TLR4-dependent effects of ISAg treatment on conventional T cell polarization in vivo

We recently demonstrated that the polysaccharide component of the Korean medicinal herb Angelica gigas (immuno-stimulatory fraction of A. gigas; ISAg) induces anticancer effects in mice by activating natural killer (NK) and natural killer T (NKT) cells. However, it is unclear whether the use of ISAg in vivo can affect the differentiation of conventional T cells. Here, we investigated the effects of ISAg on the activation of conventional CD4⁺ and CD8⁺ T cells. We found that the administration of ISAg induced the polarization of CD4⁺ T cells toward the acquisition of the Th1 phenotype in vivo. Additionally, in mice treated with ISAg, CD8⁺ T cells produced more IFNγ than in control mice treated with PBS. Moreover, treatment with ISAg activated CD4⁺ and CD8⁺ T cells as well as NK and NKT cells, resulting in the secretion of Th1-type cytokines in a toll-like receptor 4 (TLR4)-dependent manner, implying that TLR4 is critical for an optimal Th1 response. Interestingly, ISAg treatment increased the number of Foxp3⁺ Treg cells, but not of Th2 cells, compared to control mice treated with PBS, indicating that ISAg possesses an immunomodulatory capacity that can control adaptive immune responses. Taken together, our results indicate that ISAg possesses a Th1-enhancing activity that could be used to treat Th2-mediated allergic immune diseases such as atopic dermatitis.     

Caloric restriction ameliorates cardiomyopathy in animal model of diabetes

Background

The db/db mouse is an animal model of diabetes in which leptin receptor activity is deficient resulting accelerated cardiomyopathy when exposed to angiotensin (AT). Toll-like receptors 4 and 2 (TLR4, TLR2) are pattern recognition receptors, that recognize pathogen-associated molecular patterns and exacerbate and release inflammatory cytokines. Fetuin A (Fet A) is a fatty acid carrier which affects inflammation and insulin resistance in obese humans and animals through TLRs.The aim of this study was to investigate the effect of caloric restriction (CR) on free fatty acids (FFA) level and the inflammatory response in diabetic cardiomyopathy.

Genetic polymorphisms of NOD1 and IL-8, but not polymorphisms of TLR4 genes, are associated with Helicobacter pylori-induced duodenal ulcer and gastritis

BACKGROUND: Intracellular pathogen receptor NOD1 is involved in the epithelial cell sensing Helicobacter pylori, which results in a considerable interleukin (IL)-8 production. The aim of this study was to evaluate the relationship between NOD1 and IL-8 genetic polymorphisms and the development of H. pylori-induced gastritis and duodenal ulcer (DU), as compared with TLR4 polymorphisms. MATERIALS AND METHODS: Eighty-five patients with DU and 135 patients with gastritis were enrolled in the study. Seventy-five serologically H. pylori-positive subjects without gastric or duodenal symptoms served as controls. The G796A (E266K) NOD1 polymorphism was determined by restriction fragment length polymorphism, and the -251 IL-8 polymorphism by amplification refractory mutation system method. The TLR4 (ASP/299/Gly and Thr/399/Ile) gene polymorphisms were examined by melting point analysis. RESULTS: AA homozygote mutant variants of NOD1 were detected in 20% of the H. pylori-positive patients with DU versus 7% of H. pylori-positive patients with gastritis and versus 6% of the H. pylori-positive healthy controls. The IL-8 heterozygote mutant variant was detected with a significantly higher frequency among the DU patients and those with gastritis than among the H. pylori-positive controls. However, no significant correlation concerning the frequency of the TLR4 gene polymorphism could be revealed between any group of patients and the controls. CONCLUSION: E266K CARD4/NOD1, but not the TLR4 gene polymorphism increases the risk of peptic ulceration in H. pylori-positive patients. The -251 IL-8 polymorphism was significantly associated with either gastritis or DU in H. pylori-infected subjects. Host factors including intracellular pathogen receptors and IL-8 production play an important role in H. pylori-induced gastric mucosal damage.     

