Lipopolysaccharide Response Research Articles

Effects of lysine deacetylase inhibitor treatment on LPS responses of alveolar-like macrophages.

Macrophages are key immune cells that can adapt their metabolic phenotype in response to different stimuli. Lysine deacetylases are important enzymes regulating inflammatory gene expression and lysine deacetylase inhibitors have been shown to exert anti-inflammatory effects in models of chronic obstructive pulmonary disease. We hypothesized that these anti-inflammatory effects may be associated with metabolic changes in macrophages. To validate this hypothesis, we used an unbiased and a targeted proteomic approach to investigate metabolic enzymes, as well as liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry, to quantify metabolites in combination with the measurement of functional parameters in primary murine alveolar-like macrophages after lipopolysaccharide-induced activation in the presence or absence of lysine deacetylase inhibition. We found that lysine deacetylase inhibition resulted in reduced production of inflammatory mediators such as tumor necrosis factor α and interleukin 1β. However, only minor changes in macrophage metabolism were observed, as only one of the lysine deacetylase inhibitors slightly increased mitochondrial respiration while no changes in metabolite levels were seen. However, lysine deacetylase inhibition specifically enhanced expression of proteins involved in ubiquitination, which may be a driver of the anti-inflammatory effects of lysine deacetylase inhibitors. Our data illustrate that a multiomics approach provides novel insights into how macrophages interact with cues from their environment. More detailed studies investigating ubiquitination as a potential driver of lysine deacetylase inhibition will help developing novel anti-inflammatory drugs for difficult-to-treat diseases such as chronic obstructive pulmonary disease.

SAT425 Effects Of Endocrine-disrupting Polychlorinated Biphenyls On Adolescent Microglial Cytokine Expression And Responses To Secondary Inflammatory Challenge

Abstract Disclosure: S.L. Kissinger: None. A.J. Fitzgibbon: None. R.A. Mejia: None. M.R. Bell: None. Polychlorinated biphenyls (PCBs) are a group of ubiquitous environmental endocrine-disrupting chemicals (EDCs), and exposure is associated with endocrine, neuronal, and immune dysfunction in various tissue types. Innate immune cells of the nervous system called microglia are critical in responding to infection and injury, and disruption of these functions is associated with neurodegenerative disease and mental health disorders. In addition, microglia are known to regulate developmental processes during neonatal and adolescent periods, in hormone-sensitive and age- and sex-differentiated ways. Work in our lab has shown that early life PCBs can affect microglia function in ways that depend on the age and sex of the individual rat in vivo. Ongoing work uses in vitro approaches to demonstrate direct effects on microglia. In primary culture, preliminary data suggest that acute PCB exposure causes neonatal microglia to become hyperresponsive to a secondary challenge; however, effects in adolescent microglia are unknown. To test the hypothesis that PCB exposure directly alters microglia activity in an age- and sex-specific manner, we determined the effects of PCBs on cytokine gene expression in acutely isolated microglia, with or without a secondary lipopolysaccharide (LPS) challenge. Adolescent Sprague-Dawley rats underwent transcardial saline perfusion, brains were removed and homogenized into a single cell suspension with collagenase and mechanical disruption, and debris was removed. Microglia, identified as CD11b+ cells, were collected by magnetic bead isolation and plated in enriched media for 24 hours prior to experimentation. Cells were exposed to one of three concentrations of PCB 153 (0.1 µM, 1 µM, 10 µM), DMSO vehicle, or DMEM/F12 media control for 18 hours, followed by a 4-hour LPS challenge (0.5 µg/mL) or PBS vehicle control. RNA was isolated and converted to cDNA for qPCR analysis of Tnf and Il1b gene expression, analyzed by two-way (exposure x challenge) ANOVAs within both male and female cells. Results indicate that 1.0 µM PCB 153 exposure caused an increase in expression of both cytokines, more so in females than males. Preliminary results (n = 3) indicate that the 10 µM dose may also blunt the later LPS response. These results confirm that microglia are directly sensitive to PCBs, however they illustrate that cells may show differential responses depending on the stage of development. Continued work will explore acute cell toxicity and confirm release of cytokines. Adolescent microglia dysfunction from PCB exposure may be a mechanism of adolescent-onset disorders, or increased risk of neurodegenerative conditions triggered by additional challenges later in life. Presentation: Saturday, June 17, 2023

Learning single-cell perturbation responses using neural optimal transport.

