Mouse Model Of Endotoxemia Research Articles

Ethanol Extract of Potentilla supina Linne Suppresses LPS-induced Inflammatory Responses through NF-κB and AP-1 Inactivation in Macrophages and in Endotoxic mice.

In this study, we investigated the antiinflammatory effects of ethanol extracts of Potentilla. supina Linne (EPS) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and septic mice. EPS suppressed LPS-induced nitric oxide, prostaglandin E2 , TNF-α, interleukin-6 and interleukin-1β at production and mRNA levels in LPS-induced RAW 264.7 macrophages. Consistent with these observations, EPS attenuated the expressions of inducible nitric oxide synthase and cyclooxygenase-2 by downregulation of their promoter activities. Molecularly, EPS reduced the LPS-induced transcriptional activity and DNA-binding activity of nuclear factor-κB (NF-κB), and this was associated with a decrease of translocation and phosphorylation of p65 NF-κB by inhibiting the inhibitory κB-α degradation and IKK-α/β phosphorylation. Furthermore, EPS inhibited the LPS-induced activation of activator protein-1 by reducing the expression of c-Fos and c-Jun in nuclear. EPS also suppressed the phosphorylation of mitogen-activated protein kinase, such as p38 mitogen-activated protein kinase and c-Jun N-terminal kinase. In an LPS-induced endotoxemia mouse model, pretreatment with EPS reduced the mRNA levels of inducible nitric oxide synthase, cyclooxygenase-2 and proinflammatory cytokines and increased the survival rate of mice. Collectively, these results suggest that the antiinflammatory effects of EPS were associated with the suppression of NF-κB and activator protein-1 activation and support its possible therapeutic role for the treatment of endotoxemia. Copyright © 2017 John Wiley & Sons, Ltd.

Evening primrose oil or forskolin ameliorates celecoxib-enhanced upregulation of tissue factor expression in mice subjected to lipopolysaccharide-induced endotoxemia.

Celecoxib, a selective cyclooxygenase-2 inhibitor, produces thrombotic events in patients predisposed to cardiovascular risk factors. One theory reported an increase in endothelial expression of tissue factor (TF) as a predisposing factor. This work explored the effect of evening primrose oil (EPO), a source of prostaglandin E1, and forskolin (a cyclic adenosine monophosphate stimulator) against the prothrombotic effect of celecoxib in mice. Lipopolysaccharide mouse model of endotoxemia was used to induce an upregulation of TF activity. Male mice received celecoxib (25mg/kg), celecoxib plus EPO, or celecoxib plus forskolin for 4weeks and then subjected to a prothrombotic challenge in the form of an intraperitoneal injection of lipopolysaccharide. Results showed an increase in plasma TF activity, endothelial TF expression, and thrombin-antithrombin (TAT) but lower antithrombin III (ATIII) level in mice that received celecoxib in comparison to those that received the vehicle. Adding EPO or forskolin to celecoxib regimen significantly decreased the prothrombotic effect of celecoxib. A positive correlation (r=0.8501) was found between TF activity and TAT. Co-administration of EPO or forskolin decreased the activity of TF and mitigated the prothrombotic effect of celecoxib. Therefore, these combinations may have the utility to abrogate the prothrombotic adverse effect of celecoxib in clinical setting.

Acanthopanax senticosus polysaccharides-induced intestinal tight junction injury alleviation via inhibition of NF-κB/MLCK pathway in a mouse endotoxemia model.

