Toll-like Receptor 4 Mutant Research Articles

Toll-like receptor 4 mutation protects the kidney from Ang-II-induced hypertensive injury

Cellular autophagy is a protective mechanism where cells degrade damaged organelles to maintain intracellular homeostasis. Apoptosis, on the other hand, is considered as programmed cell death. Interestingly, autophagy inhibits apoptosis by degrading apoptosis regulators. In hypertension, an imbalance of autophagy and apoptosis regulators can lead to renal injury and dysfunction. Previously, we have reported that toll-like receptor 4 (TLR4) mutant mice are protective against renal damage, in part, due to reduced oxidative stress and inflammation. However, the detailed mechanism remained elusive. In this study, we tested the hypothesis of whether TLR4 mutation reduces Ang-II-induced renal injury by inciting autophagy and suppressing apoptosis in the hypertensive kidney. Male mice with normal TLR4 expression (TLR4N, C3H/HeOuJ) and mutant TLR4 (TLR4M, C3H/HeJLps-d) aged 10–12 weeks were infused with Ang-II (1000 ng/kg/d) for 4 weeks to create hypertension. Saline infused appropriate control were used. Blood pressure was increased along with increased TLR4 expression in TLR4N mice receiving Ang-II compared to TLR4N control. Autophagy was downregulated, and apoptosis was upregulated in TLR4N mice treated with Ang-II. Also, kidney injury markers plasma lipocalin-2 (LCN2) and kidney injury molecule 1 (KIM-1) were upregulated in TLR4N mice treated with Ang-II. Besides, increased nuclear translocation and activity of NF-kB were measured in Ang-II-treated TLR4N mice. TLR4M mice remained protected against all these insults in hypertension. Together, these results suggest that Ang-II-induced TLR4 activation suppresses autophagy, induces apoptosis and kidney injury through in part by activating NF-kB signaling, and TLR4 mutation protects the kidney from Ang-II-induced hypertensive injury.

Differentiation expression of toll-like receptor4 (TLR4) caused by the dysregulation of microRNA-140-5p is responsible for the development of postoperation infection.

Toll-like receptor4 (TLR4) has proven to be an important factor that's responsible for the development of postoperation infection. MicroRNAs (miRNAs) are widely regarded as key mediators of gene expression. The objectives of our study were to identify miRNA(s) and the target genes differentially expressed in monocytes in the individuals with postoperation infection. MiRNA microarrays were performed to identify and compare miRNA expression in monocytes from those with or without postoperative infection. In-silico analysis was used to further investigate the target miRNAs and finally, luciferase assay and real-time polymerase chain reaction (PCR) were performed to confirm the target miRNA identified. Enzyme-linked immunosorbent assay, real-time PCR and Western-blot were performed to explore the role of miR-140 involved in postoperation infection. MiRNA microarray results showed that ten miRNAs were upregulated in the postoperation infection group, while six miRNAs were downregulated, compared with those in the postoperation group without infection. Computational analysis was further performed to reveal that four miRNAs (miR-140, miR-7, miR-448, and miR-217) targeted the 3'-untranslated region (UTR) of TLR4 mRNA. The luciferase assay showed that only miR-140 inhibited luciferase activity of wild-type TLR4 3'-UTR and the luciferase activity of the cells cotransfected with miR-7, miR-448 or miR-217 and wild-type or mutant TLR4 3'-UTR was comparable with the control. Furthermore, only miR-140 levels were significantly lower in the postoperation infection group, while levels of miR-217, miR-7, and miR-448 showed no obvious difference between the postoperation infection and postoperation without infection groups. TLR4, tumor necrosis factor-α (TNF-α), and IL-6 levels were much higher in the postoperation infection group. In comparison with the control group, TLR4, TNF-α and Interleukin 6 (IL-6) levels in cells were decreased following transfection with miR-140 mimics and TLR4 small interfering RNA. However, the cells treated with lipopolysaccharides increased TLR4, TNF-α, and IL-6 levels. This study demonstrates that miR-140 is differentially expressed in monocytes collected from patients diagnosed with postoperation infection. The downregulation of miR-140 cause upregulation of toll-like receptor4 (TLR4), a proinflammatory factor, and is associated with infection risk in patients received surgery.

