Depletion of neutrophil extracellular traps in vivo results in hypersusceptibility to polymicrobial sepsis in mice

Introduction

Although the formation of neutrophil (PMN) extracellular traps (NETs) has been detected during infection and sepsis, their role in vivo is still unclear. This study was performed in order to evaluate the influence of NETs depletion by administration of recombinant human (rh)DNase on bacterial spreading, PMN tissue infiltration and inflammatory response in a mouse model of polymicrobial sepsis.

Methods

In a prospective controlled double-armed animal trial, polymicrobial sepsis was induced by cecal ligation and puncture (CLP). After CLP, mice were treated with rhDNase or phosphate buffered saline, respectively. Survival, colony forming unit (CFU) counts in the peritoneal cavity, lung, liver and blood were determined. PMN and platelet counts, IL-6 and circulating free (cf)-DNA/NETs levels were monitored. PMN infiltration, as well as organ damage, was analyzed histologically in the lungs and liver. Capability and capacity of PMN to form NETs were determined over time.

Results

cf-DNA/NETs were found to be significantly increased 6, 24, and 48 hours after CLP when compared to the levels determined in sham and naïve mice. Peak levels after 24 hours were correlated to enhanced capacity of bone marrow-derived PMN to form NETs after ex vivo stimulation with phorbol-12-myristate-13-acetate at the same time. rhDNase treatment of mice resulted in a significant reduction of cf-DNA/NETs levels 24 hours after CLP (P < 0.001). Although overall survival was not affected by rhDNase treatment, median survival after 24 hours was significantly lower when compared with the CLP group (P < 0.01). In mice receiving rhDNase treatment, CFU counts in the lung (P < 0.001) and peritoneal cavity (P < 0.05), as well as serum IL-6 levels (P < 0.001), were found to be already increased six hours after CLP. Additionally, enhanced PMN infiltration and tissue damage in the lungs and liver were found after 24 hours. In contrast, CFU counts in mice without rhDNase treatment increased later but more strongly 24 hours after CLP (P < 0.001). Similarly, serum IL-6 levels peaked after 24 hours (P < 0.01).

Conclusions

This study shows, for the first time, that depletion of NETs by rhDNase administration impedes the early immune response and aggravates the pathology that follows polymicrobial sepsis in vivo.     

Therapeutic efficacy of CXCR3 blockade in an experimental model of severe sepsis

Introduction

In our previous studies we demonstrated that CXC chemokine receptor 3 (CXCR3) participates in the regulation of lymphocyte trafficking during cecal ligation and puncture (CLP)-induced sepsis. In this study, we evaluated the effects of treatment with anti-CXCR3 immunoglobulin (IgG) and antibiotics on outcome during septic shock caused by CLP.

Methods

C57BL/6J mice were treated with neutralizing IgG against CXCR3 plus Primaxin either 24 hours prior to, 2 hours after or 6 hours after CLP. Control mice received nonspecific IgG plus Primaxin in the same regimen. Survival, core body temperature, bacterial clearance and systemic cytokine production were evaluated.

Results

Our results show that treatment with anti-CXCR3 IgG plus Primaxin significantly improved survival when administered 24 hours prior to CLP (50% vs. 10%), 2 hours after CLP (55% vs. 10%) or 6 hours after CLP (55% vs. 25%) compared with mice receiving nonspecific IgG plus Primaxin. Treatment with anti-CXCR3 plus Primaxin 24 hours prior to CLP attenuated hypothermia and IL-6 and macrophage inflammatory protein 2 (MIP-2) production but did not alter bacterial clearance. Treatment with anti-CXCR3 IgG and Primaxin 2 hours after CLP did not improve bacterial clearance and systemic cytokine production compared with mice treated with IgG and Primaxin, whereas 6 hours after CLP the bacterial clearance and IL-6 and MIP-2 concentrations, both in plasma and peritoneal lavage fluid, were significantly improved in mice receiving anti-CXCR3 IgG and Primaxin compared with mice that only received nonspecific IgG and Primaxin.

Conclusion

The results from this study indicate that neutralization of CXCR3 prior to, 2 hours after or 6 hours after the initiation of CLP-induced septic shock improves survival and attenuates CLP-induced inflammation and physiologic dysfunction.     

Levosimendan attenuates multiple organ injury and improves survival in peritonitis-induced septic shock: studies in a rat model

Introduction

The aim of this study was to investigate the effects of levosimendan on rodent septic shock induced by cecal ligation and puncture (CLP).

Methods

Three hours after peritonitis-induced sepsis, male Wistar rats were randomly assigned to receive an intravenous infusion of levosimendan (1.2 μg/kg/min for 10 min and then 0.3 μg/kg/min for 6 h) or an equivalent volume of saline and vehicle (5% dextrose) solution.

