Hr post-LPS Research Articles

Dynamic changes in Holstein heifer circulatory stress biomarkers in response to lipopolysaccharide immune challenge

Dairy cattle routinely face a variety of stressors. For example, climate change has resulted in more frequent heat stress events that increase the incidence of bacterial infections by inducing conditions like leaky gut syndrome, whereby the integrity of the intestinal epithelium is compromised allowing for luminal bacteria and their membrane components to infiltrate the host’s bloodstream resulting in systemic inflammation. Lipopolysaccharide (LPS) is a well-characterized and biologically relevant microbe-associated molecular pattern (MAMP) that makes up the outer membrane of pathogenic and commensal Gram-negative bacteria and is known to contribute to inflammatory disorders including mastitis, acidosis and septicemia. In the present study, Holstein heifers (n = 20) were randomly allocated into different treatment groups receiving saline, 100, 200 or 400 ng/kg of LPS intramuscularly to create an experimentally induced endotoxemic state. Serum was collected hourly for 8 hr and then again at 24 hr to profile changes in circulatory stress biomarkers. All LPS -challenged animals demonstrated distinct cortisol responses 2 hr post-LPS challenge, and in the 200 ng/kg and 400 ng/kg of LPS treatments cortisol concentrations remained significantly induced for up to 4 hr. Rectal temperature was significantly increased for heifers challenged with 100 and 200 ng/kg of LPS at 2 and 4 hr as compared to their pre-challenge temperature. All LPS-challenged animals demonstrated marked leukopenia and thrombocytopenia as compared to the saline control animals. A total of 8 cytokines, including TNFα, and IL-10, were found to be induced between 2 and 4 hr. Finally, we report that miR-1246, miR-223, miR-29 and miR-31 were significantly induced in animals challenged with LPS as compared to the saline controls. The present study demonstrated that the stress response in dairy heifers is dynamic and there are peak windows of time when cortisol, cytokines and also miRNA are induced, and blood cells are sequestered as part of the systemic inflammatory response. Variability in the response to LPS warrants further investigation in dairy cattle to better understand the contribution of genetics and associations between LPS-induced stress and health and performance.

Genetic deletion of soluble epoxide hydrolase preserves cardiac function and limits inflammation in acute lipopolysaccharide injury

Objective Acute systemic inflammatory syndromes, such as endotoxemia, elicit detrimental multi-organ responses. The heart is particularly susceptible to an acute inflammatory response resulting in myocardial dysfunction which often leads to death. A stress-induced metabolism of N-3 and N-6 polyunsaturated fatty acids (PUFAs) will generate numerous bioactive lipid mediators with beneficial or adverse effects. Cytochrome P450 (CYP) derived epoxylipid mediators may be further hydrolyzed to diol metabolites by soluble epoxide hydrolase (sEH). Emerging evidence suggests epoxylipids can exert cardioprotective effects by modulating the NLRP3 inflammasome pathway. This study investigated whether cardiomyocyte specific sEH knockdown can preserve cardiac function and attenuate inflammation in a model of acute lipopolysaccharide (LPS) injury. Hypothesis Conditional knockdown of cardiomyocyte sEH protects the heart from acute LPS injury via modulation of the NLRP3 inflammasome pathway. Methods Cardiomyocyte targeted sEH knockdown mice were produced by crossing Ephx2 floxed mice with mice expressing Cre recombinase. Male sEHFl/Fl (knockdown) and sEH+/+ (Cre control) mice were treated with tamoxifen (45 mg/kg, 6 i.p injections over 8 days) 5 weeks prior to LPS injection (10mg/kg, i.p.). A comparator group of male wild type (WT) and global sEH null mice were subjected to LPS treatment. Echocardiography was conducted pre-injection and 6 or 24hr post-LPS followed by euthanization. Plasma cytokine and chemokine levels were determined with a multi-plex assay. In vitro studies employed neonatal rat cardiomyocytes treated with LPS (1µg/mL), 19, 20-epoxydocosapentaenoic acid (EDP, 1µM) or sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (tAUCB, 10µM), for 6hr. NLRP3 and pro-IL-1β expression was assessed using Western immunoblotting. Results Genetic deletion of sEH preserved cardiac function following 24hr LPS exposure indicating an initial early decline at 6hr which was recovered in both sEHFl/Fl and sEH null but not WT mice. The increased plasma levels of pro-inflammatory cytokines and chemokines following LPS treatment was attenuated in mice lacking sEH expression. Cardiomyocytes treated with LPS had increased NLRP3 inflammasome complex formation and pro-IL-1β expression, which was not attenuated by co-treatment with EDP or tAUCB. However, plasma levels of IL-1β were reduced in both sEHFl/Fl and global sEH null mice following LPS exposure. Conclusion Deletion of sEH protects cardiac function and limits pro-inflammatory response following exposure to LPS. Preliminary data suggest epoxylipids do not prevent the formation of the NLRP3 inflammasome complex in cardiomyocytes but may impact downstream effectors. Thus, limiting the NLRP3 inflammasome cascade to reduce LPS-induced cardiac and inflammatory injury.

