Coli Lipopolysaccharide Research Articles

Rapid and sensitive whole cell E. coli detection using deep eutectic solvents/graphene oxide/gold nanoparticles field-effect transistor

Every year, millions of people suffer from gastrointestinal inflammation caused by E. coli. The increase of antibiotic-resistant strains and similar inflammatory and infectious syndromes symptoms have made rapid and sensitive diagnosis of this pathogen challenging. This study developed a Field-Effect Transistor based on deep eutectic solvents, graphene oxide, and gold nanoparticles (DES/GO/AuNPs-FET) to detect E. coli. Comparing the output current showed DES, which was a mixture of ethylene glycol and choline chloride, with ionic behavior, in addition to improving the electrical properties of GO, also led to the formation of AuNPs by self-assembly, which significantly increased the sensor's sensing performance. E. coli lipopolysaccharide aptamer immobilized on DES/GO/AuNPs-FET; capturing E. coli and changing the conformation caused changes in the charge carrier flow in the FET. This nanobiosensor detected E. coli in a completely selective manner in complex matrices like human blood serum. The excellent sensing performance of this nanobiosensor compared to other biosensors with a low detection limit (LOD = 3 CFU/ml), label-free, fast, and real-time detection showed that DES/GO/AuNPs-FET could be a reliable alternative to existing detection methods.

Integration of MALDI glycotyping and NMR analysis to uncover an O-antigen substructure from pathogenic Escherichia coli O111

Escherichia coli O111 serotype is a critical pathogenic E. coli strain that causes severe, potentially fatal complications. Despite its reported variation, only one structure of the O-antigen polysaccharide from E. coli O111 has been reported. Here, a substructure of the O-antigen from E. coli O111 was characterized using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and NMR analysis. MALDI glycotyping revealed differing O-antigen repeating unit masses of Δm/z 787 and 828 in the E. coli strains and lipopolysaccharides from the O111 serogroup. This variation was caused by the replacement of the hexose residue with hexosamine in the repeating units, which was further confirmed by LIFT-TOF/TOF analysis. Structural elucidation of the O111 substructure by NMR analysis further demonstrated replacement of the hydroxyl group with an N-acetyl group on the terminal glucose residue of the O-antigen pentasaccharide repeating unit. To our knowledge, this study is the first to provide a detailed structural analysis of a new O-antigen substructure from the E. coli O111 serogroup.

Beta-Glucans Improve the Mammary Innate Immune Response to Endotoxin Challenge in Dairy Ewes.

This study evaluated short-term immune responses of dairy ewes supplemented with barley β-glucan (BG) following an intramammary Escherichia coli lipopolysaccharide (LPS) challenge. In the adaptation period, 36 ewes were fed an alfalfa hay diet ad libitum and barley grain cv. Hispanic (3.8% BG). Then, ewes were assigned into three experimental groups: (1) Control (CON), the same previous diet (13.3 g BG/d); (2) high β-glucans barley (HBG), new barley (cv. Annapurna) containing 10% BG (35 g BG/d); (3) intraperitoneally injected (INP) with a 1.4% BG solution dose (2 g BG/ewe). At d 9, all ewes were infused with an E. coli LPS or saline solution in each udder half. After the challenge, rectal temperature (RT), milk yield and composition, somatic cell count (SCC), and plasma interleukins (IL-1α and IL-1β) were monitored daily. The INP treatment revealed a transitory increase in RT and decreased milk yield by 38%. Milk fat, protein, and SCC increased in LPS-treated udders but not by BG treatment. The IL-1α plasma concentration was similar among groups but INP ewes showed a lower IL-1β concentration suggesting a lower inflammatory response. The BG administration appears more effective intraperitoneally than orally, which needs additional study.

Immunometabolic Chaos in Septic Shock.

