RESUMEN

Background: Recent studies suggest gut-derived lipopolysaccharide (LPS)-translocation to play a role in both systemic inflammation and in inflammatory adipose tissue. We aimed to investigate whether circulating LPS-related inflammatory markers and corresponding genetic expression in adipose tissue were associated with obesity, cardiometabolic risk factors, and dietary habits in patients with coronary artery disease. Methods: Patients (n=382) suffering a myocardial infarction 2-8 weeks prior to inclusion were enrolled in this cross-sectional study. Subcutaneous adipose tissue (SAT), taken from the gluteal region, and fasting blood samples were collected at inclusion for determination of genetic expression of LPS-binding protein (LBP), CD14, toll-like receptor 2 (TLR2), and TLR4 in SAT, and LPS, LBP, and soluble cluster of differentiation 14 (sCD14) in the circulation. All patients filled out a dietary registration form. Results: Patients (median age 74 years, 25% women), had a median body mass index (BMI) of 25.9 kg/m2. Circulating levels of LBP correlated to BMI (p=0.02), were significantly higher in overweight or obese (BMI≥25 kg/m2) compared to normal- or underweight patients (BMI<25 kg/m2), and were significantly elevated in patients with T2DM, hypertension, and MetS, compared to patients without (p≤0.04, all). In SAT, gene expression of CD14 and LBP correlated significantly to BMI (p≤0.001, both), and CD14 and TLR2 expressions were significantly higher in patients with T2DM and MetS compared to patients without (p≤0.001, both). Circulating and genetically expressed CD14 associated with use of n-3 PUFAs (p=0.008 and p=0.003, respectively). No other significant associations were found between the measured markers and dietary habits. Conclusion: In patients with established CAD, circulating levels of LBP and gene expression of CD14 and TLR2 in SAT were related to obesity, MetS, T2DM, and hypertension. This suggests that the LPS-LBP-CD14 inflammatory axis is activated in the chronic low-grade inflammation associated with cardiometabolic abnormalities, whereas no significant associations with dietary habits were observed.

RESUMEN

Lipopolysaccharide binding protein (LBP) knockout mice models are protected against the deleterious effects of major acute inflammation but its possible physiological role has been less well studied. We aimed to evaluate the impact of liver LBP downregulation (using nanoparticles containing siRNA- Lbp) on liver steatosis, inflammation and fibrosis during a standard chow diet (STD), and in pathological non-obesogenic conditions, under a methionine and choline deficient diet (MCD, 5 weeks). Under STD, liver Lbp gene knockdown led to a significant increase in gene expression markers of liver inflammation (Itgax, Tlr4, Ccr2, Ccl2 and Tnf), liver injury (Krt18 and Crp), fibrosis (Col4a1, Col1a2 and Tgfb1), endoplasmic reticulum (ER) stress (Atf6, Hspa5 and Eif2ak3) and protein carbonyl levels. As expected, the MCD increased hepatocyte vacuolation, liver inflammation and fibrosis markers, also increasing liver Lbp mRNA. In this model, liver Lbp gene knockdown resulted in a pronounced worsening of the markers of liver inflammation (also including CD68 and MPO activity), fibrosis, ER stress and protein carbonyl levels, all indicative of non-alcoholic steatohepatitis (NASH) progression. At cellular level, Lbp gene knockdown also increased expression of the proinflammatory mediators (Il6, Ccl2), and markers of fibrosis (Col1a1, Tgfb1) and protein carbonyl levels. In agreement with these findings, liver LBP mRNA in humans positively correlated with markers of liver damage (circulating hsCRP, ALT activity, liver CRP and KRT18 gene expression), and with a network of genes involved in liver inflammation, innate and adaptive immune system, endoplasmic reticulum stress and neutrophil degranulation (all with q-value<0.05). In conclusion, current findings suggest that a significant downregulation in liver LBP levels promotes liver oxidative stress and inflammation, aggravating NASH progression, in physiological and pathological non-obesogenic conditions.