Effects of sinomenine in LPS-associated diseases are related to inhibition of LBP, Mac-1, and L-selectin levels.

The aim of the research was to investigate the anti-endotoxin and anti-inflammatory effects of Sinomenine, an agent commonly found in Chinese herbal medicines. Endotoxin (i.e., 1mg lipopolysaccharide (LPS)/kg)) was administered via intraperitoneal (IP) injection to piglets in high-, middle-, and low-dose sinomenine groups. Piglets were then treated with 1, 5 or 10mg/kg sinomenine, intramuscularly (i.m.), 3hr after LPS. Vehicle was administered, as above, to drug control group piglets followed 3hr later by 10mg/kg sinomenine i.m.. LPS control group piglets were challenged with 1mg/kg LPS IP, followed by vehicle i.m., and naïve control piglets were treated with normal saline IP, followed by normal saline i.m., as above. Temperatures were measured, and blood samples were collected from the precaval veins of piglets at 12, 24, and 48hr post-LPS or vehicle injection. Clinical signs were recorded, and index levels were analyzed via ELISA. Sinomenine was found to reduce the incidence and severity of LPS-induced toxicities, including body temperature elevation, cell adhesion, and systemic inflammation. These data suggest that sinomenine may be effective for regulating inflammatory responses and has the potential for use as an anti-endotoxin therapy.

Abstract # 3149 Sex differences in the consequences of early-life immune activation on microglia-neural signaling and the ontogeny of hippocampal-dependent learning in the juvenile rat

Developmental disorders associated with learning deficits, including Autism, have been linked to early-life immune activation, and notably, are male-biased. Microglia are the immune cells of the brain and are in constant communication with neurons, thus, microglia influence the function of surrounding neurons. Recent data indicate that microglia-neuron signaling is necessary for the proper formation of neural circuits during brain development. We determined that immune activation with lipopolysaccharide (LPS; 100ug/ml/kg) on postnatal day (P) 21 produces sex-dependent deficits in the emergence of hippocampal-dependent learning on P24 in rats. Gene expression analysis in the hippocampus at 2-, 4-, 8-, and 24-h following LPS showed sex differences in IL-1 β and IL-6 expression. Males, but not females, have a persistent decrease in BDNF expression starting 4hr post-LPS that persists until 24hr and an increase in C3 expression, an immune molecule that tags synapses for elimination, at 24hr. We are investigating whether we can rescue learning deficits by administering the microglia inhibitor minocycline at the time of LPS. We found a sex difference in the effectiveness of minocycline such that minocycline inhibits microglia in males, but is not as effective in females. Behavioral results following minocycline treatment will be discussed. Our data suggest that changes in microglia-neural communication following early-life immune activation are different in males and females, thus males and females may respond differently to therapies that target microglia.

