Low Lipopolysaccharide Research Articles

Acute low temperature and lipopolysaccharide differentially modulated the innate immune and antioxidant responses in a subtropical fish, the pacu (Piaractus mesopotamicus).

Exogenous factors such as low water temperature can be stressful and elicit negative immune system effects, especially for fish, which are ectothermic. Stress and immune responses require energy overload, which can affect the cellular redox balance, causing oxidative damage. These overall responses impair the animal's health and negatively affect fish farming. To evaluate indicators of stress, immune and antioxidant systems, and oxidative stress responses in fish during thermal challenge, the present study reduced the water temperature from 29.5°C to 16°C and then inoculated pacu (Piaractus mesopotamicus) with lipopolysaccharide (LPS) from Escherichia coli. Our results revealed that acute exposure to low water temperature itself increased blood glucose, impaired the serum lysozyme concentration and increased GSH-Px activity. There was an interaction effect between low temperature and LPS inoculation. After LPS inoculation, leukocytes were initially activated (3h); glucose levels increased (3h); GST activity initially decreased (3h) but then increased (6h); SOD, CAT and GSH-Px activities decreased; and lysozyme activity remained depressed in fish subjected to cold shock. The results showed that thermal and immunological challenges impaired the maintenance of leucocyte activation and compromised the pacu oxidant response. The overall response of pacu to thermal challenge indicates that the species proved to be acutely sensitive to a drop in water temperature, reducing its ability to maintain homeostasis, especially when subjected to immunological challenge.

A new type of Pseudomonas aeruginosa phage with potential as a natural food additive for eradicating biofilms and combating multidrug-resistant strains

Pseudomonas aeruginosa is a biofilm-forming bacterial pathogen with multidrug resistance that leads to serious foodborne illnesses. Bacteriophages as alternatives for controlling P. aeruginosa and its biofilms are of great interest, particularly in the food industry. This study reported, a new type of P. aeruginosa specific phage named vB PaeM HUST 1 and evaluated antibacterial and biofilm reduction to use alternative as an additive in food industry. vB PaeM HUST 1 showed excellent specificity and selectivity in host cell lysis and stability over a wide range of pH (3-10) and temperature (4–45 °C). Transmission electron microscopy revealed that the phage had a head with a long tail (head: 146.65 nm × 133.32 nm, tail: 199.98 nm); hence, it belongs to the subfamily Straboviridae. The 276114 bp vB PaeM HUST 1 genome encodes 360 ORFs, 7 tRNAs, and an average G + C content of 36.92%. In addition, they exhibited low lipopolysaccharide (LPS) levels at a concentration of 0.133 U/mL, with a phage titer of 1.14 × 109 PFU/mL and no cytotoxicity. vB PaeM HUST 1 efficiently degraded over 95% of the biofilm within 8 h and was highly effective against P. aeruginosa in both milk and beef, with significant reductions of 68.34% and 92% in bacterial concentration after 12 and 24 h of treatment, respectively, with complete eradication achieved after 36 h. Therefore, vB PaeM HUST 1 phage has unique characteristics and can be used as a novel additive to control P. aeruginosa in food matrices.

The transcriptional regulator Fur modulates the expression of uge, a gene essential for the core lipopolysaccharide biosynthesis in Klebsiella pneumoniae

BackgroundKlebsiella pneumoniae is a Gram-negative pathogen that has become a threat to public health worldwide due to the emergence of hypervirulent and multidrug-resistant strains. Cell-surface components, such as polysaccharide capsules, fimbriae, and lipopolysaccharides (LPS), are among the major virulence factors for K. pneumoniae. One of the genes involved in LPS biosynthesis is the uge gene, which encodes the uridine diphosphate galacturonate 4-epimerase enzyme. Although essential for the LPS formation in K. pneumoniae, little is known about the mechanisms that regulate the expression of uge. Ferric uptake regulator (Fur) is an iron-responsive transcription factor that modulates the expression of capsular and fimbrial genes, but its role in LPS expression has not yet been identified. This work aimed to investigate the role of the Fur regulator in the expression of the K. pneumoniae uge gene and to determine whether the production of LPS by K. pneumoniae is modulated by the iron levels available to the bacterium.ResultsUsing bioinformatic analyses, a Fur-binding site was identified on the promoter region of the uge gene; this binding site was validated experimentally through Fur Titration Assay (FURTA) and DNA Electrophoretic Mobility Shift Assay (EMSA) techniques. RT-qPCR analyses were used to evaluate the expression of uge according to the iron levels available to the bacterium. The iron-rich condition led to a down-regulation of uge, while the iron-restricted condition resulted in up-regulation. In addition, LPS was extracted and quantified on K. pneumoniae cells subjected to iron-replete and iron-limited conditions. The iron-limited condition increased the amount of LPS produced by K. pneumoniae. Finally, the expression levels of uge and the amount of the LPS were evaluated on a K. pneumoniae strain mutant for the fur gene. Compared to the wild-type, the strain with the fur gene knocked out presented a lower LPS amount and an unchanged expression of uge, regardless of the iron levels.ConclusionsHere, we show that iron deprivation led the K. pneumoniae cells to produce higher amount of LPS and that the Fur regulator modulates the expression of uge, a gene essential for LPS biosynthesis. Thus, our results indicate that iron availability modulates the LPS biosynthesis in K. pneumoniae through a Fur-dependent mechanism.

