Local thymic caspase-9 inhibition improves survival during polymicrobial sepsis in mice

Caspase-9 is believed to play an essential role in sepsis-induced lymphocyte apoptosis. The aim of this study was therefore to evaluate its contribution within the caspase-dependent apoptosis pathway in a murine model of polymicrobial sepsis. Local injections of Z-LEHD-fmk, a specific caspase-9 inhibitor, into thymi of septic mice led to the complete inhibition of caspase-9, decreased apoptosis of resident tissue cells, and, in addition, reduced further downstream caspase-3 activity. In contrast to its systemic administration, only local injections improved the overall survival of septic mice. However, local injections of a pancaspase inhibitor (Z-VAD-fmk) did not improve survival, although caspase-3 activity was reduced to a similar degree as by the administration of Z-LEHD-fmk. These results indicate that local apoptosis of lymphatic tissue in polymicrobial sepsis is processed dependent of caspase-9 and suggests alternative caspase-dependent beneficial effects, which may determine a positive outcome.     

Disruption of tight junctions during polymicrobial sepsis in vivo

The disruption of intestinal epithelial tight junctions may result in barrier function dysfunction during polymicrobial sepsis. The pathophysiology of sepsis involves breakdown of barrier integrity, which correlates with adverse outcome during sepsis. However, the mechanisms underlying loss of barrier function in sepsis remain unknown. In the present study in mice, tight junction (TJ) structure was analysed by transmission electron microscopy; intestinal permeability was assessed using molecular tracer measurement; and the distribution of TJ proteins was investigated by immunofluorescence microscopy. The membrane microdomains of TJs were isolated using discontinuous sucrose density gradients and the expression of TJ proteins in these was determined by western blot. Immunofluorescence microscopy revealed that claudins 1, 3, 4, 5, and 8 were present predominantly in the microvillous surface of epithelial cells and along the lateral membranes of the cells; in sepsis, however, labelling of these proteins was present diffusely within cells and was no longer focused at the lateral cell boundaries. Moreover, the expression of claudin‐2 was markedly up‐regulated in sepsis. Using western blot analysis, we found that occludin and claudins were displaced from raft fractions to non‐raft fractions in membrane microdomains of TJs in sepsis. In addition, the disruption of TJ structure was accompanied by increased intestinal permeability. Our results demonstrate for the first time that redistribution of TJ proteins in TJ membrane microdomains and redistribution of claudins in epithelial cells of the colon lead to alteration of TJ architecture and TJ barrier dysfunction during the development of polymicrobial sepsis. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Ghrelin clearance is reduced at the late stage of polymicrobial sepsis

The cardiovascular response to sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase. Ghrelin, a newly-identified endogenous ligand for growth hormone secretagogue receptor (i.e., ghrelin receptor), was recently demonstrated to be a potent vasoactive peptide in addition to its effects on growth hormone release and energy homeostasis. We have shown that ghrelin (via its receptor) may play an important role in regulating cardiovascular responses in the progression of polymicrobial sepsis. However, it remains unknown whether the clearance of this peptide is altered in sepsis. To determine this, male adult rats were injected with 125I-ghrelin through the jugular vein at 5 or 20 h after cecal ligation and puncture (CLP, i.e., sepsis model) or sham operation. The blood sample was collected every 2 min for 30 min for determining half-life (t1/2). Tissue samples (i.e., kidneys, liver, brain, heart, lungs, spleen, stomach, small intestine, large intestine, skin and muscle) were then harvested. The radioactivities of samples were counted. The results indicate that 125I-ghrelin's t1/2 and its distribution were not significantly altered in early sepsis (5 h after CLP). However, the t1/2 increased significantly in late sepsis (20 h after CLP). Tissue distribution of 125I-ghrelin was far greater in the kidneys than in any other tissues tested in both sham and septic animals. Moreover, the kidneys and liver had significantly less radioactive uptake at 20 h after CLP, but the radioactivity in blood was much higher at the same time point. There were no significant changes in 125I-ghrelin distribution in other organs at the late stage of sepsis. These results indicate that the kidneys are the primary site of ghrelin clearance, which is significantly diminished in late sepsis. In addition, the liver also plays a role in the clearance of ghrelin, which was also reduced in late sepsis. The decreased clearance of ghrelin by the kidneys and liver may be due to renal and hepatic dysfunctions under such conditions.     

