Extracellular Vesicles Released from Macrophages Infected with Mycoplasma pneumoniae Stimulate Proinflammatory Response via the TLR2-NF-κB/JNK Signaling Pathway.

Mycoplasma pneumoniae (M. pneumoniae, Mp) is an intracellular pathogen that causes pneumonia, tracheobronchitis, pharyngitis, and asthma in humans and can infect and survive in the host cells leading to excessive immune responses. Extracellular vesicles (EVs) from host cells carry components of pathogens to recipient cells and play a role in intercellular communication during infection. However, there is limited knowledge on whether EVs derived from M. pneumoniae-infected macrophages play as intercellular messengers and functional mechanisms. In this study, we establish a cell model of M. pneumoniae-infected macrophages that continuously secrete EVs to further asses their role as intercellular messengers and their functional mechanisms. Based on this model, we determined a method for isolating the pure EVs from M. pneumoniae-infected macrophages, which employs a sequence of operations, including differential centrifugation, filtering, and ultracentrifugation. We identified EVs and their purity using multiple methods, including electron microscopy, nanoparticle tracking analysis, Western blot, bacteria culture, and nucleic acid detection. EVs from M. pneumoniae-infected macrophages are pure, with a 30-200 nm diameter. These EVs can be taken up by uninfected macrophages and induce the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and IL-8 through the nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPK) signals pathway. Moreover, the expression of inflammatory cytokines induced by EVs relies on TLR2-NF-κB/JNK signal pathways. These findings will help us better understand a persistent inflammatory response and cell-to-cell immune modulation in the context of M. pneumoniae infection.

Mechanism of protection from insulin resistance by toll-like receptor 2 deficiency in high-fat diet fed mice: involvement in macrophage polarization.

BACKGROUND: Toll-like receptor 2 (TLR2) deficiency can increase insulin sensitivity and improves glucose tolerance. However, it is not yet fully understood about its underlying mechanism. The regulation of M1/M2 macrophage polarization has been verified to involve in insulin resistance. Here, we evaluated whether the beneficial effect of TLR2 deficiency is mediated by TLR2-associated macrophage polarization in mice fed with high-fat diet (HFD). METHODS AND RESULTS: Wild-type and TLR2 knockout (TLR2-/-) mice received HFD for two months. Following intraperitoneal glucose tolerance and insulin resistance tests, peripheral monocytes were isolated, and in vitro induced for differentiation into M1 and M2 macrophages, respectively. Macrophages polarization was evaluated using flow cytometry. The expression of macrophage polarization marker genes and cytokine production in visceral adipose tissue were measured by qRT-PCR and ELISA. Compared to wild-type mice, TLR2-/- mice showed higher glucose tolerance and insulin sensitivity, along with significantly reduced the population of M1 and increased M2 count in vitro. Additionally, TLR2-/- mice demonstrated higher expression of M2 marker iNOS mRNA and interleukin-10 level in adipose tissues. CONCLUSIONS: Our results reveal that TLR2 knockout-mediated macrophages M2 polarization is a crucial factor for preventing against diet-induced insulin resistance in mice. These findings deepen our knowledge about the mechanism underlying HFD-induced insulin resistance.

Toll-like receptor 2 and α-Smooth Muscle Actin expressed in the tunica of a urochordate, Styela plicata.

The tunicate, Styela plicata (Lesueur, 1823) present an open circulator system with a tubular heart and blood flowing in lacunae among organs, bathing the tissues directly. Blood vascular lacunae are present in the tunica that is situated outside the epidermis and present a fibrous structure. The cells of the tunic are in straight contact with the blood vessels or are highly mobile. Ascidians are considered model organisms in comparative immunology of the chordate, and hold an important phylogenetic position as sister group of vertebrates. In recent years, numerous studies have reported the presence of Toll-like receptors (TLRs) in the genome of non-mammalian organisms including invertebrates. Two TLRs, designated Ci-TLR1 and Ci-TLR2 were expressed in the stomach, intestine and in numerous hemocytes of Ciona intestinalis, demonstrating that these key transmembrane proteins are evolutionarily conserved in ascidians. In this study for the first time, hemocytes aggregates were identified by confocal immunofluorescence techniques, using TLR2 antibody in the tunica of Styela plicata; furthermore, α-Smooth Muscle Actin (α-SMA) expression has been shown in the cells lining the vessels of the tunic. Our results support the view that the TLR-mediated innate immune functions are conserved in ascidian tissues.

