Type I interferon signaling in hematopoietic cells is required for survival in mouse polymicrobial sepsis by regulating CXCL10

Type I interferon (IFN) α/β is critical for host defense. During endotoxicosis or highly lethal bacterial infections where systemic inflammation predominates, mice deficient in IFN-α/β receptor (IFNAR) display decreased systemic inflammation and improved outcome. However, human sepsis mortality often occurs during a prolonged period of immunosuppression and not from exaggerated inflammation. We used a low lethality cecal ligation and puncture (CLP) model of sepsis to determine the role of type I IFNs in host defense during sepsis. Despite increased endotoxin resistance, IFNAR^−/− and chimeric mice lacking IFNAR in hematopoietic cells display increased mortality to CLP. This was not associated with an altered early systemic inflammatory response, except for decreased CXCL10 production. IFNAR^−/− mice display persistently elevated peritoneal bacterial counts compared with wild-type mice, reduced peritoneal neutrophil recruitment, and recruitment of neutrophils with poor phagocytic function despite normal to enhanced adaptive immune function during sepsis. Importantly, CXCL10 treatment of IFNAR^−/− mice improves survival and decreases peritoneal bacterial loads, and CXCL10 increases mouse and human neutrophil phagocytosis. Using a low lethality sepsis model, we identify a critical role of type I IFN–dependent CXCL10 in host defense during polymicrobial sepsis by increasing neutrophil recruitment and function.Despite advancements in our understanding of innate and adaptive immunity, applying this knowledge to the treatment of sepsis has proven difficult. Sepsis causes a dramatic systemic inflammatory response syndrome (SIRS) but is also capable of causing dysfunction in both the innate and adaptive branches of the immune system (Bone, 1996; Döcke et al., 1997; Hotchkiss and Karl, 2003). In patients receiving appropriate resuscitation, the early systemic SIRS phase is rarely lethal, and patients increasingly succumb to secondary infections and organ failure during a prolonged period of immunosuppression and failure in host defense (Bone, 1996; Hotchkiss and Karl, 2003). Understanding both phases of human sepsis requires the application of appropriate animal models that can produce a SIRS response that can be overcome to allow the study of host defense during the more prolonged phases in sepsis. To this end, we use the cecal ligation and puncture (CLP) model of sepsis that creates a significant but not overwhelming SIRS response, allowing us to dissect factors that control host defense during prolonged sepsis (Scumpia et al., 2006, 2007; Delano et al., 2007).Many factors modulate innate and adaptive immune responses and link the two branches of immunity during infection. Type I IFNs, including IFN-α, IFN-β, and IFN-ω, are ubiquitious cytokines originally shown to inhibit viral infection (Lindenmann et al., 1957; Isaacs and Burke, 1959). Recently, type I IFNs were shown to act downstream of Toll-like receptor signaling (Uematsu and Akira, 2007) to induce a specific gene activation signature, including induction of chemokines such as CXCL10 and MCP-5 (Toshchakov et al., 2002), and antimicrobial/antiviral response genes (Sadler and Williams, 2008). Type I IFNs also serve as a link between the innate and adaptive immune systems (Hoebe and Beutler, 2004), participating in autoimmunity (Blanco et al., 2001) and viral (Müller et al., 1994) and bacterial infection (O’Connell et al., 2004; Mancuso et al., 2007). Their functions in severe bacterial infection remain controversial, as signaling through the type I IFN pathway has detrimental consequences in certain bacterial infections (O’Connell et al., 2004; Martin et al., 2009) but protective effects in others (Mancuso et al., 2007). Importantly, in response to severe endotoxicosis or a highly inflammatory model of bacterial peritonitis, colon ascendens stent peritonitis (CASP; Maier et al., 2004), absence of the receptor for type I IFN (IFN-α/β receptor [IFNAR]; Müller et al., 1994) was shown to be protective by decreasing the systemic hyperinflammation associated with these two models (Mahieu et al., 2006; Weighardt et al., 2006).In this report, we dissected the effects of type I IFN signaling on host immunity to a model of polymicrobial sepsis that more closely approximates the magnitude and mortality seen in human disease. We find that IFNAR^−/− mice are more sensitive to CLP mortality, particularly at later time points. We attribute this increased sensitivity to a failure in hematopoietic cells, particularly neutrophils, to clear the infection, and not to an exaggerated inflammatory response. We demonstrate that when the SIRS response does not predominate, type I IFN is required for host defense in sepsis, and that impaired induction of the IFN-inducible CXCL10 is at least in part responsible for this protection.     

