Injection Of Low-dose Lipopolysaccharide Research Articles

Central innate immunization induces tolerance against post-traumatic stress disorder-like behavior and neuroinflammatory responses in male mice

Post-traumatic stress disorder (PTSD) is a severe psychiatric disorder associated with abnormally elevated neuroinflammatory responses. Suppression of neuroinflammation is considered to be effective in ameliorating PTSD-like behaviors in rodents. Since pre-stimulation of microglia prior to stress exposure can prevent neuroinflammation, we hypothesized that pre-stimulation of microglia may prevent PTSD in animals. The results show that a single injection of a classical immune stimulant, lipopolysaccharide (LPS), at 50, 100 or 500, but not 10 μg/kg, one day before stress exposure, prevented the anxiety- and fear-like behaviors induced by modified single prolonged stress (mSPS). The time-dependent analysis shows that a single injection of LPS (100 μg/kg) either one or five, but not ten, days before stress prevented mSPS-induced anxiety- and fear-like behaviors. A second low-dose LPS injection 10 days after the first injection or a repeated LPS injection (4 × ) 10 days before stress induced tolerance to mSPS. Mechanistic studies show that a single injection of LPS one day before stress stimulation prevented mSPS-induced increases in levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6 mRNA in the hippocampus and medial prefrontal cortex. Inhibition of microglia by pretreatment with minocycline or depletion of microglia by PLX3397 abolished the preventive effect of low-dose LPS pre-injection on mSPS-induced anxiety- and fear-like behavior and neuroinflammatory responses. These results suggest that pre-stimulation of microglia may prevent the development of PTSD-like behaviors by attenuating the development of neuroinflammatory responses. This could help to develop new strategies to prevent the damaging effects of harmful stress on the brain.

LECT2 Deletion Exacerbates Liver Steatosis and Macrophage Infiltration in a Male Mouse Model of LPS-mediated NASH.

Leukocyte cell-derived chemotaxin 2 (LECT2) is a protein initially isolated as a neutrophil chemotactic factor. We previously found that LECT2 is an obesity-associated hepatokine that senses liver fat and induces skeletal muscle insulin resistance. In addition, hepatocyte-derived LECT2 activates macrophage proinflammatory activity by reinforcing the lipopolysaccharide (LPS)-induced c-Jun N-terminal kinase signaling. Based on these findings, we examined the effect of LECT2 deletion on nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) caused by bacterial translocation. We created the bacterial translocation-mediated NAFLD/NASH model using LECT2 knockout mice (LECT2 KO) with 28 times a low-dose LPS injection under high-fat diet feeding conditions. LECT2 deletion exacerbated steatosis and significantly reduced p38 phosphorylation in the liver. In addition, LECT2 deletion increased macrophage infiltration with decreased M1/M2 ratios. LECT2 might contribute to protecting against lipid accumulation and macrophage activation in the liver under pathological conditions, which might be accomplished via p38 phosphorylation. This study provides novel aspects of LECT2 in the bacterial translocation-mediated NAFLD/NASH model.

Mitochondrial dysfunction precedes hippocampal IL-1β transcription and cognitive impairments after low-dose lipopolysaccharide injection in aged mice

Acute cognitive impairments termed delirium often occur after inflammatory insults in elderly patients. While previous preclinical studies suggest mitochondria as a target for reducing neuroinflammation and cognitive impairments after LPS injection, fewer studies have evaluated the effects of a low-grade systemic inflammation in the aged brain. Thus, to identify the significance of mitochondrial dysfunction after a clinically relevant systemic inflammatory stimulus, we injected old-aged mice (18–20 months) with low-dose lipopolysaccharide (LPS, 0.04 mg/kg). LPS injection reduced mitochondrial respiration in the hippocampus 24 h after injection (respiratory control ratio [RCR], state3u/state4o; control = 2.82 ± 0.19, LPS = 2.57 ± 0.08). However, gene expression of the pro-inflammatory cytokine IL-1β was increased (RT-PCR, control = 1.00 ± 0.30; LPS = 2.01 ± 0.67) at a more delayed time point, 48 h after LPS injection. Such changes were associated with cognitive impairments in the Barnes maze and fear chamber tests. Notably, young mice were unaffected by low-dose LPS, suggesting that mitochondrial dysfunction precedes neuroinflammation and cognitive decline in elderly patients following a low-grade systemic insult. Our findings highlight mitochondria as a potential therapeutic target for reducing delirium in elderly patients.

