Toll-like Receptor 4 Mutation Research Articles

Plasma TNF-α Elevation in Biologic Naive Rheumatoid Arthritis Patients Belonging to a Population with New Mutations in TLR4 and CYP51A1 genes without Association with Disease-Related Antibodies Levels.

In a system biology-based study, we previously reported that IL-6 and IL6R -specific m-RNA levels were elevated in leukocytes of patients with Rheumatoid arthritis (RA). Here, the association of toll-like receptor4 (TLR4) rs 141534085 and cytochrome P450 family 51 subfamily A member 1(CYP51A1) rs6 with tumor necrosis factor-α (TNF- α), rheumatoid factor (RF)- and Anti- cyclic citrullinated peptide (anti-CCP) antibody -positivity was investigated in almost the same subjects. Forty-six patients and 48 normal subjects were recruited in this study. The blood leucocytes TLR4 rs 141534085 and CYP51A1 rs6 -comprising DNA sequences were amplified by using tetra-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) technique and the PCR products were checked by Sanger DNA sequencing method. ELISA method was used to determine plasma levels of TNF- α, anti-CCP antibody and RF positivity of plasma was evaluated through a latex agglutination test. The TNF- α level was significantly higher in the patient group than control subjects (p= 0.001). Moreover, we were not able to find any correlation between TNF-α levels and RF as well as anti-CCP antibodies when we used the K2/ Fisher's exact test and Pearson test respectively. Our DNA sequencing data revealed the following new mutations in TLR4 rs141534085 comprising regions: A>T in position 1050, T>A in position 1052, and C>A in position 1054; and for CYP51A1 rs6 encompassing region, the new mutations were; G>A in position 21680, the T nucleotide was inserted in position 21762 and the G nucleotide was inserted in position 21763, G>T in position 21764. The data of this study showed that both TLR4 rs141534085 and CYP51A1 rs6 related DNA regions should be considered as hotspot areas in RA pathogenicity. Moreover, these data indicated that, TNF- α did not alter the production of anti-CCP and RF pathogenic antibodies in patients with long-term RA.

M2a macrophages facilitate resolution of chemically-induced colitis in TLR4-SNP mice.

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, impacts millions of individuals worldwide and severely impairs the quality of life for patients. Dysregulation of innate immune signaling pathways reduces barrier function and exacerbates disease progression. Macrophage (Mφ) signaling pathways are potential targets for IBD therapies. While multiple treatments are available for IBD, (i) not all patients respond, (ii) responses may diminish over time, and (iii) treatments often have undesirable side effects. Genetic studies have shown that the inheritance of two co-segregating SNPs expressed in the innate immune receptor, TLR4, is associated with human IBD. Mice expressing homologous SNPs ("TLR4-SNP" mice) exhibited more severe colitis than WT mice in a DSS-induced colonic inflammation/repair model. We identified a critical role for M2a "tissue repair" Mφ in the resolution of colitis. Our findings provide insight into potential development of novel therapies targeting Mφ signaling pathways that aim to alleviate the debilitating symptoms experienced by individuals with IBD.

Single Nucleotide Polymorphisms of Toll-like Receptor 4 in Hepatocellular Carcinoma-A Single-Center Study.

Hepatocellular carcinoma (HCC) is the most common primary liver tumor leading to significant morbidity and mortality; its exact genetic background is largely unrecognized. Toll-like receptor-4 (TLR4) reacts with lipopolysaccharides, molecules found in the outer membrane of Gram-negative bacteria. In damaged liver, TLR4 expression is upregulated, leading to hepatic inflammation and injury. We tried to investigate the role of the two most common single-nucleotide polymorphisms (SNPs) of TLR4 in HCC-genesis. Aged > 18 years old, cirrhotic patients were included in this study. Exclusion criteria were non-HCC tumors and HIV co-infection. TLR4 SNPs association with HCC occurrence was the primary endpoint, and associations with all-cause and liver-related mortality, as well as time durations between diagnosis of cirrhosis and HCC development or death and diagnosis of HCC and death were secondary endpoints. A total of 52 out of 260 included patients had or developed HCC. TLR4 SNPs showed no correlation with primary or secondary endpoints, except for the shorter duration between HCC development and death in patients with TLR4 mutations. Overall, TLR4 SNPs showed no correlation with carcinogenesis or deaths in patients with liver cirrhosis; patients with TLR4 SNPs that developed HCC had lower survival rates, a finding that should be further evaluated.