Asunto(s)

Cirrosis Hepática , Hígado , Enfermedad del Hígado Graso no Alcohólico , Animales , Humanos , Ratones , Modelos Animales de Enfermedad , Inflamación/genética , Cirrosis Hepática/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/genética , ARN Mensajero/metabolismo

RESUMEN

Circulating lipopolysaccharide-binding protein (LBP) is increased in individuals with liver steatosis. We aimed to evaluate the possible impact of liver LBP downregulation using lipid nanoparticle-containing chemically modified LBP small interfering RNA (siRNA) (LNP-Lbp UNA-siRNA) on the development of fatty liver. Weekly LNP-Lbp UNA-siRNA was administered to mice fed a standard chow diet, a high-fat and high-sucrose diet, and a methionine- and choline-deficient diet (MCD). In mice fed a high-fat and high-sucrose diet, which displayed induced liver lipogenesis, LBP downregulation led to reduced liver lipid accumulation, lipogenesis (mainly stearoyl-coenzyme A desaturase 1 [Scd1]) and lipid peroxidation-associated oxidative stress markers. LNP-Lbp UNA-siRNA also resulted in significantly decreased blood glucose levels during an insulin tolerance test. In mice fed a standard chow diet or an MCD, in which liver lipogenesis was not induced or was inhibited (especially Scd1 mRNA), liver LBP downregulation did not impact on liver steatosis. The link between hepatocyte LBP and lipogenesis was further confirmed in palmitate-treated Hepa1-6 cells, in primary human hepatocytes, and in subjects with morbid obesity. Altogether, these data indicate that siRNA against liver Lbp mRNA constitutes a potential target therapy for obesity-associated fatty liver through the modulation of hepatic Scd1.

RESUMEN

Osteosarcoma (OS) is a rare malignant primary bone tumours characterized by a high genetic and cell composition heterogeneity. Unfortunately, despite the use of drug combinations and the recent development of immunotherapies, the overall survival has not improved in the last four decades. Due to the key role of the tumour microenvironment in the pathogenesis of OS, a better understanding of its microenvironment is mandatory to develop new therapeutic approaches. From retrospective biological cohorts of OS, we analysed by immunohistochemistry the presence of lipopolysaccharide (LPS)-binding protein (LBP) in diagnostic biopsies with local disease and compared their level of infiltration to patients suffering from metastatic status. LBP is considered as a marker of LPS exposure and can indirectly reflect the presence of Gram-negative microbiota. LBP were detected in the cytoplasm of OS cells as well as in tumour-associated macrophage. Tumour samples of patients with local disease were significantly enriched in LBP compared to tumour tissues of patients with metastatic status. Lung metastatic tissues showed similar level of LBP compared to paired primary tumours. Overall, this study strongly suggests the presence of Gram-negative bacteria in OS tissues and demonstrated their significant differential level according the metastatic status. This tumour-associated microbiome may help in the conceptualisation of new therapeutic approach to trigger efficient therapeutic responses against cancer.

RESUMEN

Cellular and molecular mechanisms of the peripheral immune system (e.g., macrophage and monocyte) in programming endotoxin tolerance (ET) have been well studied. However, regulatory mechanism in development of brain immune tolerance remains unclear. The inducible COX-2/PGE2 axis in microglia, the primary innate immune cells of the brain, is a pivotal feature in causing inflammation and neuronal injury, both in acute excitotoxic insults and chronic neurodegenerative diseases. This present study investigated the regulatory mechanism of PGE2 tolerance in microglia. Multiple reconstituted primary brain cells cultures, including neuron-glial (NG), mixed glial (MG), neuron-enriched, and microglia-enriched cultures, were performed and consequently applied to a treatment regimen for ET induction. Our results revealed that the levels of COX-2 mRNA and supernatant PGE2 in NG cultures, but not in microglia-enriched and MG cultures, were drastically reduced in response to the ET challenge, suggesting that the presence of neurons, rather than astroglia, is required for PGE2 tolerance in microglia. Furthermore, our data showed that neural contact, instead of its soluble factors, is sufficient for developing microglial PGE2 tolerance. Simultaneously, this finding determined how neurons regulated microglial PGE2 tolerance. Moreover, by inhibiting TLR4 activation and de novo protein synthesis by LPS-binding protein (LBP) manipulation and cycloheximide, our data showed that the TLR4 signal and de novo protein synthesis are necessary for microglia to develop PGE2 tolerance in NG cells under the ET challenge. Altogether, our findings demonstrated that neuron-microglia contacts are indispensable in emerging PGE2 tolerance through the regulation of TLR4-mediated de novo protein synthesis.

