigated the impact of hepatocyte- or circulating-derived small extracellular vesicles (sEV) secreted under NAFLD conditions on liver inflammation and hepatocyte insulin signalling. MethodssEV released by primary mouse hepatocytes, characterised and analysed by lipidomics, were added to mouse macrophages/Kupffer cells (KC) to monitor internalisation and inflammatory responses. Insulin signalling was analysed in hepatocytes exposed to conditioned media from sEV-loaded macrophages/KC. Mice were i.v. injected sEV to study liver inflammation and insulin signalling. Circulating sEV from mice and humans with NAFLD were used to evaluate macrophage–hepatocyte crosstalk. ResultsNumbers of sEV released by hepatocytes increased under NAFLD conditions. Lipotoxic sEV were internalised by macrophages through the endosomal pathway and induced pro-inflammatory responses that were ameliorated by pharmacological inhibition or deletion of Toll-like receptor-4 (TLR4). Hepatocyte insulin signalling was impaired upon treatment with conditioned media from macrophages/KC loaded with lipotoxic sEV. Both hepatocyte-released lipotoxic sEV and the recipient macrophages/KC were enriched in palmitic (C16:0) and stearic (C18:0) SFAs, well-known TLR4 activators. Upon injection, lipotoxic sEV rapidly reached KC, triggering a pro-inflammatory response in the liver monitored by Jun N-terminal kinase (JNK) phosphorylation, NF-κB nuclear translocation, pro-inflammatory cytokine expression, and infiltration of immune cells into the liver parenchyma. sEV-mediated liver inflammation was attenuated by pharmacological inhibition or deletion of TLR4 in myeloid cells. Macrophage inflammation and subsequent hepatocyte insulin resistance were also induced by circulating sEV from mice and humans with NAFLD. ConclusionsWe identified hepatocyte-derived sEV as SFA transporters targeting macrophages/KC and activating a TLR4-mediated pro-inflammatory response enough to induce hepatocyte insulin resistance. Impact and ImplicationsSmall extracellular vesicles (sEV) released by the hepatocytes under non-alcoholic fatty liver disease (NAFLD) conditions cause liver inflammation and insulin resistance in hepatocytes via paracrine hepatocyte–macrophage–hepatocyte crosstalk. We identified sEV as transporters of saturated fatty acids (SFAs) and potent lipotoxic inducers of liver inflammation. TLR4 deficiency or its pharmacological inhibition ameliorated liver inflammation induced by hepatocyte-derived lipotoxic sEV. Evidence of this macrophage–hepatocyte interactome was also found in patients with NAFLD, pointing to the relevance of sEV in SFA-mediated lipotoxicity in NAFLD.

Cardiomyocyte-specific deletion of TLR4 attenuates angiotensin II-induced hypertension and cardiac remodeling.

Toll-like receptor 4 (TLR4) is an integral factor in the initiation of the innate immune response and plays an important role in cardiovascular diseases such as hypertension and myocardial infarction. Previous studies from our lab demonstrated that central TLR4 blockade reduced cardiac TLR4 expression, attenuated hypertension, and improved cardiac function. However, the contribution of cardiac specific TLR4 to the development of hypertension and cardiac remodeling is unknown. Therefore, we hypothesized that cardiomyocyte specific knockdown of TLR4 would have beneficial effects on hypertension, cardiac hypertrophy, and remodeling. To test this hypothesis, cardiomyocyte-specific TLR4 knockdown (cTLR4KO) mice were generated by crossing floxed TLR4 mice with Myh6-Cre mice, and subjected to angiotensin II (Ang II, 1 µg/kg/min or vehicle for 14 days) hypertension model. Blood pressure measurements using radio telemetry revealed no differences in baseline mean arterial pressure between control littermates and cTLR4KO mice (103 ± 2 vs. 105 ± 3 mmHg, p > 0.05). Ang II-induced hypertension (132 ± 2 vs. 151 ± 3 mmHg, p < 0.01) was attenuated and cardiac hypertrophy (heart/body weight; 4.7 vs. 5.8 mg/g, p < 0.01) was prevented in cTLR4KO mice when compared with control mice. In addition, the level of myocardial fibrosis was significantly reduced, and the cardiac function was improved in cTLR4KO mice infused with Ang II. Furthermore, cardiac inflammation, as evidenced by elevated gene expression of TNF, IL-6, and MCP-1 in the left ventricle, was attenuated in cTLR4KO mice infused with Ang II. Together, this data revealed a protective role for cardiomyocyte-specific deletion of TLR4 against Ang II-induced hypertension and cardiac dysfunction through inhibition of proinflammatory cytokines.

