Toll-like Receptor 4 Deficiency Research Articles

High mobility box 1 mediates neutrophil recruitment in myocardial ischemia–reperfusion injury through toll like receptor 4-related pathway

This study aimed to explore the role of high mobility box 1 (HMGB1) and its receptor toll like receptor 4 (TLR4) on neutrophils in myocardial ischemia reperfusion (I/R) injury. We constructed TLR4-mutant (C3H/HeJ) and control (C3H/HeN) mouse models of myocardial I/R injury and subjected the mice to 30min of ischemia and 6h of reperfusion. Light microscope was used to observe structural changes in the myocardium. HMGB1 levels were measured using quantitative real-time PCR and immunohistochemistry. Neutrophil accumulation, TNF-a expression and IL-8 levels were analyzed via myeloperoxidase (MPO) biochemical studies, quantitative real-time PCR and ELISA, respectively. The results demonstrated that fewer neutrophils infiltrated in the myocardium of TLR4-mutant mice after myocardial I/R and that TLR4 deficiency markedly decreased the ischemic injury caused by ischemia/reperfusion, and inhibited the expression of HMGB1, TNF-a, and IL-8, all of which were up-regulated by ischemia/reperfusion. These findings suggest that HMGB1 plays a central role in recruiting neutrophils during myocardial I/R leading to worsened myocardial I/R injury. This recruitment mechanism is possibly due to its inflammatory and chemokine functions based on the TLR4-dependent pathway.

Toll-Like Receptor 4 Deficiency Decreases Atherosclerosis But Does Not Protect Against Inflammation in Obese Low-Density Lipoprotein Receptor–Deficient Mice

Obesity is associated with insulin resistance, chronic low-grade inflammation, and atherosclerosis. Toll-like receptor 4 (TLR4) participates in the cross talk between inflammation and insulin resistance, being activated by both lipopolysaccharide and saturated fatty acids. The present study was undertaken to determine whether TLR4 deficiency has a protective role in inflammation, insulin resistance, and atherosclerosis induced by a diabetogenic diet. TLR4 and low-density lipoprotein (LDL) receptor double knockout mice and LDL receptor-deficient mice were fed either a normal chow or a diabetogenic diet for 24 weeks. TLR4 and LDL receptor double knockout mice fed a diabetogenic diet showed improved plasma cholesterol and triglyceride levels but developed obesity, hyperinsulinemia, and glucose intolerance equivalent to obese LDL receptor-deficient mice. Adipocyte hypertrophy, macrophage accumulation, and local inflammation were not attenuated in intraabdominal adipose tissue in TLR4 and LDL receptor double knockout mice. However, TLR4 deficiency led to markedly decreased atherosclerosis in obese TLR4 and LDL receptor double knockout mice. Compensatory upregulation of TLR2 expression was observed both in obese TLR4-deficient mice and in palmitate-treated TLR4-silenced 3T3-L1 adipocytes. TLR4 deficiency decreases atherosclerosis without affecting obesity-induced inflammation and insulin resistance in LDL receptor-deficient mice. Alternative pathways may be responsible for adipose tissue macrophage infiltration and insulin resistance that occurs in obesity.

P026 Effect of Toll-like receptor-2 deficiency on transcriptomic profile in DSS-colitis

fibrosis (p = 0.004 and 0.00004), those receiving PC/TNBS had significantly less inflammation (p = 0.009), while those treated with PC/Indo/TNBS had less inflammation and fibrosis (p = 0.001 and 0.03). At 6 weeks compared to TNBS alone, inflammation and fibrosis was not different in the Indo/TNBS and PC/Indo/TNBS-treated animals, but PC/TNBS-treated animals had less inflammation (p = 0.01) and fibrosis (p = 0.006). The addition of PC to Indo/TNBS at 2 weeks significantly decreased inflammation and fibrosis compared to Indo/TNBS alone (p = 0.03 and 0.00001), however at 6 weeks, this addition had no effect on Indo/TNBS-induced inflammation or fibrosis. In CCD-18co cells the addition of DLPC increased MMP-1 and TIMP-1 mRNA expression (p = 0.03 and 0.01), with no effect on Col1 or TGF-b1 suggesting that DLPC may decrease ECM deposition. By contrast, LPPC increased Col1 (p = 0.0008) and TIMP-1 (p = 0.03) and decreased MMP-1 expression (p = 0.003) with no effect on TGF-b1 suggesting that LPPC may increase ECM deposition. DLPC/Indo and LPPC/Indo treatment mirrored DLPC and LPPC treatment alone. Conclusions: The findings demonstrate that PC can reduce inflammation and fibrosis but may require a higher dose to completely abolish the effects of Indo/TNBS in vivo. The PC isoform appears to be important in vitro as DLPC may decrease and LPPC increase ECM deposition by isolated fibroblasts.

