Bacterial translocation markers and toll-like receptors in biliary atresia following successful portoenterostomy.

AIM: The gut-liver axis may contribute to pathophysiology of cholestatic liver disorders like biliary atresia (BA) by bacterial translocation (BT). Toll-like receptors (TLR) are pattern recognition receptors known to activate innate immunity and secretion of inflammatory cytokines. Herein, we examined BT-associated biomarkers and TLRs in relation to liver injury after successful portoenterostomy (SPE) in BA. METHODS: Serum levels of lipopolysaccharide-binding protein (LBP), CD14, LAL, TNF-α, IL-6 and FABP2 along with liver expression of TLRs (TLR1, TLR4, TLR7 and TLR9), LBP and CD14 were measured during median 4.9 (1.7-10.6) years follow-up after SPE in 45 BA patients. RESULTS: Serum LBP, CD14, TNF-α and IL-6 all increased after SPE whereas LAL and FABP-2 remained unchanged. Serum LBP correlated positively with CD14 and markers of hepatocyte injury and cholestasis, but not with Metavir fibrosis stage, transcriptional markers for fibrosis (ACTA2) or ductular reaction. Serum CD14 concentration was significantly higher in patients with portal hypertension than without. While liver expression of TLR4 and LBP remained low, TLR7 and TLR1 showed marked BA-specific increases, and TLR7 correlated with Metavir fibrosis stage and ACTA2. CONCLUSION: BT does not seem to play a significant role in liver injury after SPE in our series of BA patients.

Physiologically relevant aspirin concentrations trigger immunostimulatory cytokine production by human leukocytes.

Acetylsalicylic acid is a globally used non-steroidal anti-inflammatory drug (NSAID) with diverse pharmacological properties, although its mechanism of immune regulation during inflammation (especially at in vivo relevant doses) remains largely speculative. Given the increase in clinical perspective of Acetylsalicylic acid in various diseases and cancer prevention, this study aimed to investigate the immunomodulatory role of physiological Acetylsalicylic acid concentrations (0.005, 0.02 and 0.2 mg/ml) in a human whole blood of infection-induced inflammation. We describe a simple, highly reliable whole blood assay using an array of toll-like receptor (TLR) ligands 1-9 in order to systematically explore the immunomodulatory activity of Acetylsalicylic acid plasma concentrations in physiologically relevant conditions. Release of inflammatory cytokines and production of prostaglandin E2 (PGE2) were determined directly in plasma supernatant. Experiments demonstrate for the first time that plasma concentrations of Acetylsalicylic acid significantly increased TLR ligand-triggered IL-1ß, IL-10, and IL-6 production in a dose-dependent manner. In contrast, indomethacin did not exhibit this capacity, whereas cyclooxygenase (COX)-2 selective NSAID, celecoxib, induced a similar pattern like Acetylsalicylic acid, suggesting a possible relevance of COX-2. Accordingly, we found that exogenous addition of COX downstream product, PGE2, attenuates the TLR ligand-mediated cytokine secretion by augmenting production of anti-inflammatory cytokines and inhibiting release of pro-inflammatory cytokines. Low PGE2 levels were at least involved in the enhanced IL-1ß production by Acetylsalicylic acid.

Combination of Vinpocetine and Dexamethasone Alleviates Cognitive Impairment in Nasopharyngeal Carcinoma Patients following Radiation Injury.