TLR4 mediates the impairment of ubiquitin-proteasome and autophagy-lysosome pathways induced by ethanol treatment in brain

New evidence indicates the involvement of protein degradation dysfunctions in neurodegeneration, innate immunity response and alcohol hepatotoxicity. We recently demonstrated that ethanol increases brain proinflammatory mediators and causes brain damage by activating Toll-like receptor 4 (TLR4) signaling in glia. However, it is uncertain if the ubiquitin-proteasome and autophagy-lysosome pathways are involved in ethanol-induced brain damage and whether the TLR4 response is implicated in proteolytic processes. Using the cerebral cortex of WT and TLR4-knockout mice with and without chronic ethanol treatment, we demonstrate that ethanol induces poly-ubiquitinated proteins accumulation and promotes immunoproteasome activation by inducing the expression of β2i, β5i and PA28α, although it decreases the 20S constitutive proteasome subunits (α2, β5). Ethanol also upregulates mTOR phosphorylation, leading to a downregulation of the autophagy-lysosome pathway (ATG12, ATG5, cathepsin B, p62, LC3) and alters the volume of autophagic vacuoles. Notably, mice lacking TLR4 receptors are protected against ethanol-induced alterations in protein degradation pathways. In summary, the present results provide the first evidence demonstrating that chronic ethanol treatment causes proteolysis dysfunctions in the mouse cerebral cortex and that these events are TLR4 dependent. These findings could provide insight into the mechanisms underlying ethanol-induced brain damage.     

动物TLR4基因多态性及其抗病相关性研究进展

TLR4是天然免疫系统中的一种重要模式识别受体,可选择性识别病原微生物而启动天然免疫,在宿主天然免疫和获得性免疫中具有重要作用。此外,大量研究表明,TLR4基因的多态性与多种动物的细菌、病毒和寄生虫性疾病密切相关,说明通过TLR4基因的研究可为动物分子标记辅助选育研究提供依据。该文简要论述部分动物TLR4基因多态性及其与抗病相关性的研究进展。     

Retracted: Resistin effects on pancreatic cancer progression and chemoresistance are mediated through its receptors CAP1 and TLR4

Resistin, secreted by macrophages in tumor microenvironment, has never been investigated in pancreatic cancer models, despite a vibrant tumor microenvironment around pancreatic tumors. We evaluated serum resistin levels in healthy individuals versus pancreatic cancer patients representing different tumor grades. In vitro mechanistic analysis involved MiaPaCa-2 and SW1990 cells. Resistin signaling depends on binding of resistin to its cognitive receptors. Therefore, we silenced adenylyl cyclase-associated protein 1 (CAP1) and toll-like receptor 4 (TLR4), its two known receptors, individually as well as in combination, by short hairpin RNA (shRNA). Effect of resistin on cell proliferation, migration, invasion, cell cycle, and sensitivity to gemcitabine was studied without or with silencing of resistin receptors CAP1 and/or TLR4. The results were also confirmed in vivo in mice xenografted with MiaPaCa-2 cells without or with receptor silencing. We report high resistin levels in pancreatic cancer patients which correlate positively with tumor grades. We observed a marked reduction in the resistin-induced proliferation, migration, invasion, and cell cycle of pancreatic cancer cells MiaPaCa-2 and SW1990 when the receptors were silenced. The results were confirmed in vivo wherein resistin effects were significantly attenuated in MiaPaCa-2 xenografts with silenced receptors. The combined silencing of CAP1 and TLR4 was found to be most effective in vitro and in vivo. We found activation of STAT3 by resistin in vivo and in vitro which was dependent on the presence of CAP1 and TLR4. Further, resistin was found to induce resistance to gemcitabine through its receptors. Our results describe novel functional roles of resistin with implications toward a better understanding of pancreatic tumor microenvironment.     