Understanding and predicting molecular responses in single cells upon chemical, genetic or mechanical perturbations is a core question in biology. Obtaining single-cell measurements typically requires the cells to be destroyed. This makes learning heterogeneous perturbation responses challenging as we only observe unpaired distributions of perturbed or non-perturbed cells. Here we leverage the theory of optimal transport and the recent advent of input convex neural architectures to present CellOT, a framework for learning the response of individual cells to a given perturbation by mapping these unpaired distributions. CellOT outperforms current methods at predicting single-cell drug responses, as profiled by scRNA-seq and a multiplexed protein-imaging technology. Further, we illustrate that CellOT generalizes well on unseen settings by (1) predicting the scRNA-seq responses of holdout patients with lupus exposed to interferon-β and patients with glioblastoma to panobinostat; (2) inferring lipopolysaccharide responses across different species; and (3) modeling the hematopoietic developmental trajectories of different subpopulations.

Comparison of O-specific polysaccharide responses in patients following infection with Vibrio cholerae O139 versus vaccination with a bivalent (O1/O139) oral killed cholera vaccine in Bangladesh.

Cholera caused by Vibrio cholerae O139 emerged in the early 1990s and spread rapidly to 11 Asian countries before receding for unclear reasons. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS). V. cholerae O139 also expresses the OSP-capsule. We, therefore, assessed antibody responses targeting V. cholerae O139 OSP, LPS, capsule, and vibriocidal responses in patients in Bangladesh with cholera caused by V. cholerae O139. We compared these responses to those of age-gender-blood group-matched recipients of the bivalent oral cholera vaccine (OCV O1/O139). We found prominent OSP, LPS, and vibriocidal responses in patients, with a high correlation between these responses. OSP responses primarily targeted the terminal tetrasaccharide of OSP. Vaccinees developed OSP, LPS, and vibriocidal antibody responses, but of significantly lower magnitude and responder frequency (RF) than matched patients. We separately analyzed responses in pediatric vaccinees born after V. cholerae O139 had receded in Bangladesh. We found that OSP responses were boosted in children who had previously received a single dose of bivalent OCV 3 yr previously but not in vaccinated immunologically naïve children. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 despite the presence of capsules, that vaccination with bivalent OCV is poorly immunogenic in the short term in immunologically naïve individuals, but that OSP-specific immune responses can be primed by previous exposure, although whether such responses can protect against O139 cholera is uncertain. IMPORTANCE Cholera is a severe dehydrating illness in humans caused by Vibrio cholerae serogroups O1 or O139. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) of V. cholerae LPS. Yet, little is known about immunity to O139 OSP. In this study, we assessed immune responses targeting OSP in patients from an endemic region with cholera caused by V. cholerae O139. We compared these responses to those of the age-gender-blood group-matched recipients of the bivalent oral cholera vaccine. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 and that the OSP responses primarily target the terminal tetrasaccharide of OSP. Our results further suggest that vaccination with the bivalent vaccine is poorly immunogenic in the short term for inducing O139-specific OSP responses in immunologically naïve individuals, but OSP-specific immune responses can be primed by previous exposure or vaccination.

Network Pharmacology and Molecular Docking Analysis Exploring the Mechanism of Tripterygium wilfordii in the Treatment of Oral Lichen Planus.

Background: Oral lichen planus (OLP) is an infrequent autoimmune disease of the oral mucosa, which affects up to 2% of the world population. An investigation of Tripterygium wilfordii's mechanism of action for treating OLP was conducted, and a theoretical basis was provided for improving current treatment regimens. Materials and Methods: We used a network pharmacological approach to gain insight into the molecular mechanism of Tripterygium wilfordii in the treatment of OLP. Then, potential protein targets between Tripterygium wilfordii and OLP were analyzed through a drug-target network. This was followed by KEGG enrichment analysis and Gene Ontology (GO) classification. Finally, for molecular docking, AutoDock Vina was used. Results: A protein-protein interaction (PPI) network was constructed by analyzing the common targets of a total of 51 wilfordii-OLP interactions from different databases. The GO and KEGG enrichment analyses showed that the treatment of OLP with Tripterygium wilfordii mainly involves lipopolysaccharide response, bacterial molecular response, positive regulation of cytokine production, and leukocyte proliferation, and the signaling pathways mainly include the AGE-RAGE, NF-κB, Toll-like receptor, IL-17, HIF-1, and TNF signaling pathways. The molecular docking results showed that β-sitosterol, kaempferol, hederagenin, and triptolide have a higher affinity for AKT1, TNF, CASP3, and PTGS2, respectively. Based on the CytoNCA analysis of common targets, 19 key targets, including AKT1, TNF, VEGFA, STAT3, CXCL8, PTGS2, TP53, and CASP3, and their connections were identified. Conclusions: Preliminarily, this study reveals that Tripterygium wilfordii interferes with OLP by interacting with multiple targets through multiple accesses, as validated by molecular docking.

