Combined Inhibition of Complement and CD14 Efficiently Attenuated the Inflammatory Response Induced by<i>Staphylococcus aureus</i>in a Human Whole Blood Model

Polymorphonuclear neutrophilic granulocytes (PMN) as cellular components of innate immunity play a crucial role in the defense against systemic Candida albicans infection. To analyze stimuli that are required for PMN activity during C. albicans infection in a situation similar to in vivo, we used a human whole-blood infection model. In this model, PMN activation 10 min after C. albicans infection was largely dependent on the anaphylatoxin C5a. Most importantly, C5a enabled blood PMN to overcome filament-restricted recognition of C. albicans and allowed efficient elimination of nonfilamentous C. albicans cph1Δ/efg1Δ from blood. Major PMN effector mechanisms, including oxidative burst, release of secondary granule contents and initial fungal phagocytosis could be prevented by blocking C5a receptor signaling. Identical effects were achieved using a humanized Ab specifically targeting human C5a. Phagocytosis of C. albicans 10 min postinfection was mediated by C5a-dependent enhancement of CD11b surface expression on PMN, thus establishing the C5a-C5aR-CD11b axis as a major modulator of early anti-Candida immune responses in human blood. In contrast, phagocytosis of C. albicans by PMN 60 min postinfection occurred almost independently of C5a and mainly contributed to activation of phagocytically active PMN at later time points. Our results show that C5a is a critical mediator in human blood during C. albicans infection.

Section: Discussionsupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

A Second Stimulus Required for Enhanced Antifungal Activity of Human Neutrophils in Blood Is Provided by Anaphylatoxin C5a

Hünniger

Bieber

Martin

et al. 2015

“…2E). The mechanism for the enhanced phagocytosis is likely TLR2-mediated activation of the CR3 (CD11b/CD18), as S. aureus phagocytosis in the whole blood model is strongly Cdependent (27) and the CD11b level on both monocytes and granulocytes increased strongly upon TLR2 stimulation (Fig. 2F).…”

Section: Tlr2 Ligands Antagonize the Induction Of Ifn-b By S Aureus mentioning

confidence: 99%

TLR8 Senses Staphylococcus aureus RNA in Human Primary Monocytes and Macrophages and Induces IFN-β Production via a TAK1–IKKβ–IRF5 Signaling Pathway

Bergstrøm

Aune

Awuh

et al. 2015

Self Cite

122

162

Staphylococcus aureus may cause serious infections and is one of the most lethal and common causes of sepsis. TLR2 has been described as the main pattern recognition receptor that senses S. aureus and elicits production of proinflammatory cytokines via MyD88–NF-κB signaling. S. aureus can also induce the production of IFN-β, a cytokine that requires IFN regulatory factors (IRFs) for its transcription, but the signaling mechanism for IFN-β induction by S. aureus are unclear. Surprisingly, we demonstrate that activation of TLR2 by lipoproteins does not contribute to IFN-β production but instead can suppress the induction of IFN-β in human primary monocytes and monocyte-derived macrophages. The production of IFN-β was induced by TLR8-mediated sensing of S. aureus RNA, which triggered IRF5 nuclear accumulation, and this could be antagonized by concomitant TLR2 signaling. The TLR8-mediated activation of IRF5 was dependent on TAK1 and IκB kinase (IKK)β, which thus reveals a physiological role of the recently described IRF5-activating function of IKKβ. TLR8–IRF5 signaling was necessary for induction of IFN-β and IL-12 by S. aureus, and it also contributed to the induction of TNF. In conclusion, our study demonstrates a physiological role of TLR8 in the sensing of entire S. aureus in human primary phagocytes, including the induction of IFN-β and IL-12 production via a TAK1–IKKβ–IRF5 pathway that can be inhibited by TLR2 signaling.

“…We have earlier shown that the inflammatory reaction induced by both E. coli and S. aureus in human whole blood can be attenuated by combined inhibition of CD14 and one of the key complement components (14,16). To evaluate whether bacteria-induced EC activation also would be reduced by this regimen, we incubated whole blood on EC monolayers with either E. coli (10 5 bacteria/ml) or S. aureus (10 7 bacteria/ml) in addition to blocking mAbs to CD14 and C5, alone or in combination (Fig.…”

Section: Inhibition Of Bacteria-induced Ec Activation In Whole Bloodmentioning

confidence: 99%

“…We have earlier shown that combined inhibition of CD14 and one of the key complement components, at the level of either C3 or C5, can more or less completely attenuate cytokine release and leukocyte activation in whole blood after bacteria-induced activation both with Gram-negative and Gram-positive bacteria (14)(15)(16). Furthermore, in a murine sepsis model, we have recently shown that combined inhibition reduces both inflammation and mortality (17).…”

mentioning

confidence: 99%

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

Nymo

Gustavsen

Nilsson

et al. 2016

Self Cite

Endothelial cells (EC) play a central role in inflammation. E-selectin and ICAM-1 expression are essential for leukocyte recruitment and are good markers of EC activation. Most studies of EC activation are done in vitro using isolated mediators. The aim of the present study was to examine the relative importance of pattern recognition systems and downstream mediators in bacteria-induced EC activation in a physiological relevant human model, using EC incubated with whole blood. HUVEC were incubated with human whole blood. Escherichia coli– and Staphylococcus aureus–induced EC activation was measured by E-selectin and ICAM-1 expression using flow cytometry. The mAb 18D11 was used to neutralize CD14, and the lipid A analog eritoran was used to block TLR4/MD2. C5 cleavage was inhibited using eculizumab, and C5aR1 was blocked by an antagonist. Infliximab and canakinumab were used to neutralize TNF and IL-1β. The EC were minimally activated when bacteria were incubated in serum, whereas a substantial EC activation was seen when the bacteria were incubated in whole blood. E. coli–induced activation was largely CD14-dependent, whereas S. aureus mainly caused a C5aR1-mediated response. Combined CD14 and C5 inhibition reduced E-selectin and ICAM-1 expression by 96 and 98% for E. coli and by 70 and 75% for S. aureus. Finally, the EC activation by both bacteria was completely abolished by combined inhibition of TNF and IL-1β. E. coli and S. aureus activated EC in a CD14- and C5-dependent manner with subsequent leukocyte secretion of TNF and IL-1β mediating the effect.