Effects of low dietary calcium and lipopolysaccharide challenges on production performance, eggshell quality, and bone metabolism of laying hens.

Dietary calcium supply is essential for bone development and egg production in laying hens. This study investigated the effects of low dietary calcium and lipopolysaccharide (LPS) induced immune challenge in aged laying hens. A total of thirty-two Hy-Line Brown laying hens at 80 weeks old with an average laying rate of 62% were randomly divided into two groups and fed a normal calcium diet (3.57% Ca, NCA) or low calcium diet (2.08% Ca, LCA). At 88 weeks, the experiment was designed using a 2 × 2 factorial arrangement, and hens were intraperitoneally injected with saline (SAL) or LPS (0.5mg/kg, 0.5mg/kg, or 1.5mg/kg body weight) once every 48h intervals over 5 days. Production performance, egg quality, and bone physiology were evaluated. Results showed that LPS challenge decreased the hen-day egg production, egg mass, and eggshell traits (p < 0.05), but increased (p < 0.05) the calcium content of the tibia compared to SAL-injected hens. LCA diet decreased (p < 0.05) the hen-day egg production, and eggshell traits such as weight, percentage, strength, and thickness compared to the NCA diet. LCA diet increased the serum alkaline phosphatase (ALP) activity (p < 0.01) and tibial expression of ALP (p < 0.05) compared to NCA diet. LPS injection suppressed both the serum ALP activity (p < 0.05) and tibial expression of ALP (p < 0.001) compared to SAL injection. Furthermore, LPS injection increased (p < 0.05) the expression of both pro and anti-inflammatory cytokines in the spleen and tibia. The expression of cathepsin K ( Cts K ) and matrix metalloproteinase 9 ( MMP-9 ) were downregulated by LPS injection (p < 0.001). Broken and shell-less egg production and calcium content of eggshell, as well as tibial mRNA expression of osteocalcin ( Ocn ), tumor necrosis factor-alpha ( TNF-α ) and tartrate-resistant acid phosphatase ( TRAP ) were affected by the interaction (p < 0.05) of diet and injection. Therefore, this study demonstrated that to certain extents, low dietary calcium and LPS challenge dysregulated bone homeostasis and metabolism, with detrimental effects on the performance and eggshell quality of aged laying hens.

Lipopolysaccharide with long O-antigen is crucial for Salmonella Enteritidis to evade complement activity and to facilitate bacterial survival in vivo in the Galleria mellonella infection model

Bacterial resistance to serum is a key virulence factor for the development of systemic infections. The amount of lipopolysaccharide (LPS) and the O-antigen chain length distribution on the outer membrane, predispose Salmonella to escape complement-mediated killing. In Salmonella enterica serovar Enteritidis (S. Enteritidis) a modal distribution of the LPS O-antigen length can be observed. It is characterized by the presence of distinct fractions: low molecular weight LPS, long LPS and very long LPS. In the present work, we investigated the effect of the O-antigen modal length composition of LPS molecules on the surface of S. Enteritidis cells on its ability to evade host complement responses. Therefore, we examined systematically, by using specific deletion mutants, roles of different O-antigen fractions in complement evasion. We developed a method to analyze the average LPS lengths and investigated the interaction of the bacteria and isolated LPS molecules with complement components. Additionally, we assessed the aspect of LPS O-antigen chain length distribution in S. Enteritidis virulence in vivo in the Galleria mellonella infection model. The obtained results of the measurements of the average LPS length confirmed that the method is suitable for measuring the average LPS length in bacterial cells as well as isolated LPS molecules and allows the comparison between strains. In contrast to earlier studies we have used much more precise methodology to assess the LPS molecules average length and modal distribution, also conducted more subtle analysis of complement system activation by lipopolysaccharides of various molecular mass. Data obtained in the complement activation assays clearly demonstrated that S. Enteritidis bacteria require LPS with long O-antigen to resist the complement system and to survive in the G. mellonella infection model.

Neurosteroids mediate and modulate the effects of pro-inflammatory stimulation and toll-like receptors on hippocampal plasticity and learning.

