Low-dose Lipopolysaccharide Administration Research Articles

Acute experimental inflammation in healthy women attenuates empathy for psychological pain

Administration of low-dose lipopolysaccharide (LPS) to healthy humans is a translational approach to analyze the effects of acute systemic inflammation and sickness behavior. Although studies documented that LPS-induced inflammation can alter social behavior, its impact on empathy remains poorly understood. In this double-blind, placebo-controlled study, 52 healthy female volunteers received an intravenous injection of either LPS (0.4 ng/kg body weight) or placebo and completed the Social Interaction Empathy Task (SIET) two hours after injection. Physiological responses (blood pressure, heart rate, body temperature, cytokines, cortisol) were analyzed along with sickness symptoms and mood before and after LPS or placebo administration. LPS application led to significant increases in plasma cytokines and sickness symptoms as well as low mood. Moreover, volunteers receiving LPS showed significantly less empathy for other’s psychological pain than those who received placebo. Furthermore, LPS-injected volunteers with more severe sickness symptoms displayed higher pain ratings in the first-person perspective. Thus, low-grade inflammation reduces empathy for other’s psychological pain which might reflect an adaptive strategy to save energy by not responding empathetically when sick oneself.

PSV-5 The Effect of a Prenatal Immune Challenge on Offspring Skeletal Muscle Characteristics in the Longissimus Muscle of Weaned Pigs

Abstract An insult to the health status of an animal is associated with decreased skeletal muscle growth. Furthermore, an immune challenge to a gestating dam may inhibit pre- and postnatal muscle growth of the offspring. The objective of this study was to determine if the administration of low-dose lipopolysaccharide (LPS) to gestating sows would alter muscle characteristics of the longissimus muscle (LM) in weaned pigs exposed to LPS. Pregnant Camborough sows were randomly assigned to receive LPS (LPS; n = 7) at a dose of 2.5 μg/kg BW or saline (CON; n= 7) on 78 ± 1.8 d of gestation. At weaning (21 ± 1.3 d of age), barrows (CON n = 17; LPS n = 17) from each sow received a secondary LPS challenge. Following the postnatal challenge, barrows were euthanized (31 ± 1.3 d of age), and both LM were collected. The left LM was weighed, measured for length, and measured for circumference at the cranial, middle, and caudal portion of the muscle. Two samples from the right LM were preserved for histological analyses. Morphometric, fiber type count, primary myofiber clustering, and satellite cell counts were analyzed using the MIXED procedure of SAS 9.4 with a main effect of prenatal treatment and covariate of body weight. There were no differences (P > 0.248) between any morphometric measures. The average diameter for myosin heavy chain (MHC) type I and IIB/X fibers was increased (P < 0.048) 8.6 and 5.9% respectively in LPS offspring compared with CON offspring. Average cross-sectional area (CSA) was increased 10.6% (P = 0.030) in MHC IIB/X fibers and tended to be increased 14.9% (P = 0.080) in MHC 1 fibers of LPS offspring. Number of myofibers within the LM was increased 16.1% (P = 0.032) in CON compared with LPS offspring. Diameter, CSA, and total CSA of oxidative myofibers (MHC type I and IIA) clusters was increased (P < 0.001) 13.8, 27.6, and 22.3% respectively in LPS compared with CON offspring. There were no differences (P > 0.186) between treatment groups for total nuclei or nuclei positive for MYF5, PAX7, or MYF5 and PAX7 nuclei. Based on the timing of the maternal challenge, it can be theorized that LPS hinders hyperplasia during secondary myogenesis following an immune challenge to the dam, with a rescue response of increased hypertrophy of MHC I and IIB/X myofibers post-weaning and glycolytic myofiber profile. However, due to the reduction in total myofibers, the impact of LPS on parameters such as average daily gain, feed efficiency, and lean skeletal muscle accretion should be studied further.

Utility of In Vitro Cellular Models of Low-Dose Lipopolysaccharide in Elucidating the Mechanisms of Anti-Inflammatory and Wound-Healing-Promoting Effects of Lipopolysaccharide Administration In Vivo.

