Lipopolysaccharide Shock Research Articles

Prohibitin 1 regulates mtDNA release and downstream inflammatory responses.

Exposure of mitochondrial DNA (mtDNA) to the cytosol activates innate immune responses. But the mechanisms by which mtDNA crosses the inner mitochondrial membrane are unknown. Here, we found that the inner mitochondrial membrane protein prohibitin 1 (PHB1) plays a critical role in mtDNA release by regulating permeability across the mitochondrial inner membrane. Loss of PHB1 results in alterations in mitochondrial integrity and function. PHB1-deficient macrophages, serum from myeloid-specific PHB1 KO (Phb1MyeKO) mice, and peripheral blood mononuclear cells from neonatal sepsis patients show increased interleukin-1β (IL-1β) levels. PHB1 KO mice are also intolerant of lipopolysaccharide shock. Phb1-depleted macrophages show increased cytoplasmic release of mtDNA and inflammatory responses. This process is suppressed by cyclosporine A and VBIT-4, which inhibit the mitochondrial permeability transition pore (mPTP) and VDAC oligomerization. Inflammatory stresses downregulate PHB1 expression levels in macrophages. Under normal physiological conditions, the inner mitochondrial membrane proteins, AFG3L2 and SPG7, are tethered to PHB1 to inhibit mPTP opening. Downregulation of PHB1 results in enhanced interaction between AFG3L2 and SPG7, mPTP opening, mtDNA release, and downstream inflammatory responses.

2-Deoxy-d-glucose induces deglycosylation of proinflammatory cytokine receptors and strongly reduces immunological responses in mouse models of inflammation.

Anti‐proinflammatory cytokine therapies against interleukin (IL)‐6, tumor necrosis factor (TNF)‐α, and IL‐1 are major advancements in treating inflammatory diseases, especially rheumatoid arthritis. Such therapies are mainly performed by injection of antibodies against cytokines or cytokine receptors. We initially found that the glycolytic inhibitor 2‐deoxy‐d‐glucose (2‐DG), a simple monosaccharide, attenuated cellular responses to IL‐6 by inhibiting N‐linked glycosylation of the IL‐6 receptor gp130. Aglycoforms of gp130 did not bind to IL‐6 or activate downstream intracellular signals that included Janus kinases. 2‐DG completely inhibited dextran sodium sulfate‐induced colitis, a mouse model for inflammatory bowel disease, and alleviated laminarin‐induced arthritis in the SKG mouse, an experimental model for human rheumatoid arthritis. These diseases have been shown to be partially dependent on IL‐6. We also found that 2‐DG inhibited signals for other proinflammatory cytokines such as TNF‐α, IL‐1β, and interferon ‐γ, and accordingly, prevented death by another inflammatory disease, lipopolysaccharide (LPS) shock. Furthermore, 2‐DG prevented LPS shock, a model for a cytokine storm, and LPS‐induced pulmonary inflammation, a model for acute respiratory distress syndrome of coronavirus disease 2019 (COVID‐19). These results suggest that targeted therapies that inhibit cytokine receptor glycosylation are effective for treatment of various inflammatory diseases.

Tanshinones inhibit NLRP3 inflammasome activation by alleviating mitochondrial damage to protect against septic and gouty inflammation

Tanshinones, the active ingredients derived from the roots of Salvia miltiorrhiza, have been widely used as traditional medicinal herbs for treating human diseases. Although tanshinones showed anti-inflammatory effects in many studies, large knowledge gaps remain regarding their underlying mechanisms. Here, we identified 15 tanshinones that suppressed the activation of NLRP3 inflammasome and studied their structure–activity relationships. Three tanshinones (tanshinone IIA, isocryptotanshinone, and dihydrotanshinone I) reduced mitochondrial reactive-oxygen species production in lipopolysaccharide (LPS)/nigericin-stimulated macrophages and correlated with altered mitochondrial membrane potentials, mitochondria complexes activities, and adenosine triphosphate and protonated-nicotinamide adenine dinucleotide production. The tanshinones may confer mitochondrial protection by promoting autophagy and the AMP-activated protein kinase pathway. Importantly, our findings demonstrate that dihydrotanshinone I improved the survival of mice with LPS shock and ameliorated inflammatory responses in septic and gouty animals. Our results suggest a potential pharmacological mechanism whereby tanshinones can effectively treat inflammatory diseases, such as septic and gouty inflammation.