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.

A Translational Model for Repeated Episodes of Joint Inflammation: Welfare, Clinical and Synovial Fluid Biomarker Assessment.

This study investigates repeated low-dose lipopolysaccharide (LPS) injections in equine joints as a model for recurrent joint inflammation and its impact on animal welfare. Joint inflammation was induced in eight horses by injecting 0.25 ng of LPS three times at two-week intervals. Welfare scores and clinical parameters were recorded at baseline and over 168 h post-injection. Serial synoviocentesis was performed for the analysis of a panel of synovial fluid biomarkers of inflammation and cartilage turnover. Clinical parameters and a final synoviocentesis were also performed eight weeks after the last sampling point to assess the recovery of normal joint homeostasis. Statistical methods were used to compare the magnitude of response to each of the 3 LPS inductions and to compare the baseline and final measurements. Each LPS injection produced consistent clinical and biomarker responses, with minimal changes in welfare scores. General matrix metalloproteinase (MMP) activity and joint circumference showed greater response to the second LPS induction, but response to the third was comparable to the first. Gylcosaminoglycans (GAG) levels showed a significantly decreased response with each induction, while collagen-cleavage neoepitope of type II collagen (C2C) and carboxypropetide of type II collagen epitope (CPII) showed quicker responses to the second and third inductions. All parameters were comparable to baseline values at the final timepoint. In conclusion, a consistent, reliable intra-articular inflammatory response can be achieved with repeated injections of 0.25 ng LPS, with minimal impact on animal welfare, suggesting potential as a refined translational model of recurrent joint inflammation.

Low-dose lipopolysaccharide inducing continuous and obvious increase in urinary protein in hyperglycemic rats and the underlying mechanism.

Proteinuria is an important hallmark of diabetic nephropathy models, however it takes a long time for the proteinuria and is not stable. Therefore, low-dose lipopolysaccharide (LPS) was investigated in this work to induce rapid and stable proteinuria in hyperglycemic rats and the underlying mechanism was studied. Hyperglycemia rats was induced by high-fat feeding combined with intraperitoneal injection of streptozotocin (STZ). After 21 days, the model rats received a subinjury dose of 0.8 mg / kg LPS intraperitoneally (i.p.). We detected related biochemical indexes at different time periods after LPS injection and examined the expression of glomerular podocyte-associated proteins. Simultaneously, we measured expression of inflammatory factors, apoptotic proteins and albumin (ALB) in the renal cortex and renal medulla, respectively. PAS (Periodic Acid Schiff) staining was used to observe renal pathology. After LPS injection, urinary microalbumin (umALB) increased significantly and lasted longer. The expression of Nephrin, Podocin and necroptosis factor kappa B (NF-κB) in rennal cortex and Interleukin 18 (IL-18), Caspase-1, NF-κB and ALB in the renal medulla was significantly changed. Pathologically, the glomerular basement membrane was observed to be significantly thickened, the renal tubules were dilated, and the epithelial cells fell off in a circle. LPS promoted the continuous increase in urinary microalbumin in hyperglycemic rats, which was related to the damage to the glomerular basement membrane and renal tubular epithelial cells and to the inflammatory reaction in the kidney involved in NF-κB signaling, and this pathological damage can help to establish a stable model of diabetic nephropathy with increased proteinuria.

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.

201 Effect of mild restraint stress on lipopolysaccharide-induced preterm labor in murine model

Prenatal stress leads to a pro-inflammatory state that may prime the immune system to respond aggressively to bacterial stimuli. This robust inflammatory response may be a mechanism for preterm delivery in individuals with high levels of chronic stress. The objective of this study was to assess the impact of antenatal stress on the responsivity of mice to lipopolysaccharide (LPS)-induced preterm labor. Nulliparous mice (n=39) underwent timed breeding at 8-10 weeks of age. Dams were then randomly assigned to either stress or control groups. The mice assigned to stress underwent daily 2-hour periods of restraint in a ventilated clear 50-mL conical centrifuge tube from E1-E15. On E15.5, mice from each group were randomly assigned to a 150-microliter intraperitoneal injection with either 15 micrograms LPS (E. coli O111:B4) or phosphate buffered saline (PBS). This low dose of LPS typically induces preterm labor in about 50% of mice. Mice were monitored in individual cages for the next 48 hours. The primary outcome was the number of dams exhibiting preterm labor (defined as bleeding or delivery of at least 1 fetus) within 48 hours. Mice were then sacrificed and uterine gross pathology inspected. Secondary outcomes were maternal death and presence of resorbed fetuses in the uteri. Outcomes were compared with Fisher’s Exact Test with a significant p-value of .05. In total, 10/10 of the mice exposed to antenatal stress and 7/12 of the control mice demonstrated preterm labor when exposed to low-dose LPS (Figure 1). Fisher’s Exact Test showed the rate of preterm labor after low-dose LPS injection was significantly higher in mice exposed to antenatal stress (p=.04). There were no maternal deaths observed. Two mice in the control-PBS group demonstrated resorbed fetuses. None of the other groups demonstrated resorbed fetuses (p=.52). Chronic mild restraint stress increases the sensitivity of mice to low-dose LPS-induced preterm labor. These data provide insight into a potential cause of increased preterm delivery rates among populations experiencing high rates of chronic stress.

