An unbiased proteomic approach to identifying cell surface markers in neutrophils

Introduction: Efferocytosis refers to the engulfment of dead cells by phagocytes such as neutrophils and macrophages and culminates in the beneficial removal of inflammatory cargo and auto-reactive material. While many of the mechanisms involved in efferocytosis have been described, the critically important ‘apoptotic-cell-associated molecular patterns’ have yet to be identified. Methods: Human whole blood samples were obtained from healthy volunteers. Neutrophils were isolated and subjected to a temperature shift to facilitate a wave of synchronised apoptosis. The plasma membrane proteome was assessed using tandem-mass-tag liquid chromatography-mass spectrometry (TMT-LC/MS) of apoptotic and time- and donor- matched non-apoptotic neutrophils, which allowed the identification of the plasma membrane proteins expressed or downregulated during apoptosis. In parallel, a method was developed to quantify efferocytosis. Briefly, apoptotic neutrophils or genome-modified neutrophil-like cells overexpressing the proteins identified to be up- regulated on apoptotic neutrophils, were fed to the monocytic cell line THP-1, which had been previously primed with phorbol-12-myristate-13-acetate (PMA) to acquire the functional properties of human macrophages. Efferocytosis was assessed by confocal microscopy and flow cytometry. Results: The late-stage apoptotic neutrophil plasma membrane profile identified several protein targets to be either up- or downregulated. The expression kinetics over time of ‘targets’ were then validated using flow cytometry. TIMD-4, a never previously described protein in (human) neutrophils, is actively up- regulated in preparation for apoptosis, and links to the phagocyte’s recognition and removal abilities of apoptotic cells, suggesting that TIMD-4 is a novel ‘eat me’ signal produced by apoptotic neutrophils. Conclusions: TMT-LC/MS can be successfully used to determine functionally relevant changes in the neutrophil plasma membrane, and has provided novel insights into the processes underlying efferocytosis of human neutrophils.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Subburayalu, Julien

Advisor dc:contributor.advisor

• Summers, Charlotte

• Apoptosis
• Eat-me signal
• Efferocytosis
• HL-60
• Macrophage
• Neutrophil
• Plasma membrane profiling
• Resolution of Inflammation
• THP-1
• TIMD-4

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3f6a1904-155c-47e5-8ec1-3100edc44155/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0000-0001-9243-0558

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/359987