Hierarchical cell-type-specific functions of caspase-11 in LPS shock and antibacterial host defense.

SUMMARYCaspase-11 sensing of intracellular lipopolysaccharide (LPS) plays critical roles during infections and sepsis. However, the key cell types that sense intracellular LPS and their contributions to the host responses at the organismal level are not completely clear. Here, we show that macrophage/monocyte-specific caspase-11 plays a dominant role in mediating the pathological manifestations of endotoxemia, including gasdermin D (GSDMD) activation, interleukin (IL)-1β, IL-18, and damage-associated molecular pattern (DAMP) release, tissue damage, and death. Surprisingly, caspase-11 expression in CD11c+ cells and intestinal epithelial cells (IECs) plays minor detrimental roles in LPS shock. In contrast, caspase-11 expression in neutrophils is dispensable for LPS-induced lethality. Importantly, caspase-11 sensing of intracellular LPS in LyzM+ myeloid cells and MRP8+ neutrophils, but not CD11c+ cells and IECs, is necessary for bacterial clearance and host survival during intracellular bacterial infection. Thus, we reveal hierarchical cell-type-specific roles of caspase-11 that govern the host-protective and host-detrimental functions of the cytosolic LPS surveillance.

The Protective Effects of Melatonin Against LPS-Induced Septic Myocardial Injury: A Potential Role of AMPK-Mediated Autophagy.

Aim: Melatonin is an indolamine secreted by the pineal gland, as well as most of the organs and tissues. In addition to regulating circadian biology, studies have confirmed the multiple pharmacological effects of melatonin. Melatonin provides a strong defense against septic myocardial injury. However, the underlying mechanism has not been fully described. In this study, we investigated the protective effects of melatonin against lipopolysaccharide (LPS)-induced myocardial injury as well as the mechanisms involved.Methods: Mice were intraperitoneally injected with LPS to induce a septic myocardial injury model or an LPS shock model, depending on the dose of LPS. Melatonin was given (20 mg/kg/day, via intraperitoneal injection) for a week prior to LPS insult. 6 h after LPS injection, echocardiographic analysis, TUNEL staining, transmission electron microscopy (TEM), western blot, quantitative real-time PCR and ELISA were used to investigate the protective effects of melatonin against LPS induced myocardial injury. AMPK inhibitor, autophagy activator and inhibitor, siRNAs were used for further validation.Results: Survival test showed that melatonin significantly increased the survival rate after LPS-induced shock. In the sepsis model, melatonin markedly ameliorated myocardial dysfunction, decreased the release of inflammatory cytokines, activated AMP-activated protein kinase (AMPK), improved mitochondrial function, and activated autophagy. To confirm whether the protection of melatonin was mediated by AMPK and autophagy, Compound C, an AMPK inhibitor; 3-MA, an autophagy inhibitor; and Rapamycin (Rapa), an autophagy activator, were used in this study. AMPK inhibition down-regulated autophagy, abolished protection of melatonin, as indicated by significantly decreased cardiac function, increased inflammation and damaged mitochondrial function. Furthermore, autophagy inhibition by 3-MA significantly impaired the protective effects of melatonin, whereas autophagy activation by Rapa reversed LPS + Compound C induced myocardial injury. In addition, in vitro studies further confirmed the protection of melatonin against LPS-induced myocardial injury and the mechanisms involving AMPK-mediated autophagy signaling.Conclusions: In summary, our results demonstrated that melatonin protects against LPS-induced septic myocardial injury by activating AMPK mediated autophagy pathway.

