LPS Treatment Group Research Articles

Establishment of a flow cytometry-based test for the detection of pyroptosis of BMDM

Objective To establish a flow cytometry-based test for the detection of pyroptosis of murine bone marrow-derived macrophages (BMDM). Methods Bone marrow cells were isolated from wild type (WT) C57BL/6 mice and/or caspase-1-/- C57BL/6 mice and then stimulated with macrophage colony-stimulating factor (M-CSF) to differentiate into murine BMDM. PBS, LPS and LPS+ adenosine triphosphate (ATP) were respectively used to stimulate the BMDM. Western blot assay was performed to detect the cleavage of IL-1β and caspase-1. The levels of IL-1β in the supernatants of cell culture were measured by ELISA. Lactate dehydrogenase (LDH) released in the culture media was detected by using LDH kit. The pyroptosis of murine BMDM was detected by using flow cytometry analysis after double-staining with TMR red+ caspase-1, AnnexinⅤ+ caspase-1 and propidium iodide (PI)+ caspase-1. Results IL-1β was detected in the culture medium of BMDM treated with LPS+ ATP and the cleavage of IL-1β and caspase-1 was confirmed by Western blot assay, which indicated that the NLRP3 inflammasome was activated by LPS+ ATP treatment. Compared with the caspase-1-/- mice group, higher levels of LDH were detected in the culture medium of BMDM isolated form the WT mice. Results of the flow cytometry analysis after staining BMDM with caspase-1 plus AnnexinⅤ or PI showed that more cells undergoing pyroptosis were detected in the LPS+ ATP treatment group than that in LPS or PBS treatment group, which were consistent with the results of the reported flow cytometry with caspase-1+ TMR red staining. Conclusion The flow cytometry-based test with double-staining of caspase-1 plus AnnexinⅤ or PI could be used for the detection of pyroptosis of murine BMDM. Key words: NLRP3 inflammasome; Pyroptosis; Flow cytometry

Regulation of TNF-α and NF-κB activation through the JAK/STAT signaling pathway downstream of histamine 4 receptor in a rat model of LPS-induced joint inflammation

Histamine 4 receptor (H4R) is a novel target for the pharmacological modulation of histamine-mediated immune signals during inflammatory diseases. The purpose of this study was to assess the effects of the H4R agonist 4-methylhistamine dihydrochloride (4-MeH) and antagonist JNJ7777120 (JNJ) in the inflamed rat knee. Animals were fasted for 18h before a single dose of 4-MeH or JNJ (30mg/kg) was administered intraperitoneally (i.p.), both followed by intra-articular (i.a.) injection of LPS 2h later. Blood and synovial fluid were collected after a short incubation period and TNF-α, NF-κB, and IkB-α levels were measured via flow cytometry. Additionally, we assessed the effects of H4R engagement on the expression of IL-1β, TNF-α, and NF-κB mRNAs and the protein levels of TNF-α, NF-κB, JAK-1, and STAT-3 in the inflamed knee tissue. These results revealed increased TNF-α and NF-κB expression and decreased IkB-α levels in both the LPS alone and 4-MeH treated groups in whole blood and synovial fluid. Further, IL-1β, TNF-α, and NF-κB mRNA levels were significantly increased and western blot analysis confirmed increased expression of TNF-α, NF-κB, JAK-1, and STAT-3 in both LPS and 4-MeH treatment groups. Furthermore, these increases were completely inhibited in the inflamed knee tissue of the JNJ-treated group. Thus, the inhibition of inflammatory mediators and signaling pathways by the H4R antagonist JNJ suggests the anti-arthritic importance of this molecule.