BACKGROUND: Nasopharyngeal carcinoma (NPC) originates in the nasopharyngeal epithelium. The most common treatments for NPC rT1-4 are radiotherapy and surgery. The pathogenesis of radiation-induced cognitive impairment is complex and includes oxidative stress, mitochondrial dysfunction, neuro-inflammation, and even apoptosis and cell death. Principally, toll-like receptors (TLRs) could regulate the inflammatory/anti-inflammatory balance in patients with radiation-induced brain injury. Vinpocetine has an anti-inflammatory effect as shown in both animal and in vitro studies. Also, dexamethasone is a widely used anti-inflammatory drug. Thus, it is important to test whether addition of vinpocetine could improve the anti-inflammatory properties of dexamethasone for the treatment of NPC patients with radiation-induced brain injuries. METHODS: A total of 60 NPC patients with radiation-related brain injury were recruited for this study. All subjects were randomly and blindly assigned to the following groups: the dexamethasone group (D group, n = 30) and the vinpocetine and dexamethasone group (VD group, n = 30). Both medicine treatments were uninterrupted for 14 days of administration. RESULTS: Combined administration of vinpocetine and dexamethasone lowered the expression levels of serum inflammatory cytokines, including TLR2, TLR4, interleukin (IL)-20, IL-8, tumor necrosis factor-α, interferon-γ, monocyte chemoattractant protein 2, and interferon-induced protein 20, when compared to dexamethasone monotherapy. Notably, combination therapy increased antioxidants (superoxide dismutase, glutathione, glutathione peroxidase, and glutathione reductase) and decreased oxidants (thiobarbituric acid reactive substances). Furthermore, combination therapy significantly increased the Mini Mental State Examination score, when compared to dexamethasone monotherapy. CONCLUSION: Administration of a combination of vinpocetine and dexamethasone may enhance the anti-inflammatory and anti-oxidative effects when compared to dexamethasone monotherapy, which leads to alleviated cognitive impairment in NPC patients with radiation injury.

TLR engagement induces ARID3a in human blood hematopoietic progenitors and modulates IFNα production.

The DNA binding protein AT-rich interacting domain 3a (ARID3a)2 is expressed in healthy human hematopoietic cord blood progenitors where its modulation influences myeloid versus B lineage development. ARID3a is also variably expressed in subsets of adult peripheral blood hematopoietic progenitors where the consequences of ARID3a expression are unknown. In B lymphocytes, Toll-like receptor (TLR)3 signaling induces ARID3a expression in association with Type I interferon inflammatory cytokines. We hypothesized that TLR ligand stimulation of peripheral blood hematopoietic progenitors would induce ARID3a expression resulting in interferon production, and potentially influencing lineage decisions. Our data revealed that the TLR9 agonist CpG induces ARID3a expression with interferon alpha synthesis in human hematopoietic progenitors. However, ARID3a expression was not associated with increased B lineage development. These results demonstrate the need for further experiments to better define how pathogen-associated responses influence hematopoiesis.

Genome-wide whole blood transcriptome profiling in a large European cohort of systemic sclerosis patients.

OBJECTIVES: The analysis of annotated transcripts from genome-wide expression studies may help to understand the pathogenesis of complex diseases, such as systemic sclerosis (SSc). We performed a whole blood (WB) transcriptome analysis on RNA collected in the context of the European PRECISESADS project, aiming at characterising the pathways that differentiate SSc from controls and that are reproducible in geographically diverse populations. METHODS: Samples from 162 patients and 252 controls were collected in RNA stabilisers. Cases and controls were divided into a discovery (n=79+163; Southern Europe) and validation cohort (n=83+89; Central-Western Europe). RNA sequencing was performed by an Illumina assay. Functional annotations of Reactome pathways were performed with the Functional Analysis of Individual Microarray Expression (FAIME) algorithm. In parallel, immunophenotyping of 28 circulating cell populations was performed. We tested the presence of differentially expressed genes/pathways and the correlation between absolute cell counts and RNA transcripts/FAIME scores in regression models. Results significant in both populations were considered as replicated. RESULTS: Overall, 15 224 genes and 1277 functional pathways were available; of these, 99 and 225 were significant in both sets. Among replicated pathways, we found a deregulation in type-I interferon, Toll-like receptor cascade, tumour suppressor p53 protein function, platelet degranulation and activation. RNA transcripts or FAIME scores were jointly correlated with cell subtypes with strong geographical differences; neutrophils were the major determinant of gene expression in SSc-WB samples. CONCLUSIONS: We discovered a set of differentially expressed genes/pathways validated in two independent sets of patients with SSc, highlighting a number of deregulated processes that have relevance for the pathogenesis of autoimmunity and SSc.