Noncanonical Inflammasome-Induced release of Alarmins during Sepsis

Abstract Sepsis is a life-threatening disease caused by the immune system’s response to infection that results in excessive inflammation, organ damage and often death. In fact, sepsis is the leading cause of death in intensive care units and is responsible for more than 250,000 deaths every year in the USA. Activation of inflammatory caspases, such as caspase-11 by lipopolysaccharide (LPS) from Gram-negative bacteria, is a key mechanism of innate immune defense against infection. Intracellular sensing of LPS by caspase-11 results in pyroptosis, maturation of caspase-1, IL-1β and IL-18 and unconventional secretion of intracellular proteins that lack a leader sequence for conventional secretion via endoplasmic reticulum-Golgi route. These proteins can act as alarmins or damage associated molecular patterns (DAMPs) to regulate the inflammatory response and therefore play an important role during infectious diseases. However, caspase-11-dependent release of alarmins and DAMPs following noncanonical inflammasome activation is poorly characterized. Using a proteomics approach involving ProteomeLab’s PF2D fractionation system followed by mass spectrometry, we have profiled several new alarmins released in a caspase-11 dependent manner following Gram-negative bacterial infection. By employing various mutant mice and cells, we have defined the molecular and cellular mechanisms underlying the release of a candidate alarmin as a consequence of noncanonical inflammasome activation in vitro and in vivo. Importantly, our in vivo studies demonstrate a critical role for this alarmin in LPS shock. Overall, these findings provide new insights into potential biomarkers and therapeutic targets for blocking the lethal inflammation in sepsis.

Lipopolysaccharide shock reveals the immune function of indoleamine 2,3-dioxygenase 2 through the regulation of IL-6/stat3 signalling

Indoleamine 2,3-dioxygenase 2 (Ido2) is a recently identified catalytic enzyme in the tryptophan-kynurenine pathway that is expressed primarily in monocytes and dendritic cells. To elucidate the biological role of Ido2 in immune function, we introduced lipopolysaccharide (LPS) endotoxin shock to Ido2 knockout (Ido2 KO) mice, which led to higher mortality than that in the wild type (WT) mice. LPS-treated Ido2 KO mice had increased production of inflammatory cytokines (including interleukin-6; IL-6) in serum and signal transducer and activator of transcription 3 (stat3) phosphorylation in the spleen. Moreover, the peritoneal macrophages of LPS-treated Ido2 KO mice produced more cytokines than did the WT mice. By contrast, the overexpression of Ido2 in the murine macrophage cell line (RAW) suppressed cytokine production and decreased stat3 expression. Finally, RAW cells overexpressing Ido2 did not alter nuclear factor κB (NF-κB) or stat1 expression, but IL-6 and stat3 expression decreased relative to the control cell line. These results reveal that Ido2 modulates IL-6/stat3 signalling and is induced by LPS, providing novel options for the treatment of immune disorders.

Vasoactive intestinal peptide and methylprednisolone change intestinal Toll-like receptor mRNA expression in rats with endotoxic shock

Objective To investigate the effect of vasoactive intestinal peptide (VIP) and methylprednisolone (MP) on Toll-like receptor (TLR)2/4 mRNA expression in endotoxin (lipopolysaccharide,LPS) induced shock.Methods Ninety Sprague-Dawley rats were randomly divided into LPS group (n =20),LPS + VIP group (n =20),LPS + MP group (n =20),LPS + VIP + MP group (n =20) and control group (n =10).LPS group injected intravenously LPS (E Coli O55B5) 10 mg/kg.LPS + VIP group,LPS + MP group and LPS + VIP + MP group were injected intravenously VIP 5 nmol/kg,MP 3 mg/kg and VIP 5 nmoL/kg + MP 3 mg/kg after LPS 10 mg/kg injection.The control group injected normal saline intravenously instead of LPS.The rats were sacrificed at 6 h and 24 h after injection and the intestine samples were collected.Pathological changes of the intestine were observed by microscopy.RT-PCR was used to detect the intestinal TLR2 mRNA and TLR4 mRNA expressions.Results Intestinal mucosa showed edema or necrotic change with structure of the microvilli disappeared after LPS injection.The inestinal lesions in VIP,MP and VIP + MP groups were milder than LPS group.At 6 h after LPS injection,TLR2 mRNA and TLR4 mRNA expressions were significantly up-regulated in LPS group,LPS + VIP group,LPS + MP group and LPS + VIP + MP group (TLR2 mRNA:1.14 ±0.38,1.17 ±0.42,1.16 ±0.41,0.92 ± 0.29;TLR4 mRNA 1.21 ±0.18,1.04 ± 0.38,1.11 ± 0.34,1.01 ± 0.20) compared with the control group (0.32 ± 0.20,0.24 ± 0.17) (P ＜ 0.01).But there was no significant difference between LPS group,LPS + VIP group,LPS + MP group and LPS + VIP + MP group (P ＞ 0.05).At 24 h after LPS injection,TLR2 mRNA and TLR4 mRNA expressions in LPS + VIP group,LPS + MP group and LPS + VIP + MP group (TLR2 mRNA:0.63 ± 0.12,0.59 ± 0.13,0.52 ±0.19;TLR4 mRNA 0.67 ±0.09,0.64 ±0.09,0.51 ±0.13) were significantly lower than LPS group (1.04 ± 0.38,0.82 ±0.18) (P ＜0.01) (P ＜0.05).Conclusion VIP and/or MP can mitigate intestinal injury induced by LPS shock.The gastrointestinal protection of VIP and glucocorticoids were related to downregulation signaling TLR2 mRNA and TLR4 mRNA expression.But VIP/MP and VIP + MP have no significant effect on expression of intestinal TLR2/4 mRNA until 24 h after LPS shock. Key words: Intestine ; Toll-like receptor; Lipopolysaccharide shock; Vasoactive intestinal peptide ; Glucocorticoids ; Rat

