Dose Of LPS Research Articles

Viral infection dampens human fetal membrane type I interferon responses triggered by bacterial LPS

The maternal-fetal interface possesses innate immune strategies to protect against infections. We previously reported that prior viral infection of human fetal membranes (FMs) in vitro and mouse FMs in vivo sensitized the tissue to low dose bacterial LPS leading to augmented inflammation. The objective of this study was to examine FM production of type I interferons (IFNs) and IFN-stimulated genes (ISGs) in the context of this polymicrobial model. Human FM explants and pregnant C57BL/6 mice were treated with or without low dose LPS following exposure to media or the γ-herpes virus, MHV-68. FM RNA was analyzed by qRT-PCR for type I IFNs, ISGs, upstream signaling, and MHV-68 open reading frames (ORFs). Pre-exposure to MHV-68 followed by LPS treatment inhibited the ability of LPS to induce human FM type I IFNs (IFNA, IFNB); ISGs (OAS, MxA, APOBEC3G) and upstream signaling mediators (RIG-I, TBK-1). Signaling mediators IRF-3 and IRF-7 were also reduced. In mouse FMs, pre-exposure to MHV-68 followed by LPS treatment reduced the ability of LPS to upregulate Ifna, Ifnb, Mxa, Irf7, and also reduced Irf3. MHV-68 infection of FMs induced ORF45 which targets IRF-7, and this was further augmented in response to a combination of MHV-68 and LPS. Together, these findings indicate that a viral infection blunts FM type I IFN production and signaling in response to LPS leading to a suppressed ISG response. Our studies suggest that a viral infection inhibits this protective FM response by negatively regulating IRF-7 through ORF45, leaving the maternal-fetal interface vulnerable to further viral attack.

Evaluation of the hepatotoxic potential of Tinospora crispa and its isolated borapetosides B, C and F in a murine model

The hepatotoxic potential of the methanolic extract of the stems of Tinospora crispa and of its furano-diterpenoid-borapetosides B, C, and F-was investigated in normal and health-compromised mice. Health-compromised condition was established by a single intraperitoneal injection of LPS (6 mg/kg). Two different sets of experiments were conducted to evaluate the hepatotoxic potential. A 21-day study where the mice were dosed with the extract of T. crispa (1 gm/kg b. wt./day) or standardized combination of borapetosides B, C and F (500 mg/kg b. wt.) with or without a single dose of LPS (1 gm/kg b. wt./day). In the acute toxicity study, mice were dosed with borapetosides B, C, and F (500 mg/kg b. wt.). Results showed that the ALT levels were normal and liver histopathology unaltered. No conclusive hepatotoxicity was observed in the methanolic extract or pure compounds tested under the given experimental conditions.

Prenatal low-dose endotoxin exposure prolongs intestinal epithelial activation after birth and contributes to necrotizing enterocolitis

PurposeTo investigate the effects of low dose endotoxin on transcriptional activity in intestinal epithelium, and its role in necrotizing enterocolitis (NEC). MethodsLipopolysaccharides (LPS) were injected into the amniotic cavity of pregnant mice under ultrasound guidance. The effects of LPS on fetal and neonatal intestines were determined. Mouse pups were exposed to low dose LPS (0.01 μg per fetus) prenatally and subjected to experimental NEC after birth. The incidence and severity of NEC, as well as intestinal permeability, NF-κB activation, and IL-6 expression were studied. The signaling pathways in the intestinal epithelial cells (IECs) that were activated by LPS were also investigated. ResultsLow dose LPS did not increase apoptosis, myeloperoxidase activity, histological injury or NF-κB activity in fetal intestines. However, prenatal low dose LPS exposure disturbed the transient and self-limited activation of NF-κB in neonatal intestines after birth. Importantly, it increased the incidence and severity of experimental NEC in neonatal mice. In primary IECs, low dose LPS induced IRAK-1 expression via activation of GSK3β. Elevated IRAK-1 levels prolonged the activation of IECs upon stimulation by high dose LPS. ConclusionPrenatal low dose endotoxin exposure disturbs self-limited postnatal epithelial cell activation and predisposes the neonatal intestine to NEC. Level of evidenceNot applicable (experimental animal study).

