Anne Pharo

Human genetic deficiencies reveal the roles of complement in the inflammatory network: Lessons from nature

Complement component C5 is crucial for experimental animal inflammatory tissue damage; however, its involvement in human inflammation is incompletely understood. The responses to Gram-negative bacteria were here studied taking advantage of human genetic complement-deficiencies—natures own knockouts—including a previously undescribed C5 defect. Such deficiencies provide a unique tool for investigating the biological role of proteins. The experimental conditions allowed cross-talk between the different inflammatory pathways using a whole blood model based on the anticoagulant lepirudin, which does not interfere with the complement system. Expression of tissue factor, cell adhesion molecules, and oxidative burst depended highly on C5, mediated through the activation product C5a, whereas granulocyte enzyme release relied mainly on C3 and was C5a-independent. Release of cytokines and chemokines was mediated to varying degrees by complement and CD14; for example, interleukin (IL)-1β and IL-8 were more dependent on complement than IFN-γ and IL-6, which were highly dependent on CD14. IL-1 receptor antagonist (IL-1ra) and IFN-γ inducible protein 10 (IP-10) were fully dependent on CD14 and inversely regulated by complement, that is, complement deficiency and complement inhibition enhanced their release. Granulocyte responses were mainly complement-dependent, whereas monocyte responses were more dependent on CD14. Notably, all responses were abolished by combined neutralization of complement and CD14. The present study provides important insight into the comprehensive role of complement in human inflammatory responses to Gram-negative bacteria.

CD14 inhibition efficiently attenuates early inflammatory and hemostatic responses in Escherichia coli sepsis in pigs

Sepsis is a severe infection‐induced systemic inflammatory syndrome. Inhibition of downstream inflammatory mediators of sepsis, e.g., TNF‐α, has failed in clinical trials. The aim of this study was to investigate the effects of inhibiting CD14, a key upstream innate immunity molecule, on the early inflammatory and hemostatic responses in a pig model of gram‐negative sepsis. The study comprised two arms, whole live Escherichia coli bacteria and E. coli lipopolysaccharide (LPS) (n=25 and n=9 animals, respectively). The animals were allocated into treatment (antiCD14) and control (IgG isotype or saline) groups. Inflammatory, hemostatic, physiological, and microbiological parameters were measured. The proinflammatory cytokines TNF‐α, IL‐1β, IL‐6, and IL‐8, but not the anti‐inflammatory cytokine IL‐10, were efficiently inhibited by anti‐CD14. Furthermore, anti‐CD14 preserved the leukocyte count and significantly reduced granulocyte enzyme matrix metalloproteinase‐9 release and expression of the granulocyte membrane activation molecule wCD11R3 (pig CD11b). The hemostatic markers thrombin‐antithrombin III complexes and plasminogen activator inhibitor‐1 were significantly attenuated. Anti‐CD14 did not affect LPS or E. coli DNA levels. This study documents that CD14 inhibition efficiently attenuates the proinflammatory cytokine response and granulocyte activation and reverses the procoagulant state but does not interfere with LPS levels or bacterial counts in E. coli‐induced sepsis.— Thorgersen, E. B., Hellerud, B. C., Nielsen, E. W., Barratt‐Due, A., Fure, H., Lindstad, J. K., Pharo, A., Fosse, E., Tønnessen, T. I., Johansen, H. T., Castellheim, A., Mollnes, T. E. CD14 inhibition efficiently attenuates early inflammatory and hemostatic responses in Escherichia coli sepsis in pigs. FASEB J. 24, 712–722 (2010). www.fasebj.org

Combined inhibition of complement and CD14 abolish E. coli-induced cytokine-, chemokine- and growth factor-synthesis in human whole blood.