Cyclic AMP in human preterm infant blood is associated with increased TLR-mediated production of acute-phase and anti-inflammatory cytokines in vitro.

BACKGROUND: Preterm infants are at high risk of infection and have distinct pathogen recognition responses. Suggested mechanisms include soluble mediators that enhance cellular levels of cAMP. The aim of this study was to assess the relationship between blood cAMP concentrations and TLR-mediated cytokine production in infants during the first month of life. METHODS: Cord and serial peripheral blood samples (days of life 1-28) were obtained from a cohort of very preterm (<30 weeks' gestational age) and term human infants. Whole-blood concentrations of cAMP and FSL-1 and LPS in vitro stimulated cytokine concentrations were measured by ELISA and multiplex bead assay. RESULTS: cAMP concentrations were higher in cord than in peripheral blood, higher in cord blood of female preterm infants, and lower at Days 1 and 7 in infants exposed to chorioamnionitis, even after adjusting for leukocyte counts. TLR2 and TLR4-mediated TNF-α, IL-1ß, IL-6, IL-12p70, and IL-10 production in vitro increased over the first month of life in preterm infants and were positively correlated with leukocyte-adjusted cAMP levels and reduced by exposure to chorioamnionitis. CONCLUSIONS: The ontogeny of blood cAMP concentrations and associations with chorioamnionitis and TLR-mediated production of cytokines suggest that this secondary messenger helps shape distinct neonatal pathogen responses in early life.

Toll-like receptors, long non-coding RNA NEAT1, and RIG-I expression are associated with HBeAg-positive chronic hepatitis B patients in the active phase.

BACKGROUND: Innate immunity plays a crucial role in host-virus interactions and greatly influences viral replication including HBV infection. However, few studies have investigated the possible antiviral immune roles played by TLRs, RIG-I, and long no-coding RNA NEAT1 in chronic HBV infection (CHB) patients in clinical samples and their relationships among immune responses. In this study, we sought to investigate the mRNA expression levels of TLR1-10, RIG-I, and NEAT1 expression in HBeAg-positive CHB treatment-naïve patients with the active phase. METHODS: The expression levels of TLR1-10, RIG-I, and NEAT1 of CHB patients with the active phase and healthy controls were measured by qPCR. Serum HBV DNA and routine liver biochemistry including ALT, etc were also measured to evaluate the impaired physiological function of the liver affected by CHB. RESULTS: The expression levels of TLR1 and TLR6 in CHB with active phase were remarkably lower than that in healthy controls. The levels of TLR3 in CHB patients with active phase were remarkably higher than that in healthy controls. The total NEAT1 expression was abnormally decreased in CHB patients as compared with healthy controls. The levels of RIG-I were significantly decreased in CHB patients in the active phase when compared to healthy controls. The expression of TLR6 and RIG-I was closely correlated with NEAT1 expression. TLR6 level was positively correlated with RIG-I level. CONCLUSION: Chronic HBV infection can alter the innate immune response by downregulating functional expression of TLR1, TLR6, NEAT1.

Toll-like receptor activation as a biomarker in traumatically injured patients.

BACKGROUND: Surgical insult and trauma have been shown to cause dysregulation of the immune and inflammatory responses. Interaction of damage-associated molecular patterns (DAMPs) with toll-like receptors (TLRs) initiates innate immune response and systemic inflammatory responses. Given that surgical patients produce high levels of circulating damage-associated molecular patterns, we hypothesized that plasma-activated TLR activity would be correlated to injury status and could be used to predict pathological conditions involving tissue injury. METHODS: An observational study was performed using samples from a single-institution prospective tissue and data repository from a Level-1 trauma center. In vitro TLR 2, 3, 4, and 9 activation was determined in a TLR reporter assay after isolation of plasma from peripheral blood. We determined correlations between plasma-activated TLR activity and clinical course measures of severity. RESULTS: Eighteen patients were enrolled (median Injury Severity Score 15 [interquartile range 10, 23.5]). Trauma resulted in significant elevation in circulation high mobility group box 1 as well as increase of plasma-activated TLR activation (2.8-5.4-fold) compared to healthy controls. There was no correlation between circulating high mobility group box 1 and trauma morbidity; however, the plasma-activated TLR activity was correlated with acute physiology and chronic health evaluation II scores (R square = 0.24-0.38, P < 0.05). Patients who received blood products demonstrated significant increases in the levels of plasma-activated TLRs 2, 3, 4, and 9 and had a trend toward developing systemic inflammatory response syndrome. CONCLUSIONS: Further studies examining TLR modulation and signaling in surgical patients may assist in predictive risk modeling and reduction in morbidity and mortality.

