Interaction Of Lipopolysaccharide Research Articles

ВЗАИМОДЕЙСТВИЕ ЛИПОПОЛИСАХАРИД-СВЯЗЫВАЮЩИХ БЕЛКОВ С РАЗЛИЧНЫМИ ФОРМАМИ ЛИПОПОЛИСАХАРИДОВ

Lipopolysaccharide-binding proteins from two common jellyfish species Aurellia aurita and Ropelema asamushi were isolated and purified, and the interaction of lipopolysaccharides (LPS) of various structural types with LBP was studied. By inhibiting the interaction, it was found that both proteins specifically bind to the lipid and core fragments of the LPS molecule. There are two types of binding sites in LBP with Kd = 3,28 × 10-6 M and Kd = 0,13 × 10-6 M (for the protein from A. aurita) and Kd = 3,66 × 10-6 M and Kd = 0,27 × 10-6 M (for protein from R. asamushi). It has been shown by dynamic light scattering that the binding of LBP to R-LPS leads to the dissociation of LPS micelles. The sizes of LPS aggregates decrease from 200 nm to 25–30 nm in the composition of LPS–LSB complexes. The data of electrokinetic measurements indicate the neutralization of the negative charge of Rd-LPS (-42,2 mV) in the LPS-LSB-R. asamushi complex up to -4,4 mV. S-LPS micelles from E. coli do not disaggregate upon binding to LBP, which is apparently due to the shielding of lipid A by O-specific chains in the S-LPS molecule. The binding of LPS to LBP may affect their endotoxic properties.

180 Dietary milk fat globule membrane alters intestinal microbiota of neonatal piglet following lipopolysaccharide challenge and marker of neuroinflammation

Abstract Early life stress exposure can increase morbidity and reduce animal wellbeing throughout life. Dysregulated gut function has been associated with neurological changes, and neurological changes affect gut homeostasis. Maternal milk is the gold-standard for yielding developmental benefits to offspring. Identification of milk components has led to an understanding of the bioactive nutrients important in neonatal development. Components of the milk fat globule membrane (MFGM) contribute immensely to neonatal gut health and neurodevelopment. However, mechanisms of actions are still being defined. The objective is to understand how dietary MFGM modulates the gut microbiota, blood-brain barrier tight junctions, and brain inflammatory markers following lipopolysaccharide (LPS) challenge. Piglets (n = 24, one-d-old) were assigned to soy (CON) or MFGM phospholipid supplemented diet (0.75% wt/wt) ± LPS (100 µg/kg body weight) in a 2 × 2 factorial design (n = 6 animals•diet-1•challenge-1.). Piglets were fed for 21 d and 6 animals/diet received saline or LPS injections 4 h before euthanasia. Colonic microbiota was characterized through 16s rRNA gene sequencing, while hypothalamus samples were collected to evaluate brain barrier function and inflammation-related genes using RT-qPCR. Microbiota data were processed using QIIME2 with DADA2 plugin and analyzed for alpha-diversity, beta-diversity, and differential abundance in R software. Gene transcription data were analyzed by a 2 × 2 experimental factorial design using the PROC MIXED procedure of SAS. Principal coordinates analysis indicated that MFGM-fed groups exhibited an observable separation from CON-fed groups in terms of microbial community structures (P < 0.05). Firmicutes_A, Bacteroidota, and Firmicutes_D were the predominant phyla across all treatments, with average proportions to be 52.21 %, 24.36 %, and 18.46 %, respectively. At genus level, MFGM + Saline group down-regulated Corynebacterium by 88.00 % (q < 0.05) and Unclassified Muribaculaceae by 73.33 % (0.050.05). However, there was an MFGM × LPS interaction (P < 0.05) observed for nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha, in which MFGM attenuated the LPS-induced increase in mRNA relative abundance (3.62 and 2.39 ± 0.38). In conclusion, dietary MFGM altered neonatal piglet intestinal microbiota and an important signaling molecule of the neuroinflammatory response following LPS challenge.

Protective Effect of Silibinin on Lipopolysaccharide-Induced Endotoxemia by Inhibiting Caspase-11-Dependent Cell Pyroptosis.

