Intact Endotoxin Research Articles

Genetic variants drive altered epigenetic regulation of endotoxin response in BTBR macrophages

The BTBR T+Itpr3tf/J (BTBR) mouse has been used as a complex genetic model of Autism Spectrum Disorders (ASD). While the specific mechanisms underlying BTBR behavioral phenotypes are poorly understood, prior studies have implicated profound differences in innate immune system control of pro-inflammatory cytokines. Innate immune activation and elevated pro-inflammatory cytokines are also detected in blood of children with ASD. In this study, we examined how underlying BTBR genetic variants correspond to strain-specific changes in chromatin accessibility, resulting in a pro-inflammatory response specifically in BTBR bone marrow derived macrophages (BMDM). In response to repeated lipopolysaccharide (LPS) treatments, C57BL/6J (C57) BMDM exhibited intact endotoxin tolerance. In contrast, BTBR BMDM exhibited hyper-responsive expression of genes that were normally tolerized in C57. This failure in formation of endotoxin tolerance in BTBR was mirrored at the level of chromatin accessibility. Using ATAC-seq, we specifically identified promoter and enhancer regions with strain-specific differential chromatin accessibility both at baseline and in response to LPS. Regions with strain-specific differences in chromatin accessibility were significantly enriched for BTBR genetic variants, such that an average of 22% of the differential chromatin regions had at least one variant. Together, these results demonstrate that BTBR genetic variants contribute to altered chromatin responsiveness to endotoxin challenge resulting in hyper-responsive innate immunity in BTBR. These findings provide evidence for an interaction between complex genetic variants and differential epigenetic regulation of innate immune responses.

Altered surfactant protein A gene expression and protein metabolism associated with repeat exposure to inhaled endotoxin.

Chronically inhaled endotoxin, which is ubiquitous in many occupational and domestic environments, can adversely affect the respiratory system resulting in an inflammatory response and decreased lung function. Surfactant-associated protein A (SP-A) is part of the lung innate immune system and may attenuate the inflammatory response in various types of lung injury. Using a murine model to mimic occupational exposures to endotoxin, we hypothesized that SP-A gene expression and protein would be elevated in response to repeat exposure to inhaled grain dust and to purified lipopolysaccharide (LPS). Our results demonstrate that repeat exposure to inhaled endotoxin, either in the form of grain dust or purified LPS, results in increased whole lung SP-A gene expression and type II alveolar epithelial cell hyperplasia, whereas SP-A protein levels in lung lavage fluid are decreased. Furthermore, these alterations in SP-A gene activity and protein metabolism are dependent on an intact endotoxin signaling system.

Interferon Induction by Endotoxin-Derived Nontoxic Polysaccharides

Polysaccharide-rich preparations from Serratia marcescens-derived endotoxin and components thereof, including Lipid A, were studied in terms of their ability to induce interferon (IFN) activity in murine spleen cell cultures in vitro. Although the polysaccharide-rich derivatives, similar to intact endotoxin, were only weak stimulators for IFN induction, pretreatment of splenocyte cultures with recombinant interleukin-2 (IL-2) significantly increased IFN-inducing activity. Both an endotoxin-derived polysaccharide and a "White type" polysaccharide prepared from intact Serratia had similar ability to induce IFN in vitro, but only when spleen cells were first treated with IL-2. The polysaccharide preparations were nontoxic as compared with the high degree of toxicity of the intact endotoxin, yet induced similar IFN levels as whole endotoxin. Much of the IFN induced by these preparations was of the gamma type, since activity was either not neutralized or only incompletely neutralized by treatment with anti-alpha/beta interferon antibody.

Early-phase endotoxin tolerance: induction by a detoxified lipid A derivative, monophosphoryl lipid A.

