The anti-inflammatory effect of combined complement and CD14 inhibition is preserved during escalating bacterial load

Abstract

SummaryCombined inhibition of complement and CD14 is known to attenuate bacterial-induced inflammation, but the dependency of the bacterial load on this effect is unknown. Thus, we investigated whether the effect of such combined inhibition on Escherichia coli-and Staphylococcus aureus-induced inflammation was preserved during increasing bacterial concentrations. Human whole blood was preincubated with anti-CD14, eculizumab (C5-inhibitor) or compstatin (C3-inhibitor), or combinations thereof. Then heat-inactivated bacteria were added at final concentrations of 5 3 10 4 21 3 10 8 /ml (E. coli) or 5 3 10 7 24 3 10 8 /ml (S. aureus). Inflammatory markers were measured using enzyme-linked immunosorbent assay (ELISA), multiplex technology and flow cytometry. Combined inhibition of complement and CD14 significantly (P < 0.05) reduced E. coli-induced interleukin (IL)-6 by 40-92% at all bacterial concentrations. IL-1b, IL-8 and macrophage inflammatory protein (MIP)-1a were significantly (P < 0.05) inhibited by 53-100%, and the effect was lost only at the highest bacterial concentration. Tumour necrosis factor (TNF) and MIP-1b were significantly (P < 0.05) reduced by 80-97% at the lowest bacterial concentration. Monocyte and granulocyte CD11b were significantly (P < 0.05) reduced by 63-91% at all bacterial doses. Lactoferrin was significantly (P < 0.05) attenuated to the level of background activity at the lowest bacterial concentration. Similar effects were observed for S. aureus, but the attenuation was, in general, less pronounced. Compared to E. coli, much higher concentrations of S. aureus were required to induce the same cytokine responses. This study demonstrates generally preserved effects of combined complement and CD14 inhibition on Gram-negative and Grampositive bacterial-induced inflammation during escalating bacterial load. The implications of these findings for future therapy of sepsis are discussed.

Complement in disease: a defence system turning offensive

“…Genetic ablation or pharmacological blockade of C5a signalling or C3 activation improves outcomes in animal models of sepsis 81,83,84 . Moreover, in vitro and in vivo studies have provided evidence of synergistic effects of blocking complement and TLRs simultaneously 85,86 , underscoring the crosstalk that occurs between these systems in pathological events. In patients with severe trauma, the presentation of DAMPs after tissue injury can trigger complement activation, which induces SIRS and contributes to complications such as multi-organ dysfunction and/or death 87–89 .…”

Human Endothelial Cell Activation by Escherichia coli and Staphylococcus aureus Is Mediated by TNF and IL-1β Secondarily to Activation of C5 and CD14 in Whole Blood

“…We found that E. coli-induced whole blood-mediated activation of EC was largely CD14-dependent whereas S. aureus-induced activation was mediated by the complement system, mainly through C5a-C5aR1 interaction. We have earlier found that single inhibition is less efficient than combined inhibition, particularly with increased load of inflammatory stimuli (35). In the present study, we used fairly low concentrations of bacteria compared with previous studies in whole blood alone, which might explain the impressive effect of single inhibition.…”

Defining the optimal animal model for translational research using gene set enrichment analysis

“…For instance, congruently upregulated genes included the CD14 molecule, the lipopolysaccharide‐binding protein (LBP), and the Toll‐like receptors (TLRs) 2, 4, 6, and 8. CD14 is an accessory receptor for TLRs, and ongoing clinical trials indicate that CD14 inhibition is a promising strategy to treat sepsis (Verbon et al , ; Egge et al , ). Furthermore, LBP is an acute‐phase biomarker for sepsis (Schumann & Zweigner, ), and the TLR family was recently reported to be a promising target for the treatment of sepsis (Savva & Roger, ).…”