Septic shock is associated with over 40% mortality. The immune response in septic shock is tightly regulated by cellular metabolism and transitions from early hyper-inflammation to later hypo-inflammation. Patients are susceptible to secondary infections during hypo-inflammation. The magnitude of the metabolic dysregulation and the effect of plasma metabolites on the circulating immune cells in septic shock are not reported. We hypothesized that the accumulated plasma metabolites affect the immune response in septic shock during hypo-inflammation. Our study took a unique approach. Using peripheral blood from adult septic shock patients and healthy controls, we studied: 1. Whole blood stimulation ± E. Coli lipopolysaccharide (LPS: endotoxin) to analyze plasma TNF protein, and 2. Plasma metabolomic profile by Metabolon. Inc. 3. We exposed peripheral blood mononuclear cells (PBMCs) from healthy controls to commercially available carbohydrate, amino acid, and fatty acid metabolites and studied the response to LPS. We report that: 1. The whole blood stimulation of the healthy control group showed a significantly upregulated TNF protein, while the septic shock group remained endotoxin tolerant, a biomarker for hypo-inflammation. 2. A significant accumulation of carbohydrate, amino acid, fatty acid, ceramide, sphingomyelin, and TCA cycle pathway metabolites in septic shock plasma. 3. In vitro exposure to five metabolites repressed while two metabolites upregulated the inflammatory response of PBMCs to LPS. We conclude that the endotoxin-tolerant phenotype of septic shock is associated with a simultaneous accumulation of plasma metabolites from multiple metabolic pathways, and these metabolites fundamentally influence the immune response profile of circulating cells.

Noradrenaline and Adrenoreceptors Are Involved in the Regulation of Prostaglandin I2 Production in the Porcine Endometrium after Experimentally Induced Inflammation.

Endometritis is a common disease in animals, leading to disruption of reproductive processes and economic losses. Noradrenergic control of prostaglandin (PG)I2 formation by inflamed endometrium is unknown. We determined the involvement of α1-, α2- and β-adrenoreceptors (ARs) in noradrenaline-influenced PGI synthase (PGIS) protein abundance and PGI2 release from porcine (1) endometrial explants with Escherichia coli (E. coli)-induced inflammation in vivo, and (2) E. coli lipopolysaccharide (LPS)-treated endometrial epithelial cells. Experiment 1. E. coli suspension (E. coli group) or saline (CON group) was injected into the uterine horns. In both groups, noradrenaline increased endometrial PGIS abundance and PGI2 release versus the control values, and it was higher in the E. coli group than in the CON group. In the CON group, a noradrenaline stimulating effect on both parameters takes place through α1D-, α2C- and β2-ARs. In the E. coli group, noradrenaline increased PGIS abundance and PGI2 release via α1A-, α2(B,C)- and β(1,2)-ARs, and PGI2 release also by α2A-ARs. Experiment 2. LPS and noradrenaline augmented the examined parameters in endometrial epithelial cells versus the control value. In LPS-treated cells, β(1,2)-ARs mediate in noradrenaline excitatory action on PGIS protein abundance and PGI2 release. β3-ARs also contribute to PGI2 release. Under inflammatory conditions, noradrenaline via ARs increases PGI2 synthesis and release from the porcine endometrium, including epithelial cells. Our findings suggest that noradrenaline may indirectly affect processes regulated by PGI2 in the inflamed uterus.

Identification of novel biomarkers of acute phase response in chickens challenged with Escherichia coli lipopolysaccharide endotoxin

BackgroundThe chicken’s inflammatory response is an essential part of the bird’s response to infection. A single dose of Escherichia coli (E. coli) lipopolysaccharide (LPS) endotoxin can activate the acute phase response (APR) and lead to the production of acute phase proteins (APPs). In this study, the responses of established chicken APPs, Serum amyloid A (SAA) and Alpha-1-acid-glycoprotein (AGP), were compared to two novel APPs, Hemopexin (Hpx) and Extracellular fatty acid binding protein (Ex-FABP), in 15-day old broilers over a time course of 48 h post E.coli LPS challenge. We aimed to investigate and validate their role as biomarkers of an APR. Novel plant extracts, Citrus (CTS) and cucumber (CMB), were used as dietary supplements to investigate their ability to reduce the inflammatory response initiated by the endotoxin.ResultsA significant increase of established (SAA, AGP) and novel (Ex-FABP, Hpx) APPs was detected post E.coli LPS challenge. Extracellular fatty acid binding protein (Ex-FABP) showed a similar early response to SAA post LPS challenge by increasing ~ 20-fold at 12 h post challenge (P < 0.001). Hemopexin (Hpx) showed a later response by increasing ∼5-fold at 24 h post challenge (P < 0.001) with a similar trend to AGP. No differences in APP responses were identified between diets (CTS and CMB) using any of the established or novel biomarkers.ConclusionsHpx and Ex-FABP were confirmed as potential biomarkers of APR in broilers when using an E. coli LPS model along with SAA and AGP. However, no clear advantage for using either of dietary supplements to modulate the APR was identified at the dosage used.