To explore the protective effect and the underlying mechanism of silibinin (SIB), one of the active compounds from Silybum marianum (L.) Gaertn in endotoxemia. Mouse peritoneal macrophage were isolated via intraperitoneally injection of BALB/c mice with thioglycolate medium. Cell viability was assessed using the cell counting kit-8, while cytotoxicity was determined through lactate dehydrogenase cytotoxicity assay. The protein expressions of interleukin (IL)-1 α, IL-1 β, and IL-18 were determined by enzyme-linked immunosorbent assay. Intracellular lipopolysaccharide (LPS) levels were measured by employing both the limulus amoebocyte lysate assay and flow cytometry. Additionally, proximity ligation assay was employed for the LPS and caspase-11 interaction. Mice were divided into 4 groups: the control, LPS, high-dose-SIB (100 mg/kg), and low-dose-SIB (100 mg/kg) groups (n=8). Zebrafish were divided into 4 groups: the control, LPS, high-dose-SIB (200 εmol/L), and low-dose-SIB (100 εmol/L) groups (n=30 for survival experiment and n=10 for gene expression analysis). The expression of caspase-11, gasdermin D (GSDMD), and N-GSDMD was determined by Western blot and the expressions of caspy2, gsdmeb, and IL-1 β were detected using quantitative real-time PCR. Histopathological observation was performed through hematoxylineosin staining, and protein levels in bronchoalveolar lavage fluid were quantified using the bicinchoninicacid protein assay. SIB noticeably decreased caspase-11 and GSDMD-mediated pyroptosis and suppressed the secretion of IL-1 α, IL-1 β, and IL-18 induced by LPS (P<0.05). Moreover, SIB inhibited the translocation of LPS into the cytoplasm and the binding of caspase-11 and intracellular LPS (P<0.05). SIB also attenuated the expression of caspase-11 and N-terminal fragments of GSDMD, inhibited the relative cytokines, prolonged the survival time, and up-regulated the survival rate in the endotoxemia models (P<0.05). SIB can inhibit pyroptosis in the LPS-mediated endotoxemia model, at least in part, by inhibiting the caspase-11-mediated cleavage of GSDMD. Additionally, SIB inhibits the interaction of LPS and caspase-11 and inhibits the LPS-mediated up-regulation of caspase-11 expression, which relieves caspase-11-dependent cell pyroptosis and consequently attenuates LPS-mediated lethality.

PSIII-8 Extra-Label Bisphosphonate Effects on Intra-Articular Inflammation in Juvenile Horses Challenged with Intra-Articular Lipopolysaccharide

Abstract Anecdotal assertions that bisphosphonates, such as clodronate disodium (CD), exhibit anti-inflammatory properties has led to extra-label use, despite a lack of scientific evidence of its efficacy. The objective of this study was to determine the effects of CD on intra-articular inflammation, hypothesizing that intra-muscular administration of CD would reduce intra-articular inflammation following an acute inflammatory challenge by lipopolysaccharide (LPS). To test this, 32 yearling Quarter horses were stratified by age (500 ± 13 d), BW (336 ± 26 kg), sex (n = 16 female; n = 16 male) and initial bone optical density into 4 treatment groups. The 140-d study consisted of two phases: Phase 1 (d 0 to 83) and Phase 2 (d 84 to 140) emulated sale preparation and early performance training, respectively. Horses were housed individually (3.6 m × 7.3 m) and fed to meet requirements of growing horses undergoing moderate training. Treatments consisted of control (CON; n = 8; no CD), single-dose (1X; n = 8; d 84), two-doses (2X; n = 8; d 0, 84), and four-doses (4X; n = 8; d 0, 42, 84, 126) of CD. All horses received iso-volumetric intramuscular injections of 1.8mg/kg BW clodronate disodium (OSPHOS) or saline (placebo) on d 0, 42, 84, and 126. Following treatment administration on d 126, radial carpal joints of each horse were injected with 0.8 mL of either 0.5 ng sterile LPS derived from Escherichia coli O55:B5 or sterile lactated Ringer’s solution as a contralateral control. Synovial fluid was collected before LPS injection (h 0) and at 6, 12, 24, and 336 h post injection. Synovial fluid was analyzed using an ELISA for prostaglandin E2 (PGE2), an indicator of joint inflammation. Data were log transformed and analyzed using the MIXED procedure of SAS. There was an h × LPS interaction (P &amp;lt; 0.01) confirming an acute, transient inflammatory response with increased PGE2 at 6, 12, and 24 h compared with the contralateral control joint, and resolved (P = 0.62) by h 336 post-injection. There was a treatment × h × LPS interaction (P = 0.02) in which 1X, 2X and 4X CD groups had greater synovial PGE2 concentrations at 6 h post-injection in the LPS joint compared with CON. In summary, intra-articular LPS induced localized inflammation, and the PGE2 response was greater in all horses treated with CD. The results of this study reject the hypothesis, suggesting clodronate disodium administration does not reduce intra-articular inflammation following acute induced synovitis.

Alcohol use disorder (AUD) is associated with enhanced sensitivity to cellular lipopolysaccharide challenge.