Developing a mouse model of acute encephalopathy using low-dose lipopolysaccharide injection and hyperthermia treatment.

Acute encephalopathy (AE), mainly reported in East Asia, is classified into four categories based on clinical and neuropathological findings. Among them, AE caused by cytokine storm is known as the severest clinical entity that causes cerebral edema with poor prognosis. Because suitable and convenient model animal of AE had not been developed, the treatment of patients with AE is not established. In the present study, we established a simple and convenient protocol to mimic AE due to cytokine storm. Our model animal should be useful to elucidate the pathogenesis of AE.

Systematic Injection of Low-Dose LPS Transiently Improves the Retina Function and Structure of aMouse Model of Geographic Atrophy.

Geographic atrophy (GA), the advanced form of AMD, has been linked to oxidative stress within the RPE and with low-grade inflammation. The RPE-specific Sod2 knockout mouse model of GA develops increase oxidative stress and slow retinal degeneration. Mice of the SOD2floxed::VMD2Cre+ genotype were injected subcutaneously with either saline or 3mg/kg of lipopolysaccharide (LPS) at 8weeks of age. Mice were evaluated by electroretinography (ERG) and spectral domain optical coherence tomography. Inflammatory cells within the retina were studied by CD45 immunofluorescence. Systemic low-dose LPS transiently, but significantly, improved both function and structure of RPE-specific Sod2 KO mice retina when compared to saline-injected mice. There was no difference in CD45 positive cells between saline and LPS treatment. Low-grade activation of the immune system leads to a preconditioning effect that transiently protects the retina of a mouse model of geographic atrophy.

Saturated fatty acid combined with lipopolysaccharide stimulates a strong inflammatory response in hepatocytes in vivo and in vitro.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and consumption of high-fat diet (HFD) is a risk factor for NAFLD. The HFD not only increases intake of saturated fatty acid (SFA) but also induces metabolic endotoxemia, an HFD-associated increase in circulating lipopolysaccharide (LPS). Although it is known that SFA or LPS promote hepatic inflammation, a hallmark of NAFLD, it remains unclear how SFA in combination with LPS stimulates host inflammatory response in hepatocytes. In this study, we performed both in vivo and in vitro experiments to investigate the effect of SFA in combination with LPS on proinflammatory gene expression in hepatocytes. Our animal study showed that feeding low-density lipoprotein-deficient mice HFD enriched with SFA and injection of low-dose LPS cooperatively stimulated IL-6 expression in livers. To understand how SFA and LPS interact to promote IL-6 expression, our in vitro studies showed that palmitic acid (PA), a major SFA, and LPS exerted synergistic effect on the expression of IL-6 in hepatocytes. Furthermore, coculture of hepatocytes with macrophages resulted in a greater IL-6 expression than culture of hepatocytes without macrophages in response to the combination of PA and LPS. Finally, we observed that LPS and PA increased ceramide production by cooperatively stimulating ceramide de novo synthesis, which played an essential role in the synergistic stimulation of proinflammatory gene expression by LPS and PA. Taken together, this study showed that SFA in combination with LPS stimulated a strong inflammatory response in hepatocytes in vivo and in vitro.