Lipopolysaccharide (LPS) is a bacterial component that activates intracellular signaling pathways upon binding to the Toll-like receptor (TLR)-4/MD-2 complex. It is well known that LPS injected into animals and high-dose (100 ng/mL to 1 μg/mL) LPS treatment to innate immune cells induce an inflammatory response. In contrast, LPS is naturally present in the gastrointestinal tract, respiratory tract, and skin of humans and animals, and it has been shown that TLR-4-deficient animals cannot maintain their immune balance and gut homeostasis. LPS from commensal bacteria can help maintain homeostasis against mucosal stimulation in healthy individuals. Oral LPS administration has been shown to be effective in preventing allergic and lifestyle-related diseases. However, this effect was not observed after treatment with LPS at high doses. In mice, oral LPS administration resulted in the detection of LPS at a low concentration in the peritoneal fluid. Therefore, LPS administered at low and high doses have different effects. Moreover, the results of in vitro experiments using low-dose LPS may reflect the effects of oral LPS administration. This review summarizes the utility of in vitro models using cells stimulated with LPS at low concentrations (50 pg/mL to 50 ng/mL) in elucidating the mechanisms of oral LPS administration. Low-dose LPS administration has been demonstrated to suppress the upregulation of proinflammatory cytokines and promote wound healing, suggesting that LPS is a potential agent that can be used for the treatment and prevention of lifestyle-related diseases.

Impact of Senolytic Treatment on Gene Expression in Aged Lung.

Cellular senescence plays a key role in mediating tissue remodeling and modulation of host responses to pathogenic stimuli. Our current study was designed to gain a better understanding of the impact of short-term senolytic treatment or inflammatory stimulation on lung senescence. The results of our study demonstrate that short term treatment of aged adult mice (20 months of age) with senolytics, quercetin, and dasatinib decreases p16 and p21 expression in lung tissue. Short-term treatment with senolytics also significantly improved the expression of genes associated with genomic instability, telomere attrition, mitochondrial dysfunction, DNA binding, and the inflammatory response. In contrast, in response to low-dose LPS administration, there was increased expression of genes associated with genomic instability, mitochondrial dysfunction, and heightened inflammatory responses in young adult murine lung (3 months of age). Taken together, the results of our current study illustrate the efficacy of senolytic treatment on modulating responses in aged lung and the potential role of chronic low dose inflammation on senescence induction in the lung.

Glial functions in the blood-brain communication at the circumventricular organs.

The circumventricular organs (CVOs) are located around the brain ventricles, lack a blood-brain barrier (BBB) and sense blood-derived molecules. This review discusses recent advances in the importance of CVO functions, especially glial cells transferring periphery inflammation signals to the brain. The CVOs show size-limited vascular permeability, allowing the passage of molecules with molecular weight <10,000. This indicates that the lack of an endothelial cell barrier does not mean the free movement of blood-derived molecules into the CVO parenchyma. Astrocytes and tanycytes constitute a dense barrier at the distal CVO subdivision, preventing the free diffusion of blood-derived molecules into neighboring brain regions. Tanycytes in the CVOs mediate communication between cerebrospinal fluid and brain parenchyma via transcytosis. Microglia and macrophages of the CVOs are essential for transmitting peripheral information to other brain regions via toll-like receptor 2 (TLR2). Inhibition of TLR2 signaling or depletion of microglia and macrophages in the brain eliminates TLR2-dependent inflammatory responses. In contrast to TLR2, astrocytes and tanycytes in the CVOs of the brain are crucial for initiating lipopolysaccharide (LPS)-induced inflammatory responses via TLR4. Depletion of microglia and macrophages augments LPS-induced fever and chronic sickness responses. Microglia and macrophages in the CVOs are continuously activated, even under normal physiological conditions, as they exhibit activated morphology and express the M1/M2 marker proteins. Moreover, the microglial proliferation occurs in various regions, such as the hypothalamus, medulla oblongata, and telencephalon, with a marked increase in the CVOs, due to low-dose LPS administration, and after high-dose LPS administration, proliferation is seen in most brain regions, except for the cerebral cortex and hippocampus. A transient increase in the microglial population is beneficial during LPS-induced inflammation for attenuating sickness response. Transient receptor potential receptor vanilloid 1 expressed in astrocytes and tanycytes of the CVOs is responsible for thermoregulation upon exposure to a warm environment less than 37°C. Alternatively, Nax expressed in astrocytes and tanycytes of the CVOs is crucial for maintaining body fluid homeostasis. Thus, recent findings indicate that glial cells in the brain CVOs are essential for initiating neuroinflammatory responses and maintaining body fluid and thermal homeostasis.

Podocyte-specific deletion of miR-146a increases podocyte injury and diabetic kidney disease.