AIMTo examine the effects of Acanthopanax senticosus polysaccharides (ASPS) on intestinal tight junction (TJ) disruption and nuclear factor-kappa B (NF-κB)/myosin light chain kinase (MLCK) activation in endotoxemia.METHODSBALB/C mice (6-8-weeks-old) received continuous intragastric gavage of ASPS for 7 d before injection of lipopolysaccharide (LPS), or received ASPS once after LPS injection. Blood and intestinal mucosal samples were collected 6 h after LPS challenge. Clinical symptoms, histological injury, intestinal permeability, TJ ultrastructure, and TJ protein expression were determined.RESULTSCompared with mice in the LPS group, pretreatment with ASPS improved clinical and histological scores by 390.9% (P < 0.05) and 57.89% (P < 0.05), respectively, and gut permeability change in endotoxemic mice was shown by a 61.93% reduction in reduced leakage of fluorescein isothiocyanate-dextran 6 h after LPS injection (P < 0.05). ASPS pretreatment also prevented LPS-induced TJ ultrastructure breakdown supported by increased electron dense materials between adjoining cells, sustained redistribution and expression of occludin (0.597 ± 0.027 vs 0.103 ± 0.009, P < 0.05) and zonula occludens-1 (0.507 ± 0.032 vs 0.125 ± 0.019, P < 0.05), and suppressed activation of the NF-κB/MLCK pathway indicated by reduced expression of NF-κB, phospho-inhibitor kappa B-alpha, MLCK and phospho-myosin light-chain-2 by 16.06% (P < 0.05), 54.31% (P < 0.05), 66.10% (P < 0.05) and 64.82% (P < 0.05), respectively.CONCLUSIONASPS pretreatment may be associated with inhibition of the NF-κB/MLCK pathway and concomitant amelioration of LPS-induced TJ dysfunction of intestinal epithelium in endotoxemia.

Protective effect of hemopexin on systemic inflammation and acute lung injury in an endotoxemia model

BackgroundHemopexin (HPX) has been identified as an anti-inflammatory agent, but its role in endotoxemia is unclear. The purpose of this study was to determine whether HPX suppresses systemic and lung inflammation in a mouse model of endotoxemia. Materials and methodsAt 30 min of intraperitoneal administration of lipopolysaccharide (LPS; 10 mg/kg), either distilled water (LPS-only treated animals) or HPX (5 mg/kg) was injected into mice via the tail vein, and the survival rates were analyzed after 36 h. Furthermore, the serum levels of tumor necrosis factor-α, interleukin-6 (IL-6), and HPX were determined at 0, 3, and 6 h, and the expression levels and DNA binding activities of phosphorylated cytoplasmic inhibitor κB-α, nuclear factor-κB (NF-κB), and the p65 subunit of NF-κB were evaluated and compared with the rates of histologic lung injury after 6 h. ResultsSerum tumor necrosis factor-α and interleukin-6 levels were decreased in HPX-treated animals at 3 and 6 h (P < 0.05). HPX suppressed the NF-κB pathway (P < 0.05) and reduced acute lung injury at 6 h, and 36 h after initial treatment, the survival rate was higher in HPX-treated animals than that in LPS-treated animals (P < 0.05). ConclusionsHPX downregulated proinflammatory cytokine production and acute lung injury as well as improved survival rates in a mouse model of endotoxemia. These effects were associated with HPX-mediated suppression of the NF-κB pathway.

Effects of lipopolysaccharide on changes in red blood cells in a mice endotoxemia model.

The aim of this study was to evaluate changes in RBC aggregation and deformability over 24 hr and suggest specific shear stress values for detecting RBC deformability in a mouse endotoxemia model using lipopolysaccharide (LPS).Six-week-old male BALB/c mice received LPS (20 mg/kg) intraperitoneally. Aggregation indices (AIs) and T1/2 were measured to assess RBC aggregation, and elongation indices (EIs) were used to assess RBC deformability at shear stress values of 0.3, 0.5, 1, 3, 7, 10, 15 and 20 Pascals (Pa) 0, 30 min, 1, 2, 4, 6, 9, 12, 18 and 24 hours after the LPS injection.No significant differences were detected in the AIs during the study period, however, T1/2 shortened significantly 2, 6, 12, 18, and 24 hr after the LPS injection. The EIs increased significantly 24 hr after LPS injection at 0.5 and 1 Pa shear stress, whereas it decreased significantly at 10 Pa of shear stress 24 hr after the LPS injection.Altered RBC deformability was detectable 24 hr after the LPS injection and T1/2 may be a sensitive marker for detecting changes in RBC aggregation. The EIs should be measured at 1 Pa to detect changes in RBC deformability in LPS-induced septic mice.