Creating a “pro-survival” phenotype through epigenetic modulation

We demonstrated recently that treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, improved survival in a rodent model of lipopolysaccharide (LPS)-induced endotoxic shock. The precise mechanisms, however, have not been well-defined. The aim of this study was to investigate the impact of SAHA treatment on gene expression profiles at an early stage of shock. Male C57BL/6J mice were treated with or without SAHA (50 mg/kg, IP), followed by a lethal dose of LPS (20 mg/kg, IP) and a second dose of SAHA. Lungs of the animals (LPS and SAHA+LPS groups; n= 3 per group) were harvested 3 hours post-LPS insult. Sham mice (no LPS and no SAHA) served as controls. RNA was isolated from the tissues and gene expression was analyzed using Affymatrix microarray (23,000 genes). A lower confidence bound of fold change was determined for comparison of LPS versus SAHA + LPS, and genes with a lower confidence bound of >2 were considered to be differentially expressed. Reverse transcriptase polymerase chain reaction, Western blotting, and tissue staining were performed to verify the key changes. Network graphs were used to determine gene interaction and biologic relevance. The expression of many genes known to be involved in septic pathophysiology changed after the LPS insult. Interestingly, a number of genes not implicated previously in the septic response were also altered. SAHA treatment attenuated expression of several key genes involved in inflammation. It also decreased neutrophil infiltration in lungs and histologic evidence of acute lung injury. Further analysis confirmed genes engaged in the cellular and humoral arms of the innate immune system were specifically inhibited by SAHA. Gene network analysis identified numerous molecules for the potential development of targeted therapies. Administration of SAHA in a rodent model of LPS shock rapidly modulates gene transcription, with an attenuation of inflammatory mediators derived from both arms (cellular and humoral) of the innate immune system. This may be a novel mechanism responsible for the survival advantage seen with SAHA treatment.

Inhibition of mTOR blocks the anti-inflammatory effects of glucocorticoids in myeloid immune cells

AbstractA central role for the mammalian target of rapamycin (mTOR) in innate immunity has been recently defined by its ability to limit proinflammatory mediators. Although glucocorticoids (GCs) exert potent anti-inflammatory effects in innate immune cells, it is currently unknown whether the mTOR pathway interferes with GC signaling. Here we show that inhibition of mTOR with rapamycin or Torin1 prevented the anti-inflammatory potency of GC both in human monocytes and myeloid dendritic cells. GCs could not suppress nuclear factor-κB and JNK activation, the expression of proinflammatory cytokines, and the promotion of Th1 responses when mTOR was inhibited. Interestingly, long-term activation of monocytes with lipopolysaccharide enhanced the expression of TSC2, the principle negative regulator of mTOR, whereas dexamethasone blocked TSC2 expression and reestablished mTOR activation. Renal transplant patients receiving rapamycin but not those receiving calcineurin inhibitors displayed a state of innate immune cell hyper-responsiveness despite the concurrent use of GC. Finally, mTOR inhibition was able to override the healing phenotype of dexamethasone in a murine lipopolysaccharide shock model. Collectively, these data identify a novel link between the glucocorticoid receptor and mTOR in innate immune cells, which is of considerable clinical importance in a variety of disorders, including allogeneic transplantation, autoimmune diseases, and cancer.