猪Toll样受体4基因SNPs功能分析

Toll样受体4(Toll-like receptor 4,TLR4)在机体的免疫反应中发挥重要作用,该基因突变会影响受体的信号转导能力和机体的疾病抗性/易感性。文章在前期工作的基础上,进一步分析c.611 T>A(p.Leu204His)、c.1027C>A(p.Gln343Lys)和c.1605 G>T(p.Leu535Phe)3个错义突变对猪TLR4功能的影响。利用RT-PCR方法克隆猪TLR4基因全长编码区并引入定点突变;利用真核表达、双荧光素酶报告系统和Western blotting方法在瞬时转染的PK-15细胞内研究3个单核苷酸多态(Single nucleotide polymorphisms,SNPs)对猪TLR4配体识别和信号转导能力的影响;同时,利用创造酶切位点PCR-RFLP方法分析对TLR4活性有显著影响的点突变在民猪、大白、长白和中国东北野猪4个群体中的分布。结果,成功获得了民猪TLR4基因的全长编码区和3个单碱基变异体,构建了不同等位基因的真核表达载体,在PK-15细胞内确定了c.1605 G>T变异导致TLR4向下游传递信号的能力显著降低(P<0.01),该SNP只存在于民猪和野猪中且频率较高。猪TLR4基因c.1605 G>T变异影响Toll样受体的信号传递,可能和机体的疾病抗性/易感性有关。     

Increased circulating IL-8 is associated with reduced IGF-1 and related to poor metabolic control in adolescents with type 1 diabetes mellitus

Background: A dysregulated growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis is well-recognized in children and adolescents with type 1 diabetes mellitus (T1DM). Decreased IGF-1 levels can also be found in chronic inflammatory diseases, while hyperglycemia promotes inflammatory cytokine production. Therefore, inflammatory cytokines may link poor metabolic control with GH/IGF-1 axis changes. This study examined the relationship between serum inflammatory cytokines and IGF-1 in adolescents (age 13–18) with TIDM in chronic poor (n = 17) or favorable (n = 19) glucose control. Poor control (PC) was defined as 3, consistent HbA1C > 9% during the previous 2 years, while favorable control (FC) was consistent levels of HbA1C < 9%. Results: HbA1C (FC: 7.5 ± 0.6%; PC: 10.5 ± 0.9%, p < 0.001) and interleukin (IL)-8 (FC: 3.7 ± 4.0 pg/ml; PC: 7.4 ± 4.3 pg/ml, p = 0.01) were increased and IGF-1 (FC: 536.5 ± 164.3 ng/ml; PC: 408.9 ± 157.1 ng/ml, p = 0.03) was decreased in patients with poor control compared to patients with favorable control. Moreover, IL-8 was inversely correlated with IGF-1 (r = −0.40, p = 0.03) and positively correlated with HbA1C (r = 0.36, p = 0.03). Conclusions: In adolescents with T1DM and chronic, poor glucose control, increased serum IL-8 is associated with reduced IGF-1 suggesting a pro-inflammatory milieu that may contribute to alterations in the GH/IGF-1 axis.     

TLR4 and TLR9 polymorphism: Probable role in susceptibility among the population of Bihar for Indian visceral leishmaniasis

Genetic variations in the host TLRs genes play an important role in susceptibility and/or resistance to visceral leishmaniasis by altering the host-pathogen interaction. In this study, we investigated the association between polymorphisms of TLR4 (Asp299Gly, Thr399Ile) and TLR-9 (T-1237C), with susceptibility to visceral leishmaniasis. A bi-directional PCR amplification of specific alleles technique was used to characterize the distribution of TLR4 (Asp299Gly and Thr399Ile) and TLR9 (T-1237C) polymorphisms. A total of 60 samples were randomly selected from confirmed visceral leishmaniasis patients and 24 endemic healthy volunteers. The samples were genotyped and allele frequencies were determined. We observed that TLR4 Asp299Gly and Thr399Ile genotypes were more frequent in visceral leishmaniasis patients (10% and 15% respectively) compared to controls (4.2% and 8.3% respectively). However, the differences were not significant in TLR4 Asp299Gly and Thr399Ile alleles and genotypes. In the case of TLR9, we observed the frequency of T1237C genotype was higher in visceral leishmaniasis patients (43.3%) than in healthy controls (33.3%). Statistically significant differences were observed in TLR9 T1237C alleles and genotypes. We concluded that TLR9 T1237C, but not TLR4, gene polymorphisms can be regarded as contributors to visceral leishmaniasis susceptibility among the Indian population of Bihar state.     

