Inhibitor of apoptosis proteins as regulatory factors in the normal and inflamed airways.

Abstract

Asthma is potentiated by complex gene-environment interactions, and characterised by inflammation and degenerative changes to the conducting airways. Current therapeutics targeting the inflammation and bronchoconstriction are restricted to prophylactic effects. The airway epithelium is known to participate in the pathogenesis of asthma, and represents a therapeutic target. Airway epithelial cells (AEC) from asthmatics exhibit apoptotic changes, which correlate with disease-associated factors presented by the epithelium. However, the mechanisms which cause the apoptosis are not well defined. In particular, there has been little study of the role of the family of Inhibitor of Apoptosis Proteins (IAPs) in models of AEC apoptosis. The over-arching hypothesis in this thesis is that anomalies in one or more of the IAPs contribute to inflammation-induced AE apoptosis in asthma. Experiments in this thesis explored the role of XIAP, cIAP1 and cIAP2 in asthma, models of asthma-related inflammation, and genetic susceptibility to asthma. The major methods used in these experiments included cell culture of primary AEC from both asthmatics and controls with/without treatment with IFNγ and TNFα. siRNA knockdown, qPCR, western blotting, immunocytochemistry, functional caspase assays, and genotyping. The major findings of this study are i) surprisingly, primary AECs do not undergo apoptosis after prolonged exposure to the proinflammatory cytokines IFNγ and TNFα ex vivo; ii) rather, IFNγ elicits a proapoptotic state in AECs, evidenced by, partial processing of procaspase-3, the absence of Poly (ADP-ribose) polymerase (PARP) cleavage, an increased Bax:Bcl2 transcript ratio, and the absence of morphological changes associated with apoptosis; iii) both XIAP and cIAP1 were constitutively expressed in AEC, and protein levels were unaffected by cytokine treatment. In contrast cIAP2, initially weakly expressed, was strongly inducible by cytokine treatment; iv) No differences were observed between AEC from asthmatics and controls in terms of either basal IAP gene expression levels or their response to cytokine treatment; v) siRNA-mediated depletion of cIAP2- transcripts allows AEC to progress into apoptosis after extended culture, conditions which also resulted in a decrease in both cIAP1 and Bcl2; vi) genetic polymorphism in the genes encoding, XIAP cIAP1 and cIAP2 do not associate with susceptibility for asthma. However, cIAP1 polymorphism may modulate disease severity within asthmatics. This thesis contributes to the knowledge of IAPs and apoptosis in asthma, and provides evidence that they are important for sustaining AEC survival, and participate within a cooperative of endogenous regulators of apoptosis. There is no evidence of intrinsic dysregulation of IAPs in asthma, yet cIAP1 polymorphism may modulate asthma severity, and IAPs are central in maintaining a proapoptotic state in AECs exposed to asthma related cytokines. Epithelial activation and damage, coupled with non-progression to apoptosis may contribute to fragility of the AE observed in asthma. Therapies targeting the IAPs may therefore provide a means of ameliorating the disease by allowing AECs to progress into apoptosis.