Acetate Reduces PGE2 Release and Modulates Phospholipase and Cyclooxygenase Levels in Neuroglia Stimulated with Lipopolysaccharide

Acetate supplementation attenuates neuroglial activation, increases histone and non-histone protein acetylation, reduces pro-inflammatory cytokine expression, and increases IL-4 transcription in rat models of neuroinflammation and Lyme's neuroborreliosis. Because eicosanoid signaling is involved in neuroinflammation, we measured the effect acetate treatment had on phospholipase, cyclooxygenase, and prostaglandin E2 (PGE2) levels in BV-2 microglia and primary astrocytes stimulated with lipopolysaccharide (LPS). In BV-2 microglia, we found that LPS increased the phosphorylation-state of cytosolic phospholipase A2 (cPLA2), reduced the levels of phospholipase C (PLC) β1, and increased the levels of cyclooxygenase (Cox)-1 and -2. Acetate treatment returned PLCβ1 and Cox-1 levels to normal, attenuated the increase in Cox-2, but had no effect on cPLA2 phosphorylation. In primary astrocytes, LPS increased the phosphorylation of cPLA2 and increased the levels of Cox-1 and Cox-2. Acetate treatment in these cells reduced secretory PLA2 IIA and PLCβ1 levels as compared to LPS-treatment groups, reversed the increase in cPLA2 phosphorylation, and returned Cox-1 levels to normal. Acetate treatment reduced PGE2 release in astrocytes stimulated with LPS to control levels, but did not alter PGE2 levels in BV-2 microglia. The amount of acetylated H3K9 bound to the promoter regions of Cox-1, Cox-2, IL-1β and NF-κB p65 genes, but not IL-4 in were increased in BV-2 microglia treated with acetate. These data suggest that acetate treatment can disrupt eicosanoid signaling in neuroglia that may, in part, be the result of altering gene expression due chromatin remodeling as a result of increasing H3K9 acetylation.

Effects of LPS on NF-κB p65 and anti-inflammatory cytokines mRNA expression in MSC (P1096)

Abstract Abstract Objective To investigation the effects of Inflammatory simulated by LPS on mRNA expression of NF-κB p65, anti-inflammatory cytokines (IL-10, TGF-β3) and IDO in rat bone-marrow MSC. Methods The MSC cultured in vitro was divided into 6 groups: The normal group, the PDTC(100μmol/L) treatment group, the PDTC(100μmol/L)plus LPS(5µg/mL) treatment group and the 3 LPS dose (1, 5, 10μg/mL) treatment groups. After LPS supplement for 4 hr, the total RNA of the MSC was extracted and the mRNA expression of p65, IL-10, TGF-β3 and IDO were detected by RT-PCR. The gray scale was used to compare the mean levels (X±S) of PCR product within groups were determined. Results Compared with the control group, the mRNA expression of NF-κB Subunit p65 and anti-inflammatory cytokines IL-10, TGF-β3 were significant increased in the LPS treated groups (P <0.05). The correlation coefficient between p65 and IL-10, TGF-β3 were 0.844(P<0.05), 0.837(P<0.05) respectively. The correlation coefficient of p65 with IDO was -0.942 (P<0.05). Conclusion 1 LPS stimulation could induce MSC produce more anti-inflammatory cytokines through the activation of NF-κB. 2 IDO was constitutively expressed in normal cultured MSC, and its expression may be down regulated by inflammatory stimulation. This result indicates that there are different immune-modulation pathways in MSC under different microenvironments. Key words MSC; NF-κB; IL-10; TGF β3; IDO

Maternal Immune Challenge During Early Embryo Development Affects Blastocyst Cell Lineage Ratio and Postnatal Phenotype.