Role of TLR2 in the pathogenesis of autoimmune diabetes and its therapeutic implication

Recently, a couple of articles suggested the possibility that apoptosis of pancreatic β-cells induces inflammatory/immune responses to β-cells. Such a theory is based on the assumption that apoptotic cells can, under certain circumstances, induce immune responses, inflammatory and autoimmune disorders, which is in contrast to the dogma that apoptotic cells result in immunosuppression and necrotic cells provoke inflammation/immunity. We observed that late apoptotic β-cells with secondary necrosis elicited inflammatory responses in macrophages through the toll-like receptor 2 (TLR2)/MyD88/nuclear factor-κB signalling pathway. Late apoptotic cells also induced TLR2-dependent maturation of dendritic cells and then activation of autoreactive T-cells. TLR2 knockout mice showed defective priming of diabetogenic T-cells by apoptotic β-cells in the pancreatic lymph nodes. Furthermore, TLR2 deficiency conferred a significant protection against type 1 diabetes (T1D) and insulitis in T1D animal models. These findings present evidence suggesting that apoptosis of pancreatic β-cells could be one of the initial events in T1D and provide a novel strategy for therapeutic or preventive intervention in T1D.

Novel Toll-like receptor-4 deficiency attenuates trastuzumab (Herceptin) induced cardiac injury in mice

BackgroundCardiac inflammation and generation of oxidative stress are known to contribute to trastuzumab (herceptin) induced cardiac toxicity. Toll-like receptors (TLRs) are a part of the innate immune system and are involved in cardiac stress reactions. Since TLR4 might play a relevant role in cardiac inflammatory signaling, we investigated whether or not TLR4 is involved in trastuzumab induced cardiotoxicity.MethodsSeven days after a single injection of herceptin (2 mg/kg; i.p.), left ventricular pressure volume loops were measured in HeN compotent (TLR4+/+) and HeJ mutant (TLR4-/-) treated with trastuzumab and control mice. Immunofluorescent staining for monocyte infiltration and analyses of plasma by (ELISAs) for different chemokines including: MCP-1and tumor necrosis factor-α (TNF-α), Western immunoblotting assay for ICAM-1, and used troponin I for cardiac injury marker.ResultsTrastuzumab injection resulted in an impairment of left ventricular function in TLR-4 competent (HeN), in contrast TLR4-/- trastuzumab mice showed improved left ventricular function EF%, CO; p < 0.05, attenuation of mononuclear cell infiltration in TLR4 -/-; p < 0.05 vs.TLR-4 competent (HeN), reduced level of cytokines TNF-α, MCP-1 and ICAM-1 expression in TLR4-/-, marked reduction of myocardial troponin-I levels in TLR4-deficient mice. Data are presented as means ± SE; n = 8 in each group p < 0.05 vs.TLR-4 competent (HeN).ConclusionsTreatment with trastuzumab induces an inflammatory response that contributes to myocardial tissue TLR4 mediates chemokine expression (TNF-α, MCP-1and ICAM-1), so in experimental animals TLR4 deficiency improves left ventricular function and attenuates pathophysiological key mechanisms in trastuzumab induced cardiomyopathy.

PPAR attenuates intimal hyperplasia by inhibiting TLR4-mediated inflammation in vascular smooth muscle cells

