Dehydroepiandrosterone: a modulator of cellular immunity and heat shock protein 70 production during polymicrobial sepsis

Objective DHEA is an immunomodulatory steroid hormone that improves survival during systemic inflammation. A DHEA-induced modulation of heat shock protein response may be an alternative mechanism contributing to the beneficial effects of this hormone. We investigated the effect of DHEA administration on survival, cellular immune functions, and HSP-70 production in septic mice. Design and setting Randomized animal study, level I trauma center, university research laboratory. Subjects Male NMRI mice. Interventions Mice were subjected to sham operation (laparotomy, LAP) or sepsis (cecal ligation and puncture, CLP) with or without administration of either saline 0.9% (LAP, CLP) or 20 mg/kg DHEA subcutaneously (LAP/DHEA, CLP/DHEA). Survival was monitored over a 48-h period. Splenocyte apoptosis rate (AnnexinV binding), splenocyte proliferation ([³H]thymidine incorporation), TNF-α plasma concentration (ELISA), and HSP-70 concentration (ELISA) in tissue extracts from liver, lung, and spleen were monitored 48 h after onset of sepsis. Results DHEA administration improved the survival of septic mice (78% vs. 50%). This effect was paralleled by increased splenocyte proliferation, decreased cellular apoptosis rate of splenocytes, and attenuation of TNF-α release. Furthermore, an increased HSP-70 concentration was observed in lungs and spleens of DHEA-treated septic animals. Conclusions DHEA-treatment decreased the mortality rate of septic mice. This was accompanied by improved cellular immune functions and an augmented heat shock response (HSP-70) of lungs and spleens. Further studies are required to demonstrate a direct relationship between the improved survival and the observed alterations in the immune system in DHEA-treated animals.     

The contribution of CD4+ CD25+ T-regulatory-cells to immune suppression in sepsis

Studies have indicated that there is a development of generalized immune dysfunction after septic insult. However, the mechanisms responsible for these changes remain unclear. Recently, accumulating evidence shows that several lymphocyte subpopulations such as NKT-, CD4(+)-Th2-T-, CD8(+)-T-, gammadelta-T-, and CD4+ CD25+ T regulatory cells are capable of actively contributing to the induction of septic immune suppression. Thus, our aim was to investigate the contribution of CD4+ CD25+ cells to the immune dysfunction seen in sepsis. To study this, C57BL/6J, C57BL/6-Il6(tm1Kopf) (interleukin [IL] 6 -/-), and C57BL/6-Il10(tm1Cgn) (IL-10 -/-) mice were subjected to cecal ligation and puncture (CLP) or sham operations. Twenty-four hours later, blood was collected, and splenocytes were isolated. Phenotypic expression of CD4/CD25 (by fluorescence-activated cell sorter), cell proliferation (presented as proliferation index = [with anti-CD3]/[without anti-CD3]), and immune suppressive capacity (by in vitro add-back experiments) were assessed. The results indicate a marked elevation in CD4+ CD25+ cell levels and their proliferation index after sepsis in background mice. CD4+ CD25- cells from sham and CLP mice proliferated equally. However, coculture of CD4+ CD25- with CD4+ CD25+ cells suppressed their proliferation in both sham and CLP mice. Depletion of CD25+ cells in vivo before CLP markedly restored CD4+ CD25- proliferative capacity and Th1 cytokine release while not altering plasma proinflammatory cytokine levels. Subsequently, IL-6 -/- and IL-10 -/- mice were used to elucidate the possible mediator(s) regulating the changes seen after sepsis. Although CD4+ CD25+ cells increased after septic insult in both C57BL/6J and IL-6 -/- mice, this was not observed in IL-10 -/- mice. Similarly, in vitro proliferation studies showed that proliferation index increased in CD4+ CD25+ cells from septic C57BL/6J and IL6 -/- mice, but it remained the same in IL-10 -/- mice. Surprisingly, depletion of CD25+ cells before inducing sepsis did not alter septic mortality. Together, these findings suggest that although CD4+ CD25+ T regulatory cells induced by IL-10 seem to contribute to aspects of sepsis-induced lymphoid immune suppression, the oblation of CD25+ cells does not provide a survival advantage or disadvantage.     