Results

The levosimendan-treated CLP animals had significantly higher arterial pressure and lower biochemical indices of liver and kidney dysfunction compared to the CLP animals (P < 0.05). Plasma interleukin-1β, nitric oxide and organ superoxide levels in the levosimendan-treated CLP group were less than those in CLP rats treated with vehicle (P < 0.05). In addition, the inducible nitric oxide synthase (iNOS) in lung and caspase-3 expressions in spleen were significantly lower in the levosimendan-treated CLP group (P < 0.05). The administration of CLP rats with levosimendan was associated with significantly higher survival (61.9% vs. 40% at 18 h after CLP, P < 0.05). At postmortem examination, the histological changes and neutrophil filtration index in liver and lung were significantly attenuated in the levosimendan-treated CLP group (vs. CLP group, P < 0.05).

Conclusions

In this clinically relevant model of septic shock induced by fecal peritonitis, the administration of levosimendan had beneficial effects on haemodynamic variables, liver and kidney dysfunction, and metabolic acidosis. (1) Lower levels of interleukin-1β, nitric oxide and superoxide, (2) attenuation of iNOS and caspase-3 expressions, and (3) decreases of neutrophil infiltration by levosimendan in peritonitis-induced sepsis animals suggest that anti-inflammation and anti-apoptosis effects of levosimendan contribute to prolonged survival.     

The anti-inflammatory effect of the synthetic antimicrobial peptide 19-2.5 in a murine sepsis model: a prospective randomized study

Introduction

Increasing rates of multi-resistant bacteria are a major problem in the treatment of critically ill patients. Furthermore, conventional antibiotics lead to the release of bacterial derived membrane parts initiating pro-inflammatory cascades with potential harm to the patient. Antimicrobial peptides (AMP) may kill bacteria without releasing pro-inflammatory factors. Thus, we compared three newly developed synthetic anti-lipopolysaccharide peptides (SALPs) with a broader range of efficacy to suppress cytokine release in plasma and CD14 mRNA expression in organ tissue in a murine, polymicrobial sepsis model.

Methods

A randomized, experimental trial was conducted in an animal research facility. Male NMRI mice (n = 90; 8- to 12-weeks old) were randomized to the following six groups: (i) sham operation and parenteral vehicle (NaCl 0.9%) administration (sham); (ii) cecal ligation and puncture (CLP) and vehicle infusion (sepsis-control), (iii) CLP and polymyxin B infusion (polyB), or (iv to vi) CLP and infusion of three different synthetic antimicrobial peptides Peptide 19-2.5 (Pep2.5), Peptide 19-4 (Pep4) or Peptide 19-8 (Pep8). All animals underwent arterial and venous catheterization for hemodynamic monitoring 48 hours prior to CLP or sham-operation. Physical appearance and behavior (activity), plasma cytokine levels, and CD14 mRNA expression in heart, lung, liver, spleen and kidney tissue were determined 24 hours after CLP or sham operation.

Results

Only Pep2.5 significantly enhanced the activity after CLP, whereas none of the therapeutic regimens elevated the mean arterial pressure or heart rate. The strongly elevated IL-6, IL-10 and monocyte chemoattractant protein serum levels in septic animals were significantly reduced after Pep2.5 administration (P < 0.001, P < 0.001, and P < 0.001, respectively). Similarly, Pep2.5 significantly reduced the sepsis-induced CD14 mRNA expression in heart (P = 0.003), lung (P = 0.008), and spleen tissue (P = 0.009) but not in kidney and liver.

Conclusions

Structurally variable SALPs exhibit major differences in their anti-inflammatory effect in vivo. Continuous parenteral administration of Pep2.5 is able to reduce sepsis-induced cytokine release and tissue inflammation.     

Expression of heat shock protein 70 in lung tissues of acute paraquat poisoned rats and intervention of ulinastatin

BACKGROUND:

Paraquat (PQ) is an effective herbicide and is widely used in agricultural production, but PQ poisoning is frequently seen in humans with the lung as the target organ. Clinically pulmonary pathological changes are often used to predict the severity and prognosis of the patients. In this study, we observed the expression of heat shock protein 70 (HSP70) in rat lung after PQ poisoning and to investigate the therapeutic effects of ulinastatin.

METHODS:

Seventy-two adult healthy SD rats were randomly divided into a control group (group A, n=24), a poisoning group (group B, n=24), and an ulinastatin group (group C, n=24). The rat models of acute PQ poisoning were established by intra-gastric administration of 80 mg/kg PQ to rats of groups B and C, and the rats of group C were intra-peritoneally injected with 100 000 IU/kg ulinastatin 30 minutes after poisoning. The expression of HSP70 in lung tissue was observed, and W/D and histopathological changes in the lung tissue were compared 12, 24, 48 and 72 hours after poisoning. The expression of HSP70 in the lung tissue was assayed by using RT-PCR. All quantitative data were processed with one-way analysis of variance to compare multiple sample means.

RESULTS:

Compared to group A, the expression of HSP70 in the lung of rats in groups B and C increased significantly at all intervals (P<0.05). The pathological changes in lung tissue of rats with PQ poisoning included congestion, leukocytes infiltration and local hemorrhage, whereas those of group C were significantly lessened.