RESUMEN

Microbial translocation has been suggested as a major driver of chronic immune activation HIV infection. Thus, we compared the extent of microbial translocation in patients with acute HIV infection and patients followed after CD4-guided structured treatment interruption (STI) by measuring different circulating markers: (1) lipopolysaccharide (LPS)-binding protein (LBP), (2) bacterial DNA, (3) soluble CD14 (sCD14), and (4) mitochondrial DNA (mtDNA). Bacterial DNA and sCD14 levels were similar in all groups. Patients in acute phase showed higher levels of LBP and mtDNA. In STI, we found a positive correlation between the percentage of CD8+ T cells and bacterial DNA levels. Considering all patients, LBP was positively correlated with the percentage and the absolute count of CD8+ T cells, and with mtDNA stressing the importance of mitochondrial products in sustaining chronic immune activation.

Asunto(s)

Infecciones por VIH , Proteínas de Fase Aguda , Traslocación Bacteriana , Proteínas Portadoras , ADN Bacteriano , ADN Mitocondrial , Humanos , Receptores de Lipopolisacáridos , Lipopolisacáridos , Glicoproteínas de Membrana

RESUMEN

The sensitivity of pigs to deoxynivalenol (DON) might be influenced by systemic inflammation (SI) which impacts liver. Besides following acute-phase proteins, our aim was to investigate both the hepatic fractional albumin (ALB) synthesis rate (FSR) and the ALB concentration as indicators of ALB metabolism in presence and absence of SI induced by LPS via pre- or post-hepatic venous route. Each infusion group was pre-conditioned either with a control diet (CON, 0.12 mg DON/kg diet) or with a DON-contaminated diet (DON, 4.59 mg DON/kg diet) for 4 wk. A depression of ALB FSR was observed 195 min after LPS challenge, independent of feeding group or LPS application route, which was not paralleled by a down-regulated ALB mRNA expression but by a reduced availability of free cysteine. The drop in ALB FSR only partly explained the plasma ALB concentrations which were more depressed in the DON-pre-exposed groups, suggesting that ALB levels are influenced by further mechanisms. The abundances of haptoglobin, C-reactive protein, serum amyloid A, pig major acute-phase protein, fibrinogen and LPS-binding protein mRNA were up-regulated upon LPS stimulation but not accompanied by increases in the plasma concentrations of these proteins, pointing at an imbalance between synthesis and consumption.

Asunto(s)

Reacción de Fase Aguda/metabolismo , Albúminas/metabolismo , Inflamación/metabolismo , Hígado/metabolismo , Micotoxinas/administración & dosificación , Tricotecenos/administración & dosificación , Administración Oral , Alimentación Animal , Animales , Proteína C-Reactiva/metabolismo , Suplementos Dietéticos , Haptoglobinas/metabolismo , Lipopolisacáridos/inmunología , Micotoxinas/efectos adversos , Proteína Amiloide A Sérica/metabolismo , Porcinos , Tricotecenos/efectos adversos

RESUMEN

Metabolic endotoxemia initiates low-grade chronic inflammation in metabolic syndrome (MetS) and provokes the progression towards more advanced cardiometabolic disorders. Our recent works in obese rodent models demonstrate that catechin-rich green tea extract (GTE) improves gut barrier integrity to alleviate the translocation of gut-derived endotoxin and its consequent pro-inflammatory responses mediated through Toll-like receptor-4/nuclear factor κB (TLR4/NFκB) signaling. The objective of this clinical trial is to establish the efficacy of GTE to alleviate metabolic endotoxemia-associated inflammation in persons with MetS by improving gut barrier function. We plan a double-blind, placebo-controlled cross-over trial in persons with MetS and age- and gender-matched healthy persons (18-65 y; n = 20/group) who will receive a low-energy GTE-rich (1 g/day; 890 mg total catechins) confection snack food while following a low-polyphenol diet for 28 days. Assessments will include measures of circulating endotoxin (primary outcome) and secondary outcomes including biomarkers of endotoxin exposure, region-specific measures of intestinal permeability, gut microbiota composition, diversity, and functions, intestinal and systemic inflammatory responses, and catechins and microbiota-derived catechin metabolites. Study outcomes will provide the first report of the GTE-mediated benefits that alleviate gut barrier dysfunction in relation to endotoxemia-associated inflammation in MetS persons. This is expected to help establish an effective dietary strategy to mitigate the growing burden of MetS that currently affects ~35% of Americans.