Real-time detection of LPS induced cytokine-storm using Vium Smart Housing

Abstract The objective of this study was to use Vium Smart Housing to determine if in-life digital readouts (e.g. changes in motion and breathing rate) can correlate with cytokine response profiles in an LPS-induced mouse model of endotoxemia. Real-time detection of cytokine release has the potential to reduce or eliminate the need for expensive blood sampling and biochemical analyses. Eighty Balb/c mice were injected with 5 mg/kg of UltraPure LPS (E. coli 0111:B4, Invivogen) or vehicle control. Subjects were housed in Vium’s Digital Vivarium, where sensors and a HD camera coupled with computer vision, data algorithms, and cloud capabilities allow for continuous monitoring of animals and collection of automated metrics such as motion, % time running on wheel and breathing rate. All study data is available in real-time. As early as 1 hr post-LPS administration, breathing rate increased from baseline, peaked 7 hours post-induction (8.7%±3.5% vehicle vs 39.5±3.1% LPS treated) and remained elevated relative to vehicle for over 24 hrs (p<0.05). Breathing rate changes were accompanied by decreased motion as early as 1 hr post-LPS administration, peaked at 2 hours post-induction (28.73%±53.2% vehicle vs –82.69%±1.72% LPS treated), and remained decreased for over 24 hrs (p<0.05). Increased breathing positively correlated, while decreased motion negatively correlated with plasma levels of TNFα, and IL-10 at 1.5 hrs, and IL-6, IL-1B, and IFN-γ levels at 6 hrs post-LPS (p<0.05). We found that Vium’s automated metrics was able to detect onset of LPS-induced endotoxemia in real-time. This could be used to 1) screen compounds that prevent immune responses or alternatively 2) to identify cytokine release syndrome when screening immuno-oncology candidates.

PTU-095 Bifidobacterium Species Reduce Lipopolysaccharide-induced Small Intestinal Epithelial Cell Shedding in Vivo In A Myd88-dependent Manner And Protect Against Dss-induced Colitis

IntroductionCell shedding, the process by which intestinal epithelial cells (IECs) are extruded from the small intestinal (SI) villus is known to be elevated in patients with inflammatory bowel disease (IBD)...

Tu1777 Bifidobacterium Species Reduce Lipopolysaccharide-Induced Small Intestinal Epithelial Cell Shedding In Vivo in a MYD88-Dependent Manner and Protect Against DSS-Induced Colitis

Introduction Cell shedding, the process by which intestinal epithelial cells (IECs) are extruded from the small intestinal (SI) villus is known to be elevated in patients with inflammatory bowel disease (IBD) and is correlated with disease relapse. Importantly, there is evidence that the gut bacterial communities (microbiota) influences intestinal epithelial function including gene expression, cell division and energy balance. We thus sought to determine whether specific members of the microbiota, ‘probiotic’ bifidobacterial species, modulate rates of cell shedding and progression of Dextran sodium sulphate (DSS)-mediated colitis. Methods C57BL/6 mice (WT) or mice deficient in epithelial Myd88 (Vil-Cre +; Myd88 -/-) (Myd88 KO) were orally gavaged with 1 × 10 9 Bifidobacterium breve UCC2003 , B. longum NCIMB8809 or PBS (control) in 3x daily doses. To induce SI cell shedding, mice were injected with 1.25 mg kg-1 Lipopolysaccharide (LPS) intrapertioneally. Animals were euthanized 1.5 hr post-LPS and SI tissue sections analysed for cleaved caspase 3 (CC3) by immunohistochemistry to score shedding along the first 50 cell positions from the villus tip. For colitis studies, control mice or mice colonised with B. breve were administered 2% DSS in drinking water for 6 days and euthanized 8 days post-DSS. Disease activity index (DAI) was recorded daily and histology performed on formalin-fixed tissue sections including periodic acid/Schiff (PAS) stain (goblet cell stain). Results Mice receiving B. breve and B. longum showed less CC3 +ve shedding cells (3.6% +/-0.6, p B. breve was lost in Myd88 KO mice receiving LPS as numbers of CC3 +ve IECs were the same in mice receiving B. breve or vehicle control (13.3%+/-1.7 vs 10.4%+/1.3; ns), indicating that the protective effect may be mediated by Toll-like receptors. In our colitis model, mice colonised with B. breve had reduced DAI compared to control mice, coupled with a significant increase in numbers of PAS +ve goblet cells per crypt (8.2%+/1.6 vs 16.0%+/-0.6; p = 0.05). Conclusion Bifidobacterial species modulate a reduction in rates of cell shedding from the SI villus, potentially via the Myd88 signalling pathway. B. breve is also able to partially ameliorate the adverse effects of DSS-induced colitis through induction of goblet cells. In summary, bifidobacteria, particularly B. breve, may be beneficial as a therapeutic agent for IBD. Disclosure of Interest None Declared.