Effects of propofol on early and late cytokines in lipopolysaccharide-induced septic shock in rats

Objective

It has been reported that the intravenous anesthetic propofol (PPF) has anti-inflammatory effects in vitro and in patients. The purpose of this study was to investigate whether PPF has anti-inflammatory effects in lipopolysaccharide (LPS)-induced septic shock by inhibiting the induction of pro-inflammatory cytokines [interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)] and high mobility group box 1 (HMGB1) in rats.

Methods

Thirty six male Wistar rats were randomly assigned to one of three groups (control group, PPF + LPS group and LPS group; n = 12 per group). Control group rats received a 0.9% NaCl solution (NS) by the tail vein. The PPF + LPS group rats received PPF (10 mg/kg bolus, followed by infusion at 10 mg/(kg·h) through a femoral vein catheter) 1 h before LPS (7.5 mg/kg) administration via the tail vein. The LPS group rats received injection of LPS (7.5 mg/kg) via the tail vein. Hemodynamic effects were recorded as well as mortality rates, and plasma cytokine con-centrations (TNF-α, IL-6, HMGB1) were measured for the 24-h observation period.

Results

The mean arterial pressure and heart rate of the PPF + LPS group were more stable than those of the LPS group. The mortality at 24 h after the administration of the LPS injection was much higher in the LPS group (58.3%) compared to the PPF + LPS group (25.0%). Plasma concentrations of cytokines (IL-6 and TNF-α) and HMGB1 were significantly reduced in the PPF + LPS group compared to the LPS group (P < 0.05).

Conclusion

Pretreatment with PPF reduced the mortality rate of rats and attenuated the pro-inflammatory cytokine responses in an endotoxin shock model through an anti-inflammatory action inhibiting induction of HMGB1.     

Mechanisms of mortality in early and late sepsis

A recent hypothesis postulates that sepsis moves through different phases, with periods of enhanced inflammation alternating with periods of immune suppression. In this study we determined the levels of inflammation present during early and late septic deaths to examine whether death was due to hyperinflammation or immunosuppression. The murine model of sepsis induced by cecal ligation and puncture (CLP) was used. Complete blood counts, plasma interleukin-6 (IL-6) levels, and body weights were determined. Mice that died within the first 4 days had increased plasma levels of IL-6, indicating that there was activation of the immune system. Cecal resection on day 4 after CLP resulted in decreased abscess size, lower circulating neutrophil counts, decreased anemia, and improved survival compared to the results for mice that received only antibiotic and fluid therapy. All of the mice that died in the chronic phase of infection (after day 4) had positive peritoneal cultures containing significantly more bacteria than the cultures for surviving mice. After day 4, none of the surviving mice exhibited increases in the plasma levels of IL-6. Dying mice exhibited mixed IL-6 responses; for 41% of the mice there was never an increase in the IL-6 levels in the chronic phase, while for other mice the levels of IL-6 transiently increased prior to death. Peritoneal macrophages were obtained in the late phase of sepsis from moribund and healthy mice and were stimulated ex vivo. The cells from the moribund mice produced significantly less IL-6 than the cells obtained from healthy mice produced. These results indicate that in mice that die in the early phase there is uniformly increased inflammation. However, during the chronic phase of sepsis, some mice die with evidence of immunosuppression (increased bacterial growth and low IL-6 levels), while other mice die with immunostimulation (high IL-6 levels and bacterial growth). Determining the inflammatory status of individual patients may help guide appropriate, targeted therapy.     

