Multi-omic Studies of Neutrophils Reveal Programmed Deactivation Phenotypes After Inflammatory Stimulus
Neutrophils serve as a critical first line of defence against bacterial invasion, and their dysfunction is associated with unfavourable clinical outcomes. A detailed understanding of the activation and resolution pathways that modulate neutrophil interactions with bacteria, as well as the alterations that occur in dysfunctional states, will help develop the existing knowlege on the pathobiology of infection. This knowledge may also pave the way for the development of innovative, non-antibiotic therapeutic strategies. A comprehensive time-course multi-omics study was conducted to evaluate the neutrophil response to a common human pathogen Staphylococcus aureus. This was undertaken using a model of healthy human neutrophils exposed to heat-killed, labelled S. aureus particles with the objective of identifying candidate pathways that underpin the antimicrobial responses of these cells. The methodologies employed in this investigation include proteome, phosphoproteome, kinome, and transcriptome profiling. Data interpretation was facilitated through clustering and pathway enrichment analysis. This multi-omic approach was complemented by a multi-parametric assay of neutrophil functions. Analysis of the phosphoproteome revealed a bimodal response pattern characterized by peak phosphorylation or dephosphorylation at 15 and 120 minutes, indicating distinct early and late phases of response. Within the enriched pathways associated with phosphorylated proteins, phagocytosis and degranulation were prevalent, while mRNA processing emerged as the consensus term among dephosphorylated proteins. These findings were corroborated at the kinase level, where active kinases were predominantly engaged in bactericidal and transcriptional functions at 15 minutes. In contrast, a broad suppression of kinase activity was observed at 120 minutes. Complementary RNA sequencing data illustrated a biphasic response, with early transcripts (at 15 minutes) associated with the initiation of inflammation and late transcripts (at 120 minutes) linked to pathways promoting suppressive functions. The trajectory of inflammatory function is evident in the transcript levels, as the predominant transcripts at 30 and 60 minutes reflect the peak antimicrobial activity of neutrophils. Notably, the upstream regulator of neutrophil aging, BMAL1, was consistently expressed among the top transcripts at all time points. Functional assays indicated early phagocytic activity; however, whilst ingestion and phagosome acidification continued out to at least three hours, this occurred at a progressively slowing rate, broadly consistent with the -omics data. The initial -omics profile suggested that this slowing might represent a late deactivation phenotype. This hypothesis was evaluated through a double stimulus experiment, investigating the effect of an initial exposure on neutrophil capacity to process subsequent particulate or soluble stimuli (namely S.aureus, another human pathogen Escherichia coli and platelet activating factor(PAF)/f-met-leu-phi(fMLP). Flow cytometry results demonstrated a significant reduction in neutrophil capabilities to ingest and acidify a second particulate stimulus, with two hours of initial stimulation being the critical time frame where responses to the secondary stimulus diminished notably. This phenomenon was particularly pronounced when the initial and subsequent bacterial stimuli were homologous—i.e. S. aureus. However, when the secondary stimulus was a hetrologous, (E. coli), the attenuation was less marked. Additionally, a significant decrease in ROS production in response to PAF/fMLP was recorded. Further phosphoproteomic of the double stimulation experiments confirmed the previous findings. In summary, my findings indicate consistent signatures of neutrophil responses across three modalities, revealing early antimicrobial and transcriptional response to bacterial exposure that is subsequently followed by a programmed ‘deactivation’ state. This latter phase does not represent a return to baseline functionality; rather, it signifies a matured phenotype which depends on modified kinase activity and the active transcription of deactivating factors. The underlying mechanisms that contribute to this later state, and their implications for neutrophil function and the resolution of inflammation, will form the basis of future research.
Deactivation Phenotypes, Kinome Profiling, Multi-omics, Neutrophil, Proteomics, RNAseq, Staphylococcus aureus
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