A896G and C1196T Polymorphisms Within the TLR4 Gene Abate Toll-Like Receptor 4-Mediated Signaling in HepG2 Cells.

Toll-like receptor 4 (TLR4) appears to play an important role in the development and progression of hepatocellular carcinoma (HCC), but it is unclear whether single-nucleotide polymorphisms (SNPs) in the TLR4 gene influence HCC. In this study, we investigated the effects of TLR4 SNPs on HepG2 cell survival and proliferation, migration, and invasion. Plasmids carrying wild-type or mutant versions of the TLR4 gene (A896G and/or C1196T) were stably transfected into HepG2 cells, and cell viability and proliferation were analyzed using the Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays, whereas apoptosis was assessed using flow cytometry. Migration and invasion were measured in a transwell chamber assay, and expression of inflammatory cytokines and downstream effectors was examined using real-time PCR and western blotting. Specific inhibitors of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), or phosphatidylinositol 3-kinase (PI3K) were added to the HepG2 cultures to explore the potential role of each pathway in TLR4 signaling. TLR4 SNPs did not affect expression levels in transfected cells. Compared with wild-type TLR4, mutant TLR4 was associated with lower cell proliferation, migration, invasion, and apoptotic threshold. In addition, the mutations were associated with significantly lower expression of nuclear factor κB (NF-κB), IL-6, and TGF-β1, even after stimulation with lipopolysaccharide. The expression of p-Akt was similar in the presence of wild-type or mutant TLR4. The 896G and 1196T SNPs in the TLR4 gene are associated with reduced TLR4-mediated signaling and, therefore, with lower survival, proliferation, and metastasis in HepG2 cells.

Down-regulation of toll-like receptor 4 alleviates intestinal ischemia reperfusion injury and acute lung injury in mice.

Intestinal ischemia reperfusion (IR) injury is a critical problem, which can cause intestinal injury locally and acute lung injury (ALI) distally by inflammatory responses and oxidative stress. Toll-like receptor 4 (TLR4) is involved in innate immune and inflammatory responses. This study was to determine whether TLR4 mutant can attenuate intestinal and lung injuries after intestinal IR. Wild type (WT) and TLR4 mutant mice were submitted to intestinal IR by occluding the superior mesenteric artery. Histological assessment of the intestine and the lung were conducted by HE staining. The levels of proinflammatory cytokines, oxidative stress markers, apoptotic index and other mediators were measured. In addition, a 24-hour survival study was performed. Histological assessment showed that intestinal IR caused serious injuries in the intestine and the lung, corroborated by increased proinflammatory cytokines in the circulation. TLR4 mutant suppressed the histological injuries as demonstrated by significantly decreased pathological scores. Consistent with the morphological results, the TLR4 mutant mice exhibited remarkably lowered cytokine expressions in the intestine (TNF-α, IL-6, IL-1β, and NF-κB) and the lung (NO, iNOS, MCP-1, MIP-2, NF-κB, and Caspase-3). ALT and creatinine were also significantly dampened in the liver and kidney, respectively. Furthermore, the survival rate over the course of 24 hours was significantly improved. Collectively, the findings reveal that TLR4 mutant significantly abated the intestinal IR injury and ALI at least in part by alleviating the inflammatory response and oxidative stress.