Diabetic glomerular injury is a major complication of diabetes mellitus and is the leading cause of end stage renal disease (ESRD). Healthy podocytes are essential for glomerular function and health. Injury or loss of these cells results in increased proteinuria and kidney dysfunction and is a common finding in various glomerulopathies. Thus, mechanistic understanding of pathways that protect podocytes from damage are essential for development of future therapeutics. MicroRNA-146a (miR-146a) is a negative regulator of inflammation and is highly expressed in myeloid cells and podocytes. We previously reported that miR-146a levels are significantly reduced in the glomeruli of patients with diabetic nephropathy (DN). Here we report generation of mice with selective deletion of miR-146a in podocytes and use of these mice in models of glomerular injury. Induction of glomerular injury in C57BL/6 wildtype mice (WT) and podocyte-specific miR-146a knockout (Pod-miR146a–/–) animals via administration of low-dose lipopolysaccharide (LPS) or nephrotoxic serum (NTS) resulted in increased proteinuria in the knockout mice, suggesting that podocyte-expressed miR-146a protects these cells, and thus glomeruli, from damage. Furthermore, induction of hyperglycemia using streptozotocin (STZ) also resulted in an accelerated development of glomerulopathy and a rapid increase in proteinuria in the knockout animals, as compared to the WT animals, further confirming the protective role of podocyte-expressed miR-146a. We also confirmed that the direct miR-146a target, ErbB4, was significantly upregulated in the diseased glomeruli and erlotinib, an ErbB4 and EGFR inhibitor, reducedits upregulation and the proteinuria in treated animals. Primary miR146–/– podocytes from these animals also showed a basally upregulated TGFβ-Smad3 signaling in vitro. Taken together, this study shows that podocyte-specific miR-146a is imperative for protecting podocytes from glomerular damage, via modulation of ErbB4/EGFR, TGFβ, and linked downstream signaling.

Role of TRPM8 in switching between fever and hypothermia in adult mice during endotoxin-induced inflammation

Transient receptor potential melastatin 8 (TRPM8) functions in the sensing of noxious and innocuous colds; however, its significance in pathogen-induced thermoregulation remains unclear. In the present study, we investigated the role of TRPM8 in the regulation of endotoxin-induced body temperature control. The peripheral administration of low-dose lipopolysaccharide (LPS) at 50 ​μg/kg generated fever in wild-type (WT) mice, whereas it caused hypothermia in TRPM8 knockout (KO) animals. LPS-induced sickness responses such as decrease in body weight, and food and water intake were not different between WT and TRPM8 KO mice. TRPM8 KO mice exhibited more severe hypothermia and lower locomotor activity following the peripheral administration of high-dose LPS at 5 ​mg/kg compared with WT ones. An intracerebroventricular (i.c.v.) injection of either LPS at 3.6 ​μg/kg or interleukin-1β at 400 ​ng/kg elicited hypothermia in TRPM8 KO mice, in contrast to fever in WT animals. The peripheral administration of zymosan at 3 ​mg/kg also induced hypothermia in contrast to fever in WT mice. An i.c.v. injection of prostaglandin E2 at 16 or 160 ​nmol/kg induced normal fever in both WT and TRPM8 KO mice. Infrared thermography showed significant decline of the interscapular skin temperature that estimates temperature of the brown adipose tissue, regardless of no alteration of its temperature in WT animals. Fos immunohistochemistry showed stronger Fos activation of hypothalamic thermoregulation-associated nuclei in TRPM8 KO mice compared with WT animals following the peripheral administration of low-dose LPS. Therefore, the present study indicates that TRPM8 is necessary for switching between fever and hypothermia during endotoxin-induced inflammation.

Mid-gestation low-dose LPS administration results in female-specific excessive weight gain upon a western style diet in mouse offspring

Gestational complications, including preeclampsia and gestational diabetes, have long-term adverse consequences for offspring’s metabolic and cardiovascular health. A low-grade systemic inflammatory response is likely mediating this. Here, we examine the consequences of LPS-induced gestational inflammation on offspring’s health in adulthood. LPS was administered to pregnant C57Bl/6J mice on gestational day 10.5. Maternal plasma metabolomics showed oxidative stress, remaining for at least 5 days after LPS administration, likely mediating the consequences for the offspring. From weaning on, all offspring was fed a control diet; from 12 to 24 weeks of age, half of the offspring received a western-style diet (WSD). The combination of LPS-exposure and WSD resulted in hyperphagia and increased body weight and body fat mass in the female offspring. This was accompanied by changes in glucose tolerance, leptin and insulin levels and gene expression in liver and adipose tissue. In the hypothalamus, expression of genes involved in food intake regulation was slightly changed. We speculate that altered food intake behaviour is a result of dysregulation of hypothalamic signalling. Our results add to understanding of how maternal inflammation can mediate long-term health consequences for the offspring. This is relevant to many gestational complications with a pro-inflammatory reaction in place.