Defective immunogenic cell death of HMGB1-deficient tumors: compensatory therapy with TLR4 agonists

Immunogenic cell death induced by anticancer chemotherapy is characterized by a series of molecular hallmarks that include the exodus of high-mobility group box 1 protein (HMGB1) from dying cells. HMGB1 is a nuclear nonhistone chromatin-binding protein. It is secreted at the late stages of cellular demise and engages Toll-like receptor4 (TLR4) on dendritic cells (DCs) to accelerate the processing of phagocytic cargo in the DC and to facilitate antigen presentation by DC to T cells. The absence of HMGB1 expression by dying tumor cells exposed to anthracyclines or oxaliplatin compromises DC-dependent T-cell priming by tumor-associated antigens. Here, we show that transplantable tumors exhibiting weak expression of nuclear HMGB1 respond to chemotherapy more effectively if the treatment is combined with the local or systemic administration of a highly purified and physiochemically defined and standardized lipopolysaccharide solution, which acts as a high-potency and exclusive TLR4 agonist, called Dendrophilin (DEN). The synergistic antitumor effects mediated by the combination of chemotherapy and immunotherapy relied upon the presence of the MyD88 (myeloid differentiation primary response gene) adapter of TLR4 (but not that of the TIR-domain-containing adapter-inducing interferon-β adapter), in line with the well-characterized action of DEN on the MyD88 signaling pathway. DEN and anthracyclines synergized to induce intratumoral accumulation of interferon-γ-producing CD4⁺ and CD8⁺ T lymphocytes. Moreover, DEN could restore the immunogenicity of dying tumor cells from which HMGB1 had been depleted by RNA interference. These findings underscore the potential clinical utility of combination regimens involving immunogenic chemotherapy and certain TLR4 agonists in advanced HMGB1-deficient cancers.     

Hyperglycemia-induced O-GlcNAcylation and truncation of 4E-BP1 protein in liver of a mouse model of type 1 diabetes

4E-BP1 is a protein that, in its hypophosphorylated state, binds the mRNA cap-binding protein eIF4E and represses cap-dependent mRNA translation. By doing so, it plays a major role in the regulation of gene expression by controlling the overall rate of mRNA translation as well as the selection of mRNAs for translation. Phosphorylation of 4E-BP1 causes it to release eIF4E to function in mRNA translation. 4E-BP1 is also subject to covalent addition of N-acetylglucosamine to Ser or Thr residues (O-GlcNAcylation) as well as to truncation. In the truncated form, it is both resistant to phosphorylation and able to bind eIF4E with high affinity. In the present study, Ins2(Akita/+) diabetic mice were used to test the hypothesis that hyperglycemia and elevated flux of glucose through the hexosamine biosynthetic pathway lead to increased O-GlcNAcylation and truncation of 4E-BP1 and consequently decreased eIF4E function in the liver. The amounts of both full-length and truncated 4E-BP1 bound to eIF4E were significantly elevated in the liver of diabetic as compared with non-diabetic mice. In addition, O-GlcNAcylation of both the full-length and truncated proteins was elevated by 2.5- and 5-fold, respectively. Phlorizin treatment of diabetic mice lowered blood glucose concentrations and reduced the expression and O-GlcNAcylation of 4E-BP1. Additionally, when livers were perfused in the absence of insulin, 4E-BP1 phosphorylation in the livers of diabetic mice was normalized to the control value, yet O-GlcNAcylation and the association of 4E-BP1 with eIF4E remained elevated in the liver of diabetic mice. These findings provide insight into the pathogenesis of metabolic abnormalities associated with diabetes.     

