Investigating neutrophil phenotype and migration mechanisms in the tissue draining lymph node during acute pulmonary infection with Streptococcus pneumoniae

Abstract

Neutrophils are innate immune cells that form part of the first line of defense against pathogens. These cells are rapidly mobilised in response to infection and inflammation and are equipped with a range of pathogen destruction mechanisms. In recent years it has been demonstrated that neutrophils can also modulate innate and adaptive immune responses. Neutrophils have been found to enter secondary lymphoid organs including lymph nodes, however the phenotype and function of neutrophils in lymph nodes is not well understood. Work in this thesis utilised an acute pulmonary infection model with the lung pathogen Streptococcus pneumoniae, to investigate neutrophil recruitment, phenotype and function in the lung draining (mediastinal) lymph node. It was hypothesised that neutrophil phenotype and behaviours are not cell intrinsic, rather dictated by the localised microenvironment of the cell. To investigate this hypothesis a combination of flow cytometry, immunohistochemistry, quantitative PCR and four dimensional multiphoton explant imaging was used. Neutrophil recruitment to the lung draining lymph node was rapid, peaking at 6 - 12 hours post infection. Neutrophils were localised in the lymphatic sinus, specifically in areas with medullary macrophages. Neutrophil migration in this region was highly dynamic, with swarms of neutrophils observed. When compared to blood, lung and airway neutrophils, lymph node neutrophils showed an intermediate activation profile, similar to lung tissue neutrophils. Interestingly a small population of lymph node neutrophils expressed markers of antigen presenting cells. Neutrophils in the lymph node utilised mainly chemotactic migration mechanisms involving phosphatidylinositde 3-kinase signalling and leukotriene B4, however migration was not completely integrin independent. Thus the results support the hypothesis that neutrophil phenotype and behaviours are environment dependent as opposed to cell-intrinsic, also they demonstrate and support the evolving view of the neutrophil as a complex cell type that has multiple functional profiles and mechanisms of migration