Modulation of inflammatory cell apoptosis in infection-associated inflammation

Neutrophils are a central component of the innate immune system, whose major role is to defend the host against invading microorganisms. As such they are integral players in the process of inflammation, the response of vascular tissues to injury. They are frequently the first immune cells recruited from the systemic circulation into a site of tissue injury or infection where they themselves play a key antimicrobial role. Direct killing of microbes can be accomplished by phagocytosis, degranulation, production of reactive oxygen species (ROS) or the release of DNA and antimicrobial peptides into the extracellular milieu (NETosis). In addition neutrophils orchestrate the recruitment and activation of other leucocytes, further contributing to host defence. The central importance of neutrophils in immunity is revealed by defects in either number or function leading to recurrent life threatening infection. However, as the toxic arsenal of neutrophil constituents lack specificity they can also be damaging to surrounding host tissues causing exacerbated inflammation. It is therefore essential that neutrophil function is tightly controlled to allow an appropriate response to be mounted against invading pathogens while simultaneously minimising host tissue injury. Therefore, once the inciting inflammatory insult has been successfully cleared or controlled it is imperative that these non-tissue resident specialised immune cells are rapidly ‘switched off’ or cleared to allow the return to homeostasis. This resolution phase of the inflammatory cascade is now recognised as an energy dependent, finely controlled endogenous process, the beginnings of which are activated at the onset of inflammation. One of the main aims of resolution is to ensure efficient clearance of leucocytes that are no longer necessary. It is likely that a major clearance route is by the highly regulated and energy dependent processes of neutrophil programmed cell death (apoptosis) with subsequent uptake and disposal of apoptotic neutrophils by tissue macrophages. This process of neutrophil apoptosis renders the neutrophils nonfunctional and preserves cell membrane integrity, thus preventing further release of histotoxic neutrophil-derived inflammatory mediators into the extracellular environment. Furthermore, the recognition, uptake and disposal of apoptotic neutrophils cause a dynamic change in the phagocytosing macrophage phenotype with alterations in inflammatory mediator production. The fundamental importance of neutrophil apoptosis and subsequent efferocytosis in inflammation resolution is highlighted by the pathological consequences of neutrophil necrosis or failed apoptotic cell clearance, which leads to enhanced tissue injury and autoimmunity. Acute lung infection (pneumonia) is a common and serious condition affecting both developed and developing countries; globally, childhood pneumonia is the leading cause of death in children aged less than 5 years and pneumonia is the most common fatal infection in the developed world. In over half of patients with community acquired pneumonia no causative organism is ever isolated suggesting that although the immune response has successfully controlled infection, continued uncontrolled neutrophilic inflammation in the lung continues to cause morbidity and mortality. Indeed, pneumonia frequently progresses to acute respiratory distress syndrome (ARDS), a devastating acute inflammatory condition of the lungs characterized by inflammatory cell recruitment and accumulation of protein rich oedema fluid leading to impaired lung function. ARDS affects 200,000 critically ill patients in the USA per year, and has a substantial mortality rate of up to 40%. Despite advances in intensive care treatment and antimicrobial therapy mortality from pneumonia has not fallen since the 1950s, and at present there are no specific therapies for infection-related lung inflammation or ARDS. Understanding the mechanism behind such uncontrolled, persisting inflammation, and the need for novel approaches to target infection related lung injury are therefore both urgent and essential. This thesis examines the potential of neutrophil apoptosis-inducing pharmacological agents as potential treatments for infection-associated lung inflammation. The primary agents used include a cyclin-dependent kinase inhibitor as well as plant-derived polyphenolic flavones. The ability of these compounds to induce human neutrophil apoptosis in vitro, the key importance of the intracellular neutrophil survival protein Mcl-1 in mediating this process, and the effect of targeting Mcl-1 in human macrophages is investigated. In addition, neutrophilic inflammation is modelled in zebrafish and mice with both sterile and bacterial-driven models of inflammation. A key role for Mcl-1 is delineated in vivo, with it acting as an endogenous controller of the innate immune response by influencing neutrophil apoptosis, but without effects on macrophage apoptosis or ability to phagocytose apoptotic cells. Driving neutrophil apoptosis by down-regulation of Mcl-1 accelerates resolution of inflammation in vivo. This therapeutic approach is also found to have indirect anti-bacterial effects in a model of E. Coli induced pneumonia, in stark contrast to established anti-inflammatory approaches which routinely cause immune paresis and life threatening infection. As such, targeting inflammatory cell apoptosis by changes in Mcl-1 offers a potential new therapeutic approach for the treatment of infection-associated inflammation