Toll-like receptor 2 activation and up-regulation by high mobility group box-1 contribute to post-operative neuroinflammation and cognitive dysfunction in mice.

Post-operative cognitive dysfunction (POCD) is common and is associated with poor clinical outcome. Toll-like receptor (TLR) 3 and 4 have been implied in the development of POCD. The role of TLR2, a major brain TLR, in POCD is not clear. High mobility group box-1 (HMGB1) is a delayed inflammatory mediator and may play a role in POCD. The interaction between HMGB1 and TLRs in the perioperative period is not known. We hypothesize that TLR2 contributes to the development of POCD and that HMGB1 regulates TLR2 for this effect. To test these hypotheses, 6- to 8-week old male mice were subjected to right carotid artery exposure under isoflurane anesthesia. CU-CPT22, a TLR1/TLR2 inhibitor, at 3 mg/kg was injected intraperitoneally 30 min before surgery and 1 day after surgery. Glycyrrhizin, a HMGB1 antagonist, at 200 mg/kg was injected intraperitoneally 30 min before surgery. Mice were subjected to Barnes maze and fear conditioning tests from 1 week after surgery. Hippocampus and cerebral cortex were harvested 6 hr or 12 hr after the surgery for Western blotting, ELISA, immunofluorescent staining, and chromatin immunoprecipitation. There were neuroinflammation and impairment of learning and memory in mice with surgery. Surgery increased the expression of TLR2 and TLR4 but not TLR9 in the brain of CD-1 male mice. CU-CPT22 attenuated surgery-induced neuroinflammation and cognitive impairment. Similarly, surgery induced neuroinflammation and cognitive dysfunction in C57BL/6J mice but not in TLR2-/- mice. TLR2 staining appeared in neurons and microglia. Surgery increased HMGB1 in the cell nuclei of the cerebral cortex and hippocampus. Glycyrrhizin ameliorated this increase and the increase of TLR2 in the hippocampus after surgery. Surgery also increased the amount of tlr2 DNA precipitated by an anti-HMGB1 antibody in the hippocampus. Our results suggest that TLR2 contributes to surgery-induced neuroinflammation and cognitive impairment. HMGB1 up-regulates TLR2 expression in the hippocampus after surgery to facilitate this contribution. Thus, TLR2 and HMGB1 are potential targets for reducing POCD.

Toll-like receptors as diagnostic targets in pellucid marginal degeneration.

The main purpose of this study is to evaluate the diagnostic role of Toll-like receptors 2 (TLR2) and 4 (TLR4) expression in corneal and conjunctival epithelial cells of eyes with pellucid marginal degeneration (PMD) compared to keratoconus patients (KC) and control subjects. A prospective case-control study in 29 PMD eyes, 109 KC eyes and 72 healthy eyes was done. All participants were subjected to a clinical, topographic, aberrometric and tomographic exam with extraction of corneal and conjunctival epithelial cells through scraping. The TLR2 and TLR4 expression was measured with flow cytometry. Receiver operating characteristic (ROC) curve analysis was used to determine the most appropriate cutoff point for predicting the risk of PMD and KC. Correlations between TLR2/TLR4 expression and the severity of PMD/KC were evaluated. A TLRs follow-up review was made 19 ± 4 months after to the first review. As result, mean expression of TLR2 and TLR4 in both corneal and conjunctival epithelial cells was significantly higher in eyes with corneal ectasia (PMD and KC) than in control eyes (all p < 0.05). Conjunctival TLR4 expression showed the highest capacity to diagnose the existence of PMD (odd ratio 42.84; 95% confidence interval:6.20-296.20; p < 0.0001) after adjusting by eye rubbing and steeper corneal meridian. Moreover, we found an association between the TLR2/TLR4 overexpression with the severity of the PMD and KC measured by corneal topographic, aberrometric and tomographic quantitative parameters (all p < 0.05). Differences on TLR2/TLR4 expression between study groups were maintained during the follow-up period. In conclusion, the TLR2/TLR4 overexpression in corneal and conjunctival epithelial cells of PMD and KC patients compared to healthy control subjects have demonstrated their role as diagnostic target in both corneal ectatic disorders.