Inflammation has been associated with alcohol use disorder (AUD). A novel method to characterize AUD-related immune signaling involves probing Toll-like receptor (TLR)-4 stimulated monocyte production of intracellular cytokines (ICCs) via lipopolysaccharide (LPS). We evaluated relationships between AUD and ICC production at rest and after LPS stimulation. We analyzed blood samples from 36 participants (AUD N = 14; Controls N = 22), collected across time, with ICC expression assessed at rest (i.e., unstimulated) and following stimulation with LPS (i.e., a total of 5 repeated unstimulated or stimulated measures/participant). Markers assessed included tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), TNF-α and IL-6 co-expression, and interferon (IFN). For each marker, we constructed linear mixed models with AUD, LPS, and timepoint as fixed effects (BMI as covariate), allowing for random slope and intercept. AUD × LPS was included as an interaction. For TLR4-stimulated monocyte production of TNF-α, there were effects of AUD (p < 0.01), LPS (p < 0.001), and AUD × LPS interaction (p < 0.05), indicating that individuals with AUD showed greater unstimulated- and stimulated monocyte expression of TNF-α. Similarly, for TLR4-stimulated monocyte co-expression of TNF-α and IL-6, there were effects of AUD (p < 0.01), LPS (p < 0.001), and AUD × LPS interaction (p < 0.05). No AUD or LPS effects were found for IL-6. Timepoint effects were observed on IL-6 and TNF-α/IL-6 co-expression (p < 0.001). Finally, for IFN there were also effects of AUD (p < 0.05), LPS (p < 0.001), and AUD × LPS (p < 0.001). Individuals with AUD showed greater resting or unstimulated levels of intracellular monocyte expression of TNF-α and IL-6/TNF-α co-expression than controls. AUD was associated with increases in TLR4-stimulated monocyte production of TNF-α and co-production of IL-6 and TNF-α. This is, to our knowledge, the first study to investigate relationships between AUD and monocyte production of proinflammatory cytokines, at rest and in response to TLR4 stimulation with LPS. The study extends previous findings on the roles of proinflammatory cytokines in AUD and serves as a critical proof of concept for the use of this method to probe neuroimmune mechanisms underlying AUD.

Force Characteristics of Yersinia pestis Lipopolysaccharide Interaction with TLR4 and CD14 Receptors on J774 Macrophages: Atomic Force Microscopy

Force Characteristics of Yersinia pestis Lipopolysaccharide Interaction with TLR4 and CD14 Receptors on J774 Macrophages: Atomic Force Microscopy

Long-term antibacterial activity of guanidinium carbon dots without detectable resistance for the effective treatment of pneumonia caused by Gram-negative bacteria

The development of nanomedicine for the treatment of infection caused by resistant bacteria, especially Gram-negative bacteria, is still in the bottleneck. The long-term antibacterial activity and the low induced resistance risk need improvement. Herein, guanidinium-based carbon dots (G-CDs) were prepared from citric acid, dimethyldiallylammonium chloride, and polyhexamethyleneguanidine through melting strategy. The antibacterial properties of G-CDs against Gram-negative and resistant bacteria were investigated. G-CDs exhibit strong and long-term antibacterial activity, as well as antibiofilm activity, with less potential for inducing bacterial resistance. The antibacterial mechanism of G-CDs of the adsorption action and killing effect was elucidated, which was different from the mechanism of antibiotics. Moreover, derived from the interacting mode of nanomaterials and ssDNA, the enhanced ability of G-CDs against Gram-negative bacteria was studied based on the found interaction of G-CDs and lipopolysaccharide (LPS) for the understanding of the mode for the absorption of G-CDs on the cell wall of Gram-negative bacteria. The multimode interactions of van der Waals force, electrostatic adsorption and hydrogen bonding were clarified. Furthermore, G-CDs had excellent in vivo safety and in vivo therapeutic effects in E. coli-infected pneumonia through an intravenous approach. This study provides a hopeful strategy for developing antibacterial drugs from understanding the interacting mode of the drug and the cell wall of bacteria.

Effects of Ca2+ ions on the horseshoe crab coagulation cascade triggered by lipopolysaccharide.

The lipopolysaccharide (LPS)-triggered horseshoe crab coagulation cascade is composed of three protease zymogens, prochelicerase C (proC), prochelicerase B (proB) and the proclotting enzyme (proCE). In this study, we found that Ca 2+ ions increase the production of the clotting enzyme as a result of a cascade reaction reconstituted by recombinant proteins of wild-type (WT) proC, WT proB and WT proCE. We divided the cascade into three stages: autocatalytic activation of WT proC on the surface of LPS into WT α-chelicerase C (Stage 1); activation of WT proB on the surface of LPS into WT chelicerase B by WT α-chelicerase C (Stage 2) and activation of WT proce into WT CE by chelicerase B (Stage 3). Ca2+ ions enhanced the proteolytic activation in Stage 2, but not those in Stages 1 and 3. Moreover, we performed isothermal titration calorimetry to clarify the interaction of LPS or the recombinant zymogens with Ca2+ ions. LPS interacted with Ca2+ ions at an association constant of Ka= 4.7 × 104M-1, but not with any of the recombinant zymogens. We concluded that LPS bound with Ca2+ ions facilitates the chain reaction of the cascade as a more efficient scaffold than LPS itself.