Perinatal Breathing Patterns and Survival in Mice Born Prematurely and at Term.

Infants born prematurely, often associated with maternal infection, frequently exhibit breathing instabilities that require resuscitation. We hypothesized that breathing patterns during the first hour of life would be predictive of survival in an animal model of prematurity. Using plethysmography, we measured breathing patterns during the first hour after birth in mice born at term (Term 19.5), delivered prematurely on gestational day 18.5 following administration of low-dose lipopolysaccharide (LPS; 0.14 mg/kg) to pregnant dams (LPS 18.5), or delivered on gestational day 18.7 or 17.5 by caesarian section (C-S 18.5 and C-S 17.5, respectively). Our experimental approach allowed us to dissociate effects caused by inflammation, from effects due to premature birth in the absence of an inflammatory response. C-S 17.5 mice did not survive, whereas mortality was not increased in C-S 18.5 mice. However, in premature pups born at the same gestational age (day 18.5) in response to maternal LPS injection, mortality was significantly increased. Overall, mice that survived had higher birth weights and showed eupneic or gasping activity that was able to transition to normal breathing. Some mice also exhibited a “saw tooth” breathing pattern that was able to transition into eupnea during the first hour of life. In contrast, mice that did not survive showed distinct, large amplitude, long-lasting breaths that occurred at low frequency and did not transition into eupnea. This breathing pattern was only observed during the first hour of life and was more prevalent in LPS 18.5 and C-S 18.5 mice. Indeed, breath tidal volumes were higher in inflammation-induced premature pups than in pups delivered via C-section at equivalent gestational ages, whereas breathing frequencies were low in both LPS-induced and C-section-induced premature pups. We conclude that a breathing pattern characterized by low frequency and large tidal volume is a predictor for the failure to survive, and that these characteristics are more often seen when prematurity occurs in the context of maternal inflammation. Further insights into the mechanisms that generate these breathing patterns and how they transition to normal breathing may facilitate development of novel strategies to manage premature birth in humans.

Systemic inflammation induced by lipopolysaccharide aggravates inherited retinal dystrophy

Retinal neurodegenerative diseases involve a scenario of inflammation and cell death that leads to morphological alterations and visual impairment. Non-ocular inflammatory processes could affect neurodegenerative retinal disorders and their progression, at least in part by activating microglial cells and releasing pro-inflammatory cytokines. Our purpose was to study the consequences of a systemic inflammatory process in the progression of retinal degeneration in P23H rats, a retinitis pigmentosa (RP) model. In order to induce a mild chronic systemic inflammation, we administered low doses of lipopolysaccharide (LPS) from age P20 to P60 to dystrophic P23H rats and healthy SD rats. Visual responsiveness was assessed by electroretinography (ERG). The morphological state of the retinas was analyzed by fluorescent immunohistochemistry (IHC), evaluating the number, morphology, and connectivity of different neuronal populations by means of cell type-specific markers. Microglia density, distribution, and degree of activation were evaluated by IHC and flow cytometry. The expression levels of inflammation- and apoptosis-related genes were analyzed by qRT-PCR arrays. Low-dose LPS administration did not induce significant functional or morphological changes in the retina of SD rats, although at the molecular level, we detected expression changes in genes related to apoptosis. Otherwise, systemic injection of LPS into P23H rats induced a further deterioration in the ERG response, with greater loss of photoreceptors and worsening of synaptic connectivity, accompanied by increasing numbers of microglial cells, which also showed a more intense activation state. Several inflammation- and apoptosis-related genes were upregulated. Our results indicate that chronic exacerbation of the inflammatory response in response to LPS accelerates neurodegeneration in dystrophic P23H rats, suggesting that in patients with ocular neurodegenerative diseases, peripheral damage, as a systemic infection or chronic inflammatory process, could accelerate disease progression, and should be taken into account in order to select an appropriate therapy to revert, block or slow-down the degenerative process.

Inflammation-induced fetal growth restriction in rats is associated with increased placental HIF-1α accumulation.