Administration of AMD3100 in endotoxemia is associated with pro-inflammatory, pro-oxidative, and pro-apoptotic effects in vivo.

BackgroundChemokine receptor 4 (CXCR4) is a multifunctional G protein-coupled receptor that is activated by its natural ligand, C-X-C motif chemokine 12 (CXCL12). As a likely member of the lipopolysaccharide (LPS)-sensing complex, CXCR4 is involved in pro-inflammatory cytokine production and exhibits substantial chemo-attractive activity for various inflammatory cells. Here, we aimed to characterize the effects of CXCR4 blockade in systemic inflammation and to evaluate its impact on organ function. Furthermore, we investigated whether CXCR4 blockade exerts deleterious effects, thereby substantiating previous studies showing a beneficial outcome after treatment with CXCR4 agonists in endotoxemia.MethodsThe CXCR4 antagonist AMD3100 was administered intraperitoneally to mice shortly after LPS treatment. After 24 h, health status was determined and serum tumor necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma), and nitric oxide (NO) levels were measured. We further assessed oxidative stress in the brain, kidney, and liver as well as liver biotransformation capacity. Finally, we utilized immunohistochemistry and immunoblotting in liver and spleen tissue to determine cluster of differentiation 3 (CD3), CD8, CD68, and TNF alpha expression patterns, and to assess the presence of various markers for apoptosis and oxidative stress.ResultsMice treated with AMD3100 displayed impaired health status and showed enhanced serum levels of TNF alpha, IFN gamma and NO levels in endotoxemia. This compound also amplified LPS-induced oxidative stress in all tissues investigated and decreased liver biotransformation capacity in co-treated animals. Co-treatment with AMD3100 further inhibited expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf-2), heme oxygenase-1 (HO-1), and various cytochrome P450 enzymes, whereas it enhanced expression of CD3, inducible nitric oxide synthase, and TNF alpha, as well as the total number of neutrophils in liver tissue. Spleens from co-treated animals contained large numbers of erythrocytes and neutrophils, but fewer CD3+ cells, and demonstrated increased apoptosis in the white pulp.ConclusionsAMD3100 administration in a mouse model of endotoxemia further impaired health status and liver function and mediated pro-inflammatory, pro-oxidative, and pro-apoptotic effects. This suggests that interruption of the CXCR4/CXCL12 axis is deleterious in acute inflammation and confirms previous findings showing beneficial effects of CXCR4 agonists in endotoxemia, thereby more clearly elucidating the role of CXCR4 in inflammation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-016-0286-8) contains supplementary material, which is available to authorized users.

Local Microenvironment Controls the Compartmentalization of NK Cell Responses during Systemic Inflammation in Mice

Systemic inflammatory response syndrome is a whole-body reaction to a triggering insult that often results in life-threatening illness. Contributing to the development of this inflammatory cascade are numerous cellular partners, among which NK cells were shown to play a key role. Accumulating evidence points to organ-specific properties of systemic inflammation and NK cells. However, little is known about compartment-specific activation of NK cells during systemic inflammatory response syndrome or the relative contribution of NK cell-intrinsic properties and microenvironmental cues. In this study, we undertook a sequential characterization of NK responses in the spleen, lungs, bone marrow, peritoneum, and blood using a mouse model of endotoxemia. We report that, despite similar systemic dynamics of NK cell responses, expression of activation markers (CD69 and CD25) and effector molecules (IFN-γ, granzyme B, and IL-10) display organ-specific thresholds of maximum activation. Using adoptive transfers of spleen and lung NK cells, we found that these cells have the capacity to quickly adapt to a new environment and adjust their response levels to that of resident NK cells. This functional adaptation occurs without significant alterations in phenotype and independently of subpopulation-specific trafficking. Thus, using a dynamic in vivo-transfer system, to our knowledge our study is the first to report the compartmentalization of NK cells responses during systemic inflammation and to show that NK cell-intrinsic properties and microenvironmental cues are involved in this process, in a sequential manner.