The long noncoding RNA Meg3 mediates TLR4-induced inflammation in experimental obstructive nephropathy.

Kidney inflammation contributes to the progression of chronic kidney disease (CKD). Modulation of Toll-like receptor 4 (TLR4) signaling is a potential therapeutic strategy for this pathology, but the regulatory mechanisms of TLR4 signaling in kidney tubular inflammation remains unclear. Here, we demonstrated that tubule-specific deletion of TLR4 in mice conferred protection against obstruction-induced kidney injury, with reduction in inflammatory cytokine production, macrophage infiltration and kidney fibrosis. Transcriptome analysis revealed a marked down-regulation of long noncoding RNA (lncRNA) Meg3 in the obstructed kidney from tubule-specific TLR4 knockout mice compared with wild-type control. Meg3 was also induced by lipopolysaccharide in tubular epithelial cells via a p53-dependent signaling pathway. Silencing of Meg3 suppressed LPS-induced cytokine production of CCL-2 and CXCL-2 and the activation of p38 MAPK pathway in vitro and ameliorated kidney fibrosis in mice with obstructive nephropathy. Together, these findings identify a proinflammatory role of lncRNA Meg3 in CKD and suggest a novel regulatory pathway in TLR4-driven inflammatory responses in tubular epithelial cells.

AIBP regulates TRPV1 activation in chemotherapy-induced peripheral neuropathy by controlling lipid raft dynamics and proximity to TLR4 in dorsal root ganglion neurons.

Nociceptive afferent signaling evoked by inflammation and nerve injury is mediated by the opening of ligand-gated and voltage-gated receptors or channels localized to cholesterol-rich lipid raft membrane domains. Dorsal root ganglion (DRG) nociceptors express high levels of toll-like receptor 4 (TLR4), which also localize to lipid rafts. Genetic deletion or pharmacologic blocking of TLR4 diminishes pain associated with chemotherapy-induced peripheral neuropathy (CIPN). In DRGs of mice with paclitaxel-induced CIPN, we analyzed DRG neuronal lipid rafts, expression of TLR4, activation of transient receptor potential cation channel subfamily V member 1 (TRPV1), and TLR4-TRPV1 interaction. Using proximity ligation assay, flow cytometry, and whole-mount DRG microscopy, we found that CIPN increased DRG neuronal lipid rafts and TLR4 expression. These effects were reversed by intrathecal injection of apolipoprotein A-I binding protein (AIBP), a protein that binds to TLR4 and specifically targets cholesterol depletion from TLR4-expressing cells. Chemotherapy-induced peripheral neuropathy increased TRPV1 phosphorylation, localization to neuronal lipid rafts, and proximity to TLR4. These effects were also reversed by AIBP treatment. Regulation of TRPV1-TLR4 interactions and their associated lipid rafts by AIBP covaried with the enduring reversal of mechanical allodynia otherwise observed in CIPN. In addition, AIBP reduced intracellular calcium in response to the TRPV1 agonist capsaicin, which was increased in DRG neurons from paclitaxel-treated mice and in the naïve mouse DRG neurons incubated in vitro with paclitaxel. Together, these results suggest that the assembly of nociceptive and inflammatory receptors in the environment of lipid rafts regulates nociceptive signaling in DRG neurons and that AIBP can control lipid raft-associated nociceptive processing.

Toll-Like Receptor 4 Signaling in Neurons Mediates Cerebral Ischemia/Reperfusion Injury.

In microglia, Toll-like receptor 4 (TLR4) is well known to contribute to neuroinflammatory responses following brain ischemia. TLR4 is also expressed in neurons and can mediate the conduction of calcium (Ca2+) influx, but the mechanistic link between neuronal TLR4 signaling and brain ischemic injury is still poorly understood. Here, primary neuronal cell cultures from TLR4 knockout mice and mice with conditional TLR4 knockout in glutamatergic neurons (TLR4cKO) were used to establish ischemic models in vitro and in vivo, respectively. We found that deleting TLR4 would reduce the neuronal death and intracellular Ca2+ increasement induced by oxygen and glucose deprivation (OGD) or lipopolysaccharide treatment. Infarct volume and functional deficits were also alleviated in TLR4cKO mice following cerebral ischemia/reperfusion (I/R). Furthermore, TLR4 and N-methyl-D-aspartate receptor subunit 2B (NMDAR2B) were colocalized in neurons. Deletion of TLR4 in neurons rescued the upregulation of phosphorylated NMDAR2B induced by ischemia via Src kinase in vitro and in vivo. Downstream of NMDAR2B signaling, the interaction of neuronal nitric oxide synthase (nNOS) with postsynaptic density protein-95 (PSD-95) was also disrupted in TLR4cKO mice following cerebral I/R. Taken together, our results demonstrate a novel molecular neuronal pathway in which TLR4 signaling in neurons plays a crucial role in neuronal death and provide a new target for neuroprotection after ischemic stroke.

