A2B adenosine receptor blockade enhances macrophage-mediated bacterial phagocytosis and improves polymicrobial sepsis survival in mice.

Antimicrobial treatment strategies must improve to reduce the high mortality rates in septic patients. In noninfectious models of acute inflammation, activation of A2B adenosine receptors (A2BR) in extracellular adenosine-rich microenvironments causes immunosuppression. We examined A2BR in antibacterial responses in the cecal ligation and puncture (CLP) model of sepsis. Antagonism of A2BR significantly increased survival, enhanced bacterial phagocytosis, and decreased IL-6 and MIP-2 (a CXC chemokine) levels after CLP in outbred (ICR/CD-1) mice. During the CLP-induced septic response in A2BR knockout mice, hemodynamic parameters were improved compared with wild-type mice in addition to better survival and decreased plasma IL-6 levels. A2BR deficiency resulted in a dramatic 4-log reduction in peritoneal bacteria. The mechanism of these improvements was due to enhanced macrophage phagocytic activity without augmenting neutrophil phagocytosis of bacteria. Following ex vivo LPS stimulation, septic macrophages from A2BR knockout mice had increased IL-6 and TNF-α secretion compared with wild-type mice. A therapeutic intervention with A2BR blockade was studied by using a plasma biomarker to direct therapy to those mice predicted to die. Pharmacological blockade of A2BR even 32 h after the onset of sepsis increased survival by 65% in those mice predicted to die. Thus, even the late treatment with an A2BR antagonist significantly improved survival of mice (ICR/CD-1) that were otherwise determined to die according to plasma IL-6 levels. Our findings of enhanced bacterial clearance and host survival suggest that antagonism of A2BRs offers a therapeutic target to improve macrophage function in a late treatment protocol that improves sepsis survival.

Animal models of sepsis: setting the stage.

Sepsis is a state of disrupted inflammatory homeostasis that is often initiated by infection. The development and progression of sepsis is multi-factorial, and affects the cardiovascular, immunological and endocrine systems of the body. The complexity of sepsis makes the clinical study of sepsis and sepsis therapeutics difficult. Animal models have been developed in an effort to create reproducible systems for studying sepsis pathogenesis and preliminary testing of potential therapeutic agents. However, demonstrated benefit from a therapeutic agent in animal models has rarely been translated into success in human clinical trials. This review summarizes the common animal sepsis models and highlights how results of recent human clinical trials might affect their use.

Anoxia and reoxygenation of human endothelial cells decrease ceramide glucosyltransferase expression and activates caspases.

Endothelial oxidative stress induces cellular activation and sometimes death. Endothelial death can occur via necrosis or apoptosis. Understanding the mechanisms involved in cellular activation and death may lead to therapeutics designed to increase death or preserve cellular function. In the present study, brief periods of anoxia (3 h) followed by varying lengths of reoxygenation (0-5 h) lead to a time-dependent increase in human umbilical vein endothelial cell (HUVEC) caspase activity. Furthermore, ROCK-1 cleavage, which is dependent on caspase-3 activity, was also increased in cells undergoing oxidative stress compared with normoxic cells. Microarray data demonstrated that glucosylceramide synthase (GCS; glucosylceramide transferase), but not acid sphingomyelinase, was modulated by anoxia and reoxygenation. We confirmed that GCS mRNA and protein expression were significantly decreased in a time-dependent fashion following oxidative stress by real-time polymerase chain reaction and Western blot, respectively. Treatment of normoxic cells with the GCS-specific inhibitor, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), increased caspase activity to the same degree as cells undergoing oxidative stress. Fumonisin B1, the N-acyl-sphinganine dehydrogenase (e.g., ceramide synthase) inhibitor significantly attenuated caspase activity in HUVECs undergoing oxidative stress. These data suggest that alterations in GCS expression following brief periods of oxidative stress in human endothelial cells lead to increased caspase activity.

Inhibition of C5 or absence of C6 protects from sepsis mortality.