RESUMEN

Background: Major Depressive Disorder (MDD) is associated with both proinflammatory and adaptive immune response abnormalities. Regulatory T lymphocytes (Tregs), a subtype of CD4+ T cells, are relevant for maintaining immune-inflammatory system homeostasis and control of inflammation such as the kind potentially induced by the interactions between the intestinal microbiome and gut mucosa. We investigated the Treg population and its distribution along their stages of differentiation/activation, as well as its function in MDD patients without concomitant diseases. We also studied the potential association between Treg alterations, intestinal barrier damage, and bacterial translocation. Methods: 30 MDD patients and 20 healthy controls were studied. The levels of circulating CD25FoxP3+ Tregs and their distribution on the naïve (TN), effector (TE), central (TCM), and effector memory(TEM) differentiation/activation stages were analyzed using polychromatic flow cytometry. Chemokine receptors (CCR) 2, 5, and 6, and the intracytoplasmic IL-10 expression by the Tregs were also analyzed. The serum IL-10 was measured using Luminex. The serum levels of zonulin and the intestinal fatty acid-binding protein (I-FABP), both markers of gut barrier function, and the LPS-binding protein (LBP), a marker of bacterial translocation, were measured using an enzyme-linked immunosorbent assay. Results: MDD patients had increased number of circulating Tregs cells with enhanced number of Tregs at the TN, TE, TCM, and TEM stages. The percentage of Tregs cells at TN stage was significantly higher in MDD patients. The percentage of Tregs that expressed CCR2 and CCR6 was increased as well as those expressing IL-10. MDD patients had significantly increased levels of circulating I-FABP and LBP. MDD patients with high LBP levels had a significant reduction in the number of circulating Tregs compared to normal-LBP MDD patients. Conclusions: MDD patients showed an expansion of circulating Tregs and their CD25highFoxP3+ and CD25lowFoxP3+ subsets throughout the different stages of CD4+ T lymphocyte differentiation/activation. Tregs also showed an increased frequency of cells expressing CCR6 and CCR2. IL-10 Treg production was also enhanced in MDD patients that concurrently had increased serum IL-10 levels. However, this Treg expansion was blunted in MDD patients with gut barrier damage and increased bacterial translocation.

RESUMEN

During sepsis, the increased synthesis of circulating lipopolysaccharide (LPS)-binding protein (LBP) activates LPS/TLR4 signaling in renal resident cells, leading to acute kidney injury (AKI). Pericytes are the major source of myofibroblasts during chronic kidney disease (CKD), but their involvement in AKI is poorly understood. Here, we investigate the occurrence of pericyte-to-myofibroblast trans-differentiation (PMT) in sepsis-induced AKI. In a swine model of sepsis-induced AKI, PMT was detected within 9 h from LPS injection, as evaluated by the reduction of physiologic PDGFRß expression and the dysfunctional α-SMA increase in peritubular pericytes. The therapeutic intervention by citrate-based coupled plasma filtration adsorption (CPFA) significantly reduced LBP, TGF-ß, and endothelin-1 (ET-1) serum levels, and furthermore preserved PDGFRß and decreased α-SMA expression in renal biopsies. In vitro, both LPS and septic sera led to PMT with a significant increase in Collagen I synthesis and α-SMA reorganization in contractile fibers by both SMAD2/3-dependent and -independent TGF-ß signaling. Interestingly, the removal of LBP from septic plasma inhibited PMT. Finally, LPS-stimulated pericytes secreted LBP and TGF-ß and underwent PMT also upon TGF-ß receptor-blocking, indicating the crucial pro-fibrotic role of TLR4 signaling. Our data demonstrate that the selective removal of LBP may represent a therapeutic option to prevent PMT and the development of acute renal fibrosis in sepsis-induced AKI.

Asunto(s)

Lesión Renal Aguda/etiología , Lesión Renal Aguda/metabolismo , Proteínas de Fase Aguda/metabolismo , Proteínas Portadoras/metabolismo , Transdiferenciación Celular , Glicoproteínas de Membrana/metabolismo , Miofibroblastos/metabolismo , Pericitos/metabolismo , Receptor Toll-Like 4/metabolismo , Lesión Renal Aguda/patología , Animales , Biopsia , Transdiferenciación Celular/genética , Células Cultivadas , Modelos Animales de Enfermedad , Endotoxinas/efectos adversos , Fibrosis , Inmunohistoquímica , Modelos Biológicos , Miofibroblastos/citología , Porcinos