Intravenous surfactant protein D inhibits lipopolysaccharide-induced systemic inflammation

BackgroundSurfactant protein D (SP-D) is an innate host defense protein that clears infectious pathogens from the lung and regulates pulmonary host defense cells. SP-D is also detected in lower concentrations in plasma and many other non-pulmonary tissues. Plasma levels of SP-D increase during infection and other proinflammatory states; however, the source and functions of SP-D in the systemic circulation are largely unknown. We hypothesized that systemic SP-D may clear infectious pathogens and regulate host defense cells in extrapulmonary systems. MethodsTo determine if SP-D inhibited inflammation induced by systemic lipopolysaccharide (LPS), E.coli LPS was administered to mice via tail vein injection with and without SP-D and the inflammatory response was measured. ResultsSystemic SP-D has a circulating half-life of 6 h. Systemic IL-6 levels in mice lacking the SP-D gene were similar to wild type mice at baseline but were significantly higher than wild type mice following LPS treatment (38,000 vs 29,900 ng/ml for 20 mg/kg LPS and 100,700 vs 73,700 ng/ml for 40 mg/kg LPS). In addition, treating wild type mice with purified intravenous SP-D inhibited LPS induced secretion of IL-6 and TNFα in a concentration dependent manner. Inhibition of LPS induced inflammation by SP-D correlated with SP-D LPS binding suggesting SP-D mediated inhibition of systemic LPS requires direct SP-D LPS interactions. ConclusionsTaken together, the above results suggest that circulating SP-D decreases systemic inflammation and raise the possibility that a physiological purpose of increasing systemic SP-D levels during infection is to scavenge systemic infectious pathogens and limit inflammation-induced tissue injury.

Self-assembling micelles of lipopolysaccharides (LPS) for loading hydrophobic (bio)molecules

Lipopolysaccharide (LPS) endotoxins are the most common pyrogenic biomolecules naturally present in Gram-negative bacteria membranes. Although LPS pathophysiological features are widely reported, a number of nanobiotechnological aspects perceive importance. Therefore, herein this study reports the physicochemical properties of LPS-based micelles from Escherichia coli O113, specifically focusing on their self-assembling distribution and aptitude to interact with four different (bio)molecules [i.e., chloroquine (CQ), curcumin (CCM), vemurafenib (VEM), and doxorubicin (DOX)]. The critical micelle concentration of LPS (CMCLPS) in an aqueous environment was determined to be 0.020 mg/mL, using a pyrene fluorescence emission spectra technique. The interaction of LPS and hydrophobic (bio)molecules was evaluated as a function of encapsulation parameters at the highest LPS concentration (CLPS of 0.043 mg/mL, i.e. >CMCLPS) for namely drug loading (DL) and encapsulation efficiency (EE). The DL results for CQ, CCM, VEM, and DOX were 28, 26, 22, and 20%, with 83, 80, 76, and 74% for their EE parameter, respectively. A straightforward relationship was observed for CLPS that increased jointly with micelle size (DH at 0.043 mg/mL > DH at 0.020 mg/mL). The presence of the (bio)molecules also affected LPS micelle size where DH was 51 nm (control micelles), which increased to 58, 58, 55, and 55 nm for CQ, CCM, VEM, and DOX, respectively, at a CLPS of 0.043 mg/mL. This trend demonstrates that increasing LPS micelle size (>CMCLPS) enhances the encapsulation capacity of the hydrophobic (bio)molecules. The understanding and characterization of the LPS-based micelles and their interaction with (bio)molecules could lead to novel applications in nanobiotechnology, such as the potential use of LPS, e.g., as drug/vaccine nanocarriers in the near future.

Quantitative assessment of LPS-HBsAg interaction by introducing a novel application of immunoaffinity chromatography

Lipopolysaccharide (LPS), as a stubborn contamination, should be monitored and kept in an acceptable level during the pharmaceutical production process. Recombinant hepatitis B surface antigen (r-HBsAg) is one of the recombinant biological products, which is probable to suffer from extrinsic endotoxin due to its long and complex production process. This research aims to assess the potential interaction between LPS and r-HBsAg by recruiting immunoaffinity chromatography (IAC) as a novel tool to quantify the interaction. Molecular modeling was performed on the HBsAg molecule to theoretically predict its potential binding and interaction sites. Then dynamic light scattering (DLS) analysis was implemented on HBsAg, LPS, and mixtures of them to reveal the interaction. The virus-like particle (VLP) structure of HBsAg and the ribbon-like structure of LPS were visualized by transmission electron microscopy (TEM). Finally, the interaction was quantified by applying various LPS/HBsAg ratios ranging from 1.67 to 120 EU/dose in the IAC. Consequently, the LPS/HBsAg ratios in the eluate were measured from 1.67 to a maximum of 92.5 EU/dose. The results indicated that 77 to 100% of total LPS interacted with HBsAg by an inverse relationship to the incubated LPS concentration. The findings implied that the introduced procedure is remarkably practical in the quantification of LPS interaction with a target recombinant protein.