Plasma proteomic signatures of enteric permeability among hospitalized and community children in Kenya and Pakistan

We aimed to establish if enteric permeability was associated with similar biological processes in children recovering from hospitalization and relatively healthy children in the community. Extreme gradient boosted models predicting the lactulose-rhamnose ratio (LRR), a biomarker of enteric permeability, using 7,500 plasma proteins and 34 fecal biomarkers of enteric infection among 89 hospitalized and 60 community children aged 2-23months were built. The R2 values were calculated in test sets. The models performed better among community children (R2: 0.27 [min-max: 0.19, 0.53]) than hospitalized children (R2: 0.07 [min-max: 0.03, 0.11]). In the community, LRR was associated with biomarkers of humoral antimicrobial and cellular lipopolysaccharide responses and inversely associated with anti-inflammatory and innate immunological responses. Among hospitalized children, the selected biomarkers had few shared functions. This suggests enteric permeability among community children was associated with a host response to pathogens, but this association was not observed among hospitalized children.

Identification of a novel survival predictor, CSF2RB, for female lung cancer in never smokers (LCNS) by a bioinformatics analysis.

Lung cancer in never smokers (LCNS) has been considered as a separate disease and the 7th cause of cancer-related death worldwide. However, limited research has focused on "female" cohorts, which have presented a higher incidence rate. In this study, the microarray data of lung cancer tissues derived from 54 female lung cancer patients, consisting of 43 nonsmokers and 11 smokers, were selected from GSE2109 dataset. A total of 249 differentially expressed genes (DEGs) including 102 up- and 147 down-regulated genes were identified and further analyzed for gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment. By constructing protein-protein interaction (PPI) network and calculating key modules, 10 hub genes were screened out. The module analysis of the PPI network presented that the progression of female LCNS was significantly associated with immune response as chemokine activity and lipopolysaccharide response, and these biological processes (BP) might be mediated by chemokine signaling pathway and cytokine-cytokine receptor interaction. Then, survival analysis by Kaplan-Meier (K-M) Plotter online platform presented down-regulated gene colony stimulating factor 2 receptor beta common subunit (CSF2RB) of female LCNS might be involved in poor clinical outcome. Female LCNS with high expression of CSF2RB might be relevant with relative risk reduction of mortality, longer median survival time and higher 5-year survival rate, while female LCNS with low expression of CSF2RB might be implicated in a poor clinical outcome. In short, our results support CSF2RB to be a candidate survival predictor for female LCNS.

Association between lipid-A-producing oral bacteria of different potency and fractional exhaled nitric oxide in a Norwegian population-based adult cohort

BackgroundLipid A is the primary immunostimulatory part of the lipopolysaccharide (LPS) molecule. The inflammatory response of LPS varies and depends upon the number of acyl chains and phosphate groups in lipid A which is specific for a bacterial species or strain. Traditional LPS quantification assays cannot distinguish between the acylation degree of lipid A molecules, and therefore little is known about how bacteria with different inflammation-inducing potencies affect fractional exhaled nitric oxide (FeNO). We aimed to explore the association between pro-inflammatory hexa- and less inflammatory penta-acylated LPS-producing oral bacteria and FeNO as a marker of airway inflammation.MethodsWe used data from a population-based adult cohort from Norway (n = 477), a study center of the RHINESSA multi-center generation study. We applied statistical methods on the bacterial community- (prediction with MiRKAT) and genus-level (differential abundance analysis with ANCOM-BC) to investigate the association between the oral microbiota composition and FeNO.ResultsWe found the overall composition to be significantly associated with increasing FeNO levels independent of covariate adjustment, and abundances of 27 bacterial genera to differ in individuals with high FeNO vs. low FeNO levels. Hexa- and penta-acylated LPS producers made up 2.4% and 40.8% of the oral bacterial genera, respectively. The Bray–Curtis dissimilarity within hexa- and penta-acylated LPS-producing oral bacteria was associated with increasing FeNO levels independent of covariate adjustment. A few single penta-acylated LPS producers were more abundant in individuals with low FeNO vs. high FeNO, while hexa-acylated LPS producers were found not to be enriched.ConclusionsIn a population-based adult cohort, FeNO was observed to be associated with the overall oral bacterial community composition. The effect of hexa- and penta-acylated LPS-producing oral bacteria was overall significant when focusing on Bray–Curtis dissimilarity within each of the two communities and FeNO levels, but only penta-acylated LPS producers appeared to be reduced or absent in individuals with high FeNO. It is likely that the pro-inflammatory effect of hexa-acylated LPS producers is counteracted by the dominance of the more abundant penta-acylated LPS producers in this population-based adult cohort involving mainly healthy individuals.