Peroxisome proliferator-activated receptor γ (PPARγ) has been reported to attenuate intimal hyperplasia. This study aimed to test the hypothesis that PPARγ inhibits intimal hyperplasia through suppressing Toll-like receptor 4 (TLR4)-mediated inflammation in vascular smooth muscle cells. TLR4(-/-) mice on a C57BL/6J background were used. Increased TLR4 and pro-inflammatory cytokines were observed in wire-injury-induced carotid neointima and in platelet-derived growth factor (PDGF)-activated vascular smooth muscle cells. The TLR4 deficiency protected the injured carotid from neointimal formation and impaired the cellular proliferation and migration in response to lipopolysaccharide and PDGF. Rosiglitazone attenuated intimal hyperplasia. Overexpression of PPARγ suppressed PDGF-induced proliferation and migration and inhibited TLR4-mediated inflammation in vascular smooth muscle cells, while PPARγ silencing exerted the opposite effect. Lipopolysaccharide counteracted the inhibitory effect of PPARγ on PDGF-induced proliferation and migration. Eritoran suppressed the proliferation and migration induced by PDGF and PPARγ silencing. Vascular smooth muscle cells derived from TLR4(-/-) mice showed impaired proliferation and migration upon PDGF activation and displayed no response to PPARγ manipulation. PPARγ inhibits vascular smooth muscle cell proliferation and migration by suppressing TLR4-mediated inflammation and ultimately attenuates intimal hyperplasia after carotid injury.

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We thank Dr. Schwabe and colleagues for interest in our article.1, 2 We have carefully reviewed our published results and also the original data. Our experiments showed that carcinogen-induced apoptosis in mice can be protected by lipopolysaccharide (LPS), an endotoxin (Fig. 1), or other factors induced by diethylnitrosamine (DEN). This figure was originally included in the manuscript, but deleted in revision due to page limitations. The main question concerns why reduction of LPS by antibiotics (figure 7B)1 led to lower liver injury but LPS receptor Toll-like receptor 4 (TLR4) deficiency potentiated liver injury (figure 3A).1 Possible explanations are as follows: (1) The alanine aminotransferase level usually indicates liver damage and does not exclusively represent the degree of cell apoptosis. LPS protection as previously described takes effect by a certain time period (e.g., at 48 hours after injection; figure 6C-F),1 although it even enhances liver injury at 24 hours after DEN injection. We did not claim that no or low LPS levels and antibiotic suppression of LPS will increase liver injury. (2) Because treatment with antibiotics prevented the increase of plasma LPS (figure 7A),1 inhibition of LPS-induced inflammation upon DEN exposure might be responsible for the reduction of liver injury. We found that antibiotic-induced reduction of LPS and lack of TLR4 reduced the number and volume of tumors with the decrease of tumor necrosis factor α and interleukin-6. LPS pretreatment prevents death receptor ligands–induced apoptosis in vivo and in vitro. (A,C) Survival curves of LPS-preconditioned or control wild-type mice in response to treatment with (A) tumor necrosis factor/galactosamine (TNF/GaIN) and (C) Jo-2. (B,D) The typical gross and histological appearance of the above treated mice: (B) tumor necrosis factor/galactosamine (TNF/GalN); (D) Jo-2. Scale bars = 100 μm. (E,F) The cleavage of poly(adenosine diphosphate ribose) polymerase (PARP) protein was detected by western blot in LPS-pretreated (1μg/mL for 12 hours) or untreated primary wild-type or TLR4−/− hepatocytes (E) or Hepa1-6 cells (F) upon TNF + cycloheximide (CHX) treatment. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. We had the same concerns about this discrepancy, because it appears to contradict the hypothesis that TLR4−/− mice should have less liver injury. However, in terms of the acute liver damage induced by DEN, the TLR4 knockout mice are different from wild-type mice in response to the short-term antibiotics treatment. In addition to LPS, TLR4 interacts with other endogenous ligands, such as high mobility group box 1 (HMGB1) and heat shock proteins (HSPs). TLR4 deficiency therefore ablated the protective effects of both LPS and endogenous ligands on hepatocytes, thus leading to more severe liver damage. Treatment with antibiotics mainly reduced the acute inflammatory responses to LPS in nonparenchymal cells, which is thought to be the major contributing factor to acute hepatocyte injury. It is conceivable that reduced production of proinflammatory cytokines such as tumor necrosis factor α was responsible for alleviation of DEN-induced acute liver damage. On the other hand, during the multistage process of hepatocarcinogenesis, accumulation of endotoxin promotes chronic tumorigenic inflammation and alleviates apoptosis in precancerous and cancerous cells. So, loss of TLR4 is not equivalent to LPS ablation in terms of the responsive cell types and inflammation status. As cited by Dr. Schwabe and colleagues, Karin's group at University of California San Diego reported an increase in DEN-induced hepatocellular carcinoma development when inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ) was deleted in hepatocytes. However, the same group also found that hepatocellular carcinoma was suppressed in mice with IKKβ ablated in both hepatocytes and Kupffer cells.3 Furthermore, Pikarsky et al. found that inhibiting nuclear factor κB signaling suppressed hepatocarcinogenesis.4 Resolving these differences will lead to better understanding of hepatocarcinogenesis. Lexing Yu Ph.D.*, Hexin Yan Ph.D.*, Wen Yang Ph.D.*, Hongyang Wang M.D.* , * International Cooperation Laboratory on Signal Transduction, Liver Centre of SMMU, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China, National Laboratory for Oncogene and Related Genes, Cancer Institute of Shanghai Jiao Tong University, Shanghai, China.