Zein nanoparticles for oral delivery of quercetin: Pharmacokinetic studies and preventive anti-inflammatory effects in a mouse model of endotoxemia

Zein nanoparticles were evaluated as nanocarriers to promote the oral bioavailability of quercetin and, thus, improve its anti-inflammatory effect on a mouse model of induced endotoxemia. For this purpose, the flavonoid and 2-hydroxypropyl-β-cyclodextrin were encapsulated in zein nanoparticles. The resulting nanoparticles displayed a mean size of about 300nm and the payload was calculated to be close to 70μg/mg nanoparticle. The release of quercetin from zein nanoparticles followed a zero-order kinetic. After oral administration, nanoparticles provided high and sustained levels of quercetin in plasma and the relative oral bioavailability was calculated to be approx. 60%. Animals treated with quercetin-loaded nanoparticles (1 dose every two days; 1week) presented endotoxic symptoms less severe than those observed in animals treated with the oral solution of the flavonoid (1 dose every day; 1week). This was further corroborated by the significantly low circulating TNF-alpha in the quercetin-loaded nanoparticles treated mice.

GYY4137 ameliorates intestinal barrier injury in a mouse model of endotoxemia

Intestinal barrier injury has been reported to play a vital role in the pathogenesis of endotoxemia. This study aimed to investigate the protective effect of GYY4137, a newly synthesized H2S donor, on the intestinal barrier function in the context of endotoxemia both in vitro and in vivo. Caco-2 (a widely used human colon cancer cell line in the study of intestinal epithelial barrier function) monolayers incubated with lipopolysaccharide (LPS) or TNF-α/IFN-γ and a mouse model of endotoxemia were used in this study.The results suggested that GYY4137 significantly attenuated LPS or TNF-α/IFN-γ induced increased Caco-2 monolayer permeability. The decreased expression of TJ (tight junction) proteins induced by LPS and the altered localization of TJs induced by TNF-α/IFN-γ was significantly inhibited by GYY4137; similar results were obtained in vivo. Besides, GYY4137 promoted the clinical score and histological score of mice with endotoxemia. Increased level of TNF-α/IFN-γ in the plasma and increased apoptosis in colon epithelial cells was also attenuated by GYY4137 in mice with endotoxemia. This study indicates that GYY4137 preserves the intestinal barrier function in the context of endotoxemia via multipathways and throws light on the development of potential therapeutic approaches for endotoxemia.

HDAC6 inhibition prevents TNF-α-induced caspase 3 activation in lung endothelial cell and maintains cell-cell junctions.

Pro-inflammatory mediators such as TNF-α induce caspase activation in endothelial cells, which leads to degradation of cellular proteins, induction of apoptotic signaling, and endothelial cell dysfunction. New therapeutic agents that can inhibit caspase activation may provide protection against inflammatory injury to endothelial cells. In the present study, we examined the effects of selective histone deacetylase 6 (HDAC6) inhibition on TNF-α induced caspase 3 activation and cell-cell junction dysfunction in lung endothelial cells. We also assessed the protective effects of HDAC6 inhibition against lung inflammatory injury in a mouse model of endotoxemia. We demonstrated that selective HDAC6 inhibition or knockdown of HDAC6 expression was able to prevent caspase 3 activation in lung endothelial cells and maintain lung endothelial cell-cell junctions. Mice pre-treated with HDAC6 inhibitors exhibited decreased endotoxin-induced caspase 3 activation and reduced lung vascular injury as indicated by the retention of cell-cell junction protein VE-Cadherin level and alleviated lung edema. Collectively, our data suggest that HDAC6 inhibition is a potent therapeutic strategy against inflammatory injury to endothelial cells.