CONCLUSION:

Ulinastatin may ameliorate acute lung injury to some extent after PQ poisoning in rats by enhancing the expression of HSP70.KEY WORDS: Paraquat, Poisoning, Ulilnastatin, Heat shock protein, Acute lung injury     

Multiple gene-to-gene interactions in children with sepsis: a combination of five gene variants predicts outcome of life-threatening sepsis

Introduction

The aim of the study was to identify the dependency structure of genetic variants that can influence the outcome for paediatric patients with sepsis.

Methods

We evaluated the role of single nucleotide polymorphisms for five genes: bactericidal permeability increasing protein (BPI; rs5743507), lipopolysaccharide-binding protein (LBP; rs2232618), toll-like receptor 4 (TLR4; rs4986790), heat shock protein 70 (HSP 70; rs2227956), and interleukin 6 (IL-6; rs1800795) in 598 children aged 0 to 19 years that were admitted to a paediatric intensive care unit with fever, systemic inflammatory response syndrome, sepsis, severe sepsis, septic shock, or multiple organ dysfunction syndrome. A control group of 529 healthy individuals was included. Multi-way contingency tables were constructed and statistically evaluated using log-linear models. Typical gene combinations were found for both study groups.

Results

Detailed analyses of the five studied gene profiles revealed significant differences in sepsis survival. Stratification into high-risk, intermediate-risk, and low-risk groups of paediatric patients can predict the severity of sepsis.

Conclusions

Analysis of single nucleotide polymorphisms for five genes can be used as a predictor of sepsis outcome in children.     

Induced expression and functional effects of aquaporin-1 in human leukocytes in sepsis

Introduction

Gene expression profiling was performed via DNA microarrays in leukocytes from critically ill trauma patients nonseptic upon admission to the ICU, who subsequently developed either sepsis (n = 2) or severe sepsis and acute respiratory distress syndrome (n = 3). By comparing our results with published expression profiling studies in animal models of sepsis and lung injury, we found aquaporin-1 to be differentially expressed across all studies. Our aim was to determine how the water channel aquaporin-1 is involved in regulating the immune response in critically ill patients during infection acquired in the ICU.

Methods

Following the results of the initial genetic screening study, we prospectively followed aquaporin-1 leukocyte expression patterns in patients with ICU-acquired sepsis who subsequently developed septic shock (n = 16) versus critically ill patients who were discharged without developing sepsis (n = 13). We additionally determined aquaporin-1 expression upon lipopolysaccharide (LPS) exposure and explored functional effects of aquaporin-1 induction in polymorphonuclear granulocytes (PMNs).

Results

Leukocyte aquaporin-1 expression was induced at the onset of sepsis (median 1.71-fold increase; interquartile range: 0.99 to 2.42, P = 0.012 from baseline) and was further increased upon septic shock (median 3.00-fold increase; interquartile range: 1.20 to 5.40, P = 0.023 from sepsis, Wilcoxon signed-rank test); no difference was observed between baseline and discharge in patients who did not develop sepsis. Stimulation of PMNs by LPS led to increased expression of aquaporin-1 in vitro, which could be abrogated by the NF-κB inhibitor EF-24. PMN hypotonic challenge resulted in a transient increase of the relative cell volume, which returned to baseline after 600 seconds, while incubation in the presence of LPS resulted in persistently increased cell volume. The latter could be abolished by blocking aquaporin-1 with mercury and restored by incubation in β-mercaptoethanol, which abrogated the action of mercury inhibition.

Conclusions

Aquaporin-1 is induced in leukocytes of patients with ICU-acquired sepsis and exhibits higher expression in septic shock. This phenomenon may be due to LPS-triggered NF-κB activation that can also lead to alterations in plasma membrane permeability.     

Kinetics and protective role of autophagy in a mouse cecal ligation and puncture-induced sepsis

Introduction

It is not well understood whether the process of autophagy is accelerated or blocked in sepsis, and whether it is beneficial or harmful to the immune defense mechanism over a time course during sepsis. Our aim was to determine both the kinetics and the role of autophagy in sepsis.

Methods

We examined autophagosome and autolysosome formation in a cecal ligation and puncture (CLP) mouse model of sepsis (in C57BL/6N mice and GFP-LC3 transgenic mice), using western blotting, immunofluorescence, and electron microscopy. We also investigated the effect of chloroquine inhibition of autophagy on these processes.

Results

Autophagy, as demonstrated by increased LC3-II/LC3-I ratios, is induced in the liver, heart, and spleen over 24 h after CLP. In the liver, autophagosome formation peaks at 6 h and declines by 24 h. Immunofluorescent localization of GFP-LC3 dots (alone and with lysosome-associated membrane protein type 1 (LAMP1)), as well as electron microscopic examination, demonstrate that both autophagosomes and autolysosomes are increased after CLP, suggesting that intact autophagy mechanisms operate in the liver in this model. Furthermore, inhibition of autophagy process by chloroquine administration immediately after CLP resulted in elevated serum transaminase levels and a significant increase in mortality.