Histone Deacetylase9 Represents the Epigenetic Promotion of M1 Macrophage Polarization and Inflammatory Response via TLR4 Regulation.

Atherosclerosis is a chronic inflammatory response mediated by various factors, where epigenetic regulation involving histone deacetylation is envisaged to modulate the expression of related proteins by regulating the binding of transcription factors to DNA, thereby influencing the development of atherosclerosis. The mechanism of atherosclerosis by histone deacetylation is partly known; hence, this project aimed at investigating the role of histone deacetylase 9 (HDAC9) in atherosclerosis. For this purpose, serum was separated from blood samples following clotting and centrifugation from atherosclerotic and healthy patients (n = 40 each), and then, various tests were performed. The results indicated that toll-like receptor 4 (TLR4) was not only positively correlated to the HDAC9 gene, but was also upregulated in atherosclerosis, where it was also significantly upregulated in the atherosclerosis cell model of oxidized low-density lipoprotein-induced macrophages. Conversely, the TLR4 was significantly downregulated in instances of loss of HDAC9 function, cementing the bridging relationship between HDAC9 and macrophage polarization, where the HDAC9 was found to upregulate M1 macrophage polarization which translated into the release of higher content of proinflammatory cytokines such as interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), which tend to significantly decrease following the deletion of TLR4. Hence, this study reports novel relation between epigenetic control and atherosclerosis, which could partly be explained by histone deacetylation.

Food reward depends on TLR4 activation in dopaminergic neurons

The rising prevalence of obesity and being overweight is a worldwide health concern. Food reward dysregulation is the basic factor for the development of obesity. Dopamine (DA) neurons in the ventral tegmental area (VTA) play a vital role in food reward. Toll-like receptor 4 (TLR4) is a transmembrane pattern recognition receptor that can be activated by saturated fatty acids. Here, we show that the deletion of TLR4 specifically in DA neurons increases body weight, increases food intake, and decreases food reward. Conditional deletion of TLR4 also decreased the activity of DA neurons while suppressing the expression of tyrosine hydroxylase (TH) in the VTA, which regulates the concentration of DA in the nucleus accumbens (NAc) to affect food reward. Meanwhile, AAV-Cre-GFP mediated VTA-specific TLR4-deficient mice recapitulates food reward of DAT-TLR4-KO mice. Food reward could be rescued by re-expressing TLR4 in VTA DA neurons. Moreover, effects of intra-VTA infusion of lauric acid (a saturated fatty acid with 12 carbon) on food reward were abolished in mice lacking TLR4 in DA neurons. Our study demonstrates the critical role of TLR4 signaling in regulating the activity of VTA DA neurons and the normal function of the mesolimbic DA system that may contribute to food reward.

Cellular fibronectin promotes deep vein thrombosis in diet‐induced obese mice

BackgroundOverweight and obesity are significant risk factors for deep vein thrombosis (DVT). Cellular fibronectin containing extra domain A (Fn‐EDA), an endogenous ligand for toll‐like‐receptor 4 (TLR4), contributes to thrombo‐inflammation. The role of Fn‐EDA in the modulation of DVT is not elucidated yet. ObjectiveTo determine whether Fn‐EDA promotes DVT in the context of diet‐induced obesity. MethodsWild‐type (WT) and Fn‐EDA‐deficient mice were either fed control or high‐fat (HF) diet for 12 weeks. DVT was induced by inferior vena cava (IVC) stenosis and evaluated after 48 hours. Cellular Fn‐EDA levels in the plasma of venous thromboembolism (VTE) patients were measured by sandwich ELISA. ResultsWe found that cellular Fn‐EDA levels were significantly elevated in VTE patients' plasma and positively correlated with body mass index. HF diet‐fed WT mice exhibited increased DVT susceptibility compared with control diet‐fed WT mice. In contrast, HF diet‐fed Fn‐EDA‐deficient mice exhibited significantly reduced thrombus weight and decreased incidence (%) of DVT compared with HF diet‐fed WT mice concomitant with reduced neutrophil content and citrullinated histone H3‐positive cells (a marker of NETosis) in IVC thrombus. Exogenous cellular Fn‐EDA potentiated NETosis in neutrophils stimulated with thrombin‐activated platelets via TLR4. Genetic deletion of TLR4 in Fn‐EDA+ mice (constitutively express Fn‐EDA in plasma and tissues), but not in Fn‐EDA‐deficient mice, reduced DVT compared with respective controls. ConclusionThese results demonstrate a previously unknown role of Fn‐EDA in the DVT exacerbation, which may be an essential mechanism promoting DVT in the setting of diet‐induced obesity.

