Activation of Myocardial Phosphoinositide-3-Kinase p110α Ameliorates Cardiac Dysfunction and Improves Survival in Polymicrobial Sepsis

Phosphoinositide-3-kinase (PI3K)/Akt dependent signaling has been shown to improve outcome in sepsis/septic shock. There is also ample evidence that PI3K/Akt dependent signaling plays a crucial role in maintaining normal cardiac function. We hypothesized that PI3K/Akt signaling may ameliorate septic shock by attenuating sepsis-induced cardiac dysfunction. Cardiac function and survival were evaluated in transgenic mice with cardiac myocyte specific expression of constitutively active PI3K isoform, p110α (caPI3K Tg). caPI3K Tg and wild type (WT) mice were subjected to cecal ligation/puncture (CLP) induced sepsis. Wild type CLP mice showed dramatic cardiac dysfunction at 6 hrs. Septic cardiomyopathy was significantly attenuated in caPI3K CLP mice. The time to 100% mortality was 46 hrs in WT CLP mice. In contrast, 80% of the caPI3K mice survived at 46 hrs after CLP (p<0.01) and 50% survived >30 days (p<0.01). Cardiac caPI3K expression prevented expression of an inflammatory phenotype in CLP sepsis. Organ neutrophil infiltration and lung apoptosis were also effectively inhibited by cardiac PI3k p110α expression. Cardiac high mobility group box–1 (HMGB-1) translocation was also inhibited by caPI3K p110α expression. We conclude that cardiac specific activation of PI3k/Akt dependent signaling can significantly modify the morbidity and mortality associated with sepsis. Our data also indicate that myocardial function/dysfunction plays a prominent role in the pathogenesis of sepsis and that maintenance of cardiac function during sepsis is essential. Finally, these data suggest that modulation of the PI3K/p110α signaling pathway may be beneficial in the prevention and/or management of septic cardiomyopathy and septic shock.     

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)^1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture². In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis³. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis^1-3.Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-γ)^4-6 and late proinflammatory mediators (e.g., HMGB1)⁷. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality^8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.     

Cultured Alveolar Epithelial Cells From Septic Rats Mimic In Vivo Septic Lung

Sepsis results in the formation of pulmonary edema by increasing in epithelial permeability. Therefore we hypothesized that alveolar epithelial cells isolated from septic animals develop tight junctions with different protein composition and reduced barrier function relative to alveolar epithelial cells from healthy animals. Male rats (200–300g) were sacrificed 24 hours after cecal ligation and double puncture (2CLP) or sham surgery. Alveolar epithelial cells were isolated and plated on fibronectin-coated flexible membranes or permeable, non-flexible transwell substrates. After a 5 day culture period, cells were either lysed for western analysis of tight junction protein expressin (claudin 3, 4, 5, 7, 8, and 18, occludin, ZO-1, and JAM-A) and MAPk (JNK, ERK, an p38) signaling activation, or barrier function was examined by measuring transepithelial resistance (TER) or the flux of two molecular tracers (5 and 20 Å). Inhibitors of JNK (SP600125, 20 µM) and ERK (U0126, 10 µM) were used to determine the role of these pathways in sepsis induced epithelial barrier dysfunction. Expression of claudin 4, claudin 18, and occludin was significantly lower, and activation of JNK and ERK signaling pathways was significantly increased in 2CLP monolayers, relative to sham monolayers. Transepithelial resistance of the 2CLP monolayers was reduced significantly compared to sham (769 and 1234 ohm-cm², respectively), however no significant difference in the flux of either tracer was observed. Inhibition of ERK, not JNK, significantly increased TER and expression of claudin 4 in 2CLP monolayers, and prevented significant differences in claudin 18 expression between 2CLP and sham monolayers. We conclude that alveolar epithelial cells isolated from septic animals form confluent monolayers with impaired barrier function compared to healthy monolayers, and inhibition of ERK signaling partially reverses differences between these monolayers. This model provides a unique preparation for probing the mechanisms by which sepsis alters alveolar epithelium.     