Toll-Like Receptor 4 Is Involved in Human and Mouse Vein Graft Remodeling, and Local Gene Silencing Reduces Vein Graft Disease in Hypercholesterolemic APOE*3Leiden Mice

The goal of this study was to explore the role of Toll-like receptor 4 (TLR4) in vein graft remodeling and disease. First, expression of TLR4 was analyzed in freshly isolated human saphenous veins (huSV), in freshly isolated huSV ex vivo perfused in an extracorporeal circulation, or in huSV used as coronary vein grafts. Marked induction of focal TLR4 expression was observed in perfused fresh huSV. Moreover, TLR4 was abundantly present in lesions in fresh huSV or in intimal hyperplasia in coronary vein grafts. Second, mouse venous bypass grafting was performed. In grafts of hypercholesterolemic APOE*3Leiden mice, increased TLR4 mRNA and protein was detected over time by reverse transcription-polymerase chain reaction and immunohistochemistry. Furthermore, the local presence of the endogenous TLR4 ligands heat shock protein 60, high-mobility group box 1, tenascin-C, and biglycan in the grafts was demonstrated. TLR4 deficiency in C3H-Tlr4LPS-d (LPS indicates lipopolysaccharide) mice resulted in 48±12% less vein graft wall thickening (P=0.04) than in Balb/c controls. Moreover, local TLR4 gene silencing in hypercholesterolemic APOE*3Leiden mice using lentiviral short hairpin RNA against TLR4 administered perivascularly around vein grafts led to a 44±13% reduction of vessel wall thickening compared with controls (P=0.0059). These results indicate that TLR4 is involved in vein graft remodeling and can be used as a local therapeutic target against vein graft disease.

Atherogenic Lipids and Lipoproteins Trigger CD36-TLR2-Dependent Apoptosis in Macrophages Undergoing Endoplasmic Reticulum Stress

Macrophage apoptosis in advanced atheromata, a key process in plaque necrosis, involves the combination of ER stress with other proapoptotic stimuli. We show here that oxidized phospholipids, oxidized LDL, saturated fatty acids (SFAs), and lipoprotein(a) trigger apoptosis in ER-stressed macrophages through a mechanism requiring both CD36 and Toll-like receptor 2 (TLR2). In vivo, macrophage apoptosis was induced in SFA-fed, ER-stressed wild-type but not Cd36⁻(/)⁻ or Tlr2⁻(/)⁻ mice. For atherosclerosis, we combined TLR2 deficiency with that of TLR4, which can also promote apoptosis in ER-stressed macrophages. Advanced lesions of fat-fed Ldlr⁻(/)⁻ mice transplanted with Tlr4⁻(/)⁻Tlr2⁻(/)⁻ bone marrow were markedly protected from macrophage apoptosis and plaque necrosis compared with WT →Ldlr⁻(/)⁻ lesions. These findings provide insight into how atherogenic lipoproteins trigger macrophage apoptosis in the setting of ER stress and how TLR activation might promote macrophage apoptosis and plaque necrosis in advanced atherosclerosis.

Role of Toll-Like Receptor 4 in Intimal Foam Cell Accumulation in Apolipoprotein E–Deficient Mice