Exploring the mechanism of Ginkgo biloba L. leaves in the treatment of vascular dementia based on network pharmacology, molecular docking, and molecular dynamics simulation.

Ginkgo biloba L. leaves (GBLs) play a substantial role in the treatment of vascular dementia (VD); however, the underlying mechanisms of action are unclear. This study was conducted to investigate the mechanisms of action of GBLs in the treatment of VD through network pharmacology, molecular docking, and molecular dynamics simulations. The active ingredients and related targets of GBLs were screened using the traditional Chinese medicine systems pharmacology, Swiss Target Prediction and GeneCards databases, and the VD-related targets were screened using the OMIM, DrugBank, GeneCards, and DisGeNET databases, and the potential targets were identified using a Venn diagram. We used Cytoscape 3.8.0 software and the STRING platform to construct traditional Chinese medicine-active ingredient-potential target and protein-protein interaction networks, respectively. After gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis of potential targets using the DAVID platform, the binding affinity between key active ingredients and targets was analyzed by molecular docking, and finally, the top 3 proteins-ligand pairs with the best binding were simulated by molecular dynamics to verify the molecular docking results. A total of 27 active ingredients of GBLs were screened and 274 potential targets involved in the treatment of VD were identified. Quercetin, luteolin, kaempferol, and ginkgolide B were the core ingredients for treatment, and AKT1, TNF, IL6, VEGFA, IL1B, TP53, CASP3, SRC, EGFR, JUN, and EGFR were the main targets of action. The main biological processes involved apoptosis, inflammatory response, cell migration, lipopolysaccharide response, hypoxia response, and aging. PI3K/Akt appeared to be a key signaling pathway for GBLs in the treatment of VD. Molecular docking displayed strong binding affinity between the active ingredients and the targets. Molecular dynamics simulation results further verified the stability of their interactions. This study revealed the potential molecular mechanisms involved in the treatment of VD by GBLs using multi-ingredient, multi-target, and multi-pathway interactions, providing a theoretical basis for the clinical treatment and lead drug development of VD.

KR-12 Derivatives Endow Nanocellulose with Antibacterial and Anti-Inflammatory Properties: Role of Conjugation Chemistry.

This work combines the wound-healing-related properties of the host defense peptide KR-12 with wood-derived cellulose nanofibrils (CNFs) to obtain bioactive materials, foreseen as a promising solution to treat chronic wounds. Amine coupling through carbodiimide chemistry, thiol-ene click chemistry, and Cu(I)-catalyzed azide-alkyne cycloaddition were investigated as methods to covalently immobilize KR-12 derivatives onto CNFs. The effects of different coupling chemistries on the bioactivity of the KR12-CNF conjugates were evaluated by assessing their antibacterial activities against Escherichia coli and Staphylococcus aureus. Potential cytotoxic effects and the capacity of the materials to modulate the inflammatory response of lipopolysaccharide (LPS)-stimulated RAW 245.6 macrophages were also investigated. The results show that KR-12 endowed CNFs with antibacterial activity against E. coli and exhibited anti-inflammatory properties and those conjugated by thiol-ene chemistry were the most bioactive. This finding is attributed to a favorable peptide conformation and accessibility (as shown by molecular dynamics simulations), driven by the selective chemistry and length of the linker in the conjugate. The results represent an advancement in the development of CNF-based materials for chronic wound care. This study provides new insights into the effect of the conjugation chemistry on the bioactivity of immobilized host defense peptides, which we believe to be of great value for the use of host defense peptides as therapeutic agents.

Epithelial innate immune response to Pseudomonas aeruginosa-derived flagellin in chronic rhinosinusitis.