A novel role of NLRP3-generated IL-1\u03b2 in the acute-chronic transition of peripheral lipopolysaccharide-elicited neuroinflammation: implications for sepsis-associated neurodegeneration

BackgroundSepsis-associated acute brain inflammation, if unresolved, may cause chronic neuroinflammation and resultant neurodegenerative diseases. However, little is known how the transition from acute to chronic neuroinflammation, which is critical for the following progressive neurodegeneration, occurs in sepsis. The goal of this study was to investigate potential immune factors regulating the transition process using a widely used endotoxemia LPS mouse model. This model shows distinct acute and chronic phases of neuroinflammation and recapitulates many cardinal features of Parkinson’s disease, thus, providing a unique opportunity for studying phase transition of neuroinflammation.MethodsC57BL/6 J, NLRP3−/−, and IL-1R1−/− mice were employed. Mild and severe endotoxemia were produced by LPS ip injection at 1 or 5 mg/kg. Neuroinflammation in vitro and in vivo was assessed with proinflammatory cytokine expression by qPCR or ELISA and microglial activation by immunohistochemical analysis. Neurodegeneration was measured by manual and stereological counts of nigral dopaminergic neurons and immunohistochemical analysis of protein nitrosylation and α-synuclein phosphorylation.ResultsLPS-elicited initial increases in mouse brain mRNA levels of TNFα, IL-6, IL-1β, and MCP-1, and nigral microglial activation were not dose-related. By contrast, the delayed increase in brain mature IL-1β levels was dependent on LPS doses and protracted nigral microglial activation was only observed in high dose of LPS-treated mice. LPS-elicited increase in brain mature IL-1β but not IL-1α level was NLRP3-dependent. After high dose LPS treatment, deficiency of NLRP3 or IL-1R1 did not prevent the initiation of acute neuroinflammation but abolished chronic neuroinflammation. Genetic or pharmacological inhibition of the NLRP3-IL-1β axis repressed LPS-stimulated upregulation of chronic neuroinflammatory mediators including MHC-II, NOX2, and Mac1, and protected dopaminergic neurons. Ten months after LPS-elicited severe endotoxemia, nigral persisted microglial activation, elevated nitrosylated proteins and phosphorylated α-synuclein, and significant neuronal degeneration developed in wild-type mice but not in NLRP3−/− or IL-1R1−/− mice.ConclusionsThis study uncovers a novel role of the NLRP3-IL-1β signaling pathway in gauging the severity of sepsis-associated inflammation and determining whether acute neuroinflammation will resolve or transition to low grade chronic neuroinflammation. These findings also provide novel targets for developing therapy for severe systemic infection-related neurodegeneration.

Sequential ubiquitination of NLRP3 by RNF125 and Cbl-b limits inflammasome activation and endotoxemia.

Aberrant NLRP3 inflammasome activation contributes to the development of endotoxemia. The importance of negative regulation of NLRP3 inflammasomes remains poorly understood. Here, we show that the E3 ubiquitin ligase Cbl-b is essential for preventing endotoxemia induced by a sub-lethal dose of LPS via a caspase-11/NLRP3-dependent manner. Further studies show that NLRP3 undergoes both K63- and K48-linked polyubiquitination. Cbl-b binds to the K63-ubiquitin chains attached to the NLRP3 leucine-rich repeat domain (LRR) via its ubiquitin-associated region (UBA) and then targets NLRP3 at K496 for K48-linked ubiquitination and proteasome-mediated degradation. We also identify RNF125 as an additional E3 ubiquitin ligase that initiates K63-linked ubiquitination of the NLRP3 LRR domain. Therefore, NLRP3 is sequentially ubiquitinated by K63- and K48-linked ubiquitination, thus keeping the NLRP3 inflammasomes in check and restraining endotoxemia.