Antimicrobial and Anti-Inflammatory Activities of MAF-1-Derived Antimicrobial Peptide Mt6 and Its D-Enantiomer D-Mt6 against Acinetobacter baumannii by Targeting Cell Membranes and Lipopolysaccharide Interaction.

ABSTRACTAntibiotic resistance in Acinetobacter baumannii is on the rise around the world, highlighting the urgent need for novel antimicrobial drugs. Antimicrobial peptides (AMPs) contribute to effective protection against infections by pathogens, making them the most promising options for next-generation antibiotics. Here, we report two designed, cationic, antimicrobial-derived peptides: Mt6, and its dextroisomer D-Mt6, belonging to the analogs of MAF-1, which is isolated from the instar larvae of houseflies. Both Mt6 and D-Mt6 have a broad-spectrum antimicrobial activity that is accompanied by strong antibacterial activities, especially against A. baumannii planktonic bacteria and biofilms. Additionally, the effect of D-Mt6 against A. baumannii is stable in a variety of physiological settings, including enzyme, salt ion, and hydrogen ion environments. Importantly, D-Mt6 cleans the bacteria on Caenorhabditis elegans without causing apparent toxicity and exhibits good activity in vivo. Both Mt6 and D-Mt6 demonstrated synergistic or additive capabilities with traditional antibiotics against A. baumannii, demonstrating their characteristics as potential complements to combination therapy. Scanning electron microscopy (SEM) and laser scanning confocal microscope (LSCM) experiments revealed that two analogs displayed rapid bactericidal activity by destroying cell membrane integrity. Furthermore, in lipopolysaccharide (LPS)-stimulated macrophage cells, these AMPs drastically decreased IL-1β and TNF-a gene expression and protein secretion, implying anti-inflammatory characteristics. This trait is likely due to its dual function of directly binding LPS and inhibiting the LPS-activated mitogen-activated protein kinase (MAPK) signaling pathways in macrophages. Our findings suggested that D-Mt6 could be further developed as a novel antimicrobial/anti-inflammatory agent and used in the treatment of A. baumannii infections.IMPORTANCE Around 700,000 people worldwide die each year from antibiotic-resistant pathogens. Acinetobacter baumannii in clinical specimens increases year by year, and it is developing a strong resistance to clinical drugs, which is resulting in A. baumannii becoming the main opportunistic pathogen. Antimicrobial peptides show great potential as new antibacterial drugs that can replace traditional antibiotics. In our study, Mt6 and D-Mt6, two new antimicrobial peptides, were designed based on a natural peptide that we first discovered in the hemlymphocytes of housefly larvae. Both Mt6 and D-Mt6 showed broad-spectrum antimicrobial activity, especially against A. baumannii, by damaging membrane integrity. Moreover, D-Mt6 showed better immunoregulatory activity against LPS induced inflammation through its LPS-neutralizing and suppression on MAPK signaling. This study suggested that D-Mt6 is a promising candidate drug as a derived peptide against A. baumannii.

Effects of Dietary Amylose-Amylopectin Ratio on Growth Performance and Intestinal Digestive and Absorptive Function in Weaned Piglet Response to Lipopolysaccharide.

Simple SummaryAs one of the most important components, starch has made great contributions to feed but was wasted numerously during pig feeding. This paper is designed to explore the optimal ratio of dietary amylose to amylopectin for gut health and absorption, thereby improving starch utilization. Our results indicated that the dietary amylose/amylopectin ratio (AAR) of 0.60 could reduce feed conversion rate of piglets, having a certain positive significance for saving feed. In addition, under lipopolysaccharide (LPS) stress, a diet with an AAR of 0.40 to 0.60 significantly improved the intestinal health of piglets, which would provide data to support for the formulation of feed in weaned piglets during bacterial infection.This study investigated the effects of diet with different amylose–amylopectin ratios (AAR) on the growth performance, intestinal morphology, digestive enzyme activities and mRNA expression of nutrients transporters in piglets with short-term lipopolysaccharide (LPS) intraperitoneal injections. Sixty 21 days-old piglets (Landrace × Yorkshire; 6.504 ± 0.079) were randomly assigned based on their body weight (BW) and litters of origins to five groups with experimental diets with an AAR of 0.00, 0.20, 0.40, 0.60, or 0.80 (namely, the 0.00, 0.20, 0.40, and 0.80 groups), respectively. Each treatment included 12 piglets (one piglet per pen). This experiment lasted for 28 days. On the 28th day, six piglets in each treatment were randomly selected for an LPS intraperitoneal injection (100 μg/kg BW), and other piglets were injected with normal saline. Twelve hours after LPS injection, all piglets were sacrificed to collect small intestinal mucosa for analysis. Although different AAR did not influence the final BW in piglets, the piglets in the 0.40 group represented the poorest feed-to-gain ratio (F/G) in the first, second and fourth week (p < 0.05) and the lowest average daily gain (ADG) in the fourth week (p < 0.05) compared with other groups. In terms of the small intestinal morphology, piglets in the 0.20 and 0.60 groups showed better ileal villous width (p < 0.05). Piglets in the 0.60 group presented greater activities of jejunal maltase, sucrase and alkaline phosphatase (p < 0.05) than those of 0.20 and 0.40. However, a low amylose diet increased the mRNA expression of jejunal glucose and amino acid transporters (p < 0.05). In addition, compared to saline injection, the LPS challenge significantly lessened jejunal digestive enzyme activities (p < 0.01) and, ileal villous width and downregulated the gene expression of glucose and amino acid transporters (p < 0.05) in piglets. Interestingly, certain diet -LPS interactions on duodenal VH/CD, jejunal maltase activity (p < 0.05) and the expression of glucose transporters (p < 0.05) were observed. Taken together, in terms of small intestinal digestion and absorption capacity, these results demonstrated that a diet with an AAR of 0.60 diets could improve the intestinal digestive and absorptive capability by affecting small intestinal morphology, digestive enzymes, and nutrients absorptions in piglets. In addition, the diets containing an AAR of 0.40–0.60 were more likely to resist the damage of LPS stress to intestinal morphology and nutrient absorption.