Pseudomonas aeruginosa is a common colonizing pathogen in the upper respiratory tract and is associated with recalcitrant chronic rhinosinusitis (CRS). Herein we sought to characterize the effect of P. aeruginosa-derived flagellin on human sinonasal epithelial cell (HSNEC) immune responses and determine whether these pathways are disrupted in CRS. Air-liquid interface cultures were established from CRS and healthy control donors. Cells were incubated with P. aeruginosa-derived flagellin for 24hours and transcriptional changes were assessed using whole transcriptome RNA sequencing. Apical and basolateral secretion of the pro-inflammatory cytokines in interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 were measured after stimulation by lipopolysaccharide or flagellin and responses were compared between CRS and healthy control patients. HSNECs were weakly responsive to lipopolysaccharide, whereas flagellin stimulated a profound innate immune response dominated by TNF-α, IL-1β, and IL-17 signaling and activation of the IL-17C/IL-23 axis. CRS-derived HNSECs showed an altered innate immune response to flagellin, characterized by a profound increase in TNF-α secretion coupled with reduced IL-6 secretion. Flagellin activates a potent innate immune response in HSNECs characterized by pro-inflammatory mediators and cytokines/chemokines associated with neutrophilic inflammation. HSNECs from CRS patients have a dysregulated innate immune response to flagellin characterized by an imbalance between IL-6 and TNF-α secretion.

A LY6E-PHB1-TRIM21 assembly degrades CD14 protein to mitigate LPS-induced inflammatory response

A major theme of host against invading pathogens lies in multiple regulatory nodes that ensure sufficient signals for protection while avoiding excessive signals toward over-inflammation. The TLR4/MD-2/CD14 complex receptor-mediated response to bacterial lipopolysaccharide (LPS) represents a paradigm for understanding the proper control of anti-pathogen innate immunity. In this study, we studied the mechanism by which the glycosylphosphatidylinositol (GPI)-linked LY6E protein constrains LPS response via downregulating CD14. We first showed that LY6E downregulated CD14 via ubiquitin-dependent proteasomal degradation. The subsequent profiling of LY6E protein interactome led to the revelation that the degradation of CD14 by LY6E requires PHB1, which interacts with CD14 in a LY6E-dependent manner. Finally, we identified the PHB1-interacting TRIM21 as the major ubiquitin E3 ligase for the LY6E-mediated ubiquitination of CD14. Together, our study elucidated the molecular basis of LY6E-mediated governance of LPS response, alongside providing new insights to regulatory mechanisms controlling the homeostasis of membrane proteins.

158 Evaluating Lipopolysaccharide-Induced Behavioral and Immune Response Differences in Sheep Divergently Bred for Parasite Resistance

Abstract Katahdin lambs with decreased post weaning worm egg count estimated breeding values (PWEC EBV) generated greater antibody in response to vaccination and survive better to weaning. Thus, lambs with decreased PWEC values may be more disease resistant and immunocompetent. To test this hypothesis, parasite-resistant St. Croix sheep, (STC, n = 10), and parasite-susceptible Suffolk sheep (SUF, n = 10) were compared with Katahdin sheep divergent in PWEC EBV (HiKAT, n = 10; LoKAT, n = 10) and challenged with lipopolysaccharide (LPS). Sheep were intravenously administered LPS at a dose of 2.5 ug/kg; blood and body temperature data were collected hourly. Behavioral observations were recorded during sampling and videos within pens were obtained 30 min after blood collection. An increase of body temperature by 1 ºC was observed in all groups. Interestingly, STC and SUF temperature peaked at 4 hours whereas LoKAT and HiKAT both peaked at 6 hours. One metric of behavior recorded from video was Sheep Grimace Score (SGS; 1 = normal; 7 = extremely abnormal). Peak SGS in all groups was 3 hours after LPS administration. Main effects of group on SGS indicated a stepwise increase with STC having the least SGS (1.25) and SUF greatest score (2.45) and both groups of Katahdin sheep intermediate (1.65 LoKAT, 2.00 HiKAT; P < 0.001). Evaluation of “return to normal” (RTN), when SGS scores average between 0 and, revealed that STC and LoKAT RTN segregated from the other groups and this occurred at 5.1 and 6.6 hours after LPS treatment, respectively. HiKAT and SUF RTN was at 9.3 and 9.6 hours, respectively. Average SGS RTN in STC and LoKAT were significantly less than HiKAT and SUF (P < 0.01), indicating segregation in behavior. Typical behaviors associated with LPS response, such as fever and ill-behaviors, can be attributed to the rapid and systemic influx of proinflammatory cytokines like tumor necrosis factor alpha (TNFα). Analysis of circulating TNFα revealed similar segregation whereas STC and LoKAT have early TNFα peak at 4 hours and then gradually decline, HiKAT and SUF peak at 9hrs and remain elevated to2hrs. The transition period between these two disparate groups is 6 hours post LPS administration. Thus, TNFα was evaluated before (-2 to 6 hours) and post (7 to 12hours) TNFα shift. With STC sheep having greater TNFα before (45.96 pg/mL, P < 0.001) and SUF sheep having greater TNFα post (47.51 pg/mL, P < 0.001) indicating that despite similar average TNFα there exists timing differences of TNFα that correspond with SGS. These differences are mirrored in the production of TNFα in LoKAT and HiKAT sheep. Taken together these data indicate that early resolvement of TNFα cytokine production within the parasite-resistant groups (STC and LoKAT) is associated with reduced duration of sick behaviors.