Neutrophil IL-10 suppresses peritoneal inflammatory monocytes during polymicrobial sepsis

Septic peritonitis remains a major cause of death. Neutrophils and inflammatory monocytes are principal components of the innate immune system and are essential for defense against a range of microbial pathogens. Their role and interaction in polymicrobial sepsis have not been defined clearly. Using a murine model of CLP to induce moderate sepsis, we found that neutrophil depletion did not alter survival, whereas depletion of neutrophils and inflammatory monocytes markedly reduced survival. After neutrophil depletion, inflammatory monocytes had greater phagocytic capacity and oxidative burst, and increased expression of costimulatory molecules, TNF, and iNOS. Notably, peritoneal neutrophils produced IL-10 following CLP. Adoptive i.p. transfer of WT but not IL-10(-/-) neutrophils into septic mice reduced monocyte expression of TNF. In vitro experiments confirmed that monocyte suppression was mediated by neutrophil-derived IL-10. Thus, during septic peritonitis, neutrophils suppress peritoneal inflammatory monocytes through IL-10 and are dispensable for survival.     

Neurokinin-1 receptor antagonist treatment protects mice against lung injury in polymicrobial sepsis

Earlier work from our laboratory has suggested a role for the neuropeptide substance P (SP) in inducing lung injury in sepsis. In that study, mice lacking the preprotachykinin-A gene, which encodes for SP, were protected against lung injury in sepsis. To further substantiate the role of SP in sepsis and to study its mechanism, we have evaluated the effect of SR140333, a SP receptor antagonist, on lung injury in sepsis, which was induced in male Swiss mice by cecal ligation and puncture (CLP). Sham-operated animals received the same surgical procedure, except CLP. Vehicle or SR140333 (1 mg/kg, s.c.) was administered to CLP mice 30 min before or 1 h after the CLP. Eight hours after surgery, lung tissue was collected and analyzed for myeloperoxidase (MPO) activity, chemokines, cytokines, and adhesion molecules. The CLP procedure alone caused a significant increase in the lung levels of MIP-2, MCP-1, IL-1beta, IL-6, ICAM-1, E- and P-selectin, and MPO activity when compared with sham-operated mice. SR140333 injected 30 min before or 1 h after CLP significantly attenuated the increased lung MPO activity and levels of MIP-2, MCP-1, IL-1beta, IL-6, ICAM-1, and E- and P-selectin compared with CLP-operated mice injected with the vehicle. Histological evaluation of the lung sections further supported the beneficial effect of SR140333 on lung inflammation. Therefore, SP receptor antagonism can be a potential therapeutic target in polymicrobial sepsis, and this effect is brought about via reduction in leukocyte recruitment.     

Adrenomedullin binding protein-1 is downregulated during polymicrobial sepsis in the rat

We have previously shown that administration of adrenomedullin (AM) and AM binding protein-1 (AMBP-1) in combination maintains cardiovascular stability and reduces mortality in a rat model of sepsis. However, it is unknown whether AMBP-1 is reduced under the septic condition and, if so, whether lipopolysaccharide (LPS) plays a role in down-regulating AMBP-1. To determine this, male adult Sprague-Dawley rats were subjected to either polymicrobial sepsis by cecal ligation and puncture (CLP), or endotoxemia by intraperitoneal injection of LPS (15 mg/kg body weight). In an additional group of animals, LPS neutralizing agent polymyxin B (PMB) was given intramuscularly at 0.5 h before and 9 h after CLP. At 20 h after CLP (i.e. the late stage of sepsis) or endotoxemia, hepatic tissue and blood samples were collected. Hepatic AMBP-1 gene expression along with hepatic and plasma AMBP-1 protein levels were measured by RT-PCR and Western blot analysis, respectively. Our results showed that hepatic AMBP-1 gene expression decreased by 65%, hepatic AMBP-1 protein levels decreased by 72%, and plasma levels of AMBP-1 decreased by 59% at 20 h after CLP. Similar results were also seen in the animals receiving LPS injection. Administration of PMB, however, prevented the downregulation of AMBP-1 expression at 20 h after CLP. Thus, AMBP-1 is downregulated in the late phase of sepsis, and LPS plays a critical role in the reduction of AMBP-1.     

