Systemic CD14 inhibition attenuates organ inflammation in porcine Escherichia coli sepsis.

Sepsis is an infection-induced systemic inflammatory response syndrome. Upstream recognition molecules, like CD14, play key roles in the pathogenesis. The aim of the present study was to investigate the effect of systemic CD14 inhibition on local inflammatory responses in organs from septic pigs. Pigs (n = 34) receiving Escherichia coli-bacteria or E. coli-lipopolysaccharide (LPS) were treated with an anti-CD14 monoclonal antibody or an isotype-matched control. Lungs, liver, spleen, and kidneys were examined for bacteria and inflammatory biomarkers. E. coli and LPS were found in large amounts in the lungs compared to the liver, spleen, and kidneys. Notably, the bacterial load did not predict the respective organ inflammatory response. There was a marked variation in biomarker induction in the organs and in the effect of anti-CD14. Generally, the spleen produced the most cytokines per weight unit, whereas the liver contributed the most to the total load. All cytokines were significantly inhibited in the spleen. Interleukin-6 (IL-6) was significantly inhibited in all organs, IL-1ß and IP-10 were significantly inhibited in liver, spleen, and kidneys, and tumor necrosis factor, IL-8, and PAI-1 were inhibited only in the spleen. ICAM-1 and VCAM-1 was significantly inhibited in the kidneys. Systemic CD14-inhibition efficiently, though organ dependent, attenuated local inflammatory responses. Detailed knowledge on how the different organs respond to systemic inflammation in vivo, beyond the information gained by blood examination, is important for our understanding of the nature of systemic inflammation and is required for future mediator-directed therapy in sepsis. Inhibition of CD14 seems to be a good candidate for such treatment.

Polyvalent immunoglobulin significantly attenuated the formation of IL-1ß in Escherichia coli-induced sepsis in pigs.

Evidence suggests that adjunctive treatment with intravenous immunoglobulin preparations enriched with IgA and IgM reduce mortality in sepsis. The mode of action of polyvalent immunoglobulin is complex, including neutralization of toxins and modulation of complement activation and cytokine formation toward an anti-inflammatory profile. In this study we explored the effect of Pentaglobin, containing IgG, IgA and IgM, on the initial inflammatory reaction as well as on hemodynamics, using a well characterized and standardized porcine model of sepsis. Anesthetized and mechanically ventilated pigs, mean weight 14.9 kg, were allocated into two groups of 8 animals, receiving either Pentaglobin or saline, before sepsis was induced by intravenous Escherichia coli infusion. Five negative controls received saline only. All animals were observed for 4 h under extensive invasive monitoring. Pentaglobin significantly (p < 0.05) attenuated IL-1ß formation by 38% at the end of the experiment, and markedly increased (p < 0.05) the formation of IL-10 at 60 min. TNF-α, IL-6, IL-8 and expression of the cell surface marker wCD11R3 were lower in the Pentaglobin group, but the differences were not significant. The serum concentration of LPS was three times higher in the Pentaglobin group (p < 0.005), indicating binding of LPS to Pentaglobin. Complementary in vitro experiments showed a higher binding affinity for IgM and IgA to LPS than for IgG. LPS-induced formation of IL-6 was significantly (p < 0.05) attenuated by Pentaglobin in an in vitro whole blood model. In conclusion, Pentaglobin decreased the key inflammasome IL-1ß molecule in an E. coli-model of pigs sepsis.

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 B(4) 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.

The role of bradykinin and the effect of the bradykinin receptor antagonist icatibant in porcine sepsis.

Bradykinin (BK) is regarded as an important mediator of edema, shock, and inflammation during sepsis. In this study, we evaluated the contribution of BK in porcine sepsis by blocking BK and by measuring the stable BK metabolite, BK1-5, using anesthetized pigs. The effect of BK alone, the efficacy of icatibant to block this effect, and the recovery of BK measured as plasma BK1-5 were first investigated. Purified BK injected intravenously induced an abrupt fall in blood pressure, which was completely prevented by pretreatment with icatibant. BK1-5 was detected in plasma corresponding to the doses given. The effect of icatibant was then investigated in an established model of porcine gram-negative sepsis. Neisseria meningitidis was infused intravenously without any pretreatment (n = 8) or pretreated with icatibant (n = 8). Negative controls received saline only. Icatibant-treated pigs developed the same degree of severe sepsis as did the controls. Both groups had massive capillary leakage, leukopenia, and excessive cytokine release. The plasma level of BK1-5 was low or nondetectable in all pigs. The latter observation was confirmed in supplementary studies with pigs undergoing Escherichia coli or polymicrobial sepsis induced by cecal ligation and puncture. In conclusion, icatibant completely blocked the hemodynamic effects of BK but had no beneficial effects on N. meningitidis-induced edema, shock, and inflammation. This and the fact that plasma BK1-5 in all the septic pigs was virtually nondetectable question the role of BK as an important mediator of porcine sepsis. Thus, the data challenge the current view of the role of BK also in human sepsis.

Neisseria meningitidis and Escherichia coli are protected from leukocyte phagocytosis by binding to erythrocyte complement receptor 1 in human blood.

The initial interaction of Gram-negative bacteria with erythrocytes and its implications on leukocyte phagocytosis and oxidative burst in human whole blood were examined. Alexa-labeled Escherichia coli, wild-type H44/76 N. meningitidis and the H44/76lpxA lipopolysaccharide (LPS)-deficient mutant were incubated with whole blood using lepirudin as anticoagulant which has no adverse effects on complement. Bacteria free in plasma, bound to erythrocytes or phagocytized by granulocytes and monocytes were quantified using flow cytometry. The effects of the C3 inhibitor compstatin, a C5a receptor antagonist (C5aRa) and a complement receptor 1 (CR1)-blocking antibody (3D9) were examined. Most bacteria (80%) immediately bound to erythrocytes. The binding gradually declined over time, with a parallel increase in phagocytosis. Complement inhibition with compstatin reduced erythrocyte binding and bacterial C3 opsonization. In contrast, the C5aRa efficiently reduced phagocytosis, but did not affect the binding of bacteria to erythrocytes. The anti-CR1 blocking mAb dose-dependently reduced bacterial binding to erythrocytes to nil, with subsequent increased phagocytosis and oxidative burst. LPS had no effect on these processes since similar results were obtained using an LPS-deficient N. meningitidis mutant. In vivo experiments in a pig model of sepsis showed limited binding of bacteria to erythrocytes, consistent with the facts that erythrocyte CR1 receptors are absent in non-primates and that the bacteria were mainly found in the lungs. In conclusion, complement-dependent binding of Gram-negative bacteria to erythrocyte CR1 decreases phagocytosis and oxidative burst by leukocytes in human whole blood.

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-alpha, 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 (anti-CD14) and control (IgG isotype or saline) groups. Inflammatory, hemostatic, physiological, and microbiological parameters were measured. The proinflammatory cytokines TNF-alpha, IL-1beta, 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.