Lysophosphatidylcholine Acetyltransferase 2 (LPCAT2) Influences the Gene Expression of the Lipopolysaccharide Receptor Complex in Infected RAW264.7 Macrophages, Depending on the E. coli Lipopolysaccharide Serotype.

Escherichia coli (E. coli) is a frequent gram-negative bacterium that causes nosocomial infections, affecting more than 100 million patients annually worldwide. Bacterial lipopolysaccharide (LPS) from E. coli binds to toll-like receptor 4 (TLR4) and its co-receptor's cluster of differentiation protein 14 (CD14) and myeloid differentiation factor 2 (MD2), collectively known as the LPS receptor complex. LPCAT2 participates in lipid-raft assembly by phospholipid remodelling. Previous research has proven that LPCAT2 co-localises in lipid rafts with TLR4 and regulates macrophage inflammatory response. However, no published evidence exists of the influence of LPCAT2 on the gene expression of the LPS receptor complex induced by smooth or rough bacterial serotypes. We used RAW264.7-a commonly used experimental murine macrophage model-to study the effects of LPCAT2 on the LPS receptor complex by transiently silencing the LPCAT2 gene, infecting the macrophages with either smooth or rough LPS, and quantifying gene expression. LPCAT2 only significantly affected the gene expression of the LPS receptor complex in macrophages infected with smooth LPS. This study provides novel evidence that the influence of LPCAT2 on macrophage inflammatory response to bacterial infection depends on the LPS serotype, and it supports previous evidence that LPCAT2 regulates inflammatory response by modulating protein translocation to lipid rafts.

Abstract 2096: Role Of Divalent Cations In The Enzymatic Activation Of Coagulation Factor XII By Lipopolysaccharides

Background: Lipopolysaccharide (LPS) is the primary pathogenic factor in Gram-negative sepsis. LPS is a polyanionic molecule that may act as a site of initiation of coagulation by activating factor (F)XII. While the physicochemical properties of LPS are known to be sensitive to interactions with divalent cations, it is unknown whether divalent cations modulate the activation of FXII by LPS. Aims: To determine whether divalent cations regulate the kinetics of FXII activation by select LPS chemotypes from E. coli . Methods: Aggregates of E. Coli LPS chemotypes O26:B6 and Rd2 were prepared in ultrapure water in the presence or absence of calcium (Ca 2+ ). The biophysical characterization of LPS chemotypes were determined by dynamic light scattering (DLS) and measurement of zeta potential (ZP). Chromogenic substrate assays were used to study the effects of LPS on FXII activation. Results: The LPS O26:B6 and Rd2 in the absence of Ca 2+ produced aggregates in form of micelles with hydrodynamic diameters of 190±10 and 200±7 nm and ZP of -25±0.8 and -60±2 mV, respectively. The presence of Ca 2+ produced aggregates in the form of vesicles with hydrodynamic diameters of 400±15 and 1300±60 nm and ZP of –11±2 and –3±0.5 mV, respectively. FXII was incubated in HEPES buffer with the distinct LPS morphologies in varying salt concentrations. We found maximal autoactivation of FXII by O26:B6 and Rd2 chemotypes at lower NaCl concentrations. The presence of Ca 2+ , however, selectively reduced the ability of O26:B6 to activate FXII. Strikingly, the neutralization of the surface net charge of the LPS chemotype Rd2 by Ca 2+ ions abrogated the amidolytic activity of Rd2. Conclusions: The presence of calcium ions induces changes in the physicochemical properties of LPS that result in distinct effects on FXII activation in vitro . We are currently studying whether this phenomena is conserved for other divalent cations including Zn, Mg, Cu, and Mn.