Circadian Rhythms of Endothelial Nitric Oxide Synthase and Toll-like Receptors 2 Production in Females with Rheumatoid Arthritis Depending on NOS3 Gene Polymorphism.

BACKGROUND: Rheumatoid Arthritis (RA) is an autoimmune polygenic disease characterized by rapid disability progression and high prevalence. Progression of RA is closely associated with chronobiological changes in the production of some hormones and inflammatory mediators, influencing the disease course and therapy efficacy. The main pathogenetic mechanism of RA is angiogenesis, which is controlled by biological clock-genes. Further investigation of circadian rhythms of angiogenic mediators production in RA patients may be considered as important and relevant. The aim of this study was to establish daily variability of serum endothelial Nitric Oxide Synthase (NOS3) and toll-like receptors 2 (sTLR2) levels in female RA patients depending on the NOS3 gene polymorphism. METHODS: We examined 173 RA patients (100% female) aged 43.7 ± 7.35 years and 34 age-matched healthy women without joint diseases and autoimmune diseases (control). RA was diagnosed by ACR/EULAR 2010 criteria. Blood serum NOS3 and sTLR2 levels were determined at 08:00 and 20:00 using Cloud-Clone Corp kits (USA). NOS3 T-786С (rs2070744) polymorphism was determined by Real-Time PCR (Bio-Rad iCycler IQ5) using SNP-express kits. The SPSS22 software package was used for statistical processing of the results. RESULTS: Females with RA demonstrated oppositely directed serum NOS3 and sTLR2 daily changes: NOS3 level in the morning (08:00) was lower than in the evening (+ 45.5 ± 30.7%), and sTLR2 level in the evening (at 20:00) was lower than in the morning (-21.6 ± 13.1%). RA patients had differences in NOS3 and sTLR2 production depending on NOS3 T786C genotype. CC subjects had NOS3 level at 08:00, 20:00 and day average levels lower (16-25%), and sTLR2 level higher (24-27%) than those of TT subjects. RA patients, carriers of CC genotype, had higher chances of NOS3 and sTLR2 aberrant production compared to TT and TC genotype carriers (OR = 2.99 and 4.79, respectively). CONCLUSION: RA patients demonstrated oppositely directed circadian changes of serum NOS3 and sTLR2. CC genotype carriers had lower NOS3 and higher sTLR2 production rates than TT and TC genotype carriers.

[Increased myocardial expression of Toll-like receptors 2 and 9 as a marker of active myocarditis and a possible predictor of therapeutic effectiveness]. / Povyshennaia ékspressiia toll-like-retseptorov 2-go i 9-go tipov v miokarde kak marker aktivnogo miokardita i vozmozhnyi prediktor éffektivnosti lecheniia.

