Structural Analysis of Breast-Milk αS1-Casein: An α-Helical Conformation Is Required for TLR4-Stimulation.

Breast-milk αS1-casein is a Toll-like receptor 4 (TLR4) agonist, whereas phosphorylated αS1-casein does not bind TLR4. The objective of this study was to analyse the structural requirements for these effects. In silico analysis of αS1-casein indicated high α-helical content with coiled-coil characteristics. This was confirmed by CD-spectroscopy, showing the α-helical conformation to be stable between pH 2 and 7.4. After in vitro phosphorylation, the α-helical content was significantly reduced, similar to what it was after incubation at 80 °C. This conformation showed no in vitro induction of IL-8 secretion via TLR4. A synthetic peptide corresponding to V77-E92 of αS1-casein induced an IL-8 secretion of 0.95 ng/mL via TLR4. Our results indicate that αS1-casein appears in two distinct conformations, an α-helical TLR4-agonistic and a less α-helical TLR4 non-agonistic conformation induced by phosphorylation. This is to indicate that the immunomodulatory role of αS1-casein, as described before, could be regulated by conformational changes induced by phosphorylation.

Polysaccharides from sea buckthorn (Hippophae rhamnoides L.) berries ameliorate cognitive dysfunction in AD mice induced by a combination of d-gal and AlCl3 by suppressing oxidative stress and inflammation reaction.

BACKGROUND: The therapeutic properties of Hippophae rhamnoides L. were known in Ancient Greece and in Tibetan and Mongolian medicine, which commonly used it for the treatment of heart ailments, rheumatism, and brain disorders. Modern studies have indicated that Hippophae rhamnoides L. polysaccharide (HRP) can improve cognitive impairment in mice with Alzheimer's disease (AD) but the specific mechanisms of the protective effect of HRP have not been elucidated fully. RESULTS: Our results showed that Hippophae rhamnoides L. polysaccharide I (HRPI) improved pathological behaviors related to memory and cognition, and reduced 1 Beta-amyloid (Aß) peptide deposition and neuronal cell necrosis. Pretreatment with Hippophae rhamnoides L. polysaccharide I (HRPI) also decreased the level of Toll-like receptor 4 (TLR4) and Myeloid differentiation factor 88 (MyD88), and reduced the release of inflammatory factors Tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6) in the brains of mice with AD. Treatment with HRPI also suppressed the expression level of Recombinant Kelch Like ECH Associated Protein 1 (KEAP1), and increased the levels of Nuclear factor erythroid 2-Related Factor 2 (Nrf2), antioxidant enzymes Superoxide dismutase (SOD) and Glutathione peroxidase (GSH-Px) in the brains of AD mice. CONCLUSIONS: On the whole, these findings revealed that HRPI could improve the learning and memory ability and attenuate pathologic impairment in AD mice, and the underlying mechanisms may involve mediating oxidative stress and inflammation, possibly through the regulation of the Keap1/Nrf2 and TLR4/MyD88 signaling pathways. © 2023 Society of Chemical Industry.

Gas chromatography (GC) fingerprinting and immunomodulatory activity of polysaccharide from the rhizome of Menispermum dauricum DC.

This research aimed to establish the gas chromatography (GC) fingerprints and examine the immunomodulatory activity of the rhizome of Menispermum dauricum polysaccharides. In this study, the preparation conditions were optimized by the response surface method (RSM). GC is an effective and sensitive technique employed to measure the composition of monosaccharides; the GC fingerprints of total polysaccharides from 10 batches of the rhizome of M. dauricum (tMDP) were established, and chemometrics methods were adopted to examine the differences and similarities of tMDP from distinct regions. The similarity evaluation illustrated that the polysaccharides derived from the rhizome of M. dauricum from different origins were highly similar. The results of principal components analysis (PCA) illustrated that all the tMDPs may be integrated into one group within the 95% confidence interval, but the rhizome of M. dauricum from different origins could also be distinguished in the plot of PCA scores. Then, the major bioactive fraction MDP was purified and obtained by column chromatography. Our previous study showed that MDP exhibited significant immunomodulatory activity, but the mechanism of the in vitro immunomodulatory activity of MDP is unclear. The macrophage activation induced by MDP was abolished when Toll-like receptor 4 (TLR4) signaling was knocked down by the TLR4 inhibitor. Furthermore, western blot analysis illustrated that MDP activated RAW264.7 cells through MAPKs and NFκB pathways induced by TLR4. This research offers a theoretical foundation for quality control and additional study as a potential immunomodulator of MDP.

