The Effect of Hypoxia on Neutrophil Survival and Function

Tissue levels of oxygen are well below atmospheric levels (21% O2) and in normal conditions are around 5-10 % O2. In conditions of high oxygen consumption or at a distance from the blood vessels, oxygen can become limiting and hypoxic conditions (0-3% O2) are commonly seen. Hypoxia is also a characteristic of sites of inflammation and could affect the function of the immune cells at these sites. This thesis specifically addresses how hypoxia affects the function and lifespan of neutrophils. Neutrophils are the first immune cells to respond to inflammatory signals. These cells play a key role in protection against microbial pathogens and in the wound healing process. As part of the microbicidal process, neutrophils generate large amounts of superoxide, consuming oxygen in the formation of this oxidant. Neutrophils also contain a wide array of potentially toxic enzymes such as myeloperoxidase (MPO), elastase and neutral proteases that are able to damage the host tissues, and therefore need to be contained within the cell. Hence, proper clearance of neutrophils is needed for tissue homeostasis and for the safe resolution of inflammation. The mechanism of neutrophil cell death is an important factor in the proper resolution of inflammation. Previously, it has been shown that neutrophils undergo delayed apoptosis under hypoxic conditions but the eventual fate of hypoxic neutrophils has not been studied. It has been assumed that delayed apoptosis gives the neutrophil a survival advantage under hypoxia. However, detailed analysis of extended survival and the eventual cell death pathway has not been studied. My hypothesis is that hypoxia will alter the death pathway away from apoptosis and that hypoxic exposure may affect neutrophil cell function. The purpose of this thesis was to investigate the effect of transient and sustained hypoxia, under stimulatory and non- stimulatory conditions, on neutrophil function and survival. I found that unstimulated neutrophil survival increased under mild to severe hypoxia. The morphological features associated with apoptosis were not seen under hypoxic conditions. Phosphatidylserine exposure, a marker of early apoptosis, was also decreased, but caspase 3, which is a hallmark of apoptosis, was activated under hypoxic conditions. Time lapse microscopy for caspase 3 in hypoxic neutrophils confirmed it was activated in all neutrophils. Electron microscopy images showed clear morphological changes in hypoxic neutrophils suggesting neutrophils are directed away from apoptosis, with activation of a different pathway. Several lines of evidence support the induction of autophagy: hypoxia inducible factor-1α (HIF-1α) was stabilised in hypoxic neutrophils; there was increased expression of BNIP3, a downstream gene of HIF-1 that regulates autophagy; electron microscopy images showed autophagosome formation; Western blotting and immunofluorescence demonstrated an increase in the phosphatidylethanolamine conjugated form of Microtubule-associated protein 1A/1B-light chain 3 (LC3 II), a marker of autophagy. I also studied the effect of hypoxia on stimulated neutrophils. A novel function of neutrophils which has recently been extensively studied is the formation of Neutrophil Extracellular Traps (NETs). NETs are complex chromatin structures released from neutrophils in response to a wide variety of stimuli including phorbol myristate acetate (PMA) and opsonised bacteria. Oxidant production is an important driver for NET formation and how hypoxia affects this process was investigated in this thesis. Under sustained hypoxia NET formation was decreased, presumably due to lack of oxidant generation. Pre-exposure to hypoxia also inhibited NET formation despite there being enough oxygen to support oxidant production. This suggests that in a hypoxic in vivo situation, neutrophil NET formation might be affected due to transient hypoxia. Morphology studies showed significant vacuole formation in PMA-stimulated neutrophils under normoxic and hypoxic conditions, and a significant decrease in necrotic neutrophils was observed under hypoxic conditions. I also investigated the effect of transient hypoxia on stimulated neutrophils. There was minimal superoxide production when oxygen was limiting, and this is likely to be due to a reduction in oxygen supply for the NADPH oxidase. When the cells were returned to a normoxic environment, superoxide production was measurable. Under these conditions it was apparent that extended hypoxic incubation did not result in increased functional capacity with respect to oxidant production. Also no difference in bacterial killing was observed between normoxic and hypoxic neutrophils, immediately after exposure to hypoxia Therefore, my results have shown that although neutrophils survive for longer periods in a hypoxic environment an increase in functional capacity is not observed. In summary, the results in this thesis show that exposure of neutrophils to hypoxia results in induction of autophagy and that their functional capacity is not retained. The findings reported in this thesis are relevant to many inflammatory situations where hypoxia exists, such as acute respiratory disease, stroke and myocardial infarction. Neutrophils are present in other hypoxic tissues, including tumours and fat tissue and their function in these environments might be similarly affected by hypoxia