Endogenous Protection from Ischemic Brain Injury by Preconditioned Monocytes

Exposure to low-dose lipopolysaccharide (LPS) before cerebral ischemia is neuroprotective in stroke models, a phenomenon termed preconditioning (PC). Although it is well established that LPS-PC induces central and peripheral immune responses, the cellular mechanisms modulating ischemic injury remain unclear. Here, we investigated the role of immune cells in the brain protection afforded by PC and tested whether monocytes may be reprogrammed by ex vivo LPS exposure, thus modulating inflammatory injury after cerebral ischemia in male mice. We found that systemic injection of low-dose LPS induces a Ly6Chi monocyte response that protects the brain after transient middle cerebral artery occlusion (MCAO) in mice. Remarkably, adoptive transfer of monocytes isolated from preconditioned mice into naive mice 7 h after transient MCAO reduced brain injury. Gene expression and functional studies showed that IL-10, inducible nitric oxide synthase, and CCR2 in monocytes are essential for neuroprotection. This protective activity was elicited even if mouse or human monocytes were exposed ex vivo to LPS and then injected into male mice after stroke. Cell-tracking studies showed that protective monocytes are mobilized from the spleen and reach the brain and meninges, where they suppress postischemic inflammation and neutrophil influx into the brain parenchyma. Our findings unveil a previously unrecognized subpopulation of splenic monocytes capable of protecting the brain with an extended therapeutic window and provide the rationale for cell therapies based on the delivery of autologous or allogeneic protective monocytes in patients after ischemic stroke.SIGNIFICANCE STATEMENT Inflammation is a key component of the pathophysiology of the brain in stroke, a leading cause of death and disability with limited therapeutic options. Here, we investigate endogenous mechanisms of protection against cerebral ischemia. Using lipopolysaccharide (LPS) preconditioning (PC) as an approach to induce ischemic tolerance in mice, we found generation of neuroprotective monocytes within the spleen, from which they traffic to the brain and meninges, suppressing postischemic inflammation. Importantly, systemic LPS-PC can be mimicked by adoptive transfer of in vitro-preconditioned mouse or human monocytes at translational relevant time points after stroke. This model of neuroprotection may facilitate clinical efforts to increase the efficacy of BM mononuclear cell treatments in acute neurological diseases such as cerebral ischemia.

Angiotensin receptor-binding molecule in leukocytes in association with the systemic and leukocyte inflammatory profile

Background and aimsThe components of the renin-angiotensin system in leukocytes is involved in the pathophysiology of non-communicable diseases (NCDs), including hypertension, atherosclerosis and chronic kidney disease. Angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP) is an AT1R-specific binding protein, and is able to inhibit the pathological activation of AT1R signaling in certain animal models of NCDs. The aim of the present study was to investigate the expression and regulation of ATRAP in leukocytes. MethodsHuman leukocyte ATRAP mRNA was measured with droplet digital polymerase chain reaction system, and analyzed in relation to the clinical variables. We also examined the leukocyte cytokines mRNA in bone-marrow ATRAP-deficient and wild-type chimeric mice after injection of low-dose lipopolysaccharide. ResultsThe ATRAP mRNA was abundantly expressed in leukocytes, predominantly granulocytes and monocytes, of healthy subjects. In 86 outpatients with NCDs, leukocyte ATRAP mRNA levels correlated positively with granulocyte and monocyte counts and serum C-reactive protein levels. These positive relationships remained significant even after adjustment. Furthermore, the leukocyte ATRAP mRNA was significantly associated with the interleukin-1β, tumor necrosis factor-α and monocyte chemotactic protein-1 mRNA levels in leukocytes of NCDs patients. In addition, the leukocyte interleukin-1β mRNA level was significantly upregulated in bone marrow ATRAP-deficient chimeric mice in comparison to wild-type chimeric mice after injection of lipopolysaccharide. ConclusionsThese results suggest that leukocyte ATRAP is an emerging marker capable of reflecting the systemic and leukocyte inflammatory profile, and plays a role as an anti-inflammatory factor in the pathophysiology of NCDs.

Endotoxin tolerance induced by lipopolysaccharide preconditioning protects against surgery‑induced cognitive impairment in aging mice.