Intra-tumoral immunomodulatory therapy for advanced abdominal cancers using lipopolysaccharide: The Regional Immuno-Oncology Trial-1 (RIOT-1) protocol (NCT05751837)

BackgroundIntra-tumoral immunotherapy has shown potential in treating advanced cancers. Delivery challenges have limited exploration of these modalities in intra-abdominal tumors. In this study, we explore the safety of injecting intra-abdominal tumors with lipopolysaccharide (LPS) from Escherichia coli 0113. This agonist of toll-like receptor 4 (TLR4) holds promise as an agent to enhance the anti-tumor immune response within the tumor microenvironment. MethodsThis Phase I study will recruit adult patients with peritoneal metastases from gastrointestinal primary malignancies who have at least two suitable intra-abdominal soft tissue tumors for injection. LPS will be administered as a single 1 μg dose during diagnostic laparoscopy in patients in whom a subsequent interval laparotomy is planned. A control injection of saline will be injected into a second lesion. Primary outcome is safety, with secondary outcomes being biomarkers of the tumor immune microenvironment in pre- and post-treatment biopsies. ResultsThe primary endpoint is to determine the safety (frequency and nature of adverse events) following intra-tumoral LPS injection. Adverse events will be classified using Common Terminology Criteria for Adverse Events. Secondary endpoints include cellular and molecular biomarkers of immune response. The study will proceed until twelve patients have completed the protocol. DiscussionPatients undergoing standard-of-care laparoscopy in preparation for interval cytoreductive surgery may be ideal candidates for intra-tumoral immunotherapy. This study seeks to establish the safety of using E. coli LPS for injection into intra-abdominal tumors and to establish precedent for interval tumor immune microenvironment assessment as a window-of-opportunity concept in the context of abdominal metastatic disease. Trial RegistrationThe trial is registered at Clinical Trials.gov; NCT05751837 (registered February 28th, 2023).

T-independent B-cell effect of agents associated with swine grower-finisher diarrhea.

Swine dysentery, spirochetal colitis, and salmonellosis are production-limiting enteric diseases of global importance to the swine industry. Despite decades of efforts, mitigation of these diseases still relies on antibiotic therapy. A common knowledge gap among the 3 agents is the early B-cell response to infection in pigs. Thus, this study aimed to characterize the porcine B-cell response to Brachyspira hyodysenteriae, Brachyspira hampsonii (virulent and avirulent strains), Brachyspira pilosicoli, and Salmonella Typhimurium, the agents of the syndromes mentioned above. Immortalized porcine B-cell line derived from a crossbred pig with lymphoma were co-incubated for 8h with each pathogen, as well as E. coli lipopolysaccharide (LPS) and a sham-inoculum (n = 3/treatment). B-cell viability following treatments was evaluated using trypan blue, and the expression levels of B-cell activation-related genes was profiled using reverse transcription quantitative PCR. Only S. Typhimurium and LPS led to increased B-cell mortality. B. pilosicoli downregulated B-lymphocyte antigen (CD19), spleen associated tyrosine Kinase (syk), tyrosine-protein kinase (lyn), and Tumour Necrosis Factor alpha (TNF-α), and elicited no change in immunoglobulin-associated beta (CD79b) and swine leukocyte antigen class II (SLA-DRA) expression levels, when compared to the sham-inoculated group. In contrast, all other treatments significantly upregulated CD79b and stimulated responses in other B-cell downstream genes. These findings suggest that B. pilosicoli does not elicit an immediate T-independent B-cell response, nor does it trigger antigen-presenting mechanisms. All other agents activated at least one trigger within the T-independent pathways, as well as peptide antigen presenting mechanisms. Future research is warranted to verify these findings in vivo.

Effect of Glycosylation on Self-Assembly of Lipid A Lipopolysaccharides in Aqueous Solutions.

Lipopolysaccharides (LPSs) based on lipid A produced by bacteria are of interest due to their bioactivity in stimulating immune responses, as are simpler synthetic components or analogues. Here, the self-assembly in water of two monodisperse lipid A derivatives based on simplified bacterial LPS structures is examined and compared to that of a native Escherichia coli LPS using small-angle X-ray scattering and cryogenic transmission electron microscopy. The critical aggregation concentration is obtained from fluorescence probe experiments, and conformation is probed using circular dichroism spectroscopy. The E. coli LPS is found to form wormlike micelles, whereas the synthetic analogues bearing six lipid chains and with four or two saccharide head groups (Kdo2-lipid A and monophosphoryl lipid A) self-assemble into nanosheets or vesicles, respectively. These observations are rationalized by considering the surfactant packing parameter.