PRIMARY OPEN ANGLE GLAUCOMA: MECHANISMS OF PATHOGENESIS AND GENETIC PREDISPOSITION. Review

Relevance. Primary open-angle glaucoma (POAG) is a progressive optic neuropathy with loss of retinal ganglion cells (RGCs) and narrowing of the visual fields in the eyes with a gonioscopic open angle. The main mechanisms of this are increased intraocular pressure (IOP), circulatory disorders, trabecular meshwork (TM), ischemic metabolic disorders and chronic inflammation. However, questions about the role of POAG genetic predisposition remain open.
 Objective: analysis of current data on the mechanisms of pathogenesis of progressive neuropathy in POAG and the role of genetic predisposition.
 Methods. The analysis of scientific publications in open international electronic scientometric databases: Scopus, PubMed, Web of Science, Google Scholar, SID, MagIran, IranMedex, IranDoc, ScienceDirect, Embase by keywords (a total of 67 sources). Search depth – 10 years (2012-2022).
 Results. There are more than 60 million glaucoma patients in the world, 20% of whom have an incurable stage. By 2040, the number of patients is projected to increase to 112 million, with POAG accounting for 75% of cases. Among the main mechanisms of glaucoma, an important role belongs to chronic inflammation and immune damage, which occur in response to ischemic injury. Prolonged inflammatory process leads to hypersecretion of inflammatory mediators and infiltration of inflammatory cells into ischemic tissue, which aggravates the effects of increased IOP and ischemia. It is known that mutations in the gene of Toll-like receptor 4 (TLR4) are associated with both infectious and non-infectious diseases, including POAG: activation of TLR4 initiates TM fibrosis, causes increased IOP, activates RGCs apoptosis in the model of acute glaucoma. TLR4 ligands, such as heat shock proteins and lipopolysaccharides are candidate antigens for glaucoma. TLR4 overexpression at retinal microglia and astrocytes induce an innate immune response through NF-κB activation, which enhances the expression of proinflammatory cytokines.
 Conclusions. A promising direction is to study the contribution of TLR4 mutations to the POAG mechanisms, which will identify the mechanisms of immune disorders and establish the genetic risk of individual mutations in different ethnic groups.

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.

Toll-like Receptor 4 Gene Polymorphisms in Chinese Population After Allogeneic Hematopoietic Stem Cell Transplantation

Objectives:: Graft-versus-host disease (GVHD) is the most common complication after hematopoietic stem cell transplantation (HSCT) and remains to be a major cause of mortality. Activation of toll-like receptor 4 (TLR-4) by lipopolysaccharide induces the NF-κB signaling pathway to release critical proinflammatory cytokines and increases the recipient response to GVHD. In order to clarify the role of TLR-4 in the occurrence of acute GVHD after HSCT, we collected 208 samples from HSCT recipients and their human lecucyte antigen identical donors to test the hypothesis that TLR-4polymorphism in the recipients or donors influence the risk of acute GVHD in allogeneic HSCT recipients. Methods:: TLR-4 Asp299Gly and Thr399Ile polymorphisms of each sample were examined by using DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism methods. Results:: No homozygous or heterozygous variant alleles of the Asp299Gly or Thr339Ile polymorphism were detected in any samples in our study. Our results demonstrate that TLR-4 Asp299Gly and Thr399Ile polymorphisms might be very rare in the Chinese population (Eastern China and Taiwan region). Conclusion:: The results of this study cannot confirm the role of TLR-4 mutations in the pathogenesis of GVHD in humans, yet we reach a definite conclusion by a TLR-4 knockout murine GVHD model in our ongoing project.

Toll-like receptor 4 mediates vascular remodeling in hyperhomocysteinemia.