Androstenediol administration after trauma-hemorrhage attenuates inflammatory response, reduces organ damage, and improves survival following sepsis

Although androstenediol (adiol or 5-androstene-3beta,17beta-diol), a metabolite of dehydroepiandrosterone (DHEA), has protective effects following trauma-hemorrhage (T-H), it remains unknown whether administration of adiol has any salutary effects on the inflammatory response and outcome following a combined insult of T-H and sepsis. Male rats underwent T-H shock [mean arterial pressure (MAP) 40 mmHg for 90 min] followed by resuscitation. Adiol (1 mg/kg body wt) or vehicle was administered at the end of resuscitation. Sepsis was induced by cecal ligation and puncture (CLP) at 20 h after T-H or sham operation. Five hours after CLP, plasma and tissue samples were analyzed for cytokines (IL-6 and IL-10), MPO, neutrophil chemotactic factor (CINC-3), and liver injury (alanine aminotransferase and lactate dehydrogenase). In another group of rats, the gangrenous cecum was removed at 10 h after CLP, the cavity was irrigated with warm saline and closed in layers, and mortality was recorded over 10 days. T-H followed by CLP produced a significant elevation in plasma IL-6 and IL-10 levels, enhanced neutrophil cell activation, and resulted in liver injury. Adiol administration prevented the increase in cytokine production, neutrophil cell activation, and attenuated liver injury. Moreover, rats subjected to the combined insult, receiving vehicle or adiol, had a 50% and 6% mortality, respectively. Since adiol administration suppresses proinflammatory cytokines, reduces liver damage, and decreases mortality after the combined insult of T-H and sepsis, this agent appears to be a novel adjunct to fluid resuscitation for decreasing T-H-induced septic complications and mortality.     

Anti-septic activity of α-cubebenoate isolated from Schisandra chinensis

Sepsis is a life-threatening, infectious, systemic inflammatory disease. In this study, we investigated the therapeutic effect of α-cubebenoate, a novel compound isolated from Schisandra chinensis against polymicrobial sepsis in a cecal ligation and puncture (CLP) experimental model. Administration of α-cubebenoate strongly enhanced survival in the CLP model. α-cubebenoate administration also markedly blocked CLP-induced lung inflammation and increased bactericidal activity by enhancing phagocytic activity and hydrogen peroxide generation in mouse bone marrow-derived macrophages and neutrophils. Expression of two important inflammatory cytokines, IL-1β and IL-6, was strongly increased in the CLP model, and this was dramatically blocked by α-cubebenoate. Lymphocyte apoptosis and caspase-3 activation, which are associated with immune paralysis during sepsis, were markedly attenuated by α-cubebenoate. Taken together, our findings indicate that α-cubebenoate, a natural compound isolated from Schisandra chinensis, is a powerful potential anti-septic agent. [BMB Reports 2015; 48(6): 336-341]     

Differential Role for CD80 and CD86 in the Regulation of the Innate Immune Response in Murine Polymicrobial Sepsis

Background

Inflammation in the early stages of sepsis is governed by the innate immune response. Costimulatory molecules are a receptor/ligand class of molecules capable of regulation of inflammation in innate immunity via macrophage/neutrophil contact. We recently described that CD80/86 ligation is required for maximal macrophage activation and CD80/86^−/− mice display reduced mortality and inflammatory cytokine production after cecal ligation and puncture (CLP). However, these data also demonstrate differential regulation of CD80 and CD86 expression in sepsis, suggesting a divergent role for these receptors. Therefore, the goal of this study was to determine the individual contribution of CD80/86 family members in regulating inflammation in sepsis.

