C5a impairs phagosomal maturation in the neutrophil through phosphoproteomic remodeling.
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ABSTRACT:
SUBMITTER: Wood AJ
PROVIDER: S-EPMC7455072 | biostudies-literature | 2020 Aug
REPOSITORIES: biostudies-literature
Publications
C5a impairs phagosomal maturation in the neutrophil through phosphoproteomic remodeling.
Wood Alexander Jt AJ Vassallo Arlette M AM Ruchaud-Sparagano Marie-Hélène MH Scott Jonathan J Zinnato Carmelo C Gonzalez-Tejedo Carmen C Kishore Kamal K D'Santos Clive S CS Simpson A John AJ Menon David K DK Summers Charlotte C Chilvers Edwin R ER Okkenhaug Klaus K Morris Andrew Conway AC
JCI insight 20200806 15
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 signaling 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 s ...[more]
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Project description:Critical illness is characterised by organ failure, dysregulated immune activation and frequent secondary infections. Unbridled complement activation in these patients exposes immune cells to high levels of the anaphylotoxin, C5a. C5a suppresses antimicrobial functions of key immune cells, in particular the neutrophil, and this suppression is associated with poorer outcomes amongst critically ill adults. The intracellular signalling pathways which mediate C5a-induced neutrophil dysfunction are incompletely understood. Healthy donor peripheral blood neutrophils exposed to purified C5a demonstrated a prolonged defect in phagocytosis of the common nosocomial pathogen Staphylococcus aureus which persisted for 7 hours after exposure. Phosphoproteomic profiling of 2712 unique phosphoproteins identified persistent C5a signalling at 1 hour, 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 multi-function assay of bacterial ingestion and phagosomal acidification demonstrated C5a-induced impairment of phagosomal acidification in a whole blood model of Staphylococcal bacteraemia 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, late bacterial ingestion and killing of S. aureus in whole blood. This study provides a deep phosphoproteomic assessment of human neutrophil signalling in response to S. aureus, and demonstrates how some of these pathways are disrupted by exposure to C5a, identifying a defect in phagosomal maturation and providing new information on mechanisms of immune failure in critical illness.
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Project description:Urinary tract infections (UTIs) in men are uncommon yet carry an increased risk for severe pyelonephritis and other complications. In models of Escherichia coli UTI, C3H/HeN mice develop high-titer pyelonephritis (most with renal abscesses) in a testosterone-dependent manner, but the mechanisms underlying this phenotype are unknown. Here, using female mouse models, we show that androgen exposure impairs neutrophil maturation in the upper and lower urinary tract, compounded by a reduction of neutrophil function within the infected kidney, enabling persistent high-titer infection and promoting abscess formation. Following intravesical inoculation with uropathogenic E. coli (UPEC), kidneys of androgen-exposed C3H mice showed delayed local pro-inflammatory cytokine responses while robustly recruiting neutrophils. These were enriched for an end-organ-specific population of aged but immature neutrophils (CD49d+, CD101-). Compared to their mature counterparts, these aged immature kidney neutrophils exhibited reduced function in vitro, including impaired degranulation and diminished phagocytic activity, while splenic, bone marrow, and bladder neutrophils did not display these alterations. Furthermore, aged immature neutrophils manifested little phagocytic activity within intratubular UPEC communities in vivo. Experiments with B6 conditional androgen receptor (AR)-deficient mice indicated rescue of the maturation defect when AR was deleted in myeloid cells. We conclude that the recognized enhancement of UTI severity by androgens is attributable, at least in part, to local impairment of neutrophil maturation in the urinary tract (largely via cell-intrinsic AR signaling) and a kidney-specific reduction in neutrophil antimicrobial capacity.IMPORTANCEAlthough urinary tract infections (UTIs) predominantly occur in women, male UTIs carry an increased risk of morbidity and mortality. Pyelonephritis in androgen-exposed mice features robust neutrophil recruitment and abscess formation, while bacterial load remains consistently high. Here, we demonstrate that during UTI, neutrophils infiltrating the urinary tract of androgen-exposed mice exhibit reduced maturation, and those that have infiltrated the kidney have reduced phagocytic and degranulation functions, limiting their ability to effectively control infection. This work helps to elucidate mechanisms by which androgens enhance UTI susceptibility and severity, illuminating why male patients may be predisposed to severe outcomes of pyelonephritis.
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