Conclusions

All autophagy-related processes are properly activated in the liver in a mouse model of sepsis; autophagy appears to play a protective role in septic animals.     

Inhibition of complement C5a prevents breakdown of the blood-brain barrier and pituitary dysfunction in experimental sepsis

Introduction

Septic encephalopathy secondary to a breakdown of the blood-brain barrier (BBB) is a known complication of sepsis. However, its pathophysiology remains unclear. The present study investigated the effect of complement C5a blockade in preventing BBB damage and pituitary dysfunction during experimental sepsis.

Methods

Using the standardised caecal ligation and puncture (CLP) model, Sprague-Dawley rats were treated with either neutralising anti-C5a antibody or pre-immune immunoglobulin (Ig) G as a placebo. Sham-operated animals served as internal controls.

Results

Placebo-treated septic rats showed severe BBB dysfunction within 24 hours, accompanied by a significant upregulation of pituitary C5a receptor and pro-inflammatory cytokine expression, although gene levels of growth hormone were significantly attenuated. The pathophysiological changes in placebo-treated septic rats were restored by administration of neutralising anti-C5a antibody to the normal levels of BBB and pituitary function seen in the sham-operated group.

Conclusions

Collectively, the neutralisation of C5a greatly ameliorated pathophysiological changes associated with septic encephalopathy, implying a further rationale for the concept of pharmacological C5a inhibition in sepsis.     

Kinetics of circulating immunoglobulin M in sepsis: relationship with final outcome

Introduction

The aim of this study was to investigate the kinetics of immunoglobulin M (IgM) during the different stages of sepsis.

Methods

In this prospective multicenter study, blood sampling for IgM measurement was done within the first 24 hours from diagnosis in 332 critically ill patients; in 83 patients this was repeated upon progression to more severe stages. Among these 83 patients, 30 patients with severe sepsis progressed into shock and IgM was monitored daily for seven consecutive days. Peripheral blood mononuclear cells (PBMCs) were isolated from 55 patients and stimulated for IgM production.

Results

Serum IgM was decreased in septic shock compared to patients with systemic inflammatory response syndrome (SIRS) and patients with severe sepsis. Paired comparisons at distinct time points of the sepsis course showed that IgM was decreased only when patients deteriorated from severe sepsis to septic shock. Serial measurements in these patients, beginning from the early start of vasopressors, showed that the distribution of IgM over time was significantly greater for survivors than for non-survivors. Production of IgM by PBMCs was significantly lower at all stages of sepsis compared with healthy controls.

Conclusions

Specific changes of circulating IgM occur when patients with severe sepsis progress into septic shock. The distribution of IgM is lower among non-survivors.     

Cardioprotective effect of erythropoietin on sepsis-induced myocardial injury in rats

BACKGROUND:

Sepsis-induced myocardial injury is one of the major predictors of morbidity and mortality of sepsis. The cytoprotective function of erythropoietin (EPO) has been discovered and extensively studied. However, the cardioprotective effects of EPO on sepsis-induced myocardial injury in the rat sepsis model has not been reported.

METHODS:

The rat models of sepsis were produced by cecal ligation and perforation (CLP) surgery. Rats were randomly (random number) assigned to one of three groups (n=8 for each group): sham group, CLP group and EPO group (1000 IU/kg erythropoietin). Arterial blood was withdrawn at 3, 6, 12, and 24 hours after CLP. cTnI, BNP, CK-MB, LDH, AST, TNF-α, IL-6, IL-10, and CRP were tested by the ELISA assay. Changes of hemodynamic parameters were recorded at 3, 6, 12, 24 hours after the surgery. Histological diagnosis was made by hematoxylin and eosin. Flow cytometry was performed to examine cell apoptosis, myocardium mitochondrial inner membrane potential, and NF-κB (p65). Survival rate at 7 days after CLP was recorded.

RESULTS:

In the CLP group, myocardial enzyme index and inflammatory index increased at 3, 6, 12 and 24 hours after CLP compared with the sham group, and EPO significantly blocked the increase. Compared with the CLP group, EPO significantly improved LVSP, LV +dp/dt_max, LV −dp/dt_min, and decreased LVEDP at different time. EPO blocked the reduction of mitochondrial transmembrane potential, suppressed the cardiomyocyte apoptosis, inhibited the activation of NF-κB, and reduced the production of proinflmmatory cytokines. No difference in the survival rate at 7 days was observed between the CLP group and the EPO group.

CONCLUSION:

Exogenous EPO has cardioprotective effects on sepsis-induced myocardial injury.KEY WORDS: Sepsis, Sepsis-induced myocardial injury, Apoptosis, Chondriosome membrane potential, Nuclear factor κB p65, Erythropoietin, Inflammatory cytokines, Rat     

Towards personalized medicine in sepsis: Quest for Shangri-La?

