Phagocytosis of live and dead Escherichia coli and Staphylococcus aureus in human whole blood is markedly reduced by combined inhibition of C5aR1 and CD14

Skjeflo, Espen Waage; Christiansen, Dorte; Landsem, Anne; Stenvik, Jørgen; Woodruff, Trent M.; Espevik, Terje; Mollnes, Tom Eirik

doi:10.1016/j.molimm.2019.03.014

Cited by 9 publications

(7 citation statements)

References 26 publications

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“…Similarly, distinct complement inhibitors from scabies increased the growth of S. pyogenes in blood 39 . Phagocytosis of S. aureus was also reduced by C5‐ or C5aR‐inhibition, in agreement with our previous findings 40 . Still, C5a–C5aR1 signaling gradually becomes dispensable for phagocytosis of this bacterium as time progresses, and C5 inhibition did not impair the resolution of infection in blood.…”

Section: Discussionsupporting

confidence: 90%

“…Still, C5a–C5aR1 signaling gradually becomes dispensable for phagocytosis of this bacterium as time progresses, and C5 inhibition did not impair the resolution of infection in blood. Although costimulation with a TLR2 agonist increases the uptake of S. aureus by monocytes but not PMN in blood, 8 anti‐CD14 has only modest effects 40 . Sensing of bacteria via TLR8 occurs downstream of phagocytosis and bacterial degradation, and it is delayed relative to TLR2 activation.…”

Section: Discussionmentioning

confidence: 99%

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TLR8 and complement C5 induce cytokine release and thrombin activation in human whole blood challenged with Gram-positive bacteria

Ehrnström

Kojen

Giambelluca

et al. 2020

Journal of Leukocyte Biology

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We recently showed that TLR8 is critical for the detection of Gram-positive bacteria by human monocytes. Here, we hypothesized that TLR8 and complement together regulate antibacterial responses in human blood. Anticoagulated blood was treated with selective inhibitors of TLR8 and/or complement C5, and then challenged with live Streptococcus agalactiae (Group B streptococcus, GBS), Staphylococcus aureus, or Escherichia coli. Cytokine production, plasma membrane permeability, bacterial survival, phagocytosis, and activation of coagulation was examined. GBS and S. aureus, but not E. coli, triggered TLR8-dependent production of IL-12p70, IL-1 , TNF, and IL-6 in fresh human whole blood. In purified polymorphonuclear neutrophils (PMN), GBS and S. aureus induced IL-8 release in part via TLR8, whereas PMN plasma membrane leakage and extracellular DNA levels increased independently of TLR8. TLR8 was more important than C5 for bacteria-induced production of IL-12p70, IL-1 , and TNF in blood, whereas IL-8 release was more C5 dependent. Both TLR8 and C5 induced IL-6 release and activation of prothrombin cleavage, and here their combined effects were additive. Blocking of C5 or C5aR1 attenuated phagocytosis and increased the extracellular growth of GBS in blood, whereas TLR8 inhibition neither reduced phagocytosis nor intracellular killing of GBS and S. aureus. In conclusion, TLR8 is more important than C5 for production of IL-12p70, IL-1 , and TNF upon GBS and S. aureus infection in blood, whereas C5 is central for IL-8 release and phagocytosis. Both TLR8 and C5 mediate IL-6 release and activation of coagulation during challenge with Gram-positive bacteria in blood.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

TLR8 and complement C5 induce cytokine release and thrombin activation in human whole blood challenged with Gram-positive bacteria

Ehrnström

Kojen

Giambelluca

et al. 2020

Journal of Leukocyte Biology

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“…Using a human whole blood in vitro model, we showed that anti-CD14 attenuates both phagocytosis and oxidative burst. 43 Anti-CD14 treatment in vivo may functionally protect the phagocytes as indicated by lower levels of MPO and faster recovery of circulating leukocytes.…”

Section: Discussionmentioning

confidence: 99%

CD14 inhibition improves survival and attenuates thrombo‐inflammation and cardiopulmonary dysfunction in a baboon model of Escherichia coli sepsis