Pro-inflammatory changes contribute to multiple neuropsychiatric illnesses. Understanding how these changes are involved in illnesses and identifying strategies to alter inflammatory responses offer paths to potentially novel treatments. We previously found that acute pro-inflammatory stimulation with high (μg/ml) lipopolysaccharide (LPS) for 10-15 min dampens long-term potentiation (LTP) in the hippocampus and impairs learning. Effects of LPS involved non-canonical inflammasome signaling but were independent of toll-like receptor 4 (TLR4), a known LPS receptor. Low (ng/ml) LPS also inhibits LTP when administered for 2-4 h, and here we report that this LPS exposure requires TLR4. We also found that effects of low LPS on LTP involve the oxysterol, 25-hydroxycholesterol, akin to high LPS. Effects of high LPS on LTP are blocked by inhibiting synthesis of 5α-reduced neurosteroids, indicating that neurosteroids mediate LTP inhibition. 5α-Neurosteroids also have anti-inflammatory effects, and we found that exogenous allopregnanolone (AlloP), a key 5α-reduced steroid, prevented effects of low but not high LPS on LTP. We also found that activation of TLR2, TLR3 and TLR7 inhibited LTP and that AlloP prevented the effects of TLR2 and TLR7, but not TLR3. The enantiomer of AlloP, a steroid that has anti-inflammatory actions but low activity at GABAA receptors, prevented LTP inhibition by TLR2, TLR3 and TLR7. In vivo, both AlloP enantiomers prevented LPS-induced learning defects. These studies indicate that neurosteroids play complex roles in network effects of acute neuroinflammation and have potential importance for development of AlloP analogues as therapeutic agents.

Lipopolysaccharide-binding protein in follicular fluid is associated with the follicular inflammatory status and granulosa cell steroidogenesis in dairy cows.

Metabolic stress and subsequent hepatic dysfunction in high-producing dairy cows are associated with inflammatory diseases and declining fertility. Lipopolysaccharide (LPS)-binding protein (LBP) is produced by hepatocytes and controls the immune response, suggesting that it is involved in the pathophysiology of inflammation-related attenuation of reproductive functions during metabolic stress. This study investigated the effect of LBP on the inflammatory status, oocyte quality, and steroidogenesis in the follicular microenvironment of dairy cows. Using bovine ovaries obtained from a slaughterhouse, follicular fluid and granulosa cells were collected from large follicles to evaluate the follicular status of metabolism, inflammation, and steroidogenesis. Cumulus-oocyte complexes were aspirated from small follicles and subjected to in vitro embryo production. The results showed that follicular fluid LBP concentrations were significantly higher in cows with fatty livers and hepatitis than in those with healthy livers. Follicular fluid LBP and LPS concentrations were negatively correlated, whereas LPS concentration showed a positive correlation with the concentrations of non-esterified fatty acids (NEFA) and β-hydroxybutyric acid in follicular fluid. The blastulation rate of oocytes after in vitro fertilization was impaired in cows in which coexisting large follicles had high NEFA levels. Follicular fluid NEFA concentration was negatively correlated with granulosa cell expression of the estradiol (E2) synthesis-related gene (CYP19A1). Follicular fluid LBP concentration was positively correlated with follicular fluid E2 concentration and granulosa cell CYP19A1 expression. In conclusion, follicular fluid LBP may be associated with favorable conditions in the follicular microenvironment, including low LPS levels and high E2 production by granulosa cells.

Energetic costs of mounting an immune response in a coral reef damselfish (Pomacentrus amboinensis)

While immune responses can be energetically costly, quantifying these costs is challenging. We tested the metabolic costs of immune activation in damselfish ( Pomacentrus amboinensis Bleeker, 1868) following a mass-adjusted injection of lipopolysaccharide (LPS) endotoxin. Fish were divided into eight treatments: two controls (handling and saline injection) and six LPS groups with concentrations ranging from 3 to 100 mg kg−1. We used intermittent flow respirometry to measure differences in oxygen uptake (delta ṀO2) 20 h before versus 20 h after LPS injection and changes in metabolic traits (lowest, routine, and peak metabolic rates) as proxies of the aerobic costs of metabolism. Spleen somatic index (SSI) and gene expression in spleens were measured to assess immune activation. We found no difference in metabolic traits or SSI but observed different nonlinear patterns of delta ṀO2 in fish exposed to 50 and 100 mg kg−1 LPS compared to lower doses and controls. Fish exposed to high doses of LPS also had lower residual aerobic scope compared to controls and lower LPS doses. Fish exposed to doses of 3, 50, and 100 mg kg−1 showed altered gene expression compared to the handling control. Overall, our results suggest that immune activation has measurable effects on metabolic traits that are both dose- and time-dependent.