IntroductionHypoxia-inducible factor 1-alpha (HIF-1α) is the oxygen-sensitive subunit of the transcription factor HIF-1, and its expression is increased in placentas from pregnancies complicated by pre-eclampsia (PE). Fetal growth restriction (FGR) and PE often share a common pathophysiology; however, it is unknown whether increased placental HIF-1α occurs in FGR. We previously demonstrated that aberrant maternal inflammation in rats resulted in altered utero-placental perfusion and FGR, both of which were prevented by administration of the nitric oxide mimetic glyceryl trinitrate (GTN). Our aim here was to determine whether abnormal maternal inflammation causing FGR is linked to placental HIF-1α accumulation and whether GTN administration could prevent increases in placental HIF-1α.MethodsLevels of inflammatory factors in maternal plasma were measured using a multiplex assay after an injection of low-dose lipopolysaccharide (LPS) to rats on gestational day (GD) 13.5. Following three additional daily LPS injections from GD14.5–16.5, GD17.5 placentas were harvested for HIF-1α immunolocalisation; serial sections were also stained for the hypoxia marker pimonidazole. A subset of rats received LPS injections along with GTN delivered continuously (25 μg/h via a transdermal patch) on GD12.5-GD17.5.ResultsWithin two hours of LPS administration, levels of maternal pro-inflammatory cytokines were increased compared with saline-treated controls. GD17.5 placentas of growth-restricted fetuses exhibited increased HIF-1α accumulation; however, this did not correlate with pimonidazole staining for which no differences were observed between groups. Furthermore, the LPS-mediated increases in maternal inflammatory cytokine levels and placental HIF-1α accumulation did not occur in rats treated with GTN.DiscussionOur results demonstrate that inflammation-induced FGR is associated with increased placental HIF-1α accumulation; however, expression of this transcription factor may not correlate with regions of hypoxia in late-gestation placentas. The GTN-mediated attenuation of placental HIF-1α accumulation in LPS-treated rats provides insight into the mechanism by which GTN improves inflammation-induced complications of pregnancy.

Spleen contributes significantly to increased circulating levels of fibroblast growth factor23 in response to lipopolysaccharide-induced inflammation.

Background: Circulating levels of fibroblast growth factor 23 (FGF23) increase progressively and correlate with systemic inflammation in chronic kidney disease (CKD). The aim of this study was to identify and characterize the causal relationship between FGF23 and inflammation in CKD. Methods: Circulating FGF23 and inflammatory cytokines were correlated in healthy subjects and patients with varying levels of CKD. In addition, FGF23 expression in blood and solid organs was measured in normal mice that were exposed acutely (one time) or chronically (2-week) to low-dose lipopolysaccharide (LPS); chronic exposure being either sustained (subcutaneous pellets), intermittent (daily injections) or combined sustained plus acute (subcutaneous pellets plus acute injection on the day of sacrifice). Blood was analyzed for both terminal (cFGF23) and intact (iFGF23) FGF23 levels. Solid tissues were investigated with immunohistochemistry, enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction. Results: FGF23 levels correlated significantly with neutrophil gelatinase–associated lipocalin (r = 0.72, P < 0.001), C-reactive protein (r = 0.38, P < 0.001), tumor necrosis factor-α (r = 0.32, P = 0.001) and interleukin-6 (r = 0.48, P < 0.001). Acute LPS administration increased tissue FGF23 mRNA and plasma levels of cFGF23 but not iFGF23. Neither chronic sustained nor chronic pulsatile LPS increased the tissue or circulating levels of FGF23. However, acute on chronic LPS raised tissue FGF23 mRNA and both circulating cFG23 and iFGF23. Interestingly, the spleen was the major source of FGF23. Conclusion: Acute on chronic exposure to LPS stimulates FGF23 production in a normal mouse model of inflammation. We provide the first evidence that the spleen, under these conditions, contributes substantially to elevated circulating FGF23 levels.

Spleen contributes significantly to increased circulating levels of fibroblast growth factor 23 in response to lipopolysaccharide-induced inflammation