Dissociation between plasma and monocyte-associated cytokines during sepsis

We report our investigations of circulating interleukin (IL) 1 beta, IL 6 and tumor necrosis factor (TNF)-alpha, as well as cell-associated IL 1 alpha, IL 1 beta and TNF-alpha in plasma and monocytes of 21 patients with sepsis syndrome and 6 patients with non-septic shock. Longitudinal studies reveal that (a) the most frequent detectable plasma cytokines were TNF-alpha and IL 6, (b) the presence and the kinetics of circulating cytokines were independent of one other, (c) detectable levels of cytokines could be found for a long period of time, and (d) significantly higher levels of IL 6 were found for non-surviving patients. Because of the in vivo half-life of cytokines and of the existence of numerous specific high-affinity receptors, it is quite probable that detectable plasma cytokines represent the excess of produced mediators which have not been trapped by the target cells. TNF-alpha (410 +/- 65 pg/10(6) monocytes) and IL 1 beta (153 +/- 60 pg/10(6) monocytes) were frequently found associated to monocyte lysates (88% and 50%, respectively). Despite the fact that IL 1 alpha is the most abundant cytokine found associated to monocytes following in vitro activation, IL 1 alpha was rarely found in monocytes of intensive care unit patients (29%). No correlation was found to exist between the levels of plasma cytokines and cell-associated cytokines. Some patients had plasma TNF-alpha or IL 1 beta in the absence of the corresponding monocyte-associated cytokine. This observation suggests that cells other than monocytes can participate in the production of circulating cytokines. At the end of the longitudinal study (day 14 +/- 2), only 2/12 surviving patients still had plasma TNF-alpha, whereas 8/12 had monocyte-associated TNF-alpha. These results indicate that activation of monocytes still occurs in patients for whom no plasma cytokines can be detected. Thus, in addition to the measurement of plasma cytokine, measurement of cell-associated cytokine appears useful to assess cytokine production and monocyte activation in vivo.     

Expression and characterization of anionic components in the tubulointerstitial compartment of rat kidney during polymicrobial sepsis

The aim of the study was to evaluate sialic acids and hyaluronan expression, anionic components important for the structure and function of the renal tubulointerstitial compartment, in the early stages of sepsis. Two groups of rats were used: (1) sham-operated controls; (2) cecal ligation and puncture (CLP) (polymicrobial sepsis model). A search for microbial growth was made in the peritoneal fluid to document infection. Tubular function was evaluated by means of urinary protein loss, urinary Na⁺ and urea excretion. Kidney samples were processed to analyze histology, sialic acids (lectin histochemistry) and hyaluronan (immunohistochemistry) expression. Results showed increased urinary protein loss and fractional excretion of Na⁺ and urea reduction in the CLP group. Histological changes, particularly in the cortex and in proximal tubules of the CLP group, were observed. In septic rats, compared to controls, sialic acids decreased in amount and their acetylation increased in the tubules, although to a lesser extent in the proximal portion. Hyaluronan was expressed in the medullary interstitium and in a few areas of cortex in controls. In septic rats it increased in the cortical interstitium and appeared in proximal tubules. These results suggest correlation between expression changes of anionic components and tubulointerstitium morphofunctional alterations during sepsis. A role of these molecules in protection/defense and repair processes may be suggested.     

Effects of ethyl pyruvate on leukocyte-endothelial interactions in the mesenteric microcirculation during early sepsis treatment