PXR mediated cardiac protection after sepsis through TLR4 modulation pathway

Cardiac dysfunction is a vital component of multi-organ failure during severe sepsis. The primary function of pregnane X receptor (PXR) is to sense the presence of foreign toxic substances and in response up regulate the expression of proteins involved in the detoxification and clearance of these substances from the body. Further, Toll-like receptor 4 (TLR4), the signal-transducing molecule of the LPS receptor complex, plays a fundamental role in the sensing of LPS from Gram-negative bacteria. Activation of TLR4 signaling pathways by LPS is a critical upstream event in the pathogenesis of Gram-negative sepsis, making TLR4 an attractive target for novel antisepsis therapy. TLR4 mutant (TLR4-/-) and wild type TLR4 (TLR4+/+) mice underwent a sterile (lipopolysaccharide; LPS) or infectious (Streptococcus pneumoniae or Klebsiella pneumoniae) septic challenge. Production of cytokines, TNF, IL-1β, IL-6 and IL-10, in the blood and from cardiomyocytes was exaggerated in the TLR4+/+ mice compared to responses measured in mutant TLR4-/- type mice given an identical septic challenge. This enhanced compartmentalized myocardial inflammation was associated with significantly decreased cardiac contraction and diminished relaxation in the TLR4+/+ mice. Furthermore, PXR expression has critical role in down-regulation of sepsis via TLR4 pathway, and more reduction in cytokines, improved myocardial function in PXR+/+suggesting that PXR was a major cause of the greater myocardial contractile improvement in TLR4-/- mice. Taken together, our results suggest that PXR plays a role in the down-regulation of myocardium during sepsis. Further studies are warranted to further define the precise mechanisms of PXR mediated cardiac protection.

LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis.

Colorectal carcinoma (CRC) is characterized by unlimited proliferation and suppression of apoptosis, selective advantages for tumor survival, and chemoresistance. Lipopolysaccharide (LPS) signaling is involved in both epithelial homeostasis and tumorigenesis, but the relative roles had by LPS receptor subunits CD14 and Toll-like receptor 4 (TLR4) are poorly understood. Our study showed that normal human colonocytes were CD14(+)TLR4(-), whereas cancerous tissues were CD14(+)TLR4(+), by immunofluorescent staining. Using a chemical-induced CRC model, increased epithelial apoptosis and decreased tumor multiplicity and sizes were observed in TLR4-mutant mice compared with wild-type (WT) mice with CD14(+)TLR4(+) colonocytes. WT mice intracolonically administered a TLR4 antagonist displayed tumor reduction associated with enhanced apoptosis in cancerous tissues. Mucosa-associated LPS content was elevated in response to CRC induction. Epithelial apoptosis induced by LPS hypersensitivity in TLR4-mutant mice was prevented by intracolonic administration of neutralizing anti-CD14. Moreover, LPS-induced apoptosis was observed in primary colonic organoid cultures derived from TLR4 mutant but not WT murine crypts. Gene silencing of TLR4 increased cell apoptosis in WT organoids, whereas knockdown of CD14 ablated cell death in TLR4-mutant organoids. In vitro studies showed that LPS challenge caused apoptosis in Caco-2 cells (CD14(+)TLR4(-)) in a CD14-, phosphatidylcholine-specific phospholipase C-, sphingomyelinase-, and protein kinase C-ζ-dependent manner. Conversely, expression of functional but not mutant TLR4 (Asp299Gly, Thr399Ile, and Pro714His) rescued cells from LPS/CD14-induced apoptosis. In summary, CD14-mediated lipid signaling induced epithelial apoptosis, whereas TLR4 antagonistically promoted cell survival and cancer development. Our findings indicate that dysfunction in the CD14/TLR4 antagonism may contribute to normal epithelial transition to carcinogenesis, and provide novel strategies for intervention against colorectal cancer.

TLR4-dependent immune response promotes radiation-induced liver disease by changing the liver tissue interstitial microenvironment during liver cancer radiotherapy.