Toll-like receptors and fungal infections: the role of TLR2, TLR4 and MyD88 in paracoccidioidomycosis

The aim of this minireview is to present a concise view of the most important pattern recognition receptors used by the innate immune system to sense and control pathogen growth into host tissues. A brief review of the role of Toll-like receptors (TLRs) in fungal infections followed by some recent results on the function of TLR4, TLR2 and the MyD88 adaptor molecule in the pathogenesis of paracoccidioidomycosis are presented.     

TLR4在四氯化碳诱导的肝硬化大鼠骨髓血窦内皮细胞的表达

目的：检测Toll样受体4（TLR4）在四氯化碳诱导的肝硬化大鼠骨髓血窦内皮细胞的表达,为进一步研究肝硬化时骨髓损伤的发生机制提供实验依据。方法：选择Wistar大鼠给予腹腔注射CCl4,一周两次,建立肝硬化大鼠模型。分别于建模8周和12周检测大鼠血浆内毒素的水平,免疫组化检测大鼠骨髓血窦内皮细胞上TLR4的表达情况,RT-PCR测定骨髓组织中TLR4mRNA的表达,分析TLR4的表达与内毒素血症间的关系。结果：给予CCl4 8周和12周时,对照组大鼠血浆内毒素水平分别为（0．216±0．024）Eu／ml和（0．133±0．022）Eu／ml,模型组大鼠血浆内毒素水平分别为（0．626±0．021）Eu／ml和（0．725±0．031）Eu／ml,分别较对照组显著升高,差异均有统计学意义（P〈0．001）;骨髓血窦内皮细胞TLR4蛋白表达及骨髓组织中TLR4mRNA的表达均显著高于对照组,差异均有统计学意义（P〈0．05）。大鼠骨髓TLR4蛋白和mRNA表达与血浆内毒素水平均呈显著正相关（r=0．841,0．803,P均〈0．001）。结论：CCl4诱导的肝硬化大鼠骨髓血窦内皮细胞TLR4表达升高,并伴随大鼠内毒素血症的发生,提示肝硬化时肠源性内毒素血症可能参与了骨髓的造血功能的损害和病变。     

TLR4 在四氯化碳诱导的肝硬化大鼠骨髓血窦内皮细胞的表达

目的：检测Toll样受体4(TLR4)在四氯化碳诱导的肝硬化大鼠骨髓血窦内皮细胞的表达,为进一步研究肝硬化时骨髓损伤 的发生机制提供实验依据。方法：选择Wistar大鼠给予腹腔注射CCl4,一周两次,建立肝硬化大鼠模型。分别于建模8 周和12 周 检测大鼠血浆内毒素的水平,免疫组化检测大鼠骨髓血窦内皮细胞上TLR4 的表达情况,RT-PCR 测定骨髓组织中TLR4 mRNA 的表达,分析TLR4 的表达与内毒素血症间的关系。结果：给予CCl4 8 周和12 周时,对照组大鼠血浆内毒素水平分别为(0.216± 0.024) Eu/ml 和(0.133± 0.022) Eu/ml,模型组大鼠血浆内毒素水平分别为(0.626± 0.021) Eu/ml 和(0.725± 0.031) Eu/ml,分别较对 照组显著升高,差异均有统计学意义(P<0.001);骨髓血窦内皮细胞TLR4蛋白表达及骨髓组织中TLR4 mRNA的表达均显著高于 对照组,差异均有统计学意义(P<0.05)。大鼠骨髓TLR4 蛋白和mRNA表达与血浆内毒素水平均呈显著正相关（r=0.841,0.803,P 均<0.001）。结论：CCl4 诱导的肝硬化大鼠骨髓血窦内皮细胞TLR4 表达升高,并伴随大鼠内毒素血症的发生,提示肝硬化时肠源 性内毒素血症可能参与了骨髓的造血功能的损害和病变。     

Role of TLR2- and TLR4-mediated signaling in Mycobacterium tuberculosis-induced macrophage death

