Complement C5a impairs phagosomal maturation in the neutrophil through phosphoproteomic remodelling

Abstract

Critical illness is accompanied by the release of large amounts of the anaphylotoxin, C5a. C5a suppresses antimicrobial functions of neutrophils which is associated with adverse outcomes. The signalling pathways that mediate C5a-induced neutrophil dysfunction are incompletely understood. Healthy donor neutrophils exposed to purified C5a demonstrated a prolonged defect (7 hours) in phagocytosis of Staphylococcus aureus. Phosphoproteomic profiling of 2712 phosphoproteins identified persistent C5a signalling and selective impairment of phagosomal protein phosphorylation on exposure to S. aureus. Notable proteins included early endosomal marker ZFYVE16 and V-ATPase proton channel component ATPV1G1. A novel assay of phagosomal acidification demonstrated C5a-induced impairment of phagosomal acidification which was recapitulated in neutrophils from critically ill patients. Examination of the C5a-impaired protein phosphorylation indicated a role for the phosphatidylinositol 3-kinase VPS34 in phagosomal maturation. Inhibition of VPS34 impaired neutrophil phagosomal acidification and killing of S. aureus. This study provides a phosphoproteomic assessment of human neutrophil signalling in response to S. aureus and its disruption by C5a, identifying a defect in phagosomal maturation and new mechanisms of immune failure in critical illness.

Pulmonary infections complicating ARDS

“…Patients with suspected VAP, including those with ARDS, demonstrated impaired phagocytic function of alveolar neutrophils, which interestingly appeared to be mediated by different mediators than those driving dysfunction in the peripheral blood [ 9 ]. While we have a growing understanding of the mediators driving dysfunction, and the intracellular mechanisms which drive them [ 14 ], we do not as yet have proven therapies although there are multiple potential agents [ 7 ].…”

Contrasting effects of linezolid on healthy and dysfunctional human neutrophils: reducing C5a-induced injury

“…This effect is independent of CD88. In the current study it seems reasonable to assume that co-infection with linezolid must influence this critical C5a signalling period and its effect is independent of cell surface CD88, but dependent on effects produced intracellularly on pHi or maturation of the phagosome 20 , 25 .…”

“…Thus, ligation of C5aR1 (CD88) by C5a, activates its inherent G-protein activity resulting in phosphoinositide 3-kinase delta (PI3Kδ) activation inhibiting activation of the small GTPase RhoA, actin polymerisation and phagocytosis 13 . At the time of writing Wood and co-workers confirmed that C5a induced decreases in the phagocytosis of S. aureus are associated with impaired phagosomal maturation by phosphoproteomic remodelling through selective impairment of phagosomal protein phosphorylation, including endosomal marker ZFYVE16 and V-ATPase protein channel component ATPV1G1 25 . In addition, Denk and co-workers proposed that C5aR1 ligation also leads to selective activation of the Na + /H + exchanger causing increased intracellular alkalinisation (pHi), increased glycolytic flux, glucose uptake and lactate and proton excretion 20 .…”

“…There is now very clear scientific evidence, defining the metabolic and signalling pathways underlying functional neutrophils 24 and those subjected to C5a-induced dysfunction 12 , 13 , 20 , 25 . Thus, ligation of C5aR1 (CD88) by C5a, activates its inherent G-protein activity resulting in phosphoinositide 3-kinase delta (PI3Kδ) activation inhibiting activation of the small GTPase RhoA, actin polymerisation and phagocytosis 13 .…”

“…Thus, speculation on linezolid’s targets must draw evidence from C5a signalling processes and emphasise the importance of the C5aR1 (CD88) cell surface receptor, the PI3Kδ-RhoA pathway, phagosome maturation and modulators of glycolytic flux such as glucose transporters and pHi 12 , 13 , 20 , 25 . Critically, Wood et al alluded to the timing of exposure to C5a as important in downstream effects 25 . They find that C5a only impairs phagocytosis if the cells are exposed to it before they encounter S. aureus —not at the same time or after initial exposure.…”