Decreases in Podocin, CD2-Associated Protein (CD2AP) and Tensin2 May Be Involved in Albuminuria during Septic Acute Renal Failure

Podocytes have a peculiar structure constituting slit diaphragm (SD) and foot process (FP), and play essential roles in the glomerular filtration barrier. There is now ample evidence that SD- and FP-associated molecules, such as podocin and CD2-associated protein (CD2AP), are down-regulated during albuminuria of chronic kidney disease. However, it is still unclear whether these molecules are altered during acute renal failure (ARF) with albuminuria. Using lipopolysaccharide (LPS)-treated mice as a model of septic ARF, we provide evidence that the expression of SD- and FP-associated molecules becomes faint, along with albuminuria. In the LPS-treated mice, urinary albumin levels gradually increased, associated with the elevation of blood urea nitrogen levels, indicating the successful induction of albuminuria during septic ARF. In this pathological process, glomerular podocin expression became faint, especially at 36 hr post-LPS challenge (i.e., a peak of albuminuria). Likewise, LPS treatment led to a significant decrease in CD2AP, an anchorage between podocin and F-actin. With regard to this, tensin2 is a novel molecule that stabilizes F-actin extension. Interestingly, glomerular tensin2 expression levels were also decreased during the albuminuric phase, associated with losses of glomerular F-actin and synaptopodin under septic states. As a result, there were some lesions of podocytic FP effacement, as shown by electron microscopy. Based on these data, we emphasize the importance of concomitant decreases in podocin, CD2AP and tensin2 during septic ARF-associated proteinuria.

KL4‐surfactant prevents hyperoxic and LPS‐induced lung injury in mice

KL(4)-surfactant contains the novel KL(4) peptide, sinapultide, which mimics properties of the hydrophobic pulmonary surfactant protein SP-B, in a phospholipid formulation and may be lung protective in experimental acute respiratory distress syndrome/acute lung injury. Our objective was to determine the protective role of airway delivery of KL(4)-surfactant in murine models of hyperoxic and lipopolysaccharide (LPS)-induced lung injury and further explore the mechanisms of protection. For the hyperoxic injury model, mice exposed to 80% O(2) for 6 days received an intranasal bolus of vehicle, beractant, or KL(4)-surfactant on days 3, 4, 5, and 6 of the exposure, and lungs were evaluated on day 7. Mice in the LPS-induced lung injury model received an intratracheal bolus of LPS followed by an intranasal bolus of KL(4)-surfactant or control at 1, 3, and 19 hr post-LPS challenge, and lungs were evaluated after 24 hr. To explore the mechanisms of protection, in vitro assays were performed with human and murine endothelial cell monolayers, and polymorphonuclear leukocyte (PMN) transmigration in the presence or absence of KL(4)-surfactant or lipid controls was evaluated. Based on morphology, histopathology, white blood cell count, percentage of PMNs, and protein concentration in bronchoalveolar lavage fluid, our data showed KL(4)-surfactant, unlike vehicle or beractant, blocked neutrophil influx into alveoli and suppressed lung injury. Furthermore, in vitro assays showed KL(4)-surfactant decreased neutrophil transmigration at the endothelial cell level. KL(4)-surfactant decreased inflammation and lung permeability compared with controls in both mouse models of lung injury. Evidence suggests the anti-inflammatory mechanism of the KL(4)-peptide is through inhibition of PMN transmigration through the endothelium.