Oral Delivery of Bioactive Glass-Loaded Core-Shell Hydrogel Microspheres for Effective Treatment of Inflammatory Bowel Disease.

Resolving inflammation and promoting intestinal tissue regeneration are critical for inflammatory bowel disease (IBD) treatment. Bioactive glass (BG) is a clinically approved bone graft material and has been shown to modulate inflammatory response, but it is unknown whether BG can be applied to treat IBD. Here, it is reported that BG attenuates pro-inflammatory response of lipopolysaccharide (LPS)-stimulated macrophages and hence reduces inflammatory damage to intestinal organoids in vitro. In addition, zein/sodium alginate-based core-shell microspheres (Zein/SA/BG) are developed for oral delivery of BG, which helps prevent premature dissolution of BG in the stomach. The results show that Zein/SA/BG protects BG from a gastric-simulated environment while dissolved in an intestinal-simulated environment. When administered to acute and chronic colitis mice model, Zein/SA/BG significantly reduces intestinal inflammation, promotes epithelial tissue regeneration, and partially restores microbiota homeostasis. These findings are the first to reveal the therapeutic efficacy of BG against IBD, which may provide a new therapeutic approach at low cost for effective IBD treatment.

Neferine ameliorates nonalcoholic steatohepatitis through regulating AMPK pathway

BackgroundNonalcoholic fatty liver disease (NAFLD), peculiarly nonalcoholic steatohepatitis (NASH), has become the main cause of liver transplantation and liver-related death. However, the US Food and Drug Administration has not approved a specific medication for treating NASH. Neferine (NEF), a natural bisbenzylisoquinoline alkaloid separated from the traditional Chinese medicine Nelumbinis plumula, has a variety of pharmacological properties, especially on metabolic diseases. Nevertheless, the anti-NASH effect and mechanisms of NEF remain unclear. PurposeThis study aimed to investigate the amelioration of NEF on NASH and the potential mechanisms. Study DesignHepG2 cells, hepatic stellate cells (HSCs) and high-fat diet (HFD)+carbon tetrachloride (CCl4) induced C57BL/6 mice were used to observe the effect of NEF against NASH and investigate the engaged mechanism. MethodsHSCs and HepG2 cells stimulated by oleic acid (OA) were treated with NEF. C57BL/6 mice were fed with HFD+CCl4 to induce NASH mouse model and treated with or without NEF (5 mg/kg or 10 mg/kg, once daily, i.p) for 4 weeks. ResultsNEF significantly attenuated the accumulation of lipid droplets, intracellular triglyceride (TG) levels and hepatocytes apoptosis in OA-exposed HepG2 cells. NEF not only enhanced the AMPK and ACC phosphorylation in OA-stimulated HepG2 cells, but also reduced inflammatory response and fibrosis in lipopolysaccharide (LPS)-stimulated HepG2 and in LX-2, respectively. In HFD+CCl4-induced NASH mice, pathological staining confirmed NEF treatment mitigated hepatic lipid deposition, inflammatory cell infiltration as well as hepatic fibrosis. Furthermore, the liver weight, serum and hepatic TG and total cholesterol (TC) and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were decreased compared with the model group. HFD+CCl4 also induced the upregulation of specific proteins and genes associated to inflammation (ILs, TNF-α, NLRP3, ASC, CCL2 and CXCL10) and hepatic fibrosis (collagens, α-SMA, TGF-β and TIPM1), which were also suppressed by NEF treatment. ConclusionOur results demonstrated that NEF played a protective role in hepatic steatosis via the regulation of AMPK pathways, which may serve as an attractive candidate for a potential novel strategy on prevention and treatment of NASH.