Although hyperhomocysteinemia (HHcy) is known to promote downstream pro-inflammatory cytokine elevation, the precise mechanism is still unknown. One of the possible receptors that could have significant attention in the field of hypertension is toll-like receptor 4 (TLR-4). TLR-4 is a cellular membrane protein that is ubiquitously expressed in all cell types of the vasculature. Its mutation can attenuate the effects of HHcy-mediated vascular inflammation and mitochondria- dependent cell death that suppresses hypertension. In this review, we observed that HHcy induces vascular remodeling through immunological adaptation, promoting inflammatory cytokine up-regulation (IL-1β, IL-6, TNF-α) and initiation of mitochondrial dysfunction leading to cell death and chronic vascular inflammation. The literature suggests that HHcy promotes TLR-4-driven chronic vascular inflammation and mitochondria-mediated cell death inducing peripheral vascular remodeling. In the previous studies, we have characterized the role of TLR-4 mutation in attenuating vascular remodeling in hyperhomocysteinemia. This review includes, but is not limited to, the physiological synergistic aspects of the downstream elevation of cytokines found within the vascular inflammatory cascade. These events subsequently induce mitochondrial dysfunction defined by excessive mitochondrial fission and mitochondrial apoptosis contributing to vascular remodeling followed by hypertension.

Structure-Activity Relationship in TLR4 Mutations: Atomistic Molecular Dynamics Simulations and Residue Interaction Network Analysis

Toll-like receptor 4 (TLR4), a vital innate immune receptor present on cell surfaces, initiates a signaling cascade during danger and bacterial intrusion. TLR4 needs to form a stable hexamer complex, which is necessary to dimerize the cytoplasmic domain. However, D299G and T399I polymorphism may abrogate the stability of the complex, leading to compromised TLR4 signaling. Crystallography provides valuable insights into the structural aspects of the TLR4 ectodomain; however, the dynamic behavior of polymorphic TLR4 is still unclear. Here, we employed molecular dynamics simulations (MDS), as well as principal component and residue network analyses, to decipher the structural aspects and signaling propagation associated with mutations in TLR4. The mutated complexes were less cohesive, displayed local and global variation in the secondary structure, and anomalous decay in rotational correlation function. Principal component analysis indicated that the mutated complexes also exhibited distinct low-frequency motions, which may be correlated to the differential behaviors of these TLR4 variants. Moreover, residue interaction networks (RIN) revealed that the mutated TLR4/myeloid differentiation factor (MD) 2 complex may perpetuate abnormal signaling pathways. Cumulatively, the MDS and RIN analyses elucidated the mutant-specific conformational alterations, which may help in deciphering the mechanism of loss-of-function mutations.

Toll-like receptor 4 mutation suppresses hyperhomocysteinemia-induced hypertension.

Hyperhomocysteinemia (HHcy) has been observed to promote hypertension, but the mechanisms are unclear. Toll-like receptor 4 (TLR-4) is a cellular membrane protein that is ubiquitously expressed in all cell types of the vasculature. TLR-4 activation has been known to promote inflammation that has been associated with the pathogenesis of hypertension. In this study we hypothesize that HHcy induces hypertension by TLR-4 activation, which promotes inflammatory cytokine (IL-1β, IL-6, and TNF-α) upregulation and initiation of mitochondria-dependent apoptosis, leading to cell death and chronic vascular inflammation. To test this hypothesis, we used C57BL/6J (WT) mice, cystathionine β-synthase (CBS)-deficient (CBS+/-) mice with genetic mild HHcy, C3H/HeJ (C3H) mice with TLR-4 mutation, and mice with combined genetic HHcy and TLR-4 mutation (CBS+/-/C3H). Ultrasonography of the superior mesenteric artery (SMA) detected an increase in wall-to-lumen ratio, resistive index (RI), and pulsatility index (PI). Tail cuff blood pressure (BP) measurement revealed elevated BP in CBS+/- mice. RI, PI, and wall-to-lumen ratio of the SMA in CBS+/-/C3H mice were similar to the control group, and BP was significantly alleviated. TLR-4, IL-1β, IL-6, and TNF-α expression were upregulated in the SMA of CBS+/- mice and reduced in the SMA of CBS+/-/C3H mice. Molecules involved in the mitochondria-mediated cell death pathway (BAX, caspase-9, and caspase-3) were upregulated in CBS+/- mice and attenuated in CBS+/-/C3H mice. We conclude that HHcy promotes TLR-4-driven chronic vascular inflammation and mitochondria-mediated cell death, inducing hypertension. TLR-4 mutation attenuates vascular inflammation and cell death, which suppress hypertension.