Atherosclerosis encompasses a conspicuously maladaptive inflammatory response that might involve innate immunity. Here, we compared the role of Toll-like receptor 4 (TLR4) with that of TLR2 in intimal foam cell accumulation and inflammation in apolipoprotein E (ApoE) knockout (KO) mice in vivo and determined potential mechanisms of upstream activation and downstream action. We measured lipid accumulation and gene expression in the lesion-prone lesser curvature of the aortic arch. TLR4 deficiency reduced intimal lipid by ≈75% in ApoE KO mice, despite unaltered total serum cholesterol and triglyceride levels, whereas TLR2 deficiency reduced it by ≈45%. TLR4 deficiency prevented the increased interleukin-1α (IL-1α) and monocyte chemoattractant protein-1 mRNA levels seen within lesional tissue, and it also lowered serum IL-1α levels. Smooth muscle cells (SMC) were present within the intima of the lesser curvature of the aortic arch at this early lesion stage, and they enveloped and permeated nascent lesions, which consisted of focal clusters of foam cells. Cholesterol enrichment of SMC in vitro stimulated acyl-coenzyme A:cholesterol acyltransferase-1 mRNA expression, cytoplasmic cholesterol ester accumulation, and monocyte chemoattractant protein-1 mRNA and protein expression in a TLR4-dependent manner. TLR4 contributes to early-stage intimal foam cell accumulation at lesion-prone aortic sites in ApoE KO mice, as does TLR2 to a lesser extent. Intimal SMC surround and penetrate early lesions, where TLR4 signaling within them may influence lesion progression.

Altered TLR2 Expression and Signaling contribute to the Hyperinflammation in diabetic wounds

Toll‐like receptor‐2 (TLR2) is a pivotal pathogen recognition signaling receptor and plays a key role in inflammation and injury. Hyperglycemia, inflammation, and oxidative stress induce TLR2, and are major pathophysiological mechanisms in impaired diabetic wound healing process. However, an in vivo contribution of TLR2 to the inflammatory state of diabetic wounds remains elusive. The aim of the study is to examine the contribution of TLR2 to the hyperinflammation in wounds of diabetic mice. Diabetes was induced in male C57BL/6J (WT) and TLR2 Knockout mice (KO) using streptozotocin with suitable non diabetic controls (n=6/group). Full thickness excisional wounds were made on the backs of all the mice aseptically. Total RNA and protein were subjected to RT‐PCR, Western blots, and wound sizes were recorded. TLR2 mRNA expression increased 2.5 fold in diabetic wounds of WT mice (P&lt;0.05) with corresponding Myd88 expression, IRAK‐1phosphroylation, and NF‐κB activation compared to non diabetic wounds. Wounds of diabetic TLR2KO mice showed less lipid peroxidation (−70%), Myd88 signaling, NF‐κB(−62%), IL‐1β(−82%), and smaller wound sizes compared to diabetic WT mice after 10 days. Collectively, our findings demonstrate that increased TLR2 expression and signaling contribute to the diabetic wound hyper inflammatory state and TLR2 deficiency decreases inflammation and improves diabetic wound healing.

Nucleotide-binding oligomerization domain 2 and Toll-like receptor 2 function independently in a murine model of arthritis triggered by intraarticular peptidoglycan

Blau syndrome is an autoinflammatory disease resulting from mutations in the NOD2 gene, wherein granulomatous arthritis, uveitis, and dermatitis develop. The mechanisms by which aberrant NOD2 causes joint inflammation are poorly understood. Indeed, very few studies have addressed the function of nucleotide-binding oligomerization domain 2 (NOD-2) in the joint. This study was undertaken to investigate NOD-2 function in an experimental model of arthritis and to explore the potential interplay between Toll-like receptor 2 (TLR-2) and NOD-2 in joint inflammation. Mice deficient in TLR-2, myeloid differentiation factor 88 (MyD88), or NOD-2 and their wild-type controls were given an intraarticular injection of muramyl dipeptide (MDP), peptidoglycan (PG; a metabolite of which is MDP), or palmitoyl-3-cysteine-serine-lysine-4 (Pam(3)CSK(4)), a synthetic TLR-2 agonist. Joint inflammation was assessed by near-infrared fluorescence imaging and histologic analysis. Locally administered PG resulted in joint inflammation, which was markedly reduced in mice deficient in either TLR-2 or the TLR signaling mediator MyD88. In addition to TLR-2 signaling events, NOD-2 mediated joint inflammation, as evidenced by the fact that mice deficient in NOD-2 showed significantly reduced PG-induced arthritis. TLR-2 or MyD88 deficiency did not influence arthritis induced by the specific NOD-2 agonist MDP. In addition, NOD-2 deficiency did not alter the TLR-2-dependent joint inflammation elicited by the synthetic TLR-2 agonist Pam(3)CSK(4). Whereas NOD-2 and TLR-2 are both critical for the development of PG-induced arthritis, they appear to elicit inflammation independently of each other. Our findings indicate that NOD-2 plays an inflammatory role in arthritis.