After a sublethal exposure to lipopolysaccharide (LPS) or to lipid A, which is that portion of the LPS molecule associated with endotoxicity, a transient period ensues during which a normally responsive individual is rendered hyporesponsive to LPS-induced toxicity. This period has been defined as early-phase endotoxin tolerance. Recently, a nontoxic derivative of lipid A from Salmonella typhimurium, monophosphoryl lipid A (MPL), was isolated and purified. In this study, we assessed the ability of MPL to induce early endotoxin tolerance. Initial injection of MPL resulted in a dose-dependent stimulation of both serum colony-stimulating factor and serum interferon, indicators of in vivo LPS responsiveness. In contrast, MPL failed to induce the symptoms of endotoxicity which are normally seen after injection of even sublethal amounts of intact endotoxin or lipid A preparations. Injection of MPL on day 0 reduced significantly the amount of LPS-induced serum colony-stimulating factor and interferon produced upon challenge with Escherichia coli LPS 3 days later and also mitigated toxic manifestations, as evidenced by a marked increase in the 50% lethal dose. Like the early tolerance induced by wild-type (toxic) LPS, MPL-induced tolerance was characterized by an accompanying elevation in the number of bone marrow-derived macrophage progenitor cells and by an alteration in bone marrow cell sizing profiles. These results indicate that MPL is effective in inducing a state of LPS-hyporesponsiveness without the toxic side effects of endotoxin and that the structural component(s) necessary for induction of early-phase endotoxin tolerance is contained within MPL.

Novel biochemical avenues for enhancingBacillus thuringiensis endotoxin potency againstSpodoptera littoralis [Lep.: Noctuidae

Simple biochemical means were used to enhance the endotoxin effect ofBacillus thuringiensis Berliner through optimization of conditions present in the insect gut required for release of intoxicating fragments from nonpoisonous intact endotoxin. This was achieved through the addition of either alkaline compounds naturally occurring in the insect midgut, general proteolytic activators or midly toxic compounds of inorganic (e.g. borax) and organic (e.g. tannic acid) nature along with endotoxin in the diet. A marked increase in the potency ofB.t. vars.entomocidus HD-635 andaizawai HD-133 againstSpodoptera littoralis (Boisduval) was obtained in the presence of 0.5% concentration of potassium carbonate. On the other hand, sodium carbonate was less effective at the tested concentrations. Among the tested proteolytic activators, the effect of magnesium ion was proportional to the concentration of the incorporated salt. Thus the potencies of δ-endotoxin preparations of HD-635 and HD-133 were increased 2–3 times after the addition of 1% concentration of magnesium chloride. A lesser effect was noted at 0.5%, whereas virtually the potency did not change at 0.25% concentration of the salt. Similar trends were noted with the incorporation of calcium chloride along with the endotoxin in the insect diet. The addition of sodium borate or tannic acid was highly effective in enhancing the potency of the tested sporeδ-endotoxin preparations with 2–4 fold increase.

Bacterial cell wall components decrease the number of guinea-pig lung β-adrenoceptors

Infections of the deeper respiratory airways can contribute to the progression of chronic asthmatic bronchitis. In the present report a number of microorganisms affecting the number of β-adrenoceptors in guinea-pig lung homogenates are described. Haemophilus influenzae, Streptococcus pneumoniae, Bordetella pertussis and Escherichia coli O 111B 4 induced a significant decrease of the number of β-adrenoceptors (by approximately 20%). Staphylococcus aureus, influenza A virus and Escherichia coli J 5 were not active. These data point to a common factor shared by gram-negative bacilli; i.e. endotoxin. Purified endotoxin of E. coli O 111B 4 also decreased the number of β-adrenoceptors, while E. coli J 5-LPS did not. This suggests that neural polysaccharides of bacterial cell walls, especially those in the ‘O’-antigenic side chain of gram-negative endotoxins may be responsible for the decrease of β-adrenoceptor number and therefore contribute to the pathogenesis of chronic asthmatic bronchitis. Intact endotoxin seems to be necessary since neither the isolated lipid nor the polysaccharide part of E. coli O 111B 4 LPS affected the number of β-adrenoceptors in the lung.