AIM: to investigate the myocardial expression of some structural proteins and markers of cellular proliferation and innate immunity for assessing their possible diagnostic and prognostic role in patients with chronic myocarditis. SUBJECTS AND METHODS: The investigation enrolled 23 patients (16 men; mean age, 52.0±12.4 years (range, 27 to 73) with various forms of noncoronarogenic myocardial injury who underwent right ventricular endomyocardial biopsy (n=4), intraoperative left ventricular biopsy (n=17) or autopsy (n=2). Prior to their morphological examination, the patients were divided into two groups: 1) 10 patients with dilated cardiomyopathy and presumptive myocarditis; 2) 13 patients with valvular heart disease, hypertrophic cardiomyopathy, myxoma, and chronic pulmonary thromboembolism, presumptively without myocarditis. Along with myocardial histological and immunohistochemical (IHC) examinations, the expression of vimentin, desmin, c-kit, Ki-67, and Toll-like receptors (TLR) 2 and 9 was determined. Polymerase chain reaction was used to identify whether herpes viruses of and parvovirus B19 genomes were present in the blood and myocardial samples; indirect ELISA was applied to estimate the blood level of antibodies against various cardiac antigens. RESULTS: According to the histological findings, active/borderline lymphocytic myocarditis was diagnosed in all the patients (Group 1) and in 6 patients (Group 2) in conjunction with the underlying disease (only in 9 and 7 patients, respectively), viral genome was detected in the myocardium of 15 patients, including in 5 without morphological signs of myocarditis (parvovirus B19 (n=11), herpesvirus 6 (n=4), herpes simplex virus types 1 and 2 (n=1), Epstein-Barr virus (n=2), and cytomegalovirus (n=1)), and in the blood (n=4). A marked correlation was found between TLR2 and TLR9 expressions and the morphological pattern of active myocarditis in the absence of this correlation with the expression level of other studied markers. The expression level of TLR2 in patients with and without borderline myocarditis was 0 [0; 0,75] and in those with active myocarditis was 1.5 [1; 1,5] points; that of TLR9 was 2 [2; 2] and 4 [3; 4] points, respectively (p<0.001). The expression of TLR2 and TLR9 in patients with borderline myocarditis was lower than in those without myocarditis (0 [0; 0] versus 0 [0; 1] and 2 [1,5; 2] versus 2 [2; 3] points), which can reflect cardiomyocyte destruction/depletion at later stages of the disease. There was also a close correlation between the expression level of TLR2 and that of TLR9 (r=0.824; p<0.001) and with Ki-67 levels (r=-0.531 and r=-0.702; p<0.01). There was also a correlation of the expression of the studied markers with viral persistence (desmin), the degree of myocardial dysfunction and cardiosclerosis (c-kit), which calls for further investigations. CONCLUSION: Determination of the myocardial expression level of TLR2 and TLR9 may serve as an immunohistochemical marker for myocarditis and preservation of its activity, which is especially valuable in patients with borderline forms. The marked expression of these markers for innate immunity may reflect both one of the mechanisms of genetic predisposition to myocarditis and its severe course and their secondary activation in the pathogenesis of the disease and is a potential target of therapy.

Paeoniflorin attenuates incipient diabetic nephropathy in streptozotocin-induced mice by the suppression of the Toll-like receptor-2 signaling pathway.

Toll-like receptors (TLRs) may be involved in diabetic nephropathy (DN). Paeoniflorin (PF) is an effective Chinese traditional medicine with anti-inflammatory and immunoregulatory effects that may inhibit the TLR2 signaling pathway. In this study, we investigated the effects of PF on the kidneys of mice with streptozotocin-induced type 1 diabetes mellitus using TLR2 knockout mice (TLR2-/-) and wild-type littermates (C57BL/6J-WT). After 12 weeks of intraperitoneal injection of PF at doses of 25, 50, and 100 mg/kg once a day, diabetic mice had significantly reduced albuminuria and attenuated renal histopathology. These changes were associated with substantially alleviated macrophage infiltration and decreased expression of TLR2 signaling pathway biomarkers. These data support a role of TLR2 in promoting inflammation and indicate that the effect of PF is associated with the inhibition of the TLR2 pathway in the kidneys of diabetic mice. PF thus shows therapeutic potential for the prevention and treatment of DN.

MEK inhibition enhances efficacy of bacillus Calmette-Guérin on bladder cancer cells by reducing release of Toll-like receptor 2-activated antimicrobial peptides.

Bacillus Calmette-Guérin (BCG) is one of the standard treatment options for non-muscle-invasive bladder cancer. The details of the biological defense mechanisms against BCG remain unclear. Here, we investigated whether BCG-induced release of antimicrobial peptides (AMPs; e.g., human ß-defensin-2, -3, and cathelicidin) is involved with mitogen-activated protein kinase (MAPK) pathways, and investigated the enhanced anticancer effect of BCG through the down-regulation of Toll-like receptors (TLRs) and MAPK pathways in bladder cancer cells. BCG-infected bladder cancer cells produced AMPs as a defense mechanism against BCG, which were reduced by MEK inhibitors by blocking phosphorylation of extracellular signal-regulated kinase (ERK1/2 or MEK) and c-Jun. MEK inhibitors enhanced inhibition of bladder cancer cell growth by decreased binding of c-Jun, p65 and Pol II to the activated protein-1 promoter. Knockdown of TLR2 and TLR4 reduced ERK phosphorylation. Knockdown of TLR 2 decreased release of AMPs, which was similar to the efficacy of MEK inhibitor on BCG-infected cells. BCG-infected bladder cancer cells were more prone to induction of AMP release following TLR2 activation via ERK and c-Jun pathway mediators. In conclusion, our data suggest that the BCG-induced release of AMPs in bladder cancer cells is a promising molecular target for enhancing the immunotherapeutic efficacy of BCG in bladder cancer patients.

