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oai:www.repository.cam.ac.uk:1810/3599872024-11-08T01:45:09Zcom_1810_219479com_1810_34581col_1810_219488

An unbiased proteomic approach to identifying cell surface markers in neutrophils https://doi.org/10.17863/CAM.103277 Subburayalu, Julien 0000000192430558 Summers, Charlotte 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. University of Cambridge 2022-07-03 Thesis Doctoral Doctor of Philosophy (PhD) eng AstraZeneca/MedImmune (RCAG/891) NIHR Cambridge Biomedical Research Centre German Academic Scholarship Foundation (Studienstiftung des deutschen Volkes) https://www.repository.cam.ac.uk/handle/1810/359987 embargo 2026-11-09 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/53a38d4f-0f77-4f15-9697-9c93b44ab76f/download 07ed17e8fa584e0f0c19ec1f0d86709b https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3f6a1904-155c-47e5-8ec1-3100edc44155/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ Apoptosis Eat-me signal Efferocytosis HL-60 Macrophage Neutrophil Plasma membrane profiling Resolution of Inflammation THP-1 TIMD-4