Contribution of the mutation rs8193069 in TLR4 to mastitis resistance and performance in Holstein cows in southern China.

Bovine mastitis has become increasingly important issues for farmers and consumers, leading to large economic losses in the dairy industry worldwide. Because treatment of mastitis is difficult and costly, improved mastitis resistance through selective breeding would be advantageous. The toll-like receptor 4 (TLR4) is an important player in recognising pathogens and activating immune responses. However, its roles in mastitis occurrence and the underlying molecular mechanisms are unclear. In this study, a single nucleotide polymorphism, rs8193069 (T â†’ C) in TLR4 gene was detected in a Holstein cow resource population in southern China. Association analysis with 5-year production traits, haematology, and biochemistry parameters revealed that individuals with genotype CC had significantly lower somatic cell counts (SCC), lower fat percentage, but higher 305-day milk (p < 0.05) and total milk yield (p < 0.01). Both genotypes CC and CT had lower lymphocyte counts (#LYMPH) (p < 0.01) and basophil counts (#BASO) (p < 0.05) than TT. Genotype CC had a less level of triglyceride (p < 0.01) and creatine kinase (p < 0.05) than CT. Further analysis based on the production data revealed significant positive correlations between SCC and #LYMPH. Analysis of TLR4 protein structure and properties suggested that the missense mutation on the 674th amino acid from Thr to Ile reduced the flexibility and hydrophilicity of TIR domain, implying a weakened binding ability of TLR4 to its adaptors. In conclusion, allele C of rs8193069 was the major allele in Holstein cows that indicated a greater genetic potential to mastitis resistance and milk yields, probably via the LPS-TLR4 inflammatory signalling. This study offers a marker to improve mastitis resistance in the dairy cow population in southern China.

Mechanism of dexmedetomidine preconditioning on spinal cord analgesia in rats with functional chronic visceral pain

Purpose: To analyze the effect and mechanism of dexmedetomidine (DEX) analgesia pretreatment on functional chronic visceral pain in rats. Methods: Rats were divided into six groups: W1, W2, W3, W4, W5, and W6. The behavioral changes and electrophysiological indexes of rats in each group before and after DEX treatment were detected. Results: The levels of abdominal withdrawal reflex (AWR) in W5 and W6 groups were significantly lower than those in group W3, while the levels of thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were significantly higher than those in group W3 (p < 0.05). The electromyographic signals of W1, W5, and W6 groups showed little fluctuation, while those of groups W2, W3, and W4 showed obvious fluctuation. TLR4 mRNA expression, IRF3, P65, and phosphorylation levels in W4, W5, and W6 groups were significantly lower than those in group W2 (p < 0.05). Conclusions: Dexmedetomidine epidural anesthesia pretreatment could significantly inhibit visceral pain response in rats with functional chronic visceral pain, and its mechanism was related to the activation of TLR4 in spinal dorsal horn tissue of rats and the activation inhibition of IRF3 and P65 in the downstream key signals.

Acute kidney injury in a mouse model of meningococcal disease.

INTRODUCTION: Meningococcal disease is associated with high mortality. When acute kidney injury (AKI) occurs in patients with severe meningococcal disease, it is typically attributable to sepsis, although meningococcal disease and lipopolysaccharide release are rarely investigated. Therefore, we evaluated renal tissue in a mouse model of meningococcal disease. METHODS: Female BALB/c mice were induced to AKI by meningococcal challenge. Markers of renal function were evaluated in infected and control mice. RESULTS: In the infected mice, serum concentrations of tumor necrosis factor alpha, interferon gamma, interleukins (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12), and granulocyte-macrophage colony-stimulating factor were elevated, as was renal interstitial infiltration with lymphocytes and neutrophils (p < 0.01 for the latter). Histological analysis showed meningococcal microcolonies in the renal interstitium, without acute tubular necrosis. Infected mice also showed elevated renal expression of toll-like receptor 2, toll-like receptor 4, and Tamm-Horsfall protein. The expression of factors in the intrinsic pathway of apoptosis was equal to or lower than that observed in the control mice. Urinary sodium and potassium were also lower in infected mice, probably due to a tubular defect. CONCLUSION: Our findings corroborate those of other studies of AKI in sepsis. To our knowledge, this is the first time that meningococci have been identified in renal interstitium and that the resulting apoptosis and inflammation have been evaluated. However, additional studies are needed in order to elucidate the mechanisms involved.