Keshari

Silasi‐Mansat

Popescu

et al. 2021

Journal of Thrombosis and Haemostasis

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Background During sepsis, gram‐negative bacteria induce robust inflammation primarily via lipopolysacharride (LPS) signaling through TLR4, a process that involves the glycosylphosphatidylinositol (GPI)‐anchored receptor CD14 transferring LPS to the Toll‐like receptor 4/myeloid differentiation factor 2 (TLR4/MD‐2) complex. Sepsis also triggers the onset of disseminated intravascular coagulation and consumptive coagulopathy. Objectives We investigated the effect of CD14 blockade on sepsis‐induced coagulopathy, inflammation, organ dysfunction, and mortality. Methods We used a baboon model of lethal Escherichia (E) coli sepsis to study two experimental groups (n = 5): (a) E coli challenge; (b) E coli challenge plus anti‐CD14 (23G4) inhibitory antibody administered as an intravenous bolus 30 minutes before the E coli. Results Following anti‐CD14 treatment, two animals reached the 7‐day end‐point survivor criteria, while three animals had a significantly prolonged survival as compared to the non‐treated animals that developed multiple organ failure and died within 30 hours. Anti‐CD14 reduced the activation of coagulation through inhibition of tissue factor‐dependent pathway, especially in the survivors, and enhanced the fibrinolysis due to strong inhibition of plasminogen activator inhibitor 1. The treatment prevented the robust complement activation induced by E coli, as shown by significantly decreased C3b, C5a, and sC5b‐9. Vital signs, organ function biomarkers, bacteria clearance, and leukocyte and fibrinogen consumption were all improved at varying levels. Anti‐CD14 reduced neutrophil activation, cell death, LPS levels, and pro‐inflammatory cytokines (tumor necrosis factor, interleukin (IL)‐6, IL‐1β, IL‐8, interferon gamma, monocyte chemoattractant protein‐1), more significantly in the survivors than non‐surviving animals. Conclusions Our results highlight the crosstalk between coagulation/fibrinolysis, inflammation, and complement systems and suggest a protective role of anti‐CD14 treatment in E coli sepsis.

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“…Phagocytosis is a fundamental process by which cells engulf extracellular particles with a size of >0.5 μm [1]. This uptake can be tracked by using particles labeled with different fluorescent dyes, such as hexidium iodide and Alexa Fluor 488, or genetically engineered bacteria expressing enhanced green fluorescent protein [4][5][6]. However, particles sticking to the cell membrane of a phagocyte are difficult to distinguish from those which are completely internalized by the cell.…”

Section: Methods Summarymentioning

confidence: 99%

Phagocytosis Assays with Different pH-Sensitive Fluorescent Particles and Various Readouts

2020

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Phagocytosis is a fundamental mechanism of innate immunity and its impairment is associated with severe chronic diseases, for example, chronic obstructive pulmonary disease. Investigating phagocytosis requires flexible tools and assay conditions, such as different fluorescent particle types, detection colors and readouts. We comprehensively evaluated and optimized phagocytosis assays using particles labeled with fluorescent pH-sensitive pHrodo® dyes, facilitating the specific detection of phagocytosed particles. Beads, bacterial and yeast particles labeled with pHrodo red and green were tested for their uptake by THP-1 cells and primary human macrophages by flow cytometry and high-content imaging. Whereas the latter allowed kinetic phagocytosis measurement, the former demonstrated the feasibility of using cell sorting for periods of up to 6 h, enabling downstream applications such as pooled genetic screens.

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Phagocytosis of live and dead Escherichia coli and Staphylococcus aureus in human whole blood is markedly reduced by combined inhibition of C5aR1 and CD14

Cited by 9 publications

References 26 publications

TLR8 and complement C5 induce cytokine release and thrombin activation in human whole blood challenged with Gram-positive bacteria

TLR8 and complement C5 induce cytokine release and thrombin activation in human whole blood challenged with Gram-positive bacteria

CD14 inhibition improves survival and attenuates thrombo‐inflammation and cardiopulmonary dysfunction in a baboon model of Escherichia coli sepsis

Phagocytosis Assays with Different pH-Sensitive Fluorescent Particles and Various Readouts

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