Toll-like Receptor 4 Mutant Mice Research Articles

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.

Improved Survival and Neurological Outcomes after Cardiopulmonary Resuscitation in Toll-like Receptor 4-mutant Mice

Background:Toll-like receptor 4 (TLR4) is a crucial receptor in the innate immune system and noninfectious immune responses. It has been reported that TLR4 participates in the pathological course of ischemia/reperfusion (I/R) injury. However, the role of TLR4 in the process of I/R injury after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) is still unknown. In this study, we investigated the effects of TLR4 mutation on survival and neurological outcome in a mouse model of CA/CPR.Methods:A model of potassium-induced CA was performed on TLR4-mutant mice (C3H/HeJ) and wild-type mice (C3H/HeN). After 3 min of untreated CA, resuscitation was attempted with chest compression, ventilation, and intravenous epinephrine. Behavioral tests were performed on mice on day 3 after CPR. The morphological changes in hippocampal neurons were assessed by light and electron microscopy. Expressions of TLR4 and intercellular adhesion molecule-1 (ICAM-1) were detected by Western blot. Levels of tumor necrosis factor-α (TNF-α) and myeloperoxidase (MPO) were measured with enzyme-linked immunosorbent assay (ELISA).Results:On day 3 after resuscitation the overall mortality was 33.33% in C3H/HeJ group compared with 53.33% in C3H/HeN group (P < 0.05). And there was much higher central tendency in C3H/HeJ group than C3H/HeN group during open field test (P < 0.05). Meanwhile, the percentage of nonviable neurons was 21.16% in C3H/HeJ group compared with 53.11% in C3H/HeN group (P < 0.05). And there were significantly lower levels of hippocampal TNF-α and MPO in C3H/HeJ mice (TNF-α: 6.85±1.19 ng/mL, MPO: 0.33±0.11 U/g) than C3H/HeN mice (TNF-α: 11.36±2.12 ng/mL, MPO: 0.54±0.17 U/g) (all P < 0.01). CPR also significantly increased the expressions of TLR4 and ICAM-1 in C3H/HeN group. However, the expression of ICAM-1 was much lower in C3H/HeJ group than in C3H/HeN group after CPR (P < 0.01).Conclusion:TLR4 signaling is involved in brain damage and in inflammation triggered by CA/CPR.

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 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.

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.

Reduced hind limb ischemia-reperfusion injury in Toll-like receptor-4 mutant mice is associated with decreased neutrophil extracellular traps

Ischemia-reperfusion (IR) injury is a significant problem in the management of patients with acute limb ischemia. Despite rapid restoration of blood flow after technically successful open and endovascular revascularization, complications secondary to IR injury continue to occur and limit clinical success. Our aim was to create a murine model of hind limb IR injury to examine the role of Toll-like receptor-4 (TLR4) and to determine whether inactive TLR4 led to a decrease in the detection of neutrophil extracellular traps (NETs), which are known to be highly thrombogenic and may mediate microvascular injury. A calibrated tension tourniquet was applied to unilateral hind limb of wild-type (WT) and TLR4 receptor mutant (TLR4m) mice for 1.5 hours to induce ischemia and then removed to initiate reperfusion. At the end of 48 hours of reperfusion, mice were euthanized and hind limb tissue and serum specimens were collected for analysis. Hematoxylin and eosin-stained sections of hind limb skeletal muscle tissue were examined for fiber injury. For immunohistochemistry, mouse monoclonal antihistone H2A/H2B/DNA complex antibody to detect NETs and rabbit polyclonal antimyeloperoxidase antibody were used to identify infiltrating cells containing myeloperoxidase. Muscle adenosine triphosphate levels, nuclear factor (NF)-κB activity, the α-subunit of inhibitor of NF-κB light polypeptide gene enhancer, poly (adenosine diphosphate-ribose) polymerase activity, and inducible nitric oxide synthase expression were measured. Systemic levels of keratinocyte-derived chemokine, monocyte chemotactic protein-1, and vascular endothelial growth factor in the serum samples were also examined. IR injury in the hind limb of WT mice demonstrated significant levels of muscle fiber injury, decreased energy substrates, increased NF-κB activation, decreased levels of α-subunit of inhibitor of NF-κB light polypeptide gene enhancer, increased inducible nitric oxide synthase expression, and increased poly (adenosine diphosphate-ribose) polymerase activity levels compared with the TLR4m samples. Additionally, there was marked decrease in the level of neutrophil and monocyte infiltration in the TLR4m mice, which corresponded to similar levels of decreased NET detection in the interstitial space and in microvascular thrombi. In situ nuclease treatment of WT tissue sections significantly diminished the level of NET immunostaining, demonstrating the specificity of the antibody to detect NETs and suggesting a potential role for nuclease treatment in IR injury. These results suggest a pivotal role for TLR4 in mediating hind limb IR injury and suggest that NETs may contribute to muscle fiber 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.