Esculetin Alleviates Acute Liver Failure following Lipopolysaccharide/D-Galactosamine in Male C57BL/6 Mice.

Background: Acute liver failure (ALF) is a fatal clinical situation that rapidly leads to the loss of normal liver function. Esculetin is a natural herbal compound used for the management of various diseases such as cardiovascular and renal disorders. In this study, we evaluated the protective effects of esculetin in a mouse model of ALF. Methods: This article is a report on an experimental study that was conducted at Iran University of Medical Sciences in 2019. Forty-eight male C57BL/6 mice were randomly divided into control, LPS/D-Gal, and LPS/D-Gal+Esculetin (40 mg/kg) groups (n=16 per group). ALF was induced with an intraperitoneal injection of lipopolysaccharide (LPS)/D-galactosamine (D-Gal).The LPS/D-Gal group received a mixture of LPS (50 µg/kg) and D-Gal (400 mg/kg). The LPS/D-Gal+Esculetin group received esculetin by gavage 24 hours and one hour before receiving LPS/D-Gal. Six hours after LPS/D-Gal injection, the mice were sacrificed. Liver injury markers, including alanine aminotransferase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP), were measured in the serum. Oxidative stress indices and inflammatory markers such as interleukin-1 beta (IL-1ß), IL-6, and tumor necrosis factor-alpha (TNF-α) were measured in hepatic tissue. The histopathology of liver tissue was also assessed. The data were analyzed using one-way ANOVA, followed by the post hoc Tukey test. Results: Esculetin lowered oxidative stress and myeloperoxidase activity (P<0.001); reduced the serum levels of ALT (P=0.037), AST (P=0.032), and ALP (P=0.004); and decreased the hepatic levels of IL-1ß (P=0.002), IL-6 (P=0.004), toll-like receptor 4 (P<0.001), TNF-α (P=0.003), and nuclear factor-kappa B (P<0.001) as compared with LPS/D-Gal. Additionally, esculetin ameliorated hepatic tissue injury following LPS/D-Gal challenge. Conclusion: Esculetin can reduce liver injury through the mitigation of oxidative burden, inflammation, and neutrophil infiltration and also exerts hepatoprotective effects against ALF.

Therapeutic effects of dihydroartemisinin in multiple stages of colitis-associated colorectal cancer.

Colitis-associated colorectal cancer (CAC) develops from chronic intestinal inflammation. Dihydroartemisinin (DHA) is an antimalarial drug exhibiting anti-inflammatory and anti-tumor effects. Nonetheless, the therapeutic effects of DHA on CAC remain unestablished. Methods: Mice were challenged with azoxymethane (AOM) and dextran sulfate sodium (DSS) to establish CAC models. DHA was administered via oral gavage in different stages of CAC models. Colon and tumor tissues were obtained from the AOM/DSS models to investigate inflammatory responses and tumor development. Inflammatory cytokines in the murine models were detected through qRT-PCR and ELISA. Toll-like receptor 4 (TLR4) signaling-related proteins were detected by western blot. Macrophage infiltration was measured using immunostaining analysis, and apoptosis in the colon cancer cells was detected by flow cytometry and western blot. Results: DHA inhibited inflammatory responses in the early stage of the AOM/DSS model and subsequent tumor formation. In the early stage, DHA reversed macrophage infiltration in colon mucosa and decreased the expression of pro-inflammatory cytokines. DHA inhibited the activation of macrophage by suppressing the TLR4 signal pathway. In the late stage of CAC, DHA inhibited tumor growth by enhancing cell cycle arrest and apoptosis in tumor cells. Administration of DHA during the whole period of the AOM/DSS model generated an addictive effect based on the inhibition of inflammation and tumor growth, thereby improving the therapeutic effect of DHA on CAC. Conclusion: Our study indicated that DHA could be a potent agent in managing the initiation and development of CAC without obvious side effects, warranting further clinical translation of DHA for CAC treatment.