Genetic influence of Toll-like receptors on non-HIV cryptococcal meningitis: An observational cohort study.

BACKGROUND: Cryptococcal meningitis (CM) is a significant source of mortality, the pathogenesis of which has not been fully understood, especially in non-HIV infected populations. We aimed to explore the potential genetic influence of Toll-like receptor (TLR) on non-HIV CM. METHODS: This observational cohort study was done in two stages: a discovery stage and a validation stage. A case-control genetic association study was conducted between 159 non-HIV CM patients and 468 healthy controls. TLR SNPs significantly related to susceptibility went further validation in a second cohort of 583 subjects from a certain district. Associations among TLR SNPs, cerebrospinal fluid (CSF) cytokine concentrations, and clinical severity were explored in a third cohort of 99 previously untreated non-HIV CM patients. Logistic regression model was used to determine the independent predictors for disease severity. FINDINGS: In the discovery stage, eight TLR SNPs exhibited significant genetic susceptibility to non-HIV CM, one of which was validated in a population validation of HIV-infected cases while none survived in non-HIV cases. CSF cytokine detections showed that 18 cytokines were significantly over-expressed in severely ill patients. Two of the 8 SNPs (rs5743604 and rs3804099) were also significantly associated with disease severity. Specifically, the rs3804099 C/T genotype was further found to be correlated to 12 of the 18 up-regulated cytokines in severe patients. In addition, high levels of interleukin (IL)-10 in CSF (OR 2·97, 95% CI 1·49-5·90; p = 0·002) was suggested as an independent predictor for severity after adjusted for possible confounders. INTERPRETATION: TLR participates in both the occurrence and the pathogenesis of non-HIV CM. The in situ immune responses of CM were under genetic influence of TLR and contributed to disease severity. FUND: National Natural Science Foundation of China and National Key Basic Research Program of China (973 Program).

Resveratrol supplementation decreases blood glucose without changing the circulating CD14+CD16+ monocytes and inflammatory cytokines in patients with type 2 diabetes: a randomized, double-blind, placebo-controlled study.

Chronic low-grade inflammation is the hallmark of type 2 diabetes (T2D). Although in vitro and animal studies have shown that resveratrol exerts anti-inflammatory effects, clinical trials addressing these effects in patients with T2D are limited. Therefore, in the present study, we hypothesized that supplementation of resveratrol might improve inflammatory markers in patients with T2D in a randomized, double-blind, placebo-controlled clinical trial. A total of 45 T2D patients were supplemented with either of 800 mg/d resveratrol or placebo capsules for 8 weeks. Percentage of CD14+CD16+ monocytes, plasma levels of inflammatory cytokines (tumor necrosis factor α, interleukin [IL] 1ß, IL-6, and monocyte chemoattractant protein-1), the expression levels of genes involved in the inflammatory responses (toll-like receptor 2, toll-like receptor 4, and nuclear factor κB), lipopolysaccharide-stimulated cytokine (tumor necrosis factor α, IL-1ß, and IL-6) secretion from peripheral blood mononuclear cells, and metabolic and anthropometric parameters were assessed at both the baseline level and the end of the study. Compared with the placebo group, we could not detect any significant difference in the percentage of CD14+CD16+ monocytes, lipopolysaccharide-induced cytokine secretion, plasma levels of inflammatory cytokines, and the expression of inflammatory genes in resveratrol group. Moreover, we did not find any significant change in the metabolic and anthropometric parameters except for a significant reduction in fasting blood glucose and blood pressure. In conclusion, 8-week supplementation of resveratrol reduces blood glucose level in patients with T2D without improving their inflammatory markers.