Inhibiting complement anaphlytoxin C5a during sepsis may prevent sepsis mortality. Although human anti-C5 antibodies exist, their therapeutic use in microbial sepsis has been avoided because of the hypothesis that inhibiting C5b will prevent formation of the bactericidal membrane attack complex (MAC) and worsen clinical outcome. We wished to test the hypothesis that inhibition of C5 would improve outcomes in sepsis. Sepsis was induced in rats by laparotomy and cecal ligation and puncture (CLP) by an IACUC-approved protocol. Sham animals underwent laparotomy without CLP. Following CLP rats were randomized to receive a single IV dose of purified IgG ant-C5 antibody (Ab) or control IgG Ab. Anti-C5 Ab treated rats (n = 20) had significantly lower mortality vs. controls (n = 21), 20% vs. 52% (P = 0.019, log-rank). Analysis of bacterial load by culture of spleen and liver homogenates showed a reduction in colony forming units in anti-C5 Ab treated rats vs. control IgG (P = 0.003 and 0.009, respectively). Anti-C5 treatment reduced lung injury as measured by total MPO content of lung tissue (P = 0.024). Finally, rats genetically deficient in C6 production, unable to form MAC but capable of producing C5a and C5b, were protected from CLP-induced sepsis mortality. Our results show that in anti-C5 antibody therapy prevents CLP sepsis-induced mortality and improves lung injury. Inhibition of the complement MAC does not increase bacterial load or mortality, therefore, the use of anti-C5 therapy may be beneficial rather than detrimental in sepsis.

Hyperbaric oxygen protects from sepsis mortality via an interleukin-10-dependent mechanism.

OBJECTIVE: This study was performed to determine whether hyperbaric oxygen (HBO2) therapy is protective in cecal ligation and puncture (CLP)-induced sepsis and if protection is dependent on oxygen dosing. We also wished to determine whether HBO2 affected bacterial clearance or altered macrophage production of interleukin-10 (IL-10)s in the setting of CLP sepsis. Finally, we wished to determine whether the mechanism of HBO2 protection in sepsis was dependent on IL-10 production. DESIGN: Prospective, experimental study. SETTING: University experimental research laboratory. SUBJECTS: C57BL/6 and C57BL/6 IL-10 mice. INTERVENTIONS: Sepsis was induced by CLP. Mice were randomized to receive a 1.5-hr HBO2 treatment at either 1, 2.5, or 3 atmospheres absolute every 12 hrs or HBO2 at 2.5 atmospheres absolute every 24 hrs. Mice were also harvested at 24 hrs for determination of bacterial load and isolation and study of CD11b peritoneal macrophages. MEASUREMENTS AND MAIN RESULTS: Survival was monitored for 100 hrs after CLP +/- HBO2 treatment. HBO2 significantly improved survival when administered at 2.5 atmospheres absolute every 12 hrs. Other treatment schedules were not protective, and treatment at 3.0 atmospheres absolute significantly worsened survival outcome. Bacterial load was significantly reduced in splenic homogenates but not peritoneal fluid at 24 hrs. Macrophages isolated from HBO2-treated mice demonstrated enhanced IL-10 secretion in response to lipopolysaccharide as compared with CLP controls. Mice genetically deficient in IL-10 expression treated with HBO2 at 2.5 atmospheres absolute every 12 hrs were not protected from CLP-induced mortality. CONCLUSION: HBO2 may be protective in CLP sepsis within a window of oxygen dosing. The mechanism of HBO2 protection may be potentially linked in part to expression of IL-10, as peritoneal macrophages demonstrated enhanced IL-10 expression and IL-10 mice were not protected by HBO2 treatment.

Hyperbaric oxygen reduces acetaminophen toxicity and increases HIF-1alpha expression.