Postoperative cognitive dysfunction (POCD) is a clinical syndrome characterized by varying degrees of cognitive functional decline in patients following major surgery. Inflammation and a dysregulated innate immune system exert broad effects in the periphery and central nervous system, yet the mechanisms underlying POCD remain poorly understood and without effective therapy. It has been reported that modulation of the dysregulated inflammatory response with low‑dose lipopolysaccharide (LPS) preconditioning, a phenomenon additionally referred to as endotoxin tolerance, has the potential to reduce neuroinflammation, blood‑brain barrier disruption, and cognitive impairment in a number of disease states. Therefore, it was hypothesized that endotoxin tolerance induced by LPS preconditioning may protect against surgery‑induced cognitive impairment in aging mice. Using a mouse model of surgery‑induced cognitive decline, the present study demonstrated that exploratory laparotomy caused a significant impairment in hippocampal‑dependent memory. Notably, one application of low‑dose LPS preconditioning at 24h prior to surgery improved the cognitive impairment and abolished the signs of neuroinflammation in the hippocampus following surgery. However, LPS injection at 6h or immediately prior to surgery did not confer such beneficial effects, suggesting that the effects of LPS‑induced endotoxin tolerance may depend on the time of application. Inconclusion, the results of the present study suggested that low‑dose LPS preconditioning may markedly alleviate surgery‑induced neuroinflammation and cognitive impairment in aging mice, which may provide a novel approach to preventing POCD and, potentially, other forms of memory impairment.

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.

Osteopontin Is a Blood Biomarker for Microglial Activation and Brain Injury in Experimental Hypoxic-Ischemic Encephalopathy.

Clinical management of neonatal hypoxic-ischemic encephalopathy (HIE) suffers from the lack of reliable surrogate marker tests. Proteomic analysis may identify such biomarkers in blood, but there has been no proof-of-principle evidence to support this approach. Here we performed in-gel trypsin digestion of plasma proteins from four groups of 10-d-old mice [untouched and 24 h after low-dose lipopolysaccharide (LPS) exposure, hypoxia-ischemia (HI), or LPS/HI injury; n = 3 in each group) followed by liquid chromatography-tandem mass spectrometry and bioinformatics analysis to search for HI- and LPS/HI-associated brain injury biomarkers. This analysis suggested the induction of plasma osteopontin (OPN) by HI and LPS/HI, but not by sham and injury-free LPS exposure. Immunoblot confirmed post-HI induction of OPN protein in brain and blood, whereas Opn mRNA was induced in brain but not in blood. This disparity suggests brain-derived plasma OPN after HI injury. Similarly, immunostaining showed the expression of OPN by Iba1+ microglia/macrophages in HI-injured brains. Further, intracerebroventricular injection of LPS activated microglia and up-regulated plasma OPN protein. Importantly, the induction of plasma OPN after HI was greater than that of matrix metalloproteinase 9 or glial fibrillary acid protein. Plasma OPN levels at 48 h post-HI also parallel the severity of brain damage at 7-d recovery. Together, these results suggest that OPN may be a prognostic blood biomarker in HIE through monitoring brain microglial activation.

Metformin protects against infection-induced myocardial dysfunction

Background and PurposeMetformin administration is associated with myocardial protection during ischemia and/or reperfusion, possibly via inhibition of inflammatory responses in the heart. Exposure to pathogens, in addition to the activation of the immune system and the associated metabolic dysfunction, often results in compromised myocardial function. We examined whether metformin administration could maintain the normal myocardial function in experimental moderate Gram negative infection, induced by lipopolysaccharide (LPS) administration. Experimental Approach129xC57BL/6 mice were divided into control groups that received either vehicle or a single intraperitoneal (i.p.) injection of low dose LPS (5mg/kg body wt), and metformin treated groups that received either daily metformin (4mg/kg/animal) i.p. injections for five days prior to LPS administration [Experiment 1], or a single metformin injection following same dose of LPS [Experiment 2]. Key ResultsLPS alone caused cardiac dysfunction, as confirmed by echocardiography, whereas metformin administration, either before or after LPS, rescued myocardial function. LPS caused marked reduction of the cardiac metabolism-related genes tested, including Prkaa2, Cpt1b, Ppargc1a and Ppargc1b; reduction of fatty acid oxidation, as reflected by the regulation of Ppara, Acaca and Acacb; increased glucose transport, as shown by Slc2a4 levels; reduction of ATP synthesis; significant increase of inflammatory markers, in particular IL6; and reduction of autophagy. Pretreatment with metformin normalized the levels of all these factors. Conclusions and ImplicationsWe show for the first time that metformin protects the myocardium from LPS-associated myocardial dysfunction mainly by supporting its metabolic activity and allowing efficient energy utilization. Metformin can be a potential cardioprotective agent in individuals susceptible to exposure to pathogens.