Expanded abstract

Citation

Boomer JS, To K, Chang KC, Takasu O, Osborne DF, Walton AH, Bricker TL, Jarman SD 2nd, Kreisel D, Krupnick AS, Srivastava A, Swanson PE, Green JM, Hotchkiss RS: Immunosuppression in patients who die of sepsis and multiple organ failure. JAMA 2011, 306:2594-2605.

Background

Severe sepsis is typically characterized by initial cytokine-mediated hyper-inflammation. Whether this hyper-inflammatory phase is followed by immunosuppression is a subject of controversy. Animal studies suggest that multiple immune defects occur in sepsis, but data from humans remain conflicting.

Methods

Objectives

To determine the association of sepsis with changes in host innate and adaptive immunity and to examine potential mechanisms for putative immunosuppression.

Design

Rapid prospective postmortem spleen and lung tissue harvest and analysis within 120 minutes of death.

Setting

Intensive care units (ICUs) of academic medical centers.

Subjects

A convenience sample of 40 patients who died with active severe sepsis was taken over the course of 2 years to characterize their immune status at the time of death. Control spleens (n = 29) were obtained from patients who were declared brain dead or had emergent splenectomy due to trauma; control lungs (n = 20) were obtained from transplant donors or from lung cancer resections.

Interventions

None.

Outcomes

Cytokine secretion assays and immunophenotyping of cell surface receptor-ligand expression profiles were performed to identify potential mechanisms of immune dysfunction. Immunohistochemical staining was performed to evaluate the loss of immune effector cells.

Results

The mean ages (standard deviations) of patients with sepsis and controls were 71.7 (15.9) and 52.7 (15.0) years, respectively. Patients with sepsis were in the ICU for a median of 8 days (range of 1 to 195 days), whereas control patients were in the ICU for not more than 4 days. The median duration of sepsis was 4 days (range of 1 to 40 days). Anti-CD3/anti-CD28-stimulated splenocytes from patients with sepsis, compared with those from controls, had significant reductions in cytokine secretion at 5 hours: tumor necrosis factor, 5,361 (95% confidence interval (CI) 3,327 to 7,485) pg/mL versus 418 (95% CI 98 to 738) pg/mL; interferon-gamma, 1,374 (95% CI 550 to 2,197) pg/mL versus 37.5 (95% CI -5 to 80) pg/mL; interleukin-6, 3,691 (95% CI 2,313 to 5,070) versus 365 (95% CI 87 to 642) pg/mL; and interleukin-10, 633 (95% CI -269 to 1,534) versus 58 (95% CI -39 to 156) pg/mL (P < 0.001 for all). There were similar reductions in 5-hour lipopolysaccharidestimulated cytokine secretion. Cytokine secretion in patients with sepsis was generally less than 10% of that in controls, independently of age, duration of sepsis, corticosteroid use, and nutritional status. Despite differences between spleen and lung, flow cytometric analysis showed increased expression of selected inhibitory receptors and ligands and expansion of suppressor cell populations in both organs. Unique differences in cellular inhibitory molecule expression existed in immune cells isolated from lungs of patients with sepsis versus patients with cancer and versus transplant donors. Immunohistochemical staining showed extensive depletion of splenic CD4, CD8, and HLA-DR cells and expression of ligands for inhibitory receptors on lung epithelial cells.

Conclusions

Patients who die in the ICU following sepsis compared with patients who die of non-sepsis etiologies have bio-chemical, flow cytometric, and immunohistochemical findings consistent with those of immunosuppression. Targeted immune-enhancing therapy may be a valid approach in selected patients with sepsis.     

Early alterations of the innate and adaptive immune statuses in sepsis according to the type of underlying infection

Introduction

Although major changes of the immune system have been described in sepsis, it has never been studied whether these may differ in relation to the type of underlying infection or not. This was studied for the first time.

Methods

The statuses of the innate and adaptive immune systems were prospectively compared in 505 patients. Whole blood was sampled within less than 24 hours of advent of sepsis; white blood cells were stained with monoclonal antibodies and analyzed though a flow cytometer.

Results

Expression of HLA-DR was significantly decreased among patients with severe sepsis/shock due to acute pyelonephritis and intraabdominal infections compared with sepsis. The rate of apoptosis of natural killer (NK) cells differed significantly among patients with severe sepsis/shock due to ventilator-associated pneumonia (VAP) and hospital-acquired pneumonia (HAP) compared with sepsis. The rate of apoptosis of NKT cells differed significantly among patients with severe sepsis/shock due to acute pyelonephritis, primary bacteremia and VAP/HAP compared with sepsis. Regarding adaptive immunity, absolute counts of CD4-lymphocytes were significantly decreased among patients with severe sepsis/shock due to community-acquired pneumonia (CAP) and intraabdominal infections compared with sepsis. Absolute counts of B-lymphocytes were significantly decreased among patients with severe sepsis/shock due to CAP compared with sepsis.