The relative role of complement and CD14 in E. coli-induced cytokine synthesis in an in vitro human whole blood model of sepsis was examined. Fresh lepirudin-anticoagulated whole blood was incubated with E. coli for 2h. Monoclonal antibodies or a C5a receptor antagonist were used to block complement. Inflammatory mediators (n=27) were measured by multiplex technology, selected cytokine mRNA by real time PCR, and CD11b, oxidative burst and phagocytosis by flow cytometry. E. coli significantly increased 18 of the 27 inflammatory mediators, including proinflammatory cytokines (TNF-alpha, IL-6, INF-gamma and IL-1beta), chemokines (IL-8, MCP-1, MIP-1alpha, MIP-1beta, eotaxin and IP-10), growth factors (VEGF, FGF-basic, G-CSF and GM-CSF) and other interleukins (IL-9, IL-15 and IL-17). Notably, the increases in all mediators were abolished by a combined inhibition of CD14 and complement using anti-C2 and anti-factor D in combination, whereas the relative effect of the inhibition of complement and CD14 varied. In comparison, a C5a receptor antagonist and anti-CD14 in combination reduced cytokine synthesis less efficiently. Real time PCR analysis confirmed that the cytokine synthesis was blocked at the mRNA level. Similarly, E. coli-induced CD11b up-regulation, oxidative burst and phagocytosis was totally inhibited by CD14, anti-C2 and anti-factor D in combination after 2h incubation. In conclusion, the combined inhibition of complement using anti-C2, anti-factor D and CD14 almost completely inhibits the E. coli-induced inflammatory response. The combined approach may therefore be a new treatment regimen in Gram-negative sepsis.

Biomarker Profile in Off-Pump and On-Pump Coronary Artery Bypass Grafting Surgery in Low-Risk Patients

BACKGROUND The purpose of this study was to investigate the cytokine and chemokine profile in low-risk patients undergoing off-pump and on-pump coronary artery bypass grafting (CABG) surgery by use of a broad panel of cytokines and chemokines. METHODS Eight consecutive blood samples were obtained from patients enrolled into a prospective, randomized study comparing off-pump and on-pump CABG in a low-risk population. Eleven patients from each group were randomly selected for analysis of 25 different cytokines and chemokines using multiplex technology. Data were compared using two-way repeated measures analysis of variance. RESULTS Of the 25 biomarkers analyzed, 11 were not detected while 14 increased significantly in both groups. Only three mediators, eotaxin, macrophage inflammatory protein (MIP)-1beta, and interleukin (IL)-12 were significantly different between the two groups, increasing more in the on-pump than in the off-pump group (p < 0.001, p < 0.01, and p < 0.05, respectively). There was a marked, comparable increase in the concentrations of the cytokines IL-6, IL-10, IL-15, and IL-1Ra as well as the chemokines inducible protein (IP)-10, monokine induced by interferon gamma (MIG), monocyte chemoattractant protein 1 (MCP-1), and regulated on activation, normal T cell expressed and secreted (RANTES) in both groups (p < 0.001 for all). There was only a modest, but still statistically significant, increase in IL-8, tumor necrosis factors alpha, and IL-2R, without any intergroup differences. When corrected for hemodilution the production of the antiinflammatory biomarkers IL-1Ra and IL-10 were significantly higher in the on-pump group (p < 0.001 for both). CONCLUSIONS The cytokine and chemokine production profile of the inflammatory response associated with CABG is largely similar using the off-pump and on-pump techniques in low-risk patients, but slightly higher concentrations of eotaxin, MIP-1beta, and IL-12 were found in the on-pump group.

Microdialysis for Monitoring Inflammation: Efficient Recovery of Cytokines and Anaphylotoxins Provided Optimal Catheter Pore Size and Fluid Velocity Conditions

Microdialysis emerges as a useful tool to evaluate tissue inflammation in a number of clinical conditions, like sepsis and transplant rejection, but systematic methodological studies are missing. This study was undertaken to determine the recovery of relevant inflammatory mediators using the microdialysis system, comparing microdialysis membranes with two different molecular weight cut‐offs at different flow rates. Twenty and 100 kDa pore sizes CMA microdialysis catheters were investigated using velocities of 0.3, 1.0 and 5.0 μl/min. Reference preparations for cytokines [tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6 and IL‐10; m.w. 17–28 kDa] and chemokines (IL‐8, MCP‐1, IP‐10 and MIG; m.w. 7–11 kDa) were prepared from plasma after incubating human whole blood with lipopolysaccharide. Reference preparation for complement anaphylatoxins (C3a, C4a, C5a; m.w. 9–11 kDa) was prepared by incubating human plasma with heat‐aggregated immunoglobulin G. The reference preparations were quantified for the respective inflammatory molecules and used as medium for the microdialysis procedure. Through the 20 kDa filter only the four chemokines passed, but with low recovery (3–7%) and limited to the 1.0 μl/min velocity. The recovery with the 100 kDa filter was as follows: IL‐1β = 75%, MCP‐1 = 55%, MIG = 50%, IL‐8 = 38%, C4a = 28%, IP‐10 = 22%, C5a = 20%, C3a = 16%, IL‐6 = 11, IL‐10 = 8% and TNF‐α = 4%. The highest recovery for all chemokines and anaphylatoxins were consistently at velocity 1.0 μl/min, whereas IL‐1β and IL‐10 recovered most efficiently at 0.3 μl/min. Thus, microdialysis using catheters with a cut‐off of 100 kDa is a reliable method to detect inflammation as judged by a defined panel of inflammatory markers. These findings may have important implications for future clinical studies.