Imipramine reverses the depressive symptoms in sepsis survivor rats

Objective To evaluate the antidepressant effect of imipramine on depressive symptoms observed in sepsis survivors rats. Design and setting Prospective, controlled experiment in an animal basic science laboratory. Subjects Male Wistar rats weighing 300–350 g. Interventions The rats underwent cecal ligation and perforation (CLP; sepsis group) with “basic support” (saline at 50 ml/kg immediately and 12 h after CLP plus ceftriaxone at 30 mg/kg and clindamycin at 25 mg/kg 6, 12, and 18 h after CLP) or sham-operated (control group). After 10 days of recovery rats received intraperitoneal injections of imipramine 10 mg/kg or saline and were subjected to the forced swimming test. Measurements and results The observed increase in the immobility time in the forced swimming test in animals subjected to CLP, as a parameter of depressive behavior, was reversed by imipramine. Conclusions The depressive symptoms evaluated by forced swimming test had been reversed after imipramine administration. Our data provide evidence that CLP-induced depressive symptoms are sensitive to antidepressants. This research was supported in part by UNESC (Brazil), FAPESC (Brazil), and CNPq (Brazil).     

Soluble triggering receptor expressed on myeloid cells 1 as an anti-inflammatory mediator in sepsis

Objective To define the significance of soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) in the septic cascade by comparing its kinetics to those of other proinflammatory mediators and of interleukin (IL) 10. Design Prospective study in a tertiary unit. Patients Blood was sampled from 90 patients with septic syndrome due to ventilator-associated pneumonia for 7 days after the appearance of symptoms. Concentrations of tumor necrosis factor (TNF) α, IL-6, IL-8, IL-10, and sTREM-1 were determined by enzyme-linked immunosorbent assay. Results Serum levels of TNFα, IL-6, IL-10, and sTREM-1 were higher in nonsurvivors than in survivors; similar differences were not found for IL-8. Positive correlations were found between the ratios IL-10/TNFα and sTREM-1/TNFα, between IL-10/IL-6 and sTREM-1/IL-6, and between IL-10/IL-8 and sTREM-1/IL-8. Median values of IL-10/TNFα upon presentation of sepsis, severe sepsis, and septic shock were 3.21, 2.16, and 2.86, respectively (NS). Respective values for sTREM-1/TNFα were 21.28, 7.33, and 27.78 (p = 0.047 between sepsis and severe sepsis, p = 0.003 between severe sepsis and septic shock). Conclusions sTREM-1 follows the kinetics of IL-10 and should therefore be considered an anti-inflammatory mediator in sepsis. Decreased ratios of sTREM-1/TNFα might determine transition from sepsis to severe sepsis and from severe sepsis to septic shock. This article refers to the editorial .     

Polymicrobial sepsis induces divergent effects on splenic and peritoneal dendritic cell function in mice

Dendritic cells (DCs) are professional antigen-presenting cells that act as sentinels in the cell-mediated response against invading pathogens associated with septic challenge. The purpose of the present study was to determine whether there is a loss of dendritic cells and/or changes in function of these cells in septic mice. Here we report that the number of DCs, in both spleen and peritoneum, decreased over 24 h postsepsis [cecal ligation and puncture (CLP)] when compared with sham. The most dramatic change was seen in the peritoneal cavity. This decrease appeared to be caused mainly by the depletion of immature DCs rather than mature DCs. This change was LPS independent and minimally affected by FasL; however, overexpression of human Bcl-2 gene provides protection of the septic peritoneal DCs. Moreover, although the level of IL-12 release decreased significantly in splenic DCs obtained from CLP mice, IL-12 secretion was markedly elevated by peritoneal DCs as well as in both plasma and peritoneal fluid at 24 h post-CLP. In peritoneal cells, the expression of CD40, CD80, and CD86 was unchanged, but their respective ligands CD40L, CD28, and CD152 all increased in mice 24 h after CLP, although no such change was observed in splenocytes. Regardless of the presence or absence of antigen, peritoneal DCs from CLP mice showed higher capacity to stimulate T-cell proliferation than those cells from the sham control. However, splenic DCs from CLP mice only showed augmented capacity to induce antigen-dependent stimulation of T-cell proliferation. Together, these data indicate that sepsis produces divergent functional changes in splenic and peritoneal DC populations.     

beta-Adrenergic blockade during systemic inflammation: impact on cellular immune functions and survival in a murine model of sepsis