Pathophysiology of reflux oesophagitis: role of Toll-like receptors 2 and 4 and Farnesoid X receptor.

The pathogenesis of gastroesophageal reflux disease (GERD) is not fully understood. It involves the activation of mucosal immune-mediated and inflammatory responses. Toll-like receptors (TLR) 2 and TLR4 are pattern-recognition receptors of the innate immune system; they recognize microbial and endogenous ligands. Farnesoid X receptor (FXR) is a bile acid receptor that regulates the inflammatory response. We aimed to evaluate TLR2, TLR4 and FXR expression patterns in GERD. We re-evaluated 84 oesophageal biopsy samples according to the global severity (GS) score, including 26 cases with histologically normal oesophagus, 28 with histologically mild oesophagitis and 30 with severe oesophagitis. We used immunohistochemistry and in situ hybridization to assess the expression patterns of TLR2, TLR4 and FXR in oesophageal squamous cells. Immunohistochemistry showed that nuclear and cytoplasmic TLR2 was expressed predominantly in the basal layer of normal oesophageal epithelium. In oesophagitis, TLR2 expression increased throughout the epithelium, and the superficial expression was significantly more intensive compared to normal epithelium, p <0.01. Nuclear and cytoplasmic TLR4 was expressed throughout the thickness of squamous epithelium, with no change in oesophagitis. FXR was expressed in the nuclei of squamous cells, and the intensity of the expression increased significantly in oesophagitis (p <0.05). FXR expression correlated with basal TLR2. In situ hybridization confirmed the immunohistochemical expression patterns of TLR2 and TLR4. In GERD, TLR2, but not TLR4, expression was upregulated which indicates that innate immunity is activated according to a specific pattern in GERD. FXR expression was increased in GERD and might have a regulatory connection to TLR2.

Higher Levels of Triglyceride, Fatty Acid Translocase, and Toll-Like Receptor 4 and Lower Level of HDL-C in Pregnant Women with GDM and Their Close Correlation with Neonatal Weight.

OBJECTIVES: In this study, we aimed to compare the levels of maternal blood lipids, placental and venous blood lipid transporters, and inflammatory factor receptors in pregnant women with and without gestational diabetes mellitus (GDM). We also aimed to figure out the relationship between these values and neonatal weight. METHODS: Fifty pregnant women with GDM under blood glucose control belong to the case group, and 50 pregnant women with normal glucose tolerance in concurrent delivery belong to the control group. Fasting venous blood of these pregnant women was taken 2 weeks before delivery, and umbilical cord blood was collected after delivery. The levels of triglyceride (TG), serum total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol (HDL-C) in maternal blood and umbilical cord blood were tested in the laboratory department of our hospital. The level of toll-like receptor 4 (TLR4) in serum of umbilical veins was detected by the double-antibody sandwich ELISA. Western blot and RT-PCR were used to detect the protein and mRNA expressions of TLR4, LPL, and FAT/CD36 in the placenta. RESULTS: The level of TG in maternal blood in the case group was remarkably higher than that in the control group, which was opposite to the level of HDL-C. In the umbilical cord blood of women with GDM, the expression of TLR4 increased and was closely correlated with neonatal weight. In the placenta of women with GDM, the expressions of FAT/CD36 and TLR4 increased, and both of them were closely correlated with neonatal weight. Besides, TLR4 in umbilical cord blood increased and was closely correlated with neonatal weight. Although the expression of LPL in the placenta decreased, it had no obvious correlation with neonatal weight. CONCLUSIONS: TG in maternal blood, TLR4 in the placenta and umbilical cord blood, and FAT/CD36 in the placenta were positively correlated with neonatal weight. However, HDL-C in maternal blood was negatively correlated with neonatal weight. Although the expression of LPL in the placenta reduced due to GDM, it had no correlation with neonatal weight.

Measurement of Macrophage Toll-Like Receptor 4 Expression After Morphine Treatment.