Modulation of the Acute Inflammatory Response Induced by the Escherichia coli Lipopolysaccharide through the Interaction of Pentoxifylline and Florfenicol in a Rabbit Model.

Experimental reports have demonstrated that florfenicol (FFC) exerts potent anti-inflammatory effects, improving survival in a murine endotoxemia model. Considering the anti-inflammatory and immunomodulatory properties of pentoxifylline (PTX) as an adjuvant to enhance the efficacy of antibiotics, the anti-inflammatory effects of the interaction FFC/PTX over the E. coli Lipopolysaccharide (LPS)-induced acute inflammatory response was evaluated in rabbits. Twenty-five clinically healthy New Zealand rabbits (3.8 ± 0.2 kg body weight: bw), were distributed into five experimental groups. Group 1 (control): treated with 1 mL/4 kg bw of 0.9% saline solution (SS) intravenously (IV). Group 2 (LPS): treated with an IV dose of 5 µg/kg of LPS. Group 3 (pentoxifylline (PTX) + LPS): treated with an oral dose of 30 mg/kg PTX, followed by an IV dose of 5 µg/kg of LPS 45 min after PTX. Group 4 (Florfenicol (FFC) + LPS): treated with an IM dose of 20 mg/kg of FFC, followed by an IV dose of 5 µg/kg of LPS 45 min after FFC administration. Group 5 (PTX + FFC + LPS): treated with an oral dose of 30 mg/kg of PTX, followed by an IM dose of 20 mg/kg of FFC, and, 45 min after an IV dose of 5 µg/kg of LPS was administered. The anti-inflammatory response was evaluated through changes in plasma levels of interleukins (TNF-α, IL-1β and IL-6), C-reactive protein (CRP), and body temperature. It has been shown that each drug produced a partial inhibition over the LPS-induced increase in TNF-α, IL-1β, and CRP. When both drugs were co-administered, a synergistic inhibitory effect on the IL-1β and CRP plasma concentrations was observed, associated with a synergic antipyretic effect. However, the co-administration of PTX/FFC failed to modify the LPS-induced increase in the TNF-α plasma concentrations. We concluded that the combination of FFC and PTX in our LPS sepsis models demonstrates immunomodulatory effects. An apparent synergistic effect was observed for the IL-1β inhibition, which peaks at three hours and then decreases. At the same time, each drug alone was superior in reducing TNF-α levels, while the combination was inferior. However, the peak of TNF-α in this sepsis model was at 12 h. Therefore, in rabbits plasma IL-1β and TNF-α could be regulated independently, thus, further research is needed to explore the effects of this combination over a more prolonged period.

Stimulation of shrimp (Penaeus monodon) hemocytes by lipopolysaccharide-like molecules derived from Novacq™

Immune stimulation through feed additives is a promising strategy that can help to combat disease in shrimp farming and reduce the use of antibiotics and other chemotherapeutics. The present study investigated the in vitro immunostimulatory effects of lipopolysaccharide (LPS)-like molecules isolated from the microbial based feed additive Novacq™ (N-LPS). The presence of LPS-like molecules was confirmed and quantified Novacq™ using a HEK-TLR4 reporter cell line. Primary hemocytes isolated from adult Penaeus monodon were used to measure the immunostimulatory of N-LPS compared with the control group that were treated with E. coli derived LPS (E-LPS). The N-LPS stimulated a rapid and significant induction of the phenoloxidase (PO) response in the hemocytes. The PO response increased with exposure time and LPS concentration and was significantly higher compared with an E. coli LPS (E-LPS) control. In addition, using gene expression data, we quantified the transcriptome response of the hemocytes at 15, 30 and 60 mins post stimulation. Compared with the controls, the N-LPS treated hemocytes had a significant up-regulation of genes involved in the immune system modulation and control at all time-points. Most noteworthy was the significant induction of transcripts that function as serine protease inhibitors (namely SERPINs), that regulate the overexpression of the PO system. Transcription factors from the Notch family which directly regulate the expression of many immune genes were also induced within the hemocytes. Additionally, we also saw a strong up-regulation of crustacean hyperglycemic hormone (CHH) transcripts, an important neuropeptide involved in immune function. Overall, the transcriptome profile of the hemocytes suggests that the LPS component of Novacq™ is highly immunostimulatory and generates a strong PO response in vitro. The subsequent transcriptional response appears to be directed towards preventing further activation of the PO system most likely in an attempt to limit cytoxicity to the host. Our study highlights the immunostimulatory ability of Novacq™ and provides further evidence of the positive health benefits this microbial based feed additive can have in shrimp.

