Light microscopy applied to study defective macrophage phagocytosis in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a progressive lung condition characterised by airflow limitation, primarily caused by cigarette smoking and other air pollutants, which results in irreversible damage to the lungs. This leads to alveolar destruction, reduced lung elasticity, narrowing of the small airways and fibrosis, and is associated with an increased number of phagocytic immune cells, including macrophages and neutrophils. Despite increased number of phagocytes, approximately half of COPD patients exhibit bacterial colonisation that are associated with increased numbers of exacerbations that often lead to hospitalisation. Bacterial colonisation may be explained, in part, by defective phagocytosis of pathogens by macrophages, although the mechanism involved is unclear. Light microscopy is widely used to study cell biology, although the ability to image cellular processes within cells and microorganisms is conventionally limited by the diffraction of light, as well as additional considerations of signal to noise that impacts the imaging speed and contrast. However, advances in light microscopy, including computational and physical techniques, to improve spatial resolution and image contrast prompted the investigation of their potential use in exploring phagocytosis. This project initially explored the potential of super-resolved microscopy and computational techniques, to allow image acquisition at a resolution of less than 20nm. These techniques can be used to identify and image sub-cellular proteins and cellular processes previously unobtainable, including phagocytosis. This thesis aimed to utilise live, cell imaging and super-resolved microscopy techniques to identify differences in morphology, cytoskeletal proteins, and mitochondria in macrophages. The most practically useful technique was found to be wide-field epifluorescence imaging with computational deconvolution, which was applied to study macrophages from COPD subjects, smokers and non-smoking control subjects with single cell analysis. This work confirmed that COPD macrophages were poorer at phagocytosing bacteria, and this was associated with structural changes in COPD macrophages. Using high-resolution microscopy, COPD macrophages were shown to be larger compared to non-smoker cells, with significantly longer microtubules observed in both lung tissue-derived and blood monocyte-derived macrophages (MDM), together with a reduction in actin-containing podosomes. Mitochondria function can be a key factor that affects cytoskeletal restructuring in cells, with the mitochondria membrane potential being important in the function of ATP generation, with mitochondria under normal conditions displaying a polarised phenotype, whereby they are highly negatively charged, due to the influx of electrons required for the electron transport chain. However, a significantly depolarised mitochondria is the result of a more positively charged membrane potential, which can result in impaired ATP production and cause cell death, which is associated with several pathologies. Work performed in this thesis using microscopy explored mitochondrial differences in COPD MDM, with a significantly depolarised mitochondrial membrane potential (ΔΨm) compared to non-smoker controls, but no difference in mitochondria reactive oxygen species (mROS). Mitochondrial antioxidants, SkQ1 and PQQ, were able to improve bacterial phagocytosis in COPD MDM by up to 60% but had no effect on ΔΨm or mROS. Work in this thesis has shown the use of microscopy and single cell image analysis can detect differences in key structural proteins and the mitochondria, whilst capable of being used as a screening tool for phagocytic correctors such as mitochondrial antioxidants, which were shown to increase phagocytosis with no alterations in mitochondrial function.Open Acces

A novel Streptococcus pneumoniae human challenge model demonstrates Treg lymphocyte recruitment to the infection site

To investigate local tissue responses to infection we have developed a human model of killed Streptococcus pneumoniae challenge by intradermal injection into the forearm. S. pneumoniae intradermal challenge caused an initial local influx of granulocytes and increases in TNF, IL6 and CXCL8. However, by 48 h lymphocytes were the dominant cell population, mainly consisting of CD4 and CD8 T cells. Increases in local levels of IL17 and IL22 and the high proportion of CD4 cells that were CCR6+ suggested a significant Th17 response. Furthermore, at 48 h the CD4 population contained a surprisingly high proportion of likely memory Treg cells (CCR6 positive and CD45RA negative CD4+CD25highCD127low cells) at 39%. These results demonstrate that the intradermal challenge model can provide novel insights into the human response to S. pneumoniae and that Tregs form a substantial contribution of the normal human lymphocyte response to infection with this important pathogen

A novel Streptococcus pneumoniae human challenge model demonstrates Treg lymphocyte recruitment to the infection site

To investigate local tissue responses to infection we have developed a human model of killed Streptococcus pneumoniae challenge by intradermal injection into the forearm. S. pneumoniae intradermal challenge caused an initial local influx of granulocytes and increases in TNF, IL6 and CXCL8. However, by 48 h lymphocytes were the dominant cell population, mainly consisting of CD4 and CD8 T cells. Increases in local levels of IL17 and IL22 and the high proportion of CD4 cells that were CCR6+ suggested a significant Th17 response. Furthermore, at 48 h the CD4 population contained a surprisingly high proportion of likely memory Treg cells (CCR6 positive and CD45RA negative CD4+CD25highCD127low cells) at 39%. These results demonstrate that the intradermal challenge model can provide novel insights into the human response to S. pneumoniae and that Tregs form a substantial contribution of the normal human lymphocyte response to infection with this important pathogen