398 Subclinical Lipopolysaccharide Exposure During Oocyte Maturation Disrupts Early Bovine Embryonic Development

Abstract Reduced conception rates and infertility are associated with bacterial infections in cattle. Chronic and acute gram-negative bacteria release of lipopolysaccharide (LPS) endotoxin elicits robust immune responses capable of disrupting folliculogenesis, steroidogenesis, oocyte competence, and embryo development. However, the effects of subclinical or non-detectable infections on oocyte competence and subsequent embryo development remain to be fully elucidated. Therefore, the objective of this study was to investigate varying concentrations of LPS on oocyte and embryo competence. Bovine oocytes were collected from slaughterhouse-derived ovaries and incubated with vehicle control or increasing doses of LPS (0, 0.01, 0.1, and 1 µg/mL) for 21 h during in vitro maturation. Oocytes (n = 252 in 3 replicates) were fixed and stained for evaluation of nuclear maturation. A subset of oocytes (n = 153 in 3 replicates) was fertilized and cultured in a time-lapse incubator where individual well images were captured every 5 min for 8 d for video compilation and subsequent analysis. A set of embryos from LPS-matured oocytes (n = 1,126 in 5 replicates) were cultured for 8 d to evaluate blastocyst cell counts (n = 163 in 5 replicates). This evaluation included total, trophectoderm, and inner cell mass cell numbers. Results demonstrate no significant difference among 0.01, 0.1, or 1 µg/mL LPS treatment groups and the percent of oocytes at metaphase II (MII), meiosis I (MI), or germinal vesicle (GV) when compared with vehicle-treated controls after 21 h maturation (P = 0.44, 0.26, and 0.90, respectively). Likewise, no differences were detected among 0.01, 0.1, or 1 µg/mL LPS treatment groups and presumptive zygotes that did not cleave after fertilization (P = 0.84), those that cleaved but arrested at the 2-cell stage (P = 0.50), 4-cell (P = 0.76), prior to morula (P = 76), or morula stage (P = 0.60). However, the percentage of blastocysts derived from oocytes matured in 0.01 µg/mL LPS tended to decrease compared with vehicle-treated controls (11.38 ± 8.74 and 25.45 ± 2.73%, respectively, P = 0.09). Similarly, the proportion of oocytes that developed to the blastocyst stage was greater in vehicle-treated controls (25.45%) compared with embryos derived from oocytes matured in 0.1 (5.92 ± 3.62% P = 0.03) and 1 µg/mL (6.55 ± 3.62%, P = 0.03) LPS. Lipopolysaccharide treatment did not affect total blastocyst cell count (P = 0.73), as indicated by the number of cells of the trophectoderm (P = 0.45), and inner cell mass (P = 0.30). Therefore, these data suggest that although nuclear maturation was not affected by low LPS concentrations, LPS-matured oocytes that subsequently underwent in vitro fertilization, experienced decreased competence to develop to the blastocyst stage. Therefore, non-detectable or subclinical bacterial infections have the capability to decrease embryonic development in cattle.

Low-dose lipopolysaccharide exposure during oocyte maturation disrupts early bovine embryonic development

Gram-negative bacteria release of lipopolysaccharide (LPS) endotoxin elicits robust immune responses capable of disrupting normal ovarian function contributing to female infertility. However, effects of subclinical or non-detectable infections on oocyte competence and subsequent embryo development remain to be fully elucidated. The aim of this study was to investigate the effects of exposing bovine oocytes to low LPS doses on oocyte and embryo competence. Bovine oocytes were collected from slaughterhouse-derived ovaries and matured with vehicle-control or increasing doses of LPS (0.01, 0.1, and 1 μg/mL) for 21 h. Oocytes (n = 252) were evaluated for nuclear maturation. A set of embryos from LPS-matured oocytes (n = 300) were cultured for 8 d to evaluate day 3 cleavage rates and day 8 blastocyst rates along with blastocyst cell counts. A subset of oocytes (n = 153) was fertilized and cultured for time-lapse image capture and analysis of embryo development. Results demonstrate no significant treatment differences among treatment groups in percent of oocytes at germinal vesicle (GV; P = 0.90), germinal vesicle breakdown (GVBD; P = 0.13), meiosis I (MI; P = 0.26), or metaphase II (MII; P = 0.44). Likewise, treatment differences were not observed in cleavage rates (P = 0.97), or blastocyst rates (P = 0.88) evaluated via traditional microscopy. Treatment with LPS did not affect total blastocyst cell count (P = 0.68), as indicated by trophectoderm (P = 0.83), and inner cell mass (P = 0.21) cell counts. Time-lapse embryo evaluation demonstrated no differences among control or LPS matured oocytes in number of zygotes that did not cleave after fertilization (P = 0.84), or those that cleaved but arrested at the 2-cell stage (P = 0.50), 4-cell (P = 0.76), prior to morula (P = 0.76). However, embryos derived from oocytes challenged with 0.1 μg/mL LPS tended to have reduced development to the morula stage compared with vehicle-treated controls (P = 0.06). Additionally, the percentage of blastocysts derived from oocytes matured in 0.01 μg/mL LPS tended to decrease compared to vehicle-treated controls (11.38 and 25.45 %, respectively; P = 0.09). Similarly, the proportion of oocytes that developed to the blastocyst stage was greater in vehicle-treated controls (25.45 %) compared with embryos derived from oocytes matured in 0.1 and 1 μg/mL (5.92 and 6.55 %, respectively; P = 0.03) LPS. These data suggest LPS-matured oocytes that subsequently underwent in vitro fertilization, experienced decreased competence to develop to the blastocyst stage.