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.

Chandipura Virus infection in mice: the role of toll like receptor 4 in pathogenesis

BackgroundThe susceptibility of mice and humans to Chandipura virus infection is age-dependent. Upon experimental infection, mice secrete significant amounts of proinflammatory cytokines. Similarly, children who recover from natural infection with the virus show significant amounts of TNF-α production, suggesting that innate immunity plays a major role in the response to Chandipura virus. Toll-like receptors (TLR) are key host molecules involved in innate immune responses in infections. Therefore, the aim of this study was to examine the role of TLR in the response to Chandipura virus infection.MethodsThe mouse monocyte-macrophage cell line, RAW 264.7, and C3H/HeJ mice were used as models. Micro array techniques were used to identify the type of TLR involved in the response to infection. The results were validated by examining TLR expression using flow cytometry and by measuring the levels of proinflammatory cytokines and nitric oxide (NO) in the culture supernatants using bead assays and the Griess method, respectively. The pathogenic role of Toll-like receptor 4 (TLR4) was studied in a TLR4 mutant strain of mice -C3H/HeJ and the results compared with those from wild-type mice- C3H/CaJ. The pathogenic effects of NO were studied by treating experimentally infected mice with the NO inhibitor, aminoguanidine (AG).ResultsThe micro array results showed that TLR4 was regulated after Chandipura virus infection. At high multiplicities of infection (10 MOI), RAW cells up- regulated cell surface expression of TLR4 and secreted significant amounts of TNF-α, MCP-1, IL-10 and IL-12 and NO. The survival rate of C3H/HeJ mice was higher than those of wild-type C3H/CaJ mice. The survived C3H/HeJ mice secreted significant quantity of MCP-1 and IFN-γ cytokines and cleared virus from brain. Similarly, the survival rate of AG-treated mice was higher than those of the untreated controls.ConclusionsChandipura virus regulates TLR4, which leads to the secretion of proinflammatory cytokines and NO by infected RAW cells. Difference in survival rate in TLR4 mutant mice and nitric oxide inhibitor treated mice, confirmed the role of these molecules in disease pathogenesis. The result is significant in clinical management and designing antiviral intervention for Chandipura virus infection.

Acinetobacter baumannii virulence requires LPS activation of TLR4 (55.12)