Dehydroepiandrosterone decreases mortality rate and improves cellular immune function during polymicrobial sepsis

OBJECTIVE: Sepsis is associated with a marked depression of cellular immune function. The steroid hormone dehydroepiandrosterone (DHEA) is proposed to have immunoenhancing activities. We, therefore, investigated the effect of DHEA on the mortality rate and cellular immune functions in an experimental model of sepsis. DESIGN: Randomized animal study. SETTING: Level I trauma center, university research laboratory. SUBJECTS: Male NMRI mice. INTERVENTIONS: Mice were subjected to laparotomy (sham) or cecal ligation and puncture (CLP). Mice were treated with (sham/DHEA; CLP/DHEA) or without (sham; CLP) the steroid hormone DHEA (30 mg/kg sc). Animals were killed 48 hrs after the onset of sepsis. MEASUREMENTS AND MAIN RESULTS: The survival rate of septic mice was determined 24 and 48 hrs after onset of sepsis. Forty-eight hours after the septic challenge, a white blood cell count was performed and serum tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta concentrations were monitored using ELISA. Furthermore, the delayed type of hypersensitivity (DTH) reaction was evaluated on the basis of ear pinna swelling after dinitrofluorobenzene (DNFB) administration, and clinical variables (body weight, temperature, heart rate, fluid input/output, food intake) were monitored using metabolic cages. DHEA administration improved the survival rate (87% vs. 53% after 48 hrs; p <.001). This was accompanied by a restoration of the depressed DTH reaction and a reduction in TNF-alpha serum concentrations (20.7 +/- 1.4 pg/mL vs. 32.4 +/- 6.6 pg/mL). CONCLUSIONS: These results demonstrate that DHEA administration leads to an increased survival following a septic challenge. The immunoenhancing effect of DHEA is accompanied by a reduction of TNF-alpha release and an improved activity of T-cellular immunity. DHEA administration may, therefore, be beneficial in systemic inflammation.     

Induction of endotoxin tolerance enhances bacterial clearance and survival in murine polymicrobial sepsis

The fundamental mechanisms that underlie endotoxin tolerance remain to be elucidated, and the clinical significance of endotoxin tolerance in the context of active systemic infection remains in question. We hypothesized that the endotoxin tolerance phenotype would result in decreased inflammation at the expense of altered bacterial clearance and, thus, higher mortality in a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Endotoxin tolerance was induced in C57Bl/6 mice with 5 mg/kg LPS or vehicle 18 h before subsequent CLP. Lung tissue, peritoneal fluid, and blood were collected at 1, 3, 6, and 18 h after surgery for subsequent analysis. Peritoneal macrophages were isolated for ex vivo phagocytosis assay. In separate experiments, mice were allowed to recover, and survival was monitored for 7 days. Endotoxin tolerance attenuated plasma TNF-alpha and IL-6 at 6 h after CLP. Peritoneal fluid cytokines were significantly attenuated as well. Endotoxin tolerance significantly improved bacterial clearance in both blood and peritoneal fluid after CLP. Similarly, ex vivo phagocytosis by primary peritoneal macrophages and RAW264.7 murine peritoneal macrophages was significantly improved after induction of the endotoxin tolerance phenotype. Contrary to our original hypothesis, we conclude that endotoxin tolerance significantly attenuates the host inflammatory response, augments bacterial clearance, and improves survival in this murine model of polymicrobial sepsis.     