The immune system is a complex and finely orchestrated system, and many soluble molecules and receptors contribute to its regulation.Recent studies have suggested that many of the modulatory effects induced by morphine on innate immunity, and in particular the effects on macrophage activation and function, can be due to the modulation of an important macrophage surface receptor, the toll-like receptor (TLR), that is primarily involved in early regulatory steps. In this chapter we describe a RT-real-time PCR method for assessing TLR expression in macrophage after in vivo morphine treatment.

Cerebellar and cortical TLR4 activation and behavioral impairments in Wernicke-Korsakoff Syndrome: Pharmacological effects of oleoylethanolamide.

Wernicke-Korsakoff Syndrome (WKS) is a neuropsychiatric disorder whose etiology is a thiamine deficiency (TD), with alcoholism being the main underlying cause. Previous evidence suggests the presence of initial neuroinflammation and oxidative/nitrosative stress in the physiopathology, although the specific molecular mechanisms underlying TD-induced brain damage and behavioral disabilities are unknown. We explored the specific role of the innate immune receptor TLR4 in three murine models of WKS, based on the combination of a thiamine-deficient diet and pyrithiamine injections (0.25 mg/kg, i.p.) over time. The Symptomatic Model (SM) allowed us to describe the complete neurological/neurobehavioral symptomatology over 16 days of TD. Animals showed an upregulation of the TLR4 signaling pathway both in the frontal cortex (FC) and cerebellum and clear motor impairments related with cerebellar dysfunction. However, in the Pre-Symptomatic Model (PSM), 12 days of TD induced the TLR4 pathway upregulation in the FC, which correlated with disinhibited-like behavior, but not in the cerebellum, and no motor impairments. In addition, we tested the effects of the biolipid oleoylethanolamide (OEA, 10 mg/kg, i.p., once daily, starting before any symptom of the pathology is manifested) through the Glucose-Precipitated Model (GPM), which was generated by glucose loading (5 g/kg, i.v., last day) in thiamine-deficient animals to accelerate damage. Pretreatment with OEA prevented the TLR4-induced signature in the FC, as well as an underlying incipient memory disability and disinhibited-like behavior. This study suggests a key role for TLR4 in TD-induced neuroinflammation in the FC and cerebellum, and it reveals different vulnerability of these brain regions in WKS over time. Pre-treatment with OEA counteracts TD-induced TLR4-associated neuroinflammation and may serve as co-adjuvant therapy to prevent WKS-induced neurobehavioral alterations.

TLR4 signals through islet macrophages to alter cytokine secretion during diabetes.

Toll-like receptors (TLRs), particularly TLR4, may act as immune sensors for metabolic stress signals such as lipids and link tissue metabolic changes to innate immunity. TLR signalling is not only tissue-dependent but also cell-type dependent and recent studies suggest that TLRs are not restricted to innate immune cells alone. Pancreatic islets, a hub of metabolic hormones and cytokines, respond to TLR signalling. However, the source of TLR signalling within the islet remain poorly understood. Uncovering the specific cell source and its role in mediating TLR signalling, especially within type 2 diabetes (T2D) islet will yield new targets to tackle islet inflammation, hormone secretion dysregulation and ultimately diabetes. In the present study, we immuno-characterised TLRs linked to pancreatic islets in both healthy and obese diabetic mice. We found that while TLRs1-4 and TLR9 were expressed in mouse islets, these TLRs did not co-localise with insulin-producing ß-cells. ß-Cells from obese diabetic mice were also devoid of these TLRs. While TLR immunoreactivity in obese mice islets increased, this was driven mostly by increased islet endothelial cell and islet macrophage presence. Analysis of human islet single-cell RNA-seq databases revealed that macrophages were an important source of islet TLRs. However, only TLR4 and TLR8 showed variation and cell-type specificity in their expression patterns. Cell depletion experiments in isolated mouse islets showed that TLR4 signalled through macrophages to alter islet cytokine secretome. Together, these studies suggest that islet macrophages are a dominant source of TLR4-mediated signalling in both healthy and diabetic islets.

The prognostic role of tissue TLR2 and TLR4 in colorectal cancer.