Semaphorin 3E Regulates the Response of Macrophages to Lipopolysaccharide-Induced Systemic Inflammation.

Semaphorin 3E (Sema3E) is a secreted protein that was initially discovered as a neuronal guidance cue. Recent evidence showed that Sema3E plays an essential role in regulating the activities of various immune cells. However, the exact role of Sema3E in macrophage function, particularly during inflammation, is not fully understood. We studied the impact of Sema3E gene deletion on macrophage function during the LPS-induced acute inflammatory response. We found that Sema3E-deficient (Sema3e-/- ) mice were better protected from LPS-induced acute inflammation as exemplified by their superior clinical score and effective temperature control compared with their wild-type littermates. This superior control of inflammatory response in Sema3e-/- mice was associated with significantly lower phosphorylation of ERK1/2, AKT, STAT3, and NF-κB, and a concomitant reduction in inducible NO synthase expression and production of TNF and IL-6 compared with their Sema3e+/+ littermates. Sema3e-/- mice also contained significantly higher numbers of activated macrophages compared with their Sema3e+/+ littermates at both baselines and after LPS challenge. In vivo-specific deletion of the Sema3E high-affinity receptor, plexinD1, on macrophages led to the improvement in clinical disease following exposure to a lethal dose of LPS. Collectively, our data show that Sema3E plays an essential role in dampening the early inflammatory response to LPS by regulating macrophage function, suggesting an essential role of this pathway in macrophage inflammatory response.

Time and sex dependency of hemodynamic, renal, and survivability effects of endotoxemia in rats

Widely different exposure times to endotoxic insults have been employed in reported studies. The current experimental study systematically evaluated the time-course and sex influences of endotoxic insult on survivability and cardiovascular and renal functions. Rats received i.p. lipopolysaccharide (LPS, 5 mg/kg) once or twice (over 2 successive days). Systolic blood pressure (SBP), biomarkers of renal function and inflammation, and vasodilator responsiveness of isolated perfused kidneys to acetylcholine (ACh) or N-ethylcarboxamidoadenosine (NECA) were evaluated 6 hr after first LPS injection or 1, 2, or 6 days later. A single 6-hr LPS challenge caused (i) sex-unrelated elevations in serum urea and creatinine and reductions in NECA, but not ACh, vasodilations, (ii) more increases in renal NF-κB/iNOS expressions in male than in female rats, and (iii) hypotension and tachycardia only in male rats. These parameters, except for hemodynamic changes, were restored to near-control levels 1 day after single LPS dosing. The 2-days dosing with LPS had no effects on renal function biomarkers, but caused hypotension, tachycardia, and increases in renal NF-κB/iNOS expression and NECA and ACh vasodilations in both rat sexes. None of these parameters were different from control values when measured 6 days after the endotoxic insult. Alternatively, the rat mortality was observed during first 2 days of the study and was notably higher in male than in female rats. Our data suggest that the frequency and time elapsed after LPS exposure as well as rat sex are important determinants of the magnitude and direction of detrimental effects of endotoxemia.

Memory-Like Inflammatory Responses of Microglia to Rising Doses of LPS: Key Role of PI3Kγ.

Trained immunity and immune tolerance have been identified as long-term response patterns of the innate immune system. The causes of these opposing reactions remain elusive. Here, we report about differential inflammatory responses of microglial cells derived from neonatal mouse brain to increasing doses of the endotoxin LPS. Prolonged priming with ultra-low LPS doses provokes trained immunity, i.e., increased production of pro-inflammatory mediators in comparison to the unprimed control. In contrast, priming with high doses of LPS induces immune tolerance, implying decreased production of inflammatory mediators and pronounced release of anti-inflammatory cytokines. Investigation of the signaling processes and cell functions involved in these memory-like immune responses reveals the essential role of phosphoinositide 3-kinase γ (PI3Kγ), one of the phosphoinositide 3-kinase species highly expressed in innate immune cells. Together, our data suggest profound influence of preceding contacts with pathogens on the immune response of microglia. The impact of these interactions—trained immunity or immune tolerance—appears to be shaped by pathogen dose.