Effects of oral Lactobacillus administration on antioxidant activities and CD4+CD25+forkhead box P3 (FoxP3)+ T cells in NZB/W F1 mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterised by a dysregulation of the immune system, which causes inflammation responses, excessive oxidative stress and a reduction in the number of cluster of differentiation (CD)4+CD25+forkhead box P3 (FoxP3)+ T cells. Supplementation with certain Lactobacillus strains has been suggested to be beneficial in the comprehensive treatment of SLE. However, little is known about the effect and mechanism of certain Lactobacillus strains on SLE. To investigate the effects of Lactobacillus on SLE, NZB/W F1 mice were orally gavaged with Lactobacillus paracasei GMNL-32 (GMNL-32), Lactobacillus reuteri GMNL-89 (GMNL-89) and L. reuteri GMNL-263 (GMNL-263). Supplementation with GMNL-32, GMNL-89 and GMNL-263 significantly increased antioxidant activity, reduced IL-6 and TNF-α levels and significantly decreased the toll-like receptors/myeloid differentiation primary response gene 88 signalling in NZB/W F1 mice. Notably, supplementation with GMNL-263, but not GMNL-32 and GMNL-89, in NZB/W F1 mice significantly increased the differentiation of CD4+CD25+FoxP3+ T cells. These findings reveal beneficial effects of GMNL-32, GMNL-89 and GMNL-263 on NZB/W F1 mice and suggest that these specific Lactobacillus strains can be used as part of a comprehensive treatment of SLE patients.

Pentoxifylline inhibits TLR- and inflammasome-mediated in vitro inflammatory cytokine production in human blood with greater efficacy and potency in newborns.

BACKGROUND: Toll-like receptor (TLR)-mediated inflammation may contribute to neonatal sepsis, for which pentoxifylline (PTX), a phosphodiesterase inhibitor that raises intracellular cAMP, is a candidate adjunctive therapy. We characterized the anti-inflammatory effects of PTX toward TLR-mediated production of inflammatory (tumor necrosis factor (TNF) and interleukin (IL)-1ß) and proresolution (IL-6 and IL-10) cytokines in human newborn and adult blood. METHODS: Newborn cord and adult blood were treated with PTX (50-400 µmol/l) before, during or after stimulation with LPS (TLR4 agonist), R848 (TLR7/8 agonist) or LPS/ATP (inflammasome activation). Cytokines were measured by multiplex assay (supernatants), intracellular cytokines and signaling molecules by flow cytometry, and mRNA by quantitative real-time PCR. RESULTS: Whether added 2 h pre-, simultaneously to, or 2 h post-TLR stimulation, PTX inhibited TLR-mediated cytokine production in a concentration-dependent manner, with greater efficacy and potency in newborn blood, decreasing intracellular TNF and IL-1ß with relative preservation of IL-10 and IL-6. PTX decreased TLR-mediated TNF mRNA while increasing IL-10 mRNA. Neonatal plasma factors contributed to the anti-inflammatory effects of PTX in newborn blood that were independent of soluble TNF receptor concentrations, p38 MAPK phosphorylation and IĸB degradation. CONCLUSION: PTX is a potent and efficacious inhibitor of TLR-mediated inflammatory cytokines in newborn cord blood and a promising neonatal anti-inflammatory agent.

Maternal Vitamin D Level Is Associated with Viral Toll-Like Receptor Triggered IL-10 Response but Not the Risk of Infectious Diseases in Infancy.