Abstract Background: New treatments are needed for lethal infections caused by extreme-drug resistant (XDR)/pan-drug resistant (PDR) Acinetobacter baumannii. Toll-like receptor 4 (TLR4) was previously reported to enhance bacterial clearance during non-lethal A. baumannii pulmonary infection in mice. We sought to better to understand the role of LPS-TLR4 interactions in A. baumannii pathogenesis. Methods: LPS was harvested from nine clinical isolates of A. baumannii and then assayed for TLR4 activity using the HEK-Blue cells. Wild type and TLR4 mutant mice were infected via the tail-vein with A. baumannii. Results: After A. baumannii systemic infection, 100% of wild type vs. 0% of TLR4-mutant mice died. Wild type mice developed septic shock, typified by hypothermia, acidemia, and elevated inflammatory cytokine production, wherease TLR4 mutant mice did not, despite having similar bacterial burden in organs. Density of LPS in bacteria (ng/bacillus) and TLR4 activation by LPS extracted from the bacteria did not correlate with in vivo virulence. Amount of LPS shed during growth was the key correlate with in vivo virulence. Summary: LPS activation of TLR4 is a key pathogenetic factor of A. baumannii during systemic infection. Abrogation of TLR4 function protects mice from invasive A. baumannii infection by preventing induction of septic shock. These results suggest new areas of focus for developing novel therapeutics targeting these XDR/PDR infections.

Intestinal decontamination inhibits TLR4 dependent fibronectin-mediated cross-talk between stellate cells and endothelial cells in liver fibrosis in mice

Liver fibrosis is associated with angiogenesis and leads to portal hypertension. Certain antibiotics reduce complications of liver failure in humans, however, the effects of antibiotics on the pathologic alterations of the disease are not fully understood. The aim of this study was to test whether the non-absorbable antibiotic rifaximin could attenuate fibrosis progression and portal hypertension in vivo, and explore potential mechanisms in vitro. The effect of rifaximin on portal pressure, fibrosis, and angiogenesis was examined in wild type and Toll-like receptor 4 (TLR4) mutant mice after bile duct ligation (BDL). In vitro studies were carried out to evaluate the effect of the bacterial product and TLR agonist lipopolysaccharide (LPS) on paracrine interactions between hepatic stellate cells (HSC) and liver endothelial cells (LEC) that lead to fibrosis and portal hypertension. Portal pressure, fibrosis, and angiogenesis were significantly lower in BDL mice receiving rifaximin compared to BDL mice receiving vehicle. Studies in TLR4 mutant mice confirmed that the effect of rifaximin was dependent on LPS/TLR4 pathway. Fibronectin (FN) was increased in the BDL liver and was reduced by rifaximin administration and thus, was explored further in vitro as a potential mediator of paracrine interactions of HSC and LEC. In vitro, LPS promoted FN production from HSC. Furthermore, HSC-derived FN promoted LEC migration and angiogenesis. These studies expand our understanding of the relationship of intestinal microbiota with fibrosis development by identifying FN as a TLR4 dependent mediator of the matrix and vascular changes that characterize cirrhosis.

TLR4 Mediates Lung Injury and Inflammation in Intestinal Ischemia-Reperfusion

Splanchnic ischemia is common in critically ill patients, and it can result in injury not only of the intestine but also in distant organs, particularly in the lung. Local inflammatory changes play a pivotal role in the development of acute lung injury after intestinal ischemia, but the underlying molecular mechanisms are not fully understood. We sought to examine the role of Toll-like receptor 4 (TLR4) in the mouse model of intestinal ischemia-reperfusion (I/R)-induced lung injury and inflammation. Adult male TLR4 mutant (C3H/HeJ) mice and TLR4 wild-type (WT) (C3H/HeOuJ) mice were subjected to 40 min of intestinal ischemia by clamping the superior mesenteric artery followed by 6 h of reperfusion. Lung histology was assessed and parameters of pulmonary microvascular permeability, inflammatory cytokine expression, and neutrophil infiltration were measured. Activation of mitogen-activated protein kinases (MAPKs) and the transcription factors nuclear factor κB (NF-κB) and activator protein-1 (AP-1) in the lungs were also detected. After intestinal I/R, lungs from TLR4 mutant mice demonstrated a significantly lower histological injury, a marked reduction of epithelial apoptosis associated with the decreased level of cleaved caspase-3 and the increased ratio of Bcl-xL to Bax proteins, and a large reduction in pulmonary vascular permeability and myeloperoxidase (MPO) activity in comparison with WT mice. TLR4 mutant mice also displayed marked decreases in tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2) expression. Following intestinal I/R, phosporylation of p38 MAPK and activation of NF-κB and AP-1 were significantly inhibited in lung tissue from TLR4 mutant mice compared with WT controls. These data suggest that TLR4 plays an important role in the pathogenesis of intestinal I/R-induced acute lung injury and inflammation and that p38 kinase and NF-κB may be involved in TLR4 signaling-mediated lung inflammatory processes during intestinal I/R.