Expression of Cyp2c/Cyp2j subfamily members and oxylipin levels during LPS-induced inflammation and resolution in mice.

Inflammatory stimuli, such as bacterial LPS, alter the expression of many cytochromes P450. CYP2C and CYP2J subfamily members actively metabolize fatty acids to bioactive eicosanoids, which exhibit potent anti-inflammatory effects. Herein, we examined mRNA levels of the 15 mouse Cyp2c and 7 mouse Cyp2j isoforms in liver, kidney, duodenum, and brain over a 96-h time course of LPS-induced inflammation and resolution. Plasma and liver eicosanoid levels were also measured by liquid chromatography with tandem mass spectrometry. Expression changes in Cyp2c and Cyp2j isoforms were both isoform and tissue specific. Total liver Cyp2c and Cyp2j mRNA content was reduced by 80% 24 h after LPS but recovered to baseline levels by 96 h. Total Cyp2c and Cyp2j mRNA in kidney (-19%) and duodenum (-64%) were reduced 24 h after LPS but recovered above baseline by 72 h. Total Cyp2c and Cyp2j mRNA content in brain was elevated at all time points after LPS dosing. Plasma eicosanoids transiently increased 3-6 h after administration of LPS. In liver, esterified oxylipin levels decreased during acute inflammation and before recovering. The biphasic suppression and recovery of mouse Cyp2c and Cyp2j isoforms and associated changes in eicosanoid levels during LPS-induced inflammation and resolution may have important physiologic consequences.-Graves, J. P., Bradbury, J. A., Gruzdev, A., Li, H., Duval, C., Lih, F. B., Edin, M. L., Zeldin, D. C. Expression of Cyp2c/Cyp2j subfamily members and oxylipin levels during LPS-induced inflammation and resolution in mice.

Phenylbutyrate facilitates homeostasis of non-resolving inflammatory macrophages.

Non-resolving inflammatory monocytes/macrophages are critically involved in the pathogenesis of chronic inflammatory diseases. However, mechanisms of macrophage polarization are not well understood, thus hindering the development of effective strategies to promote inflammation resolution. In this study, we report that macrophages polarized by subclinical super-low dose LPS preferentially expressed pro-inflammatory mediators such as ccl2 (which encodes the protein monocyte chemo attractant protein-1) with reduced expression of anti-inflammatory/homeostatic mediators such as slc40a1 (which encodes the protein ferroportin-1). We observed significantly elevated levels of the autophagy-associated and pro-inflammatory protein p62 in polarized macrophages, closely correlated with the inflammatory activation of ccl2 gene expression. In contrast, we noted a significant increase of ubiquitinated/inactive nuclear-erythroid-related factor 2 (NRF2), consistent with reduced slc40a1 gene expression in polarized macrophages. Addition of the homeostatic restorative agent phenylbutyrate (4-PBA) effectively reduced cellular levels of p62 as well as ccl2 gene induction by super-low dose LPS. On the other hand, application of 4-PBA also blocked the accumulation of ubiquitinated NRF2 and restored anti-inflammatory slc40a1 gene expression in macrophages. Together, our study provides novel insights with regard to macrophage polarization and reveals 4-PBA as a promising molecule in restoring macrophage homeostasis.

Locally instructed CXCR4hi neutrophils trigger environment-driven allergic asthma through the release of neutrophil extracellular traps.