Increased supply of methionine during a heat-stress challenge in lactating holstein cows alters mammary tissue mTOR signaling and its response to lipopolysaccharide.

The first objective was to investigate the effects of feeding rumen-protected methionine (RPM) during a heat stress (HS) challenge on abundance and phosphorylation of mechanistic target of rapamycin (mTOR)-related signaling proteins in mammary gland. The second objective was to investigate how HS and RPM may modulate the response of mammary gland explants to an inflammatory challenge using lipopolysaccharide (LPS). Thirty-two multiparous, lactating Holstein cows (184 ± 59 DIM) were randomly assigned to 1 of 2 environmental treatment groups, and 1 of 2 dietary treatments [TMR with RPM (Smartamine M; Adisseo Inc.; 0.105% DM as top dress) or TMR without RPM (CON)] in a crossover design. There were two periods with two phases per period. In phase 1 (9 d), all cows were in thermoneutral conditions (TN) and fed ad libitum. During phase 2 (9 d), group 1 (n = 16) cows were exposed to HS using electric heat blankets, whereas group 2 cows (n = 16) remained in TN but were pair-fed to HS counterparts to control for DMI decreases associated with HS. After a washout period (14 d), the study was repeated (period 2). Environmental treatments were inverted in period 2 (sequence), whereas dietary treatments remained the same. Mammary tissue was harvested via biopsy at the end of both periods. Tissue was used for protein abundance analysis and also for incubation with 0 or 3 μg/mL of LPS for 2 h and subsequently used for mRNA abundance. Data were analyzed using PROC MIXED in SAS. Analysis of protein abundance data included the effects of diet, environment and their interaction, and period and sequence to account for the crossover design. The explant data model also included the effect of LPS and its interaction with environment and diet. Abundance of phosphorylated mTOR and ratio of phosphorylated eukaryotic translation elongation factor 2 (p-EEF2) to total EEF2 in non-challenged tissue was greater with RPM supplementation (P = 0.04 for both) and in both cases tended to be greater with HS (P = 0.08 for both). Regardless of RPM supplementation, incubation with LPS upregulated mRNA abundance of IL8, IL6, IL1B, CXCL2, TNF, NFKB1, and TLR2 (P < 0.05). An environment × LPS interaction was observed for NFKB1 (P = 0.03); abundance was greater in LPS-treated explants from non-HS compared with HS cows. Abundance of CXCL2, NFKB1, NOS2, NOS1, and SOD2 was lower with HS (P < 0.05). Although LPS did not alter mRNA abundance of the antioxidant transcription factor NFE2L2 (P = 0.59), explants from HS cows had lower abundance of NFE2L2 (P < 0.001) and CUL3 (P = 0.04). Overall, RPM supplementation may alter mTOR activation in mammary tissue. Additionally, although HS reduced explant immune and antioxidant responses, RPM did not attenuate the inflammatory response induced by LPS in vitro.

A Fluorescence-Polarization-Based Lipopolysaccharide-Caspase-4 Interaction Assay for the Development of Inhibitors.