Liver tissue interstitial fluid (TIF) a special microenvironment around liver cells, which may play a vital role in cell communication during liver injury. Moreover, toll-like receptor 4 (TLR4) is an important trigger of the immune response that may also play a role in liver injuries, including radiation-induced liver disease (RILD). Therefore, the purpose of this study was to identify the roles of the TLR4-dependent immune response and TIFs in RILD after radiation therapy (RT) for liver cancer. This study consisted of two phases, and in the primary phase, the livers of TLR4 mutant (TLR4(-)) and normal (TLR4(+)) mice were irradiated with 30 Gy. TIF was then obtained from mouse livers and assessed by cytokine array analysis 20 days after irradiation, and cytokines in the TIFs, TLR4 and RILD were analyzed. In the second or validation phase, hepatocytes were isolated from TLR4(+) or TLR4(-) mice irradiated with 8 Gy and were co-cultured with TIFs from mouse livers, apoptosis of the hepatocytes was then measured using flow cytometry. We found that severe RILD was accompanied by higher expression of granulocyte macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and vascular endothelial growth factor receptor 2(VEGFR-2) in liver TIFs, from in TLR4(+) mice compared with TLR4(-) mice (P < 0.05). In both TLR4(+) and TLR4(-) hepatocytes, apoptosis after irradiaton was increased significantly after co-culture in TIFs from TLR4(+) mice that had their livers irradiated, compared with TIFs from TLR4(-) mice that had their livers irradiated or TIFs from unirradiated mice (P < 0.05). In summary, these findings indicate that the TLR4-dependent immune response may promote RILD by enhancing the expression of GM-CSF, VEGFR-2 and TRAIL in liver TIFs.

Toll-like receptor-4 signalling in the progression of non-alcoholic fatty liver disease induced by high-fat and high-fructose diet in mice.

The aim of the present study was to investigate Toll-like receptor-4 (TLR4) signalling at different stages of non-alcoholic fatty liver disease (NAFLD) induced by a high-fat, high-fructose (HFHFr) diet in mice. Both TLR4 wild-type (WT) and mutant (TLR4(mut) ) mice were fed either standard chow (SC) or the HFHFr diet for different periods of time from 4 to 16 weeks. Pathological characteristics and function of the liver were assessed. Simple steatosis, steatohepatitis and hepatic fibrosis occurred sequentially in Week 4, 8 and 16 in WT mice fed with the HFHFr. Expression of TLR4, myeloid differentiation factor 88 (MyD88), interferon regulatory factor (IRF) 3 and IRF7 started to increase at Week 4, peaked at Week 8 and then declined to basal levels at Week 16. This pattern was consistent with changes in inflammation in the liver revealed by haematoxylin and eosin staining. However, lipid accumulation, inflammation and fibrosis in livers of TLR4(mut) mice fed the HFHFr diet were significantly alleviated. In addition, the expression of activin A in WT mice fed the HFHFr diet increased at Week 16. The data suggest that TLR4 signalling mediates non-alcoholic steatohepatitis before fibrosis and that activin A is subsequently involved in NAFLD.

Toll-like receptor 4 mutant and null mice retain morphine-induced tolerance, hyperalgesia, and physical dependence.

The innate immune system modulates opioid-induced effects within the central nervous system and one target that has received considerable attention is the toll-like receptor 4 (TLR4). Here, we examined the contribution of TLR4 in the development of morphine tolerance, hyperalgesia, and physical dependence in two inbred mouse strains: C3H/HeJ mice which have a dominant negative point mutation in the Tlr4 gene rendering the receptor non-functional, and B10ScNJ mice which are TLR4 null mutants. We found that neither acute antinociceptive response to a single dose of morphine, nor the development of analgesic tolerance to repeated morphine treatment, was affected by TLR4 genotype. Likewise, opioid induced hyperalgesia and opioid physical dependence (assessed by naloxone precipitated withdrawal) were not altered in TLR4 mutant or null mice. We also examined the behavioural consequence of two stereoisomers of naloxone: (−) naloxone, an opioid receptor antagonist, and (+) naloxone, a purported antagonist of TLR4. Both stereoisomers of naloxone suppressed opioid induced hyperalgesia in wild-type control, TLR4 mutant, and TLR4 null mice. Collectively, our data suggest that TLR4 is not required for opioid-induced analgesic tolerance, hyperalgesia, or physical dependence.