Reports on the effect of prenatal vitamin D status on fetal immune development and infectious diseases in childhood are limited. The aim of this study was to investigate the role of maternal and cord blood vitamin D level in TLR-related innate immunity and its effect on infectious outcome. Maternal and cord blood 25 (OH)D level were examined from 372 maternal-neonatal pairs and their correlation with TLR-triggered TNF-α, IL-6, and IL-10 response at birth was assessed. Clinical outcomes related to infection at 12 months of age were also evaluated. The result showed that 75% of the pregnant mothers and 75.8% of the neonates were vitamin deficient. There was a high correlation between maternal and cord 25(OH)D levels (r = 0.67, p < 0.001). Maternal vitamin D level was inversely correlated with IL-10 response to TLR3 (p = 0.004) and TLR7-8 stimulation (p = 0.006). However, none of the TLR-triggered cytokine productions were associated with cord 25(OH)D concentration. There was no relationship between maternal and cord blood vitamin D status with infectious diseases during infancy. In conclusion, our study had shown that maternal vitamin D, but not cord vitamin D level, was associated with viral TLR-triggered IL-10 response.

Transcriptome Analysis of Peripheral Blood in Chronic Inflammatory Demyelinating Polyradiculoneuropathy Patients Identifies TNFR1 and TLR Pathways in the IVIg Response.

We have studied the response to intravenous immunoglobulins (IVIg) by a transcriptomic approach in 11 chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) patients (CIDP duration = 6 [0.83-6.5] years). RNA was extracted from cells in whole blood collected before and 3 weeks after IVIg treatment, and hybridized on Illumina chips. After RNA quality controls, gene expression was analyzed using statistical tests fitted for microarrays (R software, limma package), and a pathway analysis was performed using DAVID software. We identified 52 genes with expression that varied significantly after IVIg (fold change [FC] > 1.2, P < 0.001, false discovery rate [FDR] <0.05). Among these 52 genes, 7 were related to immunity, 3 were related to the tumor necrosis factor (TNF)-α receptor 1 (TNFR1) pathway (inhibitor of caspase-activated DNase (ICAD): FC = 1.8, P = 1.7E-7, FDR = 0.004; p21 protein-activated kinase 2 [PAK2]: FC = 1.66, P = 2.6E-5, FDR = 0.03; TNF-α-induced protein 8-like protein 1 [TNFAIP8L1]: P = 1.00E-05, FDR = 0.026), and 2 were related to Toll-like receptors (TLRs), especially TLRs 7 and 9, and were implicated in autoimmunity. These genes were UNC93B1 (FC = 1.6, P = 2E-5, FDR = 0.03), which transports TLRs 7 and 9 to the endolysosomes, and RNF216 (FC = 1.5, P = 1E-05, FDR = 0.03), which promotes TLR 9 degradation. Pathway analysis showed that the TNFR1 pathway was significantly lessened by IVIg (enrichment score = 24, Fischer exact test = 0.003). TNF-α gene expression was higher in responder patients than in nonresponders; however, it decreased after IVIg in responders (P = 0.04), but remained stable in nonresponders. Our data suggest the actions of IVIg on the TNFR1 pathway and an original mechanism involving innate immunity through TLRs in CIDP pathophysiology and the response to IVIg. We conclude that responder patients have stronger inflammatory activity that is lessened by IVIg.

Prenatal exposure to bisphenol-A is associated with Toll-like receptor-induced cytokine suppression in neonates.

BACKGROUND: Despite widespread human exposure to biphenol A (BPA), limited studies exist on the association of BPA with adverse health outcomes in young children. This study aims to investigate the effect of prenatal exposure to BPA on toll-like receptor-induced cytokine responses in neonates and its association with infectious diseases later in life. METHODS: Cord bloods were collected from 275 full-term neonates. Production of TNF-α, IL-6, and IL-10 were evaluated after stimulating mononuclear cells with toll-like receptor ligands (TLR1-4 and 7-8). Serum BPA concentrations were analyzed by enzyme-linked immunosorbent assay. Bacteria from nasopharyngeal specimens were identified with multiplex PCR and culture method. RESULT: Result showed significant association between cord BPA concentration and TLR3- and TLR4-stimulated TNF-α response (P = 0.001) and that of TLR78-stimulated IL-6 response (P = 0.03). Clinical analysis did not show prenatal BPA exposure to be correlated with infection or bacterial colonization during the first year of life. CONCLUSION: This is the first cohort study that indicated prenatal BPA exposure to play a part in TLR-related innate immune response of neonatal infants. However, despite an altered immune homeostasis, result did not show such exposure to be associated with increased risk of infection during early infancy.