Methodology/Principal Findings

CD80^−/− mice had improved survival after CLP when compared to WT or CD86^−/− mice. This was associated with preferential attenuation of inflammatory cytokine production in CD80^−/− mice. Results were confirmed with pharmacologic blockade, with anti-CD80 mAb rescuing mice when administered before or after CLP. In vitro, activation of macrophages with neutrophil lipid rafts caused selective disassociation of IRAK-M, a negative regulator of NF-κB signaling from CD80; providing a mechanism for preferential regulation of cytokine production by CD80. Finally, in humans, upregulation of CD80 and loss of constitutive CD86 expression on monocytes was associated with higher severity of illness and inflammation confirming the findings in our mouse model.

Conclusions

In conclusion, our data describe a differential role for CD80 and CD86 in regulation of inflammation in the innate immune response to sepsis. Future therapeutic strategies for blockade of the CD80/86 system in sepsis should focus on direct inhibition of CD80.     

Gastrin-Releasing Peptide Receptor Antagonism Induces Protection from Lethal Sepsis: Involvement of Toll-like Receptor 4 Signaling

In sepsis, toll-like receptor (TLR)-4 modulates the migration of neutrophils to infectious foci, favoring bacteremia and mortality. In experimental sepsis, organ dysfunction and cytokines released by activated macrophages can be reduced by gastrin-releasing peptide (GRP) receptor (GRPR) antagonist RC-3095. Here we report a link between GRPR and TLR-4 in experimental models and in sepsis patients. RAW 264.7 culture cells were exposed to lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-α and RC-3095 (10 ng/mL). Male Wistar rats were subjected to cecal ligation and puncture (CLP), and RC-3095 was administered (3 mg/kg, subcutaneously); after 6 h, we removed the blood, bronchoalveolar lavage, peritoneal lavage and lung. Human patients with a clinical diagnosis of sepsis received a continuous infusion with RC-3095 (3 mg/kg, intravenous) over a period of 12 h, and plasma was collected before and after RC-3095 administration and, in a different set of patients with systemic inflammatory response syndrome (SIRS) or sepsis, GRP plasma levels were determined. RC-3095 inhibited TLR-4, extracellular-signal–related kinase (ERK)-1/2, Jun NH₂-terminal kinase (JNK) and Akt and decreased activation of activator protein 1 (AP-1), nuclear factor (NF)-κB and interleukin (IL)-6 in macrophages stimulated by LPS. It also decreased IL-6 release from macrophages stimulated by TNF-α. RC-3095 treatment in CLP rats decreased lung TLR-4, reduced the migration of cells to the lung and reduced systemic cytokines and bacterial dissemination. Patients with sepsis and systemic inflammatory response syndrome have elevated plasma levels of GRP, which associates with clinical outcome in the sepsis patients. These findings highlight the role of GRPR signaling in sepsis outcome and the beneficial action of GRPR antagonists in controlling the inflammatory response in sepsis through a mechanism involving at least inhibition of TLR-4 signaling.     

Norepinephrine-induced hepatocellular dysfunction in early sepsis is mediated by activation of alpha2-adrenoceptors

Gut-derived norepinephrine (NE) has been shown to play a critical role in producing hepatocellular dysfunction in early sepsis, but it is not known whether alpha2-adrenoceptor activation mediates this dysfunction. We infused normal male adult rats with NE, NE plus the specific alpha2-adrenergic antagonist rauwolscine (RW), or vehicle (normal saline) for 2 h. Hepatocellular function was determined by in vivo indocyanine green (ICG) clearance. An isolated perfused liver preparation was also used to assess hepatocellular function by in vitro ICG clearance; NE alone or with RW was added to the perfusate. Rats were subjected to sepsis by cecal ligation and puncture (CLP). At 1 h after CLP, RW was infused for 15 min. At 5 h after CLP, we measured hepatocellular function and serum tumor necrosis factor-alpha (TNF-alpha) levels. Intraportal NE infusion in normal rats produced hepatocellular dysfunction, which was prevented by RW and NE infusion. This is confirmed by findings with the isolated perfused liver preparation. RW administration in early sepsis maintained hepatocellular function and downregulated TNF-alpha production at 5 h after CLP. These results suggest that NE-induced hepatocellular dysfunction in early sepsis is mediated by alpha2-adrenoceptor activation, which appears to upregulate TNF-alpha production. Modulation of hepatic responsiveness to NE by alpha2-adrenergic antagonists should provide a novel approach for maintaining cell and organ functions during sepsis.     