OBJECTIVES: To investigate the effect of hyperbaric oxygen (HBO2) on acetaminophen (APAP)-induced hepatotoxicity. The authors further evaluated the effects of APAP poisoning and HBO2 on the expression and function of hypoxia-inducible factor 1-alpha (HIF-1alpha) in an effort to further describe the mechanisms of APAP-induced hepatotoxicity. In vitro assays were performed to better understand the effects of HBO2 on HIF-1alpha function. METHODS: In vivo, four groups of C57BL/6 mice were treated as follows: APAP only, APAP followed by HBO2, HBO2 only, and untreated shams. Plasma alanine aminotransferase activity was measured, and hepatic HIF-1alpha induction was determined by Western blot. In vitro, cultured HEP G2 hepatocytes were exposed to HBO2, hypoxia (2.5% O2), or normoxia. HIF-1alpha DNA-binding and transcriptional activity were assessed. RESULTS: Alanine aminotransferase activity was reduced in the APAP+HBO2 group (2,606 IU/L +/- 4,080; vs. APAP: 6,743 +/- 3,397, p = 0.01 at 6 hours). APAP-only, HBO2-only, and APAP+HBO2 treatments all increased HIF-1alpha expression relative to shams (p = 0.02, p = 0.02, and p < 0.01, respectively). HBO2 increased HIF-1alpha DNA binding 5.7 (+/- 1.2)-fold relative to controls (p < 0.01); however, a parallel increase in HIF functional transcriptional activity did not occur. CONCLUSIONS: Hyperbaric oxygen reduced early APAP-induced hepatocellular injury. APAP poisoning increases HIF-1alpha protein levels and functional activity. HBO2 increases HIF-1alpha protein levels and DNA binding without a corresponding increase in transcriptional activity.

Endothelially derived nitric oxide affects the severity of early acetaminophen-induced hepatic injury in mice.

OBJECTIVES: The precise mechanism of hepatocellular toxicity following acetaminophen (APAP) poisoning remains unclear. Nitric oxide is implicated in APAP toxicity as an inflammatory signaling molecule and as a precursor to the free radical peroxynitrate. The effects of inducible nitric oxide synthase (iNOS)-derived NO in APAP toxicity are known; however, the role of endothelial nitric oxide synthase (eNOS)-derived NO is unknown. The authors sought to evaluate the effect of eNOS-derived NO during APAP toxicity. METHODS: C57BL6/J mice deficient in eNOS (eNOS KO) or iNOS (iNOS KO) and wild-type mice (WT) were treated with 300 mg/kg APAP. Alanine aminotransferase levels and plasma nitrate and nitrite levels were measured. Hypoxia inducible factor (HIF)-1alpha and Glucose Transporter 1 (Glut-1) levels were determined by Western blot. RESULTS: Alanine aminotransferase levels were significantly elevated in all treated animals. Alanine aminotransferase levels were significantly lower in eNOS KO and iNOS KO than in treated WT animals. Plasma nitrate/nitrite levels were significantly higher in WT animals than in iNOS KO and eNOS KO animals. HIF-1alpha expression was increased in WT mice and decreased in iNOS KO mice. Glut-1 is a downstream, indirect marker of HIF function. Glut-1 expression was increased in WT and eNOS KO mice. CONCLUSIONS: Deficiency of either iNOS or eNOS results in decreased NO production and is associated with reduced hepatocellular injury following APAP poisoning. HIF-1alpha and Glut-1 levels are increased following APAP poisoning, implying that HIF-1alpha is functional during the pathogenic response to APAP poisoning.

Gastrointestinal ischemia-reperfusion injury is lectin complement pathway dependent without involving C1q.

Complement activation plays an important role in local and remote tissue injury associated with gastrointestinal ischemia-reperfusion (GI/R). The role of the classical and lectin complement pathways in GI/R injury was evaluated using C1q-deficient (C1q KO), MBL-A/C-deficient (MBL-null), complement factor 2- and factor B-deficient (C2/fB KO), and wild-type (WT) mice. Gastrointestinal ischemia (20 min), followed by 3-h reperfusion, induced intestinal and lung injury in C1q KO and WT mice, but not in C2/fB KO mice. Addition of human C2 to C2/fB KO mice significantly restored GI/R injury, demonstrating that GI/R injury is mediated via the lectin and/or classical pathway. Tissue C3 deposition in C1q KO and WT, but not C2/fB KO, mice after GI/R demonstrated that complement was activated in C1q KO mice. GI/R significantly increased serum alanine aminotransferase, gastrointestinal barrier dysfunction, and neutrophil infiltration into the lung and gut in C1q KO and WT, but not C2/fB KO, mice. MBL-null mice displayed little gut injury after GI/R, but lung injury was present. Addition of recombinant human MBL (rhuMBL) to MBL-null mice significantly increased injury compared with MBL-null mice after GI/R and was reversed by anti-MBL mAb treatment. However, MBL-null mice were not protected from secondary lung injury after GI/R. These data demonstrate that C2 and MBL, but not C1q, are necessary for gut injury after GI/R. Lung injury in mice after GI/R is MBL and C1q independent, but C2 dependent, suggesting a potential role for ficolins in this model.