MyD88 is pivotal for immune recognition of Citrobacter koseri and astrocyte activation during CNS infection†

Citrobacter koseri (C. koseri) is a Gram-negative bacterium that can cause a highly aggressive form of neonatal meningitis, which often progresses to establish multi-focal brain abscesses. The roles of Toll-like receptor 4 (TLR4) and its signaling adaptor MyD88 during CNS C. koseri infection have not yet been examined, which is important since recent evidence indicates that innate immune responses are tailored towards specific pathogen classes. Here TLR4 WT (C3H/FeJ) and TLR4 mutant (C3H/HeJ) mice as well as MyD88 KO animals were infected intracerebrally with live C. koseri, resulting in meningitis and ventriculitis with accompanying brain abscess formation. MyD88 KO mice were exquisitely sensitive to C. koseri, demonstrating enhanced mortality rates and significantly elevated bacterial burdens compared to WT animals. Interestingly, although early proinflammatory mediator release (i.e. 12 h) was MyD88-dependent, a role for MyD88-independent signaling was evident at 24 h, revealing a compensatory response to CNS C. koseri infection. In contrast, TLR4 did not significantly impact bacterial burdens or proinflammatory mediator production in response to C. koseri. Similar findings were obtained with primary astrocytes, where MyD88-dependent pathways were essential for chemokine release in response to intact C. koseri, whereas TLR4 was dispensable; implicating the involvement of alternative TLRs since highly enriched astrocytes did not produce IL-1 upon bacterial exposure, which also signals via MyD88. Collectively, these findings demonstrate the importance of MyD88-dependent mechanisms in eliciting maximal proinflammatory responses, astrocyte activation, and bacterial containment during CNS C. koseri infection, as well as a late-phase MyD88-independent signaling pathway for cytokine/chemokine production.

Protective effect of lipopolysaccharide preconditioning in hepatic ischaemia reperfusion injury

BackgroundPreconditioning using lipopolysaccharide (LPS), a toll-like receptor4 (TLR4) ligand, has been demonstrated to reduce ischaemia/reperfusion injury (IRI) in some organs, but its effect in the liver has not been elucidated. We examined the liver protective mechanism and correlated signalling pathway of LPS preconditioning in mice. MethodsBALB/c and TLR4 mutant mice underwent 90min of 70% hepatic ischaemia. Lipopolysaccharide (100µg/kg) was injected intraperitoneally 20h or 30min before ischaemia. Liver damage after reperfusion was examined using serum samples and liver specimens. To analyse the mechanism of preconditioning in detail, phosphorylation of representative signalling mediators to nuclear factor-κB (NF-κB) activation, Akt and interleukin-1 receptor-associated kinase-1 (IRAK-1), and expression of a negative feedback inhibitor, suppressor of cytokine signalling-1 (SOCS-1), were evaluated by Western blotting. ResultsPretreatment with LPS only 20h before ischaemia elicited a preconditioning effect; however, preconditioning was absent in TLR4 mutant mice. Lipopolysaccharide significantly decreased serum alanine aminotransferase, tumour necrosis factor-α, hepatocyte necrosis and NF-κB activity after reperfusion. Phosphorylated IRAK-1 was suppressed by LPS, whereas no difference was observed in phosphorylated Akt. Pre-ischaemic LPS provided early induction of SOCS-1. DiscussionLate-phase LPS preconditioning provided liver protection against IRI through the downregulation of the TLR4 cascade derived from early induction of SOCS-1 during ischaemia/reperfusion.