Myeloid cell-specific inositol polyphosphate-4-phosphatase type I knockout mice impair bacteria clearance in a murine peritonitis model

Phosphatidylinositol 3-kinase (PI3K)/Akt signaling has been implicated in the anti-inflammatory response in a mouse model of endotoxemia and sepsis. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a), which dephosphorylates PtdIns(3,4)P2 to PtdIns(3)P, in bacterial infections. We prepared myeloid cell-specific Inpp4a-conditional knockout mice. Macrophages from these mice showed increased Akt phosphorylation and reduced production of inflammatory cytokines in response to LPS or Escherichia coli invitro The Inpp4a knockout mice survived for a shorter time than wild type mice after i.p. infection with E. coli, with less production of inflammatory cytokines. Additionally, E. coli clearance from blood and lung was significantly impaired in the knockout mice. A likely mechanism is that the Inpp4a-catalyzed dephosphorylation of PtdIns(3,4)P2 down-regulates Akt pathways, which, in turn, increases the production of inflammatory mediators. This mechanism at least fits the decreased E. coli clearance and short survival in the Inpp4a knockout mice.

Saikosaponin a inhibits LPS-induced inflammatory response by inducing liver X receptor alpha activation in primary mouse macrophages.

The aim of this study was to investigate the effects of SSa on LPS-induced endotoxemia in mice and clarify the possible mechanism. An LPS-induced endotoxemia mouse model was used to confirm the anti-inflammatory activity of SSa in vivo. The primary mouse macrophages were used to investigate the molecular mechanism and targets of SSa in vitro. In vivo, the results showed that SSa improved survival during lethal endotoxemia. In vitro, our results showed that SSa dose-dependently inhibited the expression of TNF-α, IL-6, IL-1β, IFN-β-and RANTES in LPS-stimulated primary mouse macrophages. Western blot analysis showed that SSa suppressed LPS-induced NF-κB and IRF3 activation. Furthermore, SSa disrupted the formation of lipid rafts by depleting cholesterol and inhibited TLR4 translocation into lipid rafts. Moreover, SSa activated LXRα, ABCA1 and ABCG1. Silencing LXRα abrogated the effect of SSa. In conclusion, the anti-inflammatory effects of SSa is associated with activating LXRα dependent cholesterol efflux pathway which result in disrupting lipid rafts by depleting cholesterol and reducing translocation of TLR4 to lipid rafts, thereby attenuating LPS mediated inflammatory response.

Selective HDAC6 inhibition prevents TNF-α-induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema.

Lung endothelial damage contributes to the pathogenesis of acute lung injury. New strategies against lung endothelial barrier dysfunction may provide therapeutic benefits against lung vascular injury. Cell-cell junctions and microtubule cytoskeleton are basic components in maintaining endothelial barrier integrity. HDAC6, a deacetylase primarily localized in the cytoplasm, has been reported to modulate nonnuclear protein function through deacetylation. Both α-tubulin and β-catenin are substrates for HDAC6. Here, we examined the effects of tubastatin A, a highly selective HDAC6 inhibitor, on TNF-α induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema. Selective HDAC6 inhibition by tubastatin A blocked TNF-α-induced lung endothelial cell hyperpermeability, which was associated with increased α-tubulin acetylation and microtubule stability. Tubastatin A pretreatment inhibited TNF-α-induced endothelial cell contraction and actin stress fiber formation with reduced myosin light chain phosphorylation. Selective HDAC6 inhibition by tubastatin A also induced β-catenin acetylation in human lung endothelial cells, which was associated with increased membrane localization of β-catenin and stabilization of adherens junctions. HDAC6 knockdown by small interfering RNA also prevented TNF-α-induced barrier dysfunction and increased α-tubulin and β-catenin acetylation in endothelial cells. Furthermore, in a mouse model of endotoxemia, tubastatin A was able to prevent endotoxin-induced deacetylation of α-tubulin and β-catenin in lung tissues, which was associated with reduced pulmonary edema. Collectively, our data indicate that selective HDAC6 inhibition by tubastatin A is a potent approach against lung endothelial barrier dysfunction.

