Anti-inflammatory mechanisms of the neutrophilreleased antimicrobial peptide α-defensins
Tomlinson, Gareth Hugh
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Tissue homeostasis is necessary for optimal organ functioning. The onset of tissue trauma compromises the homeostatic environment resulting in widespread cell death with the likelihood of exposure to invading micro-organisms. Early stage elimination of microbes and immunomodulation is co-ordinated by leukocytes of the innate immune system of which neutrophils and macrophages play a pivotal role. Leukocyte-released pro-inflammatory factors are vital in the containment of infection but bring with it a degree of collateral tissue destruction. Thus cascading stages during inflammation must be tightly regulated to bring about timely tissue regeneration and regained homeostasis. However, chronic inflammatory diseases e.g. rheumatoid arthritis highlights the existence of defective regulation at numerous stages during this transition, often leading to debilitating disease progression. Recently published findings by our research group identified the anti-inflammatory properties of α-defensin - an anti-microbial peptide released from dying human neutrophils - on stimulated macrophages. Thus the main objective of my research was to gain an understanding into the molecular actions of α-defensins which inhibit the macrophage inflammatory potential. Strong evidence supported the propensity of α-defensins to inhibit both intracellular and secreted protein synthesis, as assessed by de novo 35S-radiolabeled Methionine incorporation. Inhibition was not attributed to endoplasmic reticulum stress events, a common diagnosis in the regulation of global translation. Supporting evidence using cell-free systems identified a fundamental block in translation with the inclusion of α-defensin. Biochemical studies linked the ability of α-defensin to bind non-specifically to oligonucleotide sequences. This binding potential was also demonstrated on ribosomal RNA (rRNA), impeding its migration through electrophoretic gels. Immunocytochemical assays proposed an emerging suggestion of α-defensins in macrophages concentrated in close proximity to ribosomes around the perinuclear region. Evidence of suggested defensin/ribosome accumulation after 24hrs after treatment were attempted but to date remained unconfirmed. Attempts to determine the fate of these proposed accumulations were inconclusive, assessed by autophagy assays and ribosome semiquantitation. This thesis describes for the first time an enhanced understanding into the intracellular inhibitory mechanisms of α-defensins on macrophages and possibly other cell types. Understanding the molecular impact of α-defensins provide key insights into this novel inflammatory regulator, with the potential to be utilized in future immunotherapies.