Impact of toll-like receptor 4 deficiency on cerebrocardiac syndrome

In order to investigate the role of Toll-like receptor 4 (TLR4) in cerebrocardiac syndrome (CCS), the partial cerebral ischemia/reperfusion (I/R) models in mice with different TLR4 genotypes were established in the present study. TLR4 wild-type (C3H/HeN) and mutant (C3H/HeJ) mice of 6-8 weeks of age were divided into 4 groups at random: C3H/HeN sham group (n=10), C3H/HeJ sham group (n=10), C3H/HeN model group (n=10) and C3H/HeJ model group (n=10). Partial cerebral I/R was caused by the middle cerebral artery occlusion (MCAO) to duplicate CCS models in mice. After the operation, the electrocardiogram (ECG), the level of tumor necrosis factor-alpha (TNF-α) in myocardial tissue and the cardiac pathological changes were observed in each group. It was shown that the brain infarct volume in C3H/HeN model group was larger than that in C3H/HeJ model group (P<0.01). The ST segment change and T wave inversion occurred frequently in model groups. Moreover, the TNF-α level in C3H/HeN model group was higher than that in C3H/HeJ model group (P<0.01). The myocardial injury was aggravated in C3H/HeN group as compared with C3H/HeJ group. It was concluded that TLR4 was implicated in the development of CCS.

Lipopolysaccharide Induces Inflammatory Hyperalgesia Triggering a TLR4/MyD88-Dependent Cytokine Cascade in the Mice Paw

Inflammatory pain can be triggered by different stimuli, such as trauma, radiation, antigen and infection. In a model of inflammatory pain caused by infection, injection in the mice paw of lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) agonist, produces mechanical hyperalgesia. We identify here the TLR4 linked signaling pathways that elicit this response. Firstly, LPS paw injection in wild type (WT) mice produced mechanical hyperalgesia that was not altered in TRIF-/- mice. On the other hand, this response was absent in TLR4 mutant and MyD88 null mice and reduced in TNFR1 null mice. Either an IL-1 receptor antagonist, anti-KC/CXCL1 antibody, indomethacin or guanethidine injection also lessened this response. Moreover, LPS-induced time dependent increases in TNF-α, KC/CXCL1 and IL-1β expression in the mice paw, which were absent in TLR4 mutant and MyD88 null mice. Furthermore, in TNFR1 deficient mice, the LPS-induced rises in KC/CXCL1 and IL-1β release were less than in their wild type counterpart. LPS also induced increase of myeloperoxidase activity in the paw skin, which was inhibited in TLR4 mutant and MyD88 null mice, and not altered in TRIF-/- mice. These results suggest that LPS-induced inflammatory pain in mice is solely dependent on the TLR4/MyD88 rather than the TLR4/TRIF signaling pathway. This pathway triggers pronociceptive cytokine TNF-α release that in turn mediates rises in KC/CXCL1 and IL-1β expression. Finally, these cytokines might be involved in stimulating production of directly-acting hyperalgesic mediators such as prostaglandins and sympathomimetic amine.

Role of innate immunity and altered intestinal motility in LPS- and MnCl2-induced intestinal intussusception in mice

Intestinal intussusception (ISS) commonly causes intestinal obstruction in children. One mechanism that has been proposed to cause ISS is inflammation-induced alteration of intestinal motility. We investigated whether innate inflammatory factors or altered motility is required for induction of ISS by LPS. We compared rates of ISS among BALB/c and C57BL/6 mice, mice lacking lymphocytes or depleted of phagocytes, or mice with defects in the Toll-like receptor 4 (TLR4) signaling pathway following administration of LPS or the Ca(2+) analog MnCl2. At 6 or 2 h after administration of LPS or MnCl2, respectively, mice underwent image analysis to assess intestinal contraction rate or laparotomy to identify ISS. LPS-induced ISS (LPS-ISS) was observed in BALB/c mice, but not in C57BL/6 mice or any BALB/c mice with disruptions of TLR4 signaling. LPS-induced serum TNF-α, IL-6, and nitric oxide (NO) and intestinal NO levels were similar in BALB/c and C57BL/6 mice. The rate of LPS-ISS was significantly reduced in phagocyte-depleted, but not lymphocyte-deficient, mice. Intestinal contraction rates were reduced in LPS-ISS-susceptible BALB/c mice, but not in LPS-ISS-resistant C57BL/6 or TLR4 mutant mice, suggesting a role for reduced intestinal contraction rate in LPS-ISS susceptibility. This was tested with MnCl2, a Ca(2+) antagonist that reduced intestinal contraction rates and induced ISS, irrespective of mouse strain. Therefore, LPS-ISS is initiated by innate immune signaling that requires TLR4 and phagocytes but may be independent of TNF-α, IL-6, and NO levels. Furthermore, alteration of intestinal motility, specifically, reduced intestinal contraction rate, is a key factor in the development of ISS.