Low exposure to microbial products, respiratory viral infections and air pollution is a major risk factor for allergic asthma, yet the mechanistic links between such conditions and host susceptibility to type 2 allergic disorders remain unclear. Through the use of single-cell RNA sequencing (scRNA-seq), we characterized lung neutrophils in mice exposed to a pro-allergic, low dose of lipopolysaccharides (LPSlo) or a protective, high dose of LPS (LPShi) before exposure to house dust mite (HDM). Unlike exposure to LPShi, exposure to LPSlo instructed recruited neutrophils to upregulate the expression of the chemokine receptor CXCR4 and to release neutrophil extracellular traps (NETs). The LPSlo-induced neutrophils and NETs potentiated the uptake of HDM by CD11b+Ly-6C+ dendritic cells (DCs) and type 2 allergic airway inflammation in response to HDM. NETs derived from CXCR4hi neutrophils were also needed to mediate allergic asthma triggered by infection with influenza virus or exposure to ozone. Our study indicates that apparently unrelated environmental risk factors can shape recruited lung neutrophils to promote the initiation of allergic asthma.

Female- and Intruder-induced Ultrasonic Vocalizations in C57BL/6J Mice as Proxy Indicators for Animal Wellbeing.

Female urine-induced male mice ultrasonic vocalizations (FiUSV) are ultrasonic vocalizations produced by adult male mice after presentation of adult female urine, whereas intruder-induced ultrasonic vocalizations (IiUSV) are produced by resident adult female mice when interacting with an intruder female mouse. These affiliative behaviors may be reduced when mice have decreased wellbeing or are in pain and distress. To determine whether FiUSV and IiUSV can be used as proxy indicators of animal wellbeing, we assessed FiUSV produced by male C57BL/6J mice in response to female urine and IiUSV produced by female C57BL/6J mice in response to a female intruder at baseline and 1 and 3 h after administration of a sublethal dose of LPS (6 or 12.5 mg/kg IP) or an equal volume of saline. Behavior was assessed by evaluating orbital tightness, posture, and piloerection immediately after USV collection. We hypothesized that LPS-injected mice would have a decreased inclination to mate or to interact with same-sex conspecifics and therefore would produce fewer USV. At baseline, 32 of 33 male mice produced FiUSV (149 ± 127 USV in 2 min), whereas all 36 female mice produced IiUSV (370 ± 156 USV in 2 min). Saline-injected mice showed no change from baseline at the 1- and 3-h time points, whereas LPS-injected mice demonstrated significantly fewer USV than baseline, producing no USV at both 1 and 3 h. According to orbital tightness, posture, and piloerection, LPS-injected mice showed signs of poor wellbeing at 3 h but not 1 h. These findings indicate that FiUSV and IiUSV can be used as proxy indicators of animal wellbeing associated with acute inflammation in mice and can be detected before the onset of clinical signs.

Fetal exposure to maternal inflammation interrupts murine intestinal development and increases susceptibility to neonatal intestinal injury.

ABSTRACTFetal exposure to chorioamnionitis can impact the outcomes of the developing fetus both at the time of birth and in the subsequent neonatal period. Infants exposed to chorioamnionitis have a higher incidence of gastrointestinal (GI) pathology, including necrotizing enterocolitis (NEC); however, the mechanism remains undefined. To simulate the fetal exposure to maternal inflammation (FEMI) induced by chorioamnionitis, pregnant mice (C57BL/6J, IL-6−/−, RAG−/− or TNFR1−/−) were injected intraperitoneally on embryonic day (E)15.5 with lipopolysaccharide (LPS; 100 µg/kg body weight). Pups were delivered at term, and reared to postnatal day (P)0, P7, P14, P28 or P56. Serum and intestinal tissue samples were collected to quantify growth, inflammatory markers, histological intestinal injury, and goblet and Paneth cells. To determine whether FEMI increased subsequent susceptibility to intestinal injury, a secondary dose of LPS (100 µg/kg body weight) was given on P5, prior to tissue harvesting on P7. FEMI had no effect on growth of the offspring or their small intestine. FEMI significantly decreased both goblet and Paneth cell numbers while simultaneously increasing serum levels of IL-1β, IL-10, KC/GRO (CXCL1 and CXCL2), TNF and IL-6. These alterations were IL-6 dependent and, importantly, increased susceptibility to LPS-induced intestinal injury later in life. Our data show that FEMI impairs normal intestinal development by decreasing components of innate immunity and simultaneously increasing markers of inflammation. These changes increase susceptibility to intestinal injury later in life and provide novel mechanistic data to potentially explain why preterm infants exposed to chorioamnionitis prior to birth have a higher incidence of NEC and other GI disorders.