Effect of anti-lipopolysaccharide of Escherichia coli antibody feeding for Holstein calves on ruminal lipopolysaccharide activity and plasma metabolites concentrations during pre- and post-weaning periods.

This study was performed to examine the effects of anti- lipopolysaccharide (LPS) of Escherichia coli chicken egg Yolk immunoglobulin (IgY) provided to calves for 7 weeks during the pre- and post-weaning periods on rumen LPS activity, plasma acute phase protein (APP) concentrations, and metabolic parameters. A total of 30 Holstein calves were randomly assigned to two groups of 15 each: an IgY group fed Anti-E. coli LPS IgY, and a control group fed whole egg powder as a placebo. The study was conducted on calves aged 3-10 weeks, weaned at 7 weeks. The ruminal LPS activity of the IgY group was approximately 60% lower than the control group at 10 weeks of age. Plasma APP and cytokine concentrations in the IgY group did not differ from those in the control group. The daily weight gain in the IgY group was significantly higher than the control group for the whole experimental period. Plasma albumin/globulin was lower (P<0.05), and plasma aspartate transferase concentration was higher (P<0.05) in the IgY group than in the control group during the experimental period. In conclusion, feeding Anti-E. coli LPS IgY for 7 weeks pre- and post-weaning remarkably reduced the rumen LPS activity and improved the daily weight gain. The impact of Anti-E. coli LPS IgY on LPS activities in the lower gastrointestinal tract, and elucidation as to the mechanism responsible for the improvement in daily weight gain require further investigation.

The Effects of Lipopolysaccharide Derivatives in Rodent Models of Cardiac Arrhythmia.

Several previous studies have suggested that sublethal doses of Escherichia coli lipopolysaccharide (endotoxin) and monophosphoryl lipid A Re595, a nonpyrogenic derivative of Salmonella minnesota lipopolysaccharide, exhibit antiarrhythmic effects in the rat model of ischemia-reperfusion arrhythmias. In this study, the protective effect of lipopolysaccharide derivatives was also further investigated in drug (aconitine or ouabain)-induced arrhythmia models, and conclusions were drawn with particular emphasis on the molecular characteristics of different types of lipopolysaccharide. The importance of the molecular structure for the antiarrhythmic effect of monophosphoryl lipid A and E. coli lipopolysaccharide was tested in the ischemia-reperfusion arrhythmia model. In contrast to monophosphoryl lipid A from Salmonella typhimurium SL 684 which has only monophosphoryl residue in its structure, monophosphoryl lipid A Re595, obtained from S. minnesota, and E. coli lipopolysaccharide which have both mono and diphosphoryl residue reduced the duration of ventricular tachycardia (e.g., during reperfusion: vehicle: 176 ± 22.8; monophosphoryl lipid A Re595: 132.83 ± 12.1, as second, n=8-10, P < .05) and the incidence of ventricular fibrillation. The antiarrhythmic effects of E. coli lipopolysaccharide and monophosphoryl lipid A Re595 in ischemia-reperfusion arrhythmia model were absent in either aconitine- (e.g., onset time for ventricular ectopic beats: saline 25.3 5.0, E. coli lipopolysaccharide 24.3 ± 7.1; vehicle: 24.0 ± 4.5, monophosphoryl lipid A SL684 23.8 ± 4.3, as second, n=6, P > .05) or ouabain-induced arrhythmia models in mice. Therefore, we conclude that lipopolysaccharide derivatives exhibit antiarrhythmic effect only in ischemia-reperfusion arrhythmias, and lipopolysaccharide should possess diphosphoryl groups in its subcomponent composition for this antiarrhythmic effect.