Circulating levels of fibroblast growth factor 23 (FGF23) increase progressively and correlate with systemic inflammation in chronic kidney disease (CKD). The aim of this study was to identify and characterize the causal relationship between FGF23 and inflammation in CKD. Circulating FGF23 and inflammatory cytokines were correlated in healthy subjects and patients with varying levels of CKD. In addition, FGF23 expression in blood and solid organs was measured in normal mice that were exposed acutely (one time) or chronically (2-week) to low-dose lipopolysaccharide (LPS); chronic exposure being either sustained (subcutaneous pellets), intermittent (daily injections) or combined sustained plus acute (subcutaneous pellets plus acute injection on the day of sacrifice). Blood was analyzed for both terminal (cFGF23) and intact (iFGF23) FGF23 levels. Solid tissues were investigated with immunohistochemistry, enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction. FGF23 levels correlated significantly with neutrophil gelatinase-associated lipocalin ( r = 0.72, P < 0.001), C-reactive protein ( r = 0.38, P < 0.001), tumor necrosis factor-α ( r = 0.32, P = 0.001) and interleukin-6 ( r = 0.48, P < 0.001). Acute LPS administration increased tissue FGF23 mRNA and plasma levels of cFGF23 but not iFGF23. Neither chronic sustained nor chronic pulsatile LPS increased the tissue or circulating levels of FGF23. However, acute on chronic LPS raised tissue FGF23 mRNA and both circulating cFG23 and iFGF23. Interestingly, the spleen was the major source of FGF23. Acute on chronic exposure to LPS stimulates FGF23 production in a normal mouse model of inflammation. We provide the first evidence that the spleen, under these conditions, contributes substantially to elevated circulating FGF23 levels.

A combined cumulative threshold spectra and digital reconstruction analysis reveal structural alterations of microglia within the prefrontal cortex following low-dose LPS administration

Sickness behaviors have become the focus of great interest in recent years as they represent a clear case of how peripheral disturbances in immune signaling can disrupt quite complex behaviors. In the current study, we were interested in examining whether we could identify any significant morphological disturbances in microglia associated with these sickness-like behaviors in adult male Sprague–Dawley rats. We chose lipopolysaccharide (LPS 100μg/kg/i.p.), to induce sickness-like behaviors as it is the most well-validated approach to do so in rodents and humans. We were particularly interested in examining changes in microglia within the prefrontal cortex (PFC) as several recent neuroimaging studies have highlighted significant functional changes in this region following peripheral LPS administration. Paraformaldehyde-fixed tissue was collected from animals 24h post LPS administration and labeled immunohistochemically with an antibody directed to bind to Iba-1, a protein known to be involved in the structural remodeling of microglia. To analyze changes, we have made use of two recently described image analysis procedures. The first is known as cumulative threshold spectra (CTS) analysis. The second involves the unsupervised digital reconstruction of microglia. We undertook these complementary analysis of microglial cells in the both the pre- and infralimbic divisions of the PFC. Our results indicated that microglial soma size was significantly enlarged, while cell processes had contracted slightly following LPS administration. To our knowledge this study is to first to definitely demonstrate substantial microglial disturbances within the PFC following LPS delivered at a dose that was sufficient to induce significant sickness-like behavior.

Inflammation-induced pain sensitization in men and women: does sex matter in experimental endotoxemia?

A role of the innate immune system is increasingly recognized as a mechanism contributing to pain sensitization. Experimental administration of the bacterial endotoxin lipopolysaccharide (LPS) constitutes a model to study inflammation-induced pain sensitization, but all existing human evidence comes from male participants. We assessed visceral and musculoskeletal pain sensitivity after low-dose LPS administration in healthy men and women to test the hypothesis that women show greater LPS-induced hyperalgesia compared with men. In this randomized, double-blind, placebo-controlled crossover study, healthy men (n = 20) and healthy women using oral contraceptives (n = 20) received an intravenous injection of 0.4 ng/kg body weight LPS or placebo. Pain sensitivity was assessed with established visceral and musculoskeletal pain models (ie, rectal pain thresholds; pressure pain thresholds for different muscle groups), together with a heartbeat perception (interoceptive accuracy) task. Plasma cytokines (tumor necrosis factor-α and interleukin-6) were measured along with state anxiety at baseline and up to 6-hour postinjection. Lipopolysaccharide application led to significant increases in plasma cytokines and state anxiety and decreased interoceptive awareness in men and women (P < 0.001, condition effects), with more pronounced LPS-induced cytokine increases in women (P < 0.05, interaction effects). Although both rectal and pressure pain thresholds were significantly decreased in the LPS condition (all P < 0.05, condition effect), no sex differences in endotoxin-induced sensitization were observed. In summary, LPS-induced systemic immune activation leads to visceral and musculoskeletal hyperalgesia, irrespective of biological sex. These findings support the broad applicability of experimental endotoxin administration as a translational preclinical model of inflammation-induced pain sensitization in both sexes.