Colorectal cancer (CRC), the second most common cancer globally, resulted in 881,000 deaths in 2018. Toll-like receptors (TLRs) are crucial to detecting pathogen invasion and inducing the host's immune response. This study aimed to explore the prognostic value of TLR2 and TLR4 tumor expressions in colorectal cancer patients. We studied the immunohistochemical expressions of TLR2 and TLR4 using tissue microarray specimens from 825 patients undergoing surgery in the Department of Surgery, Helsinki University Hospital, between 1982 and 2002. We assessed the relationships between TLR2 and TLR4 expressions and clinicopathological variables and patient survival. We generated survival curves using the Kaplan-Meier method, determining significance with the log-rank test. Among patients with lymph node-positive disease and no distant metastases (Dukes C), a strong TLR2 immunoactivity associated with a better prognosis (p < 0.001). Among patients with local Dukes B disease, a strong TLR4 immunoactivity associated with a worse disease-specific survival (DSS; p = 0.017). In the multivariate survival analysis, moderate TLR4 immunoactivity compared with strong TLR4 immunoactivity (hazard ratio (HR) 0.66, 95% confidence interval (CI) 0.49-0.89, p = 0.007) served as an independent prognostic factor. In the multivariate analysis for the Dukes subgroups, moderate TLR2 immunoactivity (HR 2.63, 95% CI 1.56-4.44, p < 0.001) compared with strong TLR2 immunoactivity served as an independent negative prognostic factor in the Dukes C subgroup. TLR2 and TLR4 might be new prognostic factors to indicate which CRC patients require adjuvant therapy and which could spare from an unnecessary follow-up, but further investigations are needed.

The TLR4/ERK/PD­L1 axis may contribute to NSCLC initiation.

Infection and inflammation serve an important role in tumor development. Toll­like receptor 4 (TLR4) is a pivotal component of the innate and adaptive immune response during infection and inflammation. Programmed­death ligand 1 (PD­L1) is hypothesized as an important factor for non­small cell lung cancer (NSCLC) immune escape. In the present study, the relationship between TLR4 and PD­L1, in addition to the associated molecular mechanism, were investigated. TLR4 and PD­L1 expression in lung cancer tissues were detected using immunohistochemistry, whilst overall patient survival was measured using the Kaplan­Meier method. The A549 cell line stimulated using lipopolysaccharide (LPS) was applied as the in vitro inflammatory NSCLC model. Associated factors were investigated using reverse transcription­quantitative PCR and western blotting. Lung cancer tissues exhibited increased PD­L1 and TLR4 levels compared with those of adjacent para­cancerous tissues, where there was a positive correlation between TLR4 and PD­L1 expression. In addition, increased expression of these two proteins was found to be linked with poorer prognoses. Following the stimulation of A549 cells with LPS, TLR4 and PD­L1 expression levels were revealed to be upregulated in a dose­dependent manner, where the ERK and PI3K/AKT signaling pathways were found to be activated. Interestingly, in the presence of inhibitors of these two pathways aforementioned, upregulation of PD­L1 expression was only inhibited by the MEK inhibitor PD98059, which can inhibit ERK activity. These data suggested that the ERK signaling pathway is necessary for the TLR4/PD­L1 axis. In conclusion, data from the present study suggest that TLR4 and PD­L1 expression can serve as important prognostic factors for NSCLC, where TLR4 activation may induce PD­L1 expression through the ERK signaling pathway.

Variants in XPC, Noxa and TLR4 genes are not associated with bleomycin-induced lung injury in Hodgkin lymphoma patients.

Aim: Pulmonary toxicity is a well-known adverse reaction of bleomycin. In this study, we investigated the influence of XPC, PMAIP1/Noxa and TLR4 genetic variants on the development of bleomycin-induced lung injury (BILI) in south Indian patients with Hodgkin lymphoma. Materials & methods: Hodgkin lymphoma patients receiving adriamycin, bleomycin, vinblastine and dacarbazine regimen were recruited for the study and BILI was diagnosed based on symptoms and/or radiological signs. DNA samples were genotyped using real-time PCR. Results: A total of 78 patients were recruited in the study and BILI was observed in 17 (21.8%) patients. Polymorphisms in XPC, PMAIP1/Noxa and TLR4 genes were not associated with the development of BILI. Conclusion: The selected genetic polymorphisms do not predict the risk of BILI in south Indian population.

Expression profile of Toll-like receptor 4 in rat testis and epididymis throughout postnatal development.