Effect of deoxynivalenol on the levels of toll-like receptors 2 and 9 and their mRNA expression in enterocytes in the porcine large intestine: a preliminary study.

Deoxynivalenol (DON), one of the most prevalent mycotoxins in the world, and is capable of inducing immune disorders in humans and animals. The aim of this study was to determine the effect of feed contaminated with DON on the number of TLR2- and TLR9-positive cells and their mRNA expression in the porcine large intestine. The experiment was conducted on two equal groups of pigs (n=4). The experimental group (E) was administered feed contaminated with DON (1008 µg/kg of feed) for 6 weeks, and the control group (C) was administered non-contaminated feed over the same period of time. A decrease in the expression of TLR2 mRNA was noted in the cecum. The percentage of TLR9-positive enterocytes increased in the ascending colon and decreased in the cecum. The results of this study indicate that DON can modify the local immune response by changing the expression of TLRs.

Regulation of kindling epileptogenesis by hippocampal Toll-like receptors 2.

This study examined whether Toll-like receptors 2 (TLR2) contribute to rapid kindling epileptogenesis. A TLR2 agonist, lipoteichoic acid (LTA), LTA antibody (LTA-A), or normal saline (control) was administered daily over 3 consecutive days, unilaterally into ventral hippocampus of adult male Wistar rats. Thirty minutes after the last injection, the animals were subjected to a rapid kindling procedure. The ictogenesis was gauged by comparing afterdischarge threshold (ADT) and afterdischarge duration (ADD) before the treatments, after the treatments prior to kindling, and 24 h after kindling. Kindling progression and retention were analyzed using video recording. The results showed that before kindling, LTA produced an ADT reduction. Neither LTA nor LTA-A affected baseline ADD. On kindling progression, LTA accelerated occurrence of generalized seizures, whereas LTA-A delayed this effect. Treatment with LTA-A reduced the number of secondary generalized complex partial seizures. Twenty-four hours after kindling, the rats of both the saline and LTA groups showed increased hippocampal excitability as compared with prekindling parameters. Administration of LTA-A prevented kindling-induced increase of hippocampal excitability. Immunostaining revealed that LTA-A attenuated the inflammatory response produced by seizures. These findings suggest that the activation of TLR2 in the hippocampus may facilitate limbic epileptogenesis.

Chronic hypersensitivity pneumonitis caused by Saccharopolyspora rectivirgula is not associated with a switch to a Th2 response.

Hypersensitivity pneumonitis (HP) is an immune-mediated interstitial lung disease that develops following repeated exposure to inhaled environmental antigens. The disease results in alveolitis and granuloma formation and may progress to a chronic form associated with fibrosis; a greater understanding of the immunopathogenic mechanisms leading to chronic HP is needed. We used the Saccharopolyspora rectivirgula (SR) mouse model of HP to determine the extent to which a switch to a Th2-type immune response is associated with chronic HP. Exposure of wild-type (WT) and tlr2/9(-/-) mice to SR for 14 wk resulted in neutrophilic and lymphocytic alveolitis that was not dependent on Toll-like receptors (TLRs) 2 and 9. Long-term exposure of WT mice to SR resulted in a significant increase in collagen deposition, protein leakage, and IL-1α accompanied by a decrease in quasistatic compliance and total lung capacity compared with unexposed mice. This was associated with an increase in IL-17 but not IL-4 production or recruitment of Th2 cells. tlr2/9(-/-) mice exhibited an increase in protein leakage but less IL-1α and collagen deposition in the lungs compared with WT mice, yet they still displayed a decrease in quasistatic compliance, although total lung capacity was not affected. These mice exhibited an increase in both IL-13 and IL-17, which suggests that IL-13 may ameliorate some of the lung damage caused by long-term SR exposure. Our results suggest that lung pathology following long-term SR exposure in WT mice is associated with the IL-17 response and that TLRs 2 and 9 may inhibit the development of the IL-13/Th2 response.