Maternal infection during gestation is an established cause of abortion, premature birth and intrauterine growth restriction. In addition, there is a strong association between activation of the maternal immune response during mid-gestation and neurological abnormalities in the offspring. At present little is known about the consequences of maternal immune activation during early gestation. The preimplantation embryo has been identified as a critical developmental stage during which environment can program changes to postnatal phenotype. Here, we investigated the sensitivity of preimplantation mouse embryos to maternal immune challenge in the short- and long-term. Virgin female MF-1 mice were mated and randomly assigned to one of four treatment groups; control (saline only), 10, 50 or 150µg/kg of bodyweight lipopolysaccharide (LPS; Salmonella enterica enteritidis ). On gestational day 0.5 (GD0.5), successfully mated females were injected intraperitoneally with saline or LPS dose. Mice were either culled at GD3.5 to assess short-term consequences in blastocysts or allowed to go to term to investigate potential long-term changes in offspring body mass, behaviour and systolic blood pressure (SBP). All postnatal data were analysed using a multilevel random effects regression model (SPSS) which takes into account the hierarchical nature of the dataset. Blastocysts were differentially labelled to assess inner cell mass (ICM) and trophectoderm (TE) cell numbers. Blastocysts from 10µg/kg LPS mothers had more TE cells than those from control mothers (p<0.05). Blastocysts from 50 and 150µg/kg LPS mothers had fewer ICM cells than blastocysts from control mothers (p<0.05). These effects lead to a reduced ICM:TE cell ratio at each LPS dose compared with control (p<0.05). At birth, pups were weighed and litter size adjusted to 3 males and 3 females where possible. Offspring were weighed weekly thereafter and underwent a series of behavioural tests and SBP measurements (tail cuff) until 30 weeks of age. No difference in gestation length or male:female offspring ratio was observed between treatment groups. Interestingly, 10µg/kg LPS mothers had larger litters than control mothers (p<0.01). Body weight was not different between treatment groups at any time point. In the open field behavioural test, both male and female offspring from LPS treated mothers displayed reduced levels of exploratory behaviour compared with controls. For example, male offspring from 150µg/kg LPS mothers displayed fewer rears and jumps than control males at 20 weeks (p<0.05). Furthermore, males from 10µg/kg LPS mothers had elevated SBP at 28 weeks (p<0.05), but females showed no difference in SBP at any time point. These data indicate the sensitivity of the preimplantation mouse embryo to maternal immune activation in vivo leading to altered blastocyst cell allocation, increased litter size (in the lowest dose LPS treatment group) and altered developmental potential; including abnormal behaviour and elevated blood pressure in adult life (also in the lowest dose LPS treatment group). This work serves to highlight maternal immune activation during preimplantation developmentas an important route by which adverse changes in postnatal phenotype can potentially arise; and emphasises the importance of further study into this emerging area of research. This research is funded by a BBSRC studentship (to CLW) and by NIH and MRC grants (to TPF).

Role of liver X receptors alpha agonist on expressions of LPS-induced inflammatory response associated factor IRAK-4 and NF-kappaB in Kupffer cells

Objective To explore the role of activated liver X receptor α (LXRα) on the expressions of interleukin-1 receptor associated kinase-4 (IRAK-4) and NF-kappaB (NF-κB) in the inflammatory response which induced by LPS in the Kupffer cells and to investigate the possible mechanisms of LXRα negative regulation of inflammatory response. Methods The Kupffer cells were isolated from male Kunming mice by collagen perfusion in situ. And these cells were divided into 4 groups: normal control group, LPS treatment group, LXRα agonist T0901317 treatment group, LPS and T0901317 combined treatment group. The LPS treatment group were treated with a final concentration of 1 μg/ml LPS in RPMI 1640 and cultured for 6 h, the T0901317 treatment group were treated with a final concentration of 5 μg/ml in RPMI 1640 and cultured for 24 h, and the combined treatment group received pre-culture for 24 h with a final concentration of 1 μg/ml T0901317 in RPMI 1640 and then cultured for 6 h with a final concentration of 5 μg/ml LPS in RPMI 1640. All groups were cultured for 30 h. The expression of LXRα, IRAK-4 and NF-κB at mRNA and protein levels were detected by real-time PCR and Western blotting, and the TNF-α and IL-1β levels were detected by ELISA. Results The levels of LXRα mRNA and protein were highest in T0901317 group, and lowest in LPS group ( P<0.05). The level of IRAK4 and NF-κB mRNAs and proteins were evidently lower in the Combined-treated group than in LPS group ( P<0.05). And the level of TNF-α and IL-1 were observed highest in LPS group ( P<0.05), but no difference among the Control group, T0901317 group and Combined-treated group ( P>0.05). Conclusion These date suggest that the LXR agonists can effectively up-regulate the expressions of LXRα mRNA and protein and inhibit the inflammatory response. This may be via down-regulating the expressions of IRAK4 and NF-κB at mRNA and protein levels.