Recognition of intracellular lipopolysaccharide (LPS) by Caspase-4 (Casp-4) is critical for host defense against Gram-negative pathogens. LPS binds to the N-terminal caspase activation and recruitment domain (CARD) of procaspase-4, leading to auto-proteolytic activation followed by pro-inflammatory cytokine release and pyroptotic cell death. Aberrant hyper-activation of Casp-4 leads to amplification of the inflammatory response linked to sepsis. While the active site of a caspase has been targeted with peptide inhibitors, inhibition of LPS–Casp-4 interaction is an emerging strategy for the development of selective inhibitors with a new mode of action for treating infectious diseases and sepsis induced by LPS. In this study, a high-throughput screening (HTS) system based on fluorescence polarization (FP) was devised to identify inhibitors of the LPS and Casp-4 interaction. Using HTS and IC50 determination and subsequently showing inhibited Casp-4 activity, we demonstrated that the LPS–Casp-4 interaction is a druggable target for Casp-4 inhibition and possibly a non-canonical inflammatory pathway.

Metabolome and microbiome multi-omics integration from a murine lung inflammation model of bronchopulmonary dysplasia.

Respiratory tract microbial dysbiosis can exacerbate inflammation and conversely inflammation may cause dysbiosis. Dysbiotic microbiome metabolites may lead to bronchopulmonary dysplasia (BPD). Hyperoxia and lipopolysaccharide (LPS) interaction alters lung microbiome and metabolome, mediating BPD lung injury sequence. C57BL6/J mice were exposed to 21% (normoxia) or 70% (hyperoxia) oxygen during postnatal days (PND) 1-14. Pups were injected with LPS (6 mg/kg) or equal PBS volume, intraperitoneally on PND 3, 5, and 7. At PND14, the lungs were collected for microbiome and metabolomic analyses (n = 5/group). Microbiome alpha and beta diversity were similar between groups. Metabolic changes included hyperoxia 31 up/18 down, LPS 7 up/4 down, exposure interaction 8. Hyperoxia increased Intestinimonas abundance, whereas LPS decreased Clostridiales, Dorea, and Intestinimonas; exposure interaction affected Blautia. Differential co-expression analysis on multi-omics data identified exposure-altered modules. Hyperoxia metabolomics response was integrated with a published matching transcriptome, identifying four induced genes (ALDOA, GAA, NEU1, RENBP), which positively correlated with BPD severity in a published human newborn cohort. We report hyperoxia and LPS lung microbiome and metabolome signatures in a clinically relevant BPD model. We identified four genes correlating with BPD status in preterm infants that are promising targets for therapy and prevention. Using multi-omics, we identified and correlated key biomarkers of hyperoxia and LPS on murine lung micro-landscape and examined their potential clinical implication, which shows strong clinical relevance for future research. Using a double-hit model of clinical relevance to bronchopulmonary dysplasia, we are the first to report integrated metabolomic/microbiome landscape changes and identify novel disease biomarker candidates.

Pediatric traumatic brain injury and a subsequent transient immune challenge independently influenced chronic outcomes in male mice

Traumatic brain injury (TBI) is a major contributor to death and disability worldwide. Children are at particularly high risk of both sustaining a TBI and experiencing serious long-term consequences, such as cognitive deficits, mental health problems and post-traumatic epilepsy. Severe TBI patients are highly susceptible to nosocomial infections, which are mostly acquired within the first week of hospitalization post-TBI. Yet the potential chronic impact of such acute infections following pediatric TBI remains unclear. In this study, we hypothesized that a peripheral immune challenge, such as lipopolysaccharide (LPS)—mimicking a hospital-acquired infection—would worsen inflammatory, neurobehavioral, and seizure outcomes after experimental pediatric TBI. To test this, three-week old male C57Bl/6J mice received a moderate controlled cortical impact or sham surgery, followed by 1 mg/kg i.p. LPS (or 0.9% saline vehicle) at 4 days TBI. Mice were randomized to four groups; sham-saline, sham-LPS, TBI-saline or TBI-LPS (n = 15/group). Reduced general activity and increased anxiety-like behavior were observed within 24 h in LPS-treated mice, indicating a transient sickness response. LPS-treated mice also exhibited a reduction in body weights, which persisted chronically. From 2 months post-injury, mice underwent a battery of tests for sensorimotor, cognitive, and psychosocial behaviors. TBI resulted in hyperactivity and spatial memory deficits, independent of LPS; whereas LPS resulted in subtle deficits in spatial memory retention. At 5 months post-injury, video-electroencephalographic recordings were obtained to evaluate both spontaneous seizure activity as well as the evoked seizure response to pentylenetetrazol (PTZ). TBI increased susceptibility to PTZ-evoked seizures; whereas LPS appeared to increase the incidence of spontaneous seizures. Post-mortem analyses found that TBI, but not LPS, resulted in robust glial reactivity and loss of cortical volume. A TBI × LPS interaction in hippocampal volume suggested that TBI-LPS mice had a subtle increase in ipsilateral hippocampus tissue loss; however, this was not reflected in neuronal cell counts. Both TBI and LPS independently had modest effects on chronic hippocampal gene expression. Together, contrary to our hypothesis, we observed minimal synergy between TBI and LPS. Instead, pediatric TBI and a subsequent transient immune challenge independently influenced chronic outcomes. These findings have implications for future preclinical modeling as well as acute post-injury patient management.