Neuroprotective role of microglia expressing interleukin‐4

Little is known about the underlying mechanisms responsible for the death of activated microglia and the functional consequences of the death of these cells, especially in vivo. We show here that intracortical injection of lipopolysaccharide (LPS) led to upregulation of interleukin-4 (IL-4) immunoreactivity, followed by a substantial loss of microglia 3 days later, as visualized by complement receptor type 3 (OX-42) immunostaining and tomato lectin staining. Cells positive for caspase-3 and terminal deoxynucleotidyl transferase mediated fluorescein-dUTP nick-end labeling (TUNEL) were also localized within LPS-activated microglia. IL-4 immunoreactivity was detected as early as 12 hr post-LPS, disappearing at 72 hr. Surprisingly, IL-4 immunoreactivity was detected exclusively in microglia, but not in astrocytes or neurons. In addition, IL-4-neutralizing antibodies markedly increased the survival of activated microglia at 3 days post-LPS. The expression of inducible nitric oxide synthase (iNOS) and tumor-necrosis factor (TNF)-alpha was sustained in parallel in activated microglia, consequently increasing neuronal cell death. To our knowledge, this study is the first to show the endogenous expression of IL-4 in LPS-activated microglia in vivo. Our findings suggest that IL-4 may regulate brain inflammation by inducing the death of activated microglia in vivo and increasing neuronal survival.

Peroxisome Proliferator-Activated Receptor γ1 Expression in Porcine White Blood Cells: Dynamic Regulation with Acute Endotoxemia

Peroxisome proliferator-activated receptor γ (PPARγ), a primary regulator of adipocyte differentiation, has been implicated in the regulation of monocyte and macrophage function in vitro. We report that PPARγ protein is expressed in porcine peripheral white blood cells (WBC), and that PPARγ1 but not γ2 mRNA predominates. Additionally, we provide the first evidence that in vivo lipopolysaccharide challenge (LPS, 25 μg/kg BW) causes a dynamic increase in PPARγ protein expression in peripheral WBC (P < 0.05). PPARγ expression was increased 2-fold over basal (1 hr post-LPS), was maximal by 4 hr (3-fold), and was normalized to control by 8 hr post-LPS. Changes in PPARγ expression coincided with or closely followed LPS-induced changes in plasma cortisol, TNF-α, insulin, IGF-1, glucose, and free fatty acids. These data suggest that induction of PPARγ expression in WBC may play a role in host response to acute inflammatory challenge and may prove to be an important target of anti-inflammatory therapies.

Chemokine and Cell Adhesion Molecule mRNA Expression and Neutrophil Infiltration in Lipopolysaccharide‐Induced Hepatitis in Ethanol‐Fed Rats

Neutrophil infiltration is a feature of alcoholic hepatitis (AH), and although the mechanism by which this occurs is unclear, it may involve a chemotactic gradient. We used lipopolysaccharide (LPS) to induce, in ethanol-fed rats, liver damage similar to that seen in AH. To our knowledge, this study is the first to examine the effect of ethanol on LPS-stimulated chemokine mRNA expression in this model. Hepatic cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1beta, MIP-2, and eotaxin mRNA levels were elevated 1 to 3 hr post-LPS in both groups. Maximal expression of MIP-2 and MCP-1 mRNA was higher in ethanol-fed rats 1 hr post-LPS, whereas CINC-2 mRNA expression was elevated above controls at 12 to 24 hr. Hepatic intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 mRNA levels were elevated in both groups at 1 hr, whereas L-selectin expression in ethanol-fed rats was elevated above controls at 12 to 24 hr. Hepatic neutrophil infiltration was highest during maximal hepatocyte necrosis. These data suggest that cell adhesion molecules, in conjunction with elevated cytokines and the subsequently induced chemokines, may assist in the formation of a chemotactic gradient within the liver, causing the neutrophil infiltration seen both in this model and possibly in AH.

Estriol: A potent regulator of TNF and IL-6 expression in a murine model of endotoxemia