Characterization of inflammation and immune cell modulation induced by low-dose LPS administration to healthy volunteers

Human in vivo models of systemic inflammation are used to study the physiological mechanisms of inflammation and the effect of drugs and nutrition on the immune response. Although in vivo lipopolysaccharide (LPS) challenges have been applied as methodological tool in clinical pharmacology studies, detailed information is desired on dose-response relationships, especially regarding LPS hyporesponsiveness observed after low-dose in vivo LPS administration. A study was performed to assess the in vivo inflammatory effects of low intravenous LPS doses, and to explore the duration of the induced LPS hyporesponsiveness assessed by subsequent ex vivo LPS challenges. This was a randomized, double-blind, placebo-controlled study with single ascending low doses of LPS (0.5, 1 and 2 ng/kg body weight) administered to healthy male volunteers (3 cohorts of 8 subjects, LPS:placebo 6:2). The in vivo inflammatory response was assessed by measurement of cytokines and CRP. Ex vivo LPS challenges were performed (at −2, 6, 12, 24, 48 and 72 hours relative to in vivo LPS administration) to estimate the duration and magnitude of LPS hyporesponsiveness by assessment of cytokine release (TNF-α, IL-1β, IL-6, IL-8). LPS administration dose-dependently increased body temperature (+1.5°C for 2 ng/kg LPS), heart rate (+28 bpm for 2 ng/kg LPS), CRP and circulating cytokines which showed clearly distinctive increases from placebo already at the lowest LPS dose level tested (0.5 ng/kg, contrast for timeframe 0–6 hours: TNF-α +413%, IL-6 +288%, IL-8 +254%; all p ≤ 0.0001). In vivo LPS administration dose-dependently induced a period of hyporesponsiveness in the ex vivo LPS-induced cytokine release (IL-1β, IL-6 and TNF-α), with maximal hyporesponsiveness observed at 6 hours, lasting no longer than 12 hours. For IL-6 and IL-8, indications for immune cell priming were observed. We demonstrated that an in vivo LPS challenge, with LPS doses as low as 0.5 ng/kg, elicits a cytokine response that is clearly distinctive from baseline cytokine levels. This study expanded the knowledge about the dose-effect relationship of LPS-induced hyporesponsiveness. As such, the low-dose LPS challenge has been demonstrated to be a feasible methodological tool for future clinical studies exploring pharmacological or nutritional immune-modulating effects.

Oral microbiome link to neurodegeneration in glaucoma.

BackgroundGlaucoma is a progressive optic nerve degenerative disease that often leads to blindness. Local inflammatory responses are implicated in the pathology of glaucoma. Although inflammatory episodes outside the CNS, such as those due to acute systemic infections, have been linked to central neurodegeneration, they do not appear to be relevant to glaucoma. Based on clinical observations, we hypothesized that chronic subclinical peripheral inflammation contributes to neurodegeneration in glaucoma.MethodsMouthwash specimens from patients with glaucoma and control subjects were analyzed for the amount of bacteria. To determine a possible pathogenic mechanism, low-dose subcutaneous lipopolysaccharide (LPS) was administered in two separate animal models of glaucoma. Glaucomatous neurodegeneration was assessed in the retina and optic nerve two months later. Changes in gene expression of toll-like receptor 4 (TLR4) signaling pathway and complement as well as changes in microglial numbers and morphology were analyzed in the retina and optic nerve. The effect of pharmacologic blockade of TLR4 with naloxone was determined.FindingsPatients with glaucoma had higher bacterial oral counts compared to control subjects (p<0.017). Low-dose LPS administration in glaucoma animal models resulted in enhancement of axonal degeneration and neuronal loss. Microglial activation in the optic nerve and retina as well as upregulation of TLR4 signaling and complement system were observed. Pharmacologic blockade of TLR4 partially ameliorated the enhanced damage.ConclusionsThe above findings suggest that the oral microbiome contributes to glaucoma pathophysiology. A plausible mechanism by which increased bacterial loads can lead to neurodegeneration is provided by experiments in animal models of the disease and involves activation of microglia in the retina and optic nerve, mediated through TLR4 signaling and complement upregulation. The finding that commensal bacteria may play a role in the development and/or progression of glaucomatous pathology may also be relevant to other chronic neurodegenerative disorders.

Systemic injection of low-dose lipopolysaccharide fails to break down the blood-brain barrier or activate the TLR4-MyD88 pathway in neonatal rat brain.