Blood-borne extracellular vesicles of bacteria and intestinal cells in patients with psychotic disorders

Background Human cells and bacteria secrete extracellular vesicles (EV) which play a role in intercellular communication. EV from the host intestinal epithelium are involved in the regulation of bacterial gene expression and growth. Bacterial EV (bactEV) produced in the intestine can pass to various tissues where they deliver biomolecules to many kinds of cells, including neurons. Emerging data indicate that gut microbiota is altered in patients with psychotic disorders. We hypothesized that the amount and content of blood-borne EV from intestinal cells and bactEV in psychotic patients would differ from healthy controls. Methods We analyzed for human intestinal proteins by proteomics, for bactEV by metaproteomic analysis, and by measuring the level of lipopolysaccharide (LPS) in blood-borne EV from patients with psychotic disorders (n = 25), tested twice, in the acute phase of psychosis and after improvement, with age- and sex-matched healthy controls (n = 25). Results Patients with psychotic disorders had lower LPS levels in their EV compared to healthy controls (p = .027). Metaproteome analyses confirmed LPS finding and identified Firmicutes and Bacteroidetes as dominating phyla. Total amounts of human intestine proteins in EV isolated from blood was lower in patients compared to controls (p = .02). Conclusions Our results suggest that bactEV and host intestinal EV are decreased in patients with psychosis and that this topic is worthy of further investigation given potential pathophysiological implications. Possible mechanisms involve dysregulation of the gut microbiota by host EV, altered translocation of bactEV to systemic circulation where bactEV can interact with both the brain and the immune system.

The herbicides glyphosate and glufosinate and the cyanotoxin β-N-methylamino-l-alanine induce long-term motor disorders following postnatal exposure: the importance of prior asymptomatic maternal inflammatory sensitization

BackgroundPrenatal maternal immune activation (MIA) and/or perinatal exposure to various xenobiotics have been identified as risk factors for neurological disorders, including neurodegenerative diseases. Epidemiological data suggest an association between early multi-exposures to various insults and neuropathologies. The “multiple-hit hypothesis” assumes that prenatal inflammation makes the brain more susceptible to subsequent exposure to several kinds of neurotoxins. To explore this hypothesis and its pathological consequences, a behavioral longitudinal procedure was performed after prenatal sensitization and postnatal exposure to low doses of pollutants.MethodsMaternal exposure to an acute immune challenge (first hit) was induced by an asymptomatic lipopolysaccharide (LPS) dose (0.008 mg/kg) in mice. This sensitization was followed by exposing the offspring to environmental chemicals (second hit) postnatally, by the oral route. The chemicals used were low doses of the cyanotoxin β-N-methylamino-l-alanine (BMAA; 50 mg/kg), the herbicide glufosinate ammonium (GLA; 0.2 mg/kg) or the pesticide glyphosate (GLY; 5 mg/kg). After assessing maternal parameters, a longitudinal behavioral assessment was carried out on the offspring in order to evaluate motor and emotional abilities in adolescence and adulthood.ResultsWe showed that the low LPS immune challenge was an asymptomatic MIA. Even though a significant increase in systemic pro-inflammatory cytokines was detected in the dams, no maternal behavioral defects were observed. In addition, as shown by rotarod assays and open field tests, this prenatal LPS administration alone did not show any behavioral disruption in offspring. Interestingly, our data showed that offspring subjected to both MIA and post-natal BMAA or GLA exposure displayed motor and anxiety behavioral impairments during adolescence and adulthood. However, this synergistic effect was not observed in the GLY-exposed offspring.ConclusionThese data demonstrated that prenatal and asymptomatic immune sensitization represents a priming effect to subsequent exposure to low doses of pollutants. These double hits act in synergy to induce motor neuron disease-related phenotypes in offspring. Thus, our data strongly emphasize that multiple exposures for developmental neurotoxicity regulatory assessment must be considered. This work paves the way for future studies aiming at deciphering cellular pathways involved in these sensitization processes.

The Clinical Significance of Serum Biomarkers of the Intestinal Barrier in Systemic Sclerosis: A Cross-Sectional Study.