Effect of PEGylation on Host Defense Peptide Complexation with Bacterial Lipopolysaccharide

Conjugation with poly(ethylene glycol) ("PEGylation") is a widely used approach for improving the therapeutic propensities of peptide and protein drugs through prolonging bloodstream circulation, reducing toxicity and immunogenicity, and improving proteolytic stability. In the present study, we investigate how PEGylation affects the interaction of host defense peptides (HDPs) with bacterial lipopolysaccharide (LPS) as well as HDP suppression of LPS-induced cell activation. In particular, we investigate the effects of PEGylation site for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), a peptide displaying potent anti-inflammatory effects, primarily provided by its N-terminal part. PEGylation was performed either in the N-terminus, the C-terminus, or in both termini, keeping the total number of ethylene groups (n = 48) constant. Ellipsometry showed KYE28 to exhibit pronounced affinity to both LPS and its hydrophobic lipid A moiety. The PEGylated peptide variants displayed lower, but comparable, affinity for both LPS and lipid A, irrespective of the PEGylation site. Furthermore, both KYE28 and its PEGylated variants triggered LPS aggregate disruption. To investigate the peptide structure in such LPS complexes, a battery of nuclear magnetic resonance (NMR) methods was employed. From this, it was found that KYE28 formed a well-folded structure after LPS binding, stabilized by hydrophobic domains involving aromatic amino acids as well as by electrostatic interactions. In contrast, the PEGylated peptide variants displayed a less well-defined secondary structure, suggesting weaker LPS interactions in line with the ellipsometry findings. Nevertheless, the N-terminal part of KYE28 retained helix formation after PEGylation, irrespective of the conjugation site. For THP1-Xblue-CD14 reporter cells, KYE28 displayed potent suppression of LPS activation at simultaneously low cell toxicity. Interestingly, the PEGylated KYE28 variants displayed similar or improved suppression of LPS-induced cell activation, implying the underlying key role of the largely retained helical structure close to the N-terminus, irrespective of PEGylation site. Taken together, the results show that PEGylation of HDPs can be done insensitively to the conjugation site without losing anti-inflammatory effects, even for peptides inducing such effects through one of its termini.

Interaction between extracellular ATP5A1 and LPS alleviates LPS-induced neuroinflammation in mice

Neuroinflammation is one of the main causes of Alzheimer’s disease (AD). The presence of Lipopolysaccharide (LPS) in senile plaques (SP) of AD suggests that it plays a role in AD pathogenesis. ATP5A1 (F1F0-ATP synthase F1 α subunit) is abundant in SP. Further, the protein has recently been found to have an anti-infection role in zebrafish embryos. In the present study, we observed that LPS levels were higher in the brains of APP/PS1 mice than in control mice, and LPS co-localised with ATP5A1 in amyloid plaques. The interaction of recombinant ATP5A1(rATP5A1) and LPS was evidenced by cellular thermal shift assay and enzyme-linked immunosorbent assay-based binding assay in vitro. Neuroinflammation in the brain of a mouse model was induced by intracerebroventricular injection of LPS. The addition of rATP5A1 relieved LPS-induced reduction of spontaneous locomotor ability, depressive-like behaviour, and working memory impairment. Furthermore, rATP5A1 suppressed the activation of astrocytes and microglia, IL-1β accumulation, and tau phosphorylation induced by LPS. Taken together, findings suggest that ATP5A1 is involved in the regulation of LPS-mediated neuroinflammation in AD.

MicroRNA Modulation of Host Immune Response and Inflammation Triggered by Helicobacter pylori.

Helicobacter pylori (H. pylori) remains the most-researched etiological factor for gastric inflammation and malignancies. Its evolution towards gastric complications is dependent upon host immune response. Toll-like receptors (TLRs) recognize surface and molecular patterns of the bacterium, especially the lipopolysaccharide (LPS), and act upon pathways, which will finally lead to activation of the nuclear factor-kappa B (NF-kB), a transcription factor that stimulates release of inflammatory cytokines. MicroRNAs (MiRNAs) finely modulate TLR signaling, but their expression is also modulated by activation of NF-kB-dependent pathways. This review aims to focus upon several of the most researched miRNAs on this subject, with known implications in host immune responses caused by H. pylori, including let-7 family, miRNA-155, miRNA-146, miRNA-125, miRNA-21, and miRNA-221. TLR–LPS interactions and their afferent pathways are regulated by these miRNAs, which can be considered as a bridge, which connects gastric inflammation to pre-neoplastic and malignant lesions. Therefore, they could serve as potential non-invasive biomarkers, capable of discriminating H. pylori infection, as well as its associated complications. Given that data on this matter is limited in children, as well as for as significant number of miRNAs, future research has yet to clarify the exact involvement of these entities in the progression of H. pylori-associated gastric conditions.