Conclusions

Major differences of the early statuses of the innate and adaptive immune systems exist between sepsis and severe sepsis/shock in relation to the underlying type of infection. These results may have a major impact on therapeutics.     

Effects of intra-abdominal sepsis on atherosclerosis in mice

Introduction

Sepsis and other infections are associated with late cardiovascular events. Although persistent inflammation is implicated, a causal relationship has not been established. We tested whether sepsis causes vascular inflammation and accelerates atherosclerosis.

Methods

We performed prospective, randomized animal studies at a university research laboratory involving adult male ApoE-deficient (ApoE^−/−) and young C57B/L6 wild-type (WT) mice. In the primary study conducted to determine whether sepsis accelerates atherosclerosis, we fed ApoE^−/− mice (N = 46) an atherogenic diet for 4 months and then performed cecal ligation and puncture (CLP), followed by antibiotic therapy and fluid resuscitation or a sham operation. We followed mice for up to an additional 5 months and assessed atheroma in the descending aorta and root of the aorta. We also exposed 32 young WT mice to CLP or sham operation and followed them for 5 days to determine the effects of sepsis on vascular inflammation.

Results

ApoE^−/− mice that underwent CLP had reduced activity during the first 14 days (38% reduction compared to sham; P < 0.001) and sustained weight loss compared to the sham-operated mice (−6% versus +9% change in weight after CLP or sham surgery to 5 months; P < 0.001). Despite their weight loss, CLP mice had increased atheroma (46% by 3 months and 41% increase in aortic surface area by 5 months; P = 0.03 and P = 0.004, respectively) with increased macrophage infiltration into atheroma as assessed by immunofluorescence microscopy (0.52 relative fluorescence units (rfu) versus 0.97 rfu; P = 0.04). At 5 months, peritoneal cultures were negative; however, CLP mice had elevated serum levels of interleukin 6 (IL-6) and IL-10 (each at P < 0.05). WT mice that underwent CLP had increased expression of intercellular adhesion molecule 1 in the aortic lumen versus sham at 24 hours (P = 0.01) that persisted at 120 hours (P = 0.006). Inflammatory and adhesion genes (tumor necrosis factor α, chemokine (C-C motif) ligand 2 and vascular cell adhesion molecule 1) and the adhesion assay, a functional measure of endothelial activation, were elevated at 72 hours and 120 hours in mice that underwent CLP versus sham-operations (all at P <0.05).

Conclusions

Using a combination of existing murine models for atherosclerosis and sepsis, we found that CLP, a model of intra-abdominal sepsis, accelerates atheroma development. Accelerated atheroma burden was associated with prolonged systemic, endothelial and intimal inflammation and was not explained by ongoing infection. These findings support observations in humans and demonstrate the feasibility of a long-term follow-up murine model of sepsis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13054-014-0469-1) contains supplementary material, which is available to authorized users.     

Neutrophils with myeloid derived suppressor function deplete arginine and constrain T cell function in septic shock patients

Introduction

Impaired T cell function in sepsis is associated with poor outcome, but the mechanisms are unclear. In cancer, arginase-expressing myeloid derived suppressor cells (MDSCs) deplete arginine, impair T cell receptor CD3 zeta-chain expression and T cell function and are linked to poor clinical outcome, but their role during acute human infectious disease and in particular sepsis remains unknown. Hypoarginemia is prevalent in sepsis. This study aimed to determine whether neutrophils that co-purify with PBMC express arginase, and if arginine depletion constrains T cell CD3 zeta-chain expression and function in human sepsis.

Methods

Using flow cytometry, cell culture, HPLC, arginase activity and mRNA detection, our study examined whether neutrophils, with reduced buoyant density isolated in the Ficoll interface, metabolise L-arginine and suppress T cell proliferation in sepsis. A total of 35 sepsis patients (23 with septic shock) and 12 hospital controls in a tertiary referral hospital in tropical Australia were evaluated.

Results

Only sepsis patients had interphase neutrophils, neutrophils co-purifying with mononuclear cells (≤1.077 specific gravity). The percentage of interphase neutrophils in sepsis was proportional to sepsis severity and correlated with plasma IL-6 concentrations. Ex vivo, sepsis-derived interphase neutrophils expressed arginase, metabolised culture L-arginine and suppressed T cell proliferation and CD3 zeta-chain expression. In vivo, in septic shock there was a longitudinal inverse association between interphase neutrophil number and CD3 zeta-chain expression. Depletion or inhibition of interphase neutrophils in vitro restored zeta-chain expression and T cell function.

Conclusions

For the first time during an acute human infection, interphase neutrophils that express arginase were found to circulate in sepsis, in proportion to disease severity. These neutrophil-MDSCs impair T cell CD3 zeta-chain expression and T cell function via L-arginine metabolism, and likely contribute to the T cell dysfunction seen in sepsis. Modulation of neutrophil-MDSC or their downstream effects warrant consideration as targets for novel adjunctive therapies in sepsis.

Electronic supplementary material

The online version of this article (doi:10.1186/cc14003) contains supplementary material, which is available to authorized users.     