Cerivastatin improves survival of mice with lipopolysaccharide-induced sepsis

Development of severe sepsis is thought to result from the overproduction of cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), and nitric oxide. Recently, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, which are antihypercholesterolemic agents, have been reported to inhibit lipopolysaccharide (LPS)-induced production of cytokines and nitric oxide in vitro. In this study, we tested these effects in vivo. After LPS administration (15 mg/kg i.p.) to CD-1 mice, serum levels of both TNF-alpha and IL-1beta transiently increased, and peaked at 2 h. After the peak responses of TNF-alpha and IL-1beta, serum levels of nitrite and nitrate increased until at least 8 h. Pretreatment of the mice with cerivastatin (20 mg/kg i.p. 12 and 1 h before LPS injection) reduced serum levels of TNF-alpha and IL-1beta at 2 h, and nitrite and nitrate at 8 h, by 93, 60, and 44%, respectively. In this model of sepsis, cerivastatin significantly (P =.016) improved the rate of 7-day survival from 26.7 to 73.3%. These results cast new light on the usefulness of cerivastatin in preventing severe sepsis.     

Ketamine improves survival and suppresses IL-6 and TNFalpha production in a model of Gram-negative bacterial sepsis in rats

OBJECTIVE: In a previous study, ketamine suppressed Escherichia coli-induced production of the cytokines interleukin (IL)-6 and tumor necrosis factor alpha (TNF). In other previous studies ketamine improved survival after E. coli inoculation. However, the relationship between cytokines and survival following ketamine treatment is uncertain because no study has examined both cytokines and survival after E. coli inoculation. METHODS: Rats were given E. coli (0.4 x 10(9) colony forming unit (CFU)) at time 0, followed by ketamine (50 mg/kg, n=30) or saline (n=30) at 5 min or 2 h. IL-6 and TNF were measured in serum at 6 h, and mortality was recorded for 7 days. RESULTS: Survival rate with ketamine was 57% (17/30) and was significantly increased compared to saline (27%, 8/30, P=0.01). IL-6 and TNF were lower with ketamine than saline (15,197 +/- 3444 versus 30,725 +/- 4623 pg/ml [mean +/- S.E.M.], P=0.013 and 38.5 +/- 9.5 versus 122.5 +/- 14.0 pg/ml, P=0.001, respectively). With ketamine, IL-6 (but not TNF) concentrations were lower in the survivors (10,900 +/- 776 pg/ml) as compared to the non-survivors (P=0.01). IL-6 in ketamine-treated survivors was not different from that in saline-treated survivors. Conclusion: We conclude that ketamine given 5 min or 2 h after induction of E. coli sepsis significantly improves survival, possibly by interfering with the inflammatory cascade (as evidenced by attenuation of cytokine production).     

Bim siRNA decreases lymphocyte apoptosis and improves survival in sepsis

To assess the degree of lymphocyte apoptosis and survival in mice treated with small interfering RNA (siRNA) targeted to Bim, a proapoptotic molecule from the Bcl-2 family, within a clinically relevant model of sepsis. C57BL/6 mice were treated with a single dose of Bim siRNA complexed in cationic liposomes via tail vein injection. Approximately 24 h later, mice were subjected to either cecal ligation and puncture (CLP) or sham surgery. Animals were killed at 20 h postsurgery, and spleens were harvested for fluorescence-activated cell sorting analysis using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling as a marker for apoptosis. A second cohort of mice was followed for survival for 7 days. The degree of lymphocyte apoptosis in Bim siRNA-treated mice was markedly decreased compared with controls. Fluorescent activated cell sorter analysis demonstrated 13.1% +/- 1.2% B-cell apoptosis and 11.5% +/- 1.5% T-cell apoptosis in control mice compared with 2.7% +/- 0.4% B-cell apoptosis and 3.9% +/- 0.3% T-cell apoptosis in Bim siRNA-treated mice after CLP (P < 0.001 and P < 0.01, respectively). This striking difference in lymphocyte apoptosis correlated with a significant survival advantage in Bim siRNA-treated mice. At 7 days, there was 90% overall survival in Bim siRNA-treated septic mice compared with 50% overall survival in control septic mice (P < 0.05). Treatment with Bim siRNA in vivo has the potential to be an effective therapy in the treatment of sepsis.     