Systemic sclerosis (SSc) is an immune-mediated connective tissue disease. Recent studies reported differences in the composition of intestinal microbiota (dysbiosis) in patients with SSc compared to nonsclerodermic subjects. Dysbiosis may disrupt the intestinal barrier, which leads to immunological activation via microbial antigen and metabolite translocation. The study aimed to assess the differences in intestinal permeability between SSc patients and controls and to examine the correlation between intestinal permeability and complications of SSc. The study comprised 50 patients with SSc and 30 matched subjects. Serum intestinal permeability markers: intestinal fatty acid binding protein, claudin-3, and lipopolysaccharides (LPS) were determined using an enzyme-linked immunosorbent assay. SSc patients had a significantly increased concentration of LPS compared to control subjects (232.30 [149.00-347.70] versus 161.00 [83.92-252.20] pg/mL, p < 0.05). The patients with shorter SSc duration (≤6 years) had an increased concentration of LPS and claudin-3 compared to the subgroup with longer disease length: LPS (280.75 [167.30-403.40] versus 186.00 [98.12-275.90] pg/mL, p < 0.05), and claudin-3 (16.99 [12.41-39.59] versus 13.54 [10.29-15.47] ng/mL, p < 0.05). The patients with esophageal dysmotility had a decreased LPS level compared to those without this complication (188.05 [102.31-264.40] versus 283.95 [203.20-356.30] pg/mL, p < 0.05). Increased intestinal permeability in SSc may exacerbate the course of the disease and increase the risk of developing complications. Lower LPS levels in SSc might be a hallmark of esophageal dysmotility.

Lactate transport and metabolism in rumen epithelial cells in SARA condition

Lactate, a main product of rumen microbial fermentation from dietary starch, has been recognised as an important mediator of rumen microbiota regulation in rumen acidosis. However, the mechanism of lactate transport and metabolism in the rumen remains unclear. This study aimed to evaluate the significance of the absorption, transport, and metabolism of lactic acid in the pH and LPS-induced subacute ruminal acidosis (SARA) model of rumen epithelial cells (RECs). In the current study, RECs were obtained from two adult Saanen goats (44.5 ± 4.6 kg BW) and using Lipopolysaccharide (LPS) and low pH to co-stimulate RECs to construct a microenvironment under SARA conditions, to further analyse the lactate transport and metabolism by RECs. The results showed that the LPS and low pH could significantly reduce cell viability by 29.36% (p < 0.05), induce inflammation in RECs, destroy tight junctions between RECs, and increase cell permeability. In addition, stimulation leads to changes in cellular metabolic activity and inhibits the expression of lactate transporters (p < 0.05), promoting an increased risk of lactate accumulation in the intracellular and intestinal lumen. This study hopefully provides a better understanding of lactate metabolic regulation and new perspectives for the nutritional prevention and treatment of SARA. HIGHLIGHTS Lactic acid is an important fermentation product in the rumen of ruminants, and its transport and metabolic regulation mechanisms are still unclear. To understand transport uptake in the rumen epithelium, a SARA model was therefore used. Low pH and high LPS trigger epithelial inflammation, increasing the risk of lactic acid entering the bloodstream.

Characterization of IMG Microglial Cell Line as a Valuable In Vitro Tool for NLRP3 Inflammasome Studies.

Microglial cells constantly surveil the cerebral microenvironment and become activated following injury and disease to mediate inflammatory responses. The nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) inflammasome, which is abundantly expressed in microglial cells, plays a key role in these responses as well as in the development of many neurological disorders. Microglial cell lines are a valuable tool to study the causes and possible treatments for neurological diseases which are linked to inflammation. Here, we investigated whether the mouse microglial cell line IMG is suitable to study NLRP3 inflammasome by incubating cells with different concentrations of NLRP3 inflammasome priming and activating agents lipopolysaccharide (LPS) and ATP, respectively, and applying short (4h) or long (24h) LPS incubation times. After short LPS incubation, the mRNA levels of most pro-inflammatory and NLRP3 inflammasome-associated genes were more upregulated than after long incubation. Moreover, the combination of higher LPS and ATP concentrations with short incubation time resulted in greater levels of active forms of caspase-1 and interleukin-1 beta (IL-1β) proteins than low LPS and ATP concentrations or long incubation time. We also demonstrated that treatment with NLRP3 inflammasome inhibitor glibenclamide suppressed NLRP3 inflammasome activation in IMG cells, as illustrated by the downregulation of gasdermin D N-fragment and mature caspase-1 and IL-1β protein levels. In addition, we conducted similar experiments with primary microglial cells and BV-2 cell line to determine the similarities and differences in their responses. Overall, our results indicate that IMG cell line could be a valuable tool for NLRP3 inflammasome studies. In IMG cells, 4-h incubation with lipopolysaccharide (LPS) induces a stronger upregulation of NLRP3 inflammasome-associated pro-inflammatory genes compared to 24-h incubation. NLRP3 inflammasome is robustly activated only after the addition of 3mM of ATP following short LPS incubation time.