Effects of Lipopolysaccharide from Porphyromonas gingivalis and Escherichia coli on Gene Expression Levels of Toll-like Receptors and Inflammatory Cytokines in Human Dental Pulp Stem Cells.

Periodontal diseases originate from a group of oral inflammatory infections initiated by oral pathogens. Among these pathogens, Gram-negative bacteria such as p. gingivalis play a major role in chronic periodontitis. P. gingivalis harbours lipopolysaccharide (LPS) which enables it to attach to TLR2. Evaluating the effects of P. gingivalis and E. coli LPS on the gene expression of TLRs and inflammatory cytokines in human dental pulp stem cells (hDPSCs). We evaluated the expression level of TLR2, TLR4, IL-6, IL-10, and 1L-18 in hDPSCs treated with 1μg/mL of P. gingivalis lipopolysaccharide and E. coli LPS at three different exposure times using Real-time RT-PCR. The test group treated with P. gingivalis LPS showed a high level of TLR4 expression in 24 hours exposure period and the lowest expression in 48 hours of exposure time. In the case of IL-10, the lowest expression was in the 24 hours exposure period. Although in the E.coli LPS treated group, IL-10 showed the highest expression in 24 and lowest in 48 hours exposure period. Moreover, IL-18 in P. gingivalis LPS treated group showed a significant difference between 6, 24, and 48-time periods of exposure, but not in the E. coli LPS treated group. Both types of LPS stimulate inflammation through TLR4 expression. P. gingivalis LPS performs more potentially than E. coli in terms of stimulating inflammation at the first 24 hours of exposure. Nevertheless, our study confirmed that increasing P. gingivalis and/or the E.coli LPS exposure time, despite acting as an inflammatory stimulator, apparently showed anti-inflammatory properties.

Immune hyporeactivity to bacteria and multiple TLR-ligands, yet no response to checkpoint inhibition in patients just after meeting Sepsis-3 criteria.

RationaleThe immune profile of sepsis patients is incompletely understood and hyperinflammation and hypoinflammation may occur concurrently or sequentially. Immune checkpoint inhibition (ICI) may counter hypoinflammation but effects are uncertain. We tested the reactivity of septic whole blood to bacteria, Toll-like receptor (TLR) ligands and to ICI.MethodsWhole blood assays of 61 patients’ samples within 24h of meeting sepsis-3 criteria and 12 age and sex-matched healthy volunteers. Measurements included pattern/danger-associated molecular pattern (P/DAMP), cytokine concentrations at baseline and in response to TLR 2, 4, and 7/8 ligands, heat-inactivated Staphylococcus aureus or Escherichia coli, E.coli lipopolysaccharide (LPS), concentration of soluble and cellular immune checkpoint molecules, and cytokine concentrations in response to ICI directed against programmed-death receptor 1 (PD1), PD1-ligand 1, or cytotoxic T-lymphocyte antigen 4, both in the absence and presence of LPS.Main resultsIn sepsis, concentrations of P/DAMPs and inflammatory cytokines were increased and the latter increased further upon incubation ex vivo. However, cytokine responses to TLR 2, 4, and 7/8 ligands, heat-inactivated S. aureus or E. coli, and E. coli LPS were all depressed. Depression of the response to LPS was associated with increased in-hospital mortality. Despite increased PD-1 expression on monocytes and T-cells, and monocyte CTLA-4 expression, however, addition of corresponding checkpoint inhibitors to assays failed to increase inflammatory cytokine concentrations in the absence and presence of LPS.ConclusionPatients first meeting Sepsis-3 criteria reveal 1) depressed responses to multiple TLR-ligands, bacteria, and bacterial LPS, despite concomitant inflammation, but 2) no response to immune checkpoint inhibition.