Restoring Antiendotoxin Activities of Inactive Antimicrobial Peptides

Gram‐negative bacteria cell wall is composed of two layers: the inner phospholipid membrane and the outer membrane with a highly conserved unique lipid called lipopolysaccharide (LPS; endotoxin). LPS acts as a permeability barrier against a number of bactericidal agents. Furthermore, LPS is well known as a potent inducer of the immune system when it is released to blood and often causes septic shock syndromes in human. Some but not every antimicrobial peptide can neutralize LPS stimulated proinflammatory responses. We have developed an easy method to restore antiendotoxin activities of inactive antimicrobial peptides by linking two inactive α‐helical peptides with the AGP sequence. Antibacterial activities were determined by the standard broth micro‐dilution method of National Committee for Clinical Laboratory Standards. Toxicities of the peptides were determined from measuring cell death by the MTT assay against human fibroblast (HFW cells). Dynamic light scattering (DLS) was used to measure the size increase of LPS in the presence of the peptides. The ability of LPS neutralization in vitro was measured by Limulus Amebocyte Lysate (LAL) assay. LPS can induce nitrite oxide (NO) and TNF‐α production in macrophage cells. Experimental mouse endotoxemia model was used to test the antiendotoxin activities of the peptides. Significant antiendotoxin effects were observed in vitro and in vivo with the AGP linked variant of the short α‐helical peptides KR12 and WR6. The results will be used to help us design more potent antimicrobial peptides for clinical application in the future.Support or Funding InformationGrant Number 103‐2113‐M‐007‐009‐MY2, Ministry of Science and Technology, Taiwan

Changes in the levels of liver HSP70, plasma nitric oxide, and the antioxidative system in an experimentally induced endotoxemia mouse model and the role of reduced glutathione

Endotoxin molecules in lipopolysaccharides are among most important molecules that initiate a cascade of events in sepsis/endotoxemia. Lipopolysaccharide exposure may result in strong immune responses, disrupt the intracellular oxidant/antioxidant balance, and cause excessive reactive oxygen species generation. The purpose of the study was to examine if reduced glutathione (GSH) has a protective role against lipopolysaccharides. The effects of lipopolysaccharide (LPS) or GSH alone or in combination on the levels of the plasma antioxidant system, NO, and liver HSP70 were investigated. A total of 100 Swiss albino mice were divided into 4 groups as Group I (control), Group II (20 μg/kg LPS), Group III (10 mg/kg GSH), and Group IV (20 μg/kg LPS + 10 mg/kg GSH). Blood and liver samples both pre- and post-LPS and/or GSH injections after 1, 3, and 6 h were collected. Total antioxidant capacity was demonstrated with a reduction in response to lipopolysaccharide. Total oxidant capacity was higher after the injection of lipopolysaccharide alone or in combination with GSH. NO levels were elevated in response to lipopolysaccharide. The liver HSP70 level was determined to be higher in the lipopolysaccharide-treated group. These results indicate that exogenously administered GSH may have regulatory effects on liver HSP70 and plasma NO levels, and GSH treatment might have beneficial effects on antioxidant status by inhibiting the increase of oxidant molecules in endotoxemia-induced mice.

Levosimendan affects oxidative and inflammatory pathways in the diaphragm of ventilated endotoxemic mice.