IL-25 directly modulates adipocyte function and inflammation through the regulation of adiponectin.

This study aimed to investigate the direct role of IL-25 in modulating adipocyte function during homeostasis and low-grade inflammation induced by lipopolysaccharide (LPS). The 3T3-L1 preadipocyte cell lines and primary cultures of adipose-derived stromal vascular precursor cells of wild-type and IL-17RB-deficient mice were used to determine the direct function of IL-25. The expression of IL-17RB in differentiating adipocyte was determined using real-time PCR and flow cytometry analysis. The effect of IL-25 on lipid accumulation, triglyceride content, lipolysis, glucose uptake, and adipokine expression in the mature adipocytes was evaluated. IL-25 modulating the expression of inflammatory cytokines in adipocytes induced by low dose LPS was determined using real-time PCR and ELISA. The receptor for IL-25 was up-regulated during adipocyte differentiation and IL-25 directly modulated adipocyte function by reducing lipid accumulation and triglyceride concentration and enhancing lipolysis without affecting an insulin-stimulated glucose uptake. Interestingly, IL-25 induced adiponectin secretion through the PI3K/AKT signaling pathway. In 3T3-L1 adipocytes under low-grade inflammation, IL-25 attenuated the expression of IL-6 and CCL5 through the induction of adiponectin. Our studies suggest that IL-25 directly regulates adipocyte function by maintaining the adiponectin level during homeostasis and by alleviating inflammatory response through the regulation of adiponectin during low-grade inflammation in adipocytes.

Delayed Reperfusion of the Renal Outer Medulla Attenuates Ischemia Reperfusion‐Induced Vascular Congestion

Vascular congestion, or red blood cell (RBC) trapping, of the renal outer medulla (OM) is a common finding in acute tubular necrosis caused by ischemia reperfusion (IR). Work from our laboratory suggests that vascular congestion originates in the renal venous vasculature of the cortex and OM during the ischemic phase. Following reperfusion, RBCs then fill the OM plexus capillaries as the venous drainage vessels of this region remain blocked. We have also previously reported that pretreatment with low dose lipopolysaccharide (LPS) attenuates IR‐induced vascular congestion, however the mechanisms remain unknown. In the current study, we hypothesized that pretreatment with LPS prevents vascular congestion by delayed reperfusion of blood to the OM following ischemia.To test this hypothesis, WKY rats (male, 12 weeks) were pretreated (i.p) with 1mg/kg LPS (n=6) or saline control (n=7) once daily for 3 days. Rats were then anesthetized, and Transonic Laser Doppler probes were inserted in the renal cortex and OM. Doppler flux, as a determinate of regional kidney blood flow was then measured over 10 minutes of baseline, 45 minutes of renal artery clamping and 30 minutes of reperfusion.We found no differences in baseline blood flow of the cortex (pTREATMENT=0.0543) or OM (pTREATMENT=0.5085) between rats pretreated with low dose LPS or saline control. Following removal of the renal artery clamp, the return of blood flow to the cortex was gradual over the first several minutes of reperfusion (pTIME&lt;0.0001), reaching a plateau following 10 minutes of reperfusion in both treatment groups (pINTERACTION=0.9999) (Fig. A). The reperfusion of blood to the OM, however, rapidly returned to baseline levels within 1 minute of reperfusion in control animals (1 min: 0 to 0.43AU) (Fig. B). In contrast, OM blood flow returned slowly in LPS treated rats (1 min: 0 to ‐0.08AU) and did not return to baseline levels during the 30‐minute reperfusion period (pINTERACTION=0.0127) (Fig. B).These data indicate that LPS pretreatment, paradoxically, delayed early reperfusion of the renal OM via a regional vasoconstriction response. We speculate that delayed reperfusion of the OM attenuates medullary plexus congestion by allowing time for the RBCs in the shared venous circulation to clear. These findings support the hypothesis that reperfusion of the renal outer medullary plexus before cortical perfusion is restored, is responsible for the development of prolonged vascular congestion. New therapeutics focused on renal hemodynamics to prevent congestion after ischemia may prevent much of the injury associated with acute kidney injury.