CpG oligodeoxynucleotide protection in polymicrobial sepsis is dependent on interleukin-17.

CpG oligodeoxynucleotides (ODNs) may prevent mortality from infection. We have identified a therapeutic benefit in treating sepsis with phosphorothioate ODN sequences containing the CpG motif. Sepsis was induced in rats by cecal ligation and puncture (CLP), and treatment with CpG ODNs reduced sepsis mortality from 80% to 15% during a 108-h period. Protection from mortality was dose dependent. Bacterial load in peritoneal fluid was reduced in CpG ODN-treated versus non-CpG ODN-treated rats after CLP. Lung injury, as determined by total myeloperoxidase activity, was also reduced in CpG ODN-treated versus non-CpG ODN-treated rats after CLP. Indirect evidence suggests that CpG-induced expression of interleukin (IL)-23 as levels of p40--but not p35--were significantly increased in both plasma and peritoneal lavage fluid in CpG ODN-treated versus non-CpG ODN-treated rats 24 h after CLP. Anti-IL-17 antibody inhibited the CpG-mediated prevention of mortality. These data suggest that IL-17 may mediate CpG-inducible host defenses during intraabdominal sepsis.

Role for the alternative complement pathway in ischemia/reperfusion injury.

The terminal complement components play an important role in mediating tissue injury after ischemia and reperfusion (I/R) injury in rats and mice. However, the specific complement pathways involved in I/R injury are unknown. The role of the alternative pathway in I/R injury may be particularly important, as it amplifies complement activation and deposition. In this study, the role of the alternative pathway in I/R injury was evaluated using factor D-deficient (-/-) and heterozygote (+/-) mice. Gastrointestinal ischemia (GI) was induced by clamping the mesenteric artery for 20 minutes and then reperfused for 3 hours. Sham-operated control mice (+/- versus -/-) had similar baseline intestinal lactate dehydrogenase activity (P = ns). Intestinal lactate dehydrogenase activity was greater in -/- mice compared to +/- mice after GI/R (P = 0.02) thus demonstrating protection in the -/- mice. Intestinal myeloperoxidase activity in +/- mice was significantly greater than -/- mice after GI/R (P < 0.001). Pulmonary myeloperoxidase activity after GI/R was significantly higher in +/- than -/- mice (P = 0.03). Addition of human factor D to -/- animals restored GI/R injury and was prevented by a functionally inhibitory antibody against human factor D. These data suggest that the alternative complement pathway plays an important role in local and remote tissue injury after GI/R. Inhibition of factor D may represent an effective therapeutic approach for GI/R injury.

Neutrophil-derived glutamate regulates vascular endothelial barrier function.

Endothelial barrier function is altered by the release of soluble polymorphonuclear leukocyte (PMN)-derived mediators during inflammatory states. However, endogenous pathways to describe such changes are only recently appreciated. Using an in vitro endothelial paracellular permeability model, cell-free supernatants from formylmethionylleucylphenylalanine-stimulated PMNs were observed to significantly alter endothelial permeability. Biophysical and biochemical analysis of PMN supernatants identified PMN-derived glutamate in modulating endothelial permeability. Furthermore, novel expression of metabotropic glutamate receptor 1 (mGluR1), mGluR4, and mGluR5 by human brain and dermal microvascular endothelial cells was demonstrated by reverse transcription-PCR, in situ hybridization, immunofluorescence, and Western blot analysis. Treatment of human brain endothelia with glutamate or selective, mGluR group I or III agonists resulted in a time-dependent loss of phosphorylated vasodilator-stimulated phosphoprotein (VASP) and significantly increased endothelial permeability. Glutamate-induced decreases in brain endothelial barrier function and phosphorylated VASP were significantly attenuated by pretreatment of human brain endothelia with selective mGluR antagonists. These observations were extended to an in vivo hypoxic mouse model in which pretreatment with mGluR antagonists significantly decreased fluorescein isothiocyanate-dextran flux across the blood-brain barrier. We conclude that activated human PMNs release glutamate and that endothelial expression of group I or III mGluRs function to decrease human brain endothelial VASP phosphorylation and barrier function. These results identify a novel pathway by which PMN-derived glutamate may regulate human endothelial barrier function.