Investigation of the role of Mcl-1 and Mer in the regulation of eosinophil apoptosis and efferocytosis

Abstract

Regulation of the inflammatory response is essential for the successful resolution of inflammation, and restoration of normal tissue homeostasis. Eosinophils are granulocytic cells of the innate immune system historically considered to be primarily involved in the defence against parasitic infection. Eosinophils are also key effector cells in the allergic inflammatory response, initiation of which is associated with the recruitment and activation of eosinophils culminating in the release of their intracellular granule contents. Eosinophil granules contain a range of cytotoxic proteins (major basic protein, eosinophil cationic protein and eosinophil peroxidase) that act to destroy infectious and parasitic organisms. However, these cytotoxic proteins can also cause damage to surrounding host tissue cells.

The resolution of the inflammatory response acts to limit the extent of eosinophil-mediated tissue damage. Programmed cell death (apoptosis) of eosinophils represents an important component of this resolution process, limiting release of granule contents and triggering efferocytosis (the removal of apoptotic cells by phagocytes). Apoptosis is initiated by the activation of intracellular caspases, a family of cysteine proteases. Caspase activation primarily occurs as a result of changes in the balance of intracellular pro- and anti-apoptotic Bcl-2 family proteins. Mcl-1, an anti-apoptotic Bcl-2 protein has been shown to play a pivotal role in the regulation of neutrophil apoptosis. Pharmacological down-regulation of Mcl-1 initiates apoptosis and promotes the resolution of neutrophil-dominant inflammation. The importance of Mcl-1 in the regulation of apoptosis was shown using cyclin-dependent kinase inhibitors (CDKis), where induction of neutrophil apoptosis by CDKis was due to down-regulation of intracellular Mcl-1. Apoptotic cells display distinct surface molecules known as ‘eat-me’ signals that identify them for phagocytosis by macrophages and other phagocytes. One key receptor involved in the removal of apoptotic cells from tissue is the receptor tyrosine kinase Mer, a member of the Tyro3/Axl/Mer (TAM) family, which recognises the ‘eat me’ signal phosphatidylserine expressed on apoptotic cells. In the absence of Mer expression, clearance of apoptotic cells is compromised delaying the resolution of neutrophil-dominant inflammation. However, the roles of Mcl-1 and Mer in eosinophil apoptosis and clearance, respectively, and the resolution of allergic inflammation are not known.

Asthma is a chronic inflammatory lung disease characterised by shortness of breath, airway obstruction, wheeze, non-specific bronchial hyper-responsiveness, excessive airway mucus production and an eosinophil dominant inflammatory infiltrate. The persistent presence of eosinophils in the lung, in chronic asthma, is likely due to a combination of excessive eosinophil recruitment and activation together with impaired eosinophil apoptosis. Investigation into the underlying mechanisms of these processes in allergic airway disease is of critical importance, as blocking eosinophil recruitment and/or promoting eosinophil apoptosis could provide a therapeutic approach to reduce associated eosinophil-mediated tissue damage. Understanding the regulation of eosinophil apoptosis and phagocytic clearance may identify novel pharmacological targets to enhance the resolution of allergic inflammation.

We hypothesise that Mcl-1 and Mer play vital roles in the successful resolution of allergic airway inflammation. To investigate this hypothesis, we have used pharmacological and genetic manipulation of intracellular eosinophil Mcl-1 levels, and phagocyte Mer expression, to determine the role they play in the regulation of eosinophil apoptosis and phagocytic clearance of apoptotic eosinophils, respectively. Human and mouse eosinophils were cultured, and rates of constitutive and CDKi-induced apoptosis were determined, to investigate eosinophil apoptosis in vitro. Mice expressing human Mcl-1 (hMcl-1) were used to determine the effect of over-expression of Mcl-1 on eosinophil viability in vitro. The effect of hMcl-1 on eosinophil viability and disease severity in vivo was determined using an ovalbumin-induced model of allergic airway inflammation, which mimicked the symptoms of human asthma. Apoptotic eosinophils were co-incubated with macrophages in vitro to investigate the capacity for phagocytosis by different macrophage populations. Apoptotic cell clearance was further investigated using a Mer-kinase-dead mouse, which lacked Mer expression, to determine the role of Mer-dependent phagocytosis on the process of resolution of inflammation in vivo. Over-expression of Mcl-1 in eosinophils significantly delayed both constitutive and CDKi-induced apoptosis in vitro. In vivo in the ovalbumin-induced model of allergic airway inflammation, over-expression of Mcl-1 resulted in a significantly increased number of eosinophils in the lung and delayed rate of resolution of allergic airway inflammation. Alveolar and bone marrow-derived macrophages exhibited Mer-dependent phagocytosis of eosinophils, which was significantly reduced by an inhibitor of Mer kinase activity (BMS777607) or lack of macrophage Mer expression. The absence of Mer expression resulted in a significant increase in the number of apoptotic eosinophils in the lung together with a delayed rate of resolution of allergic airway inflammation in vivo.

Together this work has shown that delayed rates of eosinophil apoptosis and impaired phagocytic clearance both delayed the resolution of allergic airway inflammation. These data suggest that both Mcl-1 and Mer are pivotal for the successful regulation of eosinophil apoptosis and phagocytic clearance of apoptotic eosinophils in asthma and may provide attractive novel therapeutic targets.