Production and characterization of exopolysaccharides from salinity-induced Auxenochlorella protothecoides and the analysis of anti-inflammatory activity

The set scenario of this work was to investigate the production, physicochemical characteristics, and anti-inflammatory activities of exopolysaccharides from salinity-induced Auxenochlorella protothecoides. The results demonstrated that 10 ‰ salinity manipulation endowed preferable exopolysaccharide production by A. protothecoides. Under this salinity stress, ACPEPS1A and ACPEPS2A were purified from exopolysaccharide production by anion chromatography and molecular exclusion chromatography. ACPEPS1A exhibited a molecular weight (Mw) of 132 kDa and mainly consisted of galactose. ACPEPS2A was a heteropolysaccharide with an Mw of 170 kDa and the main monosaccharides of galactose and rhamnose with separate molar percents of 42.41 % and 35.29 %, respectively. FTIR, 1H and 13C NMR supported that monosaccharide components of ACPEPS1A and ACPEPS2A possessed both α- and β-configuration pyranose rings. Further evidence indicated that ACPEPS1A and ACPEPS2A could effectively inhibit the inflammatory response in lipopolysaccharide (LPS) induced RAW264.7 cells by quenching inflammatory factor levels such as ROS, iNOS, TNF-α, and IL-6. The potential anti-inflammatory possibilities were that the monosaccharides of ACPEPS1A and ACPEPS2A possessed higher affinity with receptors on the macrophage surface than LPS and hampered LPS-induced inflammation. The findings of this work would favor innovative applications of exopolysaccharides from microalgae in complementary medicines or functional foods.

The Regulatory Roles of Ezh2 in Response to Lipopolysaccharide (LPS) in Macrophages and Mice with Conditional Ezh2 Deletion with LysM-Cre System.

The responses of macrophages to lipopolysaccharide (LPS) might determine the direction of clinical manifestations of sepsis, which is the immune response against severe infection. Meanwhile, the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, might interfere with LPS response. Transcriptomic analysis on LPS-activated wild-type macrophages demonstrated an alteration of several epigenetic enzymes. Although the Ezh2-silencing macrophages (RAW264.7), using small interfering RNA (siRNA), indicated a non-different response to the control cells after a single LPS stimulation, the Ezh2-reducing cells demonstrated a less severe LPS tolerance, after two LPS stimulations, as determined by the higher supernatant TNF-α. With a single LPS stimulation, Ezh2 null (Ezh2flox/flox; LysM-Crecre/-) macrophages demonstrated lower supernatant TNF-α than Ezh2 control (Ezh2fl/fl; LysM-Cre-/-), perhaps due to an upregulation of Socs3, which is a suppressor of cytokine signaling 3, due to the loss of the Ezh2 gene. In LPS tolerance, Ezh2 null macrophages indicated higher supernatant TNF-α and IL-6 than the control, supporting an impact of the loss of the Ezh2 inhibitory gene. In parallel, Ezh2 null mice demonstrated lower serum TNF-α and IL-6 than the control mice after an LPS injection, indicating a less severe LPS-induced hyper-inflammation in Ezh2 null mice. On the other hand, there were similar serum cytokines after LPS tolerance and the non-reduction of serum cytokines after the second dose of LPS, indicating less severe LPS tolerance in Ezh2 null mice compared with control mice. In conclusion, an absence of Ezh2 in macrophages resulted in less severe LPS-induced inflammation, as indicated by low serum cytokines, with less severe LPS tolerance, as demonstrated by higher cytokine production, partly through the upregulated Socs3.

Effects of dietary chlorogenic acid on intestinal barrier function and the inflammatory response in broilers during lipopolysaccharide-induced immune stress

Immune stress exerts detrimental effects on growth performance and intestinal barrier function during intensive animal production with ensuing serious economic consequences. Chlorogenic acid (CGA) is used widely as a feed additive to improve the growth performance and intestinal health of poultry. However, the effects of dietary CGA supplementation on amelioration of the intestinal barrier impairment caused by immune stress in broilers are unknown. This study investigated the effects of CGA on growth performance, intestinal barrier function, and the inflammatory response in lipopolysaccharide (LPS) mediated immune-stressed broilers. Three hundred and twelve 1-day-old male Arbor Acres broilers were divided randomly into 4 groups with 6 replicates of thirteen broilers. The treatments included: i) saline group: broilers injected with saline and fed with basal diet; ii) LPS group: broilers injected with LPS and fed with basal diet; iii) CGA group: broilers injected with saline and feed supplemented with CGA; and iv) LPS+CGA group: broilers injected with LPS and feed supplemented with CGA. Animals in the LPS and LPS+CGA groups were injected intraperitoneally with an LPS solution prepared with saline from 14 d of age for 7 consecutive days, whereas broilers in the other groups were injected only with saline. LPS induced a decrease in feed intake of broilers during the stress period, but CGA effectively alleviated this decrease. Moreover, CGA inhibited the reduction of villus height and improved the ratio of villus height to crypt depth in the duodenum of broilers 24 and 72 h after LPS injection. In addition, dietary CGA supplementation significantly restored the expression of cation-selective and channel-forming Claudin2 protein 2 h after LPS injection in the ileum. LPS enhanced the expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the small intestine, but this enhancement was blocked by CGA supplementation. The expression of interleukin-10 (IL-10) increased with LPS injection and CGA promoted the production of IL-10. CGA addition downregulated the expression of intestinal interleukin-6 (IL-6) of broilers under normal rearing conditions. However, CGA supplementation upregulated the expression of IL-6 of broilers 72 h after LPS injection. The data demonstrate that dietary supplementation with CGA alleviates intestinal barrier damage and intestinal inflammation induced by LPS injection during immune stress thereby improving growth performance of broilers.