Saturated Fatty Acids and Inflammation: Who Pays the Toll?

Since the discovery that obesity is associated with macrophage accumulation in adipose tissue,1,2 mechanisms by which adipose tissue becomes inflamed, resulting in insulin resistance, have remained elusive. Several studies have demonstrated that saturated fatty acids (SFAs) stimulate adipose tissue inflammation by a process that involves Toll-like receptor 4 (TLR4), a receptor that binds bacterial lipopolysaccharide (LPS). TLR4 is a pattern recognition receptor that plays a key role in the innate immune response. The observation that TLR4 deficiency protected against insulin resistance in obesity induced by a diet high in SFAs3 suggested that TLR4 was the link between diet excess and insulin resistance.3 Attenuation of diet-induced insulin resistance4,5 and adipose tissue inflammation6 also have been observed in C3H/HeJ mice with a loss of function mutation in TLR4. See accompanying article on page 802 In vitro studies have shed light on how SFAs lead to adipose tissue inflammation. TLR4 activation by SFAs increases the expression of a number of inflammatory genes in adipocytes by a nuclear factor κB–dependent mechanism,7–11 similar to TLR4 activation by LPS. SFAs also stimulate inflammatory molecules in macrophages.3,10–12 TLR4’s critical role has been demonstrated by silencing TLR4 expression with short …

Toll-like receptor 4–deficient mice are resistant to chronic hypoxia-induced pulmonary hypertension

ABSTRACTCurrent data suggest that Toll-like receptor 4 (TLR4), a key molecule in the innate immune response, may also be activated following tissue injury. Activation of this receptor is known to induce the production of several proinflammatory cytokines. Given that pulmonary inflammation has been shown to be a key contributor to chronic hypoxia-induced pulmonary vascular remodeling, the authors hypothesized that TLR4-deficient mice would be less susceptible to pulmonary hypertension (PH) as compared to mice with intact TLR4. TLR4-deficient and TLR4-intact strains of inbred mice were exposed to 4, 8, and 16 weeks of hypoxia (0.10 FiO2) or normoxia (0.21 FiO2) in a normobaric chamber. After chronic hypoxic exposure, TLR4-intact mice developed significant PH evidenced by increased right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary artery medial thickening. In contrast, TLR4-deficient mice had no significant change in any of these parameters and this was associated with decreased pulmonary vascular inflammatory response as compared to the TLR4-intact mice. These results suggest that TLR4 deficiency may decrease the susceptibility to developing PH by attenuating the pulmonary vascular inflammatory response to chronic hypoxia.

Toll-like receptor 2 is required for opioids-induced neuronal apoptosis

Toll-like receptor 2 (TLR2), a key immune receptor in the TLR family, is widely expressed in various systems, including the immune and nervous systems and plays a critical role in controlling innate and adaptive immune responses. We previously reported that opioids inhibit cell growth and trigger apoptosis. However, the underlying mechanism by which TLR2 mediates apoptosis in response to opioids is not yet known. Here we show that chronic morphine treatment in primary neurons dramatically increased the expression of TLR2 at both the messenger RNA and protein levels. In addition, TLR2 deficiency significantly inhibited chronic morphine-induced apoptosis in primary neurons. Activation of caspase-3 after morphine treatment is impaired in TLR2 deficient primary neurons. Moreover, morphine treatment failed to induce an increased level of phosphorylated glycogen synthase kinase 3 beta (GSK3β) in TLR2 deficient primary neurons, suggesting an involvement of GSK3β in morphine-mediated TLR2 signaling. These results thus demonstrate that opioids prime neurons to undergo apoptosis by inducing TLR2 expression. Our data suggest that inhibition of TLR2 is capable of preventing opioids-induced damage to neurons.