RESUMEN

BACKGROUND & AIMS: Dysbiosis of the gut microbiota is associated with increased levels of circulating lipopolysaccharide (LPS) and subsequent activation of systemic inflammation. Diet is an important modulator of the gut microbiome. We aimed to investigate whether circulating markers of gut-related inflammation, LPS binding protein (LBP) and soluble CD14 (sCD14) can be modulated by n-3 PUFA supplementation and/or diet counselling, and whether these markers are related to cardiovascular (CV) outcome. METHODS: 484 men aged 65-75 years, at high CV-risk, were included and randomized in a 2 × 2 factorial design to 36-month intervention with dietary counselling, n-3 PUFA supplementation, or both. N-3 PUFA supplementation was placebo-controlled. ELISAs were used for determination of the biomarkers measured at baseline and study-end. A composite endpoint was defined as new CV-events and CV-mortality after 36 months. RESULTS: There were no significant differences in changes of either LBP or sCD14 in the intervention groups compared to their respective controls (n-3 PUFA vs. placebo: p = 0.58, p = 0.15, diet vs. no-diet: p = 0.53, p = 0.59, respectively). The group with LBP levels above median had about 2-fold unadjusted risk of suffering an endpoint compared to the group below (HR 2.22, 95% CI 1.25-3.96; p = 0.01). A similar tendency was seen for sCD14 (HR 1.72, 95% CI 0.97-3.03; p = 0.06). After adjusting for covariates, LBP remained significantly associated with a two-fold CV-risk, whereas sCD14 gained statistical significance, however, lost when hsCRP was added to the model. CONCLUSIONS: In our population, markers of gut-related inflammation associated with 36-month CV outcome. However, neither n-3 PUFA nor diet intervention had an effect on these markers.

Asunto(s)

Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/epidemiología , Proteínas Portadoras/sangre , Dieta , Suplementos Dietéticos , Consejo Dirigido , Ácidos Grasos Omega-3/administración & dosificación , Inflamación/sangre , Receptores de Lipopolisacáridos/sangre , Glicoproteínas de Membrana/sangre , Proteínas de Fase Aguda , Anciano , Biomarcadores/sangre , Microbioma Gastrointestinal , Humanos , Masculino , Estudios Prospectivos , Medición de Riesgo

RESUMEN

Fish oil is a natural product that has shown efficacy for managing inflammatory conditions with few side effects. There is emerging evidence that crosstalks between gut epithelial cells and immune cells contribute to chronic infectious diseases. HIV-infected (HIV+) older adults show age-related co-morbidities at a younger age than their uninfected counterparts. Persistent inflammation related to the chronic viral infection and its sequelae is thought to contribute to this disparity. However, little is known about whether fish oil reduces intestinal inflammation in HIV + patients. We measure inflammation and gut barrier function in HIV + older adults (median age = 52, N = 33), following 12 weeks of fish oil supplementation (a total daily dose of 1.6 g of omega-3 fatty acids). We showed a reduction in inflammation and gut permeability as measured by CD14, inflammatory cytokines, lipopolysaccharide, and lipopolysaccharide binding protein. The results indicate that older HIV + adults may benefit from a diet supplemented with the omega-3 fatty acids found in fish oil.

RESUMEN

Toll-like receptor 4 (TLR4) is an indispensable immune receptor for lipopolysaccharide (LPS), a major component of the Gram-negative bacterial cell wall. Following LPS stimulation, TLR4 transmits the signal from the cell surface and becomes internalized in an endosome. However, the spatial regulation of TLR4 signaling is not fully understood. Here, we investigated the mechanisms of LPS-induced TLR4 internalization and clarified the roles of the extracellular LPS-binding molecules, LPS-binding protein (LBP), and glycerophosphatidylinositol-anchored protein (CD14). LPS stimulation of CD14-expressing cells induced TLR4 internalization in the presence of serum, and an inhibitory anti-LBP mAb blocked its internalization. Addition of LBP to serum-free cultures restored LPS-induced TLR4 internalization to comparable levels of serum. The secretory form of the CD14 (sCD14) induced internalization but required a much higher concentration than LBP. An inhibitory anti-sCD14 mAb was ineffective for serum-mediated internalization. LBP lacking the domain for LPS transfer to CD14 and a CD14 mutant with reduced LPS binding both attenuated TLR4 internalization. Accordingly, LBP is an essential serum molecule for TLR4 internalization, and its LPS transfer to membrane-anchored CD14 (mCD14) is a prerequisite. LBP induced the LPS-stimulated phosphorylation of TBK1, IKKϵ, and IRF3, leading to IFN-ß expression. However, LPS-stimulated late activation of NF-κB or necroptosis were not affected. Collectively, our results indicate that LBP controls LPS-induced TLR4 internalization, which induces TLR adaptor molecule 1 (TRIF)-dependent activation of the TBK1-IKKϵ-IRF3-IFN-ß pathway. In summary, we showed that LBP-mediated LPS transfer to mCD14 is required for serum-dependent TLR4 internalization and activation of the TRIF pathway.