Glucocorticoid receptor expression on acute lung injury induced by endotoxin in rats

BACKGROUND:

In cases of severe sepsis and septic shock, a series of pathophysiological changes lead to multiple organ dysfunction syndrome. This study aimed to investigate the expression of glucocorticoid receptor mRNA in the rat lung following endotoxin (LPS) induced shock.

METHODS:

Totally 56 SD rats were randomly divided into 4 groups: LPS shock group (n=16), LPS+vasoactive intestinal peptide group(VIP) group, (n=16), LPS+VIP+ glucocorticoid (GC) group, (n=16),and control group (n=8). LPS shock was induced by intravenous injection of LPS (10 mg/kg) in rats. Within 15 minutes after LPS injection, rats in the treatment groups received VIP (5 nmol/kg) or VIP and methylprednisolone (3 mg/kg). The control group was given normal saline instead of LPS. The rats of the four groups were sacrificed at 6 hours,24 hours after injection respectively, and the lung tissues were collected. Pathological changes of the lungs were examined by light microscopy and electron microscopy. GRmRNA expression in the lung tissues was evaluated by RT-PCR.

RESULTS:

In the LPS shock group, lung histopathology demonstrated destruction of the alveolar space,widening of the inter-alveolar space, inflammatory cell infiltration and interstitial edema. However,pathological changes in the LPS+ VIP group and LPS+ VIP+GC group were milder than those in the LPS shock group. Six hours after LPS injection, GR mRNA expression was down-regulated in the LPS group (0.72± 0.24) and LPS+ VIP group (0.88±0.27) (P<0.05) as compared with the control group (1.17±0.22). The LPS shock group showed a more significant down-regualtion than the LPS+VIP group, but the difference was not statistically significant (P>0.05). In contrast, GRmRNA expression in the LPS+ VIP+GC group was significantly up-regulated at 6 hours and further at 24 hours (1.45±0.32 and 1.91±0.46 respectively) (P<0.05).

CONCLUSION:

GrmRNA expression decreased in LPS induced lung injury in rats. Combined treatment with VIP and GC mitigated lung injury ang inflammation. The mechanism may be related to up-regulation of GR mRNA expression.KEY WORDS: Glucocorticoid, GRmRNA, Vasoactive intestinal peptide, LPS, Shock, Inflammation, Lung injury, Rat     

Changes of the immunological barrier of intestinal mucosa in rats with sepsis

BACKGROUND:

Sepsis has become the greatest threat to in-patients, with a mortality of over 25%. The dysfunction of gut barrier, especially the immunological barrier, plays an important role in the development of sepsis. This dysfunction occurs after surgery, but the magnitude of change does not differentiate patients with sepsis from those without sepsis. Increased intestinal permeability before surgery is of no value in predicating sepsis. The present study aimed to observe the changes of intestinal mucosal immunologic barrier in rat models of sepsis induced by cecal ligation and puncture.

METHODS:

Sixty Sprague-Dawley rats were randomly divided into a sepsis group (n=45) and a control group (n=15). The rats in the sepsis group were subjected to cecal ligation and puncture (CLP), whereas the rats in the control group underwent a sham operation. The ileac mucosa and segments were harvested 3, 6 and 12 hours after CLP, and blood samples were collected. Pathological changes, protein levels of defensin-5 (RD-5) and trefoil factor-3 (TFF₃) mRNA, and lymphocytes apoptosis in the intestinal mucosa were determined. In an additional experiment, the gut-origin bacterial DNA in blood was detected.

RESULTS:

The intestinal mucosa showed marked injury with loss of ileal villi, desquamation of epithelium, detachment of lamina propria, hemorrhage and ulceration in the sepsis group. The expression of TFF₃ mRNA and level of RD-5 protein were decreased and the apoptosis of mucosal lymphocyte increased (P<0.05) in the sepsis group compared with the control group. Significant differences were observed in RD-5 and TFF₃ mRNA 3 hours after CLP and they were progressively increased 6 and 12 hours after CLP in the sepsis group compared with the control group (P<0.05, RD-5 F=11.76, TFF₃ F=16.86 and apoptosis F=122.52). In addition, the gut-origin bacterial DNA detected in plasma was positive in the sepsis group.

CONCLUSION:

The immunological function of the intestinal mucosa was impaired in septic rats and further deteriorated in the course of sepsis.KEY WORDS: Sepsis, Mucosal immunology, Defensin-5, Trefoil factor family 3, Cecal ligation and puncture     

Repair of damaged intestinal mucosa in a mouse model of sepsis

BACKGROUND:

The intestine is not only the main target attacked by sepsis but also the vital organ which mediated sepsis. The recovery of the damaged intestinal barrier structure and function is related to the occurrence and outcome of multiple organ dysfunction syndrome (MODS). How to protect and reduce the damage of the intestinal mucosa and how to promote the reconstruction of the intestinal mucosa have been the important topics in sepsis for many years. This study aimed to investigate the influential factors of intestinal mucosal reconstruction after intestinal epithelial injury in vivo in a mouse model of sepsis.