Deletion of TLR4 attenuates lipopolysaccharide-induced acute liver injury by inhibiting inflammation and apoptosis.

Septic acute liver injury is one of the leading causes of fatalities in patients with sepsis. Toll-like receptor 4 (TLR4) plays a vital role in response to lipopolysaccharide (LPS) challenge, but the mechanisms underlying TLR4 function in septic injury remains unclear. In this study, we investigated the role of TLR4 in LPS-induced acute liver injury (ALI) in mice with a focus on inflammation and apoptosis. Wild-type (WT) and TLR4-knockout (TLR4-/-) mice were challenged with LPS (4 mg/kg) for 6 h. TLR4 signaling cascade markers (TLR4, MyD88, and NF-κB), inflammatory markers (TNFα, IL-1β, and IL-6), and apoptotic markers (Bax, Bcl-2, and caspase 3) were evaluated. We showed that LPS challenge markedly increased the levels of serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) and other liver pathological changes in WT mice. In addition, LPS challenge elevated the levels of liver carbonyl proteins and serum inflammatory cytokines, upregulated the expression of TLR4, MyD88, and phosphorylated NF-κB in liver tissues. Moreover, LPS challenge significantly increased hepatocyte apoptosis, caspase 3 activity, and Bax level while suppressing Bcl-2 expression in liver tissues. These pathological changes were greatly attenuated in TLR4-/- mice. Similar pathological responses were provoked in primary hepatic Kupffer cells isolated from WT and TLR4-/- mice following LPS (1 μg/mL, 6 h) challenge. In summary, these results demonstrate that silencing of TLR4 attenuates LPS-induced liver injury through inhibition of inflammation and apoptosis via TLR4/MyD88/NF-κB signaling pathway. TLR4 deletion confers hepatoprotection against ALI induced by LPS, possibly by repressing macrophage inflammation and apoptosis.

Cellular Fibronectin Promotes Deep Vein Thrombosis in Obese Mice

Objective: Obesity is a significant risk factor for deep vein thrombosis (DVT). The mechanisms of increased DVT in preexisting comorbid condition of obesity remain poorly understood. Cellular fibronectin containing extra domain A (Fn-EDA), an endogenous ligand for toll-like-receptor 4 (TLR4), is known to contribute to thrombo-inflammation in the experimental models. However, the role of Fn-EDA in modulation of venous thrombosis in context of obesity is not elucidated yet. Approach and Results: We found that cellular Fn-EDA levels were significantly elevated in plasma of venous thromboembolism (VTE) patients that positively correlated with body mass index (BMI). To investigate whether Fn-EDA promotes venous thrombosis in obese condition, WT and Fn-EDA-/- mice were either fed a control or high-fat diet (HF-diet) for 12-weeks. DVT was induced by inferior vena cava stenosis and evaluated after 48 hours. We found that HF diet-fed WT mice exhibited increased DVT susceptibility compared with control diet-fed WT mice. In contrast, HF-fed Fn-EDA-/- mice exhibited significantly reduced thrombus weight and decreased incidence (%) of DVT compared with HF-fed WT mice that was concomitant with improved blood flow, reduced neutrophil content and citrullinated histone H3-positive cells (a marker of NETosis) in IVC thrombus. Exogenous Fn-EDA potentiated NETosis in neutrophils stimulated with thrombin-activated platelets via TLR4. Genetic deletion of TLR4 in Fn-EDAfl/fl mice, which constitutively express Fn-EDA, reduced DVT compared with Fn-EDAfl/fl mice. Conclusion: These results demonstrate a previously unknown role of Fn-EDA in the modulation of DVT, which may be an important mechanism promoting DVT in the setting of obesity. Figure Disclosures No relevant conflicts of interest to declare.