Macrophage-dependent polyclonal activation of splenocytes by Bordetella pertussis endotoxin and its isolated polysaccharide and Lipid A regions

Mitogenic and polyclonal activating properties of the Bordetella pertussis endotoxin and its derived fragments, pertussis polysaccharide-1 (PS-1) and pertussis Lipid A, were tested with five other endotoxins, using rabbit spleen cells. Bordetella pertussis, Proteus mirabilis, Escherichia coli, and Shigella flexneri endotoxins and pertussis Lipid A were mitogenic; Klebsiella pneumoniae and Salmonella senftenberg endotoxins and pertussis PS-1 were not. All endotoxins induced similar numbers of immunoglobulin-secreting cells in a protein A plaque-forming cell assay (Ig PFC) and thus had comparable Ig PFC polyclonal activating potencies. Both isolated fragments of the B. pertussis endotoxin, Lipid A and PS-1 induced similar numbers of Ig PFCs, but their potencies were lower than that of the intact endotoxin. Cell stimulation with a mixture of pertussis Lipid A and PS-1 induced higher numbers of Ig PFCs than stimulation by one or the other fragment alone. Removal of adherent and phagocytic cells from the spleen cell population caused a large decrease in numbers of Ig PFCs induced by B. pertussis or its fragments; although response was only partially (40%) restored upon addition of resident peritoneal macrophages to the adherent and phagocytic cell-depleted splenocytes, these experiments were taken to suggest a macrophage requirement for the polyclonal activation of Ig PFC. The stimulatory properties of pertussis endotoxin, PS-1, and Lipid A seem to be mediated by different signals at the cellular level.

The hepatocellular uptake and biliary excretion of endotoxin in the rat.

Endotoxin is known to cause a dose-dependent impairment of hepatic bile secretion, organic anion excretion, and activity of Na+, K+-activated ATPase. Since it is possible that this impaired excretory function is a result of direct interaction of endotoxin with hepatocytes, we examined: (a) the excretion of endotoxin into bile, and (b) its association with an hepatocyte-enriched, Kupffer cell-depleted population of liver cells. Escherichia coli 0127:B8 endotoxin (17 to 54 μg per 100 gm body weight) was given i.v. to male Sprague Dawley rats and bile collected up to 48 hr. The expected significant decrease in bile secretion was seen, confirming the biological effectiveness of the endotoxin. Endotoxin was quantitated by a gas chromatography/mass spectroscopy method which detects the β-hydroxymyristic acid in the lipid A moiety of endotoxin. Approximately 7% of the administered dose of endotoxin was recovered as β-hydroxymyristic acid in bile in the 48 hr following injection. Approximately two thirds of the administered dose of endotoxin was recovered as β-hydroxymyristic acid in the whole liver. Further analysis of the β-hydroxymyristic acid in bile and liver by Folch extraction and thin-layer chromatography was performed. The β-hydroxymyristic acid in bile was associated with polar and nonpolar metabolites of endotoxin. The β-hydroxymyristic acid recovered in the liver distributed similarly in Folch extraction to intact endotoxin added in vitro. Endotoxin quantitated as β-hydroxymyristic acid was measured in whole liver homogenate and hepatocyte-enriched preparations 3 hr following i.v. administration. The mass abundance by gas chromatography/mass spectrometry of β-hydroxymyristic acid per 106 hepatocytes was similar in crude liver homogenate and the hepatocyte-enriched fractions indicating the presence of substantial amounts of β-hydroxymyristic acid in association with parenchymal liver cells. Thus, our studies indicate that the bulk of the β-hydroxymyristic acid of endotoxin given i.v. to rats is found in hepatocytes in a form suggesting minimal degradation of the endotoxin. A small proportion of the β-hydroxymyristic acid associated with the endotoxin in the liver is then released slowly into bile as a complex mixture of polar and nonpolar metabolites of endotoxin.