Structure-Activity Studies and Therapeutic Potential of Host Defense Peptides of Human Thrombin

Peptides of the C-terminal region of human thrombin are released upon proteolysis and identified in human wounds. In this study, we wanted to investigate minimal determinants, as well as structural features, governing the antimicrobial and immunomodulating activity of this peptide region. Sequential amino acid deletions of the peptide GKYGFYTHVFRLKKWIQKVIDQFGE (GKY25), as well as substitutions at strategic and structurally relevant positions, were followed by analyses of antimicrobial activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive bacterium Staphylococcus aureus, and the fungus Candida albicans. Furthermore, peptide effects on lipopolysaccharide (LPS)-, lipoteichoic acid-, or zymosan-induced macrophage activation were studied. The thrombin-derived peptides displayed length- and sequence-dependent antimicrobial as well as immunomodulating effects. A peptide length of at least 20 amino acids was required for effective anti-inflammatory effects in macrophage models, as well as optimal antimicrobial activity as judged by MIC assays. However, shorter (>12 amino acids) variants also displayed significant antimicrobial effects. A central K14 residue was important for optimal antimicrobial activity. Finally, one peptide variant, GKYGFYTHVFRLKKWIQKVI (GKY20) exhibiting improved selectivity, i.e., low toxicity and a preserved antimicrobial as well as anti-inflammatory effect, showed efficiency in mouse models of LPS shock and P. aeruginosa sepsis. The work defines structure-activity relationships of C-terminal host defense peptides of thrombin and delineates a strategy for selecting peptide epitopes of therapeutic interest.

Hemodynamics and aortic tension induced by two septic shock models in rats

To compare the ventricular-dynamic parameters and thoracic aorta tension induced by two septic shock models in rats. Septic shock models were induced by cecal ligation or puncture (CLP) and intraperitoneal injection of lipopolysaccharide (LPS) in rats. The carotid artery was cannulated and connected to a pressure transducer to determine mean arterial blood pressure (MABP). Ventricular dynamic parameters, including heart rate (HR), left ventricular developed pressure (LVDP) and maximal rise/fall velocity of ventricular pressure (± dP/dtmax) were determined. Isolated thoracic rings were mounted on an organ bath and the tension of the vessel was recorded. The mortality was 65.2% in CLP shock rats, but no death in LPS shock rats. The MABP and HR of CLP rats were decreased more prominently than those of LPS rats (P < 0.01). Contraction induced by high K(+) (60 mmol/L) or 10⁻⁶ mol/L phenylephrine (PE) in endothelium-intact and endothelium-denuded aortic rings was all attenuated, but in LPS rats it was more prominent (P < 0.01). Two rat septic shock models can decrease ventricular-dynamic parameters and vasoconstriction responsiveness of aorta. The ventricular-dynamic parameters decrease more prominently in CLP model, while vasoconstriction responsiveness of aorta changes more in LPS model.

A probiotic strain of Escherichia coli, Nissle 1917, given orally exerts local and systemic anti‐inflammatory effects in lipopolysaccharide‐induced sepsis in mice

Escherichia coli Nissle 1917 is a probiotic strain used in the treatment of intestinal immune diseases, including ulcerative colitis. The aim of the present study was to test if this probiotic bacterium can also show systemic immunomodulatory properties after oral administration. The probiotic strain was administered to rats or mice for 2 weeks before its assay in two experimental models of altered immune response, the trinitrobenzenesulphonic acid (TNBS) model of rat colitis, localized in the colon, and the lipopolysaccharide (LPS) model of systemic septic shock in mice. Inflammatory status was evaluated both macroscopically and biochemically after 1 week in the TNBS model or after 24 h in the LPS shock model. In addition, splenocytes were obtained from mice and stimulated, ex vivo, with concanavalin A or LPS to activate T or B cells, respectively, and cytokine production (IL-2, IL-5 and IL-10) by T cells and IgG secretion by B cells measured. E. coli Nissle 1917 was anti-inflammatory in both models of altered immune response. This included a reduction in the pro-inflammatory cytokine tumour necrosis factor-alpha both in the intestine from colitic rats, and in plasma and lungs in mice treated with LPS. The systemic beneficial effect was associated with inhibited production of the T cell cytokines and by down-regulation of IgG release from splenocyte-derived B cells. The anti-inflammatory effects of E. coli Nissle 1917 given orally were not restricted to the gastrointestinal tract.