TLR4 signaling and the inhibition of liver hepcidin expression by alcohol.

To understand the role of toll-like receptor 4 (TLR4) signaling in the regulation of iron-regulatory hormone, hepcidin by chronic alcohol consumption. For chronic alcohol intake studies, TLR4 mutant mice on C3H/HeJ background and wildtype counterpart on C3H/HeOuJ background were pair-fed with regular (control) and ethanol-containing Lieber De Carli liquids diets. Gene expression was determined by real-time quantitative PCR. Protein-protein interactions and protein expression were determined by co-immunoprecipitation and western blotting. The occupancy of hepcidin gene promoter was determined by chromatin immunoprecipitation assays. Chronic alcohol intake suppressed hepcidin mRNA expression in the livers of wildtype, but not TLR4 mutant, mice. The phosphorylation and nuclear translocation of nuclear factor (NF)-κB p65 subunit protein was observed in alcohol-fed wildtype, but not in alcohol-fed TLR4 mutant, mice. Similarly, alcohol induced the binding of NF-κB p50 subunit protein to hepcidin gene promoter in wildtype, but not in TLR4 mutant, mice. In contrast, the phosphorylation of Stat3 in the liver was stronger in alcohol-treated TLR4 mutant mice compared to alcohol-treated wildtype mice. The occupancy of hepcidin gene promoter by Stat3 was observed in alcohol-fed mutant, but not in wildtype, mice. An interaction between NF-κB p65 subunit protein and small heterodimer partner protein (SHP) was observed in the livers of both wildtype and TLR4 mutant mice fed with the control diet, as shown by co-immunoprecipitation studies. Alcohol intake elevated cytosolic SHP expression but attenuated its interaction with NF-κB in the liver, which was more prominent in the livers of wildtype compared to TLR4 mutant mice. Activation of TLR4 signaling and NF-кB are involved in the suppression of hepcidin gene transcription by alcohol in the presence of inflammation in the liver.

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.

TLR4-dependant immune response, but not hepatitis B virus reactivation, is important in radiation-induced liver disease of liver cancer radiotherapy.

Toll-like receptor 4 (TLR4) is an important trigger of the immune response against hepatitis B virus (HBV) infection and liver injuries. The roles of HBV reactivation versus TLR4-dependant immune response may be critical factors in preventing radiation-induced liver diseases (RILDs) after liver cancer radiotherapy. This study consists of three phases. In the primary phase, livers of mutant TLR4 (TLR4(-)) mice were irradiated with 30 Gy in either the absence or presence of HBV infection. The latter was done by introduction of plasmid pAAV/HBV 1.2. In the advanced phase, RILDs were compared in normal TLR4 (TLR4(+)) versus TLR4(-) mice. In the validation phase, 28 liver cancer patients who had undergone radiotherapy before hepatectomy were enrolled. Liver biopsies near tumors, irradiated with 35-48 Gy, were used to construct tissue microarrays. HBV reactivation, TLR4 expression, and severity of RILDs were studied in both mouse and human. More HBV reactivation, without significant RILD, was observed in irradiated versus unirradiated TLR4(-) mice. RILD scores of TLR4(+) mice were higher than TLR4(-) mice. In humans, serious RILDs tended to develop in patients with high TLR4 expression, but not in patients with low TLR4 or high HBV surface antigen expression. High TLR4 expression was seen in only 2 of 12 HBV-reactive patients, but in HBV-nonreactive patients, it was seen in 6 of 9 (P < 0.03). In summary, RILDs correlated with high TLR4 expression, but not with HBV reactivation, which is inhibited in liver with high TLR4 expression after liver cancer radiotherapy.