Effects of lipid A and liposomes containing lipid A on platelet and fibrinogen production in rabbits

The effect of the lipid A moiety of endotoxin on platelet and fibrinogen production was studied in rabbits. Lipid A was infused intravenously in doses ranging from 1 to 100 micrograms/kg body mass; 18 hr later, selenomethionine-75Se was injected intravenously and its incorporation into fibrinogen and platelets determined. Lipid A in saline stimulated fibrinogen and platelet production, but the dose required was 50--100 times that required for an intact endotoxin. Although lipid A solubilized in triethylamine (TEA) was at least 60 times more active in the Limulus amebocyte lysate assay than was lipid A suspended in saline, the sensitivity of platelet and fibrinogen production to solubilized lipid A was increased only twofold. Incorporation of lipid A into liposomes had no effect on its Limulus activity. Lipid A in liposomes continued to stimulate platelet, but not fibrinogen, production. Leukopenia that was induced by lipid A in TEA did not occur when rabbits received the same dose of lipid A in liposomes. Lipid A, like intact endotoxin, can stimulate platelet and fibrinogen production and induce leukopenia but the doses required are high. The low solubility of lipid A in aqueous solutions may be only one factor that determines its biologic activity.

Effects of Lipid A and Liposomes Containing Lipid A on Platelet and Fibrinogen Production in Rabbits

The effect of the lipid A moiety of endotoxin on platelet and fibrinogen production was studied in rabbits. Lipid A was infused intravenously in doses ranging from 1 to 100 micrograms/kg body mass; 18 hr later, selenomethionine-75Se was injected intravenously and its incorporation into fibrinogen and platelets determined. Lipid A in saline stimulated fibrinogen and platelet production, but the dose required was 50--100 times that required for an intact endotoxin. Although lipid A solubilized in triethylamine (TEA) was at least 60 times more active in the Limulus amebocyte lysate assay than was lipid A suspended in saline, the sensitivity of platelet and fibrinogen production to solubilized lipid A was increased only twofold. Incorporation of lipid A into liposomes had no effect on its Limulus activity. Lipid A in liposomes continued to stimulate platelet, but not fibrinogen, production. Leukopenia that was induced by lipid A in TEA did not occur when rabbits received the same dose of lipid A in liposomes. Lipid A, like intact endotoxin, can stimulate platelet and fibrinogen production and induce leukopenia but the doses required are high. The low solubility of lipid A in aqueous solutions may be only one factor that determines its biologic activity.

Biological activities of fragments derived from Bordetella pertussis endotoxin: isolation of a nontoxic, Shwartzman-negative lipid A possessing high adjuvant properties.

Endotoxin from fresly sedimented Bordetella pertussis cells, isolated by the phenol/water procedure when submitted to kinetically controlled, mild acidic hydrolysis released a polysaccharide (polysaccharide 1), a complex lipid (lipid X), and a glycolipid. When treated with somewhat stronger acid, the glycolipid yielded a second polysaccharide (polysaccharide 2) and another complex lipid (lipid A). The intact pertussis endotoxin had all the usual properties of endotoxins extracted from enteric bacteria. Lipid X and the intermediary glycolipid retained all the endotoxic properties of the unfractionated endotoxin. In lipid A, pyrogenicity was reduced to a very low level and toxicity and Shwartzman reactivity were absent; however, this fraction retained most of the endotoxin's antiviral activity, and its adjuvant power was considerably higher than that of the intact endotoxin. Lipid A elicited nonspecific resistance against challenge with certain bacteria, but not against others.

Comparison of limulus amebocyte lysates and correlation with the United States Pharmacopeial pyrogen test

Six limulus amebocyte lysate (LAL) preparations obtained from five different suppliers were evaluated for sensitivity, dependability, cost, convenience of use, and correlation with the United States Pharmacopeial (USP) rabbit pyrogen test method. Endotoxins from various gram-negative microorganisms were used for the evaluation. Major differences among the LAL preparations lie in the area of sensitivity. Differences, up to 100-fold, exist in the sensitivity of the various LAL preparations to the same endotoxin. The LAL tests in general were 3 to 300 times more sensitive than was the USP rabbit pyrogen test method. The LAL and the USP rabbit pyrogen test data correlated well when the endotoxin in a relatively pure and undegraded form was examined. However, large discrepancies in correlation were found when partially degraded endotoxins were compared. One LAL preparation responded to both intact and degraded endotoxin, whereas others responded only to intact endotoxin; the latter closely correlated with the febrile response of the rabbit. Therefore, proper selection of an LAL preparation is important for its application in clinical, pharmaceutical, public health, and environmental areas.