Effects of intravenous infusion of lipopolysaccharide on plasma micromineral, magnesium, and cytokine concentrations and serum cortisol concentrations in lactating goats

To assess the effects of various doses of lipopolysaccharide (LPS) administered IV on plasma microminerals, magnesium, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-6 concentrations and serum cortisol concentrations in lactating goats. 6 lactating goats. Goats were allotted to 3 LPS-treatment groups: control (0 microg/kg), low LPS (10 microg/kg), and high LPS (50 microg/kg). Rectal temperatures and behaviors of goats were recorded immediately before a 10-minute IV infusion of LPS and at 0.5, 1, 2, 4, 6, 8, and 24 hours after infusion. Blood samples were obtained before IV infusion and at 0.5, 1, 2, 4, 6, 8, and 24 hours after infusion. Plasma zinc, copper, iron, and magnesium concentrations were determined by atomic absorption spectrometry; plasma TNF-alpha and IL-6 concentrations were measured by use of an ELISA; and serum cortisol concentrations were determined by use of a radioimmunoassay. A monophasic fever developed in low-LPS and high-LPS groups. In the low-LPS and high-LPS group, plasma zinc concentrations decreased at 6 hours after infusion; compared with control groups. Plasma iron concentrations were lower at 24 hours after infusion in low-LPS and high-LPS groups than in the control group. Plasma TNF-alpha and IL-6 concentrations were higher in low-LPS and high-LPS groups than in the control group at 1, 2, and 4 hours after infusion. In low-LPS and high-LPS groups, serum cortisol concentrations increased from 0.5 hours onward and peaked at 1 (high-LPS group) and 2 (low-LPS group) hours after infusion. Following IV infusion of LPS, the immune system is activated, which might affect micromineral homeostatic regulation and, subsequently, the metabolic health of lactating goats.

Marginal Zinc Deficiency Increased the Susceptibility to Acute Lipopolysaccharide-Induced Liver Injury in Rats

Lipopolysaccharide (LPS) triggers a global activation of inflammatory responses leading to liver injury in humans. Zinc pretreatment has been shown to prevent LPS-induced hepatic necrosis. In North America, suboptimal zinc status is more common than once realized. However, the effect of inadequate zinc nutrition on the host's susceptibility to LPS-induced liver injury is not known. The objective of this study was to determine whether marginal zinc deficiency would render rats more susceptible to LPS-induced liver injury. Weanling Sprague-Dawley rats were assigned to one of three dietary treatment groups: marginally low zinc ad libitum (Z3; 3 mg zinc/kg diet), adequate zinc ad libitum (Z30; 30 mg zinc/kg diet), or adequate zinc pair-fed (Z30P) group. After 6 weeks, each dietary treatment group was further divided into LPS-control (saline) groups (C-Z3, C-Z30P, C-Z30) and LPS-treatment (1 mg/kg body weight, intraperitoneal, 8 hrs) groups (LPS-Z3, LPS-Z30P, LPS-Z30). LPS reduced the serum zinc concentration and increased the liver zinc concentration regardless of dietary zinc intake. Serum alanine aminotransferase level was higher in the LPS-Z3 rats than in the LPS-Z30P and LPS-Z30 rats. LPS also induced hepatocyte necrosis and neutrophil infiltration into the liver sinusoids. This LPS-induced liver damage was more severe in the LPS-Z3 rats than in the LPS-Z30P and LPS-Z30 rats. Together these findings have demonstrated that marginal zinc deficiency increased the susceptibility to LPS-induced liver injury in rats. These results indicate that patients with sepsis who have suboptimal zinc nutrition status may be at higher risk of developing greater liver damage.