METHODS:

Mice were subjected to cecal ligation and puncture (CLP) for induction of sepsis to assess intestinal mucosal damage, epithelial cell apoptosis, and transformed number of goblet cells, and to detect the concentration of TNF-α, IL-1 and TGF-β1 and TFF3 (trefoil factor 3) expression in the small intestinal mucosa. All above were performed by HE staining, western blot, ELISA and immunohistochemistry respectively. The experimental animals were divided into a sepsis group and a sham-operation group. The animals with sepsis were separately killed at 6 (7 animals), 24 (7 animals) and 48 hours (7 animals) after CLP.

RESULTS:

Injured intestinal mucosa was observed in the 3 groups under a light microscope, in which damage scores in the 24-hour and 48-hour groups were higher than in the 6-hour group and no difference was found between the two groups. Moreover, less of goblet cells or other epithelial cells adjacent to the injured surface migrated into the wound to cover the denuded area. The number of goblet cells was substantially decreased in the three CLP groups compared with the sham-operation group. Protein levels of IL-1 and TNF-α were significantly increased by 3–4 fold at all time points when compared with the sham-operation group, and cleaved caspase-3 by 4 fold. Although TFF3 expression was modestly increased for 6 hours after the onset of CLP, it appeared to decline at 24 hours and 48 hours as shown by Western blot. A similar tendency was observed upon TGF-β1, i.e. the protein level was not elevated at 24 hours and 48 hours, but increased modestly at 6 hours.

CONCLUSIONS:

Sepsis from CLP shows less restitution on the surface of injured intestinal mucosa. There is evidence that both constant inflammatory reaction and epithelial cell apoptosis may affect mucosal reestablishment of the intestine at the onset of sepsis. Mucosa after severe sepsis showed the state of high inflammation, and declined goblet cell function and mucosal reconstruction, which affected the repair of damaged intestinal barrier. Constant inflammatory reaction, and declined goblet cell function and mucosal reconstruction ability may affect the reestablishment of intestinal mucosa at the onset of sepsis.KEY WORDS: Sepsis, Cecal ligation and puncture, Intestinal mucosa, Restitution, Goblet cells, Intestinal trefoil factor 3, Transforming growth factor β1, Cysteine-containing aspartate-specific proteases     

A touch of cooling may help

Expanded abstract

Citation

Schortgen F, Clabault K, Katsahian S, Devaquet J, Mercat A, Deye N, Dellamonica J, Bouadma L, Cook F, Beji O, Brun-Buisson C, Lemaire F, Brochard L: Multicenter randomized controlled clinical trial of fever control by external cooling to diminish vasopressor requirements in septic shock. Assistance Publique-Hôpitaux de Paris, France. Am J Respir Crit Care Med 2012, 185:1088-1095.

Background

Fever control may improve vascular tone and decrease oxygen consumption; however, fever may help combat infection.

Methods

Objective

To determine whether fever control by external cooling diminishes vasopressor requirements in septic shock.

Design

A multicenter randomized controlled trial.

Setting

Seven ICUs in France.

Subjects

Febrile patients with septic shock requiring vasopressors, mechanical ventilation, and sedation.

Intervention

Patients were randomly allocated to external cooling to achieve normothermia (36.5 to 37.8°C) for 48 hours.

Outcomes

The primary outcome was the number of patients with a 50% decrease in baseline vasopressor dose after 48 hours. Secondary outcomes were the numbers of patients with a 50% baseline vasopressor dose decrease after 2, 12, 24, and 36 hours, the percentage of patients requiring a vasopressor dose increase within 48 hours of baseline, the percentage of patients with shock reversal in the ICU, the change in Sequential Organ Failure Assessment score (ΔSOFA) versus baseline, and all-cause mortality on day 14 and at ICU and hospital discharge.

Results

There were 200 patients randomized, 101 to the cooling group and 99 to the no-cooling group. The percentage of patients with a 50% vasopressor dose decrease versus baseline was not significantly different at 48 hours of treatment (72% vs. 61%; absolute difference, 11%; 95% confidence interval (CI), -23 to 2; P = 0.4), although it was at 12 hours (54% vs. 20%; absolute difference, 34%; 95% CI, -46 to -21; P < 0.001). External cooling significantly reduced the number of patients needing a vasopressor dose increase (34% vs. 52%; absolute difference, -18%; 95% CI, -4 to -31%; P = 0.011) and significantly increased the shock reversal during the study period (86% vs. 73%; absolute difference, 13%; 95% CI, 2 to 25%; P = 0.021). Day 14 mortality was significantly lower in the cooling group (19% vs. 34%; absolute difference, -16%; 95% CI, -28 to -4; P = 0.013), but mortality was not different at ICU and hospital discharge.

Conclusions

Fever control using external cooling was safe, and decreased vasopressor requirements and early mortality in septic shock.