Exogenous Glucose Administration Impairs Glucose Tolerance and Pancreatic Insulin Secretion during Acute Sepsis in Non-Diabetic Mice

Objectives

The development of hyperglycemia and the use of early parenteral feeding are associated with poor outcomes in critically ill patients. We therefore examined the impact of exogenous glucose administration on the integrated metabolic function of endotoxemic mice using our recently developed frequently sampled intravenous glucose tolerance test (FSIVGTT). We next extended our findings using a cecal ligation and puncture (CLP) sepsis model administered early parenteral glucose support.

Methods

Male C57BL/6J mice, 8-12 weeks, were instrumented with chronic indwelling arterial and venous catheters. Endotoxemia was initiated with intra-arterial lipopolysaccharide (LPS; 1 mg/kg) in the presence of saline or glucose infusion (100 µL/hr), and an FSIVGTT was performed after five hours. In a second experiment, catheterized mice underwent CLP and the impact of early parenteral glucose administration on glucose homeostasis and mortality was assessed over 24 hrs.

Measurements

And MAIN RESULTS: Administration of LPS alone did not impair metabolic function, whereas glucose administration alone induced an insulin sensitive state. In contrast, LPS and glucose combined caused marked glucose intolerance and insulin resistance and significantly impaired pancreatic insulin secretion. Similarly, CLP mice receiving parenteral glucose developed fulminant hyperglycemia within 18 hrs (all > 600 mg/dl) associated with increased systemic cytokine release and 40% mortality, whereas CLP alone (85 ± 2 mg/dL) or sham mice receiving parenteral glucose (113 ± 3 mg/dL) all survived and were not hyperglycemic. Despite profound hyperglycemia, plasma insulin in the CLP glucose-infused mice (3.7 ± 1.2 ng/ml) was not higher than sham glucose infused mice (2.1 ± 0.3 ng/ml).

Conclusions

The combination of parenteral glucose support and the systemic inflammatory response in the acute phase of sepsis induces profound insulin resistance and impairs compensatory pancreatic insulin secretion, leading to the development of fulminant hyperglycemia.     

Increasing Blood Glucose Variability Is a Precursor of Sepsis and Mortality in Burned Patients

High glycemic variability, rather than a mean glucose level, is an important factor associated with sepsis and hospital mortality in critically ill patients. In this retrospective study we analyze the blood glucose data of 172 nondiabetic patients 18–60 yrs old with second and third-degree burns of total body surface area greater than 30% and 5%, respectively, admitted to ICU in 2004–2008. The analysis identified significant association of increasing daily glucose excursion (DELTA) accompanied by evident episodes of hyperglycemia (>11 mmol/l) and hypoglycemia (<2.8 mmol/l), with sepsis and forthcoming death, even when the mean daily glucose was within a range of acceptable glycemia. No association was found in sepsis complication and hospital mortality with doses of intravenous insulin and glucose infusion. A strong increase in DELTA before sepsis and death is treated as fluctuation amplification near the onset of dynamical instability.     