Deletion of toll-like receptor 4 ameliorates diabetic retinopathy in mice

Background Diabetic retinopathy is a common and specific microvascular complication of diabetes, which is also the leading cause of preventable blindness. Therefore, we aimed to find a promising therapeutic strategy for diabetic retinopathy. Methods To investigate the role of toll-like receptor 4 (TLR4) in the diabetic retinopathy, we injected streptozotocin (STZ) into wild-type (wt) and TLR4 knock-out mice to induce diabetes. Results While STZ induced diabetes both in wt and TLR4−/− mice, deletion of TLR4 in diabetic mice significantly improved diabetic retinopathy compared to diabetic wt mice, as judged by the enhanced thickness of retinal tissue. Furthermore, TLR4-dependent NF-κB pathway, inflammatory cytokine release and the expressions of vascular endothelial growth factor (VEGF) and glial fibrillary acidic protein (GFAP), which were all remarkably stimulated in STZ-injected wt mice, were inhibited in STZ-injected TLR4−/− mice. Conclusion TLR4 could serve as an independent target for treating diabetic retinopathy.

CRISPR-Cas9-based gene editing of human mesenchymal stromal cells to improve the outcome of cell therapy

Abstract Background The environment of the failing and infarcted myocardium drives resident and transplanted mesenchymal stromal cells (MSCs) toward a pro-inflammatory phenotype, thus restricting their survival and their reparative effects in a mechanism mediated by toll-like receptor 4 (TLR4). Hence, new approaches are needed to improve the efficacy of cell therapy for heart failure. CRISPR is a promising tool for genome editing, which raises the hope for therapeutic genome editing in the clinic. Purpose We aimed to provide a new strategy to enhance MSC-based cell therapy to improve cardiac remodeling and function. We hypothesized that ex vivo knockout (KO) of the human TLR4 gene by CRISPR would switch human-cardiac MSCs (hMSCs) to an anti-inflammatory, reparative phenotype that could prevent remodeling of the left ventricle after myocardial infarction (Fig. 1A). Methods and results We achieved up to 68% (out of 4x105 cells, R2=0.93) success rate in editing the genome of primary cardiac hMSCs taken from patients with ischemic heart disease. The deletion of TLR4 in hMSCs significantly reduced the secretion of inflammatory and extracellular-matrix (ECM) proteins, compared with unedited hMSCs, by protein mass spectrometry (Fig. 1B) and by multiplex ELISA (Fig. 1C). Additionally, edited cells secreted significantly more extracellular vesicles (EVs) than unedited hMScs (Fig. 1D, p&amp;lt;0.001). These EVs from edited hMSCs stimulated faster migration of hMSCs in a “wound healing” assay (p&amp;lt;0.001). Conclusions We show, for the first time, that CRISPR-based deletion of the TLR4 gene in hMSCs inhibits inflammatory and ECM protein secretion and facilitates a reparative response by hMSCs in vitro. This precise and efficient ex vivo gene editing could provide a newly engineered cell line to improve the outcome of hMSC-based cell therapy. Figure 1 Funding Acknowledgement Type of funding source: Public hospital(s). Main funding source(s): The Foundation in Memory of Seymour Fefer, Sheba Medical Center

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.

Toll-like receptor 4 deficiency ameliorates β2-microglobulin induced age-related cognition decline due to neuroinflammation in mice

Toll-like receptor 4 (TLR4) is a crucial receptor in neuroinflammation and apoptotic neuronal death, and increasing evidences indicated that β2-microglobulin (B2M) is thought to be a major contributor to age-related cognitive decline. In present study, we designed to investigate the effects of TLR4 on B2M-induced age-related cognitive decline. Wild-type (WT) C57BL/6, TLR4 knockout (TLR4 -KO) mice and hippocampal neurons from the two type mice were respectively divided into two groups: (1) Veh group; (2) B2M-treated group. The behavioral responses of mice were measured using Morris Water Maze. Hippocampal neurogenesis and neuronal damage, inflammatory response, apoptosis, synaptic proteins and neurotrophic factors, and TLR4/MyD88/NF-κB signaling pathway proteins were examined using molecular biological or histopathological methods. The results showed that WT mice received B2M in the DG exhibited age-related cognitive declines, increased TLR4 mRNA expression and high levels of interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and apoptotic neuronal death in the hippocampus, which were partially attenuated in TLR4-KO mice. Moreover, in absence of TLR4, B2M treatment improved hippocampus neurogenesis and increased synaptic related proteins. Our cell experiments further demonstrated that deletion of TLR4 could significantly increase synaptic related protein, decrease neuroinflammatory fators, inhibited apoptotic neuronal death, and regulated MyD88/NF-κB signal pathway after B2M treatment. In summary, our results support the TLR4 contributes to B2M-induced age-related cognitive decline due to neuroinflammation and apoptosis through TLR4/MyD88/NF-κB signaling pathway via a modulation of hippocampal neurogenesis and synaptic function. This may provide an important neuroprotective mechanism for improving age-related cognitive decline.

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

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

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.