Asunto(s)

Proteínas de Fase Aguda/metabolismo , Proteínas Portadoras/metabolismo , Quinasa I-kappa B/metabolismo , Factor 3 Regulador del Interferón/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Toll-Like 4/metabolismo , Humanos , Lipopolisacáridos/farmacología , Antígeno 96 de los Linfocitos/metabolismo , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos

RESUMEN

Lipopolysaccharide (LPS) is an essential element of nearly all Gram-negative bacterial outer membranes and serves to protect the cell from adverse environmental stresses. Seven members of the lipopolysaccharide transport (Lpt) protein family function together to transport LPS from the inner membrane (IM) to the outer leaflet of the outer membrane of bacteria such as Escherichia coli. Each of these proteins has a solved crystal structure, including LptC, which is a largely periplasmic protein that is associated with the IM LptB2 FG complex and anchored to the membrane by an N-terminal helix. LptC directly binds LPS and is hypothesized to be involved in the transfer of LPS to another periplasmic protein, LptA. Purified and in solution, LptC forms a dimer. Here, point mutations designed to disrupt formation of the dimer are characterized using site-directed spin labeling double electron electron resonance (DEER) spectroscopy, light scattering, circular dichroism, and computational modeling. The computational studies reveal the molecular interactions that drive dimerization of LptC and elucidate how the disruptive mutations change this interaction, while the DEER and light scattering studies identify which mutants disrupt the dimer. And, using electron paramagnetic resonance spectroscopy and comparing the results to the previous quantitative characterization of the interactions between dimeric LptC and LPS and LptA, the functional consequences of monomeric LptC were also determined. These results indicate that disruption of the dimer does not affect LPS or LptA binding and that monomeric LptC binds LPS and LptA at levels similar to dimeric LptC.

Asunto(s)

Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas Portadoras/genética , Espectroscopía de Resonancia por Spin del Electrón , Proteínas de Escherichia coli/genética , Proteínas de la Membrana/genética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual/genética , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

Lipopolysaccharide (LPS, endotoxin) is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium. LPS is a large lipid containing several acyl chains as its hydrophobic base and numerous sugars as its hydrophilic core and O-antigen domains, and is an essential element of the organisms' natural defenses in adverse environmental conditions. LptC is one of seven members of the lipopolysaccharide transport (Lpt) protein family that functions to transport LPS from the inner membrane (IM) to the outer leaflet of the outer membrane of the bacterium. LptC is anchored to the IM and associated with the IM LptFGB2 complex. It is hypothesized that LPS binds to LptC at the IM, transfers to LptA to cross the periplasm, and is inserted by LptDE into the outer leaflet of the outer membrane. The studies described here comprehensively characterize and quantitate the binding of LPS to LptC. Site-directed spin labeling electron paramagnetic resonance spectroscopy was utilized to characterize the LptC dimer in solution and monitor spin label mobility changes at 10 sites across the protein upon addition of exogenous LPS. The results indicate that soluble LptC forms concentration-independent N-terminal dimers in solution, LptA binding does not change the conformation of the LptC dimer nor appreciably disrupt the LptC dimer in vitro, and LPS binding affects the entire LptC protein, with the center and C-terminal regions showing a greater affinity for LPS than the N-terminal domain, which has similar dissociation constants to LptA.

Asunto(s)

Proteínas de Escherichia coli/química , Escherichia coli/genética , Lipopolisacáridos/química , Proteínas de la Membrana/química , Multimerización de Proteína , Secuencias de Aminoácidos , Sitios de Unión , Transporte Biológico , Cristalografía por Rayos X , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Lipopolisacáridos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Transporte de Proteínas

RESUMEN

The antimicrobial peptide, epinecidin-1 (Epi), was identified from Epinephelus coioides and may have clinical application for treating sepsis. Epi has been shown to ameliorate antibiotic-resistant bacteria-induced sepsis in mice, but further evaluation in mixed-flora models and a description of the protective mechanisms are essential to establish this peptide as a potential therapeutic. Therefore, we first tested the protective effects of Epi against polymicrobial sepsis-induced bactericidal infection, inflammation and lung injury that result from cecal ligation and puncture in mice. Furthermore, since lipopolysaccharide (LPS) is a key inducer of inflammation during bacterial infection and sepsis, we also tested the LPS-antagonizing activity and related mechanisms of Epi-mediated protection in mice with LPS-induced endotoxemia and LPS-treated Raw264.7 mouse macrophage cells. Epi rescued mice from both polymicrobial sepsis and endotoxemia after delayed administration and suppressed both lung and systemic inflammatory responses, while attenuating lung injury and diminishing bacterial load. In vitro studies revealed that Epi suppressed LPS-induced inflammatory cytokine production. Mechanistically, Epi disrupted the interaction between LPS and LPS binding protein, competed with LPS for binding on the cell surface, and inhibited Toll-like receptor 4 endocytosis, resulting in inhibition of LPS-induced reactive oxygen species/p38/Akt/NF-κB signaling and subsequent cytokine production. Overall, our results demonstrate that Epi is a promising therapeutic agent for endotoxemia and polymicrobial sepsis.