Differential Effects of GdCl3‐ or MDP Treatment on Rat Liver Microcirculation and Gene Expression in the Hepatic Non‐Parenchymal Cell Fraction in LPS Shock

Dichloromethylenebisphosphonate (MDP) and gadolinium chloride (GdCl(3)) are substances frequently used for experimental depletion of Kupffer Cells (KC) in models of endotoxin shock. The aim was to determine whether depletion of KC through pretreatment with GdCl(3) or MDP alters the hepatic microcirculation during lipopolysaccharide (LPS)-induced shock in rats and to test if there are substance-specific differences. Rats received either MDP or GdCl(3) or saline prior to induction of LPS shock. Hepatic microcirculation was evaluated by intravital microscopy (sinusoidal diameter, sinusoidal bloodflow, leukocyte adhesion), and the gene expression in the hepatic non-parenchymal cell fraction was determined by RT-PCR. GdCl(3) pretreatment prevented sinusoidal narrowing but did not restore sinusoidal blood flow and did not normalize leukocyte-endothelial interaction time after LPS. In contrast, MDP pretreatment improved hepatic microcirculation consistently for all parameters measured compared to GdCl(3) pretreated animals. In the non-parenchymal cell fraction, eNOS gene expression was preserved and gene expression of TNF-alpha was blocked after MDP but not after GdCl(3) application prior to LPS shock. The results show that GdCl(3) and MDP cannot be used equivalently for experimental KC depletion in the condition of LPS-induced shock. These findings should be taken into consideration in studies that evaluate the role of Kupffer cells in models of endotoxin-induced shock.

Heat shock inhibits lipopolysaccharide-induced tissue factor activity in human whole blood

BackgroundDuring gram-negative sepsis, lipopolysaccharide (LPS) induces tissue factor expression on monocytes. The resulting disseminated intravascular coagulation leads to tissue ischemia and worsens the prognosis of septic patients. There are indications, that fever reduces the mortality of sepsis, the effect on tissue factor activity on monocytes is unknown. Therefore, we investigated whether heat shock modulates LPS-induced tissue factor activity in human blood.MethodsWhole blood samples and leukocyte suspensions, respectively, from healthy probands (n = 12) were incubated with LPS for 2 hours under heat shock conditions (43°C) or control conditions (37°C), respectively. Subsequent to further 3 hours of incubation at 37°C the clotting time, a measure of tissue factor expression, was determined. Cell integrity was verified by trypan blue exclusion test and FACS analysis.ResultsIncubation of whole blood samples with LPS for 5 hours at normothermia resulted in a significant shortening of clotting time from 357 ± 108 sec to 82 ± 8 sec compared to samples incubated without LPS (n = 12; p < 0.05). This LPS effect was mediated by tissue factor, as inhibition with active site-inhibited factor VIIa (ASIS) abolished the effect of LPS on clotting time. Blockade of protein synthesis using cycloheximide demonstrated that LPS exerted its procoagulatory effect via an induction of tissue factor expression. Upon heat shock treatment, the LPS effect was blunted: clotting times were 312 ± 66 s in absence of LPS and 277 ± 65 s in presence of LPS (n = 8; p > 0.05). Similarly, heat shock treatment of leukocyte suspensions abolished the LPS-induced tissue factor activity. Clotting time was 73 ± 31 s, when cells were treated with LPS (100 ng/mL) under normothermic conditions, and 301 ± 118 s, when treated with LPS (100 ng/mL) and heat shock (n = 8, p < 0.05). Control experiments excluded cell damage as a potential cause of the observed heat shock effect.ConclusionHeat shock treatment inhibits LPS-induced tissue factor activity in human whole blood samples and isolated leukocytes.