3-Deoxy-2-octulosonic Acid 5-Phosphate: a Component of the Endotoxin of Bordetella pertussis

Upon hydrolysis with 2 N hydrochloric acid for 2 h, a 3-deoxy-octulosonic acid 5-phosphate was released from the endotoxin of Bordetella pertussis. The structure of the compound was established through chemical degradation. By periodate treatment of the intact endotoxin it was shown that positions 7 and 8 of the bound octulosonic acid phosphate were free, which, if present in a cyclic form, must be a pyranoside.

Studies on the protein moiety of endotoxin from gram-negative bacteria. Characterization of the protein moiety isolated by phenol treatment of endotoxin from Serratia marcescens 08 and Escherichia coli 0 141:K85(B).

The so‐called simple proteins isolated from the endotoxin of Serratia marcescens 08 and whole cells Escherichia coli 0 141 :K85(B) by aqueous phenol treatment were characterized by the determination of hydrodynamic properties, electrophoretic behavior, immunochemical specificity and chemical analysis. The chemical composition of both proteins revealed the presence of small amount of lipid A constituents such as glucosamine, phosphorus and fatty acids; the β‐hydroxymyristic acid regarded as the characteristic marker for lipid A was present in all protein preparations. Since the treatment of intact endotoxin or bacterial cells with aqueous phenol results in the formation of two fragments (a lipopolysaccharide and a lipid A‐protein), both of which are characterized by the presence of lipid A constituents, this dissociation seems to be caused by cleavage of a phenol‐sensitive linkage within the molecule of lipid A rather than at its point of attachment to the protein moiety. Thus, the “simple” protein preparations consist of the protein moiety and a small segment of lipid A. Contrary to previous views, such protein preparations are not simple proteins if this designation implies absence of constituents other than amino acids. However, for a historical reason we suggest that the term simple protein be retained for protein fragments isolated by aqueous phenol treatment of intact endotoxins.The simple protein from Serratia marcescens 08 was treated successively with trypsin and pronase. The resulting trypsin and pronase cores contained increasing amounts of lipid A constituents firmly bound to the residual protein moiety. Since lipid A was separated as an entity from the protein moiety only after acid hydrolysis of the pronase core, it is proposed that in intact endotoxin lipid A is covalently linked to the protein moiety. The absence of any detectable glucosamine and fatty acids in the mixture of peptides and amino acids released by trypsin and pronase, indicated a single point attachment between the lipid A and protein moieties.The protein moieties from Serratia marcescens and Escherichia coli were immunogenic and possessed a common antigenic determinant. Both trypsin and pronase cores retained the immunogenicity and revealed the presence of a common antigen with the parent simple protein. This second set of antigenic determinant(s) different from the specific O‐antigens seems to be located in the protein moiety close to lipid A.

Subcellular Localization of Salmonella enteritidis Endotoxin in Liver and Spleen of Mice and Rats

Salmonella enteritidis(14)C-endotoxin was recovered predominantly from the nuclear and mitochondrial subcellular fractions of livers and spleens of mice and rats, 3.5 hr and 3 days after intravenous administration. Of the recovered radioactivity, 10 to 20% was present in the liver mitochondrial fraction as high-molecular-weight, biologically active material, suggesting the presence of intact endotoxin. Autoradiographic studies demonstrated nuclear and cytoplasmic labeling in the liver and at least nuclear label in spleen cells. The resistance of rats, as compared to mice, to the induction of amyloidosis does not appear to be based on a difference in subcellular localization of endotoxin within the reticuloendothelial system.