Asunto(s)

Péptidos Catiónicos Antimicrobianos/farmacología , Endotoxemia/tratamiento farmacológico , Proteínas de Peces/farmacología , Sustancias Protectoras/farmacología , Animales , Antiinfecciosos/farmacología , Carga Bacteriana , Ciego/microbiología , Ciego/cirugía , Línea Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Endotoxemia/etiología , Femenino , Ligadura , Receptores de Lipopolisacáridos/efectos de los fármacos , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Receptor Toll-Like 4/metabolismo

RESUMEN

Excessive inflammatory response is critical event in the pathogenesis of acute lung injury (ALI). Previous study has shown that activating transcription factor 3 (ATF3) plays a role in downregulate inflammatory responses including ventilation-induced ALI. We hypothesized that ATF3 have a protective effect in ALI induced by pseudomonas aeruginosa. PA was intra-tracheally administrated to ATF3 knock-out (KO) mice to establish ALI model. Inflammatory factors, BALF protein, lung wet to dry ratio, lung injury score and mortality were determined. The activation of NF-κB was detected by western blot and Co-immunoprecipitation (Co-ip) was used to determinate the binding of ATF3 to LBP. Peritoneal macrophages were isolated from ATF3 KO mice and stimulated by PA. PA increased the expression of ATF3 in the lung tissues in ATF3 wild type (WT) mice. ATF3 deficiency significantly increased the concentration of TNFα, IL-6 and IL-1ß in the supernatant of peritoneal macrophages, lung tissue and BALF after PA stimulation and also enhanced the activity of NF-κB. ATF3 deficiency also enhanced the BALF protein concentration and increased the lung wet to dry ratio. The lung injury score and mortality were higher in ATF3 KO mice treated with PA. Moreover, ATF3 was demonstrated to bind to LBP These finding suggest ATF3 protect mice against ALI induced by PA partly due to the binding to LBP.

Asunto(s)

Factor de Transcripción Activador 3/metabolismo , Lesión Pulmonar Aguda/prevención & control , Proteínas de Fase Aguda/metabolismo , Proteínas Portadoras/metabolismo , Macrófagos Peritoneales/inmunología , Glicoproteínas de Membrana/metabolismo , Infecciones por Pseudomonas/prevención & control , Factor de Transcripción Activador 3/genética , Lesión Pulmonar Aguda/microbiología , Animales , Líquido del Lavado Bronquioalveolar/química , Células Cultivadas , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Tripalmitoyl-S-glyceryl-l-Cys-Ser-(Lys)4 (Pam3CSK4) is a highly conserved molecular motif found in various classes of lipoproteins. The requirement for leukocyte to respond to synthetic Pam3CSK4 were studied. Pam3CSK4 primed neutrophils for a respiratory burst in a serum-dependent manner. Pam3CSK4 upregulated CD11b, CD14, and cytochrome b558, and downregulated Leu-8. Treatment of neutrophils with anti-CD14 antibodies and treatment of serum with anti-LPS binding protein (LBP) antibodies resulted in the inhibition of priming for respiratory burst by Pam3CSK4. It should be noted that LBP could not replicate the effects of serum in priming of neutrophils for respiratory burst by Pam3CSK4. Serum LBP bound to immobilized Pam3CSK4. Pam3CSK4 induced the interleukin-8 (IL-8) production by leukocytes in a serum-dependent manner. Further, Pam3CSK4-induced priming of neutrophils for respiratory burst was not inhibited by the LPS antagonists LA-14-PP, Rhodobacter sphaeroides LPS, or E5531, and Pam3CSK4-induced IL-8 production by leukocytes was not affected by LPS antagonist, E5531, indicating that Pam3CSK4 was recognized by a different receptor than LPS. Thus, Pam3CSK4 and LPS had similar biological activities and similar requirement to act on leukocytes, but were recognized by different receptors. Serum in the action of Pam3CSK4 on leukocytes was not replicated by LBP, suggesting that Pam3CSK4 might be disaggregated by serum to result in the activation of leukocytes.