AIM OF THE STUDY: Adrenergic immuno-modulation mediated by beta-adrenergic receptors has been demonstrated. Pharmacological blockade of beta-adrenergic receptors is a therapeutic intervention frequently used in critically ill patients. The effect of beta-adrenergic blockade on cellular immune functions in a critical illness, such as polymicrobial sepsis, has not been investigated. METHODS: Male NMRI-mice were subjected to sham operation or to sepsis (caecal ligation and puncture, CLP) following administration of either the non-selective beta-adrenergic antagonist propranolol (0.5 mg/kg s.c. every 12 h in 1 ml vehicle) or saline 0.9% (1 ml s.c. every 12 h). Mice were kept in metabolic cages and were sacrificed 48 h after induction of sepsis. Survival rate, clinical situation (body weight and temperature, fluid and food intake, urine output), and immunological variables (splenocyte proliferation, apoptosis, and IFN-gamma and IL-6 release) were determined. RESULTS: Administration of propranolol in septic mice increased the splenocyte apoptosis rate, reduced the proliferative capacity of splenocytes, and modulated cellular cytokine release (IL-6, IFN-gamma). This was paralleled by a higher loss of body weight and temperature, and a decreased urine output. Furthermore, treatment with propranolol increased the sepsis-induced lethality from 47% up to 68%, respectively. CONCLUSION: beta-Adrenergic blockade was accompanied by alterations of cellular immune functions, a deterioration in the clinical situation and a reduced survival in a murine model of sepsis. These data demonstrate the potential immuno-modulatory effects of beta-adrenergic antagonists.     

Persistent inflammation and T cell exhaustion in severe sepsis in the elderly

Introduction

Sepsis is known as a complex immunological response with hyperinflammation in the acute phase followed by immunosuppression. Although aging is crucial in sepsis, the impact of aging on inflammation and immunosuppression is still unclear. The purpose of this study was to investigate the relationship between inflammation and immunosuppression in aged patients and mice after sepsis.

Methods

Fifty-five patients with severe sepsis and 30 healthy donors were prospectively enrolled, and 90-day survival was compared between elderly (≥65 years) and adult (18–64 years) septic patients with serial measurement of serum interleukin (IL)-6. Within 24 h after diagnosis of severe sepsis, peripheral blood mononuclear cells were stimulated ex vivo to measure expression of the activation maker CD25 in T cells, IL-2 levels in the supernatant, and proliferation. In the mouse study, young (6–8 weeks) and aged (20–22 months) C57/B6 mice were subjected to cecal ligation and puncture (CLP), and survival was compared after 7 days with serial measurement of serum IL-6. Expression of the negative co-stimulatory molecules, CD25, and IL-2 in CD4+ T cells was measured.

Results

The survival rate in elderly sepsis patients and aged septic mice was significantly lower than that in adult patients and young septic mice (60% vs. 93% in septic patients, 0% vs. 63% in septic mice, P < 0.05). Serum IL-6 levels in elderly sepsis patients and aged septic mice were persistently higher than those in adult patients and young septic mice. Expression of negative co-stimulatory molecules in CD4+ T cells in the spleen, lymph nodes, and peripheral blood was significantly higher in aged mice than in young mice (P < 0.01). Ex vivo stimulation decreased CD25 expression, IL-2 production, and proliferation to a greater extent in CD4+ T cells from elderly patients and aged septic mice than in those from adult patients and young septic mice. Elderly patients demonstrated increased detection of gram-negative bacteria at days 14–16 and 28–32 after sepsis (P < 0.05).

Conclusions

Persistent inflammation and T cell exhaustion may be associated with decreased survival in elderly patients and mice after sepsis.     

The effect of normal saline resuscitation on vital organ blood flow in septic sheep

Objective To study the effect of resuscitation with normal saline on vital organ blood flow and renal function in sepsis.Design and setting Randomized controlled cross-over animal study in the animal laboratory of university physiology institute.Subjects Six merino cross-ewes.Interventions Chronic implantation of flow probes around aorta, coronary, renal and mesenteric arteries. Intravenous administration of live Escherichia coli. Random allocation to normal saline resuscitation (20 ml/kg over 15 min) or observation (control) for 210 min. Continuous measurement of central haemodynamics, organ blood flow and renal function.Results Live E. coli induced hyperdynamic sepsis with oliguria (28.3 ± 12.6 to 16.7 ± 11.9 ml/30min) and reduced creatinine clearance (87.9 ± 24.5 to 44.3 ± 34.5 ml/min). During this septic state mesenteric, coronary and renal blood flow increased. During the first hour (early effect) after saline resuscitation, central venous pressure, cardiac output, stroke volume, coronary blood flow, mesenteric blood flow, urine output and creatinine clearance increased, but there was no change in renal blood flow. In the following 2 h these increments were significantly attenuated, but urine output and creatinine clearance remained greater than controls; renal blood flow decreased slightly and the fractional excretion of sodium increased significantly.Conclusion In hyperdynamic sepsis resuscitation with normal saline increases central venous pressure, cardiac output, mesenteric blood flow, urine output, creatinine clearance, and fractional excretion of sodium despite a lack of effect on renal blood flow. These effects, however, are transient.     

