Record, Julien Arnaud; (2018) Neutrophil migration in primary immunodeficiency. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Neutrophils are the first cells to reach infection sites and are able to kill pathogens. Therefore, their ability to quickly migrate to infection loci is essential and rely on actin structures called the lamellipodium at the front of the cell and the uropod at the rear. Actin cytoskeleton dynamics are crucial for the formation of these structures, and mutations in actin cytoskeleton regulators in immune cells can result in severe primary immunodeficiencies. These defects are good models to understand the role of actin regulators in neutrophil migration. To allow accurate monitoring of neutrophil migration, a new protocol, based on the Dunn chamber system, was developed. In particular, the stabilization of the chemoattractant gradient over long durations was achieved by embedding it in agarose, which allowed to reliably monitor neutrophil migration and was used throughout the different projects. It was first employed to assess the effect of constitutively activated Wiskott-Aldrich syndrome protein (CA-WASp) which is responsible for an excess of polymerized actin throughout the cytoplasm. Neutrophils expressing CA-WASp displayed a reduced migration speed together with an increased contractile activity. The development of this system further allowed us to identify a neutrophil migratory defect and correlate it to a mutation in Megakaryoblastic Leukemia 1 (MKL1). MKL1 is a transcription cofactor which regulates the expression of numerous actin genes. Study of MKL1 knockdown in neutrophils showed that MKL1 deficient neutrophils displayed severely impaired migration and dramatically reduced levels of globular and polymerised actin. MKL1 deficiency also altered the expression of numerous other actin cytoskeleton regulators indicating that the migratory defect was the consequence of the alteration of several actin cytoskeleton components. Finally, the potential for the modified Dunn chamber protocol to be used as a routine assay to monitor the migration of neutrophils from patients with unknown causes of immunodeficiency was assessed.

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