Asunto(s)

Lipopéptidos/farmacología , Neutrófilos/inmunología , Suero/metabolismo , Proteínas de Fase Aguda/metabolismo , Anticuerpos/farmacología , Proteínas Portadoras/metabolismo , Células Cultivadas , Humanos , Inmunidad Innata , Interleucina-8/metabolismo , Lípido A/análogos & derivados , Lípido A/farmacología , Receptores de Lipopolisacáridos/inmunología , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/inmunología , Glicoproteínas de Membrana/metabolismo , Activación Neutrófila , Estallido Respiratorio

RESUMEN

Lipopolysaccharide (LPS) and the periplasmic protein, LptA, are two essential components of Gram-negative bacteria. LPS, also known as endotoxin, is found asymmetrically distributed in the outer leaflet of the outer membrane of Gram-negative bacteria such as Escherichia coli and plays a role in the organism's natural defense in adverse environmental conditions. LptA is a member of the lipopolysaccharide transport protein (Lpt) family, which also includes LptC, LptDE, and LptBFG2 , that functions to transport LPS through the periplasm to the outer leaflet of the outer membrane after MsbA flips LPS across the inner membrane. It is hypothesized that LPS binds to LptA to cross the periplasm and that the acyl chains of LPS bind to the central pocket of LptA. The studies described here are the first to comprehensively characterize and quantitate the binding of LPS by LptA. Using site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy, data were collected for 15 spin-labeled residues in and around the proposed LPS binding pocket on LptA to observe the mobility changes caused by the presence of exogenous LPS and identify the binding location of LPS to LptA. The EPR data obtained suggest a 1:1 ratio for the LPS:LptA complex and allow the first calculation of dissociation constants for the LptA-LPS interaction. The results indicate that the entire protein is affected by LPS binding, the N-terminus unfolds in the presence of LPS, and a mutant LptA protein unable to form oligomers has an altered affinity for LPS.

Asunto(s)

Proteínas Portadoras/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Lipopolisacáridos/química , Periplasma/química , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Cinética , Lipopolisacáridos/metabolismo , Modelos Moleculares , Mutación , Periplasma/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Marcadores de Spin

RESUMEN

Lipid A comprises the active region of lipopolysaccharide (LPS), and its phosphate group is required for LPS activities. Additionally, it is essential for effects of inhibitors of LPS-induced coagulation activity in limulus amebocyte lysate (LAL) tests. Lipid A has phosphorylated glucosamine residues, which are structurally similar to glucose 1-phosphate (G1P) and glucose 6-phosphate (G6P). This study focused on the antagonistic effects of glucose phosphates on the action of protein or non-protein inhibitors against LAL coagulation, LPS-LPS-binding protein (LBP) interaction, and LPS bioactivities. These effects of glucose phosphates were evaluated and compared with those of other charged sugars such as fructose 6-phosphate and glucuronic acid by LAL tests, ELISA-based LPS-LBP binding assay, cell-based assay, and using a mouse endotoxin shock model. G6P neutralized the interfering actions of drug substances and plasma proteins on LPS coagulation activity in LAL tests. Compared to other sugars, G6P more strongly inhibited LPS binding to LBP, leading to significant inhibition of LPS-induced cellular responses in human umbilical vein endothelial cells and in the THP-1 human leukemic line. Consistent herewith, G6P inhibited inflammatory cytokine release and decreased serum alanine aminotransferase and hepatic caspase-3/7 activities and mortality in LPS-stimulated d-galactosamine-sensitized mice. These data indicated that the structural properties of G6P, such as its glucose moiety and phosphorylation on carbon 6, are important for suppressing the interaction of proteins with LPS. Therefore, G6P is useful to improve sensitivity and accuracy of plasma and drug LPS assays, and such structural property is more suitable to antagonize LPS activities.

Asunto(s)

Proteínas de Fase Aguda/metabolismo , Proteínas Portadoras/metabolismo , Glucosa/metabolismo , Lípido A/metabolismo , Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana/metabolismo , Nefelometría y Turbidimetría/métodos , Animales , Carbono , Caspasa 3/metabolismo , Glucosa/química , Cangrejos Herradura , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfatos/química , Fosforilación , Unión Proteica , Factor de Necrosis Tumoral alfa/metabolismo