Ornithodoros moubata Complement Inhibitor Is an Equally Effective C5 Inhibitor in Pigs and Humans

Experimental evidence suggests that C inhibition and more particularly combined inhibition of C and the TLR coreceptor CD14 may be of therapeutic benefit in sepsis and other inflammatory conditions. A barrier to the testing and further development of many inhibitors is that their activity is species specific. Pig is a relevant species for experimental models of human disease, and this study undertakes a comprehensive comparison of the inhibitory efficacy of the C5 inhibitor Ornithodoros moubata C inhibitor (OmCI) in human and porcine whole blood ex vivo models of Escherichia coli-induced sepsis. The effect of OmCI on complement activity in pigs undergoing E. coli sepsis was also examined. Porcine and human serum, and whole blood anticoagulated with lepirudin, was incubated with E. coli and the effect of OmCI investigated. The ex vivo results were virtually identical in pig and human. OmCI completely ablated the activity of all three C pathways at 0.64 μM. E. coli-induced C activation and expression of CD11b (wCD11R3 in the pig), was abolished ex vivo at 0.32 μM OmCI. Combining anti-CD14 and OmCI reduced the formation of IL-8 and TNF-α more potently than the single inhibitors. OmCI also efficiently bound E. coli-induced leukotriene B4 in pig and human plasma. In support of our ex vivo findings, in vivo the activity of all C pathways was inhibited at 0.6 mg OmCI/kg pig. In conclusion, OmCI efficiently inhibited pig and human C activation, has accompanying anti-inflammatory effects and is a promising candidate inhibitor for further in vivo studies of sepsis.

New biomarkers in an acute model of live Escherichia coli-induced sepsis in pigs.

We developed a live Escherichia coli model of acute sepsis in pigs with emphasize on biomarkers reflecting the early inflammatory response of sepsis. Healthy pigs, 25–35 kg, were challenged intravenously (IV) (n = 12) or intrapulmonary (n = 6) with live E. coli and observed for 3 and 5 h respectively. Control pigs received culture medium (n = 6 + 3). Haemodynamic parameters and a broad panel of inflammatory mediators were measured. The dose of bacteria was carefully titrated to obtain a condition resembling the early phase of human septic shock. The IV group displayed a pro‐inflammatory response [significant increase in tumour necrosis factor‐α, interleukin (IL)‐6 and IL‐8] and an early anti‐inflammatory response (significant increase in IL‐10). For the first time, we demonstrate a significant increase in IL‐12 and matrix metalloproteinase‐9 (MMP) early in pig sepsis. Coagulation was activated (significant increase in thrombin–antithrombin complexes) and there was a significant decrease in the serum proteins suggesting capillary leakage. Haemodynamic parameters reflected a septic condition with significant decrease in systemic blood pressure, increases in heart rate, pulmonary artery pressure and base deficit. None of these changes was observed in the control group. Interleukin‐1β and vascular endothelial growth factor increased in both groups. Nitric oxide measurements suggested an initial pulmonary vascular endothelial inflammatory response. The intrapulmonary group, which did not resemble septic condition, showed a substantial increase in MMP‐9. In this porcine model of sepsis, IL‐12 and MMP‐9 were detected for the first time. These biomarkers may have an impact in the understanding and future treatment of sepsis.

Meconium is a potent activator of complement in human serum and in piglets.

Meconium aspiration syndrome (MAS) is a clinical condition in the newborn infant with a significant morbidity and mortality. The complex pathophysiology of MAS, leading to both pulmonary and systemic complications, is characterized by an incompletely understood inflammatory reaction. Treatment is symptomatic, mainly limited to airway cleaning and ventilatory support. In this study, we show for the first time that meconium is a potent activator of complement, a key mediator of inflammation. In vitro, meconium activated the alternative complement pathway in human umbilical cord serum as judged by a substantial increase in the alternative pathway convertase C3bBbP. The activation proceeded through C3 (C3bc) and the terminal C5-9 pathway (terminal SC5b-9 complement complex), whereas the classical and lectin pathways were not activated (C1rs-C1-inhibitor complexes and C4bc). The lipid fraction, containing, e.g. free fatty acids, and the water fraction, containing, e.g. bile acids, contributed equally to the complement activation. A blocking antibody to factor D (alternative pathway) completely inhibited the meconium-induced complement activation, whereas blocking antibodies to mannose-binding lectin (lectin pathway) and C2 (classical and lectin pathway) had no effect. In vivo, meconium induced systemic complement activation in a piglet model of MAS, paralleling the increase in lung dysfunction. In conclusion, meconium is a potent activator of the complement system both in vitro and in vivo. Complement may be important in the pathogenesis of MAS, and specific complement inhibition might be a possible treatment approach in MAS.