Cecal Ligation Puncture Procedure

Human sepsis is characterized by a set of systemic reactions in response to intensive and massive infection that failed to be locally contained by the host. Currently, sepsis ranks among the top ten causes of mortality in the USA intensive care units ¹. During sepsis there are two established haemodynamic phases that may overlap. The initial phase (hyperdynamic) is defined as a massive production of proinflammatory cytokines and reactive oxygen species by macrophages and neutrophils that affects vascular permeability (leading to hypotension), cardiac function and induces metabolic changes culminating in tissue necrosis and organ failure. Consequently, the most common cause of mortality is acute kidney injury. The second phase (hypodynamic) is an anti-inflammatory process involving altered monocyte antigen presentation, decreased lymphocyte proliferation and function and increased apoptosis. This state known as immunosuppression or immune depression sharply increases the risk of nocosomial infections and ultimately, death. The mechanisms of these pathophysiological processes are not well characterized. Because both phases of sepsis may cause irreversible and irreparable damage, it is essential to determine the immunological and physiological status of the patient. This is the main reason why many therapeutic drugs have failed. The same drug given at different stages of sepsis may be therapeutic or otherwise harmful or have no effect ^2,3. To understand sepsis at various levels it is crucial to have a suitable and comprehensive animal model that reproduces the clinical course of the disease. It is important to characterize the pathophysiological mechanisms occurring during sepsis and control the model conditions for testing potential therapeutic agents. To study the etiology of human sepsis researchers have developed different animal models. The most widely used clinical model is cecal ligation and puncture (CLP). The CLP model consists of the perforation of the cecum allowing the release of fecal material into the peritoneal cavity to generate an exacerbated immune response induced by polymicrobial infection. This model fulfills the human condition that is clinically relevant. As in humans, mice that undergo CLP with fluid resuscitation show the first (early) hyperdynamic phase that in time progresses to the second (late) hypodynamic phase. In addition, the cytokine profile is similar to that seen in human sepsis where there is increased lymphocyte apoptosis (reviewed in ^4,5). Due to the multiple and overlapping mechanisms involved in sepsis, researchers need a suitable sepsis model of controlled severity in order to obtain consistent and reproducible results.     

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.     

HMGB1 Mediates Cognitive Impairment in Sepsis Survivors

Severe sepsis, a syndrome that complicates infection and injury, affects 750,000 annually in the United States. The acute mortality rate is approximately 30%, but, strikingly, sepsis survivors have a significant disability burden: up to 25% of survivors are cognitively and physically impaired. To investigate the mechanisms underlying persistent cognitive impairment in sepsis survivors, here we developed a murine model of severe sepsis survivors following cecal ligation and puncture (CLP) to study cognitive impairments. We observed that serum levels of high mobility group box 1 (HMGB1), a critical mediator of acute sepsis pathophysiology, are increased in sepsis survivors. Significantly, these levels remain elevated for at least 4 wks after CLP. Sepsis survivors develop significant, persistent impairments in learning and memory, and anatomic changes in the hippocampus associated with a loss of synaptic plasticity. Administration of neutralizing anti-HMGB1 antibody to survivors, beginning 1 wk after onset of peritonitis, significantly improved memory impairments and brain pathology. Administration of recombinant HMGB1 to naïve mice recapitulated the memory impairments. Together, these findings indicate that elevated HMGB1 levels mediate cognitive decline in sepsis survivors, and suggest that it may be possible to prevent or reverse cognitive impairments in sepsis survivors by administration of anti-HMGB1 antibodies.     

Decoy Receptor 3 Improves Survival in Experimental Sepsis by Suppressing the Inflammatory Response and Lymphocyte Apoptosis

Purpose

Unbalanced inflammatory response and lymphocyte apoptosis is associated with high mortality in septic patients. Decoy receptor 3 (DcR3), a member of the tumor necrosis factor receptor superfamily, is an anti-inflammatory and anti-apoptotic factor. Recently, DcR3 expression was found to be increased in septic patients. This study evaluated the therapeutic effect and mechanisms of DcR3 on cecal ligation and puncture (CLP)-induced sepsis in mice.

Methods

C57BL/6 mice were subjected to CLP-induced polymicrobial sepsis. DcR3 Fc was intravenously injected 30 min before and 6 h after CLP. Bacterial clearance, cytokine production, histology, lymphocyte apoptosis and survival were evaluated. Furthermore, we investigated the systemic effects of DcR3 in in vitro lymphocyte apoptosis regulation.