We aimed to investigate whether peripheral low-dose lipopolysaccharide (LPS) induces the breakdown of the blood–brain barrier (BBB) and/or the activation of toll-like receptor 4 (TLR4) in the neonatal rat brain. Neonatal rats received intraperitoneal injections of low-dose LPS (0.3 mg/kg∙bw), and the BBB compromise was detected by Evans Blue extravasation and electron microscopy. Meanwhile, TLR4, adaptin myeloid differentiation factor 88 (MyD88), nuclear transcription factor kappa-B (NF-κB) p50 and tumor necrosis factor alpha (TNFα) in the neonatal rat brain were determined by quantitative real-time polymerase chain reaction (PCR) and Western Blot. Immunohistochemistry was used to determine the distribution and activation of microglia in the brain after LPS administration. It was demonstrated that Evans Blue extravasation was not observed in the brain parenchyma, and that tight junctions of cerebral endothelial cells remained intact after systemic injections of LPS in neonatal rats. Although intracerebroventricular injections of LPS activated microglia and up-regulated the expression of TLR4, MyD88, NF-κB p50 and TNFα in the neonatal rat brain, systemic LPS did not induce these responses. These findings indicate that while the neonatal rat brain responds to the direct intra-cerebral administration of LPS through robust TLR4 activation, systemic low-dose LPS does not induce the innate immune reaction or compromise the BBB in neonatal rats.

Lipopolysaccharide preconditioning attenuates apoptotic processes and improves neuropathologic changes after spinal cord injury in rats

We have shown earlier that administration of low-dose lipopolysaccharide (LPS) significantly contributed to recovery of motor function after traumatic spinal cord injury in the adult female rat. Using the same standardized animal model, we have now designed a set of experiments to test the hypothesis that LPS preconditioning attenuates stress-related apoptotic processes early after spinal cord trauma. The lower thoracic spinal cord injury in adult female Sprague-Dawley rats was caused by a 10 g weight rod drop from 25 mm on the dural surface of the exposed spinal cord at T10. The rats were randomly assigned to three groups: Sham injury, control (received normal saline alone), and LPS preconditioning (0.2 mg/kg, ip; 72 h prior to the injury). The animals were euthanized at 72 h postinjury. Neuropathologic changes were assessed using hematoxylin and eosin staining. SCI-induced apoptosis were observed by transmission electron microscopy. Caspase-3, cleaved caspase-3, Bax, and Bcl-2 were examined with immunohistochemistry or Western blotting. Compared with the control group, LPS preconditioning group showed significant improvement in the SCI-induced morphology changes. Furthermore, LPS preconditioning reduced the expressions of apoptotic markers caspase-3, cleaved caspase-3, and Bax, upregulated the expression of antiapoptotic marker Bcl-2 in the samples of spinal cord. Low-dose LPS attenuated the recruitment of inflammatory cells and the proliferation of glial cells in the site of injury. LPS preconditioning has neuroprotective effects against TSCI in rats due to its antiapoptosis properties as shown by the inhibition of caspase pathway and the upregulation of antiapoptotic protein.

Temporal Metabolic Profiling of Plasma During Endotoxemia in Humans

Endotoxemia induced by the administration of low-dose lipopolysaccharide (LPS) to healthy human volunteers is a well-established experimental protocol and has served as a reproducible platform for investigating the responses to systemic inflammation. Because metabolic composition of a tissue or body fluid is uniquely altered by stimuli and provides information about the dominant regulatory mechanisms at various cellular processes, understanding the global metabolic response to systemic inflammation constitutes a major part in this investigation complementing the studies undertaken so far in both clinical and systems biology fields. This article communicates the first proof-of-principle metabonomic analysis, which comprised global biochemical profiles in human plasma samples from healthy subjects given intravenous endotoxin at 2 ng/kg. Concentrations of a total of 366 plasma biochemicals were determined in archived blood samples collected from 15 endotoxin-treated subjects at five time points within 24 h after treatment and compared with control samples collected from four saline-treated subjects. Principal component analysis within this data set determined the sixth hour as a critical time point separating development and recovery phases of the LPS-induced metabolic changes. Consensus clustering of the differential metabolites identified two distinct subsets of metabolites that displayed common coherent profiles with opposing directionality. The first group of metabolites, which were mostly associated with pathways related to lipid metabolism, was upregulated within the first 6 h and downregulated by the 24th hour following LPS administration. The second group of metabolites, in contrast, was first downregulated until the sixth hour, then upregulated. Metabolites in this group were predominantly amino acids or their derivatives. In summary, nontargeted biochemical profiling and unsupervised multivariate analyses highlighted the prominent roles of lipid and protein metabolism in regulating the response to systemic inflammation while also revealing their dynamics in opposite directions.