Platelet recruitment to lungs following inhalation of bacterial lipopolysaccharides

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract


Background 
Platelets contribute to disease-relevant inflammatory processes, and research indicates that platelet recruitment to inflamed lungs might be important in the platelet-dependence of inflammatory responses. Compared to platelet recruitment to lesions in blood vessel walls or to thrombi, the mechanisms of platelet recruitment to lungs following inflammatory responses in lung airpaces are poorly understood, so methods were sought to enable the characterisation of this response in mouse and human models. 

Methods 
Bright field and fluorescence microscopy for quantifying immunostained platelets, non-invasive radiolabelled platelet tracking, radiolabelled platelet biodistribution, lung intravital multiphoton microscopy with genetic platelet labelling, and blood microsample platelet counts were assessed as methods for measuring and describing lung platelet recruitment in a mouse model of lung inflammation induced by intranasal challenge with bacterial lipopolysaccharides (LPS). Platelets were also immunostained in human lung samples exposed to LPS via intrabronchial installation during ex vivo blood-perfused lung experiments. The effects of neutrophil depletion, blockade of the adhesion molecules P-selectin or PSGL-1, and treatment with the anti-platelet drugs MRS2500, AR-C 66096 or aspirin on LPS-induced lung platelet recruitment were then investigated in mice. 

Results 
Immunostaining and radiolabelled platelet tracking methods showed that LPS inhalation caused platelet recruitment to lungs of mice, and intravital microscopy revealed that LPS inhalation increased platelet adhesion without causing thrombosis. Radiolabelled and immunostained platelets were also found in airspaces of LPS-challenged mice and human lungs. The lung platelet recruitment response was preceded by a decrease in blood platelet counts associated with recruitment of platelets to the liver and spleen. Platelet recruitment to mouse lungs was not detectably altered by neutrophil depletion, blockade of PSGL-1 or P-selectin, or treatment with MRS2500, AR-C 66096, or aspirin. In this model, lung neutrophil recruitment was reduced with neutrophil depletion or PSGL-1 blockade but not with other interventions tested. Platelet depletion did not significantly alter neutrophil counts in bronchoalveolar lavage in the mouse model used but did reduce the quantity of neutrophil extracellular traps detected in lung airspaces. 

Conclusions 
Platelet recruitment to mouse lungs following LPS inhalation showed no clear evidence of dependence on neutrophils or PSGL-1, suggestive that platelet recruitment occurs through mechanisms distinct from those driving lung neutrophil recruitment. Platelet recruitment to lungs inflamed by LPS inhalation involved increased platelet adhesion not extending to thrombosis, and migration of platelets into lung airspaces in mice and in human lungs, with interactions of platelets with the release of neutrophil extracellular traps in lung airspaces identified. The immunostaining, radiolabelling and intravital microscopy methods described in this thesis allow for further investigation into the molecular mediators and physiological consequences of platelet recruitment to inflamed lungs.
Date of Award1 Dec 2017
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorSimon Pitchford (Supervisor) & Clive Page (Supervisor)

Cite this

'