Results

Our results demonstrated that DcR3 protein treatments significantly improved survival in septic mice (p <0.05). Treatment with DcR3 protein significantly reduced the inflammatory response and decreased lymphocyte apoptosis in the thymus and spleen. Histopathological findings of the lung and liver showed milder impairment after DcR3 administration. In vitro experiments showed that DcR3 Fc inhibited Fas-FasL mediated lymphocyte apoptosis.

Conclusions

Treatment with the DcR3 protein protects mice from sepsis by suppressing the inflammatory response and lymphocyte apoptosis. DcR3 protein may be useful in treatment of sepsis.     

Anisomycin protects against sepsis by attenuating IκB kinase-dependent NF-κB activation and inflammatory gene expression

Anisomycin is known to inhibit eukaryotic protein synthesis and has been established as an antibiotic and anticancer drug. However, the molecular targets of anisomycin and its mechanism of action have not been explained in macrophages. Here, we demonstrated the anti-inflammatory effects of anisomycin both in vivo and in vitro. We found that anisomycin decreased the mortality rate of macrophages in cecal ligation and puncture (CLP)- and lipopolysaccharide (LPS)-induced acute sepsis. It also declined the gene expression of proinflammatory mediators such as inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-1β as well as the nitric oxide and proinflammatory cytokines production in macrophages subjected to LPS-induced acute sepsis. Furthermore, anisomycin attenuated nuclear factor (NF)-κB activation in LPS-induced macrophages, which correlated with the inhibition of phosphorylation of NF-κB-inducing kinase and IκB kinase, phosphorylation and IκBα proteolytic degradation, and NF-κB p65 subunit nuclear translocation. These results suggest that anisomycin prevented acute inflammation by inhibiting NF-κB-related inflammatory gene expression and could be a potential therapeutic candidate for sepsis.     

Contributory Role of BLT2 in the Production of Proinflammatory Cytokines in Cecal Ligation and Puncture-Induced Sepsis

BLT2 is a low-affinity receptor for leukotriene B₄, a potent lipid mediator of inflammation generated from arachidonic acid via the 5-lipoxygenase pathway. The aim of this study was to investigate whether BLT2 plays any role in sepsis, a systemic inflammatory response syndrome caused by infection. A murine model of cecal ligation and puncture (CLP)-induced sepsis was used to evaluate the role of BLT2 in septic inflammation. In the present study, we observed that the levels of ligands for BLT2 (LTB₄ [leukotriene B₄] and 12(S)-HETE [12(S)-hydroxyeicosatetraenoic acid]) were significantly increased in the peritoneal lavage fluid and serum from mice with CLP-induced sepsis. We also observed that the levels of BLT2 as well as 5-lipoxygenase (5-LO) and 12-LO, which are synthesizing enzymes for LTB₄ and 12(S)-HETE, were significantly increased in lung and liver tissues in the CLP mouse model. Blockade of BLT2 markedly suppressed the production of sepsis-associated cytokines (IL-6 [interleukin-6], TNF-α[[tumor necrosis factor alpha], and IL-1β [interleukin-1β] as well as IL-17 [interleukin-17]) and alleviated lung inflammation in the CLP group. Taken together, our results suggest that BLT2 cascade contributes to lung inflammation in CLP-induced sepsis by mediating the production of inflammatory cytokines. These findings suggest that BLT2 may be a potential therapeutic target for sepsis patients.     