Abstract 5675: Local Gene Silencing of TLR4 Expression in vivo Improves Vein Graft Patency in Mice

Introduction: Patency of vein grafts is limited by vascular remodelling processes such as neointima formation and accelerated atherosclerosis. Toll Like Receptor 4 (TLR4) is associated with neointima formation and atherosclerosis. Hypothesis: We hypothesize that TLR4 can serve as a local therapeutic target against vein graft disease. Methods: In human saphenous veins (huSV) immunohistochemical staining for TLR4 was performed on cross-sections of either directly harvested huSV (n=6), in vitro perfused huSV (n=6) and coronary graft huSV derived at autopsy (n=5). Mouse venous bypass grafting was performed in Balb/c mice (n=8) and in TLR4 deficient (C3H/Hej) mice (n=7) using a venous interposition model in the carotid artery. Five lentiviral TLR4 shRNAs were created and validated for local TLR4 gene silencing by measurement of murine TLR4/MD2 expression by FACS analysis and the best vector was selected for in vivo analysis. To test the effect of local TLR4 deficiency on vein graft disease virus was administered in pluronic gel and lubricated around vein grafts after vein grafting in APOE3*Leiden mice fed a high cholesterolemic diet. Results: A marked induction of focal TLR4 expression was observed in HuSV, reperfused ex vivo with whole blood under pulsatile arterial pressure. Moreover, TLR4 was abundantly present in the neointima formed in autopsy grafts and preexisting intimal lesions in directly harvested huSV. Vein grafts in C3H/Hej mice had 48±12% less vessel wall thickening (p=0.04) than Balb/c controls, 28 days after surgery. Hypercholesterolemic APOE3*Leiden mice showed massive vein graft disease due to neointima formation and accelerated atherosclerosis after vein grafting. Local gene silencing of TLR4 led to a significant reduction of vessel wall thickening (38±10%, n=10, p=0.03) in the graft segment and superior graft patency (p=0.013) as compared to the control. In conclusion, the upregulation of TLR4 expression in huSV during remodelling, the reduced vein graft thickening in TLR deficient C3H/Hej mice and reduction of vessel wall thickening after local interfering in the TLR4 pathway indicate the potential of TLR4 as a local therapeutic target against vein graft disease to increase graft patency.

Potential role of the TLR4/IRAK-4 signaling pathway in the pathophysiology of acute pancreatitis in mice

Toll-like receptor 4 (TLR4) is potentially associated with acute pancreatitis (AP), but its exact role remains controversial. IL-1 receptor-associated kinase 4 (IRAK-4) is a common mediator of Toll-like receptors pathways, with an essential role in transducing downstream signals. This study investigates the potential role of the TLR4 pathway, in particular IRAK-4, in a murine model of AP. Acute pancreatitis was induced in wild-type and TLR4-deficient mice by intraperitoneal injections of caerulein (50 microg/kg). Pancreatic pathological scores and myeloperoxidase activity were dynamically measured, along with pancreatic TLR4 and IRAK-4 mRNA and protein. In wild-type mice, pathological scores and myeloperoxidase activity were rapidly increased at 1, 2 and 4 h, followed by alleviation at 12 and 24 h. In TLR4-deficient mice, they were slightly increased within 2 h, but became more severe at 12 and 24 h. IRAK-4 mRNA and protein were significantly down-regulated at 1, 2 and 4 h in wild-type mice. Unexpectedly, TLR4-deficient mice showed more profound reductions of IRAK-4 mRNA and protein at the same time. TLR4 deficiency delayed the initiation of pancreatitis, implying a potential role for TLR4 during AP. IRAK-4 might function during AP, but independently of TLR4.

Regulation of chemokine receptor by Toll-like receptor 2 is critical to neutrophil migration and resistance to polymicrobial sepsis

Patients with sepsis have a marked defect in neutrophil migration. Here we identify a key role of Toll-like receptor 2 (TLR2) in the regulation of neutrophil migration and resistance during polymicrobial sepsis. We found that the expression of the chemokine receptor CXCR2 was dramatically down-regulated in circulating neutrophils from WT mice with severe sepsis, which correlates with reduced chemotaxis to CXCL2 in vitro and impaired migration into an infectious focus in vivo. TLR2 deficiency prevented the down-regulation of CXCR2 and failure of neutrophil migration. Moreover, TLR2(-/-) mice exhibited higher bacterial clearance, lower serum inflammatory cytokines, and improved survival rate during severe sepsis compared with WT mice. In vitro, the TLR2 agonist lipoteichoic acid (LTA) down-regulated CXCR2 expression and markedly inhibited the neutrophil chemotaxis and actin polymerization induced by CXCL2. Moreover, neutrophils activated ex vivo by LTA and adoptively transferred into naïve WT recipient mice displayed a significantly reduced competence to migrate toward thioglycolate-induced peritonitis. Finally, LTA enhanced the expression of G protein-coupled receptor kinases 2 (GRK2) in neutrophils; increased expression of GRK2 was seen in blood neutrophils from WT mice, but not TLR2(-/-) mice, with severe sepsis. Our findings identify an unexpected detrimental role of TLR2 in polymicrobial sepsis and suggest that inhibition of TLR2 signaling may improve survival from sepsis.