Effect of maternal exposure to lipopolysaccharide during pregnancy on allergic asthma in offspring in mice

Objective: To investigate the effect of maternal exposure to lipopolysaccharide during pregnancy on allergic asthma in offspring in mice. Methods: Animal experimental research was carried out from June 2019 to June 2021.Pregnant C57BL/6J mice were randomly divided into 2 groups by intraperitoneal injection with 7 μg/kg lipopolysaccharide (LPS) or phosphate buffered saline (PBS) at day 15.5 of gestation. After birth, 6 offspring were randomly chosen from each group at the age of 4 weeks, and stimulated with house dust mites (HDM) or PBS, further divided into 4 groups, such as LPS+PBS group, LPS+HDM group, PBS+PBS group, PBS+HDM group, with 3 mice in each group. The cough and wheezing were observed, the histological changes in lung tissue were examined after HE staining, and the expression of inflammatory factors including interleukin (IL)-4, IL-6, IL-17A, IL-23, interferon (IFN)-α and IFN-β in the lung tissue were detected by high-throughput liquid protein chip detection. T test or rank sum test was used for the comparison among these groups. Results: The asthma-like airway inflammation was more obvious in PBS+HDM group after stimulated by HDM than that in PBS+PBS group, nevertheless, this manifestation in LPS+HDM group was milder than that in PBS+HDM group. HE staining showed that inflammatory cell aggregation in the lung tissue in PBS+HDM group was significantly higher than that in PBS+PBS group (4.0 (3.5, 4.0) vs. 0 (0, 0.5), Z=2.02, P=0.043), while it was much lower in LPS+HDM group compared to PBS+HDM group (1.0 (0.5, 1.5) vs. 4.0 (3.5, 4.0), Z=1.99, P=0.046). High-throughput liquid protein chip detection of lung tissue showed that IL-6, IL-23 and IFN-β levels were significantly higher in PBS+HDM group when compared to those in PBS+PBS group ((114±3) vs. (94±4) ng/L, (210±4) vs. (173±7) ng/L, (113±2) vs. (94±4) ng/L, t=4.37, 4.84, 3.96, all P<0.05), while the levels of IL-6, IL-23, IFN-α, IFN-β in LPS+HDM group were significantly lower than those in PBS+HDM group ((87±5) vs. (114±3) ng/L, (171±7) vs. (210±4) ng/L, (16.1±0.6) vs. (20.9±0.3) ng/L, (95±1) vs. (113±2) ng/L, t=5.07, 5.07, 7.28, 7.47, all P<0.05). Conclusions: Prenatal low dose LPS exposure can reduce offspring's airway inflammatory reactions and prevent the development of allergic disease. Maternal infection during pregnancy may affect the occurrence and development of allergic asthma in offspring.

Possible Cytoprotection of Low Dose Lipopolysaccharide in Rat Thioacetamide-Induced Liver Lesions, Focusing on the Analyses of Hepatic Macrophages and Autophagy.

Lipopolysaccharide (LPS) may influence hepatic macrophages and autophagy. We evaluated the potential participation of macrophages and autophagosomes in thioacetamide (TAA)-induced rat liver injury under pretreatment of a low dose LPS (0.1 mg/kg BW, intraperitoneally; nonhepatotoxic dose). F344 rats were pretreated with LPS (LPS + TAA) or saline (TAA alone) at 24 hours before TAA injection (100 mg/kg BW, intraperitoneally); rats were examined on Days 0 (controls), 1, 2, and 3 after TAA injection. Data were compared between TAA alone and LPS + TAA rats. LPS pretreatment significantly reduced TAA-induced hepatic lesion (centrilobular necrosis with inflammation) on Days 1 and 2, being reflected by declined hepatic enzyme values and decreased number of apoptotic cells. LC3B-immunoreacting autophagosomes (as cytoplasmic fine granules) were significantly increased on Days 1 and 2 in hepatocytes of LPS + TAA rats. In LPS + TAA rats, hepatic macrophages reacting to CD68, CD163, and MHC class II mainly on Day 2 and mRNA levels of macrophage-related factors (MCP-1, IL-1β, and IL-4) on Day 1 were significantly decreased. Collectively, the low-dose LPS pretreatment might act as cytoprotection against TAA-induced hepatotoxicity through increased autophagosomes and decreased hepatic macrophages, although the dose/time-dependent cytoprotection of LPS should be further investigated at molecular levels.

Induction of TLR4/TLR2 Interaction and Heterodimer Formation by Low Endotoxic Atypical LPS.

The Toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2) complex is considered the major receptor of the innate immune system to recognize lipopolysaccharides (LPSs). However, some atypical LPSs with different lipid A and core saccharide moiety structures and compositions than the well-studied enterobacterial LPSs can induce a TLR2-dependent response in innate immune cells. Ochrobactrum intermedium, an opportunistic pathogen, presents an atypical LPS. In this study, we found that O. intermedium LPS exhibits a weak inflammatory activity compared to Escherichia coli LPS and, more importantly, is a specific TLR4/TLR2 agonist, able to signal through both receptors. Molecular docking analysis of O. intermedium LPS predicts a favorable formation of a TLR2/TLR4/MD-2 heterodimer complex, which was experimentally confirmed by fluorescence resonance energy transfer (FRET) in cells. Interestingly, the core saccharide plays an important role in this interaction. This study reveals for the first time TLR4/TLR2 heterodimerization that is induced by atypical LPS and may help to escape from recognition by the innate immune system.