Mechanisms of polymicrobial sepsis-induced ileus

Sepsis frequently occurs after hemorrhage, trauma, burn, or abdominal surgery and is a leading cause of morbidity and mortality in severely ill patients. We performed experiments to delineate intestinal molecular and functional motility consequences of polymicrobial sepsis in the clinically relevant cecal ligation and puncture (CLP) sepsis model. CLP was performed on male Sprague-Dawley rats. Gastrointestinal transit, colonic in vivo pressure recordings, and in vitro muscle contractions were recorded. Histochemistry was performed for macrophages, monocytes, and neutrophils. Inflammatory gene expressions were quantified by real-time RT-PCR. CLP delayed gastrointestinal transit, decreased colonic pressures, and suppressed in vivo circular muscle contractility of the jejunum and colon over a 4-day period. A leukocytic infiltrate of monocytes and neutrophils developed over 24 h. Real-time RT-PCR demonstrated a significant temporal elevation in IL-6, IL-1beta, monocyte chemoattractant protein-1, and inducible nitric oxide synthase, with higher expression levels of IL-6 and inducible nitric oxide synthase in colonic extracts compared with small intestine. Polymicrobial CLP sepsis induces a complex inflammatory response within the intestinal muscularis with the recruitment of leukocytes and elaboration of mediators that inhibit intestinal muscle function. Differences were elucidated between endotoxin and CLP models of sepsis, as well as a heterogeneous regional response of the gastrointestinal tract to CLP. Thus the intestine is not only a source of bacteremia but also an important target of bacterial products with major functional consequences to intestinal motility and the generation of cytokines, which participate in the development of multiple organ failure.     

Carbon Monoxide-Releasing Molecule-2 Reduces Intestinal Epithelial Tight-Junction Damage and Mortality in Septic Rats

Objective

Damage to intestinal epithelial tight junctions plays an important role in sepsis. Recently we found that Carbon Monoxide-Releasing Molecule-2 (CORM-2) is able to protect LPS-induced intestinal epithelial tight junction damage and in this study we will investigate if CORM-2 could protect intestinal epithelial tight junctions in the rat cecal ligation and puncture (CLP) model.

Materials and Methods

The CLP model was generated using male Sprague-Dawley (SD) rats according to standard procedure and treated with CORM-2 or inactive CORM-2 (iCORM-2), 8 mg/kg, i.v. immediately after CLP induction and euthanized after 24h or 72h (for mortality rate only). Morphological changes were investigated using both transmission electron and confocal microscopy. The levels of important TJ proteins and phosphorylation of myosin light chain (MLC) were examined using Western blotting. Cytokines, IL-1β and TNF-α were measured using ELISA kits. The overall intestinal epithelial permeability was evaluated using FD-4 as a marker.

Results

CORM-2, but not iCORM-2, significantly reduced sepsis-induced damage of intestinal mucosa (including TJ disruption), TJ protein reduction (including zonula occludens-l (ZO-1), claudin-1 and occludin), MLC phosphorylation and proinflammatory cytokine release. The overall outcomes showed that CORM-2 suppressed sepsis-induced intestinal epithelial permeability changes and reduced mortality rate of those septic rats.

Conclusions

Our data strongly suggest that CORM-2 could be a potential therapeutic reagent for sepsis by suppressing inflammation, restoring intestinal epithelial barrier and reducing mortality.     

Anti-inflammatory response is associated with mortality and severity of infection in sepsis

Using a murine model of sepsis, we found that the balance of tissue pro- to anti-inflammatory cytokines directly correlated with severity of infection and mortality. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). Liver tissue was analyzed for levels of IL-1beta, IL-1 receptor antagonist (IL-1ra), tumor necrosis factor (TNF)-alpha, and soluble TNF receptor 1 by ELISA. Bacterial DNA was measured using quantitative real-time PCR. After CLP, early predominance of proinflammatory cytokines (6 h) transitioned to anti-inflammatory predominance at 24 h. The elevated anti-inflammatory cytokines were mirrored by increased tissue bacterial levels. The degree of anti-inflammatory response compared with proinflammatory response correlated with the bacterial concentration. To modulate the timing of the anti-inflammatory response, mice were treated with IL-1ra before CLP. This resulted in decreased proinflammatory cytokines, earlier bacterial load, and increased mortality. These studies show that the initial tissue proinflammatory response to sepsis is followed by an anti-inflammatory response. The anti-inflammatory phase is associated with increased bacterial load and mortality. These data suggest that it is the timing and magnitude of the anti-inflammatory response that predicts severity of infection in a murine model of sepsis.