Toll-like receptors (TLRs) belonging to pattern recognition receptors are involved in maintaining testicular and epididymal immune homeostasis. The purpose of the current study was to investigate TLR4 expression in rat testis and epididymis throughout postnatal development. Weak staining was detected in peritubular myoid cells and immature Sertoli cells while no staining was observed in gonocytes during prepubertal period. However, TLR4 expression began to appear in spermatocytes in pubertal period and gradually increased in spermatids. An intense staining was observed in steps 5-19 spermatids in post pubertal and mature periods. Similarly, TLR4 expression in the testes steadily increased from pubertal period to mature period. Puberty also caused a significant increase in TLR4 expression in epididymis. TLR4 expression in cauda epididymis was lower as compared to those of other epididymal segments. The majority of epididymal epithelial cells exhibited apical TLR4 expression, whereas basal cells showed intense intracytoplasmic immunoreaction. We detected an intense staining in epididymal smooth muscle cells. The expression levels of TLR4 showed dynamic changes in both spermatogenic cells, and entire testicular and epididymal tissues during postnatal development. These results suggest that TLR4 expression contributes not only to inflammation but also to the development of spermatogenic cells.

TLR2 and TLR4 Surface and Gene Expression in White Blood Cells after Fasting and Oral Glucose, Lipid and Protein Challenges: Influence of Obesity and Sex Hormones.

We studied if macronutrients of the diet have different effects on leukocyte activation, and if these effects are influenced by sex hormones or obesity. We analyzed leukocyte cell surface and gene expression of toll-like receptors 2 and 4 (TLR2 and TLR4) during fasting and after macronutrient loads in women with polycystic ovary syndrome and female and male controls. Fasting TLR2 surface expression in neutrophils was higher in men than in women. Obese subjects presented higher TLR2 gene expression than nonobese individuals, particularly in men. In contrast, surface TLR4 expression was lower in men and in obese individuals. Postprandial cell-surface expression decreased similarly after all macronutrient loads. Neutrophil TLR2 decreased only in obese subjects whereas TLR4 showed a greater decrease in nonobese individuals. However, TLR2 gene expression increased after glucose ingestion and decreased during the lipid load, while TLR4 was induced in response to lipids and mostly to glucose. Postprandial TLR gene expression was not influenced by group of subjects or obesity. Both cell-surface and gene postprandial expression inversely correlated with their fasting levels. These responses suggest a transient compensatory response aiming to prevent postprandial inflammation. However, obesity and sex hormones showed opposite influences on surface expression of TLR2 and TLR4, but not on their gene expression, pointing to regulatory posttranscriptional mechanisms.

The effect of fenofibrate, a PPARα activator on toll-like receptor-4 signal transduction in melanoma both in vitro and in vivo.

BACKGROUND: The anti-cancer effect of peroxisome proliferator-activated receptor (PPAR) α ligands on growth and metastatic potential of melanoma cells has been shown previously. However, the mechanism underlying these effects remains to be elucidated. Here, we investigated the effects of fenofibrate (PPAR ligand) on Toll-like receptor-4 (TLR-4) signaling in mice melanoma. METHODS: Mice melanoma cells (B16F10) were treated with fenofibrate or LPS or LPS + fenofibrate or pre-treated with CLI-095 (a TLR4 inhibitor), followed by fenofibrate. In in vivo model, C57BL/6 mice were subcutaneously injected with B16F10 cells (with/without LPS pre-treatment), and fenofibrate was administrated after development of palpable tumors. Cell proliferation, the expression level of Tlr4, Myd88, Nf-κb1 genes, TLR-4 protein expression, TNF-α levels, and tumor volume were measured. RESULT: Our results indicated that fenofibrate significantly inhibited the Tlr-4, Myd-88, and Nf-kb1 mRNA expression and TNF-α concentration in B16F10 LPS-stimulated cells. In addition, blocking TLR-4 signaling increased the anti-inflammatory potential of fenofibrate. Also fenofibrate can reduce LPS-induced tumor volume, Tlr-4, Myd-88, Nf-kb1 mRNA, and TLR-4 protein expression in tumor tissue and also TNF-α level in tumor tissue lysate. CONCLUSION: Our data indicate that fenofibrate may exert its anti-melanoma effects via interaction with TLR4-dependent signaling pathway (TLR-4/MyD-88/ NF-kB).