Systemic LPS-induced neuroinflammation increases the susceptibility for proteasome inhibition-induced degeneration of the nigrostriatal pathway

IntroductionBesides proteasome dysfunction, neuroinflammation is a common feature in the pathogenesis of Parkinson's disease (PD). Accordingly, peripheral inflammation has been shown to increase the susceptibility of the brain for nigrostriatal degeneration by inducing activation of glial cells and release of pro-inflammatory cytokines in the brain. Given that current animal models of PD fail to recapitulate the pathophysiology occurring in idiopathic PD, the aim of this study was to combine two pathogenic mechanisms (i.e. neuroinflammation and proteasome inhibition) to create a dual-hit mouse model of PD. MethodsWe repeatedly injected mice with a low dose of LPS (250 μg/kg/day i. p. for four days) to induce neuroinflammation, followed by a unilateral intranigral injection of lactacystin (LAC; 3 μg). Seven days later, mice were evaluated behaviorally to assess locomotion, anxiety- and depressive-like behavior. Nigrostriatal degeneration was analyzed by measuring striatal dopamine loss as well as loss of nigral dopaminergic neurons. Neuroinflammation was confirmed by quantifying microglial cells in the substantia nigra (SN) and cytokine expression in the striatum. ResultsRepeated systemic LPS injections increase the number of microglial cells in the SN and induce a mixed profile of pro- and anti-inflammatory cytokines in the striatum without affecting the integrity of the nigrostriatal pathway. Systemic LPS-induced neuroinflammation, however, increases the susceptibility of the nigrostriatal pathway for LAC-induced degeneration. ConclusionRecapitulating two relevant etiopathogenic mechanisms of PD - neuroinflammation and proteasome inhibition-, we propose this dual-hit model as a relevant mouse model for PD that could be used to investigate potential therapeutic targets.

Magnesium sulfate inhibits inflammation through P2X7 receptors in human umbilical vein endothelial cells.

Introduction:Magnesium sulfate (MgSO4) is utilized for fetal neuroprotection in preterm birth but its mechanism of action is still poorly understood. P2X7 receptor (P2X7R) is required for secretion of IL-1β, and can be blocked by divalent cations such as magnesium (Mg) and its own antagonist, Brilliant Blue G (BBG). We sought to determine, whether during inflammation MgSO4 can block endothelial IL-1β secretion, using an in-vitro model.Methods:Human umbilical vein endothelial cell (HUVEC) cultures were treated with varying doses of LPS, 2’(3)-O-(4-Benzoylbenzoyl) adenosine-5’-triphosphate (BzATP), BBG and MgSO4 for 3- or 24-hours. We determined cell cytotoxicity, apoptosis, IL-1β mRNA expression, IL-1β production and secretion and P2X7R expression on HUVECs.Results:We demonstrated that MgSO4 is efficacious in blocking IL-1β-mediated-inflammation in HUVECs, at both the initiation and propagation phases of inflammation. MgSO4 exerts these anti-inflammatory effects via downregulation of P2X7Rs on HUVECs.Conclusion:LPS-exposure increases IL-1β production and secretion in HUVECs, which is further intensified by P2X7R agonist, BzATP while MgSO4 inhibits IL-1β in both presence and absence of BzATP. This effect is similar to the results of P2X7R antagonist, BBG, suggesting that the anti-inflammatory effects of MgSO4is through P2X7R.