Early interleukin-10 treatment improves survival and enhances immune function only in males after hemorrhage and subsequent sepsis

Recent studies have demonstrated gender differences in the immune response following hemorrhagic shock with an enhanced immune function and lower mortality following subsequent sepsis in females. Early interleukin-10 (IL-10) treatment has been shown to have beneficial effects on the depressed immune function in males, but not in females following shock. However, it remains unclear if the observed gender-related effect of IL-10 treatment results in an advantage following subsequent polymicrobial sepsis. To study this, male and female CBA/J mice (age 2-3 months) were subjected to hemorrhage (35 +/- 5 mmHg for 90 min and fluid resuscitation). At resuscitation, each received either 10 microg of recombinant murine IL-10 or placebo i.p.. At 48 h after resuscitation, either peritoneal macrophages (pMphi) and plasma were harvested, or polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Following CLP, either survival over 10 days was measured, or pMphi and plasma were harvested 4 h after CLP to assess TNF-alpha, IL-6, IL-10, and prostaglandin E2 (PGE2) release of pMphi and plasma levels of IL-10, free testosteron, and 17-beta estradiol. Early IL-10 treatment restored depressed proinflammatory immune response in males (TNF-alpha and PGE2), which was associated with an enhanced survival (P < 0.05) following subsequent sepsis as compared with placebo-treated mice (8/20 and 1/20, respectively). In contrast, the immune response and survival in females receiving IL-10 was not significantly changed, although females treated with IL-10 had a trend towards higher mortality (7/15 and 2/15, respectively; P = 0.08). Thus, early IL-10 anti-inflammatory treatment following hemorrhage has potential beneficial effects only in males associated with enhanced survival following subsequent sepsis.     

Magnolol attenuates peroxidative damage and improves survival of rats with sepsis

Reactive oxygen species and peroxidative damage are implicated in the pathophysiology of sepsis. Magnolol is a compound extracted from the Chinese medicinal herb Magnolia officinalis and has multiple pharmacological effects, notably antioxidant functions. To determine whether magnolol can modulate the course of sepsis, survival rate and biochemical parameters were analyzed in rats with sepsis with various treatment protocols. Magnolol at doses ranging from 10(-9) g/kg to 10(-5) g/kg was administered either before or after induction of sepsis by cecal ligation and puncture. Magnolol did not modulate the course of sepsis induced by two cecal punctures. When one cecal puncture was performed, a moderately evolving type of sepsis was induced, and the survival rate of affected rats was significantly improved by pretreatment with 10(-7) g/kg magnolol. The beneficial effect was partially retained if magnolol was administered 6 hours after onset of sepsis when a higher dose (10(-5) g/kg) was used. The intensity of lipid peroxidation in plasma, liver, and lung of septic rats was also attenuated in a treatment-dependent manner. Magnolol at this dose range exerted these beneficial effects probably through its antioxidant efficacy. These significant results may suggest magnolol as a candidate agent for the treatment of sepsis.     

Activated protein C improves survival in severe sepsis patients with elevated troponin

Objective Multiple studies in sepsis have demonstrated that elevated troponin is associated with poor outcome. The elevated troponin in this situation is thought to be secondary to microthrombi. We hypothesized that recombinant human activated protein C (APC) treatment would improve outcomes in severe sepsis patients who have elevated troponin. Methods Patients with severe sepsis by consensus criteria in a university ICU were divided into a troponin elevated group (cTnI⁺) and a normal troponin (cTnI⁻) group. Outcome was compared using Fisher's exact test. APACHE II and MODS were calculated by standard methods. Patients We identified 105 patients with severe sepsis and troponin measured, of which 48 (46%) were in the cTnI⁺ group. The two groups were similar in terms of age and other comorbid conditions. Results APACHE II (28 ± 8 vs. 25 ± 8) was slightly higher and MODS (11 ± 4 vs. 9 ± 3) was significantly higher in the cTnI⁺ group. Mortality was 52% (25/48) in cTnI⁺ group and 30% (17/57) in cTnI⁻ group. Mortality was 30% in cTnI⁺ patients treated with APC and 72% in untreated cTnI⁺ patients. Conclusions Patients with severe sepsis who have elevated troponin have increased mortality. In patients with severe sepsis who have elevated troponin, treatment with APC improves outcome. Further study is needed to determine whether troponin can serve as a simple, readily available marker to identify which patients with severe sepsis will benefit from APC. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Peritoneal lavage with activated protein C alters compartmentalized coagulation and fibrinolysis and improves survival in polymicrobial peritonitis