Expression and function of toll-like receptors in human circulating endothelial colony forming cells.

Mature endothelial cells are known to sense microbial products through toll-like receptors (TLRs), a family of membrane proteins which serve as pathogen recognition and signaling elements; however, there are limited data in the literature about the expression and function of TLRs in human circulating endothelial colony forming cells (ECFCs), which are considered the most likely endothelial precursors. We expanded and differentiated in vitro umbilical cord blood (UCB) and adult peripheral blood (PB) ECFCs and studied the expression of TLR1 to TLR10 mRNA by qPCR analysis, and we further characterized TLR function in ECFCs through functional assays including in vitro ECFC growth and differentiation, assessment of cytokine production, and measurement of intracellular Ca(2+) signals. Both UCB- and PB-ECFCs had detectable mRNA levels of all the TLRs from 1 to 10; TLR4, a sensor of Gram-negative bacterial lipopolysaccharide (LPS), had a higher level compared to other TLRs. Exposure to LPS induced cytokine production, although with less efficiency compared to PB-mononuclear cells. However, no effect of LPS was seen on ECFC growth and differentiation, and no increase in intracellular Ca(2+) concentrations, which is essential for ECFC proliferation, was observed after exposure to increasing amounts of LPS. Our data show that all TLRs from 1 to 10 are constitutively expressed in ECFCs, and suggest that TLR4 is functional in ECFCs, but its activation through its ligand LPS does not affect ECFC growth and differentiation.

The role of platelet MyD88 in host response during gram-negative sepsis.

BACKGROUND: Beside their role in hemostasis, platelets serve as sentinel cells in host defense during infection. In sepsis, platelets have been implicated in both beneficial (antibacterial) and detrimental responses (thrombosis and organ damage). Toll-like receptors and their common adaptor, myeloid differentiation factor 88 (MyD88), are essential for pathogen recognition and protective immunity. Platelets express functional Toll-like receptors and MyD88, which participate in platelet responsiveness to bacterial agonists. OBJECTIVE: Considering the pivotal involvement of platelets and MyD88 in the host response to bacteria, we studied the role of platelet MyD88 in gram-negative sepsis using intravenous and airway infections with the common human sepsis pathogen Klebsiella pneumoniae. METHODS: Platelet-specific Myd88(-/-) mice were generated by crossing mice with a conditional Myd88 flox allele with mice expressing Cre recombinase controlled by the platelet factor 4 promoter. In a reverse approach, full Myd88(-/-) mice were transfused with wild-type platelets. RESULTS: In both settings, platelet MyD88 did not impact on bacterial growth or dissemination. In addition, platelet MyD88 did not influence hallmark sepsis responses such as thrombocytopenia, coagulation or endothelial activation, or distant organ injury. Platelet MyD88 played no role in lung pathology during pneumonia-derived sepsis. CONCLUSION: Despite known literature, platelet MyD88-dependent TLR signaling does not contribute to the host response during gram-negative sepsis.

Leishmania donovani influenced cytokines and Toll-like receptors expression among Sudanese visceral leishmaniasis patients.

Leishmaniasis remains a serious health problem. The outcome of Leishmania infection depends on the early innate response. In this study, whole blood samples of 40 patients with visceral leishmaniasis (VL), 10 leishmanin skin test-negative (LST-ve) controls and 10 leishmanin skin test-positive (LST+ve) controls were stimulated by live L. donovani promastigotes. Also, THP1 human cell line was infected with L. donovani. The production of interleukin 10 (IL-10), tumour necrosis factor alpha (TNF) and interferon gamma (IFNG) cytokines was measured, and the expression of Toll-like receptors (TLR2, TLR4 and TLR9) was done in the blood samples and also in the THP1 cell line. IL-10 was found to be higher in LST+ve controls compared with VL patients. TNF was moderately produced with no variation between patients, controls and THP1 cells. IFNG was higher in LST+ve controls also in THP1 cells. TLR4 and TLR9 were found to be highly expressed in patients with VL. L. donovani increases the expression of TLR4 and TLR9 in patients with VL and TLR2 in THP1 cells, suggesting a TLRs relation in induction of a mixed cytokine response. TLR9 was markedly recognized by L. donovani DNA.