OBJECTIVES:Experimental studies on sepsis have demonstrated that ethyl pyruvate is endowed with antioxidant and anti-inflammatory properties. This study aimed to investigate the effects of ethyl pyruvate on leukocyte-endothelial interactions in the mesenteric microcirculation in a live Escherichia coli-induced sepsis model in rats.METHODS:Male Wistar rats were administered an intravenous suspension of E. coli bacteria or were subjected to a sham procedure. Three hours after bacterial infusion, the rats were randomized into the following groups: a control group without treatment, a group treated with lactated Ringer''s solution (4 mL/kg, i.v.), and a group treated with lactated Ringer''s solution (4 mL/kg, i.v.) plus ethyl pyruvate (50 mg/kg). At 24 h after bacterial infusion, leukocyte-endothelial interactions were investigated using intravital microscopy, and the expression of P-selectin and intercellular adhesion molecule-1 was evaluated via immunohistochemistry. White blood cell and platelet counts were also determined at baseline and 3 h and 24 h after E. coli inoculation.RESULTS:The non-treated and lactated Ringer''s solution-treated groups exhibited increases in the numbers of rolling leukocytes (∼2.5-fold increase), adherent cells (∼3.0-fold), and migrated cells (∼3.5-fold) compared with the sham group. In contrast, treatment with Ringer''s ethyl pyruvate solution reduced the numbers of rolling, adherent and migrated leukocytes to the levels observed in the sham group. Additionally, the expression of P-selectin and intercellular adhesion molecule-1 was significantly increased on mesenteric microvessels in the non-treated group compared with the sham group (p<0.001). The expression of both adhesion molecules was reduced in the other groups, with ethyl pyruvate being more effective than lactated Ringer''s solution. Infusion of bacteria caused significant leukopenia (3 h), followed by leukocytosis with granulocytosis (24 h). There was also an intense and progressive reduction in the number of platelets. However, no differences were observed after treatment with the different solutions.CONCLUSIONS:The presented data suggest that ethyl pyruvate efficiently reduces the inflammatory response in the mesenteric microcirculation in an experimental model of sepsis induced by live E. coli and is associated, at least in part, with down-regulation of P-selectin and intercellular adhesion molecule-1.     

Lipopeptides rather than lipopolysaccharide favor the development of dendritic cell dysfunction similar to polymicrobial sepsis in mice

Objective

We investigated whether the dysfunction of dendritic cells (DC) that develops during polymicrobial sepsis is mimicked by systemic administration of the Toll-like receptor (TLR) 4 agonist lipopolysaccharide (LPS) or of the TLR2 agonist Pam3-Cys-Ser-Lys4 (P₃CSK₄).

Materials and methods

BALB/c mice underwent cecal ligation and puncture (CLP) or sham operation or received a single i.p. injection of LPS (30 mg/kg body weight), P₃CSK₄ (10 mg/kg body weight), or saline as control. Purified splenic DC and in-vitro-generated DC from bone marrow were analyzed in terms of surface marker expression, cytokine secretion, and antigen-specific T-cell activation in vivo.

Results

Splenic DC were suppressed in IL-12 secretion 12 h after LPS and P₃CSK₄ administration but released increased levels of IL-12 4 days after TLR agonist application, unlike DC from CLP mice. Polymicrobial sepsis and TLR agonists caused a loss of DC in the spleen but led to the expansion of diverse DC subsets. DC that differentiated from bone marrow after P₃CSK₄ but not after LPS application resembled DC from CLP mice regarding cytokine secretion and impaired Th1-cell polarization.

Conclusions

The development of DC dysfunction during sepsis is at least partly mimicked by TLR2 agonists rather than TLR4 agonists.     

CC chemokine receptor 2 regulates leukocyte recruitment and IL-10 production during acute polymicrobial sepsis

Chemokine receptors are important for recruiting leukocytes to sites of infection and may contribute to immune cell activation. The present study investigated the role of the chemokine receptor CCR2 in polymicrobial septic peritonitis. The results showed that peritoneal production of the CCR2 ligands CCL2 and CCL12 in septic mice was largely independent of the common Toll-like receptor signaling adaptor MyD88. Antibody blockade of CCR2 reduced the recruitment of macrophages and neutrophils to the infected peritoneal cavities of both wild-type and MyD88-deficient mice, suggesting that CCR2 engagement contributes to the MyD88-independent cellular response against polymicrobial septic peritonitis. Notably, administration of blocking CCR2 antibodies markedly increased local and systemic IL-10 levels in septic wild-type mice, whereas IL-10 was not detected in MyD88-deficient mice irrespective of whether CCR2 was blocked or not. Inhibition of CCR2 directly augmented Toll-like receptor-induced IL-10, but not TNF and IL-6, production of macrophages in vitro. Concomitant with enhanced IL-10 production, CCR2 blockade caused impaired bacterial clearance and aggravated kidney injury in wild-type, but not MyD88-null mice. These results indicate that CCR2 engagement modulates the innate immune response to polymicrobial septic peritonitis by both MyD88-dependent and -independent processes and suggest that a major function of CCR2 in sepsis is to attenuate IL-10 production and IL-10-mediated suppression of host defense.     