Cigarette Smoke Amplifies Inflammatory Response and Atherosclerosis Progression Through Activation of the H1R-TLR2/4-COX2 Axis.

Emerging evidence suggests that infection and persistent inflammation are key players in the pathogenesis of atherosclerotic cardiovascular disease (CVD). Although it is well established that cigarette smoke (CS) promotes atherosclerotic CVD, very little is known about the potential impact of the collective effects of CS and intermittent or chronic subclinical infection on atherosclerosis. Our previous studies demonstrated that mast cell-derived histamine and lipopolysaccharide (LPS) synergistically enhance endothelial cell inflammatory response. We further noted that the synergy between histamine and LPS was due to reciprocal upregulation of histamine receptor and Toll-like receptor 4 (TLR4) expression and functions. These results suggest that the combined and persistent effects of mast cell mediators and bacterial agents on the vasculature are risk factors of CVD. Our recent data demonstrated that CS extract enhances histamine- and LPS-induced expression of cyclooxygenase-2 (COX-2) in endothelial cells, suggesting that CS and mast cell mediators may collectively amplify inflammatory response in the vessel wall. We hypothesize that CS enhances histamine-mediated upregulation of TLR2/TLR4 signaling in the endothelium and promotes progression of atherosclerosis. This article presents our perspective on the modulatory effects of CS and nicotine on the "histamine-TLR-COX-2 axis."

Decreased TLR2 signal expression in peripheral blood mononuclear cell from patients with cryptococcal meningitis.

Toll-like receptors are the most important pattern recognition receptors that can recognize conserved molecular structures shared by large groups of pathogens. Here, the aim was to determine the expression and role of TLR2 in peripheral blood mononuclear cells (PBMCs) from patients with cryptococcal meningitis and healthy controls. TLR2 expression was measured using RT-PCR and western blotting. The role of TLR2 in cytokine production by PBMCs after Cryptococcus neoformans exposure was assessed in healthy controls prior to incubation with anti-TLR2. TLR2 mRNA and protein expression were both weaker in patients with cryptococcal meningitis than in healthy controls. Furthermore, pre-incubation of PBMCs from healthy donors with anti-TLR2 led to reduced expression of IFN-γ and IL-12p70, but not of IL-4 and IL-10, following C. neoformans stimulation. Our results suggest that impaired expression of TLR2 may be involved in defective host defense to C. neoformans through an attenuated Th1 response.

Signal pathway defects of Toll-like receptors 2-p38 mitogen activated protein kinase signal pathway defects and immune suppression in children with measles / 中华实用儿科临床杂志

Objective To investigate the expression of Toll-like receptors 2 (TLR2) mRNA,p38 mitogen activated protein kinase(p38 MAPK) mRNA and cytokine in peripheral blood of children with measles and to study the effect and possible mechanism for TLR2-p38 MAPK signal pathway defects on immune suppression in the children with measles during acute phase.Methods Thirty children with measles hospitalized in the department of infectious diseases from June 2012 to July 2013 were enrolled into the measles group,and 30 healthy children were chosen as the healthy control group.The mRNA expressions of TLR and p38 MAPK in peripheral blood mononuclear cells (PBMC) were detected by reverse transcription-polymerase chain reaction (RT-PCR).The protein levels of interferon-γ (IFN-γ),tumor necrosis factor-β (TNF-β),interleukin (IL)-12,IL-6 and IL-10 in plasma were measured by using enzyme linked immunosorbent assay (ELISA),and flow cytometry (FCM)was applied to detect the percentage of lymphocyte subpopulation.The serum IgG,IgA and IgM levels were detected by velocity scatter turbidimetry.Results (1) The expressions of TLR2 mRNA and p38 MAPK mRNA in the measles group were both significantly lower than those in the healthy control group (all P ＜ 0.05).(2) Compared with the healthy control group,the protein levels of IFN-γ,TNF-β and IL-12 in the plasma of the measles group decreased significantly (all P ＜ 0.05),and the levels of IL-6 and IL-10 increased significantly(all P ＜ 0.05).(3)Compared with the healthy control group,the percentage of CD3 +,CD4 +,CD4 +/CD8 + ratio and the level of IgG and IgA in the measles group decreased significantly(all P ＜ 0.05),and the percentage of CD19 + increased significantly(P ＜ 0.05),but there was no any significant change in the percentage of CD8 + and the level of IgM (all P ＞ 0.05).Conclusions The mRNA expressions of TLR2 and p38 MAPK are low in PBMC in the measles children during acute phase.There are different degrees of suppression of cell immunity,humoral immune and cytokines disorder in children with measles.Defects of TLR2-p38 MAPK signal pathway may cause the formation of measles immune suppression.