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.

Critical Role of Toll-Like Receptor 4 in Hypoxia-Inducible Factor 1α Activation During Trauma/Hemorrhagic Shock–Induced Acute Lung Injury After Lymph Infusion in Mice

AB The nuclear transcription factor hypoxia-inducible factor 1[alpha] (HIF-1α) is a key regulator of gene expression under hypoxic and inflammatory conditions. The germline-encoded pattern recognition receptor toll-like receptor 4 (TLR4) recognizes molecular motifs shared by large groups of microorganisms as well as by endogenous ligands released from stressed and/or injured tissues. We have previously demonstrated that local inhibition of HIF-1α ameliorates lung injury induced by trauma/hemorrhagic shock (T/HS) in rats. In the current study, we directly determined the role of TLR4 in HIF-1α activation during T/HS-induced acute lung injury in mice. C3H/HeJ mice that harbor a TLR4 mutation and wild-type (WT) mice were infused T/HS or trauma/sham shock (T/SS) lymph from Sprague-Dawley rats. Evans blue dye lung permeability, lung water content, myeloperoxidase levels, and lung histological analysis confirmed that TLR4-deficient mice are resistant to lung injury after T/HS lymph infusion. Lungs from WT and TLR4mut mice after T/SS lymph infusion expressed negligible levels of HIF-1α. The induction of HIF-1α in lung homogenates from TLR4mut mice after T/HS or T/HS lymph infusion was markedly reduced as compared with their WT counterparts but remained elevated as compared with TLR4mut mice after T/SS lymph infusion. Endothelial cells from TLR4mut mice and silence of TLR4 in cells from WT mice showed a remarkable reduction of HIF-1α on T/HS lymph stimulation. Blocking of nuclear factor-κB activity by SN50 or Bay 11-7085 in WT cells diminished T/HS lymph-induced HIF-1α accumulation when compared with T/SS lymph incubation. Thus, our data suggest that TLR4 activation by T/HS is necessary for T/HS-induced lung injury and an augmented pulmonary HIF-1α response, which will provide more insights into the pathogenesis of shock-induced acute lung injury and identify potential therapeutic targets.

Molecular Characterization and Expression Profile of Partial TLR4 Gene in Association to Mastitis in Crossbred Cattle

Crossbred cattle are more prone to mastitis in comparison to indigenous cattle. Toll-like receptor 4 (TLR4) recognizes pathogen ligands, for example, lipopolysaccharide (LPS) endotoxin from Escherichia coli and mediates signaling to initiate innate and adaptive immune responses. Mutations in TLR4 can compromise the host immune response to certain pathogens, so it may be a potential candidate for marker assisted selection to enhance mastitis resistance in dairy cattle. Hence, in this study role of bovine TLR4 gene in mastitis resistance was investigated by association as well as expression profiling analysis in crossbred cattle. The animals were divided into mastitis affected and unaffected groups on the basis of history of animals and California Mastitis Test (CMT). PCR-SSCP and Sequence analysis revealed three genotypes of coreceptor binding region 1 (CRBR1) fragment of TLR4 gene namely AA, AB, and BB in both groups of cattle. The logistic regression model did not show any significant effect of these genotypes on the occurrence of clinical mastitis. Moreover, in vitro challenge of peripheral blood mononuclear cells (PBMCs) with LPS failed to show any association of the genotypes with TLR4 gene expression. In a nutshell, in the present study enough evidence was not found for association of the SNP variants of CRBR1 fragment of TLR4 gene with mastitis susceptibility in crossbred cattle.

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.

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.