Bone Marrow and Nonbone Marrow Toll Like Receptor 4 Regulate Acute Hepatic Injury Induced by Endotoxemia

BackgroundToll-like receptors (TLRs) are expressed in immune cells and hepatocytes. We examined whether hepatic Toll-like receptor 4 (TLR4) is involved in the acute hepatic injury caused by the administration of lipopolysaccharide (LPS) (septic shock model).MethodsWild type (WT), TLR4-deficient and chimera mice underwent myeloablative bone marrow transplantation to dissociate between TLR4 expression in the liver or in the immune-hematopoietic system. Mice were injected with LPS and sacrificed 4 hours later.ResultsCompared to TLR4 deficient mice, WT mice challenged with LPS displayed increased serum liver enzymes and hepatic cellular inflammatory infiltrate together with increased serum and hepatic levels of interleukin 1β (IL-1β), tumor necrosis factor α (TNFα) ,Up-regulation of hepatic mRNA encoding TLR4, IκB and c-jun expressions. TLR4 mutant mice transplanted with WT bone marrow were more protected than WT chimeric mice bearing TLR4 mutant hemopoietic cells from LPS, as seen by IL-1β and TNFα levels. We then used hepatocytes (Huh7) and macrophages from monocytic cell lines to detect TLR mRNA expression. Macrophages expressed a significantly higher level of TLR4 mRNA and TLR2 (more than 3000- and 8000-fold respectively) compared with the hepatocyte cell line. LPS administration induced TLR4 activation in a hepatocyte cell line in a dose dependent manner while TLR2 mRNA hardly changed.ConclusionsThese results suggest that TLR4 activation of hepatocytes participate in the immediate response to LPS induced hepatic injury. However, in this response, the contribution of TLR4 on bone marrow derived cells is more significant than those of the hepatocytes. The absence of the TLR4 gene plays a pivotal role in reducing hepatic LPS induced injury.

Toll-like receptor 4 activity protects against hepatocellular tumorigenesis and progression by regulating expression of DNA repair protein Ku70 in mice

Hepatocellular carcinoma (HCC) is a devastating consequence of chronic inflammatory liver diseases. The goal of this study was to investigate whether Toll-like receptor 4 (TLR4) activity contributes to HCC initiation and progression in mice. A mouse model of diethylnitrosamine (DEN)-induced HCC was generated with wild-type and TLR4 mutant mice, and the development and progression of HCC and senescent responses were assessed using morphologic, immunological, and biochemical criteria. We found that genetic or pharmacologic blocking of TLR4 increased susceptibility to DEN-induced HCC carcinogenesis and progression, which was indicated by increases in number of tumor nodules, tumor volume, and animal death. The enhanced HCC was associated with a broad-spectrum reduction of immune response to DEN liver injury, as indicated by decreases in the liver-infiltrating F4/80+ macrophages, the apoptosis signal-regulating kinase 1/p38 mitogen-activated protein kinase/NF-κB and IRF3 signaling activities, and the expression of inflammatory cytokines. Suppressed immune networks resulted in a halt of cellular senescence induction in TLR4 mutant liver tissue, which promoted proliferation and suppressed programmed cell death. Moreover, TLR4 mutation resulted in a suppressed capacity of DNA repair due to a decrease in TLR4-medicated expression of DNA repair proteins Ku70/80 in liver tissue and cells. Isotopic expression of Ku70 in TLR4 mutant mice restored senescence and interrupted the positive feedback loop of DNA damage and oxidative stress, which reversed TLR4 mutation-deteriorated HCC carcinogenesis and progression. TLR4 plays an integrated defense role against HCC carcinogenesis by enhancing the expression and function of DNA repair protein Ku70. Our studies provide novel insight into TLR4 activity in the regulation of HCC tumorigenesis, which may be useful for the prevention of HCC development.