Relationship between expression of toll-like receptors 2/4 in dendritic cells and chronic hepatitis B virus infection.

OBJECTIVE: This study is to explore the relationship between the chronic hepatitis B virus (HBV) infection and the expressions of toll-like receptor 2/4 (TLR2/4) in peripheral blood dendritic cells (DCs), to find out the immunological significance of TLR2/4 in HBV progression. METHODS: Patients had been divided into the HBV, HBV-related liver cirrhosis (HBV-LC), and HBV-related hepatocellular carcinoma (HBV-HCC) groups. Healthy individuals served as normal controls (NC). Flow cytometry was used to determine the percentage of DCs in peripheral blood, and the expression of TLR2/4 in DCs as well as the expression of HBeAg. Real-time quantitative PCR was performed to measure the content of HBV-DNA. RESULTS: The percentages of DCs in peripheral blood exhibited a slightly decreasing trend, without statistical significances, along with the disease severity in HBV patients (9.40 ± 2.05%, 7.11 ± 3.82%, 6.51 ± 4.38% and 6.00 ± 4.73% for the groups of NC, HBV, HBV-LC, and HBV-HCC, respectively). The expression of TLR2 was significantly increased in the disease progression, with the TLR2 expression rates of 2.60 ± 1.70%, 2.67 ± 2.89%, 3.53 ± 3.41% and 5.11 ± 4.93 for NC, HBV, HBV-LC, HBV-HCC, respectively. Similar results were found for TLR4 (expression rates: 45.34 ± 4.46%, 53.94 ± 5.21%, 65.16 ± 5.92% and 75.54 ± 6.12%), which was positively correlated with TLR2. Furthermore, the HBeAg level was increased, while the amount of HBV-DNA exhibited a declining trend, along with the disease severity. Correlation analysis revealed that the expression of HBeAg was positively correlated with TLR2. CONCLUSIONS: The elevated expressions of TLR2/4 on DC cell surfaces in peripheral blood may synergistically promote the disease progression of chronic HBV infection.

Toll-like receptor expression and signaling in human diabetic wounds.

AIM: To examine the contribution of toll-like receptors (TLRs) expression and activation to the prolonged inflammation often seen in human diabetic wounds. METHODS: Debridement wound tissue was collected from diabetic patients with informed consent. Total RNA and protein were isolated and subjected to real-time polymerase chain reaction and Western blot analyses. RESULTS: TLR1, 2, 4, and 6 mRNA expressions were increased significantly in wounds of diabetic patients compared with non-diabetic wounds (P < 0.05). MyD88 protein expression was significantly increased in diabetic wounds compared to non-diabetic wounds. Interleukin-1beta, tumor necrosis factor-alpha concentration nuclear factor-kappa B activation, and thiobarbituric acid reactive substances were increased in diabetic wounds compared to non-diabetic wounds (P < 0.01). CONCLUSION: Collectively, our novel findings show that increased TLR expression, signaling, and activation may contribute to the hyper inflammation in the human diabetic wounds.

Regulation of B7-H1 expression on peripheral monocytes and IFN-γ secretion in T lymphocytes by HBeAg.

