Dusart, PJ; (2014) The effects of Neisseria meningitidis infection on endothelial E-selectin, and consequences for neutrophil adhesion and transmigration. Doctoral thesis , UCL (University College London).

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Abstract

Severe septicaemia due to the bacterium Neisseria meningitidis is characterised by substantial recruitment and activation of neutrophils on inflamed endothelium, and is associated with vascular damage. N.meningitidis associates with endothelial cells, E-selectin, and neutrophils in patient biopsies, and E-selectin can co-localise beneath adherent bacteria in vitro. This thesis aims to investigate whether N.meningitidis affects E-selectin distribution on the endothelial surface and whether meningococcal-E-selectin interactions have functional consequences on neutrophil rolling, adhesion and transmigration across vascular endothelium. Lentiviral constructs were created containing full-length E-selectin (ES) or a cytoplasmic domain deficient mutant (ΔC). Transfection of primary HUVEC demonstrated that ES E-selectin forms spontaneous clusters, while ΔC is expressed in a diffuse membrane pattern. Capsulated N.meningitidis was unreliable at inducing E-selectin clustering and co-localisation. In contrast, an unencapsulated SiaD- strain could co-localise with endogenous IL-1β induced E-selectin, although not with transfected ES or ΔC. Thus the E-selectin cytoplasmic domain affects both the molecule’s distribution on the endothelial membrane, and also N.meningitidis mediated redistribution, which additionally requires some factor present on IL-1β stimulated, but not ES or ΔC transfected, endothelium. Functional aspects of N.meningitidis interactions with E-selectin on neutrophil behaviour were also investigated. Brief co-culture of N.meningitidis with ES transfected HUVEC induced increased neutrophil adhesion under static conditions and greater transmigration under physiological flow. Neutrophil adhesion to N.meningitidis stimulated ΔC transfected HUVEC however, was no greater than untransfected cells. ΔC HUVEC also only saw a small increase in transmigration despite having comparable adhesion and rolling velocity to ES cells under flow. Taken together these results show that the E-selectin cytoplasmic domain has an important yet unstudied role in neutrophil adhesion and transmigration. These data should lead to a better understanding of the underlying processes surrounding neutrophil mediated endothelial damage caused by N.meningitidis, and could have broader relevance for other inflammatory responses, particularly in sepsis.

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