Microglial activation involved in morphine tolerance is not mediated by toll-like receptor 4

Morphine is a powerful analgesic but its effect is often diminished owing to the development of tolerance. It has been suggested that morphine activates microglia through its action on the toll-like receptor 4 (TLR4) in the spinal cord, leading to suppression of the morphine effect. However, it has not been examined whether the development of morphine tolerance is affected by the deletion and mutation of the TLR4 gene. Mice were treated with morphine (60 mg/kg) or vehicle once daily for five consecutive days to induce morphine tolerance, which was assessed by the tail-flick test before and after the treatment period. The effect of the microglial inhibitor minocycline, and the effect of TLR4 mutation (C3H/HeJ mouse) and deletion (TLR4-knockout mouse) on the development of morphine tolerance were tested. The expression of the microglial activation marker, CD11b, in the spinal cords of TLR4-knockout and wild-type mice after morphine treatment for 5 days was assessed by reverse-transcription polymerase chain reaction. Minocycline attenuated the development of morphine tolerance in mice. Mutation or deletion of the TLR4 gene did not significantly affect the development of morphine tolerance. CD11b mRNA expression was increased after morphine treatment both in TLR4-knockout and wild-type mice. Microglial activation caused by a mechanism independent of TLR4 is involved in the development of morphine tolerance. Further studies are necessary to clarify the cellular mechanisms of morphine-induced microglial activation.

LPS induces IL‐8 expression through TLR4, MyD88, NF‐kappaB and MAPK pathways in human dental pulp stem cells

To evaluate the effects of lipopolysaccharide (LPS) on interleukin-8 (IL-8) and related intracellular signalling pathways in human dental pulp stem cells (hDPSCs). Human pulp tissues were isolated from human impacted third molars, and the hDPSCs were cultured and characterized. The effects of LPS on IL-8 and Toll-like receptor 4 (TLR4) gene expression in hDPSCs were investigated using real-time quantitative RT-PCR and ELISA. Whether TLR4/MyD88/NF-кB was involved in the LPS-induced up-regulation of IL-8 in hDPSCs was determined using transient transfection, luciferase assay and ELISA. The involvement of MAPKs in the LPS-induced up-regulation of IL-8 in hDPSCs was investigated via transient transfection, luciferase assay, ELISA and western blot. The data were statistically analysed using Student's t-test or one-way anova followed by the Student-Neumann-Keuls test. Cells exposed to LPS not only displayed an enhanced expression of TLR4 but also showed an elevated IL-8 gene expression; exposure to LPS also resulted in the induction of IL-8 gene transcription via promoter activation. The LPS-induced IL-8 promoter activation was inhibited through dominant-negative mutations in TLR4 and MyD88, but not in TLR2. The LPS-induced IL-8 protein release was attenuated through the administration of TLR4-neutralizing antibody or MyD88 inhibitory peptide and a dominant-negative mutation in IκBα. In contrast, IL-8 protein release was enhanced through the expression of NF-κB p65. Treatment with PDTC, TPCK or Bay117082 effectively antagonized LPS-induced IL-8 protein release. Moreover, both the promoter activity and the LPS-induced release of IL-8 were diminished upon the administration of U0126 and SB203580, but not SP600125. Moreover, the exposure to LPS activated ERK1/2 and p38 MAPK phosphorylation in cells. This study reports the LPS-mediated transcriptional and post-translational up-regulation of IL-8, which is a process that also involves TLR4, MyD88, NF-κB and MAPK.

Loss of estrogen increases leptin levels, liver dysfunction and insulin resistance in Toll‐like receptor 4 mutation mice