This study is to observe the expression of B7-H1, PD-1 and TLR2 on peripheral blood monocytes (PBMCs) regulated by HBeAg in chronic hepatitis B (CHB), and to illustrate the relation between HBeAg and persistent infection of HBV. In both CHB patients and healthy controls, the expression of B7-H7 was significantly increased on CD14(+) monocytes incubated with HBeAg, while that of TLR2 was significantly reduced; the expression of specific IFN-γ was significantly decreased in CD3(+)CD4(+) T lymphocytes incubated with HBeAg, while IL-6 and IL-10 in conditioned media were significantly increased. HBeAg is able to significantly up-regulate B7-H1, down-regulate TLR2 on monocytes, reduce IFN-γ produced by T lymphocytes and increase Th2-type cytokines secretion. These findings suggest that HBeAg suppresses the specific cellular immunity to clear the virus, and eventually lead to immune tolerance to HBV infection. Therefore, HBeAg plays an important role in immune suppression in chronic HBV patients.

The role of TLR2, TLR4 and its signal pathway in gouty arthritis / 中华风湿病学杂志

objective The roles of TLRs and their signal pathway in gouty arthritis(GA)were explored.Methods TLR2 and TLR4 mRNA was measured using real-time quantitative polymerase chain reaction(RT-PCR)in PBMCs,IL-1β level was detected using ELISA in plasma,and NF-κB p65 protein level in PBMCs was measured using Western blot.Level of TLR2 mRNA,ILR4 mRNA,IL-1β,NF-κB p65protein was compared among acute GA,non-acute GA and healthy controls.Correlation between TLR2mRNA,TLR4 mRNA and serum uric acid,IL-1β level in GA patients was analyzed.One-way ANOVA was used to analyze data between multiple groups and q-test was used for two-two comparison.Spearman's analysis was applied for correlation analysis.Resuits The expression of TLR4 mRNA,NF-KB p65 protein,IL-1β arid serum uric acid level in patients with acute GA [(5.0±1.2), (7.11±0.18), (283±83)pg/ml,[585±123)μmol/L] was significantly increased compared to non-acute GA[(2.3±0.4),(0.63±0.06),(134±29)pg/ml,(493±107)μmol/Lj and healthy controls(1.1±0.6),(0.52±0.12),(97±17)pg/ml,(326±65)μmol/L](P<0.01,respectively).Significant diffefence was also observed between non-acute GA patients and healthy controls(P<0.05,respectively).The level of TLRR4 mRNA was positively correlated with uric acid and IL-1β level in GA patients(rs=0.876,0.779;P<0.05,respectively).Conclusion Innate immunity are activated by membrane-type pattern recognition receptors in primary GA.TLR4-NFκB p65-IL-1β signat transduction may participate in the inflammatory mechanisms of gout.Urate crystals in patients with gout may:be involved in the activation of TLR4 and its signal pathway.

Lipid-associated membrane proteins of Mycoplasma genitalium activate NF-κB via Toll-like receptors 2 / 中华微生物学和免疫学杂志

Objective To investigate whether nuclear transcription factor κB(NF-κB) through Toll-like receptors 2(TLR2) was activated by lipid-associated membrane proteins(LAMPs) of Mycoplasma genitalium.Methods LAMPs were extractded and THP-1 cells were stimulated.The activation of NF-κBp65 was detected by ELISA and the expression of TLR2 mRNA was detected by RT-PCR.Effects of TLR2 neutralizing antibody on LAMPs induced the activation of NF-κBp65 was analyzed by ELISA.After LAMPs stimulated 293T cells with the co-transfection pFLAG-TLR2,pNF-κB-luc,pRL-TK,the activity of NF-κB firefly luciferase and pRL-TK Renilla luciferase were detected by the dual-luciferase reporter gene,to analyzed the role of TLR2-mediated NF-κB activation by LAMPs in 293T cells.Results The activation of NF-κBp65 was mediated in LAMPs induced THP-1 cells and was significantly increased by LAMPs in a dose dependent manner.when LAMPs was 4.0 μg/ml,the activation of NF-κBp65 was the highest level.TLR2 mRNA expression was up-regulated by LAMPs in THP-1 cells.TLR2 neutralizing antibody could inhibit the activation of NF-κB by 60% in LAMPs stimulated THP-1.NF-κB fluorescence was significantly increased by co-transfection pFLAG-TLR2 in a dose-dependent manner. ConclusionMycoplasma genitalium-derived lipid-associated membrane proteins activate NF-κB via TLR2 and the activation of TLR2-mediated play an important role in pathogenic process of LAMPs.