Different expression patterns of inflammatory cytokines induced by lipopolysaccharides from Escherichia coli or Porphyromonas gingivalis in human dental pulp stem cells

BackgroundLipopolysaccharide (LPS) is one of the leading causes of pulpitis. The differences in establishing an in vitro pulpitis model by using different lipopolysaccharides (LPSs) are unknown. This study aimed to determine the discrepancy in the ability to induce the expression of inflammatory cytokines and the underlying mechanism between Escherichia coli (E. coli) and Porphyromonas gingivalis (P. gingivalis) LPSs in human dental pulp stem cells (hDPSCs).Material and methodsQuantitative real-time polymerase chain reaction (QRT-PCR) was used to evaluate the mRNA levels of inflammatory cytokines including IL-6, IL-8, COX-2, IL-1β, and TNF-α expressed by hDPSCs at each time point. ELISA was used to assess the interleukin-6 (IL-6) protein level. The role of toll-like receptors (TLR)2 and TLR4 in the inflammatory response in hDPSCs initiated by LPSs was assessed by QRT-PCR and flow cytometry.ResultsThe E. coli LPS significantly enhanced the mRNA expression of inflammatory cytokines and the production of the IL-6 protein (p < 0.05) in hDPSCs. The peaks of all observed inflammation mediators’ expression in hDPSCs were reached 3–12 h after stimulation by 1 μg/mL E. coli LPS. E. coli LPS enhanced the TLR4 expression (p < 0.05) but not TLR2 in hDPSCs, whereas P. gingivalis LPS did not affect TLR2 or TLR4 expression in hDPSCs. The TLR4 inhibitor pretreatment significantly inhibited the gene expression of inflammatory cytokines upregulated by E. coli LPS (p < 0.05).ConclusionUnder the condition of this study, E. coli LPS but not P. gingivalis LPS is effective in promoting the expression of inflammatory cytokines by hDPSCs. E. coli LPS increases the TLR4 expression in hDPSCs. P. gingivalis LPS has no effect on TLR2 or TLR4 expression in hDPSCs.

Porphyromonas gingivalis Lipopolysaccharide-Induced B Cell Differentiation by Toll-like Receptors 2 and 4

Porphyromonas gingivalis (P. gingivalis) is a pathogenic bacterium widely present in subgingival plaques of patients with periodontitis. It induces periodontitis with bone loss as its main feature by changing the number and composition of symbiotic microorganisms, as well as inducing the natural immune response of the host. However, the mechanism of the latter remains unclear. This study aims to investigate the effect of P. gingivalis lipopolysaccharide (LPS) on regulatory B cells (Breg) in the occurrence and development of periodontitis. We detected the mRNA levels of IL-10 in B cells under the stimulation of P. gingivalis LPS and/or E. coli LPS, distinguished IL-10-producing cells from different B cell subgroups using flow cytometry. Through toll-like receptor (TLR) knockout mice, the role of TLR2 and TLR4 in this process was also evaluated. Results showed that P. gingivalis stimulated B cells to produce IL-10 via TLR2/4. CD5+B1 subset is the main source of IL-10+Breg cell. Under P. gingivalis LPS stimulation, CD5+IgM+CD93-IL-10+B cell subset increased significantly, which was regulated through TLR2/ 4. The results of this study provides new insights into the immunopathogenic mechanism of P. gingivalis, preliminarily discussed the effect of P. gingivalis on the production of Breg, and present a theoretical foundation for subsequent investigations on the occurrence and development of periodontitis.

Effect of Resin Acid and Zinc Oxide on Immune Status of Weaned Piglets Challenged With E. coli Lipopolysaccharide

With the ban of zinc oxide (ZnO) at high dosages in piglet diets in Europe by 2022, alternative nutritional solutions are being tested to support piglet immune defence during their weaning, the most critical and stressful moment of pig production. The present study evaluated the effect of zinc oxide (ZnO; 2,500 mg/kg diet) and resin acid concentrate (RAC; 200 mg/kg diet) on the immune defence of weaned piglets challenged with lipopolysaccharide (LPS). Piglets were challenged at days 7 and 21 post-weaning, and blood was sampled 1.5 and 3.0 h after each challenge to determine serum levels of pro- and anti-inflammatory cytokines. The levels of serum tumour necrosis factor alpha (TNF-α) and interleukin 8 (IL-8) increased at days 7 and 21, and those of IL-6 at day 21 when challenged piglets were fed a diet supplemented with ZnO. In challenged piglets fed with RAC, the serum levels of IL-1β, IL-6, IL-8, IL-10 and TNF-α were increased at days 7 and 21, except for that of IL-1β, which was not affected at day 21. The increased levels of these cytokines indicate the successful immune-modulatory effect of ZnO and RAC, which appears as a candidate to replace ZnO in weaned piglets' diets.