Glutamine induces heat-shock protein and protects against Escherichia coli lipopolysaccharide-induced vascular hyporeactivity in rats

IntroductionVascular hyporeactivity is an important problem associated with sepsis. Although the mechanism involves inflammatory pathway activation, specific therapeutic approaches have not been defined. Glutamine (Gln) has been shown to provide some anti-inflammatory effects and improve outcomes in sepsis. Here, we tested the hypothesis that Gln could reduce Escherichia coli lipopolysaccharide (LPS)-induced vascular hyporeactivity and evaluated the role of heat-shock protein 70 (HSP70) induction in this process.MethodsTwenty-four male Sprague-Dawley rats were divided into control, LPS shock, and alanyl-Gln dipeptide+LPS shock (Ala-Gln+LPS) groups. Six hours after administration of LPS, phenylephrine (PE) (0.5 to approximately 2.5 μg/kg) was applied intravenously to all groups, and the percentage increase in mean arterial pressure (MAP) was detected in the respective groups. The concentration-response curve of PE was obtained in tension experiments, and the average values of PE maximum efficacy (Emax) and median effective dose (EC50) were calculated. The plasma concentrations of malondialdehyde (MDA), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) were detected in all groups. The expressions of HSP70 from heart, liver, lung, and aorta were also assayed in all groups.ResultsThe maximal percentage increase in MAP induced by PE was significantly reduced to 12.7% in the LPS shock group (P < 0.05) and was restored to 15.6% in the Ala-Gln+LPS group (P < 0.05), whereas the control group was 24.7%. The average values of PE Emax and EC50 were significantly impaired in the LPS shock group (P < 0.05) but partially restored in the Ala-Gln+LPS group (P < 0.05). The expressions of HSP70 from the heart, aorta, lung, and liver were much higher in the Ala-Gln+LPS group than those in the LPS shock group (P < 0.05). The plasma concentrations of TNF-α, IL-6, and MDA were much lower in the Ala-Gln+LPS group than those in the LPS shock group.ConclusionGln effectively improves vascular reactivity by inducing the expression of HSP70, reducing inflammatory cytokine release and peroxide biosynthesis in LPS shock rats. These results suggest that Gln has a potentially beneficial therapeutic effect for septic shock patients.

Immunobiological activities of a new nontoxic lipopolysaccharide fromAcidiphiliumGS18h/ATCC55963, a soil isolate from an Indian copper mine

A novel nontoxic lipopolysaccharide (LPS) was purified from Acidiphilium strain GS18h/ATCC55963. The chemical composition of the lipid A part of this LPS is distinctly different from that of known lipid A molecules. The LPS was investigated to determine its capacity to provide protection against toxic LPS or endotoxic shock, as has been reported for other nontoxic LPSs (Rhodobacter sphaeroides and Rhodobacter capsulatus), and also the extent and type of immunomodulatory response in terms of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-beta), and IL-6 release as well as NO secretion by stimulated monocyte-macrophage systems. This study demonstrates clearly that mice immunized or primed with this LPS are fully protected against challenge with toxic Escherichia coli LPS. Unlike most of the extensively studied nontoxic LPSs, this LPS induced reactive nitrogen intermediates and released TNF-alpha, IL-beta and IL-6 in both mouse and human monocyte-macrophage systems. However, the extent of the cytokine and lymphokine releasing response was well below the range of the toxic LPS, for example that of E. coli. Owing to its capacity to provide immunostimulation of the host without causing any lethality to ensure protection against endotoxic shock, this LPS appears to have potential therapeutic value.

THE INDUCTION OF SUPER-RESISTANCE USING SYNTHETIC LIPOPOLYSACCHARIDE RECEPTOR AGONIST RESCUES FATAL ENDOTOXEMIA IN RATS WITHOUT EXCESSIVE IMMUNOSUPPRESSION

Endotoxin tolerance provides protection against mortality under various conditions of stress. However, the induction of endotoxin tolerance thus far has no clinical application because of endotoxin toxicity and the excessive immune suppression that follows the tolerance induction. In this study, we examined whether a novel, synthetic lipopolysaccharide (LPS) receptor agonist, ER-803058 (ER) can induce endotoxin tolerance with accompanying low toxicity. The stimulative effects of ER on tumor necrosis factor (TNF)-alpha production from RAW264 cells were 50% to 70% lower than those of the corresponding quantities of LPS. ER pretreatment also diminished TNF-alpha secretion induced by a subsequent LPS shock. However, the degree of desensitization with ER pretreatment (10 ng/mL, 55.5% +/- 6.7%; 100 ng/mL, 42.3 +/- 4.9%) was modest in contrast with that measured for the corresponding LPS pretreatment (10 ng/mL, 36.7% +/- 3.7%; 100 ng/mL, 20.0% +/- 3.6%). The minimum in vivo dose (0.02 mg/kg/body weight) of ER-induced negligible production of TNF-alpha and interleukin (IL)-6 in rats, and resulted in a modest endotoxin tolerance with respect to TNF-alpha secretion. Although the plasma TNF-alpha level after ER pretreatment was decreased (48.2% +/- 1.1%), the suppression was not statistically significant. Interestingly, even this minimal quantity of ER pretreatment evoked a dramatic improvement in survival (90% survival) against administration of a lethal dose of LPS, which is inconsistent with the modest TNF-alpha suppression. Furthermore, ER pretreatment preserved normal plasma albumin levels and prevented the increase of plasma blood urea nitrogen levels seen with LPS. These results indicate that pretreatment with ER can effectively induce endotoxin tolerance, with a consequent improvement in mortality without toxicity and without subsequent excessive immunosuppression.