The critical role of toll-like receptor (TLR) 4 in alcoholic liver disease is independent of the common TLR adapter MyD88.

The Toll-like receptor 4 (TLR4) that recognizes endotoxin, a trigger of inflammation in alcoholic liver disease (ALD), activates two signaling pathways utilizing different adapter molecules: the common TLR adapter, myeloid differentiation factor 88 (MyD88), or Toll/interleukin immune-response-domain-containing adaptor inducing interferon (IFN)-beta. The MyD88 pathway induces proinflammatory cytokine activation, a critical mediator of ALD. Here we evaluated the role of MyD88 in alcohol-induced liver injury in wild-type, TLR2-deficient, TLR4-deficient, or MyD88-deficient (knockout [KO]) mice after administration of the Lieber-De-Carli diet (4.5% volume/volume ethanol) or an isocaloric liquid control diet for 5 weeks. Alcohol feeding resulted in a significant increase in serum alanine aminotransferase levels, liver steatosis and triglyceride levels suggesting liver damage in WT, TLR2-KO, and MyD88-KO but not in TLR4-KO mice. Expression of inflammatory mediators (tumor necrosis factor-alpha and interleukin-6) and TLR4 coreceptors (CD14 and MD2) was significantly higher in livers of alcohol-fed WT, TLR2-KO, or MyD88-KO, but not in TLR4-KO mice, compared to controls. Reactive oxygen radicals produced by cytochrome P450 and the nicotinamide adenine dinucleotide phosphate complexes contribute to alcoholic liver damage. Alcohol feeding-induced expression and activation of cytochrome P450 and the nicotinamide adenine dinucleotide phosphate complex were prevented by TLR4-deficiency but not by MyD88-deficiency. Liver expression of interferon regulatory factor 3 (IRF3), a MyD88-independent signaling molecule, was not affected by chronic alcohol treatment in whole livers of WT mice or in any of the KO mice. However, the induction of IRF7, an IRF3-inducible gene, was found in Kupffer cells of alcohol-fed WT mice. Alcohol feeding also induced nuclear factor-kappaB activation in a TLR4-dependent MyD88-independent manner. While TLR4 deficiency was protective, MyD88 deficiency failed to prevent alcohol-induced liver damage and inflammation. These results suggest that the common TLR adapter, MyD88, is dispensable in TLR4-mediated liver injury in ALD.

Toll-Like Receptor 2 Acts as a Natural Innate Immune Receptor to Clear Amyloid β1–42and Delay the Cognitive Decline in a Mouse Model of Alzheimer's Disease

Microglia are the immune cells of the brain, they are activated in the brain of Alzheimer's disease (AD) patients and mouse models of AD, and they express the innate immune receptor toll-like receptor 2 (TLR2). The present study investigated role of this receptor in the progression of AD-like pathologies. Here we show that amyloid beta (A beta) stimulates TLR2 expression in a small proportion of microglia. We then generated triple transgenic mice that are deficient in TLR2 from mice that harbor a mutant human presenelin 1 and a chimeric mouse/human amyloid precursor protein (APP) genes. TLR2 deficiency accelerated spatial and contextual memory impairments, which correlated with increased levels of A beta(1-42) and transforming growth factor beta1 in the brain. NMDA receptors 1 and 2A expression levels were also lower in the hippocampus of APP-TLR2(-/-) mice. Gene therapy in cells of the bone marrow using lentivirus constructs expressing TLR2 rescued the cognitive impairment of APP-TLR2(-/-) mice. Indeed, lenti-green fluorescent protein/TLR2 treatment had beneficial effects by restoring the memory consolidation process disrupted by TLR2 deficiency in APP mice. These data suggest that TLR2 acts as an endogenous receptor for the clearance of toxic A beta by bone-marrow-derived immune cells. The cognitive decline is markedly accelerated in a context of TLR2 deficiency. Upregulating this innate immune receptor may then be considered as a potential new powerful therapeutic approach for AD.