Protection from Endotoxin Shock by Selective Targeting of Proinflammatory Signaling to the Nucleus Mediated by Importin Alpha 5.

Endotoxin shock is induced by LPS, one of the most potent virulence factors of the Gram-negative bacteria that cause sepsis. It remains unknown if either proinflammatory stress-responsive transcription factors (SRTFs), ferried to nucleus by importin α5, or lipid-regulating sterol regulatory element binding proteins (SREBPs), transported to the nucleus by importin β1, mediate endotoxin shock. A novel cell-penetrating peptide targeting importin α5 while sparing importin β1 protected 80% of animals from death in response to a high dose of LPS. This peptide suppresses inflammatory mediators, liver glycogen depletion, endothelial injury, neutrophil trafficking, and apoptosis caused by LPS. In d-galactosamine-pretreated mice challenged by 700-times lower dose of LPS, rapid death through massive apoptosis and hemorrhagic necrosis of the liver was also averted by the importin α5–selective peptide. Thus, using a new tool for selective suppression of nuclear transport, we demonstrate that SRTFs, rather than SREBPs, mediate endotoxin shock.

Dynamics of Platelet Behaviors as Defenders and Guardians: Accumulations in Liver, Lung, and Spleen in Mice.

Systemic platelet behaviors in experimental animals are often assessed by infusion of isotope-labeled platelets and measuring them under anesthesia. However, such procedures alter, therefore may not reveal, real-life platelet behaviors. 5-Hydroxytryptamine (5HT or serotonin) is present within limited cell-types, including platelets. In our studies, by measuring 5HT as a platelet-marker in non-anesthetized mice, we identified stimulation- and time-dependent accumulations in liver, lung, and/or spleen as important systemic platelet behaviors. For example, intravenous, intraperitoneal, or intragingival injection of lipopolysaccharide (LPS, a cell-wall component of Gram-negative bacteria), interleukin (IL)-1, or tumor necrosis factor (TNF)-α induced hepatic platelet accumulation (HPA) and platelet translocation into the sinusoidal and perisinusoidal spaces or hepatocytes themselves. These events occurred "within a few hours" of the injection, caused hypoglycemia, and exhibited protective or causal effects on hepatitis. Intravenous injection of larger doses of LPS into normal mice, or intravenous antigen-challenge to sensitized mice, induced pulmonary platelet accumulation (PPA), as well as HPA. These reactions occurred "within a few min" of the LPS injection or antigen challenge and resulted in shock. Intravenous injection of 5HT or a catecholamine induced a rapid PPA "within 6 s." Intravenous LPS injection, within a minute, increased the pulmonary catecholamines that mediate the LPS-induced PPA. Macrophage-depletion from liver and spleen induced "day-scale" splenic platelet accumulation, suggesting the spleen is involved in clearing senescent platelets. These findings indicate the usefulness of 5HT as a marker of platelet behaviors, and provide a basis for a discussion of the roles of platelets as both "defenders" and "guardians."

Naringin mitigates myocardial strain and the inflammatory response in sepsis-induced myocardial dysfunction through regulation of PI3K/AKT/NF-κB pathway