IntroductionControlled mechanical ventilation and endotoxemia are associated with diaphragm muscle atrophy and dysfunction. Oxidative stress and activation of inflammatory pathways are involved in the pathogenesis of diaphragmatic dysfunction. Levosimendan, a cardiac inotrope, has been reported to possess anti-oxidative and anti-inflammatory properties. The aim of the present study was to investigate the effects of levosimendan on markers for diaphragm nitrosative and oxidative stress, inflammation and proteolysis in a mouse model of endotoxemia and mechanical ventilation.MethodsThree groups were studied: (1) unventilated mice (CON, n =8), (2) mechanically ventilated endotoxemic mice (MV LPS, n =17) and (3) mechanically ventilated endotoxemic mice treated with levosimendan (MV LPS + L, n =17). Immediately after anesthesia (CON) or after 8 hours of mechanical ventilation, blood and diaphragm muscle were harvested for biochemical analysis.ResultsMechanical ventilation and endotoxemia increased expression of inducible nitric oxide synthase (iNOS) mRNA and cytokine levels of interleukin (IL)-1β, IL-6 and keratinocyte-derived chemokine, and decreased IL-10, in the diaphragm; however, they had no effect on protein nitrosylation and 4-hydroxy-2-nonenal protein concentrations. Levosimendan decreased nitrosylated proteins by 10% (P <0.05) and 4-hydroxy-2-nonenal protein concentrations by 13% (P <0.05), but it augmented the rise of iNOS mRNA by 47% (P <0.05). Levosimendan did not affect the inflammatory response in the diaphragm induced by mechanical ventilation and endotoxemia.ConclusionsMechanical ventilation in combination with endotoxemia results in systemic and diaphragmatic inflammation. Levosimendan partly decreased markers of nitrosative and oxidative stress, but did not affect the inflammatory response.

Kallistatin treatment attenuates lethality and organ injury in mouse models of established sepsis.

IntroductionKallistatin levels in the circulation are reduced in patients with sepsis and liver disease. Transgenic mice expressing kallistatin are resistant to lipopolysaccharide (LPS)-induced mortality. Here, we investigated the effect of kallistatin on survival and organ damage in mouse models of established sepsis.MethodsMice were rendered septic by cecal ligation and puncture (CLP), or endotoxemic by LPS injection. Recombinant human kallistatin was administered intravenously six hours after CLP, or intraperitoneally four hours after LPS challenge. The effect of kallistatin treatment on organ damage was examined one day after sepsis initiation, and mouse survival was monitored for four to six days.ResultsHuman kallistatin was detected in mouse serum of kallistatin-treated mice. Kallistatin significantly reduced CLP-induced renal injury as well as blood urea nitrogen, serum creatinine, interleukin-6 (IL-6), and high mobility group box-1 (HMGB1) levels. In the lung, kallistatin decreased malondialdehyde levels and HMGB1 and toll-like receptor-4 (TLR4) synthesis, but increased suppressor of cytokine signaling-3 (SOCS3) expression. Moreover, kallistatin attenuated liver injury, serum alanine transaminase (ALT) levels and hepatic tumor necrosis factor-α (TNF-α) synthesis. Furthermore, delayed kallistatin administration improved survival in CLP mice by 38%, and LPS-treated mice by 42%. In LPS-induced endotoxemic mice, kallistatin attenuated kidney damage in association with reduced serum creatinine, IL-6 and HMGB1 levels, and increased renal SOCS3 expression. Kallistatin also decreased liver injury in conjunction with diminished serum ALT levels and hepatic TNF-α and TLR4 expression. In cultured macrophages, kallistatin through its active site increased SOCS3 expression, but this effect was blocked by inhibitors of tyrosine kinase, protein kinase C and extracellular signal-regulated kinase (ERK), indicating that kallistatin stimulates a tyrosine-kinase-protein kinase C-ERK signaling pathway.ConclusionsThis is the first study to demonstrate that delayed human kallistatin administration is effective in attenuating multi-organ injury, inflammation and mortality in mouse models of polymicrobial infection and endotoxemia. Thus, kallistatin therapy may provide a promising approach for the treatment of sepsis in humans.

Hemopexin in severe inflammation and infection: mouse models and human diseases.