Previous studies in the VCD‐mouse model of menopause demonstrated that the factors comprising the metabolic syndrome were accelerated when combined with a high‐fat (HF) diet. Fatty acids are able to activate Toll‐like receptor 4 (TLR4) signaling, which mediates inflammatory events and insulin resistance. Previously VCD‐treated TLR4 +/+ mice developed insulin resistance on a HF diet after 12 wks. However in VCD‐treated mice heterozygous for a TLR4 mutation (TLR4 +/−) it took 20 weeks on a HF diet to develop hyperinsulinemia and insulin resistance. Leptin levels were significantly higher in VCD‐treated mice after 16 wks on a HF diet compared to all other groups (VCD/HF 33.9±10 vs HF 12.8±1.2 and Con 2.9±1.3 ng/ml). Inflammation and lipid regulation pathways were examined in both kidney and liver tissue. At 12wks HF, the stress‐response gene HO‐1 was significantly increased in livers of HF/VCD vs HF mice. SREBP1, FAS and SCID‐1 were elevated at16 and 20 wks in VCD/HF mice. After 24 wks livers of VCD/HF mice had significant lipid accumulation, per ORO staining. Renal tissue was examined for macrophage infiltration and diabetic associated structural changes. Our data suggests that loss of TLR4 (+/−) infers a resistance to insulin resistance onset; however menopause‐associated hormonal alterations promote the development of insulin resistance and liver dysfunction when paired with a HF diet.

A TLR4-MCP-1-macrophage IL-18 Cascade Plays A Major Role In Myocardial Injury And Cardiac Dysfunction After Permanent Ischemia

Introduction: Myocardial ischemia induces an inflammatory response involving in up-regulated chemokine expression and macrophage (Mf) accumulation. Toll-like receptor 4 (TLR4) has been linked to post-ischemic myocardial injury, and it mediates myocardial expression of MCP-1, a key chemokine for Mfaccumulation, after ischemia. However, the role of Mfin post-ischemic myocardial injury and heart failure is not well understood. We hypothesize that TLR4 mediates myocardial injury through up-regulation of chemokine expression and macrophage accumulation. The purposes of this study were to determine: 1) the effect of TLR4 mutation on different chemokines expression, differential leukocytes accumulation, and myocardial injury in a mouse model of permanent, 2) whether neutralizing MCP-1 reduces Mfaccumulation and myocardial injury, 3) whether monocyte/Mfdepletion reduces myocardial injury and 4)to determine the role of IL-18 expressed by accumulated macrophage, in the myocardial injury. Methods: C3H/HeJ (TLR4 mutant, with dysfunctional TLR4) and C3H/HeN (TLR4-competent) mice were subjected to left coronary ligation. Results: In TLR4-competent mice, among chemokines only MCP-1 myocardial levels increased markedly at day 1, 3 and 7, and massive Mfaccumulation in the ischemic myocardium occurred at day 3 and 7. TLR4 mutation significantly reduced these changes, resulting in smaller infarct size and improved heart function. Neutralizing MCP-1 also reduced Mfaccumulation, infarct size and heart failure in TLR4-competent mice. Further, prior depletion of monocytes/Mfusing liposomal clodronate resulted in infarct size reduction and heart function improvement. Finally, suppression of Mfdecreased myocardial IL-18 levels in ischemic myocardium, and IL-18 knockout provided significant myocardial protection against injury and dysfunction but its effect was less robust than TLR4 mutation, MCP-1 neutralization or monocytes/Mfdepletion. Conclusions: A TLR4-MCP-1-macrophage-IL-18 cascade occupies a major in myocardial injury and cardiac dysfunction after permanent ischemia and could be a therapeutic target for protection against post-ischemic myocardial injury and heart failure.

Role of TLR4 In Acute Gvhd After Allogenetic Hematopoietic Stem Cell Transplantation