LPS-Induced Imbalanced Expression of Hepatic Vascular Stress Genes in Cirrhosis: Possible Mechanism of Increased Susceptibility to Endotoxemia

Cirrhosis predisposes the liver to secondary stresses such as endotoxemia possibly via dysregulation of the hepatic portal circulation secondary to imbalanced upregulation of vascular stress genes. In this study we determined the effect of cirrhosis on hepatic vasoregulatory gene expression in response to endotoxin (LPS, i.p., 1 mg/kg). Cirrhosis was induced by bile duct ligation (BDL) for 21 days in male Sprague-Dawley rats. Plasma and liver samples were taken 6 h following an injection of LPS for alanine aminotransferase (ALT) assays and RT-PCR analysis of mRNA levels for genes of interest: endothelin (ET-1), its receptors ET(A) and ET(B), endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and heme oxygenase-1 (HO-1). ALT release increased by 5.5-fold in the BDL animals and 9.9-fold in BDL + LPS compared to sham. ET-1 mRNA was increased by either LPS or BDL treatment alone and increased significantly more in BDL + LPS compared to sham + LPS. mRNA levels for ET(B) receptors showed no change, whereas ETA transcripts decreased in BDL animals compared to sham, with no significant difference between the saline and LPS treatment groups. The resultant increased ratio of ET(B) over ET(A) in BDL animals was reflected functionally in the portal pressure responses to ET(A) and ET(B) agonists ET-1 and IRL-1620 (a specific ETB receptor agonist). The pressor response to ET-1 was attenuated, while the response to IRL-1620 was similar in BDL and sham. eNOS mRNA levels did not increase in response to either BDL or LPS or a combination of both compared to sham. The increase in iNOS mRNA was attenuated in BDL + LPS compared to sham + LPS. HO-1 expression increased significantly in sham + LPS, but failed to increase in BDL + LPS. Taken collectively, significantly greater induction of the constrictor ET-1 over the dilation forces (i.e., eNOS, iNOS, and HO-1) was observed in BDL + LPS. This suggests a compromised ability of the cirrhotic liver to upregulate sufficient dilatory forces to counterbalance the constrictive effect of ET-1 upon a secondary insult of endotoxemia. These results may partly explain the increased susceptibility of cirrhotic livers to injury as a result of endotoxemia.

Aminoethyl-isothiourea, a selective inhibitor of inducible nitric oxide synthase activity, improves liver circulation and oxygen metabolism in a porcine model of endotoxemia.

The role of nitric oxide (NO) in hepatic oxygen transport is unclear. We investigated the effects of aminoethyl-isothiourea (AE-ITU), a selective inhibitor of iNOS activity, on liver blood flow and oxygen consumption (VO2H) in the pig. Endotoxin (lipopolysaccharide, LPS) was given intraportally (1.7 microg/kg/h), followed by AE-ITU (10 mg/kg) after 3 h (n = 7), LPS controls (n = 8) received LPS for 6 h. AE-ITU controls (n = 6) received saline/AE-ITU. LPS (treatment group) caused significant reductions at 3 h in cardiac output (CO) from 4.4 +/- .4 to 2.7 +/- .3 L/min, in hepatic artery flow (Q(HA)) from 266 +/- 53 to 127 +/- 19 mL/min, and in portal venous flow (Q(PV)) from 630 +/- 50 to 323 +/- 33 mL/min. Hepatic oxygen delivery (DO2H) was reduced from 93 +/- 11 to 38 +/- 5 mL/min (p < .05), while hepatic oxygen extraction ratio (ERO2H) increased, and VO2H was maintained. Similar changes were observed in LPS controls. AE-ITU caused no changes in saline controls. After injection of AE-ITU during LPS infusion, CO was unchanged, while Q(HA) increased gradually from 127 +/- 20 to 268 +/- 40 mL/min over 3 h (p < .05) and DO2H from 38 +/- 5 to 60 +/- 5 mL/in (p < .05). ERO2H increased from .54 +/- .04 to .69 +/- .03 in 30 min, while VO2H increased from 23 +/- 4 to 35 +/- 3 mL/in in 3 h (p < .05). Thus, AE-ITU restored hepatic arterial blood flow and increased hepatic oxygen consumption in pigs with endotoxemia.