Neuronal nitric oxide synthase deficiency decreases survival in bacterial peritonitis and sepsis

Objective To investigate the role of neuronal nitric oxide synthase (NOS1) in murine polymicrobial peritonitis and sepsis. Design Randomized experimental trial. Setting Animal research facility. Subjects B6129S NOS1^+/+ and B6;129S4 NOS^−/− mice. Interventions NOS1^+/+ and NOS1^−/− animals underwent cecal ligation and puncture (CLP) or sham surgery and received the NOS1 inhibitor 7-nitroindazole (7-NI) or vehicle. Measurements and main results After CLP, genetic deficiency and pharmacologic inhibition of NOS1 significantly increased risk of mortality [8.69 (3.27, 23.1), p < 0.0001 and 1.71 (1.00, 2.92) p = 0.05, hazard ratio of death (95% confidence interval) for NOS1^−/− and 7-NI-treated NOS1^+/+ respectively] compared with NOS1^+/+ animals. In 7-NI-treated NOS1^+/+ animals, there were increases (6 h) and then decreases (24 h), whereas in NOS^−/− animals persistent increases in blood bacteria counts ( p = 0.04 for differing effects of 7-NI and NOS1^−/−) were seen compared with NOS1^+/+ animals. After CLP, NOS1^−/− had upregulation of inducible NOS and proinflammatory cytokines and greater increases in serum tumor necrosis factor-α and interleukin-6 levels compared with NOS1^+/+ mice (all p < 0.05). Following CLP, there were similar significant decreases in circulating leukocytes and lung lavage cells ( p ≤ 0.0008) and significant increases in peritoneal lavage cells ( p = 0.0045) in all groups. Over 6 h and 24 h following CLP, compared with NOS1^+/+, NOS^−/− mice had significantly higher peritoneal cell concentrations {respectively 0.40 ± 0.09 vs 0.79 ± 0.15 [log(× 10⁴cells/ml)] averaged over both times p = 0.038}. Conclusions Deficiency and inhibition of NOS1 increases mortality, possibly by increasing proinflammatory cytokine response and impairing bacterial clearance after CLP. These data suggest that NOS1 is important for survival, bacterial clearance, and regulation of cytokine response during infection and sepsis. Drs. Cui and Besch contributed equally to this work.     

Renal blood flow and function during recovery from experimental septic acute kidney injury

Objective To measure renal blood flow (RBF) and renal function during recovery from experimental septic acute kidney injury (AKI). Design Controlled experimental study. Subjects Nine merino ewes. Setting University physiology laboratory. Intervention We recorded systemic and renal hemodynamics during a 96-h observation period (control) via implanted transit-time flow probes. We then compared this period with 96 h of septic AKI (48 h of Escherichia coli infusion) and subsequent recovery (48 h of observation after stopping E. coli). Measurements and results Compared with the control period, E. coli infusion induced hyperdynamic sepsis (increased cardiac output and decreased blood pressure) and septic AKI (serum creatinine 65.4 ± 8.7 vs. 139.9 ± 33.0 μmol/l; creatinine clearance 73.8 ± 12.2 vs. 40.2 ± 17.2 ml/min; p < 0.05) with a mortality of 22%. RBF increased (278.8 ± 33.9 vs. 547.9 ± 124.8 ml/min; p < 0.05) as did renal vascular conductance (RVC). During recovery, we observed a decrease in RVC and RBF with all values returning to control levels. Indices of tubular function [fractional excretion of sodium (FENa) and urea (FEUn) and urinary sodium concentration (UNa)], which had been affected by sepsis, returned to control values after 18 h of recovery, as did serum creatinine. Conclusions Infusion of E. coli induced a hyperdynamic circulatory state with hyperemic AKI. Recovery was associated with relative renal vasoconstriction and reduction in RBF and RVC back to control levels. Indices of tubular function normalized more rapidly than changes in RBF. This article is discussed in the editorial available at: .