Dietary methionine restriction alleviates oxidative stress and inflammatory responses in lipopolysaccharide-challenged broilers at early age.

In this study, we investigated the effect of dietary methionine restriction (MR) on the antioxidant function and inflammatory responses in lipopolysaccharide (LPS)-challenged broilers reared at high stocking density. A total of 504 one-day-old male Arbor Acre broiler chickens were randomly divided into four treatments: 1) CON group, broilers fed a basal diet; 2) LPS group, LPS-challenged broilers fed a basal diet; 3) MR1 group, LPS-challenged broilers fed a methionine-restricted diet (0.3% methionine); and 4) MR2 group, LPS-challenged broilers fed a methionine-restricted diet (0.4% methionine). LPS-challenged broilers were intraperitoneally injected with 1mg/kg body weight (BW) of LPS at 17, 19, and 21days of age, whereas the CON group was injected with sterile saline. The results showed that: LPS significantly increased the liver histopathological score (p < 0.05); LPS significantly decreased the serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity at 3h after injection (p < 0.05); the LPS group had a higher content of Interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF)-α, but a lower content of IL-10 than the CON group in serum (p < 0.05). Compared with the LPS group, the MR1 diet increased catalase (CAT), SOD, and T-AOC, and the MR2 diet increased SOD and T-AOC at 3h after injection in serum (p < 0.05). Only MR2 group displayed a significantly decreased liver histopathological score (p < 0.05) at 3h, while MR1 and MR2 groups did so at 8h. Both MR diets significantly decreased serum LPS, CORT, IL-1β, IL-6, and TNF-α contents, but increased IL-10 content (p < 0.05). Moreover, the MR1 group displayed significantly increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2), CAT, and GSH-Px at 3 h; the MR2 group had a higher expression of Kelch-like ECH-associated protein 1 (Keap1), SOD, and GSH-Px at 8h (p < 0.05). In summary, MR can improve antioxidant capacity, immunological stress, and liver health in LPS-challenged broilers. The MR1 and MR2 groups experienced similar effects on relieving stress; however, MR1 alleviated oxidative stress more rapidly. It is suggested that precise regulation of methionine levels in poultry with stress may improve the immunity of broilers, reduce feed production costs, and increase production efficiency in the poultry industry.

Effect of HM-Exos on the migration and inflammatory response of LPS-exposed dental pulp stem cells

AimThe purpose of this study was to investigate the effects of human milk exosomes (HM-Exos) on the viability, migration, and inflammatory responses of lipopolysaccharide (LPS)-exposed human dental pulp stem cells (HDPSCs) in vitro.MethodsHM-Exos were isolated, and dynamic light scattering (DLS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were used to analyze their physical properties (size and shape). To construct an in vitro inflammation model, HDPSCs were exposed to LPS. The MTT test and migration assay were used to investigate the effect of HM-Exos on cell proliferation and migration, and the quantitative polymerase chain reaction (qPCR) was used to assess the expression of inflammatory genes in HDPSCs. Data were analyzed using a one-way analysis of variance (ANOVA) with Tukey's post-test.ResultsDLS measurement revealed that HM-Exos were 116.8 ± 3.6 nm in diameter. The SEM and TEM images revealed spherical shapes with diameters of 97.2 ± 34.6 nm. According to the results of the cell viability assay, the nontoxic concentration of HM-Exos (200 µg/ml) was chosen for the subsequent investigations. The migration assay results showed that HM-Exos improved the potential of LPS-exposed HDPSCs to migrate. The qPCR results indicated that HM-Exos significantly reduced the expression of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in HDPSCs after LPS stimulation.ConclusionsHM-Exos increased LPS-exposed HDPSCs migration and proliferation and reduced gene expression of inflammatory cytokines. They may be a viable candidate for pulpitis therapy.