Adiponectin inhibits mouse mammary tumor growth and reduced tumor-induced hematopoiesis

Adiponectin, one of the most abundant plasma protein produced in adipose tissue, has been shown to be decreased in the plasma of patients with various types of cancer suggesting that adiponectin may be involved in the pathogenesis of tumor progression. In addition, studies have shown that this adipokine exhibit a suppressive effect on different types of malignancy. However, the role of adiponectin in tumor progression or tumor immunity remains unknown. In this study, we assessed the effect of adiponectin on murine breast cancer progression. We implanted murine breast cancer cells (EMT6) breast cancer cells subcutaneously into the tight flank of syngeneic mice. Local administration of adiponectin to tumor tissue decreased tumor mass in a dose-dependent manner. Since intratumoral injection of lipopolysaccharide (LPS), the major component of endotoxin, is known to inhibit tumor growth, the observed anti-tumor effect of the recombinant adiponectin might be attributable to the contamination of low amounts of bacterial endotoxin. We showed that intratumoral injection of low dose LPS failed to inhibit tumor growth and heat inactivation or protease treatment completely abrogated the cytokine’s ability to induce IL-12p35 expression in mouse bone marrow-derived dendritic cells clearly demonstrating that the observed anti-tumor effect of recombinant adiponectin is not due to LPS contamination. Our data clearly suggest that adiponectin exerts anti-tumor effects in the breast cancer model of mice via reduction of tumor-induced hematopoiesis and intratumoral injection of adiponectin may be a new strategy for the treatment of established breast cancer.

Neural circuitry mediating inflammation-induced central pain amplification in human experimental endotoxemia.

To elucidate the brain mechanisms underlying inflammation-induced visceral hyperalgesia in humans, in this functional magnetic resonance imaging (fMRI) study we tested if intravenous administration of lipopolysaccharide (LPS) involves altered central processing of visceral pain stimuli. In this randomized, double-blind, placebo-controlled fMRI study, 26 healthy male subjects received either an intravenous injection of low-dose LPS (N=14, 0.4 ng/kg body weight) or placebo (N=12, control group). Plasma cytokines (TNF-α, IL-6), body temperature, plasma cortisol and mood were assessed at baseline and up to 6 h post-injection. At baseline and 2 h post-injection (test), rectal pain thresholds and painful rectal distension-induced blood oxygen level-dependent (BOLD) responses in brain regions-of-interest were assessed. To address specificity for visceral pain, BOLD responses to non-painful rectal distensions and painful somatic stimuli (i.e., punctuate mechanical stimulation) were also analyzed as control stimuli. Compared to the control group, LPS-treated subjects demonstrated significant and transient increases in TNF-α, IL-6, body temperature and cortisol, along with impaired mood. In response to LPS, rectal pain thresholds decreased in trend, along with enhanced up-regulation of rectal pain-induced BOLD responses within the posterior insula, dorsolateral prefrontal (DLPFC), anterior midcingulate (aMCC) and somatosensory cortices (all FWE-corrected p<0.05). Within the LPS group, more pronounced cytokine responses correlated significantly with enhanced rectal pain-induced neural activation in DLPFC and aMCC. No significant LPS effects were observed on neural responses to non-painful rectal distensions or mechanical stimulation. These findings support that peripheral inflammatory processes affect visceral pain thresholds and the central processing of sensory-discriminative aspects of visceral pain.

A static magnetic field attenuates lipopolysaccharide-induced neuro-inflammatory response via IL-6-mediated pathway

An effective method for controlling brain damage and neurodegeneration caused by inflammation remains elusive. Down-expression of the lipopolysaccharide (LPS)-induced inflammatory cytokines resulting in endotoxin tolerance is reported as an alternative anti-infection treatment. Nonetheless, because the dosage and action site are hard to control, endotoxin tolerance caused by low-dose LPS injection in brain tissue may induce side effects. The aim of this study was to test the hypothesis that static magnetic fields (SMF) stimulate endotoxin tolerance in brain tissue. In this study, survival rate and pathological changes in brain tissues of LPS-challenged mice were examined with and without SMF treatment. In addition, the effects of SMF exposure on growth rate and cytokine expression of LPS-challenged BV-2 microglia cells were monitored. Our results showed that SMF pre-exposure had positive effects on the survival rate and histological outcomes of LPS-treated mice. Furthermore, SMF exposure significantly decreased IL-6 expression in BV-2 cells (p < 0.05) by a phenomenon similar to endotoxin tolerance. We suggest that SMF has potential as an alternative simulation source for controlling LPS-induced excess neuro-inflammatory response.