Distinct LPS-induced signals regulate LPS uptake and morphological changes in medfly hemocytes

Recently we demonstrated that lipopolysaccharide (LPS) promotes activation of the Ras/ERK cascade in medfly hemocytes and that phagocytosis of Escherichia coli by insect hemocytes is mediated by an integrin-dependant process via the activation of FAK/Src complex (J Biol Chem 273 (1998) 14813; FEBS Letters 496 (2001) 55). In the current study we wanted to further elucidate the effects of LPS on medfly hemocytes, in order to better understand the regulation of the evolutionary conserved signaling mechanisms between insects and mammals. We initially observed that different stimuli, including LPS, E. coli, RGD, fibronectin and heat shock activate hemocyte ERK. The response of hemocytes to these stimuli denoted that hemocyte ERK is evidently stimulated by at least an LPS receptor and via an integrin-mediated process. The medfly hemocytes respond to LPS by changing their morphology, inducing the activation of several signaling pathways, including Ras/MEK/ERK, PI-3K/ERK and Rho pathways and contributing to LPS uptake. Experiments based on inhibitors of specific signaling pathways, such as manumycin A, toxin A, U0126, PD98059 and wortmannin revealed that Ras, MEK and PI-3K are involved in the activation of ERK. Whether PI-3K is an intermediate of Ras/MEK/ERK pathway or activates ERK via other signaling pathway it remains to be elucidated. ERK is not activated via Rho pathway, denoting that Rho may not be an upstream effector molecule of ERK pathway. Regarding the role(s) that these kinases play in hemocytes, it can be suggested that PI-3K and Rho GTPases can modulate hemocyte shape changes, whereas ERK, Ras and MEK cannot. In addition, PI-3K as well as Ras and MEK through ERK activation participate in LPS endocytosis. Therefore, PI-3K shares a dual role; it is involved both in cell shape changes and in LPS endocytosis. Since ERK activation appears to be independent of the integrity of actin filaments, as cytochalasin D and latrunculin A did not block ERK activation, it can be concluded that LPS endocytosis is independent of actin cytoskeleton remodeling as is the case in mammalian systems.

Comparative Study of Several Lymphocyte Functions in Two Strains of Mice with Different Models of Endotoxic Shock.

Previously, the changes in phagocyte functions such as adherence, chemotaxis or TNFa production were found to be associated with oxidative stress in endotoxin-induced septic shock. However, in this type of oxidative stress the lymphocyte involvement has rarely been studied. In the present report, we analyzed the above functions in peritoneal lymphocytes from male and female BALB/c mice with a lethal endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg), male and female Swiss mice with lethal endotoxic shock caused by intraperitoneal injection of LPS (150 and 250 mg/kg, respectively) or non-lethal endotoxic shock (100 mg/kg). In peritoneal lymphocytes obtained at 0, 2, 4, 12 or 24 h after LPS injection, the first two functions of these cells in the immune response, i.e. adherence to tissues and directed migration (chemotaxis), were studied. At 0, 0.5, 1, 1.5, 2, 4, 12 and 24 h after LPS injection, TNFa released by lymphocytes was also analyzed. The results show that endotoxic shock increases the adherence and TNFa release, and decreases the chemotaxis of peritoneal lymphocytes. These changes were more significant in mice with lethal than with non-lethal endotoxic shock, a fact that confirms the important role of lymphocytes during endotoxic shock.