OBJECTIVE: During peritonitis, intra-abdominal fibrin entraps bacteria and hampers their elimination. Systemic administration of anticoagulant activated protein C improves survival in patients with severe sepsis, but its precise mode of action is unclear. This study in polymicrobial peritonitis assessed the effects of local activated protein C administration in peritoneal lavage fluid on coagulation, fibrinolysis, and survival. DESIGN: Prospective, randomized study. SETTING: University-based research laboratory. SUBJECTS: C57BL/6 mice. INTERVENTIONS: Twenty-four hours after induction of peritonitis by cecal ligation and puncture, mice underwent peritoneal lavage with activated protein C (1.0 microg/mL) or saline. Peritoneal lavage fluid, blood, and lungs were sampled after 24, 48, or 72 hrs (n = 8/group/time point). For survival analysis, maximum observation was 96 hrs (n = 22/group). Clotting time, tissue factor expression, thrombin-antithrombin complexes, fibrin degradation products (D-dimers), plasminogen activator, and plasminogen activator inhibitor were used to assess coagulation and fibrinolysis responses. MEASUREMENTS AND MAIN RESULTS: Activated protein C lavage reduced abdominal bacterial load, abdominal and pulmonary clotting times, D-dimers (p < .05 vs. saline), pulmonary tissue factor expression, and fibrin depositions, without clear effects on systemic thrombin generation. Activated protein C lavage decreased plasma and abdominal tissue plasminogen activator levels with increased inhibitor plasminogen activator inhibitor-1 levels (p < .05) but had reverse effects on pulmonary fibrinolysis. Survival improved from 55% (saline) to 80% after intra-abdominal activated protein C administration (p = .03). CONCLUSIONS: Peritoneal lavage with activated protein C may rebalance coagulation and fibrinolysis within compartments and improve survival in polymicrobial peritonitis.     

Toll-like receptor 9 inhibition reduces mortality in polymicrobial sepsis

The high rate of mortality in patients with sepsis results from an inappropriately amplified systemic inflammatory response to infection. Toll-like receptors (TLRs) are important for the activation of innate immunity against microbial pathogens. We demonstrate a critical role of TLR9 in the dysregulated immune response and death associated with sepsis. Compared with wild-type (WT) mice, TLR9(-/-) mice exhibited lower serum inflammatory cytokine levels, higher bacterial clearance, and greater survival after experimental peritonitis induced by cecal ligation and puncture (CLP). Protection of TLR9(-/-) mice after CLP was associated with a greater number of peritoneal dendritic cells (DCs) and granulocytes than in WT controls. Adoptive transfer of TLR9(-/-) DCs was sufficient to protect WT mice from CLP and increased the influx of peritoneal granulocytes. Subsequent experiments with a depleting antibody revealed that granulocytes were required for survival in TLR9(-/-) mice. Remarkably, a single injection of an inhibitory CpG sequence that blocks TLR9 protected WT mice, even when administered as late as 12 h after CLP. Our findings demonstrate that the detrimental immune response to bacterial sepsis occurs via TLR9 stimulation. TLR9 blockade is a potential strategy for the treatment of human sepsis.     

Functional relevance of IL-10 promoter polymorphisms for sepsis development

The induced production of proinflammatory and anti-inflammatory cytokines is considered important for the development of sepsis and its sequelae. Polymorphisms in the IL-10 gene promoter could influence its expression and sepsis susceptibility. Results obtained by Dr Ling and colleagues demonstrated that the -1082A allele was significantly associated with lower lipopolysaccharide-induced IL-10 production in an allele-dose-dependent fashion. They also showed that this polymorphism was significantly associated with sepsis development after major trauma. These and other research data clearly demonstrated that the -1082 A/G polymorphism in the IL-10 gene promoter has an important impact on susceptibility of sepsis and sepsis outcome.     

Intravenous Augmentin in bacteraemia and severe invasive polymicrobial sepsis

An intravenous formulation of Augmentin was used as sole chemotherapy in the treatment of patients with severe infections. Fourteen of 17 assessable patients (82%) responded satisfactorily including six with bacteraemia. Adverse reactions occurred in 38% of patients but in all but one, withdrawn due to diarrhoea, were trivial. There was no significant intolerance.