The increased incidence of autoimmune disease in premenopausal women suggests the involvement of sex steroids in the pathogenesis of these disease processes. The effects of estrogen on autoimmunity and inflammation may involve changes in the secretion of inflammatory mediators by mononuclear phagocytes. Estradiol, for example, has been reported to regulate TNF, IL-6, IL-1 and JE expression. In the present study the effects of the estrogen agonist, estriol, on cytokine expression have been investigated in mice administered a sublethal lipopolysaccharide, LPS, challenge. Pretreatment of mice with pharmacologic doses of estriol, 0.4-2 mg/kg, resulted in a significant increase in serum TNF levels in both control and autoimmune MRL/lpr mice, following LPS challenge. This increase in TNF over the placebo group was blocked by the estrogen antagonist tamoxifen. Estriol treated mice also exhibited a rapid elevation in serum IL-6 levels following LPS challenge with the peak increase occurring 1 hr post LPS. This contrasted with the placebo group in which maximal serum IL-6 levels were detected at 3 hrs post challenge. This shift in the kinetics of IL-6 increase by estriol was inhibited by tamoxifen. The estriol mediated effects of TNF and IL-6 serum levels were consistent with the changes in TNF and IL-6 mRNA observed ex vivo in elicited peritoneal macrophages. Macrophage cultures from estriol treated animals however, did not demonstrate significant differences from the placebo group for TNF or NO secretion following in vitro LPS challenge. These results suggest that the estrogen agonist estriol can have significant quantitative, TNF, and kinetic, IL-6, effects on inflammatory monokines produced in response to an endotoxin challenge.

Tumor Necrosis Factor–Alpha Gene Expression in Human Whole Blood

Tumor necrosis factor-alpha (TNF) is recognized as a principal mediator of a variety of pathophysiologic and immunologic events. Lipopolysaccharide (LPS) challenge, either in vitro or in vivo, results in significant TNF production. In this study we present data demonstrating LPS-induced TNF mRNA expression and bioactivity using an in vitro tissue system of whole blood (WB). The kinetics of LPS-induced TNF production by WB was significantly accelerated as compared to isolated cultured peripheral blood monocytes (PBM). At post-LPS challenge, plasma from WB demonstrated a rapid rise in TNF bioactivity, peaking by 4 hr (1,021 units/ml/10(6) cells), plateauing between 4 and 8 hr, and then decreasing over the next 16 hr. In contrast, the highest measured TNF bioactivity from PBM did not occur until the 24-hr time-point (175 units/ml/10(6) cells). Whole blood buffy-coat TNF mRNA was assessed by Northern blot analysis, and demonstrated significant TNF mRNA accumulation at 1 hr and a peak 2 hr post-LPS challenge. By 8 hr TNF mRNA was undetectable. Concomitant administration of LPS with either prostaglandin E2 (10(-6)M) or Dexamethasone (10(-6)M) resulted in significant suppression of LPS-induced TNF production. This data supports WB as a useful in vitro medium for the molecular and cellular analysis of TNF. As specialized connective tissue, WB may provide an important environment to study the pharmacologic manipulation of TNF mRNA and bioactivity.

Is macrophage-induced microthrombosis during endotoxemia dependent on prostaglandin synthesis?

Steroids and nonsteroidal anti-inflammatory drugs (NSAID), in addition to inhibiting prostaglandin synthesis, have recently been shown to block production of procoagulant activity (PCA) by alveolar macrophages (A-MΦ). This inhibition may ameliorate one deleterious effect of endotoxin (LPS), since LPS-induced PCA may contribute to the diffuse microvascular thrombosis (DIC) and subsequent organ failure seen in endotoxemia. However, the results of treatment of endotoxemia with steroids of NSAID are variable or short-lived. To help elucidate the basis for these results, the effect of these agents on production of PCA by another MΦ population, the hepatic MΦ (H-MΦ PCA is functionally discrete from A-MΦ PCA) was evaluated. Four agents, (hydrocortisone, indomethacin, ibuprofen and acetylsalicylic acid (ASA)), used to both block production of prostaglandins and ameliorate the deleterious effects of LPS in vivo were tested on the production of rabbit MΦ PCA in vitro in homogeneous cultures. PCA was measured by a standardized one-step coagulation assay. Treatment with LPS (1 to 10 g/ml) produced maximal stimulation of PCA production with a peak at 8 hr with PCA levels 20- to 30+-fold higher than controls. The effect of pretreatment ( T −10 min) of MΦ cultures with the various inhibitors on PCA production at 8 hr post-LPS was evaluated, ( N = 6 to 10 experiments). The results demonstrate that (1) steroids inhibit production of PCA by both A-MΦ and H-MΦ (>90%); however, (2) NSAID, while inhibiting A-MΦ PCA (65–90%), only minimally inhibit the production of H-MΦ PCA (30–34%). This differential effect on these discrete MΦ populations may partially explain the erratic results of treatment of endotoxemia with these agents.