Sepsis-induced myocardial dysfunction (SIMD) is a manifestation of severe sepsis and is the main cause of increased mortality in sepsis patients. Naringin (Nar) has been reported to possess various biological activities and pharmacological properties. Therefore, the present study was undertaken to evaluate whether Nar can protect rats from the effects of LPS-induced SIMD. SD Rats were pre-treated with Nar (50 and 100 mg/kg) for 7 days before administration of a single dose of LPS (10 mg/kg, i.p.) on the seventh day. We found that Nar treatment markedly improved the global strain and strain rate of longitudinal, circumference, and radial direction (GLS/GLSr, GCS/GCSr, GRS/GRSr) compared to the LPS group. The layer-specific strain decreased gradually from the endocardial layer to epicardial layer, and the most serious damage occurred in the endocardial layer. Moreover, Nar significantly decreased the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and myocardial enzymes (CK, LDH, and AST) induced by LPS and attenuated the inflammation response. Finally, Nar also inhibited NF-κB nuclear translocation and the activity of iNOS in H9c2 cardiomyocytes by activating PI3K/AKT signaling pathway. These results suggest that naringin may possess novel therapeutic potential for protection against LPS-induced myocardial dysfunction.

Bacterial endotoxin decreased histone H3 acetylation of bovine mammary epithelial cells and the adverse effect was suppressed by sodium butyrate

BackgroundIn practical production, dairy cows are frequently exposed to bacterial endotoxin (lipopolysaccharide, LPS) when they are subjected to high-concentrate diets, poor hygienic environments, as well as mastitis and metritis. Histone acetylation is an important epigenetic control of DNA transcription and a higher histone acetylation is associated with facilitated transcription. LPS might reduce histone acetylation in the mammary epithelial cells, resulting in lower transcription and mRNA expression of lactation-related genes. This study was conducted to investigate the effect of LPS on histone acetylation in bovine mammary epithelial cells and the efficacy of sodium butyrate (SB) in suppressing the endotoxin-induced adverse effect. Firstly, the bovine mammary epithelial cell line MAC-T cells were treated for 48 h with LPS at different doses of 0, 1, 10, 100, and 1000 endotoxin units (EU)/mL (1 EU = 0.1 ng), and the acetylation levels of histones H3 and H4 as well as the histone deacetylase (HDAC) activity were measured. Secondly, the MAC-T cells were treated for 48 h as follows: control, LPS (100 EU/mL), and LPS (100 EU/mL) plus SB (10 mmol/L), and the acetylation levels of histones H3 and H4 as well as milk gene mRNA expressions were determined.ResultsThe results showed that HDAC activity increased linearly with increasing LPS doses (P < 0.01). The histone H3 acetylation levels were significantly reduced by LPS, while the histone H4 acetylation levels were not affected by LPS (P > 0.05). Sodium butyrate, an inhibitor of HDAC, effectively suppressed the endotoxin-induced decline of histone H3 acetylation (P < 0.05). As a result, SB significantly enhanced the mRNA expression of lactation-related genes (P < 0.05).ConclusionsThe results suggest one of the adverse effects of LPS on the lactation of bovine mammary gland epithelial cells was due to decreasing histone H3 acetylation through increasing HDAC activity, whereas the endotoxin-induced adverse effects were effectively suppressed by SB.

Perturbation of ubiquitin homeostasis promotes macrophage oxidative defenses

The innate immune system senses microbial ligands through pattern recognition and triggers downstream signaling cascades to promote inflammation and immune defense mechanisms. Emerging evidence suggests that cells also recognize alterations in host processes induced by infection as triggers. Protein ubiquitination and deubiquitination are post-translational modification processes essential for signaling and maintenance of cellular homeostasis, and infections can cause global alterations in the host ubiquitin proteome. Here we used a chemical biology approach to perturb the cellular ubiquitin proteome as a simplified model to study the impact of ubiquitin homeostasis alteration on macrophage function. Perturbation of ubiquitin homeostasis led to a rapid and transient burst of reactive oxygen species (ROS) that promoted macrophage inflammatory and anti-infective capacity. Moreover, we found that ROS production was dependent on the NOX2 phagocyte NADPH oxidase. Global alteration of the ubiquitin proteome also enhanced proinflammatory cytokine production in mice stimulated with a sub-lethal dose of LPS. Collectively, our findings suggest that major changes in the host ubiquitin landscape may be a potent signal to rapidly deploy innate immune defenses.