Combined Inhibition of Complement (C5) and CD14 Markedly Attenuates Inflammation, Thrombogenicity, and Hemodynamic Changes in Porcine Sepsis

Complement and the TLR family constitute two important branches of innate immunity. We previously showed attenuating effects on inflammation and thromogenicity by inhibiting the TLR coreceptor CD14 in porcine sepsis. In the present study, we explored the effect of the C5 and leukotriene B4 inhibitor Ornithodoros moubata complement inhibitor (OmCI; also known as coversin) alone and combined with anti-CD14 on the early inflammatory, hemostatic, and hemodynamic responses in porcine Escherichia coli–induced sepsis. Pigs were randomly allocated to negative controls (n = 6), positive controls (n = 8), intervention with OmCI (n = 8), or with OmCI and anti-CD14 (n = 8). OmCI ablated C5 activation and formation of the terminal complement complex and significantly decreased leukotriene B4 levels in septic pigs. Granulocyte tissue factor expression, formation of thrombin–antithrombin complexes (p < 0.001), and formation of TNF-α and IL-6 (p < 0.05) were efficiently inhibited by OmCI alone and abolished or strongly attenuated by the combination of OmCI and anti-CD14 (p < 0.001 for all). Additionally, the combined therapy attenuated the formation of plasminogen activator inhibitor-1 (p < 0.05), IL-1β, and IL-8, increased the formation of IL-10, and abolished the expression of wCD11R3 (CD11b) and the fall in neutrophil cell count (p < 0.001 for all). Finally, OmCI combined with anti-CD14 delayed increases in heart rate by 60 min (p < 0.05) and mean pulmonary artery pressure by 30 min (p < 0.01). Ex vivo studies confirmed the additional effect of combining anti-CD14 with OmCI. In conclusion, upstream inhibition of the key innate immunity molecules, C5 and CD14, is a potential broad-acting treatment regimen in sepsis as it efficiently attenuated inflammation and thrombogenicity and delayed hemodynamic changes.

Inhibition of Complement and CD14 Attenuates the Escherichia coli-Induced Inflammatory Response in Porcine Whole Blood

ABSTRACT The innate immune response is a double-edged sword in systemic inflammation and sepsis. Uncontrolled or inappropriate activation can damage and be lethal to the host. Several studies have investigated inhibition of downstream mediators, including tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). Emerging evidence indicates that upstream inhibition is a better therapeutic approach for attenuating damaging immune activation. Therefore, we investigated inhibition of two central innate immune pathways, those of complement and CD14/Toll-like receptor 4 (TLR4)/myeloid differentiation protein 2 (MD-2), in a porcine in vitro model of Escherichia coli-induced inflammation. Porcine whole blood anticoagulated with lepuridin, which did not interfere with the complement system, was incubated with E. coli lipopolysaccharide (LPS) or whole bacteria. Inhibitors of complement and CD14 and thus the LPS CD14/TLR4/MD-2 receptor complex were tested to investigate the effect on the inflammatory response. A broad range of inflammatory readouts were used to monitor the effect. Anti-CD14 was found to saturate the CD14 molecule on granulocytes and completely inhibited LPS-induced proinflammatory cytokines in a dose-dependent manner. Anti-CD14 significantly reduced the levels of the E. coli-induced proinflammatory cytokines TNF-α and IL-1β, but not IL-8, in a dose-dependent manner. No effect on bacterial clearance was seen. Vaccinia complement control protein and smallpox inhibitor of complement enzymes, two Orthopoxvirus-encoded complement inhibitors, completely inhibited complement activation. Furthermore, these agents almost completely inhibited the expression of wCD11R3, which is associated with CD18 as a β2 integrin, on porcine granulocytes and decreased IL-8 levels significantly in a dose-dependent manner. As expected, complement inhibition reduced bacterial clearance. We conclude that inhibition of complement and CD14 attenuates E. coli-induced inflammation and might be used as a therapeutic regimen in gram-negative sepsis along with appropriate treatment with antibiotics.