IntroductionCell-free plasma hemoglobin is associated with poor outcome in patients with sepsis. Extracellular hemoglobin and secondarily released heme amplify inflammation in the presence of microbial TLR ligands and/or endogenous mediators. Hemopexin, a plasma protein that binds heme with extraordinary affinity, blocks these effects and has been proposed as a possible treatment approach to decrease inflammation in critically ill patients.MethodsWe studied mouse models of endotoxemia, burn wound infections and peritonitis in order to assess if a repletion strategy for hemopexin might be reasonable. We also measured hemopexin in small numbers of three patient populations that might be logical groups for hemopexin therapy: patients with sepsis and ARDS, patients with severe burns, and premature infants.ResultsDespite severe disease, mean plasma hemopexin levels were increased above baseline in each murine model. However, plasma hemopexin levels were decreased or markedly decreased in many patients in each of the three patient populations.ConclusionsPotentially different behavior of hemopexin in mice and humans may be important to consider when utilizing murine models to represent acute human inflammatory diseases in which heme plays a role. The findings raise the possibility that decreased hemopexin could result in insufficiently neutralized or cleared heme in some patients with ARDS, burns, or in premature infants who might be candidates to benefit from hemopexin administration.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-0885-x) contains supplementary material, which is available to authorized users.

Deacetylation-mediated interaction of SIRT1-HMGB1 improves survival in a mouse model of endotoxemia.

Inflammatory signal-mediated release of high-mobility group box 1 (HMGB1) is a damage-associated molecular pattern or alarmin. The inflammatory functions of HMGB1 have been extensively investigated; however, less is known about the mechanisms controlling HMGB1 release. We show that SIRT1, the human homolog of the Saccharomyces cerevisiae protein silent information regulator 2, which is involved in cellular senescence and possibly the response to inflammation, forms a stable complex with HMGB1 in murine macrophage RAW264.7 cells. SIRT1 directly interacted with HMGB1 via its N-terminal lysine residues (28–30), and thereby inhibited HMGB1 release to improve survival in an experimental model of sepsis. By contrast, inflammatory stimuli such as lipopolysaccharide (LPS) and tumor necrosis factor-α promoted HMGB1 release by provoking its dissociation from SIRT1 dependent on acetylation, thereby increasing the association between HMGB1 and chromosome region maintenance 1, leading to HMGB1 translocation. In vivo infection with wild-type SIRT1 and HMGB1K282930R, a hypo-acetylation mutant, improved survival (85.7%) during endotoxemia more than infection with wild-type SIRT1 and HMGB1-expressing adenovirus, indicating that the acetylation-dependent interaction between HMGB1 and SIRT1 is critical for LPS-induced lethality. Taken together, we propose that SIRT1 forms an anti-inflammatory complex with HMGB1, allowing cells to bypass the response to inflammation.

The tyrosine kinase inhibitor imatinib prevents lung injury and death after intravenous LPS in mice.

Severe sepsis and septic shock are frequent causes of the acute respiratory distress syndrome, and important sources of human mortality. Lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls, plays a major role in the pathogenesis of severe sepsis and septic shock. LPS exposure induces the production of harmful reactive oxygen species, and the resultant oxidant injury has been implicated in the pathogenesis of both severe sepsis and ARDS. We previously showed that the tyrosine kinase inhibitor imatinib increases lung endothelial antioxidant enzymes and protects against pulmonary endothelial antioxidant injury. In the present study, we tested the hypothesis that imatinib would protect against lung injury and systemic inflammation caused by intravenous LPS in an intact mouse model of endotoxemia mimicking early sepsis. We found that intravenous LPS induced a significant increase in the activity of lung xanthine oxidoreductase (XOR), an enzyme which is a major source of reactive oxygen species and implicated in the pathogenesis of acute lung injury. Imatinib had no effect of LPS-induced XOR activity. However, pretreatment of mice with imatinib increased lung catalase activity and decreased intravenous LPS-induced lung oxidant injury as measured by γ-H2AX, a marker of oxidant-induced DNA damage, lung apoptosis, and pulmonary edema. Imatinib also attenuated systemic cytokine expression after intravenous LPS exposure. Finally, imatinib completely prevented mortality in an in vivo, intravenous LPS mouse model of endotoxemia and lung injury. These results support the testing of imatinib as a novel pharmacologic agent in the treatment of Gram-negative sepsis and sepsis-induced ARDS.