Systemic mast cell degranulation increases mortality during polymicrobial septic peritonitis in mice

MCs are required for an effective host response during septic peritonitis. Local MC degranulation facilitates neutrophil recruitment, activation, and bacterial killing. However, the role of MCs located distant from the site of infection is unknown. We studied the temporal and spacial degranulation of MCs following CLP-induced septic peritonitis. The functional importance of systemic MC degranulation during infection was evaluated by compartment-specific MC reconstitution. Serum histamine, reflecting MC degranulation, was elevated 4 h after onset of septic peritonitis. Histologic examination revealed progressive MC degranulation in select tissues during the first 24 h of infection. MC-deficient Wsh mice, reconstituted only in the peritoneal compartment, had improved survival after CLP compared with controls. However, reconstitution in peritoneal plus systemic compartments worsened survival after CLP. IL-6 contributed to the detrimental effects of systemic MCs on survival, as mice systemically reconstituted with IL-6(-/-) MCs were more likely to survive than control mice. These results indicate that in contrast to the benefits of local MC activation during infection, systemic MC activation worsens survival during CLP-induced sepsis.     

右美托咪定对脓毒血症大鼠血脑屏障的影响

目的探讨右美托咪定(dexmedetomidine,DEX)对内毒素血症大鼠模型血脑屏障及认知功能的影响。方法健康SD雄性大鼠24只,随机分为4组(n=6):空白对照组(C组)、脂多糖组(LPS组)、右美托咪定组(DEX组)和右美托咪定+脂多糖组(DEX+LPS组)。采用尾静脉注射LPS方法制备内毒素血症模型。各组大鼠进行Morris水迷宫实验,记录大鼠逃避潜伏期、穿越平台次数、在平台所在象限时间。处死大鼠,取海马组织,采用免疫组化法和Western blot检测各组大鼠海马组织血脑屏障(BBB)结构蛋白Occludin和ZO-1蛋白表达,采用ELISA方法检测各组大鼠海马组织炎症因子TNF-α、IL-1β和IL-6。结果 LPS组大鼠认知功能受损严重,海马组织Occludin和ZO-1蛋白的表达水平降低,炎症因子TNF-α、 IL-1β和IL-6含量显著升高;DEX+LPS组大鼠认知功能受损程度较LPS组轻,海马组织Occludin蛋白和ZO-1的蛋白表达升高,炎症因子TNF-α、IL-1β和IL-6含量显著降低。结论右美托咪定改善内毒素血症大鼠的认知功能,与上调Occludin以及ZO-1蛋白表达水平相关。     

Detrimental role of CC chemokine receptor 4 in murine polymicrobial sepsis

CC chemokine receptor 4 (CCR4) and its two ligands, CCL17 and CCL22, are critically involved in different immune processes. In models of lipopolysaccharide-induced shock, CCR4-deficient (CCR4^−/−) mice showed improved survival rates associated with attenuated proinflammatory cytokine release. Using CCR4^−/− mice with a C57BL/6 background, this study describes for the first time the role of CCR4 in a murine model of polymicrobial abdominal sepsis, the colon ascendens stent peritonitis (CASP). CASP-induced sepsis led to a massive downregulation of CCR4 in lymphoid and nonlymphoid tissues, whereas the expression of CCL17 and CCL22 was independent of the presence of CCR4. After CASP, CCR4^−/− animals showed a strongly enhanced bacterial clearance in several organs but not in the peritoneal lavage fluid and the blood. In addition, significantly reduced levels of proinflammatory cytokines/chemokines were measured in organ supernatants as well as in the sera of CCR4^−/− mice. CCR4 deficiency consequently resulted in an attenuated severity of systemic sepsis and a strongly improved survival rate after CASP or CASP with intervention. Thus, our data provide clear evidence that CCR4 plays a strictly detrimental role in the course of polymicrobial sepsis.