Sex differences in platelet toll-like receptors and their association with cardiovascular risk factors.

OBJECTIVE: Platelets contribute to thrombosis, and platelet toll-like receptors (TLRs) are central in pathogen detection, potentially mediating infection-induced vascular occlusion. Using a large community-based cohort study, we sought to examine if platelets express all known TLR transcripts and analyze their association with cardiovascular risk factors. APPROACH AND RESULTS: mRNA levels for TLRs were measured in isolated platelets by high-throughput quantitative reverse transcriptase polymerase chain reaction in 1625 participants (mean age, 66.6±9; 54% women) of the Framingham Heart Study. We measured circulating inflammatory and thrombotic markers (C-reactive protein, interleukin-6, monocyte chemoattractant protein 1, intracellular cell adhesion molecule 1, soluble tumor necrosis factor-α receptor 1, and soluble p-selectin) and analyzed TLRs and their association with sex and cardiovascular risk factors by multivariable logit regression model adjusted for confounding factors. Platelets expressed all 10 TLR transcripts, and all TLRs were coexpressed. Women had higher platelet TLR expression, which associated with different cardiovascular risk factors, compared with men. In women, TLR1, TLR3, TLR6, and TLR7 were associated with body mass index and TLR5, TLR7, and TLR10 were associated with total cholesterol to high-density lipoprotein ratio. In men, TLR1, TLR2, and TLR3 were associated with lipid and TLR8 with hypertension treatment. Similarly, TLR expression in men was more commonly associated with circulating inflammatory markers (soluble tumor necrosis factor-α receptor 1 and intracellular cell adhesion molecule 1), whereas in women, TLR expression was associated with soluble p-selectin levels. CONCLUSIONS: We report the first study to demonstrate that platelets express all TLR transcripts using a large community-based observational cohort. These transcripts are more abundant in women and have distinct associations with cardiovascular risk and inflammatory biomarkers that vary by sex.

Expression and function of Toll-like receptors in peripheral blood mononuclear cells from patients with ovarian cancer.

Inflammation has been implicated in the initiation and progression of ovarian cancer (OC), the underlying mechanisms of which are still unclear. We hypothesized that the abnormal expression of Toll-like receptors (TLRs), which were potential activators of nuclear factor-kappa B p65 (NF-κB p65), could promote inflammation and tumorigenesis in OC. In this study, we characterized the expression of TLRs in peripheral blood mononuclear cells (PBMCs) and found TLR2 and TLR6 mRNAs levels to be higher in PBMCs from OC patients than in those from benign disease (BC) or healthy normal controls (NC). Flow cytometry analysis showed that TLR1, TLR2 and TLR6 were highly expressed in monocytes from OC patients, but not in those from control subjects. Consistently, inflammatory cytokines interleukin (IL)-1ß and IL-6 were up-regulated in PBMCs from OC patients upon stimulation with Pam3CSK4 (TLR1 ligand) and HKLM (TLR2 ligand), compared with unstimulated PBMCs. Stimulation of PBMCs with TLR ligands led to activation of downstream signaling molecules in TLRs (MyD88, TRAF6, TANK, NF-κB p65 and p-NF-κB p65). We also discovered that SK-OV-3-secreted factors were potent PBMCs activators, leading to the production of IL-1ß, IL-6 and IL-8 through activation of TLRs and downstream signaling molecules in PBMCs. Before coculturing with SK-OV-3, pretreatment of THP-1 cells or PBMCs with monoclonal antibodies against TLR1, TLR2 or TLR6 inhibited the production of IL-1ß and IL-6 and activation of MyD88, TRAF6, TANK, NF-κB p65 and p-NF-κB p65. Our results provided new evidence that TLR1, TLR2 and TLR6 signaling was linked with inflammation in OC microenvironment.