Structural Analyses of Human Toll-like Receptor 4 Polymorphisms D299G and T399I

TLR4 polymorphism replacing Asp-299 with Gly and Thr-399 with Ile (D299G/T399I) causes LPS hyporesponsiveness. TLR4(SNPs)·MD-2·LPS exhibits an agonistic 2:2:2 architecture. Local structural differences were observed around D299G, but not around T399I, SNP site. These local differences cause the modulation of surface properties of TLR4, which may affect ligand binding. This study provides structural evidence of the functionality of the mutant TLR4 carrying the SNPs. Toll-like receptor 4 (TLR4) and its coreceptor MD-2 recognize bacterial lipopolysaccharide (LPS) and signal the innate immune response. Two single nucleotide polymorphisms (SNPs) of human TLR4, D299G and T399I, have been identified and suggested to be associated with LPS hyporesponsiveness. Moreover, the SNPs have been proposed to be associated with a variety of infectious and noninfectious diseases. However, how the SNPs affect the function of TLR4 remains largely unknown. Here, we report the crystal structure of the human TLR4 (D299G/T399I)·MD-2·LPS complex at 2.4 Å resolution. The ternary complex exhibited an agonistic "m"-shaped 2:2:2 architecture that was similar to that of the human wild type TLR4·MD-2·LPS complex. Local structural differences that might affect the binding of the ligands were observed around D299G, but not around T399I, SNP site.

Distinct responses of lung and liver macrophages to acute endotoxemia: Role of toll-like receptor 4

Exposure to excessive quantities of bacterial-derived lipopolysaccharide (LPS) is associated with injury to the lung and the liver. Macrophages are thought to play a key role in the pathogenic response to LPS by releasing proinflammatory/cytotoxic mediators. Macrophage responses to LPS are mediated in large part by toll-like receptor 4 (TLR4). In the present studies we used C3H/HeJ mice, which possess a mutated nonfunctional TLR4, to examine its role in lung and liver macrophage responses to acute endotoxemia induced by LPS administration. Treatment of control C3H/HeOuJ mice with LPS (3mg/ml, i.p.) was associated with a significant increase in the number of macrophages in both the lung and the liver. This was most prominent after 48h, and was preceded by expression of proliferating cell nuclear antigen (PCNA), suggesting that macrophage proliferation contributes to the response. In liver, but not lung macrophages, LPS administration resulted in a rapid (within 3h) increase in mRNA expression of Mn superoxide dismutase (SOD) and heme oxygenase-1 (HO-1), key enzymes in antioxidant defense. In contrast, HO-1 protein expression decreased 3h after LPS administration in liver macrophages, while in lung macrophages it increased. mRNA expression of enzymes mediating the biosynthesis of eicosanoids, including cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1), but not 12/15-lipoxygenase (LOX), was upregulated in liver macrophages 3–24h after LPS, with no effect on lung macrophages. However, COX-2 protein expression increased in both cell types. Loss of functional TLR4 significantly blunted the effects of LPS. Thus, no major changes were observed after LPS administration in the number of lung and liver macrophages recovered from TLR4 mutant mice, or on expression of PCNA. Increases in HO-1, MnSOD, COX-2 and PGES-1 mRNA expression in liver macrophages were also reduced in these mice. Conversely, in lung macrophages, loss of functional TLR4 resulted in increased expression of COX-2 protein and 12/15-LOX mRNA. These results demonstrate distinct lung and liver macrophage responses to acute endotoxemia are mediated, in part, by functional TLR4.

TLR-4-Dependent and -Independent Mechanisms of Fetal Brain Injury in the Setting of Preterm Birth

In this study, we sought to assess how essential activation of toll-like receptor 4 (TLR-4) is to fetal brain injury from intrauterine inflammation. Both wild-type and TLR-4 mutant fetal central nervous system cells were exposed to inflammation using lipopolysaccharide in vivo or in vitro. Inflammation could not induce neuronal injury in the absence of glial cells, in either wild-type or TLR-4 mutant neurons. However, injured neurons could induce injury in other neurons regardless of TLR-4 competency. Our results indicate that initiation of neuronal injury is a TLR-4-dependent event, while propagation is a TLR-4-independent event.