AbstractAbstract 2538Graft-versus-host disease (GVHD) is the most common complication after hematopoietic stem cell transplantation (HSCT). Lipopolysaccharide (LPS) has been implicated in the pathogenesis of GVHD. The toll-like receptor-4 (TLR4) has been identified as a major receptor for LPS. Here arises the question whether TLR4 mutations may increase risk of microbial infection and affect acute GVHD in allogeneic HSCT recipients. In order to clarify the role of TLR4 in the occurrence of acute GVHD, we detected the interaction of TLR4 mutations in recipient and donor cells and analyzed allogeneic lymphocyte infiltration in the liver, intestine and skin of host mice by immunohistochemistry after allogeneic HSCT. Wild type C57BL/6 (TLR4+/+) and TLR4 knockout (TLR4−/−) mice were received myeloablative total body irradiation, followed by tail vein injection of donor BALB/c bone marrow cells and splenocytes to induce acute GVHD. GVHD severity was assessed using clinical scores. In vivo the proliferation activity of allogeneic donor BALB/c T cells in TLR4−/− and TLR4+/+ transplanted mice was evaluated ex vivo by flow cytometry after labeling with CFSE. Mixed lymphocyte reaction (MLR) assays were performed to evaluate the proliferation of allogeneic donor BALB/c T cells at different times of coculture with MHC class II antigen presenting cells (APCs) obtained from bone marrow of TLR4+/+ or TLR4−/− mice with or without LPS stimulation for 24 h. When myeloablative irradiated TLR4−/− mice, instead of wild-type mice, were used as graft recipients, clinical score of acute GVHD severity were decreased and survival were increased (18/30 vs 9/30 mice still alive at day 30, GVHD clinical score 6.7 vs 4.5). The decreased mortality and morbidity in TLR4−/− mice were associated with reduced proliferation of allogeneic donor cells transplanted in these mice.We evaluated the activation of spleen APCs in TLR4+/+ or TLR4−/− mice after myeloablative conditioning. Higher expression of CD80 and CD86 costimulatory molecules on MHC class II cells was detected in wide type strain at 3 d postirradiation. Ex vivo experiments CD80, CD86 and CD40 costimulatory markers on bone marrow APCs of C57BL/6 wild-type more significant up-regulation than TLR4−/− mice after LPS stimulation 24 h. TLR4−/− recipients receiving BALB/c donors developed significantly less GVHD as measured by liver, skin and intestinal of mice histopathology compared with TLR4+/+ recipients. Cytokines IL-2/IFN-γexpression in TLR4+/+ recipients mice serum was stronger but IL-4/IL-10 expression was weaker comparing to that in TLR4−/− recipients. These results suggest that TLR-4 mutation in donor cells increases the expression of Th2-related cytokines and decreases the risk of GVHD after allogeneic bone marrow transplantation.These data reveal that TLR4 mutations in recipitents is crucial in the prevention of GVHD, while responsiveness of wide type mice APC to LPS may be an important risk factor for acute GVHD. Overall together, these results suggest that the function of TLR4 has influence on the occurrence of acute GVHD, which might provide methods to reduce this complication after allogeneic hematopoietic stem cell transplantation. Disclosures:No relevant conflicts of interest to declare.

A functional nonsynonymous toll-like receptor 4 gene polymorphism is associated with metabolic syndrome, surrogates of insulin resistance, and syndromes of lipid accumulation

Toll-like receptor 4 (TLR4) plays a key role in the activation of innate immune responses. Loss-of-function mutations in TLR4 prevent diet-induced obesity and insulin resistance (IR). We conducted a population cross-sectional study to evaluate whether Asp299Gly (rs4986790) TLR4 gene polymorphism is associated with metabolic syndrome (MS), surrogates of IR, and syndromes of lipid accumulation (SLAs) in Argentinean healthy male subjects. rs4986790 was genotyped in 621 healthy unrelated male blood donors. National Cholesterol Education Program/Adult Treatment Panel III–MS (NCEP/ATP III-MS); SLAs such as enlarged waist elevated triglyceride syndrome (EWET), hypertriglyceridemic waist (HW), and overweight-lipid syndrome (OLS); and surrogates of IR were assessed. The prevalence of MS, OLS, and EWET was significantly higher among Asp299Asp carriers ( P < .05). These findings were confirmed using 32 000 bootstrap samples. Surrogate markers of IR were also significantly higher in Asp299Asp carriers ( P < .05). Most findings were especially strengthened among individuals with C-reactive protein below the 95th percentile and/or total cholesterol to high-density lipoprotein cholesterol ratio ≥5. This is the first report to find, in Argentinean healthy male blood donors, associations between the Asp299Asp genotype of rs4986790 TLR4 gene polymorphism and high risk for NCEP/ATP III-MS, SLAs, and surrogates of IR. These findings are consistent with previous functional and observational studies showing that Asp299 allele, in comparison with Gly299, is associated with increased TLR4 activation, higher levels of inflammatory cytokines, acute-phase reactants and soluble adhesion molecules, and higher risk of atherosclerosis.