Infection of macrophages with Mycobacterium tuberculosis (Mtb) induces cell death by apoptosis or necrosis. TLRs 2 and 4 recognition of mycobacterial ligands has been independently associated to apoptosis induction. To try to understand the particular contribution of these receptors to apoptotic or necrotic signaling upon infection with live Mtb H37Rv, we used macrophage lines derived from wild-type or TLR2-, TLR4-, and MyD88-deficient mouse strains. Mtb-infection triggered apoptosis depending on a TLR2/TLR4/MyD88/p38/ERK/PI-3K/NF-kB pathway; however, necrosis was favored in absence of TLR4 signaling independently of p38, ERK1/2, PI-3K or NF-κB activity. In conclusion, our results indicate that cooperation between TLR2- and TLR4-dependent mediated signals play a critical role in macrophage apoptosis induced by Mtb and the TLR4-mediated signaling has important role in the maintenance of the balance between apoptotic vs. necrotic cell death induced by macrophage infection with Mtb.     

The BET/BRD inhibitor JQ1 attenuates diabetes-induced cognitive impairment in rats by targeting Nox4-Nrf2 redox imbalance

Diabetes-induced oxidative damage is believed to play an important role in the development of cognitive dysfunction. In this study, the involvement of the Nox4-Nrf2 redox imbalance was investigated. STZ-induced diabetic rats exhibited obvious oxidative stress and apoptosis in the hippocampus assessed by augmentation of lipid peroxidation, positive TUNEL staining, elevated ratio of Bax/Bcl-2 and increased caspase 3 activity. Furthermore, hyperglycemia markedly increased Nox4 activity and reduced the activation of Nrf2 by suppressing its up-stream regulatory Akt as well as down-stream target HO-1. Significant improvement of cognitive performance was observed after treatment with the BET/BRD inhibitor JQ1, accompanied by decreased oxidative stress, neuroinflammation and apoptosis in the hippocampus. JQ1 treatment also improved changes in the neuronal cell morphology as well as increased the expression of p-AKT, Nrf2 and HO-1. Our results provide evidence indicating that JQ1 treatment could modulate Nox4-Nrf2 redox imbalance in the hippocampus and may be a promising agent for diabetes-associated cognitive dysfunction.     

TLR7在1型糖尿病大鼠肾缺血再灌注损伤中的作用研究

目的:探讨Toll样受体7(TLR7)介导的My D88/NF-κB信号通路在1型糖尿病大鼠肾缺血再灌注损伤中的作用。方法:雄性SD大鼠随机分为3组(n=6),糖尿病假手术组(DS),糖尿病缺血再灌注组(DIR),糖尿病缺血再灌注+氯喹预处理组(DIR+CQ)。采用腹腔注射链尿佐菌素65 mg/kg建立糖尿病模型,TLR7抑制剂氯喹预处理于糖尿病模型成功后第3周0.5%氯喹40 mg/kg进行腹腔注射,连续给药7天。于第四周采用双侧肾蒂夹闭25 min,再灌注48 h建立肾缺血再灌注损伤模型。取大鼠肾脏HE染色观察大鼠病理学结果,血标本测定血尿素氮(BUN)和血肌酐(Scr)水平,ELISA法检测白细胞介素6(IL-6)和肿瘤坏死因子-α(TNF-α),TUNEL法检测细胞凋亡,Western blot检测TLR7,My D88和NF-κB蛋白表达。结果:与DS组相比,DIR组肾小管肿胀,间质水肿,刷状缘丢失,空泡变性坏死,Paller评分升高(P0.01)。与DIR组相比,氯喹预处理可以改善肾损伤(P=0.017);与DS组相比,DIR组BUN,Scr,IL-6,TNF-α,细胞调亡指数(Apoptosis%),TLR7,My D88,NF-κB增高(P0.05);与DIR组相比,DIR+CQ组BUN,Scr,IL-6,TNF-α,Apoptosis%,TLR7,My D88,NF-κB降低(P0.05)。结论:TLR7介导的My D88/NF-κB信号通路参与糖尿病肾缺血再灌注损伤,氯喹通过抑制TLR7表达,阻断My D88/NF-κB信号通路,降低炎症反应,从而减轻1型糖尿病大鼠肾缺血再灌注损伤。